Histone H3

Transcriptional Regulation

Since purified GAGA factor and TFIID interact similarly with the hsp70 and histone H3 promoters, the architecture of the endogenous H3 promoter has been analyzed to determine what interactions might be needed to establish a potentiated state containing a pause as seen in HSP70 promoter. Despite the detection of TFIID and GAGA on the H3 promoter, no paused polymerase as seen in the HSP70 promoter is evident. In addition, no proteins appear to interact with the transcription start. These results suggest that the GAGA factor and TFIID are not sufficient to establish a potentiated state containing paused polymerase and that TFIID interactions downstream from the TATA element could be important for pausing (Weber, 1995).

The abnormal oocyte (abo) gene of Drosophila is a peculiar maternal effect gene whose mutations cause a maternal-effect lethality that can be rescued by specific regions of heterochromatin during early embryogenesis. An increase in the dosage of specific regions of heterochromatin, denoted ABO, to either the mutant mother or the zygote, increases embryonic survival rates. abo encodes an evolutionary conserved chromosomal protein that localizes exclusively to the histone gene cluster and binds to the regulatory regions of such genes. A significant increase of histone transcripts occurs in eggs of abo mutant mothers and a partial rescue of the abo maternal-effect defect takes place with deficiencies of the histone gene cluster. On the basis of these results, it is suggested that the Abo protein functions specifically as a negative regulator of histone transcription and a molecular model is proposed to account for the ability of heterochromatin to partially rescue the abo maternal-effect defect. This model proposes that increased doses of specific heterochromatic regions titrate out abnormally high levels of histones present in embryos from mutant abo mothers and that a balanced pool of histones is critical for normal embryogenesis in Drosophila (Berloco, 2001).

The abo gene consists of a 1,974-bp sequence containing a putative TATA box, a CAAT box, and an ORF, interrupted by a small intron, and producing a single 1.8-kb transcript. This transcript encodes a putative 509-aa protein. The abo¹ mutation is due to the insertion of an incomplete Doc transposable element into the coding region of the abo gene, producing a larger transcript than the wild type, whereas that abo² mutation is caused by a P[ry⁺] insertion into the 5' promoter region and does not produce a detectable transcript (Berloco, 2001).

A computer database search (the BLASTP program) found no known protein motifs in the conceptually translated Abo protein. However, 25.3% identity and 51.9% similarity were found to the DET1 protein, a nuclear located negative regulator of light-mediated gene expression in Arabidopsis, whose putative homologs are present also in Oryza sativa and Lycopersicon esculentum. Intriguingly, 24% identity and 44% similarity were found to the putative human hCP43420 protein from the Celera Human Report and to a putative mouse protein. Considering the evolutionary distance, the homology between these proteins appears significant. They share stretches of homology across their entire lengths and are very similar in charge, distribution of hydrophilic residues, and overall amino acid composition. In particular, the human and mouse proteins appear strikingly identical, with few differences in the nucleotide sequences of their encoding genes (Berloco, 2001).

The homology with DET1 suggests that the Abo protein might also be a transcriptional regulator and therefore might bind specific target sequences. To test this, bacterially produced Abo protein was used as an antigen to raise a polyclonal antibody in mice. Both the polytene chromosomes from salivary glands and the mitotic chromosomes of neuroblasts from wild-type larvae stain for Abo protein. A strong signal exclusively localized on the 39E region on polytene chromosomes was seen. In mitotic metaphase chromosomes, a unique strong signal is present at the constriction on the base of the left arm of the second chromosome. In both cases, the signal is localized at the position of the histone gene cluster, as confirmed by sequential immunostaining with the anti-Abo antibodies and in situ hybridization of the cDm500 probe, which contains the histone cluster. These results clearly demonstrate that the regions with exclusive binding affinity for Abo contain the histone clusters in both the polytenes and mitotic chromosomes (Berloco, 2001).

To identify Abo-binding sites in the histone repeat unit, the X-ChIP (formaldehyde-crosslinked-chromatin immunoprecipitation) method was applied by using polyclonal anti-Abo antibodies. 12 overlapping primer pairs were designed that amplify 400- to 500-bp fragments spanning the whole Drosophila histone repeat unit and they were used to amplify the DNA immunoprecipitated from chromatin of early embryos (0-4 h old) and SL-2 cultured cells. Binding of Abo protein to the promoter regions of H2A-H2B and H3-H4 was found in early embryos. In SL-2 cells, Abo binds to an additional site in an H1 promoter fragment. These results show clearly that Abo protein binding is restricted to the three main regulatory regions of the repeat unit containing the histone gene promoters (Berloco, 2001).

The functional significance of the interaction of abo with the promoters of histone genes was addressed by a quantitation of histone transcripts in unfertilized eggs from heterozygote abo¹/abo² and abo¹/abo⁺ mothers. The results show that abo mutations affect histone transcription. Much higher levels of H2A and H2B were found in eggs from mutant mothers than in eggs from their heterozygous sisters. The amount of H3 and H4 transcripts was significantly higher, whereas variations in the amount of H1 transcripts were not detectable. These results strongly suggest that abo is a negative regulator of histone genes. This possibility was further examined by testing the genetic effects of deficiencies of the entire histone gene cluster on the abo¹ maternal effect. The results clearly show that the histone deficiencies [Df(2)DS5 and Df(2)DS6] induce a strong suppression of the abo¹ maternal-effect defect, thus giving strong support to the suggestion that Abo negatively regulates histone gene expression (Berloco, 2001).

Taken together, these studies reveal that abo is a negative regulator of H2A, H2B, H3, and H4 expression during oogenesis. Hence, the deleterious maternal-effect defect induced by the abo mutations is probably due to an excess of these histones. The regulation of histone expression has been extensively studied in different species. The 5' flanking regions contain cis elements that interact with transacting factors. These transacting factors differ among species and, more surprisingly, also differ among the different classes of histone genes. It has been proposed that the coordinate expression of the histone genes probably depends on the interaction of a protein complex with the different transacting factors. In this context, the uniqueness of the Abo protein location on the histone genes in different Drosophila species and its strong evolutionarily conservation suggest that this protein probably plays a basic role in regulating histone gene expression. However, differential histone gene expression in early embryogenesis of several species has been seen. In Drosophila, specific histone classes are also known to be differentially expressed. For example, it has been shown that the maternal histone H1 transcript is not translated in early embryogenesis and is replaced by the HMG-D chromosomal protein. Intriguingly, the lack of any effect on H1 histone maternal transcription by the abo mutations and the lack of binding to its promoter by Abo in early embryos suggest that the regulation of histone H1 in both ovaries and embryos could not involve the abo gene. However, Abo does bind to the H1 promoter in SL-2 cells (representing late embryonic tissue), suggesting that Abo is probably involved in transcriptional regulation of histone H1 later in embryogenesis. Moreover, the differential enhancement of transcripts found in eggs from abo mutant mothers suggests that Abo could be more important for H2A and H2B repression than H3 and H4 repression during oogenesis (Berloco, 2001).

The data suggest a simple direct model for explaining an intriguing aspect of this gene, namely its interaction with the specific heterochromatic regions termed ABO elements. According to the model, homozygous abo mothers produce eggs with disproportionately high levels of H2A, H2B, H3, and H4 histones, which affect egg viability. Increasing doses of the ABO regions may titrate out these histones, reducing their negative effect. It is predicted that the abo and ABO-counteracting effects are produced by modulations in chromatin structure. Histones could be involved in such effects, as suggested by growing evidence showing that modified histones have differential chromosomal distributions, and hence they could play a role in the formation of heterochromatic domains. In fact H4 histone acetylated at lysine 4 and H3 histone methylated at lysine 9 are both present along the mitotic heterochromatin of Drosophila, with patterns of distribution indicating preferential binding for some regions (Berloco, 2001).

In conclusion, the characterization of abo opens the possibility of using this gene as an entry point to dissect the regulatory machinery of histone expression by looking at Abo-interacting molecules. Moreover, it could be a paradigm for experimental approaches to study the biological role of the heterochromatin. In D. melanogaster, other maternal-effect mutations closely linked to abo have been isolated. Preliminary experiments provide evidence that these abo-like mutations produce defects that can be compensated by discrete heterochromatic elements similar to ABO. It is possible that these other genes, like abo, may also encode transregulators of histone genes or other essential genes encoding chromosomal proteins (Berloco, 2001).

Gene-specific transcriptional mechanisms at the histone gene cluster revealed by single-cell imaging

To bridge the gap between in vivo and in vitro molecular mechanisms, this study dissected the transcriptional control of the endogenous histone gene cluster (His-C) by single-cell imaging. A combination of quantitative immunofluorescence, RNA FISH, and FRAP measurements revealed atypical promoter recognition complexes and differential transcription kinetics directing histone H1 versus core histone gene expression. While H1 is transcribed throughout S phase, core histones are only transcribed in a short pulse during early S phase. Surprisingly, no TFIIB or TFIID was detectable or functionally required at the initiation complexes of these promoters. Instead, a highly stable, preloaded TBP/TFIIA 'pioneer' complex primes the rapid initiation of His-C transcription during early S phase. These results provide mechanistic insights for the role of gene-specific core promoter factors and implications for cell cycle-regulated gene expression (Guglielmi, 2013).

This study found that whereas the four core histone genes are cotranscribed, the timing and pattern of H1 expression is quite distinct despite the tight promoter arrangement of all five His genes. The core histone genes are expressed through short but intense bursts in the very beginning of S phase, whereas histone H1 is expressed throughout S phase. It is speculated that this separation in the timing, distinct factor requirements, and pattern of H1 expression is in keeping with the differential functions assigned to these gene products. The core histone genes encode proteins destined to form a tight nucleosomal octamer, whereas histone H1 operates alone by binding to internucleosomal spacer regions to mediate higher-order condensed chromatin. The differential pattern of H1 transcription versus core histones could reflect a mechanism to achieve greater flexibility and access to chromatin during DNA replication to allow S phase checkpoint control. For example, a common event that can instigate S phase checkpoint arrest is DNA damage requiring repair. The continuous transcription of H1 throughout S phase, coupled with its much shorter mRNA half-life, makes the system more responsive to checkpoint arrest. One can imagine that a tight but highly responsive control of H1 could be a simple mechanism to avoid a buildup of excess H1 protein pools, thereby limiting the formation of higher-order, less accessible chromatin for repair enzymes to reach damaged regions of DNA. On the other hand, building an abundant and stable pool of H2A, H2B, H3, and H4 mRNA early in S phase could be a way to maintain core nucleosome-mediated chromatin integrity during S phase arrest. It is speculateb that perhaps such a constant flux of nucleosome production might be a mechanism to prevent the loss of valuable epigenetic information encoded in pools of modified nucleosomes that must be transmitted to newly formed chromatin (Guglielmi, 2013).

The transcription of both H1 and core histone genes becomes activated at early stages of S phase, suggesting that they are both likely responsive to S phase signaling initiated by the CyclinE-Cdk2 complex . However, it is unclear how core histone gene transcription is shut down early in S phase while the arrest of H1 transcription only occurs at the end of S phase. It has also been proposed that the DNA binding factor HERS silences histone gene transcription by formation of heterochromatin at the His-C locus toward the end of S phase. This mechanism, if involved, could not account for the early S phase silencing of the core histone genes, but could represent a more long-term silencing of the locus outside of S phase (Guglielmi, 2013).

Another intriguing feature of core histone gene expression revealed by these studies is the unusual pattern of transcription dynamics during S phase. A number of studies have concluded that transcription is largely a stochastic process. By contrast, the current studies of the His-C cluster point to a more concerted and deterministic type of mechanism that may be at play. Various studies measured the in vivo binding rates of transcription factors. Other studies of RNA Pol II transcription in living cells have found that initiation can in some cases be rather inefficient relative to elongation. However the binding of linchpin core promoter factors such as TBP to specific endogenous genes in living cells had not been measured. These studies of the endogenous histone genes in Drosophila cells revealed highly efficient initiation by RNA Pol II and very long dwell times (hours) for TBP. Indeed, it was found that TBP is involved in an unusually stable TBP-TFIIA complex that enhances the TBP dwell time at the His locus (Guglielmi, 2013).

It is imagined that such a stable marking of the His-C locus that occurs well before the onset of transcription during S phase can act to rapidly deploy the transcription machinery by serving as a landing pad for the other PIC components, including RNA Pol II. Such a preloaded highly stable pioneer TBP-TFIIA complex may provide an efficient mechanism to rapidly direct high levels of His gene transcription activation needed during the short window of early S phase expression. A stable bookmarked TBP/TFIIA complex could also serve as an insulator protecting the His gene promoter from heterochromatin formation, thereby facilitating transcription initiation at the start of S phase. Such a mechanism could likewise be useful at certain stages of development when spikes of gene activation must occur in narrow time intervals that are followed by efficient shut down. Curiously, there has been one other example of a TBP/TFIIA complex that can be purified from P19 embryonal carcinoma cells but not from more differentiated cells. Why this dimer complex is so abundant in these cells is not clear, but it may point to a more general role of TBP-TFIIA substituting for TFIID as a physiologically relevant alternative transcription initiation complex (Guglielmi, 2013).

The rapid and coordinated mechanism of on/off switches modulating transcription initiation reported in this study is quite distinct from the popular models invoking paused polymerases and reliance on post-initiation events to regulate transcription output described in a number of recent studies. It seems likely that diverse mechanisms may have evolved to deal with timing and dynamics of transcription dependent on cell type and developmental context (Guglielmi, 2013).

Finally, the use of alternative PIC subunits even within the His-C locus was most surprising. First, the dependence of H1 transcription initiation on the TBP-related factor TRF2 was confirmed, whereas H2A, H2B, H3, and H4 depend on the prototypic TBP for their expression. This differential use of core promoter recognition factors could play a key role in the transcriptional control of the His genes, particularly in combination with other gene-specific promoter binding factors responsible for directing S phase-triggered His gene transcription. Perhaps even more surprising was the observation that no TFIIB or TAFs could be detected binding to the His gene promoters. It cannot be entirely rule out that TFIIB has such a fast on/off rate that it was not possible to measure its presence at this locus. However, an equally plausible explanation would be that the PIC at these promoters is substantially different from canonical initiation complexes and neither TFIIB nor TAFs are required to form an active PIC at these promoters. It is also possible that there is another, as yet unidentified functional substitute for TFIIB operating at these promoters. At this stage it cannot be rationalized why the His genes would have evolved to require such a seemingly distinct PIC instead of merely adapting more classical mechanisms such as gene specific DNA binding activators to orchestrate the differential expression of H1 versus core histones during S phase. In any case, the pre-eminence of a highly stable preloaded TBP/TFIIA complex and potential changes in PIC subunit composition suggest that whatever form of PIC does assemble at the His-C locus represents a significant departure in composition and likely functional specificity from the prototypic housekeeping or canonical RNA Pol II promoter recognition apparatus. Perhaps the notion that a universal invariant 'basal or general' core promoter machinery is all that is required to mediate transcriptional regulation within a single cell type in eukaryotic organisms is a concept that has outlived its usefulness (Guglielmi, 2013).

In summary, single live-cell gene expression analysis unmasked a set of unexpected mechanisms regulating transcription of an endogenous gene cluster. Single-cell imaging of core promoter factors in unsynchronized populations of living cells also revealed a surprisingly efficient temporal control of transcription executed by a unique set of stably prebound promoter recognition complexes that significantly expands understanding of the diverse molecular mechanisms that have evolved to accommodate gene-specific transcription controlling physiologically critical processes in animal cells (Guglielmi, 2013).

The assembly of chromatin during DNA synthesis

The assembly of newly synthesized DNA into chromatin is essential for normal growth, development, and differentiation. To gain a better understanding of the assembly of chromatin during DNA synthesis, the Caf1-180 and Caf1-105 subunits of Drosophila chromatin assembly factor 1 (dCAF-1: see Drosophila Chromatin assembly factor 1 subunit) have been identified, cloned, and characterized. The purified recombinant p180+p105+p55 dCAF-1 complex is active for DNA replication-coupled chromatin assembly. Furthermore, the putative 75-kDa polypeptide of dCAF-1 is a C-terminally truncated form of p105 that does not coexist in dCAF-1 complexes containing the p105 subunit. The analysis of native and recombinant dCAF-1 revealed an interaction between dCAF-1 and the Drosophila anti-silencing function 1 (dASF1) component of replication-coupling assembly factor (RCAF). The binding of dASF1 to dCAF-1 is mediated through the p105 subunit of dCAF-1. Consistent with the interaction between dCAF-1 p105 and dASF1 in vitro, dASF1 and dCAF-1 p105 colocalize in vivo in Drosophila polytene chromosomes. This interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin (Tyler, 2001).

The analysis of factors that are required in addition to CAF-1 for DNA replication-coupled chromatin assembly led to the identification of RCAF. RCAF comprises the Drosophila homolog of the yeast anti-silencing function 1 protein (dASF1) and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones that are assembled onto newly replicated DNA. RCAF functions synergistically with CAF-1 in the assembly of chromatin in DNA replication-chromatin assembly reactions. The study of yeast strains that are lacking CAF-1 and/or RCAF further suggested that CAF-1 and RCAF have both common and unique functions in the cell. RCAF-mediated chromatin assembly appears to be essential for normal progression through the cell cycle, gene expression, DNA replication, and DNA repair. Furthermore, it appears that the checkpoint kinase Rad53 may regulate the chromatin assembly function of ASF1 during DNA replication and repair (Tyler, 2001 and references therein).

To analyze the biochemical properties of dCAF-1, the p180, p105, and p55 proteins were synthesized in Sf9 cells by using baculovirus expression vectors. The p180 subunit contained a C-terminal FLAG epitope tag and was thus designated as p180-FLAG. The p105 subunit contained a C-terminal His6 tag and was therefore termed p105-His6. Different combinations of dCAF-1 subunits were synthesized and purified by either anti-FLAG or Ni(II) affinity chromatography. When p180-FLAG, p105-His6, and p55 were cosynthesized and subjected to anti-FLAG immunoaffinity chromatography, the purified p180+p105+p55 dCAF-1 complex was obtained. Similarly, cosynthesis of p180-FLAG with either p105-His6 or p55 yielded p180+p105 and p180+p55 subcomplexes. Although the three-subunit p180+p105+p55 complex can be purified by Ni(II) affinity chromatography via p105-His6, cosynthesis of p105-His6 and p55 and subsequent Ni(II) affinity chromatography yielded only p105. Hence, these findings indicate that dCAF-1 p180 interacts with both p105 and p55, but that p105 and p55 do not interact with one another (Tyler, 2001).

To test whether the p180, p105, and p55 subunits are required for chromatin assembly, DNA replication-chromatin assembly reactions were performed with partial and complete (i.e., p180+p105+p55) dCAF-1 complexes. These experiments revealed that the purified recombinant p180+p105+p55 dCAF-1 complex possesses a specific activity for DNA replication-coupled chromatin assembly that is comparable to that of native dCAF-1, as demonstrated by plasmid supercoiling analysis. It was further confirmed that dCAF-1-mediated plasmid supercoiling is a consequence of chromatin assembly by using micrococcal nuclease digestion analysis. In addition, the two-subunit p180+p105 subcomplex is fully active for chromatin assembly. In contrast, neither the p180 subunit alone nor the p105 subunit alone is sufficient for chromatin assembly. These results thus indicate that the p180 and p105 subunits are each essential for DNA replication-coupled chromatin assembly by dCAF-1 (Tyler, 2001).

It is relevant that the DNA replication extract used in these experiments contains significant amounts of hCAF-1 p60 and hCAF-1 p48 (also known as RbAp48: Drosophila homolog Caf1), which are homologous to dCAF-1 p105 and dCAF-1 p55, respectively. Based on the requirement of dCAF-1 p105 for chromatin assembly, it appears that the hCAF-1 p60 subunit cannot function with the Drosophila CAF-1 polypeptides. However, the lack of a requirement for dCAF-1 p55 may be due to the ability of the hCAF-1 p48 subunit, which is about 87% identical to dCAF-1 p55, to function with the dCAF-1 p180 and p105 subunits in lieu of dCAF-1 p55. It is also possible, however, that the dCAF-1 p180+p105 subcomplex has the intrinsic ability to mediate chromatin assembly. It has not been possible to immunodeplete the hCAF-1 p48 protein from the DNA replication extract to differentiate between these possibilities. It is noteworthy, however, that the Arabidopsis equivalent of dCAF-1 p55 is required for DNA replication-coupled chromatin assembly with the same assay (Tyler, 2001).

The assembly of newly replicated DNA into chromatin requires both dCAF-1 and the RCAF chromatin assembly factor, which comprises Drosophila ASF1 (dASF1) and specifically acetylates histones H3 and H4. To investigate this effect further, coimmunoprecipitation analyses was performed with a crude Drosophila embryo extract. In these experiments, it was observed that immunoprecipitation with anti-dASF1 results in the coimmunoprecipitation of dCAF1 p180, p105, and p55, but not dCAF-1 p75. Conversely, immunoprecipitation with anti-p105 or with anti-p55 results in the coimmunoprecipitation of dASF1. Thus, these findings indicate that native dASF1 interacts with the native p180+p105+p55 form of dCAF-1 but not with the p75-containing form of dCAF-1. Immunoprecipitation of dCAF-1 with anti-p105+p75 does not result in the coimmunoprecipitation of dASF1, which suggests that the anti-p105+p75 antibodies destabilize the interaction between dASF1 and the p180+p105+p55 form of dCAF-1 (Tyler, 2001).

To summarize, the p75 subunit of dCAF-1 appears to be a C-terminally truncated form of p105 and there are distinct forms of dCAF-1 that contain either the p105 subunit or the p75 subunit. The p105-containing form of dCAF-1 comprises the p180, p105, and p55 proteins. The purified recombinant p180+p105+p55 dCAF-1 complex is as active for DNA replication-coupled chromatin assembly as native dCAF-1. Both the p180 and p105 subunits are essential for chromatin assembly. A preexisting interaction between dCAF-1 and the dASF1 chromatin assembly factor has been discovered in crude extracts. This dCAF-1-ASF1 interaction occurs via the dCAF-1 p105 subunit, and this interaction appears to be direct. dASF1 and dCAF-1 p105 colocalize in vivo in Drosophila polytene chromosomes. These results suggest that there is physical cooperation between dCAF-1 and dASF1 during chromatin assembly. The p105 and p75 subunits of dCAF-1 are closely related, and dCAF-1 is not a single four-subunit complex but rather a three-subunit p180+p105+p55 complex and a presumed p180+p75+p55 complex. Thus, the basic three-subunit structure of CAF-1 is conserved among yeast, Drosophila, and humans. The presence of multiple forms of dCAF-1 is of particular interest. Because dCAF-1 was isolated from whole embryos instead of a specific cell line, there is potential for considerable diversity in the range of functions that may be performed by the different forms of dCAF-1. It is possible, for instance, that the p105-containing form of dCAF-1 functions in ASF1-dependent processes, whereas the p75-containing form of dCAF-1 may function in ASF1-independent processes. Alternatively, the activity of dCAF-1 may be regulated during embryogenesis by processing the p105 polypeptide into p75 (Tyler, 2001).

This physical interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin. The coupling of DNA synthesis and chromatin assembly appears to require a specific interaction between CAF-1 and PCNA. The results presented in this work further extend this model to include the binding of ASF1 to CAF-1. It is possible, for instance, that a complex of RCAF and CAF-1 is recruited to sites of DNA synthesis via the interaction of CAF-1 with PCNA. In the future, it will be interesting to study how RCAF and CAF-1 mediate the formation of nucleosomes in conjunction with the other components of the chromatin assembly machinery (Tyler, 2001).

Concerted epigenetic signatures inheritance at PcG targets through replication

Polycomb group of proteins (PcG), by controlling gene silencing transcriptional programs through cell cycle, lock cell identity and memory. Recent chromatin genome-wide studies indicate that PcG targets sites are bivalent domains with overlapping repressive H3K27me3 and active H3K4me3 marked domains. During S phase, the stability of epigenetic signatures is challenged by the replication fork passage. Hence, specific mechanisms of epigenetic inheritance might be provided to preserve epigenome structures. Recently, a critical time window before replication was identified, during which high levels of PcG binding and histone marks on BX-C PRE target sites set the stage for subsequent dilution of epigenomic components, allowing proper transmission of epigenetic signatures to the next generation. This study has extended this analysis to promoter elements, showing the same mechanism of inheritance. Furthermore, to gain insight into the inheritance of PREs bivalent marks, dynamics were analyzed of H3K4me3 deposition, a mark that correlates with transcriptionally active chromatin. Likewise, an early S-phase enrichment of H3K4me3 mark was found preceding the replication-dependent dilution. This evidence suggests that all epigenetic marks are inherited simultaneously to ensure their correct propagation through replication and to protect the 'bivalency' of PREs (Lanzuolo, 2012).

In Drosophila, genome-wide ChIP-seq and transcriptional analysis, in parallel with the detection of transcription start sites (TSS), revealed new features of Polycomb distribution along the Drosophila genome. One particularly clear feature is that Polycomb often targets TSSs with a stalled RNAPolII. These sites are also enriched in H3K4me1/me2, and these specific signatures at TSSs might serve transcriptional pausing of key developmental genes. Although the H3K27me3 and H3K4me3 marks do not generally coexist in Drosophila, these transcriptionally paused promoters could be functionally considered as the fly analogs of the 'bivalent domains' in mammals, which represent poised states for lineage-specific activation of developmental genes (Enderle, 2011). This was further confirmed in S2 cells, where repressed PcG targets show occupancy of repressive marks in combination with active marks, general transcription factors and RNA PolII. It was previously shown that the inheritance of distinct repressive marks at BX-C PREs during replication share a common timeframe (Lanzuolo, 2012). In particular, it was found that the H3K9me3 repressive mark, present on PREs, shows a trend similar to H3K27me3, being enriched in early S phase and subsequently diluted in late S phase, when PRE replication takes place. This result is in agreement with the evidence that H3K9me3 histone mark is controlled by PcG proteins and suggests that repressive epigenetic signatures are simultaneously inherited during replication. This study analyzed the S-phase dynamic of H3K4me3 deposition, a mark that correlates with transcriptionally active chromatin. As expected, the presence of H3K4me3 mark at PREs was confirmed, although at lower levels compared with H3K27me3. Surprisingly, quantification of H3K4me3 enrichment in different S-phase fractions revealed that the active mark deposition follows a similar tendency compared with H3K27me3 and H3K9me3 marks. This dynamic is specific for PREs, because H3K4me3 levels on the transcriptionally active and early replicating Gapdh promoter show a different trend, being diluted from G₁ to early S phase. Altogether, these results indicate that all three epigenetic marks responsible for the bivalent transcriptional potential of PREs are inherited at the same time to preserve their epigenetic state (Lanzuolo, 2012).

These findings determine the early S phase as the critical time point for the Polycomb cell memory system integrating recent observations. It is suggested that PcG complex's binding and enrichment for all repressive and active histone marks that determine the 'epigenetic bivalency' of PcG bound elements are uncoupled from and precede PcG targets replication, when epigenetic signatures are redistributed on daughter strands. This time-dependent dynamics would allow the local conservation of the epigenetic structures through DNA replication and is necessary for the inheritance of the epigenome (Lanzuolo, 2012).

TrxG and PcG proteins but not methylated histones remain associated with DNA through replication

Propagation of gene-expression patterns through the cell cycle requires the existence of an epigenetic mark that re-establishes the chromatin architecture of the parental cell in the daughter cells. This study devised assays to determine which potential epigenetic marks associate with epigenetic maintenance elements during DNA replication in Drosophila embryos. Histone H3 trimethylated at lysines 4 or 27 is present during transcription but, surprisingly, is replaced by nonmethylated H3 following DNA replication. Methylated H3 is detected on DNA only in nuclei not in S phase. In contrast, the TrxG and PcG proteins Trithorax and Enhancer-of-Zeste, which are H3K4 and H3K27 methylases, and Polycomb continuously associate with their response elements on the newly replicated DNA. It is suggested that histone modification enzymes may re-establish the histone code on newly assembled unmethylated histones and thus may act as epigenetic marks (Petruk, 2012).

The key logical problem that has prevented identification of epigenetic marks is the difficulty of distinguishing effects on transcriptional regulation from heritable effects. Any change to transcriptional regulation will affect inheritance, and vice versa. Reasoning that any protein or posttranslational modification (PTM) that is not stable to replication is unlikely to be the epigenetic mark, this study focused on identification of proteins or PTMs that are stable to DNA replication. To do so, it was asked what proteins or PTMs are closely associated with the RF based on association with proteins found near the RF, using proximity ligation assay (PLA) to assess in vivo protein-protein interactions and sequential chromatin immunoprecipitation (re-ChIP) to examine protein associations on specific nascent DNA sequences. To examine events at longer distances from the RF, protein and PTM association with nascent DNA labeled with EdU or BrdU was investigated using 'Chromatin Assembly Assay' (CAA or reverse re-ChIP to ensure that protein-DNA interactions were being examined rather than transient interactions with the replication machinery. Together, these approaches give consistent, repeatable results, allowing events to be assayed at different distances from the RF (Petruk, 2012).

The results of PLA assays show that unmodified H3 and H4K5Ac are in close proximity to PCNA and CAF-1, but H3K4me3 and H3K27me3 are not), agreeing with the results from re-ChIP with PCNA. Electron micrographs of replication bubbles from cleavage-stage Drosophila embryos show less than 200 bp of nucleosome-free DNA adjacent to the RF on one strand and on the other strand show partial nucleosome assembly very close to the RF. These micrographs, together with the current observation that 70% of DNA fragments after sonication are less than 200 bp, suggest that the majority of the histones detected in re-ChIP assays with PCNA are associated with DNA. However, it cannot be ruled out that protein-protein and protein-DNA interactions are being detected in the PLA assays, since small amounts of histones may be detected that are associated with CAF-1 prior to deposition rather than histones bound to nascent DNA. It is possible that parental-modified H3 forms are recruited to nascent DNA with some delay. This would not be detected in re-ChIP experiments because the fragment sizes are short (Petruk, 2012).

Therefore, the CAA assay was developed to detect direct association of modified histones on much larger fragments of nascent DNA in vivo. Surprisingly, no H3K4me3 and H4K27me3 PTMs were detected for at least 30 min after passage of the RF, and the first clear signal was detected at 1 hr. This agrees with the observation that H3K4me3 and H4K27me3 are present at low amounts at the cellular blastoderm or are undetectable in S phase during gastrulation. Taken together, the results of CAA, PLA, and re-ChIP suggest that methylated H3 forms are not associated with nascent DNA from the time of the passage of the RF to the end of the S phase. This is in contrast to unmodified H3, which is easily detected by re-ChIP and CAA on nascent DNA of any size (Petruk, 2012).

The data suggest that in embryos, CAF-1 may deposit acetylated histones and unmodified H3 but does not transfer parental H3K4me3 and H3K4me27 to nascent DNA soon after replication as proposed. In yeast, experiments in which parental histones can be distinguished from de novosynthesized histones show that parental histones are deposited within 400 bp of their original locations (Radman-Livaja, 2011). These experiments did not consider PTMs or timing of deposition but make the important point that if parental histones retaining PTMs are deposited at a different location on nascent DNA, then these would be 'epi-mutations' because the same mark would be at a different location. Considering the yeast and the current data, it is proposed that parental histones are dissociated from parental DNA, lose their trimethyl PTMs, and are then transferred to nascent DNA along with the newly synthesized histones. This suggestion is consistent with earlier results showing that methylation of lysines 9 and 27 of histone H3 is lost during DNA synthesis), and that most histone methylation occurs after deposition (Petruk, 2012).

Together these results suggest that in embryos, trimethylated parental histones are not transferred to nascent DNA and that trimethylation of H3K4 and H3K27 occurs after deposition, and they support the previous suggestion that trimethylation of bound, unmodified H3 is regulated (Scharf, 2009). These data suggest that H3K4me3 and H3K27me3 are unlikely to be epigenetic marks in Drosophila embryos (Petruk, 2012).

In contrast to methylated histones, Trx, Pc, and E(z) associated with PCNA was detected in PLA and re-ChIP assays and stably bound to labeled nascent DNA in CAA assays and re-ChIP assays with BrdU. The results show that Trx, Pc, and E(z) are stable to DNA replication and are constitutively associated with nascent DNA through the S phase, consistent with previous observations that Psc and Pc are stable to replication in an SV40 in vitro replication system (Francis, 2009). Stability to DNA replication and the ability to restore the structure of chromatin required for transcriptional regulation are prerequisites for any putative epigenetic mark. Thus Trx and E(z) fulfill these criteria for epigenetic marks, although functional analysis will be needed to confirm this suggestion. MEs have been proposed to be 'cellular memory modules' because they are sufficient to retain gene-expression pattern. The observation that Trx, E(z), and Pc are retained at the ME after DNA replication supports the model that MEs are cellular memory modules and is consistent with the possibility that these proteins could be epigenetic marks. However, the current observations do not rule out the possibility that other proteins not tested in these experiments are epigenetic marks (Petruk, 2012).

The data imply that Trx, Pc, and E(z) remain bound or rapidly rebind to nascent DNA in the absence of trimethylated histones. There are many reports of trimethylated histone-independent binding of PcG proteins and of MLL1. The current data do not preclude a role for modified histones in binding of PcG and TrxG proteins during transcriptional regulation but suggest that their retention on nascent DNA does not require trimethylation in Drosophila embryos. A recent report shows that in mammalian cell lines, EZH2 associates with PCNA and DNA labeled with BrdU for 5 min, that H3K27me3 is needed to propagate transcriptional repression at a reporter locus, and that H3K27me3 is required for recruitment of PRC2 in interphase in mammalian cells. However that paper did not directly assay the role of H3K27me3 for recruitment of EZH2 in S phase. It is possible that chromatin assembly in Drosophila embryos differs from that in mammalian cells (Petruk, 2012).

The mechanism of retention of Trx and Pc during DNA replication requires association with the ssDNA following unwinding by DNA helicase and transfer from the ssDNA to the nascent dsDNA following passage of the DNA polymerase. The preSET domains of Trx and E(z) bind very tightly to ssDNA, thus providing a plausible explanation for retention of these proteins on the ssDNA found immediately downstream of helicase. It is not known how TrxG and PcG proteins are transferred to nascent DNA. Stable association of the core components of the PRC1 PcG complex, including Pc, with replicating SV40 DNA in vitro may occur by mass-action. Alternatively, Trx, Pc, and E(z) may be retained on replicating DNA by transient interaction with components of the DNA replication complex (Petruk, 2012).

The results suggest that the appropriate amounts of Trx and Pc are replenished late in the S phase or in the interphase, but the mechanism remains unknown. One possibility is that this may occur through interactions between these proteins themselves, for example through dimerization of the SET domain of Trx that has been demonstrated previously. Interestingly, Trx binding can withstand assembly of nucleosomes and interferes with the formation of regular nucleosomal arrays. Thus, Trx may specify a particular nucleosome structure in the MEs. This is a likely possibility given the recent discovery of a specific nucleosome that associates with Gal4 at its binding sites. Importantly, this complex works as a barrier that is essential for establishing specific chromatin architecture of the region surrounding Gal4-binding sites (Petruk, 2012).

PRC2 activity is cell cycle regulated. Several authors have examined retention of PcG and TrxG proteins in mitosis with varying results. The current results suggest that in future it will be interesting to monitor DNA binding and enzymatic activity of PcG and TrxG proteins to determine how epigenetic marks are propagated in G2 and M phases of the cell cycle (Petruk, 2012).

A model is proposed for the reconstitution of chromatin structure during DNA replication in Drosophila embryos. It is suggested that Trx, Pc, and E(z), and likely other TrxG and PcG proteins, are stably associated with their response elements during the progression of the RF, potentially through direct interactions with components of the DNA polymerase complex. Importantly, the stability of the TrxG and PcG proteins during replication ensures sequence specificity in association of these proteins with their response elements after replication. During replication, methylated histones are rapidly replaced by unmethylated histones. The continuous presence of histone-modifying TrxG and PcG proteins may result in histone modification, leading to restoration of the specific chromatin structure that allows either activation or repression of the target gene in the corresponding cells (Petruk, 2012).

The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin; H2Av variant replacement is followed by H4 Lys 12 acetylation as necessary steps before H3 Lys 9 methylation and HP1 recruitment

Activation and repression of transcription in eukaryotes involve changes in the chromatin fiber that can be accomplished by covalent modification of the histone tails or the replacement of the canonical histones with other variants. The histone H2A variant of Drosophila melanogaster, Histone H2A variant (H2Av), localizes to the centromeric heterochromatin, and it is recruited to an ectopic heterochromatin site formed by a transgene array. His2Av behaves genetically as a PcG gene and mutations in His2Av suppress position effect variegation (PEV), suggesting that this histone variant is required for euchromatic silencing and heterochromatin formation. His2Av mutants show reduced acetylation of histone H4 at Lys 12, decreased methylation of histone H3 at Lys 9, and a reduction in HP1 recruitment to the centromeric region. Neither H2Av accumulation nor histone H4 Lys 12 acetylation is affected by mutations in either Su(var)3-9 or Su(var)2-5. The results suggest an ordered cascade of events leading to the establishment of heterochromatin, requiring the recruitment of the histone H2Av variant followed by H4 Lys 12 acetylation as necessary steps before H3 Lys 9 methylation and HP1 recruitment can take place (Swaminathan, 2005).

Recent results suggest that H3 trimethylated at Lys 27 facilitates Pc binding to silenced regions and this modification is carried out by the Enhacer of zeste [E(z)] protein present in the ESC-E(z) complex. Since a reduction in Pc on polytene chromosomes was observed in His2Av mutants, whether recruitment of the ESC-E(z) complex is also impaired in these mutants was examined. In wild type, E(z) can be observed at multiple sites throughout the genome. The levels and localization of E(z) do not appear to be altered in the His2Av⁸¹⁰ mutant compared to wild type. Whether H3 Lys 27 methylation is affected by mutations in His2Av was examined. The levels and distribution of this modification appear to be the same in polytene chromosomes from wild-type and His2Av⁸¹⁰ mutant larvae. This result was confirmed by Western analysis, which shows equal levels of H3 trimethylated at Lys 27 in wild-type and His2Av⁸¹⁰ mutant larvae. These results suggest that H2Av is required upstream of Pc recruitment in the process of Pc-mediated silencing. Since neither recruitment of the E(z) complex nor H3 Lys 27 methylation seem to be affected in His2Av mutants, H2Av replacement might take place after H3 Lys 27 methylation and before Pc recruitment. Alternatively, Pc repression might require at least two parallel and independent pathways, one involving H2Av recruitment and a second one leading to H3 Lys 27 methylation, both of which might be required for proper Pc recruitment (Swaminathan, 2005).

Formation of heterochromatin requires deacetylation of H3 Lys 9 followed by methylation of the same residue and recruitment of HP1. The heterochromatin of Drosophila chromosomes is enriched in dimethylated and trimethylated histone H3 in the Lys 9 residue. To analyze the possible role of H2Av in heterochromatin assembly, the localization was examined of H3 dimethylated at Lys 9 in polytene chromosomes from larvae carrying a mutation in the His2Av gene. Antibodies against histone H3 dimethylated in Lys 9 stain the pericentric heterochromatin in wild-type larvae. Interestingly, polytene chromosomes from His2Av⁸¹⁰ mutants show a decrease in the amount of methylated H3 Lys 9, whereas the presence of Su(Hw), used as a control, is the same in chromosomes from wild-type and His2Av⁸¹⁰ mutant larvae. Since modification of this residue is important for HP1 recruitment, whether localization of HP1 in heterochromatin is also affected by mutations in His2Av was examined. In wild-type larvae, HP1 localizes preferentially to the pericentric heterochromatin of the chromocenter, but accumulation of HP1 is dramatically reduced in the His2Av⁸¹⁰ mutant (Swaminathan, 2005).

To confirm these results, Western analyses of protein extracts obtained from wild-type and His2Av mutant larvae was carried out using antibodies against HP1 and histone H3 dimethylated in Lys 9. The results show little or no accumulation of histone H3 methylated in Lys 9, and lower levels of HP1 in the His2Av⁸¹⁰ mutant. Methylation of histone H3 at the Lys 9 residue is carried out by the Su(var)3-9 histone methyltransferase, and HP1 is encoded by the Su(var)2-5 gene. In order to ensure that the observed effects on the levels of HP1 or the methylation of H3 Lys 9 were not caused by alterations in transcription of Su(var)3-9 or Su(var)2-5 due to the His2Av mutation, quantitative RT-PCR analyses of RNA obtained from wild-type and His2Av⁸¹⁰ mutant third instar larvae were carried out . The results show that there are no significant changes in the levels of Su(var)3-9 or HP1 RNAs in His2Av⁸¹⁰ mutant larvae when compared to wild type. These results and those from immunocytochemistry analyses confirm a role for H2Av in the methylation of H3 Lys 9 and subsequent recruitment of HP1 (Swaminathan, 2005).

Based on the observed effects of His2Av mutations on H3 Lys 9 methylation and HP1 recruitment, it appears that the presence of H2Av in heterochromatin might be required prior to these two events. To confirm this hypothesis, the pattern of H2Av distribution on polytene chromosomes from larvae carrying mutations was examined in the Su(var)2-5 and Su(var)3-9 genes. In both cases, H2Av localization appears normal, suggesting that the presence of H2Av is required prior to H3 Lys 9 methylation and HP1 recruitment during the establishment of heterochromatin (Swaminathan, 2005).

Epigenetic Silencing of Core Histone Genes by HERS in Drosophila

Cell cycle-dependent expression of canonical histone proteins enables newly synthesized DNA to be integrated into chromatin in replicating cells. However, the molecular basis of cell cycle-dependency in the switching of histone gene regulation remains to be uncovered. This study reports the identification and biochemical characterization of a molecular switcher, HERS (histone gene-specific epigenetic repressor in late S phase), for nucleosomal core histone gene inactivation in Drosophila. HERS protein is phosphorylated by a cyclin-dependent kinase (Cdk) at the end of S-phase. Phosphorylated HERS binds to histone gene regulatory regions and anchors HP1 and Su(var)3-9 to induce chromatin inactivation through histone H3 lysine 9 methylation. These findings illustrate a salient molecular switch linking epigenetic gene silencing to cell cycle-dependent histone production (Ito, 2012).

Histones are fundamental components of chromatin that maintain and regulate appropriate chromatin conformation to support all genomic DNA-dependent processes, including transcription, replication, repair, and mitosis). It is now accepted that histone proteins play a prime role in epigenetic regulation, serving as substrates for chromatin modifications (Ito, 2012).

The genes encoding canonical histones (H1, H2A, H2B, H3, and H4) are present in multiple copies and transcriptionally inactive in the quiescent cell state. Unlike the vast majority of the genes transcribed by RNA polymerase II, multiple copies of the histone genes are organized in gene clusters. In Drosophila, the histone gene cluster is composed of about 100 copies of tandemly arranged nucleosomal core (H2A, H2B, H3, and H4) and linker (H1) histone gene cassettes. The histone gene cluster is localized at a subnuclear compartment, histone locus body (HLB) that is presumed to contain factors essential for coordinated regulation of all histone gene copies. In early S phase of the cell cycle, histone genes are activated to supply histone proteins for the integration of newly synthesized DNA into nucleosomes. Coordinated protein production is required for all of the canonical core histones to produce optimal amounts of histone octamers in response to cellular requirements. Therefore, regulatory sequences appear to be common in the nucleosomal core histone gene loci to ensure their synchronized expression (Ito, 2012).

The cell cycle-dependent expression of canonical histones might be achieved through conserted action of cell-cycle, phase-specific nuclear factors at multiple levels of regulation. Recent studies have shown that the nuclear protein ataxia-telangiectasia locus (NPAT) plays a key role in the induction of histone genes. NPAT associates with histone loci and is functionally activated in early S phase by cyclin-dependent kinase 2 (Cdk2)/cyclin E (CycE), a kinase complex known to promote G1/S transition. Recently, fly homeotic gene mxc was identified as Drosophila NPAT ortholog. At the end of S phase, histone gene transcription is rapidly turned down and histone mRNAs are destabilized, ending the histone protein production. Although it has been assumed that dephosphorylation of NPAT is critical for histone gene silencing, the mechanisms of transcriptional suppression of histone genes remain to be identified (Ito, 2012).

During genetic screening of transcriptional coregulators in Drosophila, a critical factor has been identified that switched off nucleosomal core histone gene expression at the end of S phase through epigenetic inactivation of chromatin. Therefore, this factor was designated histone gene-specific epigenetic repressor in late S-phase (HERS) and presumed to function as a repressor of canonical core histone gene loci (Ito, 2012).

In contrast to NPAT, HERS protein abundance appears to be cell cycle-dependent because HERS was undetectable at the G1/S transition and only emerged in late S phase. Cdk-dependent phosphorylation of HERS is required for its association with core histone gene regulatory regions through sequence-specific DNA binding. This study has identified Cdk1/CycA as a prospective kinase complex that may phosphorylate HERS in vivo. However, because it has not been confirmed whether the Cdk1 inhibitor that was used here does not affect Drosophila Cdk2 or Cdk1/CycB activities, the possibility cannot be excluded that these Cdk complexes may also phosphorylate HERS. Owing to the cell cycle-specific expression and phosphorylation of HERS, its localization on the HIS-C appears to be limited to the completion of replication in the late S phase. This is further supported by HERS localization on the histone gene loci in the polytene chromosomes of salivary glands, in which DNA replication is completed and histone genes are inactive. Moreover, phosphorylation also significantly enhances HERS protein stability. Therefore, it is conceivable that dephosphorylation terminates HERS action and targets the protein for degradation (Ito, 2012).

HERS shuts down core histone gene expression through recruitment of the Su(var)3-9/HP1 repressor complex, a universal and normally abundant epigenetic silencer. Molecular interaction has been confirmed only between HERS and Su(var)3-9 but not with other tested H3K9 methyltransferases, G9a and dSETDB1, although genetic interaction of HERS was also observed with G9a and dSETDB1 in the Drosophila experimental system. It has been proposed that histone H3K9-specific methyltransferases function independently at their target loci, whereas they act in a sequential manner at the same gene locus. Moreover, it has been reported that G9a and dSETDB1 are active in early development, whereas Su(var)3-9 predominantly acts from early embryogenesis to later stages of Drosophila development. Thus, the possibility cannot be excluded that other histone H3K9 methyltransferases may also support HERS function (Ito, 2012).

The motif organization of HERS protein is atypical compared to other reported cell-cycle regulators. Even though phosphorylated HERS appears to act as a DNA binding factor, it lacks known motifs associated with DNA binding, protein-protein interaction, or other molecular interactions. Instead, this protein encompasses only one known motif, the BAH domain. The BAH domain is present in many chromatin-related regulators, including ORC1, polybromo, DNMT1, and yeast Sir3. The only reported role for the BAH domains in yeast Sir3 and Orc1 is that of nonselective association with nucleosomes. Although functional and physical interactions of the BAH domains were assessed for different histones modified at particular residues (for example, H3K9 methylation), it has not yet been possible to delineate any specific role of the HERS BAH domain at this stage. Nevertheless, it is conceivable that nonselective association with nucleosomes through the BAH domain may facilitate or stabilize HERS binding to specific regulatory DNA sequences in histone gene loci (Ito, 2012).

Although it remains unclear whether Drosophila HERS acts as a regulator of other cellular events, it was found that HERS knockdown in insect S2 cells resulted in cell-cycle arrest at the G2/M phase. Furthermore, when HERS was overexpressed in mammalian cell lines (HEK293 and Y1 cells), cell-cycle arrest was observed together with polyploidy. Besides other possible implications, this suggests an existence of mammalian HERS homolog(s). Interestingly, several uncharacterized mammalian proteins bearing the BAH domain have been documented. As mammals have three HP1 protein family members (α, β, and γ) with different functions, it is conceivable that mammalian HERS may consist of multiple members or isoforms with distinct cellular functions (Ito, 2012).

Aurora kinase and phosphorylation of Histone H3 during mitosis

Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression. The segregation of chromosomes with high fidelity requires exquisite coordination of cellular processes. The mechanisms that coordinate the cycle of chromosome condensation and decondensation with the assembly, function, and subsequent disassembly of the mitotic spindle are poorly understood. Highly conserved genes essential for chromosome condensation have been found through genetic screens in yeasts and Drosophila. For example, five members of a protein complex known as condensin, have been identified that are functionally and structurally conserved. Mutants exhibit incomplete chromosome condensation associated with failure of segregation and the stretching of chromatin upon the spindle. Biochemical approaches also identified the protein complex in Xenopus and showed that it can promote chromatin condensation by directing the supercoiling of the DNA in an ATP-dependent manner. Chromosome condensation is also accompanied by phosphorylation of histones H1 and H3. Indeed, mutation of the mitotic phosphorylation site of histone H3 of Tetrahymena leads to both chromosome condensation and segregation defects. A direct link between histone H3 phosphorylation and condensin recruitment onto chromosomes has recently been suggested by the colocalization of members of the condensin complex with phosphorylated histone H3 during the early stages of mitotic chromosome condensation. However, the generality of the requirement for the phosphorylation of histone H3 for chromosome condensation and segregation must be questioned by the finding that budding yeast cells in which serine 10 of histone H3 is replaced with alanine show no apparent defects in cell cycle progression or chromosome transmission. Nevertheless, maximal chromosome condensation in meiosis does correlate with maximal levels of phospho-histone H3 in wild-type cells. The enzyme required for histone H3 phosphorylation in Saccharomyces cerevisiae is the aurora-related protein kinase Ipl1p (Hsu, 2000). Moreover, one of its two counterparts from Caenorhabditis elegans, the air-2 protein kinase, has been shown to have the same function (Giet, 2001 and references therein).

One striking effect of aurora B RNAi is to permit progression through mitosis with improperly condensed chromosomes. It was possible to account for these condensation defects by a diminution of the phosphorylation of serine 10 of histone H3 and a failure to localize condensin on the chromosomes. The former finding is consistent with several studies that now implicate a requirement for the phosphorylation of the NH₂-terminal region of histone H3 at this residue for chromosome condensation. Not only does the formation of mitotic chromosomes in a Xenopus cell-free extract by a nucleosome-associated kinase correlate with histone H3 phosphorylation, but when the serine 10 residue is mutated to alanine it results in abnormal segregation and chromosome loss during mitosis and meiosis in Tetrahymena. One enzyme credited with the ability to phosphorylate histone H3 at mitosis is the NIMA kinase of Aspergillus. However, the finding that levels of histone H3 phosphorylation are reduced after aurB RNAi in Drosophila cells is more in keeping with the report that the Aurora-like kinase homologs, Ipl1 of yeast and Air-2 (but not Air-1) of C. elegans, are required for histone H3 phosphorylation in these organisms (Hsu, 2000). The finding of some residual histone H3 phosphorylation either could reflect the incomplete elimination of Aurora B by RNAi, or could indicate that an alternative kinase has this capability, offering an explanation of the partial chromosome condensation seen in the RNAi-treated cells. The current data are important in emphasizing the importance of histone H3 phosphorylation for chromosome transmission and as such are in line with the findings in Tetrahymena. This differs from the effects seen in budding yeast cells that continue through division cycles in the absence of histone H3 phosphorylation without showing defects in chromosome transmission. As an explanation, it has been suggested that other histones could be phosphorylated in addition to the histone H3 in the yeast cell and that such phosphorylation events could be sufficient to ensure normal chromosome dynamics. A major role of the yeast enzyme Ipl1p is to regulate the function of the kinetochore-associated protein Ndc10p through its phosphorylation. Therefore, the increase in ploidy reported in ipl1 mutant cells has been attributed more to inappropriate kinetochore function, and consequently the effects of Air-2 depletion upon chromosome condensation in C. elegans have been a little overshadowed. It seems likely that the abnormal chromosome segregation in Drosophila cells after aurB RNAi is due to incomplete condensation, since a similar phenotype is seen in mutants of the condensin subunit Barren. Of course, this does not exclude the possibility that defects in the organization of the centromeric regions and kinetochores arise directly as a result of aurB RNAi or as either a direct or indirect consequence of condensation defects. The increase in ploidy seen after aurora B RNAi is reminiscent of the Ipl1 phenotype in budding yeast, but differs in that it arises from both chromosome segregation and cytokinesis defects (Giet, 2001).

The resemblance of the mitotic phenotype of cells after RNAi with aurB to that previously reported for Drosophila barren mutants (Bhat, 1996) can be further explained by the failure of Barren protein to be recruited to the mitotic chromosomes after aurB RNAi. Originally recognized through this mutant defect, it was later realized that Barren is the fly homolog of a member of the pentameric complex, condensin, first shown to be required for mitotic chromosome condensation in Xenopus. It is possible that Barren or other members of the condensin complex could themselves be directly phosphorylated by Aurora B during chromosome condensation. However, the process seems likely to involve a plethora of phosphorylation events: the nuclear A-kinase anchoring protein (AKAP95) appears to target the human hCAP-D2 condensin to chromosomes and phosphorylation of condensin subunits by cdk1 has been associated both with their nuclear accumulation and activation. It has been proposed that phosphorylation of the NH₂ terminus of histone H3 leads to the recruitment or the activation of the condensin complex to the chromosome, where it can modify DNA topology. The data presented here indicate that phosphorylation of histone H3 by the Aurora B kinase and the localization of Barren onto chromosomes are associated events in mitosis. They support and extend a recent observation that human condensin proteins hCAP-E, hCAP-C, and hCAP-D2 colocalize with phosphorylated histone H3 in clusters in partially condensed regions of chromosomes in early prophase. The similarity of the effects seen on chromosome condensation resulting from loss of either aurora B or barren function is striking and points to the value of studying these processes in a single model organism amenable to both genetic manipulation and RNAi. It is perhaps surprising that in both cases partial chromosome condensation is achieved and that there can be some degree of segregation of chromatin to the poles (Giet, 2001 and references therein).

The second major mitotic abnormality observed after aurB RNAi in Drosophila cells is a failure of cytokinesis. Thus, like its mammalian and nematode counterparts AIM-1 and AIR-2, the enzyme encoded by aurora B appears essential for this process. Two proteins that play a role in cytokinesis have recently been shown to associate with the Aurora B-like kinases: Incenp, as discussed above (Adams, 2000; Kaitna, 2000), and the Zen-4 kinesin-like protein of C. elegans (Kaitna, 2000; Severson, 2000). The localization of the latter is disrupted after disruption of air-2 function using RNAi or conditional mutant alleles. Zen-4 is the C. elegans homolog of the Pavarotti KLP of Drosophila, which likewise is mislocalized on the central spindle from anaphase onwards after aurB RNAi. Pav-KLP also cooperates with Polo kinase to achieve its localization and function in Drosophila, suggesting that multiple mitotic kinases may be required to coordinate central spindle formation before cytokinesis, just as several kinases appear to be required for centrosome maturation and separation and chromosome condensation (Giet, 2001).

It is striking that aurB RNAi cells are not arrested by a mitotic checkpoint, given the abnormalities that they show in chromosome alignment at metaphase and the subsequent disorganization of the later mitotic spindle. However, the treated cells do undergo multiple cell cycles, as is clearly demonstrated in this cell culture system in which one can monitor the shift in ploidy by FACS analysis and the increase in chromosome and centrosome complements by immunocytology. It is possible that these abnormalities arise too late in the mitotic cycle to trigger checkpoint arrest, although this seems unlikely for the chromosome segregation defect. Although it is possible that Aurora B is itself required for checkpoint functions, it could also be that the kinetochore regions of chromosomes are insufficiently well organized after aurB RNAi to promote the checkpoint activity of the complex of Bub/Mad proteins that associate with unaligned centromeres. It is noteworthy that the C. elegans baculovirus inhibitor of apoptosis (IAP)-related repeat protein Bir-1 appears to be required for the localization of Air-2. Bir-1 localizes to chromosomes and then the spindle midzone and Air-2 fails to localize to these same sites in the absence of Bir-1 (Speliotes, 2000). These IAP proteins, also known as survivin, are caspase inhibitors and as such counteract apoptosis. Is it possible that B-type Aurora kinases might play a role alongside survivin in an apoptotic checkpoint to promote mitosis? (Giet, 2001).

It is of considerable interest to know the multiple substrates of Aurora B kinase and to understand its mode of regulation in mitotic progression. It seems that subcellular localization of the enzyme could be one critical means of controlling access to its substrates. The enzyme localizes throughout condensing chromosomes when phosphorylation of histone H3 is required. Aurora B's subsequent concentration at centromeres could direct enzyme activity toward specific chromosomal proteins at these sites, but may be instrumental in its movement onto the central spindle at anaphase, thereby providing an effective way of removing the enzyme from the chromatin to facilitate chromosome decondensation at telophase. Understanding the intricacies of these processes will be a future challenge (Giet, 2001).

The availability of mitotic cells containing chromosomes with a range of levels of H3 phosphorylated on serine¹⁰ has enabled an assessment of the widely held hypothesis that H3 phosphorylation is correlated with the degree of chromatin condensation. When phospho-H3 levels and the degree of chromatin compaction were compared by quantitative fluorescence microscopy, only a weak correlation between the two values was observed. Instead, interference with Incenp and Aurora B function appears to correlate much more strongly with difficulties in assembling mitotic chromosomes of normal morphology. Mitotic chromosomes deficient in phospho-H3 have a characteristic dumpy morphology, with no evidence of resolved sister chromatids. This resembles the defects seen in Drosophila mutants in the SMC4 subunit of condensin and also those of a ts mutant in C. elegans aurora B/AIR-2 when it enters mitosis at nonpermissive temperature. Phosphorylation of histone H3 or another chromosomal substrate by Aurora B might be required for the binding of condensins or other chromosomal proteins that give mitotic chromosomes their characteristic morphology (Adams, 2001).

The chromosomal passenger complex (CPC) is a key regulator of mitosis in many organisms, including yeast and mammals. Its components co-localise at the equator of the mitotic spindle and function interdependently to control multiple mitotic events such as assembly and stability of bipolar spindles, and faithful chromosome segregation into daughter cells. This study reports the first detailed characterisation of a CPC mutation in Drosophila, using a loss-of-function allele of borealin (borr). Like its mammalian counterpart, Borr colocalises with the CPC components Aurora B kinase and Incenp in mitotic Drosophila cells, and is required for their localisation to the mitotic spindle. borr mutant cells show multiple mitotic defects that are consistent with loss of CPC function. These include a drastic reduction of histone H3 phosphorylation at serine 10 (a target of Aurora B kinase), and a pronounced attenuation at prometaphase and multipolar spindles. The evidence suggests that borr mutant cells undergo multiple consecutive abnormal mitoses, producing large cells with giant nuclei and high ploidy that eventually apoptose. The delayed apoptosis of borr mutant cells in the developing wing disc appears to cause non-autonomous repair responses in the neighbouring wild-type epithelium. These responses involve Wingless signalling, which ultimately perturbs the tissue architecture of adult flies. Unexpectedly, during late larval development, cells survive loss of borr and develop giant bristles that may reflect their high degree of ploidy (Hanson, 2005).

One crucial role of the CPC during mitosis is to mediate the H3 phosphorylation of serine 10 (P-H3) by Aurora B, as has been demonstrated in budding yeast, C. elegans and Drosophila. The numbers of P-H3-positive (dividing) cells are reduced in the VNC of borr mutant embryos. Furthermore, the P-H3 levels of individual borr mitotic nuclei are typically reduced compared with those of wild-type nuclei. Often, they exhibit blotchy P-H3 staining rather than the more 'structured' staining outlining condensed chromosomes as observed in the wild type. A similar loss of P-H3 staining has also been observed in borr RNAi-depleted Kc167 cells. This reduction of the P-H3 levels in borr mutant cells is consistent with a loss of Aurora B kinase activity and, thus, with a disruption of CPC function (Hanson, 2005).

Despite the strong reduction of the P-H3 levels in mitotic VNC cells of borr mutant embryos, these cells display only a slight undercondensation of their chromatin, although the degree of undercondensation is somewhat variable from cell to cell. These results suggest that borr may not be essential for chromatin condensation (Hanson, 2005).

To examine the effects of borr loss on actively dividing epithelial cells, FRT-FLP-mediated recombination was used to generate borr mutant clones in imaginal discs whose cells undergo cell divisions throughout larval development. If borr mutant clones are induced during early larval stages and examined in fully grown larval discs, these clones are rare and are much smaller than the corresponding wild-type twin spots, suggesting that a large fraction of the mutant cells die. Hoechst staining revealed that many of the surviving borr mutant cells are large, with giant but well-formed nuclei that appear healthy, and well integrated into the epithelial tissue (Hanson, 2005).

Imaginal discs bearing borr mutant clones were stained with antibodies against Incenp and Aurora B, to assess the effect of borr loss on these CPC components during mitosis. Wild-type cells in metaphase show characteristic well-ordered mitotic spindles, with distinct staining of Aurora B and Incenp at specific sites along condensed chromatin. By contrast, borr mutant cells invariably show abnormal mitotic spindles, including multipolar ones. Most of these mutant spindles do not show any chromatin-associated Incenp or Aurora B staining, although occasionally patches of Incenp staining can still be observed, but they do not seem to be associated with any of the spindle components. These staining patterns suggest that these CPC components fail to localise properly to mitotic spindles in the absence of borr (and their levels may also be reduced, though the low frequency of surviving borr mutant cells does not allow quantitative assessment of this). Therefore, as in mammalian cells, the correct localisation of Incenp and Aurora B to mitotic spindles of dividing imaginal disc cells depends on Borr. This is further evidence that Borr is a CPC protein, and that it interacts functionally with other known CPC components (Hanson, 2005).

Drosophila Incenp is required for cytokinesis and asymmetric cell division during development of the nervous system

The chromosomal passenger protein complex has emerged as a key player in mitosis, with important roles in chromatin modifications, kinetochore-microtubule interactions, chromosome bi-orientation and stability of the bipolar spindle, mitotic checkpoint function, assembly of the central spindle and cytokinesis. The inner centromere protein (Incenp; a subunit of this complex) is thought to regulate the Aurora B kinase and target it to its substrates. To explore the roles of the passenger complex in a developing multicellular organism, a genetic screen was performed looking for new alleles and interactors of Drosophila Incenp. A new null allele of Incenp has been isolated that has allowed a study of the functions of the chromosomal passengers during development. Homozygous incenp^EC3747 embryos show absence of phosphorylation of histone H3 in mitosis, failure of cytokinesis and polyploidy, and defects in peripheral nervous system development. These defects are consistent with depletion of Aurora B kinase activity. In addition, the segregation of the cell-fate determinant Prospero in asymmetric neuroblast division is abnormal, suggesting a role for the chromosomal passenger complex in the regulation of this process (Chang, 2006).

By embryonic stage 13, Drosophila Incenp was no longer detectable by immunostaining in incenp^EC3747 embryos. Consistent with this, phosphorylation of histone H3 on Ser10 (a known Aurora B kinase substrate) was also no longer detected. This phenotype confirms that Incenp is required for Aurora B kinase to function as a histone H3 Ser10 kinase. At this stage, cells of the central nervous system (CNS) in incenp^EC3747 embryos showed enlarged nuclei compared with wild-type as observed by DAPI staining for DNA. Staining with anti-gamma-tubulin antibody showed that these enlarged cells contain bigger than normal centrosomes or multiple centrosomes. This phenotype, detectable in mutant embryos following the complete disappearance of the Drosophila Incenp, can be explained as the result of failure of cytokinesis in the previous division and is consistent with a lack of Aurora B kinase function (Adams, 2001c; Oegema, 2001) in the early development of the CNS (Chang, 2006).

JIL-1 targets Histone H3 during dosage compensation in Drosophila

JIL-1 is part of the dosage compensation apparatus. JIL-1 colocalizes and physically interacts with male specific lethal (MSL) dosage compensation complex proteins. Ectopic expression of the MSL complex directed by MSL2 in females causes a concomitant upregulation of JIL-1 to the female X that is abolished in msl mutants unable to assemble the complex. Thus, these results strongly indicate JIL-1 associates with the MSL complex and further suggest that JIL-1 functions in signal transduction pathways regulating chromatin structure (Jin, 1999 and 2000).

To analyze the function of the chromosomal kinase JIL-1, an allelic series of hypomorphic and null mutations was generated. JIL-1 is an essential kinase for viability, and reduced levels of JIL-1 kinase activity led to a global change in chromatin structure. In JIL-1 hypomorphs, euchromatic regions of polytene chromosomes are severely reduced and the chromosome arms condensed. This is correlated with decreased levels of histone H3 Ser10 phosphorylation. These levels can be restored by a JIL-1 transgene placing JIL-1 directly in the pathway mediating histone H3 phosphorylation. A model is proposed where JIL-1 kinase activity is required for maintaining the structure of the more open chromatin regions that facilitate gene transcription (Wang, 2001).

The original EP(3)3657 line in conjunction with the newly generated JIL-1^z60 and JIL-1^z2 lines constitute an allelic series of JIL-1 hypomorphic and null mutations. This allows an anaysis of the effects of decreasing levels of JIL-1 protein on viability and male to female sex ratios. In order to measure and compare viability, homozygous pupae were collected from heterozygous crosses of the three alleles and their eclosion rates were determined. The homozygous mutant pupae could be readily identified because they did not display the Tubby marker carried on the balancer third chromosome. It was found that homozygous EP(3)3657 larvae, which have only one tenth of the level of JIL-1 protein normally found in wild-type, have an eclosion rate of 81%. However, as the level of JIL-1 protein further decreases to about 3% in JIL-1^z60/JIL-1^z60 larvae, the eclosion rate reduces to only 0.5%. Moreover, the few homozygous JIL-1^z60/JIL-1^z60 animals surviving to adulthood are unable to produce offspring and most die shortly after eclosion. The eclosion rate for the null allele JIL-1^z2/JIL-1^z2 larvae was 0%. These results strongly suggest that JIL-1 is an essential kinase for viability (Wang, 2001).

Eclosed EP(3)3657/EP(3)3657 adults are fertile and able to produce offspring, and thus embryos from homozygous parents can be analyzed for the effect of reduced levels of JIL-1 on embryonic development. The hatch rate of such embryos is only 4%, showing a significant decrease below the 83% observed in wild-type. Whereas reduced eclosion levels are observed for both males and females, male viability is more severely affected in all cases of lowered JIL-1 expression. For example, in EP(3)3657/EP(3)3657 homozygous offspring from heterozygous mothers that provide maternal levels of JIL-1 protein during early development due to the mother's wild-type allele, the number of males eclosing was 73% that of females. In adults eclosing from crosses of homozygous parents and thus developing without the increased maternal levels of JIL-1, the percentage of males relative to females was 48%. Further reduction is observed in the severe JIL-1^z60/JIL-1^z60 hypomorph, which gives rise to only 32% the expected number of males relative to females. The male to female sex ratio in EP(3)3657 flies can be rescued to near wild-type ratio by the JIL-1- GFP transgene. When this transgene is introduced into these flies, the male to female sex ratio recovers from 48% to 97% (Wang, 2001).

The phenotypic consequences of reduced levels of JIL-1 kinase were investigated in embryos from EP(3)3657 homozygous parents by labeling chromatin with Hoechst and microtubules with anti-tubulin antibody. The average expression level of JIL-1 kinase in these mutant animals is reduced to about one tenth that of wild-type. A range of phenotypes was observed from embryos appearing wild-type with regularly spaced nuclei to embryos where chromatin structure had completely disintegrated. In intermediate phenotypes, nuclei in various stages of fragmentation were still discernible. The variable penetrance and range of phenotypes are likely to be a result of different levels of JIL-1 expression in individual embryos. Some embryos have enough JIL-1 to carry them through embryogenesis as reflected in the 4% hatching rate, whereas others are below the threshold for maintaining JIL-1 kinase function. In embryos double labeled with Hoechst and anti-tubulin antibody, centrosomes were often observed to be separated from the nuclear remnants, and in other cases, the nuclear fragmentation would lead to aberrant and misaligned tubulin spindles. These data suggest that reduced levels of JIL-1 kinase lead to a disintegration of nuclear and chromatin structure during embryonic development (Wang, 2001).

The consequences of loss of JIL-1 on polytene chromosome structure in interphase nuclei was assessed. Chromosomal squashes prepared from either wild-type or homozygous hypomorphic EP(3)3657, JIL-1^z60, or null JIL-1^z2 larvae were fixed and labeled with Hoechst to visualize the DNA, anti-MSL2 antibody to identify the male X chromosome, and, in some cases, with anti-JIL-1 antibody. Labeling with anti-MSL2 antibody revealed that MSL2 protein still localizes to the X chromosome in all three JIL-1 mutant alleles, indicating that JIL-1 is not necessary for targeting of the MSL complex to the male X chromosome. Identical results were obtained using anti-MSL1, -MSL3, or histone H4Ac16 antibodies, confirming this observation. However, chromosome morphology in both males and females is markedly affected. Whereas wild-type polytene chromosomes show extended arms with a regular pattern of Hoechst-stained bands, this pattern, while relatively normal in the hypomorphic EP(3)3657 mutant animals, becomes severely perturbed in strong JIL-1^z60 hypomorphs and the null JIL-1^z2 larvae. In these latter preparations, the euchromatic interband regions are largely absent and the chromosome arms are highly condensed. Thus, these results suggest that the JIL-1 kinase is involved in both males and females in establishing or maintaining the more open chromatin structure found in the gene-active interband regions that comprise less tightly packed euchromatin (Wang, 2001).

Although all of the chromosomes from JIL-1 mutant animals display abnormalities, perturbation of the male X chromosome is relatively more severe than that of the autosomes. This can be observed in the weaker hypomorphic phenotype from EP(3)3657 preparations where although the autosomes are only subtly affected, the male X chromosome is significantly shorter and has lost a large degree of its banding pattern. In the strong JIL-1^z60 hypomorph or the null JIL-1^z2 mutant, the male X chromosome is even more condensed with no remaining observable banding pattern or structure. Further support for the fact that the reduction of JIL-1 protein level is responsible for the defects observed in the homozygous animals comes from rescue experiments in which transgenic JIL-1-GFP is introduced into JIL-1^z2/JIL-1^z2 animals. Chromosomes from these animals now appear essentially wild-type including the male X chromosome; JIL-1 antigenicity is restored and upregulated on the X chromosome as detected by JIL-1 antibody. Identical results were observed in rescue experiments employing a full-length JIL-1 transgene which did not contain the GFP moiety (Wang, 2001).

The upregulation of JIL-1 on the male X chromosome in conjunction with its ability to phosphorylate histone H3 Ser10 in vitro led to an examination of the question of whether higher levels of phosphorylated histone H3 Ser10 (pH3S10) are also present on the male X. Conventional polytene chromosome fixation and squash techniques led to inconsistent banding patterns. It was reasoned that the highly acidic fixation conditions of the conventional squash protocol might be interfering with either antibody performance or antigen stabilization during fixation. Therefore, a modified whole-mount staining technique was developed for salivary glands that gently compress nuclei beneath a coverslip before fixation in a standard paraformaldehyde/PBS solution with a physiological pH. Although the overall resolution of the bands is inferior to the normal squash technique, it does allow visualization of the chromosomes suitable for analysis. Such salivary gland preparations were double labeled with antibodies to JIL-1 and pH3S10 as well as with antibodies to JIL-1 and phosphoacetylated histone H3 (pH3S10Ac14). JIL-1 protein is upregulated on the male X chromosome. This upregulation is concomitant with an upregulation of both pH3S10 and pH3S10Ac14 labeling on the male X chromosome as compared to the autosomes. Furthermore, it is evident that the staining pattern of the antibodies in wild-type animals overlap as indicated by a predominantly yellow banding pattern. This labeling pattern was consistently observed in different experiments using different lots of pH3S10 antibody from two different companies. Such an upregulation was not observed in the female, and in homozygous JIL-1^z2/JIL-1^z2 polytene chromosomes; neither JIL-1 nor pH3S10 or pH3S10Ac14 labeling was detectable. These data suggest that levels of phosphorylated histone H3 Ser10 are increased on the male X chromosome in a pattern overlapping with that found for the JIL-1 kinase. However, it is of interest to note that Western blot analysis does not indicate higher overall levels of pH3S10 in males than females (Wang, 2001).

Recent studies have revealed a tight correlation between histone H3 Ser10 phosphorylation and proper chromosome condensation and segregation during mitosis. This mitotic phosphorylation of histone H3 is governed by the lpl1/aurora kinase in budding yeast and nematodes and by the NIMA kinase in Aspergillus. Thus, different kinases or more than one kinase may serve this function in different organisms. This raises the question whether JIL-1 regulates mitotic histone H3 Ser10 phosphorylation in Drosophila. To address this issue, pH3S10 levels were analyzed in null JIL-1^z2/JIL-1^z2 larval neuroblast mitotic chromosomes. In the null JIL-1 background, pH3S10 is not observed in interphase nuclei, but is enriched on the mitotic chromosomes at a level comparable to wild-type. Therefore, loss of JIL-1 activity does not appear to alter the mitotic phosphorylation of histone H3 Ser10 in larval neuroblasts (Wang, 2001).

Since the interphase upregulation of pH3S10 phosphorylation levels correlates with JIL-1 kinase localization and since the majority of larval cells are in interphase at any one given time, whether pH3S10 phosphorylation levels were decreased in JIL-1 hypomorphs was examined, as would be predicted if JIL-1 were involved in this process. Levels of phosphorylated histone H3 Ser10 were determined by immunoblot analysis of larval protein lysates from wild-type or homozygous JIL-1 mutant (EP(3)3657, JIL-1^z60, JIL-1^z2) animals. Lysates were fractionated and probed with anti-pH3S10, anti-tubulin, anti-lamin, and anti-histone H3 antibodies. All of the JIL-1 mutants showed lower levels of pH3S10 than observed in wild-type larvae. Furthermore, the level of reduction of pH3S10 corresponded directly to the severity of the JIL-1 allele, with the null JIL-1^z2/JIL-1^z2 allele showing the lowest level of pH3S10 phosphorylation and EP(3)3657/EP(3)3657, the weaker of the two hypomorphs showing higher pH3S10 levels than the strong JIL-1^z60/JIL-1^z60 hypomorph. In contrast, the levels of control proteins such as histone H3, tubulin, and lamin were roughly equivalent to wild-type levels in all three mutant lines. Since introduction of the JIL-1-GFP transgene on the second chromosome of JIL-1^z2/JIL-1^z2 animals rescued the chromosomal defects, it was of interest to determine whether there was also a corresponding restoration of pH3S10 levels in these animals. Western blots of larval protein lysates from wild-type, JIL-1^z2/JIL-1^z2, or JIL-1^z2/JIL-1^z2 larvae carrying the JIL-1-GFP transgene were probed with anti-pH3S10 antibody or anti-histone H3 total protein antibody. In the presence of the JIL-1-GFP transgene, pH3S10 levels are restored to essentially wild-type levels (Wang, 2001).

Thus, in a JIL-1 null mutant allele that shows no detectable JIL-1 kinase, the level of histone H3 Ser10 phosphorylation is reduced to about 5% of wild-type levels. These results suggest the existence of another kinase that can phosphorylate histone H3 Ser10 and are consistent with the recent identification of Aurora B kinase as the likely mitotic H3 Ser10 kinase in Drosophila (Giet, 2001). However, these results suggest that JIL-1 is the predominant kinase regulating the phosphorylation state of this residue at interphase. This is further supported by findings that histone H3 Ser10 phosphorylation is upregulated on the male X chromosome in a pattern similar to that of the JIL-1 kinase and that the loss of histone H3 Ser10 phosphorylation as well as aberrant chromosome structure found in JIL-1 mutants can be rescued by the presence of a JIL-1 transgene. Thus, taken together these results demonstrate that JIL-1 is in the pathway mediating histone H3 Ser10 phosphorylation and that JIL-1 kinase activity is required for the maintenance of normal chromosome architecture in Drosophila at interphase (Wang, 2001).

In embryos with dividing nuclei, a reduction in JIL-1 kinase activity leads to nuclear fragmentation and to dispersion of centrosomes and deformed mitotic spindles. However, this phenotype is thought to be a consequence of altered chromatin structure that occurs during interphase, such as that observed in polytene chromosomes. The aberrant chromosome structure interferes with proper mitotic condensation and segregation and ultimately leads to chromatin disintegration. The findings that a low level of histone H3 Ser10 phosphorylation persists in JIL-1^z2 homozygous flies, that mitotic chromosomes in neuroblasts from JIL-1 null mutant larvae show high levels of histone H3 Ser10 phosphorylation, and that the Drosophila Aurora B kinase phosphorylates histone H3 Ser10 during mitosis (Giet, 2001) further support the notion that JIL-1 is not a mitotic histone H3 Ser10 kinase in Drosophila, but rather is important for maintenance of chromatin structure at interphase. It is becoming clear that multiple kinases can phosphorylate the Ser10 residue on histone H3 and that this single histone modification can elicit diverse cellular responses. What determines which of multiple pathways are activated may be influenced by the presence of additional histone tail modifications that, used in a combinatorial fashion, mediate context-dependent signaling (Wang, 2001 and references therein).

The significant decrease in male viability beyond that observed in females argues strongly for a specific role of JIL-1 in dosage compensation in males that may be separate from its function in maintenance of global chromatin structure. Dosage compensation results in a 2-fold hypertranscription of the male's single X chromosome relative to the female's two X chromosomes. The level of H4Ac16 is increased on the male X chromosome as a consequence of MSL chromatin remodeling complex activity, and this targeted acetylation has been directly linked to transcriptional activation. Notably, levels of phosphorylated histone H3 Ser10 as well as the double H3 Ser10Ac14 modifications are also upregulated on the male X, in agreement with the model that enhanced transcription may require a combined signaling via both acetylation and phosphorylation motifs. However, that the male X continues to be hyperacetylated at histone H4Ac16 in the absence of JIL-1 indicates that JIL-1's role in dosage compensation is not likely to be effected via regulation of the MSL complex's histone acetyltransferase activity (Wang, 2001).

Characterization of the grappa gene, the Drosophila histone H3 lysine 79 methyltransferase.

A novel gene, grappa (gpp) is the Drosophila ortholog of the Saccharomyces cerevisiae gene Dot1, a histone methyltransferase that modifies the lysine (K)79 residue of histone H3. gpp is an essential gene identified in a genetic screen for dominant suppressors of pairing-dependent silencing, a Polycomb-group (Pc-G)-mediated silencing mechanism necessary for the maintenance phase of Bithorax complex (BX-C) expression. Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but also display phenotypes characteristic of trithorax-group mutants. Mutations in gpp also disrupt telomeric silencing but do not affect centric heterochromatin. These apparent contradictory phenotypes may result from loss of gpp activity in mutants at sites of both active and inactive chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns, the appearance of methylated K79 during embryogenesis coincides with the maintenance phase of BX-C expression, suggesting that there is a unique role for this chromatin modification in development (Shanower, 2005).

Recent studies on telomeric silencing in S. cerevisiae have led to the identification of a histone methlylase, DOT1, which has a number of unusual properties. First, unlike the previously identified histone methylases, DOT1 does not have a canonical SET domain. Instead, the DOT1 protein resembles a family of S-adenosyl methione methyltransferases that modify arginine residues. DOT1 methylates histone H3 at lysine 79 only when it is assembled into nucleosomes and methylation strongly depends upon prior Rad6 dependent ubiquitination of histone H2B at K123. Second, in yeast, deletion or overexpression of Dot1 disrupts TPE and also silencing of the mating-type loci. In contrast, silencing in the yeast ribosomal gene cluster is disrupted only when DOT1 is overexpressed. Third, both telomeric and mating-type silencing are disrupted by mutations in the lysine 79 residue of histone H3. Fourth, methylation of K79 appears to influence the recruitment of the SIR silencing proteins to the telomeres. The SIR silencing proteins appear to preferentially associate with chromatin that is deficient in K79 methylation, while the proteins are generally not associated with chromatin in which there is an enrichment for K79 methylated H3. Fifth, there is evidence that K79 methylation is coordinated with polymerase transcription via the COMPASS complex. Consistent with the idea that K79 methylation might be coordinated with transcription, H3meK79 is enriched in transcribed sequences in yeast and mammals. Interestingly, the distribution of H3meK79 in the β-globin locus differs from H3meK4 in that it is not found at the locus control region. These findings have led to a model in which H3meK79 serves as a marker for transcribed sequences where it functions to block the association of chromatin proteins that mediate transcriptional silencing (Shanower, 2005).

While Dot1 homologs have been identified in higher eukaryotes, little is known about their biological functions. This report characterized the Drosophila Dot1 ortholog gpp. The gpp transcription unit is >40 kb in length and it encodes a complex array of alternatively spliced transcripts that range in size from 6.5 to >9 kb and are expressed at different developmental stages. Consistent with the assignment of the gpp gene, P-element and X-ray mutations disrupt this large transcription unit and in at least one case lead to the production of truncated mRNAs. The gpp transcripts are predicted to encode 170- to 232-kD polypeptides that share a common N-terminal domain that corresponds to about two-thirds of the protein but have different C-terminal domains. The common N-terminal domain contains the Dot1 homology region including the MT methyltransferase fold required for methylation of histone H3. Mutation of conserved glycine residues in the active site of both yeast and human DOT1 protein inactivates the enzyme. GPP also contains domains that are not present in DOT1 including a coiled-coil motif also found in the human, C. elegans, D. pseudoobscura, and A. gambia DOT1-like proteins. In yeast, K79 is mono-, di-, and trimethylated and Dot1 is responsible for all three modifications. The different methylated states of H3 at K79 suggest that multiple regulatory activities are conferred on these modified nucleosomes. However, in fly tissue culture cells, the mono- and di- but not the trimethylated form is observed. Since database searches indicate that gpp is the only fly Dot1 homolog, it should also be the sole fly protein in this class that methylates histone H3 on K79. Consistent with this suggestion, discs and other tissues isolated from gpp mutant larvae have little if any H3 mono- or dimethyl K79 (Shanower, 2005).

Like its yeast counterpart, gpp is required for the silencing of reporter transgenes inserted into telomeric heterochromatin. However suppression of silencing associated with pericentric heterochromatin is unaffected by mutations in gpp. While these observations point to a role of gpp in silencing specific for telomeric heterochromatin, antibody staining experiments indicate that there is a paucity of H3dmeK79 at telomeres in polytene chromosomes compared to many other chromosomal DNA segments. In this respect it is interesting that both telomeric and mating-type chromatin in yeast are hypomethylated on K79 compared to 'bulk' chromatin even though DOT1 is required for SIR silencing in each case. It has been suggested that the meK79 modification in euchromatic nucleosomes blocks SIR protein association and that silencing is lost in the absence of DOT1 because the SIR proteins spread into euchromatin. In contrast, in flies, since many euchromatic domains in wild-type polytene chromosomes have only little H3meK79, it is difficult to see how telomeric silencing proteins would be restricted to telomeres by this modification even when gpp is fully active (Shanower, 2005).

gpp also has functions in flies besides telomeric silencing. Unlike Dot1, gpp is essential for viability. Although the underlying cause of lethality remains to be established, gpp mutant larvae grow more slowly than wild type and this potentially implicates gpp in pathways that control growth rates and size in flies. In addition, gpp mutants display defects that are characteristic of both Pc-G and trx-G genes. The first gpp alleles were recovered as dominant suppressors of mini-white silencing by two BX-C PREs. Consistent with a role in Pc-G silencing, gpp mutants enhance the segmentation defects of several Pc-G genes. In this context, it is interesting to note that several Pc-G genes have recently been shown to play a role not only in the repression of genes in the homeotic complexes but also in telomeric silencing. Thus, it is possible that gpp activity in telomeric silencing may be linked in some manner to its role in Pc-G silencing (Shanower, 2005).

gpp mutants also exhibit transformations in segment identity and genetic interactions with Abd-B that are characteristic of trx-G mutations. This would point to a role in promoting rather than repressing gene expression. Some function in transcription would be consistent with studies in other systems as well as with the enrichment of meK79 seen in many polytene interbands and puffs. However, this correlation is not complete. Thus, there are many puffs and interbands that have only little H3dmeK79. Conversely, H3dmeK79 is sometimes enriched in bands. These findings would argue that in Drosophila, meK79 is not a ubiquitous marker for transcriptionally active chromatin, but rather may have functions that are specific to particular chromatin domains. In this case, the disruptions in homeotic gene expression seen in gpp mutants could reflect a special requirement for H3meK79 in the transcription of these particular genes. Domain-specific requirements for gpp activity in transcription could also potentially account for the effects of gpp mutations on Pc-G and telomeric silencing. In this model, Pc-G and telomeric silencing would be disrupted in gpp mutants because the expression of one or more Pc-G (and/or telomeric heterochromatin) genes is downregulated when gpp activity is compromised (Shanower, 2005).

The developmental profile of H3dmeK79 also suggests that this modification cannot be a ubiquitous marker for either transcriptionally active or silenced chromatin. High levels of Pol II transcription in somatic nuclei begin in the precellular blastoderm stage around nuclear cycle 11/12. Concomitant with the activation of transcription, H3meK4 can be first be detected at this stage, and the level of meK4 then increases through cellularization. By contrast, little if any H3 mono- or dimethyl K79 is in either the transcriptionally active somatic nuclei or the transcriptionally quiescent pole cell nuclei. H3meK79 can first be readily detected only later in development in germband extended embryos. However, at this stage accumulation is restricted primarily to a subset of cells in the embryo, most of which seem to be in the process of cell division. High levels of H3meK79 are not observed until stages 13-15, long after the initial upregulation of transcription in the early zygote. This result also suggests that the homeotic transformations seen in gpp mutants are unlikely to be due to defects in the initial establishment of parasegment-specific patterns of homeotic gene expression by the gap and pair-rule genes. Rather, these transformations probably reflect a requirement for gpp activity later in development during the maintenance phase of homeotic gene regulation -- a phase that is dependent upon Pc-G and trx-G genes. In this respect it is curious that homeotic transformations are not observed in gpp embryos when they hatch as first instar larvae. Maternally derived gpp activity in homozygous mutant embryos maybe sufficient to maintain specific parasegmental patterns of homeotic gene expression through the end of embryogenesis. Alternatively, there may not be absolute requirement for H3meK79 in maintaining appropriate parasegmental patterns of homeotic expression during embryogenesis (Shanower, 2005).

The developmental profile of H3meK79 indicates that this modification is present at low levels in specific developmental stages and tissues (CNS) undergoing active cell division. In contrast, the highest levels of H3meK79 are observed in epidermal cells that have exited the cell cycle and are undergoing differentiation. Thus, it seems possible that this modification may be activated when specific chromatin configurations, active or inactive, need to be maintained for extended periods of time in the absence of de novo DNA synthesis/chromatin assembly. In this respect it is interesting that it has been reported that the highest levels of meK79 are found in a histone H3 variant, H3.3, which is assembled into chromatin by a replication-independent mechanism. Further studies of gpp in Drosophila will be required to understand the mechanisms governing the temporal and tissue-specific regulation of the K79 modification and how this relates to the functions of this particular histone modification during development. Understanding this aspect of the histone code in a multicellular organism such as Drosophila will lead to a better understanding of chromatin regulatory mechanisms during development (Shanower, 2005).

MSL complex is attracted to genes marked by H3K36 trimethylation using a sequence-independent mechanism

In Drosophila, X chromosome dosage compensation requires the male-specific lethal (MSL) complex, which associates with actively transcribed genes on the single male X chromosome to upregulate transcription 2-fold. On the male X chromosome, or when MSL complex is ectopically localized to an autosome, histone H3K36 trimethylation (H3K36me3) is a strong predictor of MSL binding. Mutants lacking Set2, the H3K36me3 methyltransferase, were isolated, and it was found that Set2 is an essential gene in both sexes of Drosophila. In set2 mutant males, MSL complex maintains X specificity but exhibits reduced binding to target genes. Furthermore, recombinant MSL3 protein preferentially binds nucleosomes marked by H3K36me3 in vitro. These results support a model in which MSL complex uses high-affinity sites to initially recognize the X chromosome and then associates with many of its targets through sequence-independent features of transcribed genes (Larschan, 2007).

MSL complex colocalizes with H3K36 trimethylation on X-linked genes: To investigate the relationship between MSL complex recruitment and histone methylation, ChIP-on-chip analysis of SL2 cells was performed with antibodies that recognize H3 trimethylated at K36 (H3K36me3) or dimethylated at K4 (H3K4me2). The SL2 cell line exhibits a male phenotype with respect to dosage compensation. NimbleGen tiling arrays were used; these contain the entire X chromosome and left arm of chromosome 2, tiled at 100 bp resolution. A general histone H3 antibody was used as a control for histone occupancy, and three biological replicates for tiling arrays indicated a high degree of reproducibility. As expected, the H3K36me3 and H3K4me2 modifications were associated with the 3' and 5' ends of transcribed genes, respectively, as previously reported for S. cerevisiae, mammals, and chicken. Close to 100% of transcribed genes on the X and 2L chromosomes were methylated at H3K36 and H3K4, largely independent of transcript level as previously reported for other organisms. Similar results were observed for MSL3-TAP, specifically on the X chromosome, but a lower fraction of transcribed genes on the X was bound (approximately 80%). With improved computational analysis, 1014 genes on the X chromosome scored positive for MSL binding in SL2 cells (up from previous estimate of 675 genes). 67% of the newly scored MSL-bound genes in SL2 cells were identified previouslyw was clearly bound in at least one cell type (Larschan, 2007).

To determine whether MSL binding colocalizes with H3K36me3 or H3K4me2, the correlation was examined between the data sets at the gene level. Of the 1014 MSL-bound genes in SL2 cells, 93% were positive for H3K36me3, and 83% were positive for H3K4me2. Interestingly, it was previously reported that a small percentage of untranscribed genes were bound by MSL3-TAP (7%), and the current study found that these genes also carried the H3K36me3 histone modification. In addition, untranscribed genes bound by MSL have significantly higher levels of H3K36me3 than untranscribed genes that are unbound by MSL complex. A likely explanation is that some nontranscribed genes are located near transcribed genes with very extensive H3K36me3 and MSL signals or within domains that have continuous strong signal over many kilobases. Specifically, 82% of MSL3-TAP-bound genes are transcribed, while 93% percent of MSL3-TAP-bound genes carry the H3K36me3 modification. Therefore, H3K36me3 is an even better predictor of MSL binding on the X than transcription state as defined by Affymetrix expression arrays. Similar results were observed for clone 8 cells, a Drosophila cell line derived from the wing disc (Larschan, 2007).

Colocalization in terms of whole genes could occur without coincident binding along the gene. It was previously reported that MSL3-TAP binds over the body of transcribed genes specifically on the X chromosome with a bias toward the 3' end. To determine whether H3K36me3 on the X chromosome and MSL complex colocalize spatially within transcription units, average gene profiles were compared for H3 methylation modifications and MSL3-TAP. It was found that H3K36me3 and MSL3-TAP exhibit a similar 3' biased profile, whereas H3 lysine 4 dimethylation is associated with the 5' end of transcription units, as reported in other organisms. Furthermore, at the probe level, a strong positive correlation is observed between MSL binding and H3K36me3 association. In contrast, a weaker correlation is observed with H3K4me2 that associates with the 5' ends of genes. These results demonstrate that H3K36 trimethylation is a 3' biased mark associated generally with active transcription units and that it is a very strong predictor of MSL binding on the X chromosome (Larschan, 2007).

MSL complex attracted to chromosome 2L by a roX2 transgene binds neighboring 2L genes marked by transcription and H3K36me3: When either a roX1 or a roX2 genomic transgene is inserted on an autosome, it attracts MSL complex to its site of insertion, with occasional signs of additional binding to neighboring regions along the autosome. Ectopic binding along the autosome is greatly increased when the X chromosome in the same nucleus is deleted for both roX1 and roX2. Such binding generally extends >1 Mb bidirectionally from the site of the roX transgene insertion, as measured by immunofluorescence for the MSL proteins. One interpretation is that nascent roX RNAs compete for attraction of the MSL proteins for assembly at their site of synthesis and that, after local assembly, MSL complex becomes competent to search for targets in its new chromosome environment. To determine whether ectopic binding on a normally untargeted chromosome would provide clues to the specificity of MSL binding, ChIP-on-chip analysis was performed on MSL3-TAP male larvae mutant for both roX1 and roX2 on the X chromosome and containing a roX2 transgene inserted at position 26D8-9 (near the CG9537 gene) on chromosome 2L. When assayed by immunostaining of polytene chromosomes, such males consistently show MSL binding in interbands along chromosome 2L, surrounding the site of the transgene insertion. At the level of genomic tiling arrays, ChIP results map this binding at high resolution. As a control, an additional array was used that contains the 3R chromosome and the entire X. It was found that the domain of MSL binding extends greater than 2 Mb in each direction from the insertion site on 2L, while binding to 3R was undetected. Importantly, the targets of binding are transcribed 2L genes, with the averaged binding profile showing enrichment over the bodies of genes, with a bias toward 3'ends. Each of these characteristics is typical of target genes on the X chromosome in wild-type larvae, cells, and embryos. Furthermore, when the 2L pattern of ectopic MSL binding in larvae was compared to the wild-type distribution of H3K36 trimethylation in tissue culture cells, a strong correlation was found between MSL binding and K36me3 within 1 Mb of the site of the roX transgenic insertion. Interestingly, although MSL-bound genes are consistently marked with H3K36me3, at greater than 1 Mb distances from the transgene insertion site, MSL complex increasingly skips some H3K36me3-bound genes while binding others. Overall, it was found that MSL targets selected on 2L were transcribed genes enriched for H3K36 trimethylation and that MSL binding showed a 3′ bias analogous to that normally found on X chromosome targets. These results raise the strong possibility that, once targeted to a chromosomal domain by a high-affinity site, MSL complex recognizes general marks for transcription such as H3K36me3 or other 3′-associated features rather than an X-specific sequence element at each individual target (Larschan, 2007).

Set2 is required for H3K36 trimethylation and for viability in both males and females in Drosophila : To investigate whether H3K36me3 plays a functional role in MSL complex targeting, a genetic approach was taken to inactivate the methyltransferase responsible for H3K36me3 in Drosophila. In S. cerevisiae, the Set2 histone methyltransferase is responsible for di- and trimethylation of H3K36. The CG1716-encoded protein has been identified as the likely functional homolog of ySet2 in Drosophila based on the presence of SRI and SET domains. Two initial tests were pursued to examine CG1716 function, the first in yeast and the second in Drosophila tissue culture cells. To test the function of CG1716 in yeast, an inducible CG1716 expression vector was transformed into set2Δ mutant S. cerevisiae that lack detectable H3K36me3. When CG1716 was induced by growth in media containing galactose, H3K36me3 (and some H3K36me2) was restored, demonstrating that a CG1716 cDNA functionally complements the yeast set2Δ. Also, the CG1716-encoded protein can interact with the RNA Pol II CTD as observed for S. cerevisiae Set2, further confirming the identity of CG1716 as the functional homolog of the S. cerevisiae SET2 gene. To test the function of CG1716 in Drosophila tissue culture cells, RNAi was used to target CG1716. A strong reduction of CG1716 mRNA was found to correlate with a significant loss of H3K36me3 by Western blot, immunostaining, and ChIP analysis. H3K4me2, a distinct chromatin mark for transcribed genes, was largely unaffected. ChIP analysis allowed quantification of a 3- to 5-fold reduction in H3K36me3 and only very small changes in H3K4me2. Based on these results, a Drosophila mutant was isolated that disrupts the CG1716 gene, henceforth referred to as the Set2 gene (Larschan, 2007).

Imprecise excision of a P element upstream of the Set2 gene was induced to create a series of Set2 deletion strains, and Set2¹ was selected for further analysis. dSet2¹ eliminates most of the coding region including the catalytic SET domain without extending bidirectionally into the neighboring CG1998 gene. Since the Set2 gene is located on the X chromosome, hemizygous males were initially isolated, and they were found to die as late third-instar larvae. To demonstrate that this lethality was due to loss of Set2, and not to any additional defects that might have been induced during P element excision, a transgene was constructed encompassing only the genomic region of Set2; it was able to fully rescue the Set2¹ mutants. Using the rescued males as fathers, homozygous mutant females were subsequently examined, and the Set2¹ mutation was found to cause late larval lethality in both sexes. To further analyze the viability of Set2 mutants at the cellular level, homozygous mutant Set2 eyes were created in the context of heterozygous mutant adult females, using the GMR-hid system. set2 mutant eyes were diminished in size and rough compared to wild-type eyes, which is a qualitative assay suggesting that Set2 is important for normal cell proliferation (Larschan, 2007).

To determine whether or not H3K36me3 was affected in the set2 mutant, polytene chromosome squashes of mutant larvae were were immunostained. H3K36me3 was significantly depleted in the Set2¹ mutant when compared to wild-type. As a control for the specificity of this defect, the same nuclei were immunostained for the interband protein Z4, which showed similar staining in wild-type and mutant. Set2¹ mutant larvae were further analyzed by ChIP to quantify the H3K36me3 levels in wild-type and Set2¹ mutants. H3K36me3 in the Set2¹ mutant was found to be dramatically decreased at the transcribed genes tested, to levels comparable to an untranscribed gene (CG15570). Changes in H3K4me2 varied from slight to none. Thus, Set2 is required for viability and methylation of H3K36 in Drosophila (Larschan, 2007).

Set2 contributes to optimal MSL complex targeting at transcribed genes, but not at high-affinity sites: To examine whether MSL complex targeting requires H3K36me3, polytene chromosomes of Set2¹ mutant larvae were immunostained with antibodies directed against MSL complex, but no difference in MSL pattern or intensity was detected at this level of resolution. Upon initial consideration, this result would appear to rule out a requirement for H3K36me3 in MSL targeting. However, when attempts were made to validate this observation with ChIP assays conducted with two independent fly stocks and ChIP protocols (both anti-MSL2 and MSL3-TAP IPs), it was found that wild-type and Set2¹ mutant larvae showed significant differences at many specific gene targets. Nine genes with high, medium, or low levels of MSL complex binding were assayed for recruitment of MSL2 and MSL3-TAP in wild-type and Set2¹ mutant third-instar larvae by ChIP analysis. Highly reproducible 2- to 10-fold decreases were observed in MSL2 and MSL3-TAP association at all nine genes assayed. In contrast, MSL complex association with previously reported 'high-affinity sites', such as roX1, roX2, and 18D11, was largely unaffected in the Set2¹ mutant (Larschan, 2007).

Such a result might be attributed to indirect effects in Set2¹ mutant larvae as opposed to specific defects in MSL targeting. To address this, roX RNA and msl2 mRNA levels were measured, and it was found that they were not affected significantly in the Set2¹ mutant, suggesting that H3K36me3 does not affect MSL complex recruitment indirectly by affecting expression of MSL components. Western and polytene staining analysis of Msl1 and Msl2 also indicate that protein levels are largely unchanged. It was also found that ChIP for H3K4me2 and RNA polymerase II were not significantly affected in set2 mutants, further supporting a direct role for H3K36me3 in stabilization of MSL complex at target genes (Larschan, 2007).

To address the functional role of H3K36me3 in transcription of genes bound by MSL complex, the transcript levels of MSL complex target genes were compared in wild-type and Set2¹ mutant larvae. Transcription of MSL target genes is not strongly affected in Set2¹ mutant larvae, although genes that exhibit the strongest loss of MSL complex binding (CG13316, CG12690, CG32555, and CG32575) exhibit decreases in transcript level. Dosage compensation involves a 2-fold upregulation of transcription, limiting the expected transcriptional changes to a 50% decrease in transcript. Furthermore, when H4K16 acetylation at these genes was examined, significant residual levels were found (10-fold over autosomal controls or untranscribed genes), even when very small amounts of MSL complex remain. Thus, residual MSL complex function may be largely sufficient for transcriptional upregulation in the Set2¹ mutant, yet MSL complex targeting is significantly reduced (Larschan, 2007).

Together, these results suggest that a subset of MSL binding sites is particularly sensitive to H3K36me3 levels, while others, including three previously defined high-affinity sites are not. Since MSL binding is diminished significantly but not ablated in the Set2¹ mutant, these results support a model in which recognition of H3K36me3 is one contributing factor to MSL complex targeting that functions with additional features of transcribed genes (Larschan, 2007).

An important caveat to the conclusion that H3K36me3 functions together with other recognition features is that the heterozygous mothers of hemizygous Set2¹ mutants carry a functional Set2 gene and thus could provide a maternal supply of wild-type Set2 mRNA or protein to the mutant embryos. This maternal contribution of H3K36me3 could be sufficient to initially establish MSL binding, which might be maintained through development, independent of the initial recognition mark. Thus, if the maternal contribution of H3K36me3 could be eliminated, it was hypothesized that an even more significant defect would be observed in MSL complex recruitment. To address this possibility genetically, a stock designed to create homozygous set2 mutant germline clones was constructed using FLP-FRT-mediated recombination in an ovo^D dominant female sterile mutant. After recombination, the set2 mutant germ cells would no longer carry ovo^D and thus should produce oocytes that would lack any maternal Set2 mRNA or protein. Despite recombination to remove ovo^D from germ cells, no functional oocytes were produced, demonstrating that Set2 is essential for oogenesis. Therefore, the maternal contribution of Set2 remains in these studies; its elimination might reveal an even more significant role or H3K36me3 in MSL recruitment than has been reported (Larschan, 2007).

Recombinant MSL3 binds preferentially to nucleosomes trimethylated at H3K36: Eaf3, the yeast member of the conserved MSL3/MRG family of proteins, has been implicated in a physical and functional interaction of Rpd3(S) complexes with H3K36me3, raising the attractive hypothesis that MSL3 plays an analogous function in MSL complex. Furthermore, the distinction between high-affinity MSL binding sites such as roX1, roX2, and 18D11 and the majority of MSL targets is that high-affinity sites are MSL3 independent. Therefore, sensitivity to loss of H3K36me3 might be a specific characteristic of MSL3-dependent targets. To test the idea that MSL3 contributes to specific recognition of H3K36me3-modified nucleosomes, gel shift analyses was performed with recombinant MSL3 protein produced in baculovirus using nucleosomes assembled in vitro. Using an EMSA assay system where specifically modified recombinant nucleosomes were assembled, it was found that purified MSL3 protein showed increased affinity to nucleosomes pretreated with active Set2, and thus marked with H3K36 methylation, as opposed to nucleosomes that were unmodified at H3K36. This preferential binding was only detected in nucleosomes bearing linker DNA, suggesting that affinity for free DNA may be contributing to the binding of MSL3 to the nucleosomes methylated at H3K36. Titrations were performed to measure the relative affinity of MSL3 association with methylated compared to unmethylated nucleosomes. The increased affinity of MSL3 for methylated nucleosomes is best observed at the 4.4 nM concentration. These results provide additional evidence supporting a model in which H3K36me3 is a 3' chromatin mark required for the robust, wild-type MSL binding pattern on the X chromosome (Larschan, 2007).

This study has found that ectopic spreading of MSL complex to the 3' ends of transcribed genes on autosomes indicates that a sequence-independent mechanism can define MSL complex target genes. Furthermore, trimethylation of H3K36 is required for optimal MSL complex targeting to transcribed genes on the male X chromosome subsequent to initial recognition of the X. In the absence of H3K36me3, MSL complex can associate with high-affinity sites on the X chromosome but exhibits reduced binding to target genes. Since MSL binding is reduced but is not eliminated, a model if favored in which association with H3K36me3 is a contributing factor that functions with recognition of one or more additional 3' features of transcribed genes such as nascent mRNAs or RNA Pol II CTD phosphorylation (Larschan, 2007).

In addition to a function for Set2 in MSL complex targeting, this study demonstrates that Set2 is essential for viability of both sexes in Drosophila. Conservation of the Set2 H3K36 methyltransferase function from S. cerevisiae to Drosophila was observed, as predicted by sequence conservation. A variety of roles have been reported for Set2 in several organisms. In Neurospora, S. pombe, and NIH 3T3 cells, Set2 is required for optimal growth rate. The S. cerevisiae set2Δ mutant suppresses the loss of positive elongation factors. In Drosophila, mutants lacking zygotic Set2 function fail to proceed through the developmental transitions from late larval to adult stages. The cause(s) of inviability in Drosophila set2 mutants remains to be determined, but eyes composed entirely of homozygous set2 mutant tissue were small and rough, indicating defects in cell proliferation (Larschan, 2007).

In vitro studies using recombinant MSL3 produced in baculovirus revealed preferential interaction with nucleosomes that were trimethylated at H3K36, suggesting that a direct interaction may occur between MSL complex and H3K36me3 chromatin on the X chromosome. In S. cerevisiae, an MSL3 homolog, Eaf3, mediates an interaction between the Rpd3(S) complex and H3K36me3 at active genes. If conserved, this function in Drosophila presumably would be played by another MSL3 family member, MRG15. In S. cerevisiae, Rpd3(S) is thought to deacetylate histones in the wake of RNA polymerase II to prevent uncontrolled activation and transcription initiation from cryptic start sites within genes. This raises the possibility that, on the X chromosome, MSL complex might compete for binding to H3K36me3 with the repressive deacetylation function of Rpd3(S). Alternatively, H3K36me3 may simply be a mark utilized by MSL complex to regulate target genes by a mechanism independent of Rpd3(S) (Larschan, 2007).

H3K36me3 marks transcribed genes independent of transcript level but is a weak modulator of endogenous transcript and RNA polymerase II levels. In S. cerevisiae, where its role is best understood, Set2 functions to suppress formation of aberrant internal transcripts by facilitating histone deacetylation yet has only small effects on endogenous transcript levels. In Drosophila, small but reproducible changes were detected in transcript levels at MSL complex target genes in set2 mutant larvae. Also, minimal changes were observed in RNA Pol II levels as previously reported for the set2Δ mutant in S. cerevisiae. Also, changes in transcription level due to loss of dosage compensation are small, with a maximal 50% decrease predicted. Thus, the combined loss of the Set2 protein and reduction in MSL complex recruitment did not cause dramatic changes in transcript level. Furthermore, levels of H4Ac16 were decreased but not eliminated at target genes, consistent with residual MSL function that can explain why more dramatic changes in transcription of MSL complex target genes were not observed (Larschan, 2007).

A defined mechanism for MSL complex targeting to hundreds of sites along the male X chromosome has remained elusive. Previous reports have posited two highly related models for MSL complex recruitment: a 'spreading' model and an 'affinities' model. Both models are based on the idea that specific MSL interaction occurs at high-affinity sites that mark the X chromosome. These sites have been mapped on polytene chromosomes, but most are not yet defined at the molecular level. roX genes and other high-affinity sites are thought to concentrate MSL complex within an X chromosome domain. In the spreading model, MSL complex creates the full MSL binding pattern by searching the X chromosome for general characteristics of active genes without necessarily requiring a specific DNA sequence at each gene. This could occur either by scanning along the chromosome in a linear manner or by releasing and rebinding chromosomal regions in close physical proximity. It has been demonstrated that roX RNAs can move in trans from one DNA molecule to another, so linear scanning is possible but not obligatory. The affinities model proposes that there is a continuum of affinity sites for MSL complex, ranging from high to low. Only when high-affinity sites are locally concentrated can low-affinity sites be recognized, similar to the spreading model. The major difference is that even low-affinity sites are predicted to contain sequence elements that direct MSL binding. It is thought that the results documenting the pattern of ectopic MSL binding on chromosome 2L surrounding a roX transgene make the existence of sequence elements at every MSL binding site on the X chromosome unlikely. That the 2L pattern was analogous to that normally found on the X chromosome, targeting transcribed genes marked by H3K36me3 and binding with a 3' bias, is strong evidence that MSL complex recognizes target genes marked by transcription. This does not exclude the possibility that transcribed genes carry common sequence elements but makes it unlikely that such sequence elements differ between autosomal genes and the majority of MSL target genes on the X chromosome (Larschan, 2007).

In summary, the data are consistent with a model in which MSL complex first recognizes nascent roX transcripts and a series of high-affinity sequences along the male X chromosome and then scans the X for target genes that exhibit H3K36 trimethylation and other marks of active transcription. Recognition may involve the MSL3 chromodomain and additional factors. Trimethylation of H3K36 marks the middle and 3' ends of transcription units, independent of absolute transcript levels in Drosophila, consistent with S. cerevisiae and mammalian systems. Thus, MSL complex recognition of H3K36me3 provides an important mechanism for identification of transcribed genes and avoidance of silenced regions (Larschan, 2007).

Transcription-coupled methylation of histone H3 at lysine 36 regulates dosage compensation by enhancing recruitment of the MSL complex in Drosophila melanogaster

In Drosophila melanogaster, dosage compensation relies on the targeting of the male-specific lethal (MSL) complex to hundreds of sites along the male X chromosome. Transcription-coupled methylation of histone H3 lysine 36 is enriched toward the 3' end of active genes, similar to the MSL proteins. This paper reports a study of the link between histone H3 methylation and MSL complex targeting using RNA interference and chromatin immunoprecipitation. Trimethylation of histone H3 at lysine 36 (H3K36me3) relies on the histone methyltransferase Hypb (Set2) and is localized promoter distal at dosage-compensated genes, similar to active genes on autosomes. However, H3K36me3 has an X-specific function; reduction specifically decreases acetylation of histone H4 lysine 16 on the male X chromosome. This hypoacetylation is caused by compromised MSL binding and results in a failure to increase expression twofold. Thus, H3K36me3 marks the body of all active genes yet is utilized in a chromosome-specific manner to enhance histone acetylation at sites of dosage compensation (Bell, 2008).

This study reports that trimethylation of histone H3 lysine 36 is required for high levels of H4K16ac at dosage-compensated genes on the male X chromosome. This function does not reflect an X-specific methylation signature, since both H3K36 methylation states have similar localization patterns at autosomal genes: dimethylation peaks promoter-proximal, and trimethylation shows a 3' bias. Furthermore, the regulation of H3K36me3 depends on the activity of Hypb, which is equally targeted to autosomal and X-linked loci, indicating a common mode of regulation (Bell, 2008).

Nevertheless, downregulation of H3K36me3 in Drosophila SL2 cells resulted in reduced levels of H4K16 hyperacetylation at X-linked genes but simultaneously increased levels at autosomal genes in the same cells. This differential effect on acetylation suggests a context-dependent readout of lysine 36 methylation. In Saccharomyces cerevisiae, H3K36me signals binding of the chromodomain-containing protein Eaf3, which in turn recruits an Rpd3 complex to deacetylate the 3' end of transcribed genes. Evidence is provided that the X-specific reduction of histone acetylation in Hypb-depleted Drosophila SL2 cells reflects compromised recruitment of MSL1 and MOF at dosage-compensated genes. This is in full agreement with reduced binding of MSL3 upon Hypb knockdown, which was recently reported by Larschan and colleagues. MSL3 is one of the Drosophila homologues of yeast Eaf3 and localizes together with MOF and MSL1 to the 3' end of dosage-compensated genes. Thus, in analogy to yeast, MSL3 is likely to associate with H3K36me3 at the 3' end of X-linked genes, leading to robust complex binding and enhanced H4K16 hyperacetylation. This is supported by evidence showing that MSL3 preferentially interacts with Set2-methylated nucleosomes in vitro. Moreover, the observation of Hypb localizing to active sites on polytene chromosomes provides further evidence for a direct role of H3K36me3 in MSL recruitment. However, not all sites enriched for Hypb were also bound by MSL1, suggesting that H3K36me3 is necessary but not sufficient for MSL complex recruitment (Bell, 2008).

Whereas proper binding of the MSL complex to Par-6, CG8173, and Ucp4A relies on the presence of H3K36me3, Hypb knockdown has been shown to not significantly decrease MSL1 recruitment at the roX2 gene. This is similar to the binding of MSL1 and MSL2 to high-affinity sites in msl3 or mof mutant flies, suggesting that strong sequence affinity can target partial MSL complexes independent of H3K36me3. Importantly, despite its presence at the roX2 locus in Hypb knockdown cells, MSL1 was insufficient for adequate MOF recruitment and transcriptional upregulation. Thus, these data indicate that H3K36me3 is necessary at high-affinity sites to facilitate robust MOF interaction and the subsequent hyperacetylation needed to double transcription (Bell, 2008).

Interestingly, roX2 transcription was unaffected by Hypb RNAi when expressed from a plasmid model system. Since the consequence of reduced H3K36me3 on H4K16ac on the roX2 plasmid was not determined in this study, it is possible that a less pronounced reduction in acetylation might account for this effect (Bell, 2008).

In contrast to the roX2 gene, H3K36me3 was required for MSL1 binding to lower-affinity genes. At these genes, transcription-dependent methylation might facilitate DNA accessibility in the 3' end by enhancing the recruitment of MOF and the hyperacetylation of H4K16 (Bell, 2008).

At autosomal genes, reduced trimethylation caused the opposite effect on H4 lysine 16 acetylation. Thus, one modification may signal two different outcomes in the same cell in a chromosome-specific fashion. It is conceivable that such differential readouts involve interaction with either distinct methyl-binding proteins or alternative subunit compositions (Bell, 2008).

The presence of antagonistic activities in the same nucleus, which are targeted to the same modification, requires spatial restriction of individual protein complexes to avoid deregulation by improper acetylation or deacetylation. Thus, the preferential interaction of MSL proteins with H3K36me3 on the X chromosome might be favored by locally accumulating MSL proteins at high-affinity sites. MSL interactions with nuclear pore proteins suggest a possible role of nuclear organization in X chromosome dosage compensation, which may further contribute to a preferential binding of MSL proteins to H3K36me3. Conversely, while this confines histone acetyltransferase activity to dosage-compensated genes on the X chromosome, it might also ensure that the same activity is not mistargeted to autosomal genes (Bell, 2008).

Drosophila histone demethylase KDM4A has enzymatic and non-enzymatic roles in controlling heterochromatin integrity

Eukaryotic genomes are broadly divided between gene-rich euchromatin and the highly repetitive heterochromatin domain, which is enriched for proteins critical for genome stability and transcriptional silencing. This study shows that Drosophila KDM4A (dKDM4A), previously characterized as a euchromatic histone H3 K36 demethylase and transcriptional regulator, predominantly localizes to heterochromatin and regulates heterochromatin position-effect variegation (PEV), organization of repetitive DNAs, and DNA repair. dKDM4A demethylase activity is dispensable for PEV. In contrast, dKDM4A enzymatic activity is required to relocate heterochromatic double-strand breaks outside the domain, as well as for organismal survival when DNA repair is compromised. Finally, DNA damage triggers dKDM4A-dependent changes in the levels of H3K56me3, suggesting that dKDM4A demethylates this heterochromatic mark to facilitate repair. It is concluded that dKDM4A, in addition to its previously characterized role in euchromatin, utilizes both enzymatic and structural mechanisms to regulate heterochromatin organization and functions (Colmenares, 2017).

Heterochromatin (HC) comprises 20% and 30% of human and Drosophila genomes, respectively, and remains condensed throughout the cell cycle. HC composition is distinct from euchromatin (EC), with its low gene count and high enrichment for repetitive sequences, di- and tri-methylated histone H3 K9 (H3K9me2 and 3), and Heterochromatin Protein 1a (HP1a). HC is concentrated at pericentromeric and telomeric regions, where it plays important roles in genome stability. Disruption of HC structure impairs chromosome segregation, replication timing, transposon silencing, gene expression, and DNA repair. However, the mechanisms by which HC components mediate these diverse processes remain poorly understood (Colmenares, 2017).

Central to HC structure is the enrichment for H3K9me2 and me3, primarily catalyzed by the histone methyltransferase (HMTase) Su(var)3-9. These methyl marks form the epigenetic basis for HC maintenance by providing binding sites for HP1a. HP1a, in turn, recruits other proteins that mediate the many chromosomal and nuclear functions of HC. Loss of Su(var)3-9, HP1a, or other HC proteins leads to defects in HC function, which can be observed as changes in the silencing of genes inserted into or near HC (position-effect variegation or PEV). Reporter genes placed in proximity to HC undergo stochastic silencing due to variable spreading of HC proteins, and become derepressed or further silenced upon disruption or augmentation of HC components, respectively. Genetic screens for PEV modifiers have identified ~150 genes that regulate HC function, but the identities of the majority of these genes and their molecular roles in HC structure and function are unknown (Colmenares, 2017).

Accumulating evidence shows that HC components are critical for genome integrity and play important roles in DNA repair. Loss of the Su(var)3-9 HMTase leads to chromosome segregation defects, repetitive DNA instability, and accumulation of DNA repair protein foci, including phosphorylated H2A variant (γH2Av). Su(var)3-9, HP1a, and the Smc5/6 complex also facilitate repair of heterochromatic double-strand breaks (DSBs) by the homologous recombination (HR) pathway, which utilizes a distinct and dynamic spatiotemporal regulatory mechanism. Early steps in HR repair, such as end resection, occur within minutes after DNA damage inside the HC domain. However, Rad51 recruitment, which is required to complete HR repair, only occurs after DSBs are relocalized outside the HC domain and associate with the nuclear periphery. This spatial partitioning of HR events enables the separation of repeats with DSBs from the rest of the HC, which likely reduces the probability of reciprocal exchange that results in genome instability (e.g., translocations that form acentric and dicentric chromosomes), and promotes less harmful HR repair from homologs or sister chromatids (Colmenares, 2017).

The Drosophila KDM4A (dKDM4A) protein belongs to the jumonji family of Fe(II)- and α-ketoglutarate-dependent lysine demethylases (Whetstine, 2006). Members of this family play vital roles in epigenetic mechanisms that govern gene expression and development, regulate DNA repair and genome stability, and are misregulated in many types of cancers. Despite its name, the closest dKDM4A mammalian homolog is KDM4D, since both contain the JmjN and JmjC domains responsible for enzymatic activity, and lack the PHD and Tudor domains found in human KDM4A. dKDM4A catalyzes the demethylation of H3K36me3 and H3K36me2 in vitro and in vivo (Lin, 2008; Crona, 2013), suggesting a transcriptional role, as these modifications are hallmarks of active gene bodies. Consequently, recent studies have focused on how dKDM4A regulates gene activity in EC (Colmenares, 2017).

dKDM4A homologs, as well as the closely related fly homolog KDM4B, have also been reported to demethylate H3K9me3. However, an in vitro study concluded that dKDM4A lacks the H3 K9 demethylase activity associated with mammalian family members and dKDM4B; thus dKDM4A-dependent changes in H3K9me3 levels observed in vivo in flies are likely indirect or require additional cofactors. KDM4A homologs have also been shown to demethylate other HC-associated modifications, including H1.4K26me3 and H3K56me3. However, Drosophila H1 lacks the lysine methylated in other species, and H3K56me3 has not been tested as a dKDM4A substrate in flies. Interestingly, dKDM4A contains a PxVxL motif that mediates its interaction with HP1a. Surprisingly, despite these links to HC, a potential role for dKDM4A in regulating HC structure or function has not been reported (Colmenares, 2017).

This study demonstrates that Drosophila KDM4A is highly enriched in HC and is required for normal HC structure and function, including repair of DNA damage. dKDM4A is show to affect transcription of some EC genes, but limited evidence was found for transcriptional regulation of heterochromatic genes and transposons. Instead, this study shows that dKDM4A is required for the spatial organization of repetitive elements, PEV, and the mobilization and repair of HC DSBs. It was further determined that dKDM4A contributes a structural, non-catalytic role in maintenance of HC integrity, as assayed by PEV. In contrast, dKDM4A catalytic activity was shown to be important for relocalization of DSBs outside the HC domain and for organismal survival of DNA repair mutants. Finally, DNA damage was found to trigger dKDM4A-dependent changes in the levels of heterochromatic H3K56me3. Altogether, it is concluded that dKDM4A is an HC component vital to the stability and organization of repetitive DNA and HC-mediated gene silencing, through a combination of structural and enzymatic functions (Colmenares, 2017).

These investigations show that Drosophila KDM4A is a structural component of HC, and regulates HC organization, PEV, and DNA repair. This study also identifies distinct dKDM4A functions in different nuclear domains (HC versus EC) and both structural and catalytic dKDM4A roles in HC, highlighting the significance of determining the enzymatic and non-enzymatic roles of dKDM4A homologs and their diversity in function and localization. dKDM4A is recruited to HC downstream of HP1a and Su(var)3-9, and is required for PEV in a non-catalytic manner. This suggests that dKDM4A contributes structurally to gene silencing and regulates the proper organization of HC complexes and sequences. dKDM4A is also required for relocalization and proper repair of heterochromatic DSBs. Intriguingly, normal HC DSB dynamics depend on dKDM4A catalytic activity and are associated with dKDM4A-dependent H3K56me3 demethylation, suggesting that this HC mark and its demethylated state(s) are important for DNA repair. Moreover, dKDM4A is required for organismal survival in the presence of mutations disrupting DNA repair and checkpoint pathways, further supporting a key role for this protein in maintaining genome stability (Colmenares, 2017).

The observation that dKDM4A is required for higher-order HC structure, specifically the organization of satellite DNAs, suggests that dKDM4A functions to maintain HC architecture. However, such defects in HC structure caused by loss of dKDM4A do not result from visible disruption of HP1a localization or dynamics, or from altered H3K36me3 levels in HC. Although changes in H3K9me3 levels after loss of dKDM4A were detected, these were not sufficient to alter transcription of the majority of HC elements. Whether dKDM4A directly or indirectly affects H3K9me3 levels in vivo is unclear. Regardless, this study shows that PEV does not require dKDM4A enzymatic activity, indicating that HC-mediated gene silencing is not directly regulated by dKDM4A demethylation of H3K9me3, or any other substrate. Although it is possible that the minor H3K9me3 perturbations are sufficient to disrupt HC, these results are more consistent with a direct structural role for dKDM4A in HC organization and gene silencing (Colmenares, 2017).

dKDM4A is directly recruited to HC by HP1a, suggesting that dKDM4A closely participates in HP1a-mediated HC functions. dKDM4A effects on variegation indicate that recruitment of factors downstream of HP1a assembly are required for full HC integrity and function. Although dKDM4A is not required for HP1a-mediated suppression of transposable element transcription, dKDM4A-dependent effects on satellite repeat organization were detected. This could suggest that suppression of PEV by dKDM4A mutants results from disruption of higher-order HC structure that increases accessibility of transcriptional machinery to HC domains. FISH studies have identified a host of regulatory proteins, including cohesins and condensins, that promote or antagonize pairing between HC domains. Whether dKDM4A participates in chromosome pairing, or influences cohesion or condensation in HC, remains to be determined. Alternatively, dKDM4A may assemble or link repetitive sequences into discrete domains within HC, which become unraveled and dispersed in the absence of dKDM4A, or dKDM4A may protect repetitive regions from being excised as extrachromosomal circles (Peng and Karpen, 2007; Colmenares, 2017 and references therein).

A role in genome organization could also account for the impact of dKDM4A on HC DNA repair. Impaired repair of IR-induced DSBs in the absence of dKDM4A could result from defects in pairing of homologous chromosomes or sister chromatids, which would normally facilitate 'safe' HR among repeats, or from problems in folding or concatenation of HC domains that inhibit efficient exit of repair foci from HC. In fact, homozygous dKDM4A mutant adult flies are underrepresented compared with their heterozygote siblings; improper repair of spontaneous DNA breaks in HC could account for this subviability. This is consistent with observations in Caenorhabditis elegans, where loss of the dKDM4A homolog results in impaired DNA replication, accumulation of DNA damage, and increased apoptosis (Colmenares, 2017 and references therein).

Support for this hypothesis comes from the synthetic lethality and infertility observed when a dKDM4A mutation is combined with mutations in the DNA damage checkpoint/DNA repair pathways, but not components of the mitotic checkpoint. Thus, spontaneous DNA damage requires dKDM4A to regulate HC structure for efficient repair. Su(var)3-9, but not dKDM4A, is also synthetically lethal with mitotic checkpoint mutants, which may reflect a higher level of spontaneous breaks that occur in the absence of Su(var)3-9, or defects in cohesin recruitment. This suggests that HC affects multiple pathways controlling genome stability, of which a subset is regulated by dKDM4A (Colmenares, 2017).

These experiments also identify a close relationship between dKDM4A and HP1a. HP1a directly recruits dKDM4A to HC, and dKDM4A overexpression can effectively sequester HP1a away from HC (Lin, 2008), which results in suppression of PEV. dKDM4A overexpression also potentially excludes other HP1a CSD-binding partners from HC, which could further exacerbate effects on HC functions. This study also identified EC genes that are co-regulated by dKDM4A and HP1a, indicating that even outside the HC domain these two proteins function together. This result contrasts with previously published data showing antagonistic effects of dKDM4A and HP1a on transcription (Crona, 2013), and may reflect the differences between immediate RNAi effects on tissue culture cells and steady-state effects that develop in mutant fly tissues (Colmenares, 2017).

Previously, HP1a binding was shown to enhance the H3K36me3 demethylase activity of dKDM4A. However, the current results suggest that dKDM4A does not exert PEV regulatory effects through demethylase activity and is not responsible for the low H3K36me3 levels in HC. Therefore, HP1a stimulation of dKDM4A H3K36me3 likely occurs in EC genes, although the possibility that local H3K36me3 levels increase at a few HC genes cannot be excluded. Instead it is proposed that the primary role of dKDM4A in HC is to ensure normal assembly of HP1a complexes that drives HC organization/structure and PEV (Colmenares, 2017).

A primarily structural role for dKDM4A in HC does not preclude the possibility that its catalytic activity regulates other HC functions. In fact, this study shows that dKDM4A enzymatic activity is required for DSBs to relocalize from HC, suggesting that a demethylated substrate facilitates HC repair dynamics and completion of DNA repair. Of three potential histone substrates tested, H3K56me3, which is enriched in HC and is demethylated by a dKDM4A homolog (KDM4D) in mammals, was identified as accumulating in the absence of dKDM4A, specifically after DNA damage induction by IR and in a Su(var)3-9-dependent manner. In mammals, KDM4D transiently localizes to DNA damage sites, but whether KDM4D demethylates H3K56me3 during DNA damage remains to be determined. Little is known about H3K56me3 function, but this residue resides at the junction between histone H3 and nucleosomal DNA and could regulate unfolding of DNA from the nucleosome. H3K56me3 demethylation also occurs during replication in mammalian cells, suggesting that the replication machinery requires removal of this mark for access to HC. Similarly, H3K56me3 demethylation by dKDM4A could also facilitate chromatin changes required for DSB relocalization and successful DNA repair (Colmenares, 2017).

Alternatively, the requirement for dKDM4A enzymatic activity in DSB relocalization may involve demethylation of other histone and non-histone proteins. Methylated peptides found in various non-histone chromatin proteins have been shown to be demethylated by a mammalian KDM4A homolog. Moreover, a fission yeast homolog lacking demethylase activity functions as an anti-silencing factor in HC, and has been proposed to act as a protein hydroxylase. Therefore, it is proposed that dKDM4A recruitment to HC by HP1a regulates heterochromatic DSB repair and genome stability through demethylation of HC-specific mark(s), such as H3K56me3, but could also involve demethylation or hydroxylation of other HC components (Colmenares, 2017).

Many cancers acquire abnormal levels of HC components, which may increase genome instability and promote misregulation of oncogenes and tumor suppressors. Overexpression of several KDM4A family members has been shown to correlate with and drive tumor progression. Although it remains to be determined if human KDM4A family members regulate HC structure and promote HC DNA repair, disruption of such functions potentially contributes to tumorigenesis. The current findings therefore expand understanding of how this demethylase family exerts a myriad of effects in cancer tissue. Overexpression/ectopic expression of human KDM4A homologs have been shown to induce transient site-specific amplification of a cytogenetic region containing satellite DNA in tumors and cell lines

Long-range spreading of dosage compensation in Drosophila captures transcribed autosomal genes inserted on X

Dosage compensation in Drosophila males is achieved via targeting of male-specific lethal (MSL) complex to X-linked genes. This is proposed to involve sequence-specific recognition of the X at approximately 150-300 chromatin entry sites, and subsequent spreading to active genes. This study asked whether the spreading step requires transcription and is sequence-independent. It was found that MSL complex binds, acetylates, and up-regulates autosomal genes inserted on X, but only if transcriptionally active. It is concluded that a long-sought specific DNA sequence within X-linked genes is not obligatory for MSL binding. Instead, linkage and transcription play the pivotal roles in MSL targeting irrespective of gene origin and DNA sequence (Gorchakov, 2009).

To ask whether autosomal genes can effectively recruit MSL complex, a construct was created that was named TrojanHorse, consisting of a 14-kb fragment from chromosome arm 2L encompassing two small genes, Rpl40 and cg3702. These genes were chosen because they are expressed at all stages of development and are associated with H3K36me3, both hallmarks of typical MSL targets on the X. The sequence of the 14-kb fragment was altered by incorporating five 0.2-kb tags of non-Drosophila origin into the 3'-untranslated regions (UTRs) of Rpl40 and cg3702, and the nontranscribed regions of TrojanHorse. This design allowed clear discrimination between transcripts derived from the endogenous Rpl40 and cg3702 genes and their transposed copies on the X. Additionally, these tags enabled direct assessment of MSL protein and histone modification profiles across the TrojanHorse insertion on the X chromosome. The TrojanHorse construct was inserted in two precise genomic positions using the recently developed attB/attP phage phiC31 integration system. One insertion site (attP18 at cytological position 6C12) was in a gene-rich region encompassing numerous MSL gene targets. In contrast, the second landing site (attP3 at 19C4) was intentionally chosen to be distant from MSL targets and active genes, with the nearest MSL target 60 kb proximal to the TrojanHorse insertion. Thus, in the second case, the two constitutively expressed autosomal genes were inserted on X within a large, MSL-depleted region (Gorchakov, 2009).

If specific DNA sequences at each gene are required to complete the MSL pattern on X, then TrojanHorse genes, originating from an autosome, should not acquire MSL binding. Alternatively, if linkage to the X chromosome and the active state are sufficient, TrojanHorse genes should be bound and acquire H4K16ac. Therefore, ChIP assays were performed to determine the binding profiles for two MSL subunits, MSL2 and TAP-tagged MSL3, within TrojanHorse placed at either attP18 or attP3. Independent of the integration site, pronounced binding was observed of MSL proteins to the tags located at the 3' ends of both cg3702 and Rpl40 of TrojanHorse in mixed sex embryos and male larvae. In contrast, the untranscribed regions of TrojanHorse did not attract the MSL complex. Furthermore, MSL complex binding led to H4K16 acetylation of TrojanHorse chromatin. In agreement with the binding profiles for MSL2 and MSL3-TAP, H4K16ac was found to peak at the 3'-UTRs of cg3702 and Rpl40 in both embryos and male larvae. Notably, the histone H4 acetylation profile was broader than that of the MSL complex, consistent with recent studies. Finally, it was confirmed that the two genes within TrojanHorse were indeed transcribed in larvae and male and female embryos by mapping RNA polymerase II and histone methylation marks expected for active genes: dimethylated H3K4 (H3K4me2) at the 5' ends, and H3K36me3 biased toward the 3' ends. Taken together, these results demonstrate that transcribed and H3K36me3-marked autosomal genes can become typical MSL targets when placed on the male X, favoring a sequence-independent model for spreading. These data also indicate that targeting can occur in the apparent absence of a nearby CES, as the closest identified entry sites are located ~54 kb and 80 kb proximal to attP18 and attP3, respectively. Furthermore, attP3 is 60 kb away from the nearest active gene cluster or MSL-bound region, indicating that targeting can occur over long distances in cis (Gorchakov, 2009).

Despite the clear association of transposed autosomal genes with MSL protein in the male X environment, it remained to be determined whether their transcription or some other features were required for the observed complex binding. In order to address this question, a 'promoterless' version of TrojanHorse was created where the 1.2-kb region, including the promoters and 5' ends of both cg3702 and Rpl40, was deleted. This TrojanHorseδ, otherwise identical to TrojanHorse, was placed in the same attP18 site by attP/attB recombination. Quantitative PCR (qPCR) analysis confirmed that transcription of both genes in TrojanHorseδ was dramatically reduced (at least 25-fold), approaching the detection limit. In addition, the genes exhibited background levels of H3K36me3 in TrojanHorseδ. It was then asked whether transcriptionally inactive genes showed any changes in binding patterns of MSL and other proteins. Indeed, MSL3-TAP no longer bound nontranscribed cg3702 and Rpl40, resulting in decreased H4K16ac levels throughout TrojanHorseδ. This direct comparison clearly supports the importance of transcription for MSL recognition of its targets (Gorchakov, 2009).

To test whether the genes within the intact TrojanHorse were not only associated with MSL complex and marked with H4K16ac but also were subject to dosage compensation, the degree of up-regulation of cg3702 and Rpl40 was directly measured in the TrojanHorse context. These genes are transcribed and dosage-compensated in male larvae so that expression of one copy in males is approximately equal to two copies in females, and about twofold higher than in heterozygous females. Likewise, up-regulation is observed in male embryos. However, in this case the dosage compensation appears less complete, perhaps due to maternal deposition of the transcripts leading to an underestimate of the male/female zygotic transcription ratio. Taken together, these data indicate that the MSL complex bound to TrojanHorse retains all its functions: It acetylates the underlying chromatin and increases transcriptional output, resulting in dosage compensation of both Rpl40 and cg3702 (Gorchakov, 2009).

To exclude the possibility that the two genes in the TrojanHorse construct were somehow exceptional in their ability to attract MSL complex, a more extensive set of active autosomal genes was tested by engineering a larger A-to-X transposition. TrojanElephant was created by inserting a 65-kb region from chromosome 2L, containing 20 genes from cg13773 to snRNP70K into the attB-P[acman] vector via recombineering. None of the TrojanHorse or TrojanElephant genes attracted MSL complex when located in their endogenous positions on autosomes. Therefore, the TrojanElephant construct was inserted at the previously characterized attP3 site on the X, which is a gene-poor region. Several possible results were hypothesized: (1) Within the 65-kb segment, the MSL complex might target all H3K36me3-positive genes. (2) MSL binding might be detected over the active genes closest to the ends of the 65-kb TrojanElephant, with a gradual decrease in binding closer to the center. (3) The MSL complex might skip genes in TrojanElephant altogether (Gorchakov, 2009).

To determine which of these scenarios occurs in vivo, MSL binding across TrojanElephant was measured in third-instar larvae by ChIP followed by qPCR. The data obtained from both experiments clearly demonstrated the ability of the MSL complex to faithfully recognize the transcribed and H3K36me3-marked genes within the transposed material, with no evidence for skipping any of the active genes. Therefore, if the MSL complex spreads linearly from the closest identified flanking CES, it can travel at least 83 kb. The fact that all active autosome-derived genes were bound by MSL complex argues strongly against the idea that each X-chromosomal gene possesses special MSL recruiting signals. Instead, it is proposed that after the initial attraction of the MSL complex by a chromatin entry sites (CES) or roX RNA gene, transcription of active genes serves as the main guiding feature for MSL complex binding (Gorchakov, 2009).

These results are consistent with early transgenic studies, indicating that single genes could become dosage-compensated when inserted on X. However, the work still needs to be reconciled with results from established transposition stocks, in which much larger inserts of autosomal material onto X, failed to acquire dosage compensation or cytologically visible MSL binding, or showed binding only a few kilobases into the insertion by ChIP analysis. One possibility is that stable stocks carrying large spontaneous or X-ray-induced rearrangements may display exceptional behavior, as they have been preselected for viability and thus possibly for the maintenance of chromosome of origin regulation. Alternatively, these results may indicate that distance to the nearest functional CES is critical for MSL targeting, but that this critical distance cannot be reached in the current experiments. It may be that once a certain size is attained, any DNA insertion will tend to associate with its chromosome of origin rather than be generally localized within the X-chromosome three-dimensional territory, thus excluding spreading. Testing these possibilities may be within reach in the near future, as improvements in transgenic technology allow larger insertions at predefined breakpoints to be obtained (Gorchakov, 2009).

The data allow further refinement of the two-step model for MSL complex recruitment to the male X chromosome. In the first step, MSLs are thought to bind 150-300 CES containing MRE motifs on X, and ignore autosomes. In the second step, MSLs recognize the active genes on the X irrespectively of their sequence and origin through a transcription-dependent mechanism. Trimethylation of H3K36 is partially responsible for this sequence-independent step. Here it is speculated on the coexistence of two modes of MSL spreading on the X: (1) long-range spreading from the roX genes, and (2) local distribution of the MSL complex from the CES scattered throughout the X (with a median distance of ~100 kb). It is only in the context of the X chromosome that both roX genes and CES are found in cis to each other, making possible both correct chromosome identification and efficient spreading. The enigmatic nature of roX spreading remains to be understood, including whether features of active genes might be recognized directly by roX RNAs (Gorchakov, 2009).

H3K36 trimethylation-mediated epigenetic regulation is activated by Bam and promotes germ cell differentiation during early oogenesis in Drosophila

Epigenetic silencing is critical for maintaining germline stem cells in Drosophila ovaries. However, it remains unclear how the differentiation factor, Bag-of-marbles (Bam), counteracts transcriptional silencing. This study found that the trimethylation of lysine 36 on histone H3 (H3K36me3), a modification that is associated with gene activation, is enhanced in Bam-expressing cells. H3K36me3 levels were reduced in flies deficient in Bam. Inactivation of the Set2 methyltransferase, which confers the H3K36me3 modification, in germline cells markedly reduced H3K36me3 and impaired differentiation. Genetic analyses revealed that Set2 acts downstream of Bam. Furthermore, orb expression, which is required for germ cell differentiation, was activated by Set2, probably through direct H3K36me3 modification of the orb locus. These data indicate that H3K36me3-mediated epigenetic regulation is activated by bam, and that this modification facilitates germ cell differentiation, probably through transcriptional activation. This work provides a novel link between Bam and epigenetic transcriptional control (Mukai, 2015).

To examine histone modifications in differentiating germ cells, wild-type ovaries were stained using monoclonal antibodies specific for histone modifications. The H3K36me3 histone modification, associated with active genes, accumulated in differentiating cystoblasts. H3K36me3 signals were increased in the differentiating cystoblasts that expressed the bam reporter gene (bam-GFP). By contrast, the H3K27me3 modification associated with gene repression accumulated in early germ cells, and its signals decreased as the cells differentiated. These results suggest that the H3K36me3 levels were upregulated in differentiating cystoblasts. Next, H3K36me3 levels were examined in the ovaries of the third instar larvae and bam⁸⁶ mutant adult females, both of which contain undifferentiated germ cells. Although H3K27me3 signals were detected in these undifferentiated germ cells, strong H3K36me3 signals were not detected. Taken together, these data supported the idea that H3K36me3-mediated epigenetic regulation may be involved in germ cell differentiation. (Mukai, 2015).

Set2 methyltransferase is responsible for the H3K36me3 modification. Immunostaining revealed that, in the germarium region, Set2 was expressed in most of the germline cells, and that nuclear Set2 levels increased in differentiating cystoblasts. To determine whether Set2 participates in H3K36me3 accumulation and differentiation, Set2 expression was inhibited by using an UAS-Set2.IR line. Set2 levels in germ cells were reduced by the expression of Set2 RNAi. Specifically, while Set2 signals in differentiating cystoblasts were detected in 100% of control (nanos-Gal4/+) germaria, the Set2 signals in the cystoblasts were significantly reduced in 57% of the germaria, when Set2 RNAi was expressed in germ cells under the control of the nanos-Gal4 driver. Next, H3K36me3 levels were investigated in the ovaries expressing Set2 RNAi. As expected, H3K36me3 levels were reduced as a consequence of Set2 RNAi treatment. In control ovaries, H3K36me3 signals in differentiating cystoblasts were detected in 97% of germaria. By contrast, when Set2 RNAi was expressed in germ cells under the control of the nanos-Gal4 driver, H3K36me3 signals in cystoblasts were severely reduced in 41% of the germaria. Moreover, germ cell differentiation was impaired because of the expression of Set2 RNAi. In 96% of the control germaria, cysts with branched fusomes were observed. However, fragmented fusomes were detected in 34% of the germaria expressing Set2 RNAi. These results indicate that Set2 was required for both H3K36me3 accumulation and cyst formation. Mosaic analysis was performed by using a Set2 null allele Set2¹. Strong H3K36me3 signals were observed in 80% of the control germline clones. By contrast, H3K36me3 levels were considerably reduced in 74% of the Set2^- cystoblasts. Furthermore, a differentiation defect was observed that was similar to that induced by Set2 RNAi treatment in 84% of Set2^- mutant cysts. These results suggest that Set2 is intrinsically required both for H3K36me3 accumulation in cystoblasts and for differentiation (Mukai, 2015).

To investigate the potential regulatory link between Set2 and Bam, their genetic interaction was analyzed. Reduction in Set2 activity by introduction of a single copy of Set2¹ dominantly increased the number of germaria with the differentiation defect in bam⁸⁶/+ flies. Fragmented fusomes were observed in 26% of germaria from the Set2¹/+; bam⁸⁶/+ females , as compared to 5% in bam⁸⁶/+ and 3% in Set2¹/+ females. These results indicated that Set2 cooperates with bam to promote cyst formation. To determine whether bam expression requires Set2 activity, Bam expression in Set2^- germline clones by immunostaining. Indeed, Set2 activity in germ cells was dispensable for bam expression. Conversely, nuclear Set2 expression in the germ cells was significantly reduced by bam mutation, suggesting that bam is involved in the regulation of Set2 in these cells. This result is consistent with the observation that H3K36me3 levels were reduced by bam mutation. Moreover, reducing of bam activity by introducing of a single copy of bam⁸⁶ dominantly increased the number of germaria with weaker H3K36me3 signals in Set2¹/+ flies. Decreased H3K36me3 signals in the cystoblasts were observed in 29% of germaria from the Set2¹/+; bam⁸⁶/+ females, as compared to 3% in Set2¹/+ and 2% in bam⁸⁶/+ females. These data prompted an exploration of the mechanism of regulation of Set2 activity by bam (Mukai, 2015).

To address whether bambam is sufficient for H3K36me3 accumulation, H3K36me3 levels were examined in the ovaries carrying the hs-bam transgene, which is used to ectopically express bam⁺ by heat shock treatment (Ohlstein and McKearin, 1997). No GSCs with a strong H3K36me3 signal were observed in germaria from wild-type females 1 hour post-heat shock (PHS; n = 42). However, H3K36me3 levels in the GSCs were significantly increased in 51% of the germaria from hs-bam females 1 hour PHS (n = 65), indicating that ectopic bam expression is sufficient for H3K36me3 accumulation. Because Set2 is responsible for H3K36me3, it is speculated that bam may regulate Set2 activity to control H3K36me3 accumulation and GSC differentiation. To determine whether Set2 activity is required for these bam-mediated processes, the effect was studied of a reduction in Set2 activity on the GSC differentiation induced by bam. When bam⁺ was ectopically expressed by heat shock, GSC differentiation was induced as previously reported. In 71% of ovaries from hs-bam flies dissected 24 hours PHS, it was found that differentiating cysts, instead of GSCs, occupied the tip of germaria. By contrast, when both bam and Set2 RNAi were ectopically expressed, GSC loss was significantly suppressed. These data suggest that Set2 activity is regulated by Bam, and that Set2 acts downstream of bam and promotes differentiation (Mukai, 2015).

Nuclear Set2 levels were increased in differentiating cystoblasts. Furthermore, nuclear Set2 levels in germ cells were reduced by bam mutation. It is speculated that bam may regulate Set2 nuclear localization. Therefore, whether bam expression is sufficient for Set2 nuclear accumulation was examined. The subcellular localization of Set2 was examined in hs-bam flies cultured at 30°C. First, H3K36me3 levels were examined in the GSCs. H3K36me3 levels in GSCs were increased in 36% of the germaria from the hs-bam females, as compared to 6% in wild-type females. This result suggests that the ectopic expression of bam is sufficient for H3K36me3 accumulation. Next, Set2 subcellular localization was examined in GSCs of hs-bam females cultured at 30°C. Nuclear Set2 levels in GSCs were increased in 54% of the germaria from the hs-bam females, as compared to 12% in wild-type females. These results suggest that bam promotes the nuclear accumulation of Set2 (Mukai, 2015).

To understand the mechanism by which Set2 regulates germ cell differentiation, the genetic interaction between Set2 and the differentiation genes A2BP1 and orb, both of which are required for cyst differentiation, were examined. Reduction of Set2 activity by introduction of a single dose of Set2¹ dominantly increased the number of germaria exhibiting a differentiation defect in orb^dec/+ flies. In 24% of germaria from the Set2¹/+; orb^dec/+ females, fragmented fusomes were observed, as compared with 4% in orb^dec/+ and 7% in Set2¹/+ females. By contrast, the reduction of Set2 activity did not significantly affect cyst formation in A2BP1^KG06463/+ ovaries). These results implied that Set2 function is required to specifically regulate orb expression and promote cyst formation. To confirm this, orb expression was examined in Set2^- cyst clones. Deletion of Set2 led to the delayed activation of orb. Although 74% of the control cyst clones located at the boundary of germarium regions 1 and 2a initiated orb expression, only 31% of Set2^- cyst clones expressed orb. Most (61%) of the Set2^- cyst clones in germarium region 2b recovered orb expression. These observations suggest that Set2 was required for the proper activation of orb in differentiating cysts. Next, the H3K36me3 state of the orb locus was investigated in the ovaries. ChIP assays demonstrated that the H3K36me3 enrichment in the 3'-UTR region of orb was significantly higher than in the 5'-UTR region. It has been reported that the H3K36me3 modification exhibits a 3'-bias, such that H3K36me3 is preferentially enriched at the 3' regions of actively transcribed genes. These results support the idea that orb expression in differentiating cysts is controlled in part by H3K36me3-mediated epigenetic regulation (Mukai, 2015).

Next, the H3K36me3 status was investigated in the orb gene in bam⁸⁶ mutant ovaries. ChIP assays showed that bam mutation reduced the amount of H3K36me3 in the 3'-UTR region of the orb gene. The H3K36me3 modification is linked to transcriptional elongation. Therefore, the results suggested that bam activates orb expression through the epigenetic control. Additionally, H3K4me3 and RNA polymerase II levels in the 5'-UTR region of the orb gene were also reduced by bam mutation, implying a role for bam in transcriptional initiation. To investigate this possibility, further investigation will be needed in order to identify the enzymes responsible for H3K4me3 and exploring the interactions between bam and those enzymes (Mukai, 2015).

These results have shown that H3K36me3 levels are regulated by bam. As a cytoplasmic protein, Bam may indirectly regulate Set2 nuclear localization. Set2 exerts its functions through the interactions with cofactors. Understanding the mechanism by which Bam regulates Set2 will require the identification of the cofactors that mediate the nuclear transport of Set2. These data suggest a link between Bam and epigenetic transcriptional control. Bam may counteract epigenetic silencing in GSCs through H3K36me3-mediated epigenetic regulation. This study showed that orb expression is activated by epigenetic regulation. Because orb encodes a cytoplasmic polyadenylation element-binding protein, Orb may control translation in differentiating cysts in a polyadenylation-associated manner. Bam antagonizes the Nanos/Pumilio complex, which suppresses the translation of target mRNAs that encode differentiation factors . However, the ientity of the target mRNAs and the mechanisms for transcriptional activation have not yet been elucidated. Because Set2 is required for bam-induced GSC differentiation, studies focused on identifying the genes marked by H3K36me3 and on their epigenetic regulation will aid in the identification of the differentiation genes. Because Set2 is linked to transcriptional elongation, differentiation genes in GSCs might be poised for expression, but may be kept awaiting bam expression for full activation. It is anticipated that these results will facilitate a better understanding of the epigenetic mechanisms that regulate gametogenesis (Mukai, 2015).

Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II

Polycomb and trithorax group proteins regulate cellular pluripotency and differentiation by maintaining hereditable states of transcription. Many Polycomb and trithorax group proteins have been implicated in the covalent modification or remodeling of chromatin, but how they interact with each other and the general transcription machinery to regulate transcription is not well understood. The trithorax group protein Kismet-L (KIS-L) is a member of the CHD subfamily of chromatin-remodeling factors that plays a global role in transcription by RNA polymerase II (Pol II). Mutations in CHD7, the human counterpart of kis, are associated with CHARGE syndrome, a developmental disorder affecting multiple tissues and organs. To clarify how KIS-L activates gene expression and counteracts Polycomb group silencing, this study characterized defects resulting from the loss of KIS-L function in Drosophila. These studies revealed that KIS-L acts downstream of P-TEFb recruitment to stimulate elongation by Pol II. The presence of two chromodomains in KIS-L suggested that its recruitment or function might be regulated by the methylation of histone H3 lysine 4 by the trithorax group proteins ASH1 and TRX. Although significant overlap was observed between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L does not bind methylated histone tails in vitro, and loss of TRX or ASH1 function does not alter the association of KIS-L with chromatin. By contrast, loss of kis function leads to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and is accompanied by increased histone H3 lysine 27 methylation - a modification required for Polycomb group repression. A similar increase in H3 lysine 27 methylation was observed in ash1 and trx mutant larvae. These findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin (Srinivasan, 2008).

The trithorax group proteins Kismet and ASH1 promote H3K36 dimethylation to counteract Polycomb group repression in Drosophila

Members of the Polycomb group of repressors and trithorax group of activators maintain heritable states of transcription by modifying nucleosomal histones or remodeling chromatin. Although tremendous progress has been made toward defining the biochemical activities of Polycomb and trithorax group proteins, much remains to be learned about how they interact with each other and the general transcription machinery to maintain on or off states of gene expression. The trithorax group protein Kismet (KIS) is related to the SWI/SNF and CHD families of chromatin remodeling factors. KIS promotes transcription elongation, facilitates the binding of the trithorax group histone methyltransferases ASH1 and TRX to active genes, and counteracts repressive methylation of histone H3 on lysine 27 (H3K27) by Polycomb group proteins. This study sought to clarify the mechanism of action of KIS and how it interacts with ASH1 to antagonize H3K27 methylation in Drosophila. Evidence is presented that KIS promotes transcription elongation and counteracts Polycomb group repression via distinct mechanisms. A chemical inhibitor of transcription elongation, DRB, had no effect on ASH1 recruitment or H3K27 methylation. Conversely, loss of ASH1 function had no effect on transcription elongation. Mutations in kis cause a global reduction in the di- and tri-methylation of histone H3 on lysine 36 (H3K36) - modifications that antagonize H3K27 methylation in vitro. Furthermore, loss of ASH1 significantly decreases H3K36 dimethylation, providing further evidence that ASH1 is an H3K36 dimethylase in vivo. These and other findings suggest that KIS antagonizes Polycomb group repression by facilitating ASH1-dependent H3K36 dimethylation (Dorighi, 2013).

Since KIS promotes transcription elongation, promotes ASH1 binding and counteracts Polycomb repression, it is suspected that these activities might be functionally interdependent. However, the loss of ASH1 function leads to an increase in repressive H3K27 trimethylation without affecting transcription elongation. Furthermore, the treatment of salivary glands with the elongation inhibitor DRB did not affect the level of ASH1 or H3K27me3 associated with polytene chromosomes. It is therefore concluded that KIS promotes transcription elongation and antagonizes Polycomb repression via distinct mechanisms (Dorighi, 2013).

These findings suggest that the major mechanism by which KIS antagonizes Polycomb group repression is by promoting the association of the trithorax group histone methyltransferases ASH1 and TRX with chromatin. Recent biochemical studies have suggested several mechanisms by which ASH1 and TRX counteract Polycomb repression. A histone modification catalyzed by TRX in vitro (H3K4 trimethylation) disrupts interactions between PRC2 and its nucleosome substrate. H3K4me3 directly interferes with the binding of the PRC2 subunit NURF55 (CAF1) to nucleosomes and inhibits the catalytic activity of E(Z) allosterically through interactions with the SU(Z)12 subunit of PRC2. The relevance of this modification to TRX function in vivo is not clear, however, as the bulk of H3K4 trimethylation in Drosophila is catalyzed by the histone methyltransferase SET1. Another mechanism by which TRX counteracts Polycomb repression was suggested by its physical association with the histone acetyltransferase CBP in the TAC1 complex. The acetylation of H3K27 by CBP directly blocks the methylation of this residue by PRC2. It is therefore tempting to speculate that the diminished binding of TAC1 to active genes contributes to the increased methylation of H3K27me3 observed in kis mutants (Dorighi, 2013).

Other histone modifications, including both the di- and tri- methylation of H3K36, also block the catalytic activity of PRC2 in vitro. In Drosophila, H3K36 trimethylation is catalyzed by SET2, which associates with the elongating RNA Pol II via its phosphorylated CTD. In this way, H3K36me3 becomes concentrated at the 3' ends of genes where it plays a role in preventing cryptic initiation. Consistent with its role in transcription elongation, kis mutations decreased the level of H3K36me3 on polytene chromosomes. Interestingly, H3K36me3 blocks the methylation of H3K27 at genes expressed in the C. elegans germline. Thus, H3K36 trimethylation might represent a conserved mechanism for antagonizing PRC2 function to maintain appropriate patterns and steady-state levels of transcription. Transcription- dependent H3K36 trimethylation is unlikely to be the sole mechanism by which KIS counteracts Polycomb repression, however, because blocking transcription elongation with DRB did not increase the level of H3K27me3 on polytene chromosomes. Furthermore, ash1 mutants display elevated levels of H3K27 methylation without a reduction in H3K36 trimethylation or transcription elongation, suggesting that additional mechanisms exist to counteract repressive H3K27 methylation (Dorighi, 2013).

An antagonism between H3K36 dimethylation and H3K27 trimethylation was suggested by the recent discovery that H3K36me2 inhibits PRC2 function in vitro. This finding, together with recent evidence that ASH1 dimethylates H3K36 in vitro, prompted an investigation of whether ASH1 also dimethylates H3K36 in vivo. The chromosomal distributions of ASH1 and H3K36me2 overlap significantly, consistent with their localization at the 5' end of active genes. Furthermore, the levels of H3K36me2 on the polytene chromosomes of both ash1 and kis mutant larvae were significantly reduced, consistent with the role of KIS in promoting ASH1 binding. Taken together, these observations strongly suggest that KIS antagonizes Polycomb repression by promoting the ASH1-dependent dimethylation of H3K36 (Dorighi, 2013).

The differences in the chromosomal distributions of ASH1 and H3K36me2 and the residual H3K36me2 observed in ash1 mutants are probably due to the presence of another H3K36 dimethylase (MES-4) in Drosophila. In addition to dimethylating H3K36, MES-4 is required for SET2-dependent H3K36 trimethylation in vivo, as revealed by RNAi knockdown of MES-4 both in larvae and in cultured cells. By contrast, this study failed to observe a significant reduction in H3K36me3 levels in ash1 mutant larvae. The findings suggest that ASH1 and MES-4 play non-redundant roles in H3K36 methylation in vivo (Dorighi, 2013).

It is becoming increasingly clear that multiple mechanisms (including the trimethylation of H3K4, the di- and tri-methylation of H3K36 and the acetylation of H3K27) antagonize repressive H3K27 methylation catalyzed by Polycomb group proteins. The current findings suggest that KIS plays a central role in coordinating these activities. By facilitating the binding of TRX and ASH1, KIS promotes H3K27 acetylation and H3K36 dimethylation in the vicinity of active promoters. By stimulating elongation, KIS promotes H3K36 trimethylation over the body of transcribed genes. Thus, KIS appears to counteract Polycomb group repression by promoting multiple histone modifications that inhibit H3K27 methylation by the E(Z) subunit of PRC2 (Dorighi, 2013).

Haploinsufficiency for CHD7, a KIS homolog in humans, is the major cause of CHARGE syndrome, a serious developmental disorder affecting ~1 in 10,000 live births (Janssen, 2012). Infants born with CHARGE syndrome often have severe health complications due to defects in the development of tissues derived from the neural crest, including coloboma of the eye, cranial nerve abnormalities, ear defects and hearing loss, congenital heart defects, genital abnormalities and narrowing or blockage of the nasal passages. Based on the phenotypes associated with kis mutations in Drosophila, it seems likely that some of these defects may stem from changes in gene expression resulting from loss of transcription elongation and inappropriate gene silencing by Polycomb group proteins. The current findings suggest that changes in histone H3 modifications resulting from the loss of CHD7 function might contribute to the broad spectrum of developmental defects associated with CHARGE syndrome (Dorighi, 2013).

Sex comb on midleg (Scm) is a functional link between PcG-repressive complexes in Drosophila

The Polycomb group (PcG) proteins are key regulators of development in Drosophila and are strongly implicated in human health and disease. How PcG complexes form repressive chromatin domains remains unclear. Using cross-linked affinity purifications of BioTAP-Polycomb (Pc) or BioTAP-Enhancer of zeste [E(z)], this study captured all PcG-repressive complex 1 (PRC1) or PRC2 core components and Sex comb on midleg (Scm) as the only protein strongly enriched with both complexes. Although previously not linked to PRC2, direct binding of Scm and PRC2 was confirmed using recombinant protein expression and colocalization of Scm with PRC1, PRC2, and H3K27me3 in embryos and cultured cells using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing). Furthermore, it was found that RNAi knockdown of Scm and overexpression of the dominant-negative Scm-SAM (sterile α motif) domain both affected the binding pattern of E(z) on polytene chromosomes. Aberrant localization of the Scm-SAM domain in long contiguous regions on polytene chromosomes revealed its independent ability to spread on chromatin, consistent with its previously described ability to oligomerize in vitro. Pull-downs of BioTAP-Scm captured PRC1 and PRC2 and additional repressive complexes, including PhoRC, LINT, and CtBP. It is proposed that Scm is a key mediator connecting PRC1, PRC2, and transcriptional silencing. Combined with previous structural and genetic analyses, these results strongly suggest that Scm coordinates PcG complexes and polymerizes to produce broad domains of PcG silencing (Kang, 2015).

One of the most interesting properties of chromatin modification is the ability, under certain circumstances, to propagate in cis independent of sequence. This ability to 'spread' may be important for the inheritance of chromatin states initially established through interactions at nucleation sites such as PREs. SAM domain-mediated polymerization is therefore an attractive model to explain the propagation of PcG silencing. From that perspective, Ph, one of the core components of PRC1, may be responsible for the spreading of PRC1 through Ph-SAM polymerization. The compact chromatin environment formed by PRC1 spreading may improve the enzymatic activity of PRC2, and the capacity of the Pc chromodomain to interact with H3K27me3 may also contribute to the synergistic spreading of PcG silencing. However, consistent with the identification of PRC1 and PRC2 as distinct complexes that purify independently, E(z) RNAi does not significantly affect binding patterns of Pc on polytene chromosomes, and E(z) binding is likewise still detected after Pc RNAi. This study found that Scm directly interacts with the PRC2 complex and colocalizes with H3K27me3 in genome-wide analyses. Scm RNAi results in the loss of major sites of E(z) binding and the redistribution of H3K27me3 on polytene chromosomes. In addition, overexpression of the Scm-SAM domain interferes with binding of endogenous Scm to chromosomes and appears to self-polymerize for long distances on polytene chromosomes independently of PRC1 and PRC2. Taken together, it is suggested that the interaction of Scm with PRC2 and polymerization by the Scm-SAM domain may be key factors contributing to PRC2 and H3K27me3 spreading (Kang, 2015).

The strong interaction that discovered between Scm and the G9a SET domain protein, an H3K9 methyltransferase, suggests a new link between H3K27 and H3K9 methylation in Drosophila. These classical histone marks, associated with silent chromatin, were once thought to be largely distinct but are now proposed to have a functional relationship in PcG silencing in mammals, most notably in X inactivation. There was also evidence for colocalization of these two marks in early ChIP analyses at the HOX gene Ubx in imaginal discs. G9a may also play a role in regulation of H3K27 methylation (Mozzetta, 2014). Alternatively, the abundance of G9a may reflect a key role for the CtBP corepressor complex in PcG function rather than for G9a itself, which is a nonessential gene. Genome-wide binding profile analyses have shown that the components of the CtBP complex such as Su(var)3-3 (LSD1) and Rpd3 (HDAC1) are mainly enriched on active genes rather than repressed genes in human cells, and L(3)mbt, one component of the LINT complex, colocalizes with insulator proteins, including CP190 and mod(mdg4), rather than with PcG proteins in Drosophila. Therefore, the possibility that the interactions of Scm with CtBP or LINT repressor complexes occur as independent complexes irrelevant to PcG silencing cannot be excluded. However, considering that PcG silencing could require dynamic interactions during development, components of these repressor complexes may not be permanently stationed in PcG silenced domains but rather participate in PcG silencing transiently. Furthermore, some of the CtBP subunits were copurified in Pc and E(z) affinity purifications, and previous studies reported that CtBP complex components can contribute to PcG silencing in Drosophila. For example, CtBP mutation causes the loss of Pc recruitment to many PREs. Furthermore, Rpd3 deacetylates H3K27ac, which is mutually exclusive with H3K27me3, and Su(var)3-3 demethylates H3K4me1 and H3K4me2, which are active marks linked to Trithorax (Trx) activity and H3K27ac (Kang, 2015).

How PcG complexes find PREs and spread to create repressive domains is not known on a mechanistic level. Perhaps repressor complexes such as CtBP help remove active chromatin marks to attract the PcG initially or enable cycles of spreading to maintain those domains. Future analyses will entail dissecting the direct interactions of Scm, including nucleosomes and their post-translational modifications. Furthermore, through iterative use of BioTAP-XL, the wealth of additional candidates in the Pc, E(z), and Scm pull-downs featured in this study will be invaluable in extending understanding of chromatin-based PcG repression (Kang, 2015).

A molecular threading mechanism underlies Jumonji lysine demethylase KDM2A regulation of methylated H3K36

The dynamic reversible methylation of lysine residues on histone proteins is central to chromatin biology. Key components are demethylase enzymes, which remove methyl moieties from lysine residues. KDM2A, a member of the Jumonji C domain-containing histone lysine demethylase family, specifically targets lower methylation states of H3K36. Structural studies reveal that H3K36 specificity for KDM2A is mediated by the U-shaped threading of the H3K36 peptide through a catalytic groove within KDM2A. The side chain of methylated K36 inserts into the catalytic pocket occupied by Ni(2+) and cofactor, where it is positioned and oriented for demethylation. Key residues contributing to K36me specificity on histone H3 are G33 and G34 (positioned within a narrow channel), P38 (a turn residue), and Y41 (inserts into its own pocket). Given that KDM2A was found to also bind the H3K36me3 peptide, it was postulated that steric constraints could prevent alpha-ketoglutarate from undergoing an 'off-line'-to-'in-line' transition necessary for the demethylation reaction. Furthermore, structure-guided substitutions of residues in the KDM2A catalytic pocket abrogate KDM2A-mediated functions important for suppression of cancer cell phenotypes. Together, these results deduce insights into the molecular basis underlying KDM2A regulation of the biologically important methylated H3K36 mark (Cheng, 2014).

Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at 'entry sites' that contain a consensus sequence motif ('MSL recognition element' or MRE). However, this motif is only ~2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. This study asked whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), it was found that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. These sites were tested for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. Ectopic MSL expression in Kc cells was shown to lead to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, these results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. This analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome (Alekseyenko, 2012).

This study considered the roles of chromatin environment and flanking sequence composition in selection of functional binding sites by a sequence-specific protein complex. It is generally not clear whether the chromatin features that are often observed at the binding sites of proteins contribute directly to binding selectivity or are simply a consequence of binding. In the dosage compensation system of the X chromosome in Drosophila, a unique opportunity is presented to address this question because it is possible to compare the chromatin environment of MSL binding sites in female cells, in the absence of the complex, to male cells, where the functional sites are bound. Binding data from an RNAi experiment were used in which a component of the sex determination pathway was knocked down in females to induce dosage compensation. Bioinformatic analysis of a large number of profiles from the modENCODE project suggests that a pre-existing active chromatin context plays a critical role in establishing the initial binding of the MSL complex on the X. The surprising discovery was made that GC content in the DNA surrounding functional binding sites has a characteristic profile (Alekseyenko, 2012).

In summary, the results strongly support a model in which an active chromatin composition helps define the initial entry sites selected by the MSL complex. Functional MSL binding results in increased lability of local nucleosomal composition, and H4K16 acetylation and JIL-1 binding along the bodies of virtually all active X-linked genes. This work provides key insights into the order of events leading to dosage compensation in Drosophila, and can also serve as a model for using genome-wide data sets to understand how sequence-specific factors find their ultimate targets (Alekseyenko, 2012).

The results support roles for local chromatin environment and flanking GC content in discrimination of true target sites of the MSL dosage compensation complex. The model (see Model for binding site selection by a chromatin associated factor) depicts the GC content and active chromatin marks surrounding MREs in female Kc cells that predict binding by MSL complex in male S2 or BG3 cells (or after MSL induction in female Kc cells). MREs that do not pre-exist in a favorable environment are not bound by MSL complex and thus are non-functional. Definition of the favorable chromatin features that pre-exist factor binding may be a general tool, in addition to DNA motif analysis, for prediction of functional binding sites (Alekseyenko, 2012).

Genome-wide phosphoacetylation of histone H3 at Drosophila enhancers and promoters

Transcription regulation is mediated by enhancers that bind sequence-specific transcription factors, which in turn interact with the promoters of the genes they control. This study shows that the JIL-1 kinase is present at both enhancers and promoters of ecdysone-induced Drosophila genes, where it phosphorylates the Ser10 and Ser28 residues of histone H3. JIL-1 is also required for CREB binding protein (CBP)-mediated acetylation of Lys27, a well-characterized mark of active enhancers. The presence of these proteins at enhancers and promoters of ecdysone-induced genes results in the establishment of the H3K9acS10ph and H3K27acS28ph marks at both regulatory sequences. These modifications are necessary for the recruitment of 14-3-3, a scaffolding protein capable of facilitating interactions between two simultaneously bound proteins. Chromatin conformation capture assays indicate that interaction between the enhancer and the promoter is dependent on the presence of JIL-1, 14-3-3, and CBP. Genome-wide analyses extend these conclusions to most Drosophila genes, showing that the presence of JIL-1, H3K9acS10ph, and H3K27acS28ph is a general feature of enhancers and promoters in this organism (Kellner, 2012).

Activation of transcription in higher eukaryotes requires the interaction between transcription factors bound to distal enhancers and proteins present at the promoter. Recent findings indicate that enhancers contain a variety of histone modifications that change during the establishment of specific cell lineages suggesting that these sequences may play a more complex role in transcription than previously thought. Given the presence of common as well as specific histone marks at enhancers and promoters, it is tempting to speculate that epigenetic modifications at these sequences serve to integrate various cellular signals required to converge in order to activate gene expression. Results described in this study support this hypothesis, demonstrating that the proteins that carry out these histone modifications are necessary to establish enhancer-promoter contacts and activate transcription of ecdysone-inducible genes (Kellner, 2012).

The execution of this process in Drosophila requires the recruitment of JIL-1 by mechanisms that are not well understood. Although the direct involvement of JIL-1 in the transcription process has been brought into question due to the failure to observe recruitment of JIL-1 to heat shock genes in polytene chromosomes, results presented in this study clearly indicate that JIL-1 affects transcription at different steps in the transcription cycle. At the promoter region, phosphorylation of H3S10 by JIL-1 results in the recruitment of 14-3-3 and, subsequently, histone acetyltransferases Elp3 and MOF (Karam, 2010). This study found that JIL-1 is also able to phosphorylate H3S28 at both promoters the enhancers. The establishment of the H3K9acS10ph and H3K27acS28ph modifications correlates with the recruitment of 14-3-3 to enhancers and promoters of ecdysone-induced genes. 14-3-3 has been implicated in numerous cellular processes, where it functions as a scaffold protein). 14-3-3 is found as dimers and multimers; each monomer is capable of binding two targets and can mediate and stabilize interactions between two phosphoproteins. Additionally, acetylation facilitates the dimerization of 14-3-3 molecules and their ability to bind certain substrates. Binding assays have demonstrated that 14-3-3 interacts weakly with H3 tail peptides phosphorylated at S10 and S28, but strong binding is detected if the peptide is both phosphorylated and acetylated on the neighboring lysine residues. Given the ability of 14-3-3 to serve as a scaffold for large protein complexes, its demonstrated interactions with H3K9acS10ph and H3K27acS28ph and the presence of these two modifications at enhancers and promoters, it is possible that contacts between these two sequences are stabilized by 14-3-3. This hypothesis is supported by 3C experiments indicating that induction of transcription of the Eip75B gene is accompanied by strong enhancer-promoter interactions. These interactions are lost in JIL-1, CBP, and 14-3-3 knockdown cells. Since these proteins act several steps downstream from transcription factor binding in the pathway leading to enhancer-promoter contacts, and loss of these proteins results in the abolishment of these contacts, it appears that these proteins, rather than specific transcription factors, may be responsible for enhancer promoter interactions at the ecdysone-inducible genes (Kellner, 2012).

Genome-wide studies using ChIP-seq clearly show the presence of JIL-1, H3K9acS10ph, and H3K27acS28ph at enhancers and promoters of most Drosophila genes. There is a clear correlation between the amount of JIL-1, H3K9acS10ph, and H3K27acS28ph at promoters and the level of transcripts associated with the gene. These three marks are also present at enhancers defined by the occurrence of H3K4me1 and H3K27ac, suggesting that the JIL-1 kinase is a regulator of histone dynamics at enhancers and promoters genome-wide. JIL-1, H3K9acS10ph, and H3K27acS28ph are found at low levels at enhancers before activation, which then increase in intensity and drop in baseline when found in combination with H3K27ac, a mark of active enhancers. These conclusions are different from those previously published examining the role of JIL-1 in transcription and dosage compensation (Regnard, 2011). This study concluded that JIL-1 binds active genes along their entire length and that the levels of JIL-1 are not associated with levels of transcription. The differences in the conclusions may be due to the different cell lines used -- male S2 cells versus female Kc cells -- and the emphasis of the analysis by Regnard on the expression of dosage-compensated genes in the male X-chromosome, which may contain JIL-1 throughout the genes as a consequence of their regulation at the elongation step. In addition, the study by Regnard used ChIP-chip on custom tiling arrays of the X chromosome plus cDNA arrays containing the whole genome. This strategy may bias the conclusions and suggest the presence of JIL-1 in the coding region of genes rather than at enhancers and promoters (Kellner, 2012).

Results presented in this study extend the previous list of histone modifications characteristic of active enhancers to include H3K9acS10ph and H3K27acS28ph. Enhancers tend to be cell type-specific and are determined during differentiation with the characteristic H3K4me1 modification. It is unclear how these regions are designated before activation and what keeps them in a poised state ready for activation upon receiving the proper signal from the cell. It is tempting to speculate that the presence of JIL-1 at enhancers prior to activation might play a role in maintaining the enhancer in this poised state. An important question for future studies is the mechanistic significance of the looping between enhancers and promoters in order to achieve transcription activation. One interesting possibility is that various signaling pathways in the cell contribute to building epigenetic signatures at enhancers and promoters in the form of histone acetylation and/or phosphorylation of various Lys/Ser/Thr residues. Acetylation marks at enhancers and promoters may then cooperate to recruit BRD4 (FS(1)H in Drosophila), which contains two bromodomains each able to recognize two different acetylated Lys residues. The requirement for acetylation of histones at enhancers and promoters in order to recruit Brd4 would ensure that several different signaling events have taken place before recruitment of P-TEFb by BRD4 can release RNAPII into productive elongation (Kellner, 2012).

Drosophila dSet2 functions in H3-K36 methylation and is required for development

Drosophila Set2 encodes a developmentally essential histone H3 lysine 36 (K36) methyltransferase. Larvae subjected to RNA interference-mediated (RNAi) suppression of Set2 lack Set2 expression and H3-K36 methylation, indicating that Set2 is the sole enzyme responsible for this modification in Drosophila. Set2 RNAi blocks puparium formation and adult development, and causes partial (blister) separation of the dorsal and ventral wing epithelia, defects suggesting a failure of the ecdysone-controlled genetic program. A transheterozygous EcR null mutation/Set2 RNAi combination produces a complete (balloon) separation of the wing surfaces, revealing a genetic interaction between the Ecdysone receptor (EcR) and Set2. Immunoprecipitation studies demonstrated that Set2 associates with the hyperphosphorylated form of RNA polymerase II (RNAPII) (Stabell, 2007).

Ash1 targets H3 and H4

The establishment and maintenance of mitotic and meiotic stable (epigenetic) transcription patterns is fundamental for cell determination and function. Epigenetic regulation of transcription is mediated by epigenetic activators and repressors, and may require the establishment, 'spreading' and maintenance of epigenetic signals. Although these signals remain unclear, it has been proposed that chromatin structure and consequently post-translational modification of histones may have an important role in epigenetic gene expression. The epigenetic activator Ash1 has been shown to be a multi-catalytic histone methyl-transferase (HMTase) that methylates lysine residues 4 and 9 in H3 and 20 in H4. Transcriptional activation by Ash1 coincides with methylation of these three lysine residues at the promoter of Ash1 target genes. The methylation pattern placed by Ash1 may serve as a binding surface for a chromatin remodelling complex containing the epigenetic activator Brahma (Brm), an ATPase, and inhibits the interaction of epigenetic repressors with chromatin. Chromatin immunoprecipitation indicates that epigenetic activation of Ultrabithorax transcription in Drosophila coincides with trivalent methylation by Ash1 and recruitment of Brm. Thus, histone methylation by Ash1 may provide a specific signal for the establishment of epigenetic, active transcription patterns (Beisel, 2002).

Acetylation/de-acetylation, ubiquitination and methylation of histones (H1, H2A, H2B, H3, H4) have been correlated with the activation and silencing of transcription. Histone methylation occurs predominantly at arginine and lysine residues in the amino-terminal tails of H3 and H4. Arginine methylation mediates transcriptional activation by hormone receptors and probably other chromatin-dependent processes. By contrast, methylation of K9 and K4 in H3 and K20 in H4 has been linked to transcriptionally inactive chromatin, and corresponding HMTases have been identified. Methylation of H3 K4 has also been detected in transcription-competent chromatin, but the functional link between histone methylation and activation has not been dissected (Beisel, 2002).

To identify HMTases that establish activation-specific methylation patterns, a biochemical screen was used that identified Ash1, a member of the trithorax group of epigenetic activators as an HMTase. Ash1 contains a SET domain -- the 'signature motif' of lysine-specific HMTases -- flanked by cysteine-rich regions (pre-SET and post-SET domains). To confirm that Ash1 has HMTase activity, the ability to methylate histones was assessed in recombinant Ash1 derivatives Ash1DeltaN (deleted N terminus) and Ash1(SET) (containing the pre-SET, post-SET and SET domains only). The Ash1 derivatives methylate H3 and, to a lesser extent, H4 in polynucleosomes and histone core octamers. By contrast, 'free' H3 and H4 were methylated to a lesser extent compared with nucleosomes, even though free histones were present at a fivefold excess over polynucleosomal histones or when supplemented with DNA. These results suggest that Ash1 methylates H3 and H4. Since Ash1 used in the described HMTase assays was purified from eukaryotic cells, the HMTase activity of Ash1 could result from an associated rather than intrinsic activity. To test this, Ash1DeltaN was subjected to protein transfer membrane assays that detect intrinsic enzymatic activities in proteins. Ash1(SET) was separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred electrophoretically onto polyvinylidene fluoride (PVDF) membrane, and denatured/re-natured. Reconstituted Ash1(SET) methylates H3 and H4, suggesting that Ash1 has intrinsic HMTase activity (Beisel, 2002).

To identify the target amino acid residue(s) of Ash1, radiolabelled H3 was subjected to Edman-degradation. Scintillation counting of the released amino acid fractions detected radiolabelling of H3 K4 and K9. To support this, the ability of Ash1DeltaN to methylate peptides consisting of amino acids 1-20 of H3 [H3(1-20)] was tested. Ash1DeltaN methylates the peptides H3(1-20), H3(1-20)K4 (which contains H3 K4 but leucine residues instead of lysine residues at positions 9, 14 and 18) and H3(1-20)K9 (which contains H3 K9 but leucine residues instead of lysine residues at positions 4, 14 and 18). By contrast, H3(1-20)L4/L9 peptides, which contain leucine residues at position 4 and 9 of H3, are not significantly methylated, indicating further that Ash1 methylates H3 K4 and K9. Owing to the weak radiolabelling, the target(s) of Ash1 in H4 could not be identified by Edman-degradation. Since H4 K20 is the only H4 residue being methylated in vivo, a monoclonal antibody was generated against dimethylated H4 K20 [anti-dim(H4-K20)] to investigate whether Ash1 methylates H4 K20. H4 that was free, in histone core octamers or polynucleosomes, was methylated by Ash1 and analysed by Western blot analysis. Anti-dim(H4-K20) antibody recognizes Ash1-methylated H4, but not un-methylated H4, indicating that Ash1 methylates H4 K20 (Beisel, 2002).

Single amino acid point mutations ash1¹⁰ and ash1²¹ abolish Ash1 activator function in Drosophila. The mutation in ash1¹⁰ (N1458I) resides within the SET domain, and in ash1²¹ (E1357K) in the pre-SET domain. To assess whether these mutations affect HMTase activity, recombinant proteins were expressed and purified containing one of these mutations (Ash1DeltaN¹⁰, Ash1DeltaN²¹) and a third mutant (Ash1DeltaN¹¹⁴²) whose mutation (H1459K) resides in the SET domain and abolishes HMTase activity of SUV39H1. HMTase assays revealed that the mutants do not significantly methylate H3 and H4, indicating that the mutations abolish HMTase activity and that both the pre-SET and SET domains of Ash1 contribute to HMTase activity and transcriptional activation by Ash1 (Beisel, 2002).

To assess whether the mutations in Ash1 specifically inactivate HMTase activity or cause a general functional inactivation, the ability of mutant Ash1DeltaN to bind the known interaction partner Trx was investigated. Ash1DeltaN and the three mutants can interacte with Trx in vitro, suggesting that the inability of mutant Ash1 proteins to methylate histones is based on a specific inactivation of HMTase activity (Beisel, 2002).

To investigate the effect of ash1¹⁰ and ash1²¹ on transcriptional activation by Ash1 in Drosophila, transgenic flies were used carrying the Ash1-dependent reporter gene N18/15, which contains a 4-kilobase (kb) regulatory element of the bxd region from the Ash1 target gene Ubx fused to the mini-white gene. Ash1 supports activation of N18/15 transcription in the Drosophila eye. By contrast, N18/15 expression is significantly reduced in ash1¹⁰/ + or ash1²¹/+ heterozygous flies. Since Ash1²¹ and Ash1¹⁰ lack HMTase activity in vitro, these results imply that HMTase activity contributes to transcriptional activation by Ash1 in vivo (Beisel, 2002).

To dissect the functional relationship between transcriptional activation and histone methylation by Ash1, transcriptional activation by Ash1 was reconstituted in Drosophila S2 cells. To monitor transcription in chromatin, S2 (BCAT5) cells were generated that carry the stable integrated reporter gene BCAT5, which contains five DNA-binding sites for the yeast activator Gal4, a core promoter and the bacterial cat gene. To recruit Ash1 to chromatin, Ash1 derivatives were fused to the Gal4 DNA-binding domain (amino acids 1-147) [Gal4(DBD)]. BCAT5 cells were transfected with plasmids expressing fusion proteins comprising Gal4(DBD) and either wild type or mutant Ash1DeltaN. Gal4(DBD)-Ash1DeltaN activates BCAT5 expression 20-fold, whereas HMTase-inactive Ash1DeltaN derivatives did not. These results support the hypothesis that HMTase activity of Ash1 mediates activation of transcription (Beisel, 2002).

To link transcriptional activation by Ash1 to histone methylation, crosslinked chromatin immunoprecipitation (XChIP), which detects protein-DNA interactions in vivo, was used. Crosslinked chromatin was isolated from BCAT5 cells expressing Gal4(DBD)-Ash1DeltaN, Gal4(DBD)-Ash1DeltaN¹⁰ or Gal4(DBD)-Ash1DeltaN²¹, and immunoprecipitated by antibodies recognizing dimethylated H3 K4, H3 K9 or H4 K20. Precipitated DNA was purified and the enhancer/promoter of target genes was detected by polymerase chain reaction. All three antibodies precipitate chromatin containing the BCAT5 enhancer/promoter from cells in which Gal4(DBD)-Ash1DeltaN activates transcription. Methylation of these lysine residues was detectable 500 bp upstream of the enhancer/promoter and at the 3'-end of the cat gene. In cells expressing Gal4(DBD)-Ash1DeltaN¹⁰, methylation of H3 K4 was undetectable, but weak methylation of H3 K9 and H4 K20 could be observed. This finding supports current models proposing that transcriptional repression correlates with methylation of H3 K9 and H4 K20. As, however, H3 K9 and H4 K20 methylation is enhanced at the transcriptionally active (active) reporter, transcriptional activation by Ash1 correlates with de novo methylation of not only H3 K4 but also H3 K9 and H4 K20 (Beisel, 2002).

Methylation of H3 K9 at the transcriptionally silent (silent) enhancer/promoter implies that BCAT5 might be associated with HP1, which binds methylated H3 K9. This was tested by XChIP using anti-HP1 polyclonal antibody. The antibody precipitated the BCAT5 enhancer/promoter from cells expressing HMTase-inactive Gal4(DBD)-Ash1DeltaN¹⁰. By contrast, the enhancer/promoter was only weakly precipitated from cells in which Gal4(DBD)-Ash1DeltaN activates reporter expression. These results suggest that HP1 binds the silent enhancer/promoter and is removed/relocated from the reporter by Ash1-mediated histone methylation (Beisel, 2002).

To investigate whether Ash1 methylates histones to activate transcription of a natural target gene, methylation of Ubx was monitored in BCAT5 cells. Ubx is not expressed in S2-cells but PCR with reverse transcription (RT-PCR) indicates that transiently expressed Gal4(DBD)-Ash1DeltaN activates expression of this gene in BCAT5 cells. XChIP experiments indicate that H3 K4 is not methylated and that H3 K9 and H4 K20 are only weakly methylated at the silent Ubx promoter. By contrast, methylation of all three lysine residues is significantly enhanced when Ash1 activates BCAT5 expression, indicating that transcriptional activation of Ubx by Ash1 coincides with methylation of H3 K4, K9 and H4 K20 (Beisel, 2002).

Genetic data indicate that Ash1 activates Ubx expression in imaginal discs of the third leg. Therefore, to investigate histone methylation by Ash1 in the natural context of the activator, the methylation pattern of Ubx was examined in third leg discs by XChIP. Crosslinked chromatin was prepared from third leg discs dissected from third instar larvae. Chromatin immunoprecipitations indicated methylation of H3 K4, K9 and H4 K20 at the Ubx promoter, suggesting that Ash1-mediated methylation of all three lysine residues coincides with epigenetic activation of Ubx transcription in Drosophila (Beisel, 2002).

On the basis of the result that methylated lysine residues facilitate or inhibit the binding of proteins, an investigation was carried out to determine whether the trivalent (H3 K4, K9 and H4 K20) methylation pattern placed by Ash1 attracts or repels proteins to establish epigenetic activation. The XChIP experiments in indicate that the trivalent methylation pattern removes/relocates HP1 from chromatin. To support this finding, the interaction was investigated of HP1 with methylated H3 peptides and histone core octamers that had been methylated by Ash1 or Drosophila SU(VAR)3-9, which methylates H3 K9. HP1 binds H3 K9-methylated peptides and histone core octamers, as well as H3 K4/K9-methylated peptides. By contrast, HP1 does not bind Ash1-methylated core octamers, suggesting that the trivalent methylation pattern inhibits the interaction of HP1 with chromatin (Beisel, 2002).

Protein-protein interaction assays using H3(1-20) peptides methylated at H3 K4, K9 or H3 K4 and K9 (H3 K4/K9), and Drosophila embryonic nuclear extract or recombinant proteins, resulted in the identification of three proteins that exhibit differential binding to methylated peptides. Two of these proteins -- the epigenetic repressor Polycomb (Pc) and Caf-1 p55, a subunit of different protein complexes involved in, for example, epigenetic repression -- bind H3 K9-methylated peptides and histone core octamers, but show significantly reduced binding to H3 K4- or H3 K4/K9-methylated peptides and trivalently methylated histone core octamers. Furthermore, protein-binding assays indicate that Brm and Moira (Mor) interact with H3 K4/K9-methylated peptides. In contrast, both proteins were not recruited to peptides methylated at H3 K4 or H3 K9. Brm and Mor are subunits of a SWI/SNF-like chromatin remodelling complex, suggesting that this complex, rather than individual proteins, is recruited to K4/K9-methylated H3. These results imply that the trivalent methylation pattern established by Ash1 facilitates or prevents the interaction of proteins with methylated H3 during epigenetic activation. To support this finding, XChIP was used to investigate the interaction of Brm and repressors with Ash1 target genes. These analyses indicate that Brm and Mor are present at the active but not at silent promoters of Ash1 target genes in cells or third leg imaginal discs. By contrast, the repressors were only detected at silent promoters. Thus, transcriptional activation by Ash1 may coincide with the recruitment of Brm and Mor and the extinction of repressor binding at the promoter of Ash1 target genes (Beisel, 2002).

Collectively, these data indicate that the epigenetic activator Ash1 activates transcription by methylation of H3 K4, K9 and H4 K20 at the promoter of target genes. This suggests that epigenetic activation and silencing, which has been linked to methylated H3 K9, may correlate with different histone methylation patterns. Each of the three lysine residues targeted by Ash1 can be individually methylated by specific HMTases, resulting in transcriptional repression and probably activation (H3 K4). Combining these three modifications results in a novel biological readout: epigenetic activation. Why does the trivalent modification pattern generated by Ash1 mediate epigenetic activation? The results indicate that each modification of the pattern fulfils a specific function. Methylation of H3 K4 prevents the interaction of repressors (Pc, p55) with Ash1 target genes. Methylation of H4 K20 in addition to H3 K4 and H3 K9 prevents the interaction of HP1 with chromatin. Inhibition of repressor binding is an important mechanism, as epigenetic activators and repressors are expressed together during Drosophila development. Finally, methylation of H3 K4 and H3 K9 generates an interaction surface for a chromatin-remodelling complex. These results imply that a specific functional interplay between the epigenetic activators Ash1 and Brm mediates epigenetic activation of transcription. Ash1 initially binds target genes and generates the trivalent histone methylation pattern, which subsequently recruits a Brm-containing chromatin-remodelling complex. The activity of this complex may contribute to the establishment of epigenetic active chromatin structures (Beisel, 2002).

ASH1 is required for methylation of lysine 4 residues on histone H3

Covalent modifications of histone tails modulate gene expression via chromatin organization. As examples, methylation of lysine 9 residues of histone H3 (H3) (H3-K9) is believed to repress transcription by compacting chromatin, whereas methylation of lysine 4 residues of H3 (H3-K4) is believed to activate transcription by relaxing chromatin. The Drosophila trithorax group protein Absent, small, or homeotic discs 1 (Ash1) is involved in maintaining active transcription of many genes. In extreme ash1 mutants, no H3-K4 methylation is detectable. This lack of detectable H3-K4 methylation implies that Ash1 is required for essentially all H3-K4 methylation that occurs in vivo. The 149-aa SET domain of ASH1 is sufficient for H3-K4 methylation in vitro. These findings support a model in which ASH1 is directly involved in maintaining active transcription by conferring a relaxed chromatin structure (Byrd, 2003).

The extent of H3-K4 methylation, observed by immunofluorescence on polytene chromosomes from ash1 mutants, correlates extremely well with that observed by immunoblotting of salivary gland extracts. Immunofluorescence on polytene chromosomes provides qualitative information about the genomic distribution of H3-K4 methylation in addition to quantitative information about the extent of H3-K4 methylation. By using immunofluorescence on polytene chromosomes from salivary glands and immunoblotting of salivary gland extracts, it has been shown that detectable H3-K4 methylation is essentially eliminated by strong ash1 mutations. This lack of detectable H3-K4 methylation in ash 1 mutants indicates that Ash1 is required for H3-K4 methylation, but it does not indicate whether that requirement is direct. One possibility is that histone integrity is destroyed in ash1 mutants, and the failure to detect H3-K4 methylation is only a secondary consequence. This possibility was ruled out by showing that in those same mutants histone acetylation and methylation of other residues is not affected. Ash1 is a component of a multimeric protein complex; another possibility is that some other component of the complex is responsible for histone methylation. In the antimorphic alleles where no full-length Ash1 protein can accumulate, the complex might not form and thus prevent another component from functioning. One argument against this possibility is that in the missense mutant ash1¹⁰, no methylation of H3-K4 is detected despite the accumulation of normal amounts of full-length Ash1 protein on polytene chromosomes. However, an even stronger argument against this possibility is that fragments of Ash1 can methylate H3-K4 in vitro, so the evidence is that Ash1 is required directly for virtually all of the H3-K4 methylation that occurs in vivo (Byrd, 2003).

In wild-type polytene chromosomes the vast majority of H3 methylated on lysine 9 residues is located in the chromocenter. In Su(var)3-9 mutants, there is strongly reduced accumulation of methylated H3-K9 in the chromocenter. It is evident, however, that Su(var)3-9 is not the sole HMTase with specificity for H3-K9 in Drosophila, because there is still some H3-K9 methylation on chromosomes from Su(var)3-9 null mutants. Su(var)3-9 null mutants are viable, affecting only position effect variegation, suggesting that the catalytic activity of Su(var)3-9 alone is not sufficient for global gene silencing. There is also significantly reduced accumulation of methylated H3-K9 at the chromocenter of ash1 mutants. This was surprising, because Ash1 is not known to play any role in heterochromatic gene silencing. Perhaps Ash1 plays a role in gene silencing in combination with SU(VAR)3-9 that has yet to be discovered. In any case, this means that Ash1 is one of at least two enzymes that can catalyze methylation of H3-K9. This is consistent the observation that a 588-aa fragment of Ash1 can methylate both K4 and K9 residues of H3 in vitro. However, little H3-K9 methylation is detected outside of the chromocenter on wild-type chromosomes. The pattern of H3-K9 methylation observed is completely different from the pattern of H3-K4 methylation, which is not consistent with the idea that Ash1 catalyzes the methylation of both K4 and K9 on the same H3 molecules. One possible explanation of this discrepancy is that Ash1 catalyzes only a small fraction of the H3-K9 that occurs in vivo, and that the amount of H3-K9 methylation in the chromocenter catalyzed by SU(VAR)3-9, Ash1, and possibly other enzymes overwhelms the ability to detect any on the chromosome arms. A widespread distribution of acetylated H3-K9 is detected on chromosome arms. If Ash1 methylates H3-K9 along chromosome arms, it might have been expected that the level of H3-K9 acetylation would increase in ash1 mutants, because the absence of methylation would increase the availability of free H3-K9 residues. However, no such increase was observed. The 588-aa Ash1 fragment can also catalyze a low level of methylation of H4-K20. A widespread distribution of methylated H4-K20 along the chromosome arms is observed. However, no loss of H4-K20 methylation was observed even in the strongest ash1 mutants. If Ash1 catalyzes H4-K20 methylation in vivo, it must catalyze only a small fraction of the total H4-K20 methylation that occurs (Byrd, 2003).

The SET domain of Ash1 is important for HMTase activity. The ash1¹⁰ allele that has a substitution within the SET domain (N1385I) causes absence of H3-K4 methylation. The significance of this conserved asparagine for the HMTase activity of the Ash1 SET domain is underscored by evidence that H3-K4 methylase activity of a 588-aa Ash1 fragment is lost when this same substitution is introduced. The finding that the ash1²¹ allele that has a substitution within the preSET domain (E1248K) causes reduction but not elimination of H3-K4 methylation suggests that the preSET domain may affect the efficiency of methylation but is not essential for activity. This conclusion is supported by data showing that the SET domain by itself (residues 1300-1448) can methylate H3-K4 in vitro. However, this conclusion is not supported by a report that the H3-K4 methylase activity of a 588-aa Ash1 fragment (1032-1619) is lost when this same substitution (E1248K) is introduced. It has also been reported that the 588-aa fragment of Ash1 can methylate H3-K9 residues. The 149-aa SET domain contained within that 588-aa fragment cannot methylate H3-K9 residues, suggesting that the preSET and postSET domains add functionality required for H3-K9 methylation (Byrd, 2003).

Lysine residues can be mono-, di-, or tri-methylated. Recent evidence suggests that, at least in Saccharomyces cerevisiae, active genes are trimethylated. The antibody used to detect lysine 4 methylation of H3 was made against a peptide with a dimethylated lysine. It has the highest specificity for peptides with dimethylated lysine residues, but it can also detect peptides with mono- and tri-methylated lysine residues. Although the 149-aa SET domain of Ash1 can methylate H3-K4 in vitro, the methylation state of the product detected is not known. The in vivo function of Ash1 may be to mono- and/or dimethylate H3-K4 residues; di- and/or trimethylation may be the function of some other HMTase. If this is the case, then the absence of H3-K4 methylation found in extreme ash1 mutants means that the methylation function of Ash1 creates a substrate essential for subsequent methylation. Support for this idea comes from data showing that in ash2 null mutants, the extent of H3-K4 methylation is reduced, but the distribution is like wild-type. The human homologue of ASH2 is in a multimeric complex with SET1, a protein shown to have H3-K4 methylase activity. If Drosophila ASH2 is also in a complex with a SET1 homologue, then such a complex may be responsible for subsequent methylation of H3-K4 residues initially methylated by Ash1 (Byrd, 2003).

Genetic evidence has indicated that Ash1 is a member of the Trx group of proteins that is involved in maintaining active transcription of many genes. The activities of Ash1 and Trx are functionally related. Mutations in ash1 and trx exhibit intergenic noncomplementation; Ash1 and Trx colocalize at multiple sites on polytene chromosomes, and Ash1 can be coimmunoprecipitated from embryonic nuclear extracts by antibodies that recognize Trx. Moreover, Trx accumulation on polytene chromosomes is reduced in an ash1 mutant and to associate with a histone acetyltransferase, dCBP. These results, together with the results showing that Ash1 functions as an HMTase, suggest a model for the sequential order to Ash1 and Trx association and in turn histone methylation and acetylation. According to this model, Ash1 binds to H3 via its SET domain and methylates K4 residues. The SET domain of Trx recognizes these methylated H3-K4 residues, which could explain the loss of Trx on chromosomes from an ash1 mutant. Trx recruits dCBP, which can then acetylate nearby lysine residues. If this model were correct, one would expect that loss of Ash1 catalyzed methylation would secondarily cause loss of acetylation. The data, however, do not fulfill this expectation. In ash1 mutants, where the levels of H3-K4 methylation are barely detectable, the levels of acetylation on both H3 and histone H4 are unchanged as compared with wild type. Thus, site-specific changes in histone methylation due to disruption of ash1 have no apparent effect on histone acetylation. It could be, however, that only acetylation of residues that do not depend on methylation of H3-K4 were examined. Moreover, ash1¹⁴, the mutant that showed reduced chromosomal Trx, has a molecular defect at nearly the same location as ash1¹⁶. It is likely that ash1¹⁴ also has a normal level of HMTase activity, which suggests that Trx requires the C-terminal domain of Ash1 rather than its HMTase activity to bind to chromosomes. Further work will be required to understand the molecular basis of the functional relationship between Ash1 and Trx and the roles of other components of the 2MDa Ash1 complex (Byrd, 2003).

Drosophila Paf1 modulates chromatin structure at actively transcribed genes

The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. The Drosophila homologs of three subunits of the yeast Paf1 complex have been identified and characterized and striking differences were found between the yeast and Drosophila complexes. Although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure (Adelman, 2006; full text of article).

Characterization of the Drosophila homologs of yeast Paf1 subunits has revealed several features in common and several critical differences between the yeast and Drosophila Paf1 complexes. The most striking similarities between the yeast and Drosophila Paf1 complexes are their association with elongating RNA Pol II and their roles in gene activation, while the nature of the Pol II association and the composition of the Paf1 complex reflect marked differences between the species (Adelman, 2006).

The global view provided by Drosophila polytene chromosomes shows that the chromosome-associated Paf1 and Rtf1 proteins colocalize with active Pol II. This result supports the idea that these proteins participate in most, if not all, Pol II transcription. Remarkably, Paf1 and Rtf1 do appear to be separable from actively elongating Pol II under conditions of heat shock. Although Paf1 and Rtf1 are recruited actively to heat shock loci upon heat stress, these factors also remain associated with a number of additional sites on the chromosome, while Pol II is localized almost exclusively at heat shock loci under these conditions. These data suggest that Paf1 and Rtf1 may remain bound to the chromosome at activated genes through interactions with additional proteins (Adelman, 2006).

It has been suggested that, in yeast, while the Paf1 complex is entirely nuclear in its localization, it has cellular functions that are independent of elongating Pol II. The nucleolar association of Paf1 and Rtf1 observed on Drosophila polytene chromosomes could possibly represent such a function. At the nucleolar organizer, Paf1 shows broad labeling while the Rtf1 signal is restricted to the nucleolar periphery in a manner that is largely nonoverlapping. Interestingly, although the yeast Paf1 complex does not show strong nucleolar association normally (Porter, 2005), in an Rtf1 mutant, the Paf1 complex shows a strong association that is postulated to be a manifestation of its normal role in nuclear processing or export (Adelman, 2006).

By using ChIP experiments, this study obtained a higher-resolution view of the localization of Paf1, Rtf1, and Cdc73 at the Hsp70 gene. The lack of a ChIP signal at Hsp70 under uninduced conditions demonstrates that the presence of engaged Ser-5-P Pol II or the associated elongation factors such as Spt5 and TFIIS is not sufficient to recruit Paf1, Rtf1, or Cdc73. Upon heat induction, recruitment of all three proteins was observe primarily within the coding regions of active Drosophila genes, rather than regions upstream of the promoter, or downstream of the site for cleavage and polyadenylation. The reduction in the Paf1 signal downstream of the polyadenylation site, which accompanies a decrease in the Pol II signal, likely signifies that Paf1 dissociates from chromatin within this region, consistent with recent results obtained with yeast. However, it is noted that the absence of a significant Paf1 signal obtained with a given primer pair may simply indicate that the interactions of Paf1 with a particular region are transient (Adelman, 2006).

The Paf1 complex in S. cerevisiae has been reported to be required for full Ser-2 phosphorylation of the Pol II CTD. This role of Paf1 in CTD phosphorylation regulation also appears consistent with the fact that rtf1Delta mutants show synthetic lethality with CTD kinase and phosphatase mutants in CTK1 and FCP1. The lack of a Ser-2-P Pol II signal detected in yeast Paf1 mutants resulted in reduced recruitment of cleavage and polyadenylation factors, causing a defect in the polyadenylation of nascent transcripts. However, although depletion of Drosophila Paf1 or Rtf1 has a marked effect on induced Hsp70 RNA levels, no change was seen in the levels of Ser2-P Pol II on the Hsp70 gene in Paf1 or Rtf1 RNAi-treated cells, indicating a difference between the functions of Paf1 in yeast and a metazoan system (Adelman, 2006).

Another fundamental difference that observed between Drosophila and yeast Paf1 complexes is the relationship of the Paf1 and Rtf1 subunits in providing anchorage of the complex to Pol II. In yeast, it has shown that the association of Paf1 with Pol II and active chromatin depends on the presence of Rtf1. In contrast, this study found that the recruitment of Paf1 to activated Drosophila Hsp70 is independent of Rtf1, while Rtf1 recruitment is dependent on Paf1. These results may reflect the evolution of a more important role for the Paf1 protein in metazoans in providing affinity of the complex for Pol II, while Rtf1 became a more loosely bound component of the complex (Adelman, 2006).

The role was investigated of Drosophila Paf1 in the modification of histones within actively transcribed regions. Whereas yeast Paf1 has been implicated in regulating the bulk levels of methylation of histone H3 at lysine residues 4 and 79, an effect was observed of Paf1 depletion on the trimethylation of H3-K4, but not on di- or trimethylation of H3-K79. Similarly, it was observed that trimethylation of H3-K4 occurred within the promoter-proximal region of Hsp70 and Hsp26 upon heat shock and could be seen to increase from 2.5 to 10 min after heat induction, but no significant levels of H3-K79 dimethylation were observed within the active Hsp70 gene. The latter result differs from results from other systems which link H3-K79 dimethylation with active transcription. However, it is consistent with recent data suggesting that both Grappa, the Drosophila H3-K79 methyltransferase, and the signal corresponding to H3-K79 dimethylation are localized to both active and intergenic regions of Drosophila polytene chromosomes. An alternative possibility is that the apparent differences between yeast and Drosophila result from the experimental systems used; RNAi treatments in Drosophila decrease, but do not completely abolish, their target, and thus the small amount of remaining protein may be sufficient to carry out certain functions. Conversely, the deletion mutants used to investigate yeast Paf1 entirely remove an important protein for many generations of cell growth, raising the possibility that some observed effects are indirect or secondary in nature (Adelman, 2006).

It is interesting that although H3-K4 trimethylation depends upon Paf1 and the recruitment of Paf1 is temporally similar to H3-K4 methylation, the distribution of Paf1 appears to be spatially distinct from the promoter region where the strongest trimethylated H3-K4 signals are observed. Thus, the results suggest that the effects of Paf1 mutants on the modification of promoter-proximal nucleosomes (including the ubiquitination of H2B-K123) may occur through indirect mechanisms. These data are consistent with reports on yeast that indicate that the distribution of Paf1 subunits does not strictly correlate with the patterns of ubiquitinated H2B or methylated histone H3 (Ng, 2003). The localization of H3-K4 trimethylation reported in this study is in agreement with the recently described distribution of Trithorax, a Drosophila H3-K4 methyltransferase (Smith, 2004). Furthermore, recent studies employing a Drosophila Trithorax mutant fly line suggest that a multiprotein complex that contains Trithorax plays a role in Hsp70 gene activation. However, whether the role of Trithorax in Hsp70 activation is direct or indirect remains to be established. It is noted that no effect of Paf1 depletion is observed on the rates of Pol II recruitment, or distribution over the gene, suggesting that H3-K4 trimethylation may serve as a mark of transcription activation rather than a prerequisite for gene activation (Adelman, 2006).

These studies have provided new insights into the increased importance of the Paf1 complex in a metazoan system. It is significant that Paf1 is recruited in a manner that is spatially and temporally identical to that of chromatin-associated factors Spt6 and FACT. In agreement with the strong colocalization of Paf1 with these nucleosome-associated factors, it was shown that depletion of Paf1 significantly reduces the recruitment of both Spt6 and the FACT subunit SSRP1. A relationship among Paf1, Rtf1, and FACT is consistent with findings that an rtf1Delta mutation shows synthetic lethality with POB3, a subunit of the yeast FACT complex. Moreover, the FACT complex has been shown to interact with the Paf1 complex and the chromodomain-containing Chd1 protein at actively transcribed genes. In vitro, FACT has been shown to function optimally to facilitate transcription through nucleosomes when it is present at approximately one molecule of FACT per two nucleosomes; the effectiveness of FACT in promoting elongation is decreased dramatically below this threshold. If these results reflect the situation in vivo, the greater than 50% decrease in FACT levels at the active Hsp70 gene in Paf1-depleted cells would result in a rather pronounced effect on transcription through nucleosomes (Adelman, 2006).

Furthermore, recent evidence obtained with yeast has shown that mutations of Spt6 or the FACT subunit Spt16 lead to aberrant chromatin architecture in the wake of elongating Pol II, presumably due to defects in reassembly of nucleosome structure. The failure to efficiently repackage transcribed DNA results in transcription initiation from cryptic sites and a reduction in levels of properly initiated and processed RNA. If a primary role of Drosophila Paf1 is to help stably recruit factors like Spt6 and FACT, then loss of Paf1 activity could also lead to the accumulation of nonfunctional or improperly processed RNA species. In support of this idea, a paper that was published during the preparation of this report states that mutations in yeast Spt6 alter the recruitment of Paf1 subunit Ctr9 and lead to defects in 3'-end processing of nascent RNA (Kaplan, 2005). It is thus tempting to speculate that the vast array of transcription elongation and RNA processing and export defects reported in yeast Paf1 mutant strains could result from perturbation of the nucleosome structure along actively transcribed genes. Moreover, it may be these chromatin and processing defects that account for the decrease in the amount of Hsp70 mRNA that accumulates in response to heat shock in Paf1- or Rtf1-depleted cells (Adelman, 2006).

Finally, the Paf1 gene in yeast is nonessential while the Paf1 gene in Drosophila is essential. This may reflect the more varied and demanding requirements of the transcription machinery in higher eukaryotes, where chromatin frequently plays a greater and more stringent role in regulation. This, in turn, may place a greater demand on the Paf1 complex, which appears to function at the interface between transcription and chromatin, perhaps serving as a platform that stimulates the association of a number of nucleosome-modifying complexes with actively elongating Pol II (Adelman, 2006).

In summary, the gene for Paf1 is a required Drosophila gene that colocalizes with actively elongating Pol II when chromatin associated and plays a critical role in the activation of stress-induced genes. Furthermore, recent data reveal that mutations in parafibromin, the human homolog of the Paf1 complex subunit Cdc73, are associated with an elevated risk of parathyroid carcinomas; thus, the Paf1 complex may be a key regulator of cellular control in metazoans (Rozenblatt-Rosen, 2005; Yart, 2005). The connection between Paf1 and trimethylation of histone H3 at lysine 4 near the promoters of active genes is particularly interesting because a human homolog of Trithorax, the histone methyltransferase implicated in this activity, is ALL-1/MLL-1, which is associated with a number of acute leukemias. Future work to define the way in which Paf1 directs the histone methyltransferase activity of this key enzyme should provide insight into the interaction between active transcription and modifications of chromatin structure. The data support a model in which the Drosophila Paf1 complex plays a key role in coordinating histone modifications and changes in nucleosome structure with transcription activation and Pol II elongation, thereby serving as a critical link between gene expression and chromatin structure (Adelman, 2006).

Drosophila Rtf1 functions in histone methylation, gene expression, and Notch signaling

The Rtf1 subunit of the Paf1 complex is required for proper monoubiquitination of histone H2B and methylation of histone H3 on lysines 4 (H3K4) and 79 in yeast Saccharomyces cerevisiae. Using RNAi, the role of Rtf1 in histone methylation and gene expression was examined in Drosophila. Drosophila Rtf1 (dRtf1) is required for proper gene expression and development. Furthermore, RNAi-mediated reduction of dRtf1 results in a reduction in histone H3K4 trimethylation levels on bulk histones and chromosomes in vivo, indicating that the histone modification pathway via Rtf1 is conserved among yeast, Drosophila, and human. Recently, it was demonstrated that histone H3K4 methylation mediated via the E3 ligase Bre1 is critical for transcription of Notch target genes in Drosophila. This study demonstrates that the dRtf1 component of the Paf1 complex functions in Notch signaling (Tenney, 2006).

Many of the subunits of COMPASS (complex of proteins associated with Set1 - consisting of the mammalian MLL complex, its yeast homolog, and the Drosophila trithorax complex) are required for the proper mono-, di-, and/or trimethylation of H3K4. In addition to COMPASS, the E2 conjugating enzyme Rad6 and its E3 ligase Bre1 are required for proper H3K4 methylation via the regulation of H2B monoubiquitination. Also, it has been shown that deletion of components of the Paf1 complex and the Bur1/Bur2 kinase can greatly reduce histone H2B monoubiquitination and, thereby, H3K4 methylation. However, deletion of RTF1, which is required for the activation of Rad6, seems to be required for mono-, di-, and trimethylation mediated by COMPASS. This observation mirrors that of effects observed when either RAD6 or BRE1 is deleted. Although loss of H2B monoubiquitination is not fully required for H3K4 monomethylation by COMPASS, this observation can be explained by the fact that Rtf1 is not only required for the regulation of H2B monoubiquitination but also plays a role in the recruitment of COMPASS to the transcribing RNA Pol II. In addition to regulation of H3K4 methylation, different components of the Paf1 complex have varying effects on other types of H3 tail methylation (Tenney, 2006).

Because Paf1 is required for histone H3K4 and K79 methylation, the effect of Paf1 loss on histone methylation stability was examined in vivo. A tetracycline-regulated PAF1 gene strain grown under normal conditions, turning off the expression of Paf1 in the presence of tetracycline, was used. Cell extracts were prepared at different time points, and histone H3 modification stability was tested in the absence of Paf1. After approximately 4 hours in the presence of tetracycline, Paf1 levels were reduced by >95%. However, even 12 hours after Paf1 loss, histone H3K4 and K79 methylation levels appear to be unaffected. This observation substantiates a report by Ng (2003), who found that H3K4 methylation seems to be a stable mark once established on the Gal gene. It was verified that histone H3K4 methylation seems to be stable, further supporting a role for the stability of this type of histone modification (Tenney, 2006).

Because Rtf1 loss in yeast results in the abrogation of H3K4 methylation, whether dRtf1 in Drosophila functions in the same pathway was tested. Extracts prepared from both actin-Gal4 driven dRtf1 progeny (Rtf1 RNAi on) and control progeny (Rtf1 RNAi off) were tested for methylated H3K4 levels. dRtf1 RNAi knockdown resulted in a significant reduction in total cellular trimethylated H3K4. To test whether this reduction occurs throughout the genome, fixed polytene chromosomes squashes were prepared from actin-Gal4 driven dRtf1 RNAi and control larvae and were immunostained with an antibody specific for trimethylated H3K4. In WT polytene chromosomes, trimethylated H3K4 is widely distributed throughout the euchromatic chromosome arms and is highly enriched at developmental puffs, sites of active transcription. In contrast, the polytene chromosomes from dRtf1 knockdown larvae consistently show reduced staining with the same antibody. Together, these data suggest that the loss of Rtf1 results in the loss of H3K4 methylation in Drosophila (Tenney, 2006).

Heat shock regulates the level of phosphorylated histone H3

Posttranslational modifications of the N-terminal tails of the core histones within the nucleosome particle are thought to act as signals from the chromatin to the cell for various processes. The experiments presented here show that the acetylation of histones H3 and H4 in polytene chromosomes does not change during heat shock. In contrast, the global level of phosphorylated H3 decreases dramatically during a heat shock, with an observed increase in H3 phosphorylation at the heat shock loci. Additional experiments confirm that this change in phosphorylated H3 distribution is dependent on functional heat shock transcription factor activity. These experiments suggest that H3 phosphorylation has an important role in the induction of transcription during the heat shock response (Nowak, 2000).

The acetylation of the N-terminal tails is the best-studied modification of the core histones. Several transcription factors, such as GCN5, and the TAF_II250 subunit of TFIID, as well as subunits of the RNA polymerase complex show intrinsic histone acetyltransferase (HAT) activity, which suggests a potential role for histone acetylation in either the activation or maintenance of transcription. The acetylation of the N-terminal tail domains of core histones H3 and H4 at various lysine residues is essential for the normal implementation of various cellular processes, such as promoter-transcription factor association, gene transcription, and dosage compensation (Nowak, 2000 and references therein).

Phosphorylation of serine 10 of the N-terminal arm of histone H3 has been shown to be essential for proper mitotic chromosomal condensation and segregation. In addition, recent studies have outlined the possibility that histone H3 phosphorylation may have a role in the regulation of transcription. Ser 10 H3 phosphorylation is found to rapidly increase in quiescent cells during mitogenic stimulation, as well as during immediate-early gene induction via the epidermal growth factor (EGF)-signaling pathway. In addition, recent experiments performed in vitro have suggested that EGF-stimulated H3 phosphorylation may act as a signal for histone acetyltransferase binding and subsequent acetylation of a particular locus during transcription initiation (Nowak, 2000 and references therein).

Acetylation of core histones H3 and H4 at lysines 14 and 8, respectively, has been linked to gene transcription. In addition, deacetylation of core histones is thought to have a role in silencing specific loci. Because of the near-total repression of cellular gene products during a heat shock, it might be expected that the distribution of acetylated H3 and H4 would radically change during thermal stress in a manner reflective of the transcriptional profile of the cell. Because acetylation of H3 at Lys 14 of the N-terminal arm has been described as essential for transcription, the distribution of acetylated H3 was examined by staining polytene chromosomes with an antibody specific for Lys 14 acetylated histone H3. Lys 14 acetylated H3 staining is observed at the puffs, which are active sites of transcription in polytene chromosomes, and distributed throughout the chromosomes in discrete bands before heat shock. One locus, subdivision 62A, which becomes puffed during larval development in response to ecdysone, is intensely labeled with the Lys 14 acetylated H3 antibody. In addition, other chromosomal subdivisions such as 89B display Lys 14 acetylated H3 staining but are not puffed before heat shock. The chromosomal subdivision 93D, which is known to become puffed during heat shock, is Lys 14 acetylated but not puffed before heat shock. Examination of polytene chromosomes from larvae that were subjected to a 20-min heat shock shows that the 87A and 87C heat shock puffs, which contain the hsp70gene cluster, are stained by the anti-Lys 14 acetylated antibody, although the staining at these puffs appears to be less intense and rather diffuse. This might not represent a reduction in the level of acetylation, but rather a decrease in signal intensity due to the large puffing at the heat shock loci. After heat shock, the overall number of discrete stained bands does not appear to change significantly and regions that were stained before heat shock, such as 89B, remain acetylated. Loci with acetylated H3 staining that were puffed before heat shock, such as 62A, are no longer puffed after heat shock but remain acetylated. The observation that the heat shock genes are acetylated before heat shock, at a time when they are not transcribed, and non-heat shock genes, which are not transcribed during heat shock, are acetylated during heat shock, suggests that the presence of Lys 14-acetylated H3 does not necessarily denote an actively transcribed locus (Nowak, 2000).

Examination of H3 acetylation during EGF stimulation raises the issue that antibodies against Lys 14 acetylated H3 may show decreased recognition of their epitope when other modifications, such as phosphorylation, coexist on the same histone tail. This problem can be overcome by using antibodies against histone H3 acetylated at lysines 9 and 14. To ensure that these results were not caused by this potential artifact, the distribution of hyperacetylated H3 was examined using antibodies against H3 acetylated at lysines 9 and 14 on the N-terminal tail before and after heat shock. The results suggest that the distribution of diacetylated H3 is similar to the distribution of Lys 14 acetylated H3 before and after heat shock. Diacetylated H3 staining appears to be more widespread than monoacetylated staining, which is probably caused by the antibody's recognition of acetylation of H3 at lysine 9. The intensity of staining of the Lys 9,14-acetylated H3 antibody at several of the heat shock puffs examined appears to be similar to that observed with the Lys 14-acetylated H3 antibody. These results suggest that the diffuse staining at the heat shock puffs is not an artifact attributed to the masking of the acetylated Lys 14 epitope by Ser 10 phosphorylation (Nowak, 2000).

H4 acetylation was also examined using antibodies specific for Lys 8-acetylated histone H4 to stain polytene chromosomes isolated from third instar larvae. The distribution of Lys 8 acetylated histone H4 is similar to that of acetylated H3, with H4 acetylation observed in discrete bands in nonpuffed regions, such as subdivision 89B, and at ecdysone-induced puffed regions, such as 62A, before heat shock. Chromosomal subdivisions 87A and 87C, which contain the hsp70 heat shock genes, are acetylated before and after heat shock. Similar to acetylated H3, heat shock does not significantly affect the observed distribution of Lys 8 acetylated H4 in polytene chromosomes. Taken together, the above results suggest that the acetylation state of H3 and H4 does not change substantially during heat shock and that a gene locus can be acetylated when it is not actively transcribed (Nowak, 2000).

The absence of a drastic change in H3 acetylation during heat shock is rather surprising, given current models that indicate that H3 acetylation is a crucial step in transcription initiation. This would lead to the expectation that the heat shock loci would not be acetylated before heat shock and should become intensely acetylated during thermal stress. To determine if other histone modifications occur during the heat shock response, whether changes in histone H3 phosphorylation occur after temperature elevation was examined. Stimulation of quiescent cells with EGF leads to rapid and transient phosphorylation of histone H3 at Ser 10 of the N-terminal arm in vivo. This EGF-mediated phosphorylation of H3 is targeted to a small subpopulation of total histone H3 that is acetylated at the Lys 14 position. In addition, in vitro studies have shown that phosphorylated H3 may serve as an affinity-increasing substrate for HAT activity in H3 acetylation, which raises the possibility that phosphorylation may be tied to transcription. If histone phosphorylation were implicated in transcription, then the distribution of phosphorylated H3 might change in response to heat shock and would most likely be localized primarily to the heat shock puffs while disappearing from other loci after heat shock. Because histone H3 phosphorylation is a robust marker for mitotic cells, analysis of the distribution of phosphorylated H3 in polytene chromosomes, rather than isolation of phosphorylated H3 from whole cell extracts, allows for the examining of phosphorylation of H3 in a nonmitotic environment. To examine whether the heat shock-induced puffs contain N-terminal phosphorylated H3 molecules, polytene chromosomes were stained with antibodies specific for Ser 10 phosphorylated histone H3. Before heat shock, phosphorylated H3 staining is found in discrete bands throughout the chromosomes, with the most intense staining observed in the naturally occurring ecdysone-induced developmental puffs. After a 20-min heat shock at 37°C, phosphorylated H3 staining is not distributed throughout the chromosomes but is instead concentrated at a few specific sites. The most prominent of these regions corresponds to chromosomal divisions 63BC, 67B, and 87AC. These regions contain the hsp83 gene, the hsp22, hsp23, hsp26, and hsp27 gene cluster, and hsp70 gene clusters, respectively. These regions become reproducibly puffed during the heat shock response. Although in some chromosomes examined there are several non-heat shock loci that remain slightly phosphorylated during heat shock, the intensity of staining at these regions is much lower than the staining observed at the heat shock loci (Nowak, 2000).

The regions of the chromosome where the heat shock genes are located do not contain histone H3 phosphorylated at Ser 10 before heat shock. After temperature elevation, the only puffs that possess phosphorylated histone H3 are the heat shock puffs. The appearance of phosphorylated histone H3 in the heat shock puffs, accompanied by the nearly complete reduction of staining at all other loci during heat shock, leads to the conclusion that the presence of the Ser 10 phosphorylated isoform of histone H3 might be required for the transcriptional activation of the heat shock genes (Nowak, 2000).

Induction of the heat shock genes and cessation of normal gene expression is rapid and reproducible in response to heat shock. Transcription run-on assays reveal that after only 1 min at 37°C, the levels of many normal cellular gene transcripts have greatly diminished, with the heat shock gene transcripts dominating the population of total mRNA in the cell. Following a heat shock, the normal pattern of gene expression within the cell is restored gradually over time. Therefore an examination was made of the change in phosphorylated histone H3 staining over time during and after heat shock, to determine whether or not the appearance of phosphorylated H3 closely parallels the induction of transcription of the heat shock genes and whether or not the non-heat shocked H3 distribution might be restored following recovery from heat shock. After only 1 min at 37°C, there is a noticeable change in the distribution of Ser 10 phosphorylated H3. The level of global H3 phosphorylation decreases, with several regions remaining intensely phosphorylated. Within 5 min of incubation at 37°C, many of the less intense regions of staining have disappeared. After 10 min at 37°C, the only remaining intense regions of staining are those at the heat shock puffs. When larvae were allowed to recover at room temperature from a 20-min heat shock at 37°C, H3 phosphorylation reappears in several non-heat shock loci after 10 min of recovery. After 30 min of recovery from heat shock, the number and distribution of loci that contained phosphorylated H3 appears to be virtually indistinguishable from normal (i.e., non-heat shocked) chromosomes. This restoration of the normal (non-heat shocked) H3 phosphorylation pattern closely mimics previously described restoration of normal gene expression in cells experiencing thermal stress (Nowak, 2000).

During heat shock, the heat shock transcription factor (HSF) rapidly trimerizes in solution, localizes to the heat shock loci, binds to heat shock response promoter elements (HSEs), and induces the expression of the heat shock gene products. The appearance of phosphorylated H3 at the heat shock loci could therefore be due to HSF recruitment of a specific histone kinase on binding to the HSEs of the heat shock genes. To test this hypothesis, the staining pattern of phosphorylated H3 was examined in polytene chromosomes isolated from hsf⁴-mutant larvae, which lack functional HSF at restrictive temperatures and do not respond to thermal stress. Before heat shock, the distribution of phosphorylated H3 in hsf⁴-mutant chromosomes is similar to wild-type chromosomes, with staining observed in discrete bands and at the developmental puffs. In contrast to wild-type chromosomes, histone H3 at the heat shock loci does not become phosphorylated in hsf⁴-mutant chromosomes during heat shock, which suggests that phosphorylation of histone H3 at the heat shock loci depends on functional HSF activity. In addition, no H3 phosphorylation was detected in the rest of the genome during heat shock in hsf⁴ mutants, suggesting that repression of normal transcription and loss of H3 phosphorylation at non-heat shock loci does not require the presence of an active HSF protein (Nowak, 2000).

To determine if the loss of the HSF transcription factor could also alter the distribution of acetylated H3 and H4 during heat shock, acetylation of each of these histones was examined in hsf⁴-mutant polytene chromosomes. The distribution of Lys 14 acetylated histone H3 before and after heat shock in hsf⁴ mutants was indistinguishable from the wild-type distribution, with staining observed at both the developmental puffs and nonpuffed regions. H3 acetylation was observed at the 87A and 87C chromosomal subdivisions, which normally are puffed during heat shock but these regions do not become puffed in hsf⁴-mutant chromosomes. Examination of acetylated H3 using antibodies for Lys 9- and Lys 14-acetylated H3 shows a pattern similar to that observed for the Lys 14 acetylated H3 antibody. In addition, H4 acetylation does not change after heat shock in hsf⁴ mutants. Because the heat shock genes are not induced in hsf⁴ mutants during thermal stress and because hsf⁴-mutant chromosomes are acetylated, but not phosphorylated after heat shock, it is concluded that H3 phosphorylation, and not acetylation, depends on the presence of a functional heat shock transcription factor (Nowak, 2000).

How might acetylation and phosphorylation of histones H3 and H4 work together to promote transcription of a particular gene? The data suggest that acetylated histones might define a particular locus that is primed for possible phosphorylation and subsequent transcription. This acetylated locus would attract transcription factors that interact with the acetylated residues on histones H3 and H4, known to be essential for proper association of several transcription factors with their promoters. Once bound to this locus, the transcription factor would then recruit a particular histone, which phosphorylates Ser 10 of the N-terminal arm of histone H3. The most logical site of phosphorylation would be an H3 molecule with a Lys 14 acetylated N-terminal arm, a species that has been shown to exist in vivo. The presence of this dimodified H3 would define that locus as 'active' for transcription (Nowak, 2000).

There are several kinases known to localize to specific loci on polytene chromosomes that phosphorylate H3 in vitro, such as JIL-1 on the X chromosome and P-TEFb kinase at the heat shock loci (Lis, 2000). This raises the possibility that the specificity of a kinase for activation of a particular gene through H3 phosphorylation might be regulated by the specific transcription factors that control expression of this gene. It has yet to be determined whether phosphorylation of H3 is required for assembly of the RNA polymerase II complex or if phosphorylation is a by-product of complex formation and polymerase procession during transcription. If phosphorylation of H3 were indeed the critical step in activating gene transcription, then a reasonable hypothesis is that deactivation of a particular gene would be dependent on either regulated or unregulated phosphatase activity to remove the activating phosphate group from the N-terminal tails of H3. The disappearance of phosphorylated H3 at nontranscribing loci and appearance of phosphorylated H3 at actively transcribing loci during heat shock suggests that a functional transcription complex might actively maintain the phosphorylated state of histone H3, which would be subject to ready dephosphorylation by either passive or regulated phosphatase activity in a nontranscribing state (Nowak, 2000).

A role for the Drosophila SU(VAR)3-9 protein in chromatin organization at the histone gene cluster and in suppression of position-effect variegation

Mutations in the gene for Su(var)3-9 are dominant suppressors of position-effect variegation (PEV). Su(var)3-9 is shown to be a chromatin-associated protein and the large multicopy histone gene cluster (HIS-C) is identified as one of its target loci. The organization of nucleosomes over the entire HIS-C region is altered in Su(var)3-9 mutants and there is a concomitant increase in expression of the histone genes. Su(var)3-9 is a histone H3 methyltransferase and, using chromatin immunoprecipitation, it has been shown that Su(var)3-9 is present at the HIS-C locus and that histone H3 at the HIS-C locus is methylated. It is proposed that Su(var)3-9 is involved in packaging HIS-C into a distinct chromatin domain that has some of the characteristics of ß-heterochromatin. It is suggested that methylation of histone H3 is important for the chromatin structure at HIS-C. The chromosomal deficiency for the HIS-C is also a suppressor of PEV. In contrast to what might be expected, it is shown that hemizygosity for the HIS-C locus leads to a substantial increase in the histone transcripts (Ner, 2002).

An antibody was raised against an Escherichia coli expressed polypeptide representing one-third of Su(var)3-9 that includes the chromodomain region. The antibody appeared to detect Su(var)3-9 localized to the 39D-E region as well as to many other sites in the euchromatin. The immunolocalization of Su(var)3-9 to the 39D-E region is especially strong. Since this is the site of the large multicopy HIS-C, it raised the possibility that Su(var)3-9 is associated with this cluster of tandemly reiterated genes. HIS-C includes ~0.5 Mb of DNA and contains 110 copies of the five histone genes, and it occupies bands 39D_2-3-39E_1-2. Since the HIS-C genomic region and its chromatin structure are well defined, and its product is very abundant as well as essential, this locus provides an almost ideal site for examining the effect, if any, of Su(var)3-9 mutations on chromatin structure and gene expression (Ner, 2002).

The data show that mutations in a Su(var)3-9 alter chromatin structure and concomitantly alter gene expression. This is the first demonstration that mutations in a Su(var) gene actually alter chromatin structure and that this alteration in structure is associated with an alteration in gene expression. Even though the Drosophila Su(var)3-9 protein is 223 amino acids larger than its mammalian counterpart, this study shows that Drosophila Su(var)3-9 also has a histone H3 methyltransferase. Finally, the ChIP data indicate that a significant proportion of the H3 histones associated with the HIS-C locus are methylated and that the Su(var)3-9 protein associates with the HIS-C locus. Collectively, these data suggest that Su(var)3-9 is a trans-regulator of histone gene expression and that it regulates histone gene packaging and expression by forming part of the HIS-C chromatin rather than by transiently associating with the region (Ner, 2002).

These data highlight an intriguing discrepancy in the localization of Su(var)3-9 protein. Su(var)3-9 has been shown to localize to many euchromatic sites and to the chromocenter of polytene chromosomes. In addition, the pattern of localization of methylated histone H3 is similar to the pattern observed for Su(var)3-9, a modification that is carried out by Su(var)3-9. The antibody behaves differently and does not detect Su(var)3-9 at the chromocenter. One clear possibility is that the epitope of Su(var)3-9 to which the antibody was raised is hidden at many binding sites, including the chromocenter. Indeed this is exactly the situation that occurs with human HP1 gamma. Antibodies that recognized the amino terminal half of the protein failed to detect HP1 gamma at centric regions of chromosomes, while an antibody that recognized the carboxy end of the protein does detect HP1 gamma in centric regions of chromosomes. This issue is being addressed by preparing antibodies to other regions of Su(var)3-9 (Ner, 2002).

The data show that the pattern of hypersensitive sites over the histone genes is dramatically altered in Su(var)3-9 mutants relative to Oregon-R. For example, both the number and the relative position of the hypersensitive sites in the noncoding region between HIS1 and HIS3 genes are drastically reduced, from 10 primary sites in Oregon-R to 1 in the Su(var)3-9 mutants. The absence of smearing in the nuclease digestion pattern suggests that the change in chromatin structure observed in Su(var)3-9 mutants is homogeneous, occurring at each of the 110 copies of the histone repeat unit. While there is a dramatic alteration in nuclease hypersensitive sites, no loss of nucleosomes is observed in Su(var)3-9 mutants vs. wild type. This is interpreted to mean that the structure of the HIS-C region is altered in Su(var)3-9 mutants but the reorganized chromatin is ordered and uniform over each of the 110 his gene units. Since the number of nuclease hypersensitive sites is reduced, especially in the intergenic regions, in virtually all of the his repeat units in Su(var)3-9 strains vs. wild type it is inferred that Su(var)3-9 mutants provide a more 'open' chromatin configuration. Indeed, the data show that P-element insertions and EMS-induced mutations in Su(var)3-9 cause an increase in the steady-state levels of the histone transcripts. The increase in histone mRNA levels varies somewhat from one mutant genotype to another, as expected, but generally averages about twofold in the various Su(var)3-9 mutants examined. The strains in which the P element is precisely excised show that a return of histone transcript amounts to wild-type levels. This is the first time it has been shown that a protein modifying histone gene expression is a component of the histone chromatin domain. This is also the first demonstration that alterations in Su(var)3-9 protein are associated with alterations in both chromatin structure and gene expression at one of its target loci (Ner, 2002).

Finally, since histone gene transcription is tightly regulated and coordinated with DNA replication, one presumes that the rate of histone gene transcription must be modulated from one tissue type or developmental stage to another, commensurate with the changes in the length of S-phase. There are two ways in which this modulation in histone gene transcription could be accomplished. Either the transcription rates of all 110 copies of the his unit are modulated similarly or, alternatively, the number of his units that are actively transcribed varies with the length of the S-phase. While the Su(var)3-9 mutants cause a dramatic change in the number of nuclease hypersensitive sites, especially in the intergenic regions, the histone units were uniformly cut (no smearing). This uniformity in packaging of the histone repeat units infers that all 110 copies of the his repeats are packaged and transcribed similarly; that is, the HIS-C is modulated as a cluster. The only other hypothesis consistent with these observations is that silent his units completely lack hypersensitive sites while active his units have hypersensitive sites. But even under this scenario the Su(var)3-9 mutants must alter the hypersensitive site pattern of the actively transcribed his units. The active his units could be distinguished from the inactive units through a replication-independent deposition of the variant histone H3.3 into the nucleosomes encompassing the active units. Such a situation is observed with the regulation of transcription of the rDNA arrays. Methylated-lysine 9 histone H3 is absent in transcriptionally active loci but is present in the inactive loci (Ner, 2002).

Histones not only are essential in all eukaryotic cells, but also must be made in the correct stoichiometric ratios to serve as the basic substrates of DNA packaging. Indeed, H2A-H2B and H3-H4 genes are generally organized as pairs and often clustered in many organisms. The conservation of histone gene organization may reflect the need for, and maintenance of, this balanced expression. Strains heterozygous for a deletion of the HIS-C region, Df(2L)DS5/+, result in a three- to fivefold increase in the histone mRNA production/template. Unlike the mutations in the Su(var)3-9 gene, hemizygosity for the HIS-C region appears to uncouple the stoichiometry in histone production. Therefore, while mutations in Su(var)3-9 and hemizygosity for the HIS-C region both result in an increase in histone gene expression, the mechanisms by which this increase occurs may differ. Consistent with this hypothesis, the effect of combining Su(var)3-9 mutants with the HIS-C deletion appears to be additive, rather than epistatic or synergistic. It is not known why hemizygosity for the HIS-C region leads to an increase in histone transcripts. It is known that response to deletion of one copy of HIS-C, the HIS-C region on the normal homolog is capable of undergoing magnification. Magnification, which requires four to five generations, is not the basis of the increase in transcript levels that is reported in this study since the increase in transcript levels is observed among the F₁ of outcrossed strains. It is hypothesized that the gene regulation of the HIS-C locus may be pairing sensitive and that the deletion of one copy of HIS-C disrupts pairing and leads to overexpression of the histone genes from the remaining copy. Consistent with this hypothesis, in Drosophila embryos the histone locus was found to pair more frequently and more rapidly than other euchromatic loci. The HIS-C deletion stocks and mutations in genes that influence transvection are currently being tested to see if any of these alter the chromatin structure and/or the expression of the HIS-C locus (Ner, 2002).

DNA microarray studies in Saccharomyces cerevisiae have shown that when the histone stoichiometry is altered, for example, by deletion of the yeast histone gene pair HTA2-HTB2 encoding H2A and H2B or by reduction of histone H4 levels, the expression profiles of ~10%-15% of the genetic loci are altered. Nuclei isolated from Su(var)3-9 mutants and wild-type strains were examined to determine whether the twofold increase in histone gene transcripts led to a detectable increase in the amount of histones associated with total chromatin. A doubling of the amount of histone proteins associated with chromatin was not expected, nor was it detected. However, an increase of ~20%-25% in histone H3, acetylated H4, and H1 was detected. Since all three histone types, which represented core, modified, and linker histones, respectively, were increased to similar levels, it is believed that the alteration in HIS-C expression caused by Su(var)3-9 mutations alters chromatin packaging at many areas of the genome. The 20% increase in the amount of nuclear histones that was detected in Su(var)3-9 mutants correlates reasonably well with the observation that the expression of 10%-15% of the yeast genome is altered when histone levels are altered in yeast strains. Studies in yeast have also shown that telomeric silencing, a gene-silencing phenomenon closely resembling PEV, is strongly reduced when one of two H3 and H4 gene pairs is deleted. Furthermore, mutations in HIR1 and SPT21 genes of yeast, which cause a misregulation of histone gene transcription in opposite directions, change the histone stoichiometry and result in suppression and enhancement of telomeric-position effect, respectively. While all these studies link alterations in histone amounts to altered gene expression at many loci in the genome, and in particular, to alterations in the gene silencing associated with TPEV, no mechanism has been proposed to explain these effects (Ner, 2002 and references therein).

Finally, the doubling in histone mRNA that was detected in Su(var)3-9 mutant strains does not result in a twofold increase in the amount of nucleosomes or histones associated with chromatin in these strains. Clearly, doubling the number of histones/unit of DNA is probably impossible and, if attempted, would no doubt result in lethality early in development. Since no significant reduction was detected in viability among Su(var)3-9^- homozygotes, HIS-C hemizygotes, or double-mutant strains, it is assumed that there is a mechanism that prevents excess formation and/or transport of the histones to the nucleus (Ner, 2002).

While there is no evidence that directly links alterations in histone levels to PEV, the histones are key packaging components and it is curious that both hemizygosity for HIS-C and mutations of Su(var)3-9 increase histone transcript levels by about twofold. This implies that alteration in histone transcript levels can suppress PEV either directly or indirectly. But clearly the Su(var)3-9 mutants are very strong dominant suppressors of PEV, whereas HIS-C deletion lines are only moderate suppressors of PEV. However, the level to which the histone transcripts are elevated in the Su(var)3-9 mutants and in the HIS-C deletion lines is similar. This indicates that enlarging the pool of histones available to the cell at the time of DNA replication cannot be the only factor influencing the packaging and/or expression of the genes subjected to PEV. Since methylated-lysine 9 H3 is associated with heterochromatin and silenced genes, the HMTase activity of Su(var)3-9 could play a significant role in determining directly or indirectly the transcriptional competency of a variegating locus. Perhaps the decrease in methylated histone H3 gives a dramatic advantage to the formation of euchromatin, which replicates early, and concomitantly impairs the formation of heterochromatin, perhaps through the extended presence and use of the variant histone H3.3. Equally, the lack of methylation may tend to position the variegating segment of the genome in a compartment of the nucleus that is more favorable to its transcription. The role of Su(var)3-9 in PEV is currently being investigated (Ner, 2002).

In summary, it is believed that the HIS-C locus provides a good target locus for dissecting the role of various chromatin-associated proteins in both chromatin architecture and gene expression. The data show that the HIS-C region in Drosophila, and perhaps also in other organisms, is an excellent domain for examining proteins involved in establishing chromatin-domain structure and in regulating gene expression. Future experiments will be directed at examining how the entire HIS-C region is packaged into chromatin with the aim of determining the status of other histone modifications and the involvement of trans-regulators of histone gene expression (Ner, 2002).

Characterization of the human EED-EZH2 complex and the role of Histone H3 lysine 27 methylation in Polycomb-Group silencing of Ubx

Polycomb group (PcG) proteins play important roles in maintaining the silent state of HOX genes. Recent studies have implicated histone methylation in long-term gene silencing. However, a connection between PcG-mediated gene silencing and histone methylation has not been established. This study reports the purification and characterization of an EED-EZH2 complex, the human counterpart of the Drosophila ESC-E(Z) complex. The complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27). Using chromatin immunoprecipitation assays, it is shown that H3-K27 methylation colocalizes with, and is dependent on, E(z) binding at an Ultrabithorax (Ubx) Polycomb response element (PRE), and that this methylation correlates with Ubx repression. Methylation on H3-K27 facilitates binding of Polycomb (Pc), a component of the PRC1 complex, to histone H3 amino-terminal tail. Thus, these studies establish a link between histone methylation and PcG-mediated gene silencing (Cao, 2002).

To understand the function of histone methylation, attempts were made to identify histone methyltransferase (HMTase) using a systematic biochemical approach. Certain fractions derived from HeLa cell nuclear pellet contained high levels of HMTase activity toward nucleosomal histone H3. To identify the enzyme(s) present in these fractions, the proteins were further fractionated in a DEAE5PW column, which separated the HMTase activities into two peaks. The present study focuses on the second peak. After fractionation on phenyl sepharose and hydroxyapatite columns, the active fractions were further purified through a gel filtration Superose 6 column. Analysis of the fractions derived from this column indicates that the HMTase activity elutes between fraction 47 and 50 with an estimated mass of about 500 kD. Silver staining of an SDS-polyacrylamide gel containing these fractions revealed that six major polypeptides copurify with the enzymatic activity. Because the largest protein band neither cofractionates with the HMTase activity in the hydroxyapatite column, nor coimmunoprecipitates with the other components, it is concluded that this largest band is not a part of the HMTase protein complex (Cao, 2002).

To identify the proteins that copurify with the HMTase activity, the protein bands were excised and analyzed by a combination of peptide mass fingerprinting and mass spectrometric sequencing. In addition to RbAp48, a polypeptide present in many protein complexes involved in histone metabolism, several human PcG proteins, including EZH2, SUZ12, and EED, were identified in the HMTase complex. A zinc finger transcriptional repressor named AEBP2 was also identified. Whether this protein is involved in targeting the complex remains to be determined. EZH2 contains a SET domain, a signature motif for all known histone lysine methyltransferases, except the H3-K79 methyltransferase DOT1, and is therefore likely to be the catalytic subunit. However, recombinant EZH2 made in Escherichia coli or baculovirus-infected SF9 cells has no detectable HMTase activity, indicating that either a posttranslational modification or other components in the complex are required for the HMTase activity. This is consistent with previous results in which a partial EZH2 protein containing the SET domain was used (Cao, 2002).

Although mammalian EZH2 and EED, and their respective homologs in Drosophila and Caenorhabditis elegans, are known to interact directly, the presence of SUZ12 in such a complex has not been previously reported. To verify that these proteins are components of the same protein complex, antibodies against each of these proteins were generated. Western blot analysis of the column fractions derived from the last two columns indicates that these proteins copurify with the HMTase activity. To further confirm that the copurified proteins exist as a single protein complex, the last column fractions were immunoprecipitated with an antibody to SUZ12. All five proteins coimmunoprecipitate. Because a protein complex containing Drosophila Esc and E(z), respective homologs of EED and EZH2, has been previously named the ESC-E(Z) complex, the human counterpart is referred to as the EED-EZH2 complex. Although both EED-EZH2 and Esc-E(z) complexes physically associate with HDACs, the purified complex neither contains any HDAC polypeptide nor possesses detectable HDAC activity. It is possible that a different protein complex containing EED, EZH2, and HDAC may exist. Alternatively, HDACs may be recruited to target sites through direct interaction with EED, yet may not exist as a stable subunit of EED-EZH2 complexes. Further work is needed to differentiate these possibilities (Cao, 2002).

To characterize the substrate specificity of the EED-EZH2 complex, equivalent amounts of histone H3 that exist alone, in complex with other core histones, and in mono- or oligo-nucleosome forms were subjected to methylation by equal amounts of the enzyme. The EED-EZH2 complex is capable of methylating all forms of histone H3, but shows a strong preference for H3 in oligonucleosome forms (Cao, 2002).

Attempts were made to identify the residue methylated by the EED-EZH2 complex. Because oligonucleosomes are preferred substrates, they were subjected to methylation by the EED-EZH2 complex in the presence of S-adenosyl-L-[methyl-³H]methionine (³H-SAM). After purification, the labeled H3 was subjected to microsequencing followed by liquid scintillation counting. Neither K4 nor K9 released numbers of counts clearly greater than background. However, a small radioactive peak was detected in cycle 27. Given that the recovery efficiency decreases with each microsequencing cycle, the detection of a small peak on cycle 27 indicates that K27 is likely to be the site targeted by the EED-EZH2 complex. To confirm this possibility, each of the five potential methylation sites on H3 were mutated and the effect of the mutation on the ability of H3 to serve as a substrate for the enzyme was compared. As a control, the ability of these H3 mutants to be methylated by SUV39H1 was also analyzed. Mutation of K27 completely abolishes the ability of H3 to serve as a substrate, whereas mutations of other sites have little effect. As expected, only mutation of K9 affects the SUV39H1-mediated H3 methylation. These data, led to the conclusion that K27 is the predominant site, if not the only site, that is targeted for methylation by the EED-EZH2 complex (Cao, 2002).

To gain insight into the function of H3-K27 methylation in vivo, a polyclonal antibody was generated against a dimethyl-K27 H3 peptide. This antibody is highly specific for mK27 when evaluated by peptide competition and enzyme-linked immunosorbent assay. Western blot analysis with the H3-mK27-specific antibody demonstrates that H3-K27 methylation occurs in a variety of multicellular organisms, including human, chicken, and Drosophila . However, it does not appear to occur in the budding yeast Saccharomyces cerevisiae (Cao, 2002).

Given that both H3-K27 methylation as well as the EED-EZH2 counterpart exist in Drosophila , whether the ESC-E(Z) complex is responsible for H3-K27 methylation was examined in this organism. Several E(z) temperature-sensitive mutant alleles have been characterized, one of which, E(z)⁶¹, contains a Cys-to-Tyr substitution (C603Y) in the cysteine-rich region immediately preceding the SET domain. When reared continuously at 18°C (permissive temperature), E(z)⁶¹ homozygotes exhibit no detectable mutant phenotype and maintain wild-type expression patterns of HOX genes, such as Ubx. However, at 29°C (restrictive temperature), E(z)⁶¹ produces multiple homeotic phenotypes due to derepression of HOX genes, which correlates with loss of polytene chromosome binding by the E(Z)⁶¹ protein and disruption of chromosome binding by Polycomb (PC) and other PRC1 components. Given that chromosome binding by E(Z)⁶¹ protein is abolished at 29°C, H3-K27 methylation should be correspondingly reduced in the mutants at 29°C, if E(Z) is responsible for H3-K27 methylation. Western blot analysis of the histones from wild-type and E(z)⁶¹ fly embryos at 18° and 29°C demonstrate that the H3-K27 methylation is abolished in the E(z)⁶¹ embryos at 29°C. However, these conditions do not have a detectable effect on H3-K9 methylation. It is therefore concluded that functional E(Z) protein is required for H3-K27 methylation in vivo (Cao, 2002).

To understand the functional relation between E(z)-mediated H3-K27 methylation and HOX gene silencing, a study was carried out of E(z) binding, H3-K27 methylation, and recruitment of PC, a core component of the PRC1 complex, to the major PRE of the Ubx gene in S2 tissue culture cells by chromatin immunoprecipitation (ChIP). Consistent with the involvement of E(z) in H3-K27 methylation, ChIP analysis of a 4.4-kb region that includes this PRE showed precise colocalization of E(z) binding and H3-K27 methylation. In contrast, similar colocalization was not observed for mK9, indicating that H3-K9 methylation, or at least K9-dimethylation, is independent of E(z) binding. To further verify the importance of E(z) binding for H3-K27 methylation, attempts were made to disrupt Esc-E(z) complex activity using RNA interference (RNAi). It was reasoned that depletion of the Esc protein, a direct binding partner of E(z) and a component of the Esc-E(z) complex, would result in disruption of PRE binding by E(z). Depletion of Esc with RNAi results in greatly reduced PRE binding by E(z), loss of H3-K27 methylation, and concomitant loss of PC binding. Depletion of PC in S2 cells has been shown to result in derepression of Ubx. Therefore, these data collectively suggest that the Esc-E(z) complex is critical not only for H3-K27 methylation, but also for PC binding to the PRE region, and that H3-K27 methylation is associated with Ubx repression (Cao, 2002).

To examine the relation between E(z) binding, H3-K27 methylation, and Ubx gene repression in vivo, wing imaginal discs were dissected from homozygous E(z)⁶¹ larvae that had been either reared continuously at 18°C or shifted from 18° to 29°C ~48 hours before dissection, and analyzed E(z) binding and H3-K27 methylation in the same Ubx PRE region by ChIP. Consistent with previous studies demonstrating disruption of polytene chromosome binding by both E(z)⁶¹ and PC proteins at 29°C, ChIP analysis showed loss of E(z)⁶¹ and PC binding to this PRE at restrictive temperature. In addition, H3-K27 methylation colocalizes with E(z) binding at permissive temperature, but is lost along with E(z) binding at 29°C. In contrast, similar changes in H3-K9 methylation were not observed under the same conditions. Under normal conditions, Ubx is not expressed in wing discs due to PcG-mediated silencing. Similar inactivation of an E(z) temperature-sensitive allele during larval development has been shown to result in derepression of Ubx in wing discs. Thus, Ubx PRE-associated nucleosomes appear to be targeted by E(z)-mediated H3-K27 methylation, which correlates with PC binding and repression of Ubx. Collectively, these data suggest that H3-K27 methylation plays an important role in the maintenance of Ubx gene silencing (Cao, 2002).

The chromodomain of the heterochromatin protein HP1 specifically binds to H3 tails that are methylated at K9 by the HMTase SUV39H1. Given that PC contains a chromodomain and that loss of E(z) function abolishes H3-K27 methylation as well as Pc binding to the Ubx PRE, it is possible that methylation of H3-K27 by Esc-E(z) facilitates PRE binding by PC, analogous to the effect of H3-K9 methylation on nucleosome binding by HP1. To test this possibility, Drosophila PC was generated using the rabbit reticulocyte transcription/translation-coupled system and it was incubated with biotinylated H3 peptides with or without K27 methylation in the presence of streptavidin-conjugated Sepharose beads. Analysis by peptide pull-down assay indicated that methylation on K27 facilitates binding of Pc to the H3 peptide. Binding of Pc to the peptides is specific because the chromodomain-containing protein HP1 fails to bind to the same peptides under the same conditions (Cao, 2002).

Previous studies strongly suggest that the chromodomain of PC is necessary and sufficient for targeting PC to specific chromosomal locations in vivo because mutations in the PC chromodomain abolish the ability of PC to bind to chromatin in vivo. In addition, a chimeric PC/HP1 protein, in which the HP1 chromodomain is replaced by the PC chromodomain, binds to both heterochromatin and PcG target sites in euchromatin. To evaluate the contribution of the chromodomain in the preferential binding of PC to K27 methylated peptide, a PC mutant was generated in which two of the highly conserved amino acids Trp-47 and Trp-50 were changed to Ala. These two amino acids were chosen because the corresponding amino acids in the HP1 chromodomain have been shown to directly contact the methyl group of an H3-mK9 peptide. The mutant PC does not preferentially bind to the K27 methylated peptide, suggesting that the chromodomain of PC is responsible for the preferential binding to the H3-mK27 (Cao, 2002).

Collectively, these studies support a model in which Esc-E(z)-mediated H3-K27 methylation serves as a signal for the recruitment of the PRC1 complex by facilitating PC binding. Recruitment of PRC1 in turn prevents the access of nucleosome remodeling factors, such as SWI/SNF, leading to the formation of a repressive chromatin state. Although this model is attractive, it does not exclude the possibility that protein-protein interaction also contributes to the recruitment of PRC1 to PREs. Indeed, a recent study indicates that PC transiently associates with the Esc-E(z) complex during early embryogenesis. These studies established a correlation between H3-K27 methylation and PcG silencing. Further work is needed to establish the exact role of H3-K27 methylation in PcG silencing (Cao, 2002).

Characterization of the methylation of Histone H3 by an Enhancer of zeste/Extra sexcombs protein complex

Recombinant E(z) has no MTase activity in vitro but acquires this activity when combined with p55 and Esc. Both are typical WD40 proteins, containing seven WD40 repeats and thought to mediate protein-protein interactions. The p55 protein has been found associated with the Retinoblastoma protein, is a constituent of Chromatin Assembly Factor CAF-1, and is thought to mediate the interactions of CAF-1 with histones. WD40 domain proteins are also found in other repressive chromatin-modifying complexes, e.g., yeast Tup-1, fly and mammalian Groucho, and mammalian TBL1, and have been shown to interact with histones and particularly with H3. Esc and p55 are therefore likely to mediate the interaction of E(z) with histone H3 while the E(z) SET domain is responsible for the catalytic activity (Czermin, 2002 and references therein).

Other components known to associate with the Esc/E(z) complex are Rpd3 and Pho. Pho is a DNA binding protein and most likely required to recruit the components to the PRE, but its association with E(z) does not necessarily persist in the later embryo; Rpd3 is a histone deacetylase whose role in the complex is presumably to deacetylate H3 lysine 9 and permit its methylation. Su(z)12 is a zinc finger protein highly conserved in vertebrates and plants. Its function is unknown but it was recently implicated in PcG silencing and position-effect variegation. SU(Z)12 is not present stoichiometrically in the purified complex, suggesting either that it can dissociate during purification or that the embryonic E(z)/Esc complex is heterogeneous. Assuming one copy of each, these components do not account for the size of the Esc/E(z) complex, estimated at around 600- 650 kDa. It is likely therefore that some components, possibly E(z) itself, are present in more than one copy. Genetic evidence suggests that E(z) function requires two or more copies of the protein acting in concert. The original E(z)¹ mutant produces a dominant enhancer of zeste phenotype when heterozygous with a wild-type copy of E(z) but acts as a null when homozygous, implying that the mutant and wild-type copies of the protein act together (Czermin, 2002 and references therein).

To test whether E(z) or Trithorax might display methyltransferase (MTase) activity that could serve as a chromatin mark, the corresponding complexes were immunoprecipitated from embryonic nuclear extracts with anti-Esc antibody or with anti-Trx antibody. The immunoprecipitated material in both cases contains a histone MTase activity specific for histone H3. To determine the site of methylation, synthetic N-terminal peptides containing the first 19 amino acids of histone H3 were used. The peptide is still methylated by the material immunoprecipitated by the anti-Esc or the anti-Trx antibodies although the activity is weaker than with the entire histone H3. The Trx-associated activity decreases to control levels if the peptide is already dimethylated at lysine 4 (me2K4), strongly implying that the Trx-associated MTase targets H3 Lysine 4. In contrast, the activity associated with the Esc/E(z) complex can still methylate the me2K4 peptide. It is also still active on the peptide dimethylated at lysine 9 (me2K9) but not on a peptide containing trimethylated lysine 9 (me3K9). These results show that the activity associated with Esc/E(z) is distinct from that associated with Trx and is probably able to add a methyl group to dimethylated lysine 9 (Czermin, 2002).

Chromatographic fractionation of Drosophila embryonic nuclear extracts reveals the presence of at least five distinct MTase activities, which have been named HIMalpha, ß, gamma, delta, and epsilon according to their elution from a resource Q column. HIMß contains the well-known MTase Su(var)3-9, which plays an important role in establishment and maintenance of methylated histones at and around the centromeric region. HIMalpha copurifies with E(z) and Esc proteins. Gel filtration chromatography indicates that the HIMalpha has an apparent size of 600- 650 kDa, in good agreement with the reported size of a E(z)/Esc complex found in embryos. By introducing a MonoQ fractionation prior to the Superdex200 column, it was possible to purify HIMalpha to apparent homogeneity. Judging from a silver-stained gel, HIMalpha consists of six components. Western blots with corresponding antibodies identify five of these as p55, Esc, Rpd3, E(z), and SU(Z)12 in order of increasing molecular weight. The sixth protein has a molecular weight of approximately 168 kDa and is not yet unambiguously identified. These results show that HIMalpha is likely to correspond to the MTase activity immunoprecipitated by the anti-Esc antibody (Czermin, 2002).

Recombinant human E(z) lacks MTase activity on H3 peptide substrates. To test whether the same is true for the Drosophila ortholog, Drosophila E(z) was expressed as an intein fusion protein in bacteria and the purified, recombinant E(z) was used in MTase assays with various substrates. As expected, no significant MTase function could be detected using the bacterially produced polypeptide. Both HIMalpha activity and endogenous E(z) reside in a high molecular weight complex containing several protein components. Histone-modifying enzymes often require other polypeptides, in addition to the catalytic subunit, to recognize their substrate. Human HAT1, for example, requires p48 (Caf1), a WD40-motif histone binding protein. The Drosophila ortholog of p48, called p55, has also been shown to interact with E(z), and Esc, another subunit of the E(z) complex, shares with p55 the WD40 domain structure, which is needed for histone binding. To test whether E(z) might require additional components to gain histone MTase activity, recombinant E(z), immobilized on chitin beads, was used to reconstitute a complex with either bacterially produced recombinant Esc, p55 produced in a baculoviral system, or both. While none of the components has appreciable activity individually, E(z) plus p55 has some MTase activity and the ternary complex of E(z), p55, and Esc displays even stronger ability to methylate histone H3. In contrast, when an E(z) protein mutated in the SET domain is used, the ternary complex has no MTase activity. This experiment demonstrates that the E(z) protein is responsible for the methylation and strongly suggests that the SET domain harbors the MTase catalytic function (Czermin, 2002).

The MTase activity of HIMalpha methylates histone H3, has a slight preference for H3 incorporated in a nucleosome, and is not active on the globular domain of H3, with a deletion of the first 26 N-terminal amino acids. A histone deacetylase activity is also present in the purified complex. To identify the target of the Esc/E(z) MTase, a peptide containing the first 19 amino acids of histone H3 was methylated in vitro with ³H-labeled S-adenosyl methionine and subjected to N-terminal degradation. When the radioactivity of the progressively released amino acids was determined, a single peak was found at lysine 9. It is concluded, therefore, that lysine 9 is a target of the Esc/E(z) MTase. Nevertheless, the N-terminal peptide already dimethylated at lysine 9 can still be methylated. The simplest explanation is that the enzymatic activity can add a third methyl group to the dimethylated lysine 9. To confirm this, new synthetic peptides with or without trimethylation at lysine 9 were obtained. With these peptides as substrates, it was found that, while the unmodified peptide is methylated, methylation of the me3K9 peptide is abrogated (Czermin, 2002).

In addition to lysine 9, other residues appear to be methylated. A tryptic digest of histone H3 methylated with HIMalpha shows that significant methyl label remains with the globular part of the histone, suggesting that it contains additional targets of methylation. However, the globular domain of H3 (residues 27-135) cannot serve as a HIMalpha substrate, suggesting that although it may harbor an additional methylation site, amino acid residues within the tail are important for substrate recognition. Lysine 27 is a good candidate site since it is present in the truncated H3 but lacks its immediate N-terminal context. A peptide comprising amino acids 21-34 of H3 is in fact methylated by HIMalpha but no methylation occurs if lysine 27 is mutated to leucine (K27L). In contrast, the Su(var)3-9 MTase is very active on a peptide comprising amino acids 1-19 of H3 but has no activity on the 21-34 peptide. Furthermore, a recombinant histone H3 carrying the point mutation K9A can still be methylated by HIMalpha, as can H3 with the K27A mutation. Methylation activity drops to background level with H3 carrying both K9A and K27A mutations (Czermin, 2002).

An antibody directed against dimethylated H3 lysine 9 (anti-me2K9) has been described. Another antibody, specifically raised against the N-terminal peptide of histone H3 trimethylated at lysine 9, is also available. Western blotting of differentially modified peptides confirms that the anti-me2K9 antibody and the me3K9 antibody have distinct and nonoverlapping specificities. When these antibodies are used to stain a Western blot of nucleosomal histones isolated from Drosophila embryos and then treated with trypsin to remove the N-terminal tail, the anti-me3K9 antibody stains strongly the intact histone H3 but still recognizes weakly the trypsinized H3. In contrast, the anti-me2K9 recognizes the intact H3 but not the trypsinized H3. This weak reaction of the me3K9 antibody with the methylated and trypsinized H3 suggests that lysine 27 may also be trimethylated in vivo and weakly recognized by the anti-me3K9 antibody (Czermin, 2002).

Polycomb group (PcG) proteins maintain transcriptional repression during development, likely by creating repressive chromatin states. The Extra Sex Combs (Esc) and Enhancer of Zeste [E(z)] proteins are partners in an essential PcG complex, but its full composition and biochemical activities are not known. A SET domain in E(z) suggests this complex might methylate histones. An Esc-E(z) complex has been purified from Drosophila embryos and four major subunits were found: Esc, E(z), NURF-55, and the PcG repressor, SU(Z)12. A recombinant complex reconstituted from these four subunits methylates lysine-27 of histone H3. Mutations in the E(z) SET domain disrupt methyltransferase activity in vitro and HOX gene repression in vivo. These results identify E(Z) as a PcG protein with enzymatic activity and implicate histone methylation in PcG-mediated silencing (Müller, 2002).

Histone H3 is an attractive candidate to be the primary functional target for the Esc-E(z) complex in vivo. Previous work on heterochromatin proteins has established a molecular model that links H3 methylation and gene repression. In this case, SUV39H HMTase methylates H3-K9 and this modification creates a binding site for the chromodomain of HP1. Similarly, H3 methylation by Esc-E(z) might provide a binding site for another chromodomain protein, such as Polycomb (PC), a component of the PRC1 complex, whose chromodomain is required for chromatin localization in vivo. Targeting of PRC1 via histone methylation performed by Esc-E(z) is consistent with several in vivo results that imply synergy between these complexes. Rather than supplying binding sites for specific proteins, Esc-E(z) mediated methylation could influence chromatin more generally by altering adjacent nucleosome interactions needed to package the chromatin fiber (Müller, 2002).

Besides histone methylation, genetic and biochemical tests link histone deacetylation to PcG-mediated repression and hyperacetylation to trxG-dependent active states. It will be important to test for regulatory interactions, or crosstalk, among the various tail modifications. For example, H3-K27 acetylation would be anticipated to antagonize H3-K27 methylation, similar to the mutual exclusion of these two modifications on H3-K9. Likewise, interplay between phospho-H3-S28 and methylation of H3-K27 could resemble the inhibitory crosstalk between H3-S10 and H3-K9. This possibility could enable kinases and phosphatases to make regulatory inputs to the PcG/trxG system. It will be important to determine whether additional PcG/trxG proteins possess or interact with histone-modifying activities, and also to define the histone modification states on PcG-repressed and trxG-activated genes in vivo (Müller, 2002).

The HOX genes of Drosophila provide the best example of heritable silencing by PcG proteins. Recent work has shown that derepression of HOX genes after removal of Pc or Psc in proliferating cells can be reversed if the depleted protein is resupplied within a few cell generations. These results led to the proposal that silenced HOX genes bear a heritable molecular mark that targets them for PcG repression and that this mark can be maintained for a few cell generations, even after HOX genes are derepressed. Could the proposed mark reflect, at least in part, H3-K27 methylation by the Esc-E(z) complex? Histone lysine methylation is a very stable modification, so is well-suited for a long-term molecular mark. E(z) mutants show an unusually long delay before HOX misexpression is detected in imaginal disc clones (72 hr for robust misexpression of UBX) and a long delay is observed in temperature-shift experiments with an E(z) allele that behaves as a null at restrictive temperature. In addition, of all the PcG mutants tested, only Su(z)12 mutants show a comparably long delay in release from silencing in imaginal disc clones. In contrast, removal of the PRC1 components Psc or Ph triggers rapid loss of repression (Müller, 2002).

Remarkably, Esc, E(z), and SU(z)12 are conserved in many organisms, where they appear to function together as repressors in a wide array of developmental processes. Mammalian complexes that resemble the Esc-E(z) complex are implicated in multiple processes including early embryonic patterning, HOX gene regulation, and hematopoiesis. Intriguingly, mouse homologs of Esc and E(z) associate with the inactive X chromosome in trophoblast stem cells, suggesting a direct role in X-inactivation. In C. elegans, the Esc- and E(z)-related proteins MES-6 and MES-2 form a stable complex and are required for germline development and gene silencing. The conserved partnership extends to plants, where proteins related to Esc, E(z), and SU(z)12 are cohort regulators in several Arabidopsis developmental pathways. A striking example is VRN2, a SU(z)12 relative, which is required for long-term gene silencing in response to vernalization. Further work in these systems should address if histone methylation by Esc-E(z) complexes represent an evolutionary ancient mechanism to mark chromatin for heritable repression during development (Müller, 2002).

Elements of the Polycomb repressor SU(Z)12 needed for histone H3-K27 methylation, the interface with E(Z), and in vivo function

Polycomb repressive complex 2 (PRC2) is an essential chromatin-modifying enzyme that implements gene silencing. PRC2 methylates histone H3 on lysine-27 and is conserved from plants to flies to humans. In Drosophila, PRC2 contains four core subunits: E(Z), SU(Z)12, ESC, and NURF55. E(Z) bears a SET domain that houses the enzyme active site. However, PRC2 activity depends upon critical inputs from SU(Z)12 and ESC. The stimulatory mechanisms are not understood. This study presents functional dissection of the SU(Z)12 subunit. SU(Z)12 contains two highly conserved domains: an ∼140 amino acid VEFS domain and a Cys2-His2 zinc finger (ZnF). Analysis of recombinant PRC2 bearing VEFS domain alterations, including some modelled after leukemia mutations, identifies distinct elements needed for SU(Z)12 assembly with E(Z) and stimulation of histone methyltransferase. The results define an extensive VEFS subdomain that organizes the SU(Z)12-E(Z) interface. Although the SU(Z)12 ZnF is not needed for methyltransferase in vitro, genetic rescue assays show that the ZnF is required in vivo. Chromatin immunoprecipitations reveal that this ZnF facilitates PRC2 binding to a genomic target. This study defines functionally critical SU(Z)12 elements, including key determinants of SU(Z)12-E(Z) communication. Together with recent findings, this illuminates PRC2 modulation by conserved inputs from its noncatalytic subunits (Rai, 2013).

Drosophila ESC-like can substitute for ESC and becomes required for Polycomb silencing if ESC is absent

The Drosophila esc-like gene (escl) encodes a protein very similar to ESC. Like ESC, ESCL binds directly to the E(Z) histone methyltransferase via its WD region. In contrast to ESC, which is present at highest levels during embryogenesis and low levels thereafter, ESCL is continuously present throughout development and in adults. ESC/E(Z) complexes are present at high levels mainly during embryogenesis but ESCL/E(Z) complexes are found throughout development. While depletion of either ESCL or ESC by RNAi in S2 and Kc cells has little effect on E(Z)-mediated methylation of histone H3 lysine 27 (H3K27), simultaneous depletion of ESCL and ESC results in loss of di- and trimethyl-H3K27, indicating that either ESC or ESCL is necessary and sufficient for di- and tri-methylation of H3K27 in vivo. While E(Z) complexes in S2 cells contain predominantly ESC, in ESC-depleted S2 cells, ESCL levels rise dramatically and ESCL replaces ESC in E(Z) complexes. A mutation in escl that produces very little protein is viable and exhibits no phenotypes but strongly enhances esc mutant phenotypes, suggesting they have similar functions. esc escl double homozygotes die at the end of the larval period, indicating that the well-known 'maternal rescue' of esc homozygotes requires ESCL. Furthermore, maternal and zygotic over-expression of escl fully rescues the lethality of esc null mutant embryos that contain no ESC protein, indicating that ESCL can substitute fully for ESC in vivo. These data thus indicate that ESC and ESCL play similar if not identical functions in E(Z) complexes in vivo. Despite this, when esc is expressed normally, escl appears to be entirely dispensable, at least for development into morphologically normal fertile adults. Furthermore, the larval lethality of esc escl double mutants, together with the lack of phenotypes in the escl mutant, further suggests that in wild-type (esc⁺) animals it is the post-embryonic expression of esc, not escl, that is important for development of normal adults. Thus escl appears to function in a backup capacity during development that becomes important only when normal esc expression is compromised (Kurzhals, 2008).

The rescue of embryos containing no ESC protein by ESCL over-expression did produce an occasional fly with a mild PcG phenotype, a hint that ESCL may not be quite as effective as ESC either in forming complexes with other PRC2 components or in promoting H3K27 methylation by PRC2 complexes. Some of the RNAi results also hint that ESCL-containing complexes may not be quite as effective at H3K27 methylation as ESC-containing complexes. In particular, depletion of ESC reduced the 3mH3K27 level more than depletion of ESCL did, in both S2 and Kc cells. This occurred despite the fact that the ESCL level increases quite substantially when ESC alone is depleted. While differences in efficiency of knockdown might contribute to this, it may also indicate that more ESCL is required to produce the same level of 3mH3K27 achieved with less ESC, i.e., ESC-containing complexes may be more effective at H3 methylation than ESCL-containing complexes. A possible basis for such a difference is suggested in previous work demonstrating that ESC and ESCL both bind to histone H3 (Tie, 2007), in which it was noticed that ESCL binding to H3 appears to be somewhat weaker (Kurzhals, 2008).

The results demonstrate that while ESCL is expressed throughout development and during adulthood, it is apparently dispensable, under normal culture conditions, at least for development of morphologically normal fertile adults. This is somewhat surprising in light of the complete lethality of esc escl double mutants, which clearly indicates that the well-known 'maternal rescue' of the embryonic lethality of esc mutants is absolutely dependent on ESCL. esc escl double homozygotes die at the end of an extended larval period and have markedly smaller brain hemispheres, wing and eye-antennal discs, while the other discs appear to be similar in size to wild-type discs. The normally large wing disc is likely to be smaller due to transformation into a haltere disc due to derepression of Ubx in the wing disc, a phenotype typically seen only as a partial transformation in adults of very strong Polycomb mutant genotypes. Transformation of the leg discs to other segmental identities, which would not be reflected in changes in disc size, is also likely in the esc escl mutants, since the signature 'extra sex combs' phenotype seen in maternally rescued adult esc homozygotes is already enhanced when these adults are also heterozygous for escl. Similarly, the decrease in the size of the eye antennal disc is consistent with the partial antenna to leg transformations observed in the esc adults that are also heterozygous for the escl mutation. Mutations in E(z) and Su(z)12 have a similar late lethal phase and also exhibit small imaginal discs and larval brains, consistent with their functional collaboration in the same complexes. Why then is ESCL largely dispensable, given the impact of the escl mutation on development to adulthood in esc escl double mutants (Kurzhals, 2008).

The demonstration that ESCL can substitute fully for ESC in vivo, provided it is expressed at adequate levels at all times, further suggests that the two proteins are qualitatively equivalent and that in the absence of the other, either one is necessary and sufficient for normal development to adulthood. Nonetheless, it appears that normally ESCL does not come into play in a substantial way unless ESC levels are compromised. A reason for this is suggested by the discovery that in S2 cells most ESCL protein is in free form when ESC is relatively abundant, and that the level of ESCL, along with the proportion of it found in the 600-kDa complex, increases dramatically in cells that have been depleted of ESC by RNAi. The amount of ESCL that is bound to the Ubx PRE also increases after ESC depletion. These observations suggest that ESCL levels are regulated in part by ESC levels through a mechanism involving competition for participation in E(Z) complexes, which serves to stabilize ESCL. Since the actual abundance of ESC and ESCL is not known, it cannot be determined to what extent this may be due to intrinsic differences in the affinity of the two proteins for these complexes or is simply a reflection of mass action favoring the more abundant of the two proteins. Abundance almost certainly plays a key role in early embryos, when the ESC level is highest and the ESCL level is at its lowest. It is harder to explain why ESCL does not become essential in postembryonic stages, when the ESC level is much lower and the ESCL level is at its highest. This might hint that even at low levels ESC has an intrinsic advantage over ESCL in competition for complex assembly. Alternatively, it is possible that the levels of ESC in postembryonic stages have been underestimated by measurements in bulk extracts and that in imaginal discs, critical tissues for development of the adult, ESC levels remain high enough to continue to out-compete ESCL for complex formation in those tissues. However, in bulk extracts from late larvae, a substantial fraction of ESCL is detected in the 600-kDa complex and ESCL is readily co-immunoprecipitated with E(Z) from these extracts. Whichever the case, it appears that ESCL has little or no role when ESC is present at normal levels. It remains possible that ESCL becomes more important in adults or under specific environmental conditions (Kurzhals, 2008).

The results of RNAi-mediated knockdown in S2 and Kc cells clearly demonstrate that depletion of either ESC or ESCL has no appreciable effect on the levels of di-methyl and tri-methyl H3K27, while simultaneous depletion of both proteins greatly reduces the tri- and di-methyl isoforms. This strongly suggests that ESC and ESCL are functionally interchangeable for this function of PRC2 and indicates that each protein is produced at sufficient levels in S2 cells to promote normal levels of H3K27 methylation in the absence of the other. This also suggests that the common role of both proteins in PRC2 complexes, i.e., binding to histone H3 and stimulating E(Z)-mediated H3K27 methylation (Tie, 2007), is the basis of their interchangeability in vivo (Kurzhals, 2008).

In summary, while the complementary temporal expression patterns of ESC and ESCL during development initially suggested that ESCL might become essential during postembryonic development, the data presented here suggest otherwise, beginning with the lack of any obvious phenotypes in an strong escl mutant that makes little residual protein. The reason for the apparent dispensability of ESCL would appear to be due simply to the sufficiency of normal ESC levels at all times to promote normal development. Ironically, this is now made even clearer by the observed late larval lethality of the esc escl double mutants, since together with the viability and normal morphology of escl mutants, it demonstrates very clearly that, while ESCL is absolutely required for survival to adulthood in the absence of zygotic ESC expression, ESC alone is sufficient to promote survival to adulthood. Even the variable extra sex combs phenotype seen in maternally rescued esc mutant adults is unlikely to reflect a postembryonic requirement for ESCL, since the escl mutants themselves do not exhibit this phenotype even at low frequency. Unless a true protein null escl allele does, this means that esc by itself is sufficient for development into normal adults (Kurzhals, 2008).

On the other hand, the data presented here do help to solve the puzzle posed by previous genetic evidence indicating that esc is unique among Polycomb Group proteins in being required only during early embryogenesis, and more recent biochemical evidence that ESC is essential for H3K27 methylation by E(Z), which is required continuously. While the maternal rescue of esc null embryos to adulthood by a single esc⁺ allele in the mother is remarkable and indicates that there is a critical early requirement for ESC, this genetic result does not reflect a requirement for ESC only in the early embryo. It requires the backup function provided by ESCL, which becomes required after the maternally deposited esc⁺ gene products are depleted in esc⁻ embryos. The elevated level of ESCL and ESCL/E(Z) complexes caused by ESC depletion in S2 cells also suggests that this may involve compensatory elevation of ESCL/E(Z) complexes as maternally derived ESC disappears in esc⁻ embryos. Similar considerations pertain to the experiments demonstrating rescue of esc null mutants by a brief pulse of zygotic esc⁺ expression from a heat-inducible hsp70-esc transgene during the first 4 h of embryogenesis, which almost certainly also requires the backup function of ESCL. Genetic experiments have been interpreted to suggest that ESC was required only in the early embryo to ensure normal development to adulthood, except for the variable partial transformation of T2 and T3 legs to T1 identity. However, while it is now evident that the existence of ESCL and its continuous expression explains the apparent requirement for ESC only during embryogenesis, inferred from maternal rescue, these genetic experiments do not necessarily reveal a normal requirement for ESCL. On the contrary, the lack of obvious phenotypes in the escl mutant, together with the inability of maternally derived ESC alone to promote normal development to adults in the absence of ESCL, leads to the conclusion that under normal circumstances, not only is ESC sufficient to promote normal development in the absence of ESCL, but also that esc expression during postembryonic stages is required for development of normal adult morphology. This conclusion was also arrived at almost 50 years ago based on genetic mosaic experiments in which esc mutant phenotypes were observed in clones of homozygous esc mutant cells induced at various times during the larval period (Hannah-Alava, 1958). The absence of phenotypes in the escl mutant, particularly the variable T2 and T3 extra sex combs phenotype seen in maternally rescued esc mutants, is consistent with the conclusion that postembryonic expression of ESC itself is required for development of normal adult morphology (Kurzhals, 2008).

Given the apparent redundancy of ESCL, at least for development of morphologically normal, fertile adults, why then has it persisted? All vertebrates for which complete genome sequences are available contain a single ESC/ESCL ortholog encoded by the EED gene, which is expressed continuously in all tissues. While there are several different EED isoforms arising from use of alternative translation initiation sites, it is not known whether their different N-termini make them functionally different. The C. elegans genome also contains a single ESC ortholog, mes-6 (Korf, 1998). Structural similarities between the esc and escl genes, particularly their intron-exon arrangements and the ESC and ESCL protein sequences, as well as the close proximity of the two genes on chromosome 2L, indicate that they arose by a gene duplication event. To date, outside of plants, the presence of two ESC/ESCL genes appears to be restricted to the Drosophilidae lineage. A single ortholog is present in the next most closely related lineages for which genome sequences are complete, including Culicidae dipterans (three mosquitoes), lepidopterans (Bombyx mori), coleopterans (Tribolium casteneum) and hymenopterans (Apis mellifera). All twelve Drosophila species for which complete genome sequence is available have two genes that are clearly recognizable ESC and ESCL orthologs. The most distant of these species diverged over 40 million years ago, sufficient time for one member of a duplicate gene pair to degenerate in the absence of selection for its function. A careful analysis of the changes that the esc and escl coding sequences have undergone in all these species should reveal whether or not the escl gene shows any signs of having begun to degenerate. However, the qualitative functional equivalence of ESC and ESCL revealed in this study suggests that the persistence of ESCL and the conservation its function in H3K27 methylation by PRC2 complexes may be due to a fitness advantage conferred by ESCL for a function that has not yet been identified, perhaps a function it performs during adulthood (Kurzhals, 2008).

Polycomb repressive complex 2 and Trithorax modulate Drosophila longevity and stress resistance

Polycomb Group (PcG) and Trithorax Group (TrxG) proteins are key epigenetic regulators of global transcription programs. Their antagonistic chromatin-modifying activities modulate the expression of many genes and affect many biological processes. This study reports that heterozygous mutations in two core subunits of Polycomb Repressive Complex 2 (PRC2), the histone H3 lysine 27 (H3K27)-specific methyltransferase E(Z) and its partner, the H3 binding protein ESC, increase longevity and reduce adult levels of trimethylated H3K27 (H3K27me3). Mutations in trithorax (trx), a well known antagonist of Polycomb silencing, elevate the H3K27me3 level of E(z) mutants and suppress their increased longevity. Like many long-lived mutants, E(z) and esc mutants exhibit increased resistance to oxidative stress and starvation, and these phenotypes are also suppressed by trx mutations. This suppression strongly suggests that both the longevity and stress resistance phenotypes of PRC2 mutants are specifically due to their reduced levels of H3K27me3 and the consequent perturbation of Polycomb silencing. Consistent with this, long-lived E(z) mutants exhibit derepression of Abd-B, a well-characterized direct target of Polycomb silencing, and Odc1, a putative direct target implicated in stress resistance. These findings establish a role for PRC2 and TRX in the modulation of organismal longevity and stress resistance and indicate that moderate perturbation of Polycomb silencing can increase longevity (Siebold, 2009).

The evidence presented in this study establishes a role for PRC2 and TRX in the modulation of life span and stress resistance. Using multiple alleles of several PRC2 subunits, evidence is provided that heterozygous mutations in the PRC2 subunits E(z) and esc extend life span and increase resistance to oxidative stress and starvation in Drosophila. Consistent with the enzymatic function of PRC2 in the methylation of H3K27, long-lived E(z) and esc mutants have reduced H3K27me3 levels. Furthermore, mutations in trx suppress the increased longevity and stress resistance phenotypes of E(z) mutants, while concomitantly increasing their reduced H3K27me3. The moderate reduction of H3K27me3 in long-lived E(z) mutants is sufficient to partially derepress some direct targets of Polycomb silencing, and this is also counteracted by mutations in trx. These results provide strong evidence that derepression of one or more Polycomb target genes is likely to be responsible for their increased longevity. Interestingly, E(z) was also recently identified as one of a number genes whose mRNA expression levels were significantly associated with variation in longevity in a large set of wild-type derived inbred lines (Siebold, 2009).

The counterbalancing effects of PRC2 and TRX on H3K27me3 levels suggest a simple model for their modulation of longevity. Although complete loss of PRC2 activity results in preadult lethality, moderately reducing H3K27me3 destabilizes Polycomb silencing sufficiently to cause partial derepression of some Polycomb target genes that can increase life span and stress resistance. Simultaneously reducing TRX and E(Z) exerts a compensatory effect, reestablishing more normal levels of H3K27me3 and Polycomb target gene expression. Based on this model, it is expected that heterozygous trx mutations would decrease longevity. However, the modestly elevated H3K27me3 level (13%) of the heterozygous trxB11 null mutant may simply be insufficient to cause this effect in a wild-type background. It will be interesting to see whether increased TRX levels, which decrease H3K27me3 levels (much like PRC2 mutants) by elevating CBP-mediated H3K27 acetylation, will promote increased longevity, as the model would predict. The evolutionary conservation of PRC2 components in metazoans and their conserved function in epigenetic silencing raises the possibility that they may play a conserved role in modulating life span in other organisms. Although histone methyl-transferases have not been previously implicated in modulating organismal longevity, several other highly conserved chromatin- modifying enzymes have been. In addition to SIR2 and RPD3, the histone H3K4 demethylase LSD-1 has also recently been implicated in modulating longevity in C. elegans. Given the roles of these enzymes in the epigenetic maintenance of transcriptional states, it seems likely that additional chromatin modifying enzymes will be found to modulate longevity. The most well-characterized targets of Polycomb silencing are the homeotic genes of the Bithorax and Antennapedia complexes (Siebold, 2009).

Although heterozygous PRC2 mutants exhibit no overt homeotic phenotypes, the elevated level of Abd-B expression in E(z) heterozygotes demonstrates that their moderately reduced H3K27me3 level is sufficient to partially derepress Polycomb target genes. Could modest derepression of one or more of the homeotic genes be responsible for the increased longevity? Given that they encode transcription factors, their potential for regulating expression of many other genes leaves this possibility open. PRC2 mutants exhibit increased resistance to oxidative stress and starvation. The elevated expression level of Odc1, a putative direct target of Polycomb silencing may contribute to this as it has been shown to mediate resistance to oxidative stress and a variety of other chemical and environmental stresses. Dietary supplementation with the polyamine spermidine was also recently shown to increase longevity in yeast, C. elegans, Drosophila, and mice, consistent with the possibility that Odc1 overexpression may contribute to the increased longevity of PRC2 mutants. Recent evidence suggests that other changes in metabolism and adult physiology might also contribute to the increased longevity of PRC2 mutants. YY1, the mammalian homolog of Drosophila PHO (a DNA-binding PcG protein involved in recruiting PRC2 to chromatin), directly regulates many genes required for mitochondrial oxidative metabolism. It will be interesting to determine whether transcriptional regulation of metabolic genes is a broader theme in the adult function of PcG proteins. PRC2 and TRX play key roles in promoting epigenetically stable transcriptional states through their mutually antagonistic effects on H3K27me3 levels. Recent work has revealed a growing number of biological processes in which they play an important role. The results presented here now point to a role for these epigenetic transcriptional regulators in modulating life span (Siebold, 2009).

Interaction of Groucho protein with H3

An investigation was carried out of the molecular mechanisms underlying the functions of Groucho and its mammalian homologs, the transducin-like Enhancer of split (TLE) proteins. A minority fraction of nuclear Groucho/TLEs are associated with chromatin in live cells; they co-purify with isolated histones. Affinity chromatography and far Western blotting studies show further that native Groucho/TLE proteins interact specifically with histone H3 and not with other core histones. This interaction is mediated by the H3 amino-terminal domain previously shown by genetic analysis in yeast to be essential for the role of H3 in transcriptional silencing. Groucho/TLEs form oligomeric structures in vivo. These combined findings suggest that transcription complexes containing Groucho/TLEs may associate with chromatin through interactions with the amino terminus of histone H3. These interactions may be propagated along the chromosome due to the ability of Groucho/TLEs to participate in higher order structures (Palaparti, 1997).

Does methylation increase the affinity of H3 for Polycomb protein?

The chromodomain of Hp1 has been shown to bind specifically both dimethyl and trimethyl lysine 9 N-terminal peptide of histone H3. The structure of the chromodomain and the sequence conservation of critical residues in Pc suggest that the Pc chromodomain might also have such a specific affinity. Immobilized H3 N-terminal peptide unmethylated or trimethylated at lysine 9 was used to see if these peptides were able to bind recombinant Pc protein. The Pc protein has some affinity for the trimethylated peptide but binds to the same extent to the unmethylated peptide. The interaction was examined of GST-Pc to full-length histone H3 either methylated with purified HIMalpha or acetylated with GCN5. Compared to the acetylated H3, methylated H3 binds five times better to GST-Pc. The binding is nearly unaffected by the K9A mutation but decreases more than 2-fold with the K27A mutant H3. These results indicate that methylation by HIMalpha significantly increases the affinity of H3 for Pc but most of this affinity is due to K27 methylation (Czermin, 2002).

Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27

The chromodomain of Drosophila Polycomb protein is essential for maintaining the silencing state of homeotic genes during development. Recent studies suggest that Polycomb mediates the assembly of repressive higher-order chromatin structures in conjunction with the methylation of Lys 27 of histone H3 by a Polycomb group repressor complex. A similar mechanism in heterochromatin assembly is mediated by HP1, a chromodomain protein that binds to histone H3 methylated at Lys 9. To understand the molecular mechanism of the methyl-Lys 27 histone code recognition, a 1.4-Å-resolution structure was determined of the chromodomain of Polycomb in complex with a histone H3 peptide trimethylated at Lys 27. The structure reveals a conserved mode of methyl-lysine binding and identifies Polycomb-specific interactions with histone H3. The structure also reveals a Pc dimer in the crystal lattice that is mediated by residues specifically conserved in the Polycomb family of chromodomains. The dimerization of Drosophila Pc can effectively account for the histone-binding specificity and provides new mechanistic insights into the function of Polycomb. It is proposed that self-association is functionally important for Polycomb (Min, 2003).

The crystal structure presented in this study shows that the chromodomains of Pc and HP1 have a common overall structure, and they also bind methyl-lysine-containing substrates similarly. The structure identifies that Arg 67 and Asp 65 of Pc are important for Polycomb-specific interactions with histone H3. Curiously, these residues interact with the main chain of the histone peptide. This is also true in the HP1 structure, where most of the chromodomain-histone interactions are through main-chain atoms. An important question concerning the recognition of the methylated histone tail by Polycomb and HP1 is what determines their binding specificities. The difference in histone-binding affinity of Polycomb and HP1 is not sufficient to account for their substrate specificities. Crystallographic analyses have identified a Pc chromodomain dimer in the crystal lattice that can account for the binding specificity of Pc, compelling reasons in support of the potential physiological significance of the observed dimeric interactions are outlined (Min, 2003).

Although dimerization of Pc was never pointed out explicitly before, self-association of the Pc chromodomain was noted in several previous studies. Interestingly, an in vivo domain-swap experiment replacing the chromodomain of HP1 with that of Pc has shown that the chimeric protein binds to both heterochromatin and Pc binding sites in polytene chromosomes. Furthermore, some endogenous Pc is misdirected to the heterochromatic center, whereas some endogenous HP1 is mislocalized to the Pc binding sites. These observations not only confirmed earlier observations that the chromodomain of Pc and the C-terminal chromo shadow domain of HP1 possess intrinsic nuclear localization and chromosomal binding properties, they also indicate that the dPC chromodomain directs the mislocalization of endogenous dPC through protein-protein contacts (Min, 2003).

The dimerization model predicts that the binding specificity of the Pc chromodomain arises from histone-histone interactions resulting from the close proximity of the two histone-tail-binding sites in the Pc chromodomain dimer. The binding of two methyl-Lys9 histone H3 tails by the Pc chromodomain dimer is disallowed because of potential steric clashes. It is possible that the Pc chromodomain dimer may bind only one histone H3 tail in some instances. In this case, the available structural information cannot exclude the binding of a methyl-Lys9 histone H3 tail to the Pc chromodomain dimer. The observation that the Pc chromodomain binds specifically to methyl-27 histone H3 peptides in vitro clearly supports the binding of two histone H3 tails to the Pc dimer. The structure also provides insights into the function of Pc proteins in the assembly of repressive higher-order chromatin structure. Because the two histone H3-binding sites are closely juxtaposed, the two histone tails are unlikely to come from the same nucleosome. In the nucleosome core particle structure, Lys 27 is ordered in one of the H3 tails, whereas the ordered residues start from Pro 38 in the other H3 molecule. The observed distance between the Calpha atoms of Pro 38 and Lys 27 of the same H3 molecule is 26 Å. The two histone-H3 Pro 38 residues are 73 Å apart (linear distance) in the nucleosome core particle structure, which coverts to ~80 Å along the arc of wrapped DNA. The two Lys 27 Calpha atoms must be within 27 Å to occupy the two binding sites in the Pc chromodomain dimer. Assuming that the two H3 tails N-terminal to Pro 38 can be maximally stretched and free to adopt any conformation, it has not been possible to model the simultaneous binding of two histone tails from the same nucleosome to the Pc chromodomain dimer without steric clashes. Thus, it is believed that the histone tails binding to the Pc chromodomain dimer must come from two separate nucleosomes. The in vivo binding of two methyl-Lys 27 histone-H3 tails, from spatially adjacent nucleosomes, will effectively lock the nucleosomes into a more compact configuration. This compaction will lead to a repressive chromatin state associated with the silencing of homeotic genes. A similar function for HP1 in the assembly of heterochromatin has been proposed, although dimerization of HP1 via the C-terminal chromo shadow domain makes the binding of two histone tails from the same nucleosome, as well as separate ones, possible (Min, 2003).

On the histone H3 tail, Lys 9 and Lys 27 are both methylation sites associated with epigenetic repression, and reside within a highly related sequence motif ARKS, a sequence common to chromodomain proteins Polycomb (Pc) and heterochromatin protein 1 (HP1). Pc and HP1 are highly discriminatory for binding to these sites in vivo and in vitro. In Drosophila S2 cells, and on polytene chromosomes, methyl-Lys 27 and Pc are both excluded from areas that are enriched in methyl-Lys 9 and HP1. Swapping of the chromodomain regions of Pc and HP1 is sufficient for switching the nuclear localization patterns of these factors, indicating a role for their chromodomains in both target site binding and discrimination. To better understand the molecular basis for the selection of methyl-lysine binding sites, the 1.8 Å structure of the Pc chromodomain was solved in complex with a H3 peptide bearing trimethyl-Lys 27, and it was compared with the structure of the HP1 chromodomain in complex with a H3 peptide bearing trimethyl-Lys 9. The Pc chromodomain distinguishes its methylation target on the H3 tail via an extended recognition groove that binds five additional residues preceding the ARKS motif (Fischle, 2003).

These studies show a clear preference of the Pc chromodomain for the H3 Lys 27 methyl mark. How does this activity of Pc contribute to PcG function? In the case of the formation of heterochromatin and the initially genetically defined pathway of suppression of variegation, it has been suggested that methylation of H3 on Lys 9 by Suv3-9 generates a docking site for the HP1 (also known as Suv2-5) chromodomain. Further recruitment of Suv3-9 by the chromo shadow domain of HP1 has been postulated to lead to a perpetuation and spreading of a heterochromatic domain until blocked by yet unknown mechanisms. Similarly, Esc-E(z)-dependent methylation of Lys 27 (and possibly Lys 9) and consecutive recruitment of Pc and Pc-containing complexes might contribute to the stability of the PcG complex, particularly in the early stages of assembly at a PRE by permitting complex formation to spread to neighboring sequences. This interpretation of the specific binding of Pc to methyl-Lys 27 is in agreement with studies demonstrating loss of chromosome binding for several components of PRC1 upon inactivation of E(z) and is consistent with several other in vivo results that imply synergy between these complexes. However, it is unclear at present if dynamic perpetuated spreading of a Lys 27 mark indeed exists and is dependent on Pc recruitment and Esc-E(z) enzymatic activity. Other possible functions for the binding of Pc to the Lys 27 methyl mark include a more static maintenance effect that could contribute to epigenetic memory. In this model, the recruitment of Esc-E(z) would be independent of and precede any involvement of Pc binding. Alternatively, although complex recruitment could be constitutive, the decision to repress or not could depend on an epigenetic switch mediated by Lys 27 methylation and its interaction with local Pc/PRC1 (Fischle, 2003 and references therein).

However, alternative routes and mechanisms of Pc and PcG recruitment to local sites of chromatin (in addition to recognition of Lys 27 methylation) might also exist. For example, studies involving the localization of a chimeric HP1 protein containing the chromodomain of Pc on polytene chromosomes imply critical interactions of the Pc chromodomain with other PcG components that are recruited to PcG sites. Therefore, the particular localization patterns observed for the wild-type and chimeric proteins in swapping experiments might be the result of additive effects, including other targeting mechanisms besides methyl-lysine binding. Nevertheless, it is intriguing to note that the subnuclear localization patterns for wild-type and chimeric Pc and HP1 proteins are coincident with the localization of specifically recognized methyl-lysine marks on the histone H3 tail. It is unclear to what extent additional regions of the proteins C-terminal to the chromodomains could contribute to subnuclear localization and function. For example, different studies have implicated the C-terminal chromo shadow domain and hinge regions of HP1 in addition to the chromodomain in the specific subnuclear targeting of this factor. However, a C-terminal truncation of Pc does not affect its specific chromosomal localization. Additional studies will have to address the exact contribution of the chromodomains and their recognition and binding of particular methyl-lysine marks to the specific functions of the Pc and HP1 proteins (Fischle, 2003 and references therein).

A mutation in the E(Z) methyltransferase that increases trimethylation of histone H3 lysine 27 and causes inappropriate silencing of active Polycomb target genes

Drosophila Polycomb Repressive Complex 2 (PRC2) is a lysine methyltransferase that trimethylates histone H3 lysine 27 (H3K27me3), a modification essential for Polycomb silencing. Mutations in its catalytic subunit, E(Z), that abolish its methyltransferase activity disrupt Polycomb silencing, causing derepression of Polycomb target genes in cells where they are normally silenced. In contrast, the unusual E(z) mutant allele Trithorax mimic (E(z)^Trm) causes dominant homeotic phenotypes similar to those caused by mutations in trithorax (trx), an antagonist of Polycomb silencing. This suggests that E(z)^Trm causes inappropriate silencing of Polycomb target genes in cells where they are normally active. This study shows that E(z)^Trm mutants have an elevated level of H3K27me3 and reduced levels of H3K27me1 and H3K27me2, modifications also carried out by E(Z). This suggests that the E(z)^Trm mutation increases the H3K27 trimethylation efficiency of E(Z). Acetylated H3K27 (H3K27ac), a mark of transcriptionally active genes that directly antagonizes H3K27 methylation by E(Z), is also reduced in E(z)^Trm mutants, consistent with their elevated H3K27me3 level causing inappropriate silencing. In 0–4 h E(z)^Trm embryos, H3K27me3 accumulates prematurely and to high levels and does so at the expense of H3K27ac, which is normally present at high levels in early embryos. Despite their high level of H3K27me3, expression of Abd-B initiates normally in homozygous E(z)^Trm embryos, but is substantially lower than in wild type embryos by completion of germ band retraction. These results suggest that increased H3K27 trimethylation activity of E(Z)^Trm causes the premature accumulation of H3K27me3 in early embryogenesis, 'predestining' initially active Polycomb target genes to silencing once Polycomb silencing is initiated (Stepanik, 2012).

Evidence presented in this study suggests that the trithorax-like phenotypes of E(z)^Trm mutants are due to the substantial bulk increase in H3K27me3, which leads to inappropriate silencing of Polycomb target genes in cells where they are normally active. How does this come about? The suppression of the high H3K27me3 levels of E(z)^Trm mutants by Su(z)12 and esc mutations as well as increased E(z)⁺ gene dosage indicates that, like wild type E(Z), the E(Z)^Trm mutant protein must be assembled into PRC2 complexes and that its activity depends on other PRC2 subunits. This means that E(Z)^Trm mutant protein is likely to be targeted to the same regions of the genome as wild type E(Z) and not mistargeted to novel sites. Developmental analysis indicates that inappropriate silencing of Abd-B in E(z)^Trm mutants begins in early embryogenesis, and is presaged by premature accumulation of H3K27me3 in early (0–4 h) embryos to much higher levels than in wild type embryos. Surprisingly, this does not prevent normal initiation of Abd-B expression on schedule, but appears to potentiate its subsequent attenuation around the time that Polycomb silencing normally becomes required (Stepanik, 2012).

A recent analysis of the in vitro enzymatic activities of recombinant human PRC2 complexes demonstrated that wild type EZH2 displays the greatest catalytic efficiency for the H3K27 monomethylation reaction and lower efficiency for the dimethylation (mono- to di-) and the trimethylation (di- to tri-) reactions (Sneeringer, 2010). The increase in H3K27me3 at the expense of H3K27me1 and H3K27me2 in E(z)^Trm mutants suggests that the R741K substitution alters E(Z) catalytic activities to preferentially increase its H3K27 trimethylation efficiency. The mono- and di-methylation activities might also be affected, but the reduced steady state levels of H3K27me1 and H3K27me2 levels must be interpreted cautiously, since they are also influenced by presence of the H3K27 demethylase UTX and since some H3K27me1 may be produced by another methyltransferase besides E(Z) (Stepanik, 2012).

The decrease in H3K27ac also indicates that not all of the increase in H3K27me3 occurs at the expense of H3K27me1/2. The reduced level of H3K27ac in E(z)^Trm mutants, a modification that directly antagonizes H3K27 methylation, strongly suggests that at least some of the increase in H3K27me3 occurs at the expense of H3K27ac, i.e., occurs at H3K27 sites that are normally acetylated and associated with transcriptionally active Polycomb target genes. Indeed, in the absence of direct evidence that the excess H3K27me3 in E(z)^Trm mutants occupies genomic sites of normally active Polycomb target genes, the lower level of bulk H3K27ac is perhaps the strongest evidence that the elevated H3K27me3 level in E(z)^Trm mutants is the cause of the inappropriate silencing of normally active Polycomb target genes (Stepanik, 2012).

The R741K substitution occurs in a region of the catalytic SET domain of E(Z) that plays an important role in the methyltransferase activity of other SET domain proteins. The adjacent Y740 residue is invariant in all SET domain proteins. Mutating the corresponding tyrosine to alanine in SET7/9 or to phenylalanine in DIM-5 all but eliminates the methyltransferase activity of these proteins. A structural study showed that this conserved tyrosine is positioned in close proximity to the substrate lysine targeted for methylation. Residue Y742 is also invariant in E(Z) orthologs, though it varies among other SET domain proteins. The residue present at the corresponding position in the H3K9 trimethylase DIM-5 is involved in recognition of the substrate peptide (Stepanik, 2012).

Of particular relevance to the current work are several structure-based in vitro mutagenesis studies of other SET domain proteins that identified a 'phenylalanine/tyrosine switch' that affects the number of methyl groups the enzymes can add to the substrate lysine. This 'switch' affects the residue corresponding to F738 in E(Z). Mutating the phenylanine present at this position to the bulkier tyrosine in the H3K9 trimethylases DIM-5 and G9a results in enzymes that can only catalyze mono- and dimethylation. Conversely, mutating the tyrosine present at this position to phenylalanine in the H4K20 monomethylase SET8 allows the enzyme to add a second methyl group to H4K20 (Stepanik, 2012).

The results of these studies suggest that the E(Z)^Trm R741K substitution may increase trimethylation efficiency by altering the dimensions of the catalytic pocket either directly, via its more compact side chain, or by altering the positioning of the adjacent Y740, Y742, F738 or other residues that affect the catalytic pocket of E(Z). This could occur by altering the rate of the reaction, the affinity of E(Z) for the substrates of the methyltransferase reaction, enhancing the release of products of the reaction, or by enabling the enzyme to better accommodate the bulkier products of the trimethylation reaction (Stepanik, 2012).

Recently, a number of other dominant mutations in human EZH2 that cause elevated H3K27me3 levels have been detected in follicular lymphomas and diffuse-large B-cell lymphomas, always in heterozygous condition (Morin, 2010). These recurring mutations, single amino acid substitutions for Y641 (Y655 in E(Z)), a conserved residue in the SET domain, were found to have increased H3K27 di- and trimethylation activity but no monomethylation activity, explaining their obligate heterozygosity for manifestation of their elevated H3K27me3 phenotype. In contrast, the E(Z)^Trm mutant enzyme produces a higher H3K27me3 level in the absence of wild type protein than when heterozygous, indicating that the R741K substitution does not abolish monomethylation activity (Stepanik, 2012).

Remarkably, 0–4 h E(z)^Trm embryos exhibit an 8-fold increase in bulk H3K27me3 and a greater than 55% reduction in the level of H3K27ac. This suggests that if the H3K27ac present in early embryos normally plays a role in preventing the premature onset of H3K27 trimethylation, the increased trimethylation efficiency of E(Z)^Trm is sufficient to overcome it. It further suggests that despite the very low level of H3K27me3 in 0–4 h wild type embryos, the PRC2 subunits that are already present in the nuclei of early cleavage stage embryos are likely to be assembled into enzymatically active PRC2 complexes (Stepanik, 2012).

In spite of the much higher H3K27me3 level in 0–4 hour E(z)^Trm embryos, the levels of ABD-B in E(z)^Trm and wild type embryos are indistinguishable until after completion of germ band elongation, i.e., after the time when Polycomb silencing of the homeotic genes is normally established. This indicates that while H3K27me3 is required for Polycomb silencing, the elevated H3K27me3 level in 0–4 hour E(z)^Trm embryos does not, by itself, exert a 'repressive' effect on early Abd-B expression or advance the time of onset of Polycomb silencing. Thus, some other factor or signal that is required for Polycomb silencing must be absent or inactive during this early stage. This might include, for example, a developmentally programmed delay in the maturation of functionally active PRC1 complexes, which bind to H3K27me3-containing nucleosomes produced by PRC2 and carry out additional activities required for Polycomb silencing. Whatever the nature of this limiting factor/signal, once Polycomb silencing begins, the premature accumulation of H3K27me3 appears to 'predestine' or potentiate the inappropriate silencing of already activated Polycomb target genes (Stepanik, 2012).

Interestingly, it has been observed that feeding the histone deacetylase inhibitor sodium butyrate to E(z)^Trm/ + females partially suppressed the trx-like phenotypes of their E(z)^Trm/ + progeny, suggesting that a deacetylase activity may be required during early embryogenesis for these phenotypes to develop. The observation that during embryogenesis, H3K27ac levels are highest in 0–4 h wild type embryos, but are more than 55% lower in 0–4 h E(z)^Trm embryos is consistent with the possibility that it is specifically the inhibition of H3K27ac deacetylation by maternal butyrate feeding that suppresses the development of trx-like phenotypes in E(z)^Trm mutants, indirectly inhibiting the premature accumulation of high levels of H3K27me3 by helping to maintain H3K27ac. The histone deacetylase RPD3/HDAC1 has been implicated in H3K27ac deacetylation in vivo by RNAi knockdown in Drosophila S2 cells. RPD3 is physically associated with PRC2 and PRC1 complexes in both Drosophila and mammals and is required in vivo for robust Polycomb silencing. RPD3 of maternal origin is already present in the early embryo, and is presumably available to deacetylate H3K27. The results thus suggest that the initial establishment of Polycomb silencing during normal development may require active deacetylation of preexisting H3K27ac as a prerequisite to at least some H3K27 trimethylation (Stepanik, 2012).

If the H3K27ac already present in 0–4 h embryos normally plays a role in inhibiting the premature accumulation of H3K27me3, then the effect of the E(z)^Trm mutation on the H3K27ac level indicates that the mutant enzyme can override this inhibition. How could it do this? It seems unlikely that simply altering the H3K27 trimethylation efficiency of E(Z) would lead to increased H3K27ac deacetylation activity. Instead, it is speculated that the higher trimethylation efficiency of the E(Z)^Trm mutant enzyme alters an ongoing dynamic balance between H3K27 acetylation/deacetylation and methylation/demethylation activities in the early embryo. It was recently shown that the CBP-mediated acetylation of H3K4me3-containing nucleosomes is highly dynamic and its dynamic nature is critical for transcriptional activation (Crump, 2011). The physical association of E(Z) and RPD3 likely increases the efficiency of the sequential deacetylation and methylation reactions required for replacement of an H3K27ac with H3K27me3, a possible advantage for efficient developmentally programmed switching from a transcriptionally active to silent state. This suggests that with no change in an already dynamic acetylation–deacetylation cycle, the presence of E(Z)^Trm would further increase the probability that an un/deacetylated H3K27 will be methylated before it can be (re)acetylated, and would have the cumulative effect of increasing the bulk H3K27me3 level at the expense of H3K27ac, as observed (Stepanik, 2012).

To gain further insight into how E(z)^Trm promotes inappropriate silencing of normally active Polycomb target genes, it will be important to determine the effect of E(z)^Trm on the genome-wide distributions of H3K27me3/2/1 and H3K27ac, as well as PcG and TrxG proteins. Understanding the effects of E(z)^Trm in further detail could provide new insights into the role of aberrant Polycomb silencing in cancer. In addition to the above EZH2 mutations with increased H3K27 trimethylation activity in B cell lymphomas, EZH2 and other PRC2 subunits have been found to be overexpressed in many solid tumors, leading to aberrant H3K27 trimethylation and silencing of tumor suppressor genes. The level of overexpression is positively correlated with poor prognosis and RNAi knockdown of elevated EZH2 levels in can halt tumor cell proliferation. While the mechanisms underlying inappropriate silencing due to EZH2 overexpression and due to E(Z)^Trm and other hyper-trimethylating mutants are likely to differ, they underscore the importance of understanding how TRX, CBP, UTX and other TrxG factors antagonize H3K27 methylation by PRC2 to prevent inappropriate silencing of normally active Polycomb target genes and maintain their active states. Further understanding of how E(z)^Trm overrides their antagonistic activities should provide additional insights into how they do so (Stepanik, 2012).

Nanos function and histone methylation

In C. elegans, mRNA production is initially repressed in the embryonic germline by a protein unique to C. elegans germ cells, PIE-1. PIE-1 is degraded upon the birth of the germ cell precursors, Z2 and Z3. A chromatin-based mechanism has been identified that succeeds PIE-1 repression in these cells. A subset of nucleosomal histone modifications, methylated lysine 4 on histone H3 (H3meK4) and acetylated lysine 8 on histone H4 (H4acetylK8), are globally lost and the DNA appears more condensed. This coincides with PIE-1 degradation and requires that germline identity is not disrupted. Drosophila pole cell chromatin also lacks H3meK4, indicating that a unique chromatin architecture is a conserved feature of embryonic germ cells. Regulation of the germline-specific chromatin architecture requires functional nanos activity in both organisms. These results indicate that genome-wide repression via a nanos-regulated, germ cell-specific chromatin organization is a conserved feature of germline maintenance during embryogenesis (Schaner, 2003).

The C. elegans nanos homologs nos-1 and nos-2 play important overlapping roles in worm primordial germ cell development. Knockdown of both NOS-1 and NOS-2 activities results in sterile animals, a population of which exhibit germ cells that begin proliferation prematurely. Loss of NOS-2 alone can also result in ectopic localization of the primordial germ cells. H3meK4 levels were examined in Z2/Z3 chromatin of embryos from nos-1(gv5);nos-2(RNAi) hermaphrodites. Animals homozygous for the nos-1(gv5) are viable and fertile; RNAi of nos-2 in these animals results in germ cell defects. nos-1 mutant animals were therefore injected with nos-2 dsRNA, and parallel broods were assessed for sterility as adults or stained for H3meK4 in Z2/Z3. In these experiments, 59% of the offspring of the injected animals grew up to be completely sterile adults. An almost identical percentage of parallel brood embryos exhibited inappropriate staining for H3meK4 in one or both of the primordial germ cells, Z2/Z3. The C. elegans NANOS homologs, NOS-1 and NOS-2, therefore play important roles in either establishing and/or maintaining the chromatin-based mode of transcriptional repression in Z2/Z3 (Schaner, 2003).

While the progenitors of the soma and germline are also separated from each other during early embryogenesis in Drosophila, the processes that give rise to the physical segregation of these two distinct cell types in flies are quite different from those in worms. In light of these differences, it was of interest to determine if distinct chromatin states are also established in the somatic and germline nuclei of fly embryos. Staining of early Drosophila embryos with the H3meK4 antibody has revealed that there is little detectable H3meK4 in any nuclei during the rapid synchronous nuclear divisions in the center of the embryo. Similarly, no H3meK4 was detected in the nuclei of newly formed pole cells, or in somatic nuclei when they first migrate to the periphery of the embryo. H3meK4 was first detected in somatic nuclei between nuclear division cycles 12 and 13, after the nuclei around the periphery of the embryo have already undergone several rounds of nuclear division. This change in the methylation status of histone H3 is coincident with, or slightly precedes, the time when transcription is broadly upregulated in the somatic nuclei of syncytial blastoderm embryos. High levels of H3meK4 are maintained through the cellular blastoderm stage, with all somatic nuclei staining with the H3meK4 antibody (Schaner, 2003).

In contrast to the somatic nuclei, little if any H3meK4 was detected in the pole cells (marked with anti-Vasa-specific antibody) of either syncytial blastoderm or cellular blastoderm embryos. Staining of embryos at later stages revealed that H3meK4 could still not be detected in germ cells in early gastrulation stage through midgut invagination. However, once the germ cells traversed the midgut wall and began migrating away from the hindgut toward the somatic gonadal precursor cells, H3meK4-specific signal became readily evident. Therefore, from the time of their formation until stage 9 of embryogenesis, there is little if any H3meK4 in the germ cells' chromatin. H3meK4 becomes readily detectable in the germ cells, however, coincident with the onset of transcription at stage 9/10. The accumulation of H3meK4 in the germ cells, as in the soma, correlates temporally with transcriptional activation in these cells (Schaner, 2003).

Embryos were stained for epitopes corresponding to H4acetylK5, H4acetylK8, H4acetylK12, H3diacetyl, and H3dimeK36. Only the antibodies specific for H3diacetyl and H4acetylK5 exhibited nuclear staining above background levels in early embryonic stages, and the appearance of these epitopes was temporally identical to that observed for H3meK4. In contrast to the H3meK4 modification, however, no differences were observed in the levels of these modifications in pole cell nuclei compared to somatic nuclei. This indicates that a global lack of H3meK4, in combination with the presence of other modifications that correlate with transcriptional competence, is a conserved feature of chromatin structure in transcriptionally inert germ cell nuclei. (Schaner, 2003).

nos function is required for either the establishment or maintenance of transcriptional quiescence in newly formed Drosophila pole cells. In light of results showing a premature appearance of H3meK4 in C. elegans germ cells in the absence of nos activity, an obvious question is whether nos is also required to block this methylation in the early fly germline. To answer this question, fly embryos produced by nos mutant mothers were probed with H3meK4- and Vasa-specific antibodies. H3meK4 can be detected in the pole cells of stage 4 nos^- embryos at nuclear division cycle 14. In contrast, H3meK4 is completely absent from pole cell chromatin in wild-type embryos at this stage. Moreover, H3meK4-specific signal was also detected in pole cells in stage 5 embryos that have just initiated gastrulation. It is interesting to note that not all nos^- pole cells (27/42) display H3meK4-specific staining. This is consistent with findings that transcription is not activated in every pole cell of nos^- blastoderm stage embryos and supports the suggestion that additional factors contribute to the establishment/maintenance of transcriptional quiescence in fly pole cells (Schaner, 2003).

While methylation of lysine 4 of histone H3 is a conserved mark of transcriptionally competent or active chromatin, methylation of lysine 9 of histone H3 (H3meK9) is a highly conserved modification that is enriched in silenced genomic regions, such as either facultative or constitutive heterochromatin. Consequently, it was of interest to determine if the transcriptionally quiescent pole cell nuclei are enriched in H3meK9 as compared to the transcriptionally active somatic nuclei. Drosophila embryos were probed with antibodies against H3meK9 and Vasa to mark the pole cells. In contrast to H3meK4, cleavage stage Drosophila embryonic nuclei exhibited readily detectable H3meK9 even prior to their migration. The modification was also detected in somatic nuclei after nuclear migration, where it appeared enriched in nuclear regions adjacent to the periphery of the embryo that correspond to telomeric and centromeric heterochromatin in the Rabl configuration. These regions, conversely, were observed to lack H3meK4. H3meK9 is also detected in pole cells as soon as they are formed, with the level of H3meK9 antibody staining in the pole cell nuclei being considerably higher than in the somatic nuclei. The observed enrichment of H3meK9 in the pole cell nuclei coincided with loss of the mitosis-specific modification, H3 phospho-Ser10, suggesting that a specific remodeling of the pole cell chromatin accompanies their exit from the cell cycle (Schaner, 2003).

Since depletion of maternal nos activity results in a premature increased accumulation of H3meK4 in early pole cells, whether nos^- pole cells exhibit a parallel reduction in H3meK9 was investigated. Indeed, nos^- pole cell nuclei do not exhibit the characteristic enrichment for H3meK9-specific signal seen in wild-type embryos. Furthermore, more than 50% of the pole cells examined show considerably reduced staining specific for nuclear H3meK9 compared to the neighboring somatic nuclei. The known dichotomous roles for H3meK4 and H3meK9 in the regulation of transcriptional competence are therefore conserved in pole cells, and their relative abundances in pole cell chromatin are responsive to nos activity (Schaner, 2003).

Identifying the mechanisms that guide the separation of somatic and germ lineages is one of the oldest pursuits in developmental biology. How the totipotent germline is maintained during development has become increasingly relevant to modern science in the search for conserved mechanisms guarding stem cell identity. These data provide new evidence for at least two modes of germline-specific repression that guard the germline during C. elegans embryogenesis, one of which is conserved in Drosophila. In the earliest phase, maternal PIE-1 activity in the germline P blastomeres prevents mRNA production through a mechanism that does not involve substantial, germline-specific alterations in chromatin architecture. After the degradation of PIE-1, however, a second mode involving a dramatic and specific remodeling of chromatin arises in the germ cell precursors, Z2/Z3. Whereas the former mode appears to be unique to C. elegans, the latter mode, which bears the hallmarks of direct transcriptional repression provided by a specific mode of chromatin organization, is a conserved feature of germ cell maintenance (Schaner, 2003).

In both worms and flies, lineage restriction to germ cell fate is marked by a global absence of H3meK4 and a more condensed chromatin structure. In the case of C. elegans, the absence of H3meK4 is the result of a specific depletion; in Drosophila the absence arises from preventing H3meK4 accumulation. This absence is maintained in both organisms until zygotic activation of the genome in germ cells. Premature activation, marked by premature accumulation of H3meK4, results in the loss of the germ cell lineage in both worms and flies. In both species, the lack of H3meK4 occurs in the presence of high levels of other histone modifications that often correlate with an open chromatin configuration, indicating a 'dominance' of H3meK4 as an indicator of transcriptional activity (Schaner, 2003).

The conserved correlation between H3meK4 presence and absence and global activation and silencing, respectively, is striking. The inactive X chromosome in mammals is globally depleted of H3meK4, as are silenced regions of the genome in fission yeast. In Tetrahymena, the germinal micronucleus is transcriptionally silent and H3meK4 is missing. Sexual conjugation causes transformation of the micronucleus into a transcriptionally active somatic macronucleus, which becomes enriched in H3meK4. Thus, even in protozoans, genome-wide diminishment of H3meK4 is a property of the 'germline' and its accumulation accompanies 'somatic' activation (Schaner, 2003).

The lack of H3meK4 in fly pole cells is mirrored by enrichment for H3meK9. This is not observed in Z2/Z3 in C. elegans. The reason for this difference is not understood but may reflect a more substantial role for this modification in genome regulation in Drosophila, which has a more highly repetitive genome than that of C. elegans, and consequently more abundant classically defined heterochromatin. Indeed, in contrast to Drosophila, H3meK9 is only cytologically enriched at telomeres in C. elegans embryos and on the highly condensed, unpaired X chromosome during male meiosis. In both examples, the enrichment for H3meK9 is always mirrored by the absence of H3meK4. (Schaner, 2003).

Posttranscriptional regulation plays a major role in maintaining the embryonic germline in both worms and flies. The conserved classes of proteins involved include the pumilio (Pufs) and nanos families. nanos activities are similarly required for proper migration of the primordial germ cells, maintenance of their mitotic quiescence, proper proliferation of the germline after hatching, and, as shown in this report, the regulation of chromatin organization in Z2/Z3 in both species. In addition, nanos has also been shown to be required to maintain transcriptional quiescence in pole cells. Similar nanos functions may also be required in mammals, where two of three nanos homologs are required for fertility. The phenotype of nanos-3 null mice is consistent with specification of primordial germ cells occurring normally, followed by an inability to maintain germ cell identity during migration. This phenotype is strikingly similar to those observed in nanos(-) worms and flies (Schaner, 2003).

nanos function is essential to maintain embryonic germline repression prior to normal activation of proliferation, presumably through its characterized roles in posttranscriptional regulation. Only a few direct targets of nanos regulation in germ cells have been identified, including maternal cyclin mRNAs. Targets that are the effectors of H3meK4 addition have yet to be identified, but conceivably include an H3 lysine 4-specific methyltransferase. The phenotypes observed upon removal of nanos are also only partially penetrant in both organisms. It is thus likely that other conserved, partially redundant systems exist. A clear candidate is germ cell-less (gcl), which is required in Drosophila to maintain transcriptional quiescence. Preliminary experiments suggest that H3meK4 regulation is also disrupted in gcl mutants. A predicted C. elegans gene with substantial homology to gcl has been identified, but its role in these processes has not as yet been assessed (Schaner, 2003).

It appears clear in flies that nos is required to maintain the repressive state of the germline and that part of this involves preventing accumulation of H3meK4. The pole cells begin life lacking this modification, and Nanos function is required to prevent its addition to pole cell chromatin. It is not as clear-cut in worms, since H3meK4 is initially present and then removed at germline restriction. The detection of H3meK4 in Z2/Z3 of nos-depleted embryos could therefore conceivably reflect a role for nos in either (presumably indirectly) promoting the removal of H3meK4 after PIE-1 and/or preventing the re-addition of H3meK4 before hatching. Given the conservation of most other phenotypes caused by the loss of nos activity in multiple organisms, the latter role for C. elegans NOS proteins is favored (Schaner, 2003).

At approximately the 100-cell stage in C. elegans, PIE-1 levels decrease and this is concurrent with a global loss of a distinct subset of histone modifications, H3meK4 and H4acetylK8, in Z2/Z3. The presence of other histone tail modifications in Z2/Z3 indicates that the loss of H3meK4 and H4acetylK8 epitopes are not due to a general cleavage of histone tails, but rather a specific chromatin-remodeling event. While removal of H4acetylK8 is likely to be performed by a histone deacetylase (HDAC), an activity that demethylates lysines has yet to be identified in any organism. The loss of H3meK4 could therefore represent either a replication-independent or a replication-coupled replacement of histone H3, perhaps by a germline-specific H3 variant. A diminishment of H3meK4 in P4 after gastrulation and prior to its entry into mitosis, which could indicate an S phase-related event, is frequently observed (Schaner, 2003).

HP1 controls telomere elongation and telomere silencing interaction with H3

HP1 is a conserved chromosomal protein, first discovered in Drosophila, which is predominantly associated with the heterochromatin of many organisms. It has been shown that HP1 is required for telomere capping, telomere elongation, and transcriptional repression of telomeric sequences. Several studies have suggested a model for heterochromatin formation and epigenetic gene silencing in different species that is based on interactions among histone methyltransferases (HMTases), histone H3 methylated at lysine 9 (H3-MeK9), and the HP1 chromodomain. This model has been extended to HP1 telomeric localization by data showing that H3-MeK9 is present at all of the telomeres. This model has been tested, and it has been found that the capping function of HP1 is due to its direct binding to telomeric DNA, while the silencing of telomeric sequences and telomere elongation is due to its interaction with H3-MeK9 (Perrini, 2004).

In heterozygous HP1 mutant stocks, over time, the telomeres show a strong elongation, and the transcription of both TART and HeT-A is significantly increased. The telomeres from the strain carrying the HP1 mutation are clearly elongated. It is clear that, compared to the wild-type telomere of the second chromosome left arm, this elongation can result from either the addition of both types of telomeric transposons or from a sort of telomeric rearrangement. Telomere elongation was also found in Hp1 mutant [Su(var)2-5⁰²/Cy and Su(var)2-5⁰⁵/Cy] stocks (Perrini, 2004).

The transcription of the telomeric transposons were examined in heterozygous and trans-heterozygous Su(var)2-5⁰⁴/Su(var)2-5⁰⁵ and Su(var)2-5⁰²/Su(var)2-5⁰⁵ mutant larvae. HeT-A transcription is very low in wild-type larvae, while these transcripts are clearly present in heterozygous (mutant/wild-type) larvae and are even more abundant in trans-heterozygous mutant larvae. Similar results were found for TART. Interestingly, similar amounts of HeT-A transcripts were found in both male and female mutant larvae, thus suggesting that the HeT-A sequences being transcribed are mainly those at the telomeres rather than those on the Y chromosome (Perrini, 2004).

Using real-time RT-PCR analysis, it was found that, in Su(var)2-5⁰²/Su(var)2-5⁰⁵ mutants, the HeT-A transcription is 95.76 times higher than in wild-type, and similar results were also observed in Su(var)2-5⁰⁴/Su(var)2-5⁰⁵. Since, the Su(var)2-5⁰² point mutation that disrupts the HP1 chromodomain also strongly derepresses both TART and HeT-A, it appears that the silencing of telomere transposons requires a functional HP1 chromodomain. Considering that this mutation does not affect protein localization or telomere stability, it is concluded that the HP1 chromodomain, although dispensable for telomere capping, is required for telomeric transposon silencing and, most probably, telomere elongation. Since it is known that the HP1 chromodomain binds H3-Me3K9, and that the presence of this modified histone seems to overlap HP1 in all the telomeres, it is possible that repressive telomeric chromatin is formed by the interaction of HP1 with the modified histone. To test this idea, it was asked if the methylation of H3-K9 at the telomeres could also be affected by the absence of HP1. It has been recently shown that HP1 mutations affect heterochromatic H3-K9 methylation, thus suggesting that HMTase SU(VAR)3-9 and HP1 are probably functionally interdependent in forming the pericentromeric heterochromatin mediated by the H3-K9 methylation in Drosophila . Polytene chromosomes from HP1 mutant larvae were immunostained with an H3-Me3K9-specific antibody. It was found that H3-Me3K9 is absent from telomeres of mutant larvae completely lacking HP1. Most importantly, it was found that H3-Me3K9 is also absent from telomeres in Su(var)2-5⁰²/Su(var)2-5⁰⁵ mutant larvae. Su(var)2-5⁰² is a mutation in the chromodomain of HP1 that is a strong dominant suppressor of variegation and is homozygous lethal, but does not affect telomeric binding of the mutant protein. As confirmation, it was found that the imaginal disks of Su(var)2-5⁰²/Su(var)2-5⁰⁵ mutant larvae are similar to wild-type imaginal disks, while those lacking HP1 show extensive apoptosis. These data clearly show that the methylation of H3-K9 at the telomeres depends on the presence of the HP1 chromodomain. The HP1 chromodomain and H3-Me3K9 are not, however, required for telomere capping, but both are necessary for the control of telomeric transposon transcription and telomere elongation (Perrini, 2004).

HP1 has the ability to directly interact with DNA in vitro. This suggests that a direct interaction of HP1 with telomeric DNA may be necessary for the telomere capping function. Direct HP1-DNA binding was tested in vivo by using a crosslinking assay with cis-diamminedichloroplatinum (cis-DDP). cis-DDP is considered a useful crosslinker to identify non-histone proteins that interact directly with DNA. The most frequent site of primary binding for cis-DDP on DNA is the N7 of guanine, exposed on the surface of the major groove. To confirm the direct DNA binding of HP1 in vitro, recombinant HP1 (500 ng) and genomic DNA (12 mg) isolated from Drosophila adults were mixed in the presence of 0.1 mM cis-DDP and incubated for 90 min at 37°C. Then, the DNA-protein complexes were subjected to hydroxyapatite purification, and DNA bound proteins were eluted by 1.5 M thiourea, subjected to SDS-gel electrophoresis, and analyzed on Western blot by the C1A9 HP1 monoclonal antibody. The presence of clear immunosignals shows that HP1 crosslinks to DNA, suggesting a direct interaction between the two molecules (Perrini, 2004).

To test the DNA binding activity of HP1 in vivo, crosslinking was induced by cis-DDP in intact nuclei purified from Drosophila larvae. This approach allows the detection of DNA-protein interactions under conditions very close to those existing in vivo, since the interactions are stabilized before the disruption of the nucleus. After purification of the crosslinked complexes, it was found that HP1 is present among the crosslinked nuclear components, suggesting that HP1 also has a DNA binding activity in vivo (Perrini, 2004).

An immunoprecipitation assay (X-ChIP assay) was used to test the capacity of HP1 to bind telomeric DNA in vivo. cis-DDP crosslinked complexes from intact nuclei were purified by gel-filtration chromatography. The nuclei used came exclusively from female larvae to avoid confusion from the tandem array of telomeric HeT-A and TART-related sequences found at the centromeric region of the Y chromosome. The complexes were then immunoprecipitated with a monoclonal HP1 antibody. To examine the presence of telomeric sequences among the immunoprecipitated DNA, a PCR analysis was performed with specific pairs of primers covering fragments of the telomeric HeT-A region. The telomeric sequence is amplified only in the DNA of the immunoprecipitated sample and in the genomic DNA, but not in the DNA of the immunoprecipitated control. Thus, HP1 is directly bound to the telomeric region of HeT-A (Perrini, 2004).

HP1 is also located at the extremity of stable terminal deletions that lack both HeT-A and TART telomeric transposons. Since some terminal deletions of the X chromosome end inside the yellow gene, whether HP1 directly binds to the yellow sequences of such terminal deletions was tested by using the immunoprecipitation assay in larvae carrying the y^Tdl4 terminal deletion. The PCR analysis of the immunoprecipitates was performed by using pairs of primers located proximal to the original breakpoint in the terminal deletion. Amplified yellow sequences were found only in the DNA of immunoprecipitates from the y^Tdl4 strain. These data show that HP1 can directly bind telomeric DNA independent of specific sequences. It was also found, by using a gel shift assay, that HP1 is capable of binding both double- and single-strand telomeric DNA, but competition experiments have also suggested that it has a major affinity for single-strand DNA. Since HP1 does not have any obvious DNA binding domain, this raises the question of what part of the protein is responsible for this binding. As discussed above, the chromodomain appears to be dispensable for telomeric binding. A COOH-terminal region corresponding to the HP1 chromoshadowdomain has been found to be required for the nuclear localization of HP1, and an additional functional domain inside the hinge portion specifies HP1 heterochromatin binding. Intriguingly, this domain also permits the HP1 telomeric localization, thus suggesting a role of the hinge region in HP1 telomeric DNA binding. To test this suggestion, a gel shift assay was done on the series of HP1 fragments. Only the HP1 fragments containing the hinge regions are capable of producing a gel shift of single-strand HeT-A DNA. These results strongly suggest that the hinge region is required for the direct binding of HP1 to telomeric DNA (Perrini, 2004).

It is concluded that the involvement of HP1 in telomere capping and in controlling telomeric DNA transcription, and probably elongation, is mediated by two different types of binding to the telomeres. Telomere capping depends on the direct binding of HP1 to telomeric sequences, while the transcriptional control of such sequences depends on the interaction of the HP1 chromodomain with H3-Me3K9. Interestingly, the observation that H3-K9 methylation depends on the presence of HP1 at the telomeres suggests that this histone modification is due to a previous interaction of HP1 with a specific HMTase (Perrini, 2004).

Together these data suggest a simple model for HP1 function at the telomeres. It is suggested that HP1 first directly binds telomeric DNA and recruits a yet unknown specific HMTase. The enzyme would then methylate H3-K9, creating an additional binding site for HP1. The spreading of HP1, HMTase, and H3-Me3K9 interactions would form the telomeric repressive chromatin. Since the present data seem to exclude an involvement of RNAi, suggesting instead a preferential affinity of HP1 for single-strand HeT-A DNA, it is proposed that HP1 is probably recruited to the telomeric DNA by its specific recognition of the protruding telomeric ends. At present, it cannot be excluded that this affinity is not potentiated by yet another telomeric component. Supporting this possibility is the recent finding that the HP1-interacting HOAP protein is also required for telomere capping. It is not known yet if the telomeric functions of HP1 are evolutionarily conserved. A suggestion of this possibility comes from recent studies showing the existence of the telomeric position effect in human cells that depends on a specific higher-order organization of telomeric chromatin in which HP1 is probably involved (Perrini, 2004).

YY1 DNA binding and PcG recruitment requires CtBP resulting in recruitment of PcG proteins and E(z) mediated methylation of histone H3

Mammalian Polycomb group (PcG) protein YY1 can bind to Polycomb response elements in Drosophila embryos and can recruit other PcG proteins to DNA. PcG recruitment results in deacetylation and methylation of histone H3. In a CtBP mutant background, recruitment of PcG proteins and concomitant histone modifications do not occur. Surprisingly, YY1 DNA binding in vivo is also ablated. CtBP mutation does not result in YY1 degradation or transport from the nucleus, suggesting a mechanism whereby YY1 DNA binding ability is masked. These results reveal a new role for CtBP in controlling YY1 DNA binding and recruitment of PcG proteins to DNA (Srinivasan, 2004).

To determine whether YY1 can recruit PcG proteins to DNA, chromatin immunoprecipitation (ChIP) assays were performed in a transgenic Drosophila embryo system consisting of hsp70-driven GALYY1 and a reporter construct containing the LacZ gene under control of the Ultrabithorax (Ubx) BXD enhancer and the Ubx promoter adjacent to GAL4-binding sites (BGUZ). The BGUZ reporter is expressed ubiquitously during embryogenesis but is selectively repressed in a PcG-dependent manner by GALYY1 and GALPc. Embryos were either left untreated or heat shocked to induce GALYY1 expression. After immunoprecipitation with various antibodies, the region surrounding the GAL4-binding sites in the BGUZ reporter was detected by PCR. Prior to heat shock, no GALYY1 could be observed at the reporter gene. After heat shock, GALYY1 binding to the reporter gene was easily detected. Interestingly, concomitant with GALYY1 binding, there was an increase in binding of the Polycomb (Pc) and Polyhomeiotic (Ph) proteins. Thus, YY1 DNA binding results in PcG recruitment to DNA (Srinivasan, 2004).

Binding of PcG proteins to PRE sequences is known to cause deacetylation of histone H3 and methylation on Lys 9 and Lys 27. Interestingly, induction of GALYY1 binding to the reporter gene resulted in loss of histone H3 acetylation on K9 and K14. Simultaneously, there was a gain of methylation on histone H3 Lys 9 and Lys 27. Therefore, YY1 binding to the BGUZ reporter results in the recruitment of PcG proteins to DNA and subsequent post-translational modifications of histones characteristic of PcG complexes (Srinivasan, 2004).

The presence of PcG proteins and the status of histone H3 modifications at the Ubx promoter region, which is 4 kb downstream of the GALYY1-binding site, were determined. To avoid amplification of the endogenous Ubx promoter, immunoprecipitated samples were amplified with primers spanning the Ubx-LacZ boundary. Interestingly, the presence of Pc and Ph was detected at the promoter after GALYY1 induction. The presence of GALYY1 at this site was also detected. The GAL4 protein alone does not bind to the Ubx promoter region, indicating specificity for YY1 sequences. The induced GAL4 protein was functional, however, because it efficiently bound to the GAL4-binding site in the BGUZ reporter. Binding by GALYY1 could, therefore, be due to either cryptic YY1-binding sites present at the promoter, physical association of GALYY1 with other proteins bound at the promoter, or interactions via looping of DNA between the GAL4-binding sites and the Ubx promoter. Again, induction of GALYY1 resulted in loss of acetylation of H3K9 and H3K14 and simultaneous gain of methylation on H3K9 and H3K27. These results are consistent with studies that have reported spreading of PcG proteins and histone modifications to flanking DNA (Srinivasan, 2004).

PHO and YY1 bind to the same DNA sequence, and PHO-binding sites have been identified in multiple PREs. Therefore, it was reasoned that YY1 would bind to endogenous PREs and perhaps increase recruitment of PcG proteins. For this, the major Ubx PRE (PRE_D), that contains multiple PHO-binding sites located in the bxd region, was examined. As expected, upon GALYY1 induction, GALYY1 was detected at this endogenous PRE site. In addition, YY1 binding was accompanied by an increase in Pc and Ph signals when compared with no heat shock controls and a loss of H3 K9 and H3 K14 acetylation and gain of H3 K9 and H3 K27 methylation. Thus, YY1 can bind to an endogenous PRE and can augment PcG recruitment (Srinivasan, 2004).

These results clearly indicated that YY1 DNA binding results in recruitment of PcG proteins, histone deacetylases (HDACs), and histone methyltransferases (HMTases) to DNA. To determine whether the Drosophila E(z) protein (which possesses HMTase activity) was involved, whether YY1 transcriptional repression was lost in an E(z) mutant background was examined. The results are consistent with the observation that E(z) specifically methylates histone H3 on Lys 27, which creates a binding site for the chromodomain of Pc. Thus, the repression observed with GALYY1 requires function of the E(z) PcG protein (Srinivasan, 2004).

A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin

Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, Suv4-20h1 and Suv4-20h2 were identified as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, Suv4-20 is a novel position-effect variegation modifier. Together, these data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin (Schotta, 2004).

These data suggest H4-K20 trimethylation is a mark of silenced chromatin domains. Therefore whether this modification would indeed be important for gene silencing in well-described PEV models in Drosophila was investigated. A single, homozygous-viable P-element insertion (P{GT1}BG00814) into the third exon of Suv4-20 has been identified in the course of the Drosophila gene disruption project. H4-K20 trimethylation at polytene chromatin is nearly lost in homozygous mutant larvae, demonstrating that the P-element insertion (Suv4-20^BG00814) represents a strong hypomorphic allele of Suv4-20. Because the Suv4-20 locus maps on the X chromosome, the classical PEV rearrangement In(1)w^m4 cannot be used to analyze a potential modifier effect of Suv4-20. Therefore, another PEV rearrangement was analyzed that translocates a different marker, Stubble (Sb), close to pericentric heterochromatin (T(2;3)Sb^V). The dominant mutation Stubble induces short bristles, but heterochromatin-induced silencing of Sb^V results in wild-type (long) bristles. Homozygous Suv4-20^BG00814 as well as control wild-type females were crossed to T(2;3)Sb^V males. In the progeny, the extent of Sb^V reactivation was determined as the ratio of short bristles (active Sb^V) to long bristles (inactive Sb^V). In males and females of the wild-type crosses, only 1%-2% of bristles show a Sb phenotype, indicating that Sb^V is largely inactivated. In contrast, Sb^V becomes derepressed in the progeny of Suv4-20^BG00814 flies, because now ~25% of the bristles are short. This result classifies Suv4-20 as a dominant PEV modifier and further indicates a functional role for Suv4-20-dependent H4-K20 trimethylation in gene silencing (Schotta, 2004).

The histone modification pattern of active genes revealed through genome-wide chromatin analysis of Drosophila

The covalent modification of nucleosomal histones has emerged as a major determinant of chromatin structure and gene activity. To understand the interplay between various histone modifications, including acetylation and methylation, a genome-wide chromatin structure analysis was performed in Drosophila. A binary pattern of histone modifications was found among euchromatic genes, with active genes being hyperacetylated for H3 and H4 and hypermethylated at Lys 4 and Lys 79 of H3, and inactive genes being hypomethylated and deacetylated at the same residues. Furthermore, the degree of modification correlates with the level of transcription, and modifications are largely restricted to transcribed regions, suggesting that their regulation is tightly linked to polymerase activity (Schübeler, 2004).

ChIP analysis followed by hybridization to DNA microarrays was used to map the pattern of six different histone modifications in the Drosophila genome. The karyotypically stable Drosophila Kc cell line was used. Chromatin was purified after formaldehyde cross-linking (= input) and immunoprecipitated either with antibodies that recognize a specific histone modification or without the addition of antisera as a control. DNA enriched for a specific modification (= bound) and DNA from the input material was isolated, labeled with different fluorescent dyes, and hybridized to a DNA microarray. Enrichment for a histone modification via immunoprecipitation results in a stronger fluorescence signal from the bound fraction, whereas absence of the modification results in a stronger signal from the input fraction. Because the observed enrichments are antibody specific, the ratio of the two dyes represents a quantitative measure of the studied modification (Schübeler, 2004).

The principal findings include the following: (1) there is a binary pattern of histone modifications for euchromatic genes, with active genes consistently marked by all of the euchromatic histone modifications analyzed and the absence of any of these modifications on nontranscribed genes; (2) the level of transcript abundance is positively correlated with the degree of euchromatic histone modifications, and (3) the chromosomal extent of the modification coincides with, and is limited to, the transcribed region. The surprising observation of an 'all-or-none' pattern of histone modification for euchromatic genes suggests a concerted mechanism for the placing of these marks. For example, the euchromatic modifications could be restricted to nucleosomes containing a certain histone H3 variant. The replication-independent deposition of the H3 variant 3.3 raises the possibility that in Metazoa the majority of euchromatic histone H3 modifications may occur on H3.3. Indeed, histone H3.3 has recently been reported to be enriched in acetylated lysines and in methylated Lys 4 and Lys 79 (Schübeler, 2004).

Although it is currently unclear whether these euchromatic modifications can be set prior to nucleosome assembly and deposition, there is ample evidence for post-deposition modification of histones. For example, a link between the elongating polymerase complex and several histone-modifying enzymes, including Set1 (an H3-K4 methylase; see Drosophila Set1), Set2 (an H3-K36 methylase; see Drosophila Set2), and Sas3 (a HAT), has been demonstrated in S. cerevisiae. Furthermore, genetic evidence from S. cerevisiae suggests that Dot1, the H3-K79 methylase, may also be recruited to chromatin by the elongating polymerase complex. These findings in budding yeast indicate a coupling of histone modifications and transcription. This genome-wide analysis in Drosophila cells strongly supports these findings and further argues that such interactions may be an integral component of transcriptional elongation in metazoans (Schübeler, 2004).

More than 25 years ago, it was observed that chromatin of active genes is more sensitive to DNaseI digestion than that of inactive genes. Although, to date, the nature of this sensitivity has been elusive, it is proposed that this sensitivity reflects the presence of euchromatic tail modifications. Why does such a 'switch' between two chromatin configurations involve a large set of histone modifications? Each modification may participate in creating a chromatin structure that facilitates transcription, either by changing nucleosomal interactions or by serving as a binding substrate for other proteins. The use of multiple modifications would make such system more robust. Regardless, these results reveal a tight coupling between transcription and euchromatic histone modifications. On recruitment, these modifications may serve to facilitate polymerase elongation and reinitiation and to propagate the transcriptional state through cell division (Schübeler, 2004).

A critical epitope for substrate recognition by the nucleosome remodeling ATPase ISWI

The ATPase ISWI is the catalytic core of several nucleosome remodeling complexes that are able to alter histone-DNA interactions within nucleosomes such that the sliding of histone octamers on DNA is facilitated. Dynamic nucleosome repositioning may be involved in the assembly of chromatin with regularly spaced nucleosomes and accessible regulatory sequence elements. The mechanism that underlies nucleosome sliding is largely unresolved. The N-terminal 'tail' of histone H4 is critical for nucleosome remodeling by ISWI. If deleted, nucleosomes are no longer recognized as substrates and do not stimulate the ATPase activity of ISWI. The H4 tail is part of a more complex recognition epitope, which is destroyed by grafting the H4 N-terminus onto other histones. The H4 tail requirement has been mapped to a hydrophilic patch consisting of the amino acids R₁₇H₁₈R₁₉ localized at the base of the tail. These residues have been shown to contact nucleosomal DNA, suggesting that ISWI recognizes an 'epitope' consisting of the DNA-bound H4 tail. Consistent with this hypothesis, the ISWI ATPase is stimulated by isolated H4 tail peptides ISWI only in the presence of DNA. Acetylation of the adjacent K₁₂ and K₁₆ residues impairs substrate recognition by ISWI (Clapier, 2002).

This study highlights the importance of the H4 N-terminal residues R₁₇H₁₈R₁₉ for substrate recognition by the nucleosome remodeling ATPase ISWI. Out of context, either as isolated tail peptides or grafted onto histone H3 or H2A, H4 tails containing this sequence element will not trigger the ATPase activity of ISWI. Position-dependent functions of the H4 tail have been observed in yeast. While nucleosome assembly and structure per se is unaffected by swapping the tail domains, the regulation of specific loci through chromatin, however, requires the H4 tail at its native position. The data are consistent with an independent analysis of the histone tail requirements for nucleosome sliding by NURF (Clapier, 2002).

The critical amino acids are part of a larger, basic patch, K₁₆R₁₇H₁₈R₁₉K₂₀, where the tail emerges from the compact globular histone domains. Early crosslinking experiments demonstrated a direct contact of histidine 18 with nucleosomal DNA. The finding that combining H4 tail peptides with DNA (but with neither of the components alone), elicits an ATPase response in ISWI that strongly supports the idea that some part of the H4 tail associates with DNA, generating an 'epitope' consisting of DNA and protein that triggers the ATPase activity of ISWI. The fact that mutagenesis of arginines 17 and 19 to alanines abolishes this effect may point to a critical role for these residues in DNA binding. However, the alternative possibility that these arginines are directly recognized by ISWI cannot be excluded (Clapier, 2002).

The crystal structure of the nucleosome yields little further insight. One H4 tail is not at all visible in the structure while the second one is ordered by extensive interactions with an acidic patch on the H2A-H2B dimer surface of an adjacent nucleosome. Since both tails are not equivalent in the crystal and the localization of the visible tail involves interactions that may be dictated by crystal packing forces (mutations in this region prevent crystallization), it is possible that the interaction with a neighboring particle leads to an artificial 'lifting' of the H4 tail base off the DNA (Clapier, 2002).

It has been suggested that interaction of the H4 tail with the minor groove of DNA may lead to the induction or stabilization of an alpha-helical conformation of the part in question. On the basis of elegant in vivo experiments using the yeast model, Ling (1996) proposed that residues 16-19 of the H4 tail might adopt an alpha-helical structure upon charge neutralization by an acidic protein or DNA. Later, Baneres (1997) concluded on the basis of CD spectroscopy and secondary structure prediction that the H4 tail sequences between 16 and 24 are in an alpha-helical conformation upon interaction with DNA. Residues 16-20, which contain the ISWI recognition epitope, are important for repression of the yeast mating type loci. In a search for extragenic suppressors of mutations in these residues SIR3 has been identifed. Direct interaction of SIR3 with this site within the H4 tail induces the heterochromatic state that characterizes the silent mating type cassettes. However, SIR3 (and its companion SIR4) are apparently able to interact with the H4 tail in the absence of DNA, although the formation of an alpha-helix in the critical region has been assumed in order to explain the results of the mutagenesis study. In contrast, the current study has been unable to identify a stable interaction between the GST-H4 tail fusion protein and ISWI in the absence of DNA (Clapier, 2002).

Several mechanisms, not mutually exclusive, may contribute to repressive function of the DNA-bound basic patch in the H4 N-terminus. Interaction of SIR3/SIR4 proteins may lead to an inaccessible higher order folding of chromatin. At the same time, binding of these proteins may stabilize the contact of the basic patch with DNA, which may restrict the flexibility of the nucleosomal fiber. It is also conceivable that the interaction of ISWI-containing nucleosome remodeling factors with the tail may be modulated by SIR protein binding (Clapier, 2002).

Most interestingly, the yeast homolog of the ATPase ISWI has recently been shown to be involved in the repression of many genes, including some that govern the meiotic programme, apparently through positioning nucleosomes in ways that are incompatible with transcription. The histone tail requirements for nucleosome remodeling by the ISW1 and ISW2 complexes and their resident ATPases have not yet been determined. If the mechanism of action of the ISWI homologs have been as conserved as the H4 tails, the deletion study would predict that the regulatory events that have been ascribed to yeast ISWI function would be compromised in strains that harbor a deletion/mutation of the critical H4 tail residues (Clapier, 2002).

In yeast and higher eukaryotes, active loci are marked by particular histone acetylation patterns whereas repressed domains are generally characterized by hypoacetylation. The reconstitution of a minimal epitope able to stimulate ISWI ATPase activity allowed addressing the question of whether acetylation of specific lysine residues within the H4 N-terminus affect ISWI function. Acetylation of those lysines that reside closest to the ISWI recognition epitope, lysines 16 and 12, interfere significantly with ISWI function. Interestingly, systematic mutational analysis of single amino acids in the H4 N-terminus in yeast has suggested that acetylation of lysine 16, but not of any of the other lysines in the H4 tail, interferes with silencing of mating type loci. Acetylation of lysine 16 is also a hallmark of the hyperactive, partially decondensed male X chromosome in Drosophila. The recent observation that the higher order chromosome structure of the male X chromosome is specifically disrupted in an ISWI homozygous mutant larvae, suggested that ISWI-containing nucleosome remodeling complexes counteract the effect of histone acetylation on higher order folding of chromatin, presumably through their nucleosome assembly/spacing functions. Such a scenario is reminiscent of the repressive functions of ISWI-containing complexes in yeast (Clapier, 2002).

Why is the activity of ISWI so sensitive to deletion and modification of the recognition epitope? Early crosslinking studies in nuclei mapped the contact of histidine 18 to a region of DNA 1.5 helical turns off the dyad axis of the nucleosome. This site is likely to be in reach for ISWI bound to DNA at the exit point from the nucleosome, suggesting that a direct interaction of ISWI may be possible. It is difficult to formulate hypotheses as to how this contact stimulates the ATPase activity of ISWI, since it is not known at which step during the nucleosome remodeling cycle ATP is hydrolyzed. ATP-dependent changes in ISWI conformation may be modulated by contact with the DNA-bound H4 motif. Conversely, disruption of the histone-DNA interactions at that site due to the remodeling process may feed back on the activity of the remodeling enzyme itself (Clapier, 2002).

Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin; H3 subdomains present within CEN chromatin are enriched for H3 Lys4-diMe, a modification associated with open but not active euchromatin

Post-translational histone modifications regulate epigenetic switching between different chromatin states. Distinct histone modifications, such as acetylation, methylation and phosphorylation, define different functional chromatin domains, and often do so in a combinatorial fashion. The centromere is a unique chromosomal locus that mediates multiple segregation functions, including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint. Centromeric (CEN) chromatin is embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with blocks of CENP-A nucleosomes, the histone H3 variant, also termed Centromere identifier (CID) that provides a structural and functional foundation for the kinetochore. This study demonstrates that the spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin. It is speculated that this distinct modification pattern contributes to the unique domain organization and three-dimensional structure of centromeric regions, and/or to the epigenetic information that determines centromere identity (Sullivan, 2004).

Post-translational modifications of histones are known to be biologically important in defining chromatin states, such as silent or active gene expression. Centromeric chromatin in flies and humans is defined as the full extent of staining for the centromere-specific histones CENP-A and CID, which contain interspersed subdomains of the CENP-A/CID and H3 nucleosomes. Immunofluorescence analysis of two-dimensional extended chromatin fibers and three-dimensional mitotic chromosomes demonstrated that H3 subdomains present within CEN chromatin are enriched for H3 Lys4-diMe, a modification associated with open but not active euchromatin. H3 subdomains within CEN chromatin do not contain the H3 Lys9 di- or trimethylation associated with heterochromatin, and lack acetylations at H3 Lys9 and H4 Lys5, Lys8, Lys12 and Lys16 that are generally found in euchromatin. Finally, the H3 Lys4-trimethylation associated with actively transcribed regions is also not present in CEN chromatin. It is concluded that the interspersed H3 present in fly and human CEN chromatin contains individual H3 and H4 modifications previously associated with both euchromatin and heterochromatin, but in a combined pattern that is distinct from each chromatin state individually. These results are unexpected; the fact that eukaryotic centromeres are embedded in heterochromatin has suggested that CEN chromatin should contain heterochromatic epigenetic imprints. This distinct pattern of histone modifications, which has been termed 'centrochromatin,' may contribute to the unique structure and function of the centromere, in combination with the presence of CENP-A/CID (Sullivan, 2004).

The regions that flank CEN chromatin in fly and human samples contained H3 Lys9-diMe and triMe, and hypoacetylation of H3 and H4, consistent with previous studies of pericentric heterochromatin. In fission yeast, tRNA genes seem to be associated with boundaries between CEN and flanking chromatin. It is unclear at this time whether the separation of CENP-A/CID and flanking heterochromatin domains in humans and Drosophila reflects the presence of a sequence-specific boundary, or a sequence-independent balance between the two epigenetic states. The pericentromeric regions of human metaphase chromosomes contained H3 Lys9-triMe, although this modification is under-represented, but is not completely deficient, in Drosophila pericentromeric heterochromatin. Fly pericentromeric regions showed a much more substantial enrichment for H3 Lys9-diMe in metaphase chromosome and chromatin fiber analysis. These results are consistent with previous reports showing that H3 Lys9-diMe is concentrated at heterochromatic chromocenters in Drosophila salivary glands. Pericentric regions in flies contain essential genes, whereas few genes have been reported in the pericentric regions of human chromosomes. Perhaps higher-order heterochromatin is regulated differently between humans and flies by other histone-modifying enzymes and heterochromatin proteins, to allow the expression of heterochromatic genes. Further investigations of the distributions of different histone modifications in pericentric heterochromatin are necessary to validate these observations, and to determine whether they have functional consequences (Sullivan, 2004).

Two recent studies have discovered correlations between distinct heterochromatic domains and different degrees of H3 Lys9 methylation in mouse embryonic stem cells and embryonic fibroblasts. It was demonstrated by indirect immunofluorescence that H3 Lys9-triMe is enriched in pericentric regions of mouse chromosomes and at DAPI-bright regions in interphase nuclei. These results agree with the current findings that H3 Lys9 methylation is present in pericentric regions of human and fly chromosomes. Chromatin immunoprecipitation (ChIP) was used analysis to identify patterns of H3 methylation within mouse chromosomes. In mice, the centromere and pericentromeric regions contain distinct, expansive arrays of satellite DNA. Major satellite comprises the largest region and is immediately adjacent to the functional kinetochore, and minor satellite is the region where mouse kinetochore proteins are located. It was recently reported that major and minor satellite DNAs are enriched primarily for H3 Lys9-triMe, and that both satellites are associated with H3 Lys9-diMe to a lesser extent (Sullivan, 2004).

The presence of H3 Lys9 methylation in mouse minor satellite suggests that CEN chromatin may be modified differently in mice, in comparison with the results reported for for humans and Drosophila in this study. However, human centromeres contain expansive, megabase-sized arrays of alpha-satellite DNA, and CENP-A localizes to only a portion of these arrays. The demonstration that CEN chromatin in humans and flies lacks H3 Lys9 methylation is consistent with a similar model for centromere organization in mice, in which minor satellite DNA contributes partly to CEN chromatin and partly to heterochromatin formation. Additional studies on mouse centromeres are necessary to specifically map H3 Lys9 methylation with respect to CENP-A and CEN chromatin (Sullivan, 2004).

These studies have focused on centromeric chromatin structure in human cells and Drosophila cultured cells and larval brains. Is CEN chromatin in other organisms marked by the same histone modifications? Centromeres in S. pombe consist of a basic unit of central core chromatin that contains CENP-A (Cnp1), flanked on both sides by heterochromatin that is marked by H3 Lys9 methylation. The initial finding that subdomains of CENP-A/CID and H3 are interspersed in fly and human centromeres produced the hypothesis that centromeres in larger eukaryotes might represent amplification of the basic CEN domain unit (heterochromatin - CENP-A - heterochromatin) found in S. pombe. However, in the present study, no H3 Lys9 methylation was observed in the regions between CENP-A/CID subdomains with the CEN regions. Thus, the current results argue that fly and human centromeres are not composed of multimers of units equivalent to S. pombe centromeres. However, the overall organization of the centromere region is conserved, such that the entire CENP-A/CID chromatin domain is flanked by heterochromatin that contains H3 Lys9 methylation (Sullivan, 2004).

Alternatively, it is possible that CEN chromatin does differ among organisms. Recently, ChIP analysis of rice centromeric regions suggested that H3 Lys9 di-Me is present within the CEN chromatin (defined by the presence of the CENP-A homolog CenH3). This result may reflect differences in CEN chromatin composition and organization between plants and flies, humans and S. pombe. However, a more extensive analysis of the spectrum of modifications, including cytological studies of the distributions of modifications in extended fibers and mitotic chromosomes, needs to be carried out in different plant species to test this hypothesis (Sullivan, 2004).

What are the functional roles of histone modifications in CEN and flanking chromatin? (1) Distinct chromatin states in the CEN region may contribute to the diverse properties of centromeric domains, such as differential replication timing of the CEN and flanking heterochromatin. Heterochromatic modifications may also maintain centromere size by creating a barrier against expansion of CEN chromatin. In Drosophila, CEN chromatin readily spreads into neighboring sequences when flanking heterochromatin is removed, allowing neocentromere activation. (2) The stacking and self-association of CENP-A nucleosomes, distinctly modified interspersed H3 nucleosomes and flanking heterochromatin may be responsible for the three-dimensional structure of CEN chromatin in mitosis. This organization could facilitate kinetochore assembly by orienting CENP-A/CID chromatin toward the outside of the chromosome, where it can interact with kinetochore proteins. CEN-specific combinations of histone modifications and the three-dimensional organization could also be important for recruitment of cohesion complexes to heterochromatin near sister kinetochores, while ensuring spatial separation of cohesion and kinetochore domains (Sullivan, 2004).

(3) Distinctly modified, interspersed H3 nucleosomes could participate in epigenetic propagation of centromere identity. As observed for other histone 'variants', CENP-A/CID assembly can be replication-independent, unlike that of canonical H3 nucleosomes. Specifically modified interspersed H3 subdomains could create a 'permissive' chromatin structure necessary for assembly of new CENP-A16 (Sullivan, 2004).

A new model for deposition of CENP-A specifically in centromeric chromatin is suggested by these observations. Perhaps the modification pattern of interspersed H3 nucleosomes and histone modification proteins (such as acetyltransferases, methyltransferases and kinases) helps propagate centromere identity, in lieu of (or in addition to) CENP-A-associated proteins. Future studies are necessary to address mechanisms responsible for formation, maintenance and separation of these distinct chromatin states, and well as their roles in centromere structure and function. It is also important to determine whether other functional domains embedded within heterochromatin, such as the nucleolus organizer - ribosomal DNA, show distinct patterns of histone modifications (Sullivan, 2004).

Modulation of heat shock gene expression by methylation and acetylation of H3 by the TAC1 chromatin-modifying complex

Rapid induction of the Drosophila melanogaster heat shock gene hsp70 is achieved through the binding of heat shock factor (HSF) to heat shock elements (HSEs) located upstream of the transcription start site. The subsequent recruitment of several other factors, including Spt5, Spt6 and FACT, is believed to facilitate Pol II elongation through nucleosomes downstream of the start site. This study reports a novel mechanism of heat shock gene regulation that involves modifications of nucleosomes by the TAC1 histone modification complex. After heat stress, TAC1 is recruited to several heat shock gene loci, where its components are required for high levels of gene expression. Recruitment of TAC1 to the 5'-coding region of hsp70 seems to involve the elongating Pol II complex. TAC1 has both histone H3 Lys 4-specific (H3-K4) methyltransferase (HMTase) activity and histone acetyltransferase activity through Trithorax (Trx) and CREB-binding protein (CBP), respectively. Consistently, TAC1 is required for methylation and acetylation of nucleosomal histones in the 5'-coding region of hsp70 after induction, suggesting an unexpected role for TAC1 during transcriptional elongation (Smith, 2004).

Characterization of the grappa gene, the Drosophila histone H3 lysine 79 methyltransferase

grappa (gpp) is the Drosophila ortholog of the Saccharomyces cerevisiae gene Dot1, a histone methyltransferase that modifies the lysine (K)79 residue of histone H3. gpp is an essential gene identified in a genetic screen for dominant suppressors of pairing-dependent silencing, a Polycomb-group (Pc-G)-mediated silencing mechanism necessary for the maintenance phase of Bithorax complex (BX-C) expression. Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but also display phenotypes characteristic of trithorax-group mutants. gpp dominantly enhances the phenotypic effects of mutations in Sex combs extra, Polycomblike, Sex combs on the midleg, and Polycomb. Mutations in gpp also disrupt telomeric silencing but do not affect centric heterochromatin. These apparent contradictory phenotypes may result from loss of gpp activity in mutants at sites of both active and inactive chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns, the appearance of methylated K79 during embryogenesis coincides with the maintenance phase of BX-C expression, suggesting that there is a unique role for this chromatin modification in development (Shanower, 2005).

S. cerevisiae DOT1 does not have a canonical SET domain. Instead, the DOT1 protein resembles a family of S-adenosyl methione methyltransferases that modify arginine residues. DOT1 methylates histone H3 at lysine 79 only when it is assembled into nucleosomes and methylation strongly depends upon prior Rad6 dependent ubiquitination of histone H2B at K123 (Briggs, 2002). Second, in yeast, deletion or overexpression of Dot1 disrupts telomeric position effect and also silencing of the mating-type loci (Singer, 1998). In contrast, silencing in the yeast ribosomal gene cluster is disrupted only when DOT1 is overexpressed (Singer, 1998). Third, both telomeric and mating-type silencing are disrupted by mutations in the lysine 79 residue of histone H3. Fourth, methylation of K79 appears to influence the recruitment of the SIR silencing proteins to the telomeres (Van Leeuwin, 2002; Ng, 2003a). The SIR silencing proteins appear to preferentially associate with chromatin that is deficient in K79 methylation, while the proteins are generally not associated with chromatin in which there is an enrichment for K79 methylated H3 (van Leeuwin, 200; Ng, 2003a). Fifth, there is evidence that K79 methylation is coordinated with polymerase transcription via the COMPASS complex (Krogan, 2003a). Consistent with the idea that K79 methylation might be coordinated with transcription, H3meK79 is enriched in transcribed sequences in yeast and mammals (Im, 2003; Ng, 2003c). Interestingly, the distribution of H3meK79 in the ß-globin locus differs from H3meK4 in that it is not found at the locus control region (Im, 2003). These findings have led to a model in which H3meK79 serves as a marker for transcribed sequences where it functions to block the association of chromatin proteins that mediate transcriptional silencing (Shanower, 2005).

While Dot1 homologs have been identified in higher eukaryotes, little is known about their biological functions. The Drosophila Dot1 ortholog gpp transcription unit is greater than 40 kb in length and it encodes a complex array of alternatively spliced transcripts that range in size from 6.5 to greater than 9 kb and are expressed at different developmental stages. Consistent with assignment of the gpp gene, P-element and X-ray mutations disrupt this large transcription unit and in at least one case lead to the production of truncated mRNAs. The gpp transcripts are predicted to encode 170- to 232-kD polypeptides that share a common N-terminal domain that corresponds to about two-thirds of the protein but have different C-terminal domains. The common N-terminal domain contains the Dot1 homology region including the MT methyltransferase fold required for methylation of histone H3 (Feng, 2002). Mutation of conserved glycine residues in the active site of both yeast and human DOT1 protein inactivates the enzyme (Feng, 2002; van Leeuen, 2002). GPP also contains domains that are not present in DOT1 including a coiled-coil motif also found in the human, C. elegans, D. pseudoobscura, and A. gambia DOT1-like proteins. In yeast, K79 is mono-, di-, and trimethylated and Dot1 is responsible for all three modifications (Feng, 2002; van Leeuen, 2002). The different methylated states of H3 at K79 suggest that multiple regulatory activities are conferred on these modified nucleosomes. However, in fly tissue culture cells, the mono- and di- but not the trimethylated form is observed (McKittrick, 2004). Since database searches indicate that gpp is the only fly Dot1 homolog, it should also be the sole fly protein in this class that methylates histone H3 on K79. Consistent with this suggestion, discs and other tissues isolated from gpp mutant larvae have little if any H3 mono- or dimethyl K79 (Shanower, 2005).

Like its yeast counterpart, gpp is required for the silencing of reporter transgenes inserted into telomeric heterochromatin. However suppression of silencing associated with pericentric heterochromatin is unaffected by mutations in gpp. While these observations point to a role of gpp in silencing specific for telomeric heterochromatin, antibody staining experiments indicate that there is a paucity of H3dmeK79 at telomeres in polytene chromosomes compared to many other chromosomal DNA segments. In this respect it is interesting that both telomeric and mating-type chromatin in yeast are hypomethylated on K79 compared to "bulk" chromatin even though DOT1 is required for SIR silencing in each case (Ng, 2003a). It has been suggested that the meK79 modification in euchromatic nucleosomes blocks SIR protein association and that silencing is lost in the absence of DOT1 because the SIR proteins spread into euchromatin (vanLeeuwen, 2002). In contrast, in flies, since many euchromatic domains in wild-type polytene chromosomes have only little H3meK79, it is difficult to see how telomeric silencing proteins would be restricted to telomeres by this modification even when gpp is fully active (Shanower, 2005).

gpp also has functions in flies besides telomeric silencing. Unlike Dot1, gpp is essential for viability. Although the underlying cause of lethality remains to be established, gpp mutant larvae grow more slowly than wild type and this potentially implicates gpp in pathways that control growth rates and size in flies. In addition, gpp mutants display defects that are characteristic of both Pc-G and trx-G genes. The first gpp alleles were recovered as dominant suppressors of mini-white silencing by two Bx-C PREs. Consistent with a role in Pc-G silencing, gpp mutants enhance the segmentation defects of several Pc-G genes. In this context, it is interesting to note that several Pc-G genes have recently been shown to play a role not only in the repression of genes in the homeotic complexes but also in telomeric silencing (Boivin, 2003). Thus, it is possible that gpp activity in telomeric silencing may be linked in some manner to its role in Pc-G silencing (Shanower, 2005).

gpp mutants also exhibit transformations in segment identity and genetic interactions with Abd-B that are characteristic of trx-G mutations. This would point to a role in promoting rather than repressing gene expression. Some function in transcription would be consistent with studies in other systems as well as with the enrichment of meK79 seen in many polytene interbands and puffs. However, this correlation is not complete. Thus, there are many puffs and interbands that have only little H3dmeK79. Conversely, H3dmeK79 is sometimes enriched in bands. These findings would argue that in Drosophila, meK79 is not a ubiquitous marker for transcriptionally active chromatin, but rather may have functions that are specific to particular chromatin domains. In this case, the disruptions in homeotic gene expression seen in gpp mutants could reflect a special requirement for H3meK79 in the transcription of these particular genes. Domain-specific requirements for gpp activity in transcription could also potentially account for the effects of gpp mutations on Pc-G and telomeric silencing. In this model, Pc-G and telomeric silencing would be disrupted in gpp mutants because the expression of one or more Pc-G (and/or telomeric heterochromatin) genes is downregulated when gpp activity is compromised (Shanower, 2005).

The developmental profile of H3dmeK79 also suggests that this modification cannot be a ubiquitous marker for either transcriptionally active or silenced chromatin. High levels of Pol II transcription in somatic nuclei begin in the precellular blastoderm stage around nuclear cycle 11/12. Concomitant with the activation of transcription, H3meK4 can be first be detected at this stage, and the level of meK4 then increases through cellularization (Schaner, 2003). By contrast, little if any H3 mono- or dimethyl K79 is in either the transcriptionally active somatic nuclei or the transcriptionally quiescent pole cell nuclei. H3meK79 can first be readily detected only later in development in germband extended embryos. However, at this stage accumulation is restricted primarily to a subset of cells in the embryo, most of which seem to be in the process of cell division. High levels of H3meK79 are not observed until stages 13–15, long after the initial upregulation of transcription in the early zygote. This result also suggests that the homeotic transformations seen in gpp mutants are unlikely to be due to defects in the initial establishment of parasegment-specific patterns of homeotic gene expression by the gap and pair-rule genes. Rather, these transformations probably reflect a requirement for gpp activity later in development during the maintenance phase of homeotic gene regulation—a phase that is dependent upon Pc-G and trx-G genes. In this respect it is curious that homeotic transformations are not observed in gpp embryos when they hatch as first instar larvae. Maternally derived gpp activity in homozygous mutant embryos may be sufficient to maintain specific parasegmental patterns of homeotic gene expression through the end of embryogenesis. Alternatively, there may not be absolute requirement for H3meK79 in maintaining appropriate parasegmental patterns of homeotic expression during embryogenesis (Shanower, 2005).

The developmental profile of H3meK79 indicates that this modification is present at low levels in specific developmental stages and tissues (CNS) undergoing active cell division. In contrast, the highest levels of H3meK79 are observed in epidermal cells that have exited the cell cycle and are undergoing differentiation. Thus, it seems possible that this modification may be activated when specific chromatin configurations, active or inactive, need to be maintained for extended periods of time in the absence of de novo DNA synthesis/chromatin assembly. In this respect it is interesting that McKittrick, (2004) has reported that the highest levels of meK79 are found in a histone H3 variant, H3.3, which is assembled into chromatin by a replication-independent mechanism. Further studies of gpp in Drosophila will be required to understand the mechanisms governing the temporal and tissue-specific regulation of the K79 modification and how this relates to the functions of this particular histone modification during development. Understanding this aspect of the histone code in a multicellular organism such as Drosophila will lead to a better understanding of chromatin regulatory mechanisms during development (Shanower, 2005).

Drosophila Ada2b is required for viability and normal histone H3 acetylation

Regulation of chromatin through histone acetylation is an important step in gene expression. The Gcn5 histone acetyltransferase, Pcaf, is part of protein complexes, e.g., the SAGA complex, that interacts with transcriptional activators, targeting the enzyme to specific promoters and assisting in recruitment of the basal RNA polymerase transcription machinery. The Ada2 protein directly binds to Gcn5 and stimulates its catalytic activity. Drosophila contains two Ada2 proteins, Drosophila Ada2a (dAda2a) and dAda2b. Flies were generated that lack dAda2b, which is part of a Drosophila SAGA-like complex. dAda2b is required for viability in Drosophila, and its deletion causes a reduction in histone H3 acetylation. A global hypoacetylation of chromatin was detected on polytene chromosomes in dAda2b mutants. This indicates that the dGcn5-dAda2b complex could have functions in addition to assisting in transcriptional activation through gene-specific acetylation. Although the Drosophila p53 protein has been shown to interact with the SAGA-like complex in vitro, p53 induction of reaper gene expression occurs normally in dAda2b mutants. Moreover, dAda2b mutant animals show excessive p53-dependent apoptosis in response to gamma radiation. Based on this result, it is speculated that dAda2b may be necessary for efficient DNA repair or generation of a DNA damage signal. This could be an evolutionarily conserved function, since a yeast ada2 mutant is also sensitive to a genotoxic agent (Qi, 2004).

The Drosophila Ada2b protein is present in a SAGA-like HAT complex. In accordance, dAda2b and dGcn5 directly interact and are expressed in similar patterns during embryogenesis. Ada2b is required for viability in Drosophila. Although dAda2b protein levels become greatly reduced in mutant embryos at stage 15, the animals survive until the pupal stage. Concomitant with reduced dAda2b protein amounts, a significant reduction is observed in acetylation of lysines 14 and 9 in histone H3. Lysine 14 acetylation is most severely affected, since a reduced staining intensity is observed even in dAda2b heterozygous embryos compared to the wild type. The preference for H3 K14, and the selective reduction in histone H3 over histone H4 acetylation, fits the substrate specificity of Gcn5. Furthermore, Gcn5's presence in the SAGA complex is dependent on Ada2 in yeast. It is therefore assumed that the loss of histone H3 acetylation in dAda2b mutants results from diminished dGcn5 activity, either as a consequence of reduced catalytic activity of the enzyme or due to a failure to target dGcn5 to chromatin (Qi, 2004).

The reduced histone H3 acetylation levels appear to persist during development; third-instar larval salivary gland chromosomes retained a similar reduction in H3 acetylation. Interestingly, chromatin acetylation is globally affected in dAda2b mutants, rather than affecting a subset of genes. Therefore, dAda2b is not likely to function solely as an adaptor that brings dGcn5 to only some promoter regions through associations with transcriptional activators. Instead, it is probable that dAda2b has a function similar to that of yeast Ada2, which is to retain Gcn5 within the SAGA complex and to stimulate its catalytic activity. This result also suggests that the SAGA complex, or at least a dAda2b-dGcn5 complex, has functions in addition to transcription-coupled gene-specific acetylation. Some heterochromatic regions with low gene density that stain with DAPI contain acetylated histone H3. These regions exhibit a decrease in H3 acetylation similar to that of other parts of the chromosomes (Qi, 2004).

Drosophila Gcn5 is present in complexes in addition to the dAda2b-containing SAGA-like complex. The fraction of dGcn5 that associates with dAda2b makes a significant contribution to the overall levels of histone H3 acetylation, and association with dAda2a cannot substitute for dGcn5's function within the SAGA complex. Interestingly, normal histone H3 acetylation appears to be dispensable for development from at least mid-embryogenesis until pupation. Furthermore, it is not known if the lethality observed at pupation is caused by reduced histone acetylation or by another function of dAda2b. In addition to its role in pupal development, dAda2b is also required for oogenesis. Females with dAda2b homozygous germ cells lack developed ovaries (Qi, 2004).

All Ada2 proteins share a ZZ zinc finger and a SANT domain in their N termini. The minimal Gcn5 interaction domain includes both the ZZ domain and the SANT domain, and the SANT domain additionally stimulates Gcn5's catalytic activity (Boyer, 2002; Sterner, 2002). The dAda2b gene is alternatively spliced, generating proteins with different C termini. Both protein isoforms contain the ZZ and SANT domains, but the longer isoform lacks another conserved element, Ada box 3. However, no evidence was found for differential usage of the two proteins during development. The Ada2-Gcn5 interaction, although conserved in evolution, is species specific. For example, human Ada2a cannot interact with yeast Gcn5, and both Drosophila Ada2a and Ada2b fail to interact with yeast Gcn5. As might be expected from these results, dAda2b fails to complement a yeast Ada2 mutant (Qi, 2004).

It is believed that the SAGA complex is recruited to target genes through interactions with sequence-specific transcription factors. At the promoter, the Gcn5 subunit acetylates histone H3 and perhaps other proteins, whereas the Spt 3 subunit facilitates preinitiation complex assembly by interacting with TATA binding protein. Several yeast activator proteins, including Gcn4, Pho4, and Gal4, require the SAGA complex for activity and recruit it to target genes in vivo. The activator proteins VP16 and Drosophila p53 can precipitate the SAGA-like complex from a Drosophila S2 cell nuclear extract (Kusch, 2003). Whether this interaction is of significance for the in vivo function of the Drosophila p53 protein was tested. p53-induced apoptosis and reaper gene expression in response to DNA damage are not impaired in dAda2b mutants. This result indicates that the Ada2/Gcn5 subcomplex of SAGA is not necessary for p53 function, but it does not exclude the possibility that p53 activity requires other SAGA components. In fact, many yeast genes show a differential requirement for the Ada2/Gcn5 and Spt3 subcomplexes of SAGA (Qi, 2004).

Unexpectedly, increased apoptosis in response to gamma radiation in dAda2b mutants was found. Irradiation causes DNA damage, which leads to activation of ATM/ATR kinases that phosphorylate a variety of substrates, including the Chk2 kinase, ultimately resulting in p53 activation. In Drosophila, p53 activation brings about apoptosis but does not lead to a G₁ cell cycle arrest, which is another possible outcome in mammalian cells. It is possible that the reason for increased apoptosis in dAda2b mutants is that dAda2b normally has an inhibitory role in the generation of a DNA damage signal or in controlling p53 function. Another possibility is that DNA repair is not as efficient in dAda2b mutants as in the wild type, leading more cells to enter the apoptosis program. The role of dAda2b in DNA repair could be indirect. For example, dAda2b could be necessary for proper expression of a DNA repair factor. Alternatively, dAda2b might have a more direct function in DNA repair, presumably through chromatin modification. In dAda2b mutants, excessive apoptosis occurs after irradiation but not before irradiation or when apoptosis is induced by an alternative means. Furthermore, the extra apoptosis is p53 dependent. For this reason, it is unlikely that increased apoptosis in dAda2b mutants is the consequence of a higher sensitivity to stress in general. Instead, it is believed that dAda2b has a role in the p53-dependent pathway that is activated by DNA damage (Qi, 2004).

All eukaryotes respond to DNA lesions with activation of ATM/ATR kinases, although the downstream component p53 is absent from unicellular organisms such as the yeast S. cerevisiae. If the involvement of dAda2b in the DNA damage response is upstream of p53 activation, one would predict this function to be conserved in evolution. In fact, yeast strains lacking Ada2p, or Gcn5p or Ada3p, are sensitive to the genotoxic agent MMS. It is possible, therefore, that histone acetylation facilitates DNA repair or generates a DNA damage signal. Intriguingly, an increase in radiation-induced apoptosis of a magnitude similar to that observed in dAda2b mutants was observed in larvae in which the C terminus of a histone variant, H2Av, is missing. The H2Av C terminus contains a conserved serine that is phosphorylated in response to DNA double-strand breaks. Apparently, the absence of this phosphorylation event reduces DNA repair efficiency, which increases the amount of apoptosis twofold. Perhaps histone acetylation likewise contributes to the generation of a DNA damage signal, and maybe this is why chromatin is globally acetylated by Ada2/Gcn5. Future studies will be aimed at investigating this possibility (Qi, 2004).

Sex-specific role of Drosophila melanogaster HP1 in regulating chromatin structure, histone modification and gene transcription

Drosophila melanogaster heterochromatin protein 1 (HP1a or HP1)1 is believed to be involved in active transcription, transcriptional gene silencing and the formation of heterochromatin. But little is known about the function of HP1 during development. Using a Gal4-induced RNA interference system, it has been shown that conditional depletion of HP1 in transgenic flies results in preferential lethality in male flies. Cytological analysis of mitotic chromosomes showed that HP1 depletion caused sex-biased chromosomal defects, including telomere fusions. The global levels of specific histone modifications, particularly the hallmarks of active chromatin, are preferentially increased in males as well. Expression analysis shows that approximately twice as many genes were specifically regulated by HP1 in males than in females. Furthermore, HP1-regulated genes showed greater enrichment for HP1 binding in males. Taken together, these results indicate that HP1 modulates chromosomal integrity, histone modifications and transcription in a sex-specific manner (Liu, 2005).

Mutations in D. melanogaster Su(var)205 (also called HP1) cause lethality at larval stages, precluding a systematic functional analysis of Su(var)205 during development. To circumvent this limitation, the role of HP1 was studied using a Gal4-inducible RNA interference (RNAi) system, which allows for depletion of HP1 in a tissue- and developmental stage-specific manner. D. melanogaster y w^67c23 embryos were transformed with a construct expressing double-stranded RNA from a Su(var)205 cDNA. To deplete HP1, four independent transgenic lines (HP1-2, HP1-11, HP1-21 and HP1-31) were crossed with an act-Gal4 line (y w; +/+; act-Gal4/TM6B) expressing Gal4 ubiquitously during development. Resulting larval progeny from lines HP1-11 and HP1-21 showed a reduction in HP1 levels of ~90%, line HP1-31 showed a 60% reduction and line HP1-2 showed no reduction. Those progeny with a 60%-90% reduction in HP1 generally survived to the third-instar larval stage, but progeny with a 90% reduction rarely survived to the adult stage. Lethality mainly occurred at the pupal stage and seemed to be due to a failure to eclose. Adult progeny of the HP1-31/act-Gal4 line were also viable, but the female:male ratio was highly skewed (2.4:1 versus 0.9:1 for this genotype at the larval stage). An alteration in the sex ratio was also evident in adult flies from the HP1-11/act-Gal4 line: all 21 survivors were female. There were no adult HP1-21/act-Gal4 survivors when progeny were grown at 25°C, but 30 'escapers' were obtained at 18°C, all of which were female. Collectively, these results suggested that there was an association between sex-biased lethality and HP1 dosage (Liu, 2005).

Because the mutated lethal allele Su(var)205⁰² does not result in telomeric fusions, however, lethality cannot be solely due to this cause. To explore whether additional mechanisms are involved in the sex-biased lethality, the impact of HP1 depletion on core histone modifications was measured, since increases in histone acetylation can cause apoptosis. Using cell extracts from larval imaginal discs of HP1 RNAi mutants and control larvae, the global levels were compared of several core histone modifications in males and females. The levels of acetylation at Lys8 of histone H4 (H4K8ac), methylation at Lys4 of histone H3 (H3K4me) and methylation at Lys79 of histone H3 (H3K79me; all hallmarks of active chromatin) were all increased in males after HP1 depletion. But levels of methylation at Lys20 of histone H4 (H4K20me) and methylation at Lys9 of histone H3 (H3K9me; both hallmarks of heterochromatin) showed a global decrease when cells were lysed in 300 mM salt buffer. No change was observed in H4K20me or H3K9me when cells were lysed in SDS buffer, suggesting that the changes in histone modifications associated with the active state may have a role in the observed lethality. These effects were not caused by misregulation of genes encoding known histone-modifying enzymes, including histone methylases, acetylases or deacetylases, as these were unaffected by HP1 depletion (Liu, 2005).

It was asked whether any change in histone H3K9me occurs on chromatin, since this histone modification is interdependent with the dynamics of HP1. In polytene chromosomes from HP1-depleted mutants, H3K9me remains at the pericentric heterochromatin region in both sexes. But the intensity of the pericentric H3K9me signal in males is lower than that in females, a modification linked to X-chromosome dosage compensation in males. This staining showed no obvious changes that were dependent on HP1. To test the possibility that the HP1-induced preferential lethality in males is linked to the disruption of specific functional genes in males, total RNA isolated from two independent populations of male and female third-instar larvae of line HP1-21/act-Gal4 was compared using microarray analysis. More than 200 predicted transcripts or genes were specifically affected in males, but only 119 were specifically affected in females; 127 genes seemed to be affected in both males and females. Among the affected genes with known function, those essential for DNA replication, such as mus209 and Mcm6, were downregulated in both sexes; wrinkled (W) and Rep4, both regulators of apoptosis, were upregulated. Notably, a number of genes encoding cell cycle regulators, such as fizzy (fzy), pimples (pim), cyclin-dependent kinase subunit (Cks30A) and the DNA replication initiation inhibitor geminin, were all specifically affected only in males, suggesting that these genes have a role in the observed differential lethality. Transcription of genes known to regulate the sex ratio, such as msl-1 (also called MSL), was not affected (Liu, 2005).

The differential change in H3K9me on chromatin may be due to an alteration in Su(var)3-9 localization, since HP1 is essential for maintaining its dynamics. Changes in global histone acetylation and phosphorylation could result from an HP1-induced global change in chromatin structure or from secondary effects; the absence of a Su(var)3-9 homolog in mammals also caused changes in different histone modifications, in addition to H3K9me. Notably, all these changes occur in a sex-biased manner. This is attributed to the sex-specific distribution of HP1 on chromatin, shown by ChIP analysis. This hypothesis suggests that the male genome, relatively enriched in HP1, is subject to more changes in histone modifications, more chromosome segregation defects and more changes in transcription in the absence of HP1, which seems to be the case. The heterochromatic Y chromosome in males may be also involved in the sex-biased distribution of HP1 in the genome, for example, by altering the distribution of remaining HP1 and other heterochromatin proteins (Liu, 2005).

The Histone H3 acetylase dGcn5 is a key player in Drosophila melanogaster metamorphosis

Although it has been well established that histone acetyltransferases (HATs) are involved in the modulation of chromatin structure and gene transcription, there is only little information on their developmental role in higher organisms. Pcaf, alternatively called Gcn5, was the first transcription factor with HAT activity identified in eukaryotes. Null alleles of Drosophila Gcn5, a component of the SAGA complex, block the onset of both oogenesis and metamorphosis, while hypomorphic Gcn5 alleles impair the formation of adult appendages and cuticle. Strikingly, the dramatic loss of acetylation of the K9 and K14 lysine residues of histone H3 in Gcn5 mutants has no noticeable effect on larval tissues. In contrast, strong cell proliferation defects in imaginal tissues are observed. In vivo complementation experiments have revealed that Gcn5 integrates specific functions in addition to chromosome binding and acetylation. Defects displayed by Gcn5 mutant adults rescued by the Gcn5DeltaPcaf variant (deficient in the N-terminal Pcaf specific motif) suggest a role of Gcn5 in the ecdysone regulatory hierarchy during metamorphosis, possibly through interactions with nuclear receptors. Surprisingly, a Gcn5 variant protein with a deletion of the bromodomain, which has been shown to recognize acetylated histones, appears to be fully functional. These results establish Gcn5 as a major histone H3 acetylase in Drosophila which plays a key role in the control of specific morphogenetic cascades during developmental transitions (Carré, 2005).

To investigate the contribution of Gcn5 to histone acetylation, polytene chromosomes from larval salivary glands were immunostained using antibodies specific for the acetylation of various lysine residues of histones H3 and H4. In order to compare their acetylation levels, polytene chromosomes from wild-type and mutant larvae were squashed and stained together on the same slide. The expression of a histone 2B yellow fluorescent fusion protein (H2b-YFP) in wild-type larvae allowed the distinguishing of their chromosomes from Gcn5 mutant chromosomes. The acetylation of the H3 K14 and K9 residues was detected in numerous loci of wild-type polytene chromosomes. In striking contrast, the acetylation of H3 K14 was undetectable and the acetylation of H3 K9 was barely detectable in Gcn5 mutant chromosomes. Antibodies against the acetylated H4 K8 and H4 K16 residues revealed unchanged acetylation of these residues in Gcn5 mutants compared to that in wild-type animals. Whether the loss of H3 K9 and K14 acetylation in Gcn5 mutants affects other modifications of histone H3 residues was analyzed. Like H3 K9 and H3 K14 acetylation, the phosphorylation of H3 S10 and di- and tri-methylation of H3 K4 have been associated with the transcriptional activation of target loci. It was found that global levels of these modifications are not affected in Gcn5 mutant polytene chromosomes. In contrast, methylation of the H3 K9 residue acts as a marker for the recruitment of the heterochromatin protein HP1 and transcriptional silencing. The loss of H3 K9 acetylation in Gcn5 mutants could have favored ectopic H3 K9 methylation and the subsequent delocalization of HP1. However, it was found that both H3 K9 methylation levels and HP1 localization in the pericentromeric regions of polytene chromosomes were unchanged in Gcn5 mutants (Carré, 2005).

The contribution of Gcn5 to histone acetylation in imaginal tissues was examined by immunostaining of imaginal discs in which Gcn5 was silenced in the P compartment by UAS-IR[gcn5] under the control of en-GAL4. The acetylation of H3 K9 and H3 K14 residues was strongly reduced in the nuclei of the Gcn5-depleted imaginal cells, while the acetylation of H4 K8 and H4 K16 residues was not affected. As seen with polytene chromosomes of Gcn5 mutants, Gcn5 depletion did not affect the level of H3 K4 or H3 K9 methylation in the nuclei of imaginal discs. When expression of the UAS-IR[Gcn5] transgene was driven by the ubiquitous da-GAL4 driver, animals arrested their development at the onset of metamorphosis. Western blot analysis of such late-third-instar larvae showed a strong depletion of histone H3 acetylated at the K9 and K14 residues, while the level of histone H4-AcK8 remained unchanged. Hence, the effect of the Gcn5 loss of function on polytene chromosomes, together with the results of Gcn5 RNAi studies with imaginal discs and larval tissues, points to dGcn5 as the major histone H3 K9 and H3 K14 acetyltransferase in Drosophila (Carré, 2005).

To analyze the functional requirement of the evolutionarily conserved domains of the Gcn5 protein, lines transgenic for Gcn5 variant genes were established under the control of the UAS promoter. The variant genes were designed in order to express Gcn5 with a deletion of the Pcaf homology domain (DeltaPcaf), the catalytic domain for acetylation (DeltaHAT), the conserved domain shown in yeast to be involved in interaction with the Ada2 protein (DeltaAda), or the bromodomain (DeltaBromo) were examined. It was verified by Western blot analysis that the variant transgenes are expressed in the presence of da-GAL4 at levels comparable to that of the UAS-Gcn5 wild-type transgene and then their ability to rescue the Gcn5 function was tested. In contrast to the UAS-Gcn5 wild-type construct, the UAS-Gcn5DeltaHAT construct did not rescue the lethal phenotype of the Gcn5^E333st/sex204 heteroallelic combination. As expected from the disruption of the catalytic acetyltransferase domain, the acetylation of polytene chromosomes at the H3 K9 and K14 residues was not restored by the Gcn5DeltaHAT variant in the mutant animals compared to the rescue of acetylation by the wild-type Gcn5 protein. However, it should be noted that the Gcn5DeltaHAT variant still localized to the interbands of the polytene chromosomes in a manner similar to that of the wild-type dGcn5 protein expressed under the control of the da-GAL4 driver (Carré, 2005).

It was recently shown that the dAda2b component of the Drosophila SAGA-like complex directly interacts with Gcn5 and is required for the acetylation of histone H3. This result suggested that the interaction with dAda2b is essential either for the HAT activity of dGcn5 or for its targeting to chromatin (Qi, 2004). Surprising, the Gcn5DeltaAda protein appeared normally distributed on polytene chromosomes and restored histone H3 acetylation in Gcn5 mutants. However, Gcn5 mutants were not rescued by Gcn5DeltaAda and still arrested at puparium formation (Carré, 2005).

The Gcn5DeltaPcaf variant protein also appeared to be normally distributed at the interbands of polytene chromosomes and to restore the acetylation of histone H3. In addition, about half of the Gcn5 mutant animals expressing Gcn5DeltaPcaf formed their puparium and completed metamorphosis but died before eclosion as pharate adults, while the other half gave rise to adult flies. However, rescued adults displayed held-out, misshapen wings, legs with a severe femur kink, and rough eyes. They died a few hours after eclosion, indicating that the UAS-Gcn5DeltaPCAF construct only partially rescues Gcn5 mutants (Carré, 2005).

Strikingly, the Gcn5DeltaBromo variant protein not only restored histone H3 acetylation but also restored complete viability of the Gcn5 mutants. Adult flies were indistinguishable from Gcn5 mutant flies rescued by the full-length Gcn5 protein. The only observed defect was female sterility, a result expected from the absence of activity of the UAS promoter in the female germ line. It was not possible to examine the chromosomal localization of the Gcn5DeltaBromo protein because the Gcn5 antibody was prepared against the Gcn5 bromodomain. Nevertheless, the full phenotypic rescue by this variant strongly suggests that its localization to chromosomes was restored (Carré, 2005).

The data demonstrate that Gcn5 is the major acetylase for two distinct histone residues, H3 K9 and H3 K14, in Drosophila. In contrast, its contribution to histone H4 acetylation could not be detected in a global analysis. The loss of Ada2b, another component of the SAGA complex, results in a partial loss of H3 K9 and H3 K14 acetylation on polytene chromosomes, while the loss of Ada2a has no detectable effect on chromosome acetylation. In light of the results, these data suggest that the Drosophila Gcn5-containing N-acetyltransferase (GNAT) complexes may retain partial Gcn5 HAT activity in the absence of the Ada2 components. Alternatively, Gcn5 could exert its HAT activity in other complexes which remain to be characterized. The substrate specificities of Drosophila and yeast Gcn5 proteins appear to be identical (Grant, 1999). However, it is interesting that in contrast to what was found for Drosophila, both the Gcn5 and Elp3 HATs must be invalidated in yeast to significantly impair histone H3 K9 and K14 acetylation (Wittschieben, 2000). Recent studies have extensively documented the relationship between histone H3 acetylation and gene transcription, but whether or not patterns of histone modification constitute a true code for the control of gene activity in eukaryotes is still a matter of debate. In either case, the results strongly suggest that specific histone acetylation profiles may be established in vivo through the activity of a very limited set of substrate-specific enzymes. The observation that Gcn5 mutant larvae survive for several days without detectable acetylation of H3 K9 and H3 K14 residues is striking and suggests that transcriptional regulation in larval versus embryonic or adult insect tissues involves distinct mechanisms (Carré, 2005).

Numerous data indicate that histone modifications can influence each other. The loss of H3 K9 and H3 K14 acetylation in imaginal discs or salivary glands has no detectable effect either on the levels of H3 S10 phosphorylation and H3 K4 methylation, both of which have been associated with transcriptional activation, or on the level of H3 K9 methylation, which marks HP1 recruitment and silencing. These results suggest that histone H3 acetylation is either a terminal or independent process in the cascade of histone H3 modifications (Carré, 2005).

Alternative ESC and ESC-like subunits of a Polycomb group histone methyltransferase complex are differentially deployed during Drosophila development

The Extra sex combs (ESC) protein is a Polycomb group (PcG) repressor that is a key noncatalytic subunit in the ESC-Enhancer of zeste [E(Z)] histone methyltransferase complex. Survival of esc homozygotes to adulthood based solely on maternal product and peak ESC expression during embryonic stages indicate that ESC is most critical during early development. In contrast, two other PcG repressors in the same complex, E(Z) and Suppressor of zeste-12 [SU(Z)12], are required throughout development for viability and Hox gene repression. A novel fly PcG repressor, called ESC-Like (ESCL), is described whose biochemical, molecular, and genetic properties can explain the long-standing paradox of ESC dispensability during postembryonic times. Developmental Western blots show that ESCL, which is 60% identical to ESC, is expressed with peak abundance during postembryonic stages. Recombinant complexes containing ESCL in place of ESC can methylate histone H3 with activity levels, and lysine specificity for K27, similar to that of the ESC-containing complex. Coimmunoprecipitations show that ESCL associates with E(Z) in postembryonic cells and chromatin immunoprecipitations show that ESCL tracks closely with E(Z) on Ubx regulatory DNA in wing discs. Furthermore, reduced escl⁺ dosage enhances esc loss-of-function phenotypes and double RNA interference knockdown of ESC/ESCL in wing disc-derived cells causes Ubx derepression. These results suggest that ESCL and ESC have similar functions in E(Z) methyltransferase complexes but are differentially deployed as development proceeds (Wang, 2006).

ESC and E(Z), and their homologs, are functional partners in the chromatin of plants, invertebrates, and mammals. Working together, they control a diverse array of developmental processes, including flower and seed differentiation in Arabidopsis thaliana, germ line development in Caenorhabditis elegans, X chromosome inactivation in mice, and Hox gene repression in flies and mammals. Recent studies show that this partnership reflects a requirement for ESC in potentiating the histone methyltransferase activity of E(Z) (Wang, 2006).

In light of this functional interdependence, a paradox is presented by developmental studies in Drosophila melanogaster, which show that ESC is primarily needed during early embryogenesis, whereas E(Z) is required throughout embryonic, larval, and pupal development. Analysis of ESCL, which can replace ESC in E(Z) HMTase complexes in vitro, provides a plausible solution to this puzzle. ESCL expression is largely complementary to that of ESC, peaking during later developmental stages, and functional studies show that ESCL is partially redundant with ESC in imaginal tissues. These results, together with prior genetic data that address esc time of action, indicate that ESC predominates in embryos, whereas both ESCL and ESC make functional contributions during postembryonic development (Wang, 2006).

Phenotypic analyses of esc loss-of-function mutants provided the original evidence that the primary time of ESC action is during embryogenesis. Although complete loss of esc⁺ product is embryonic lethal and yields wholesale misexpression of Hox genes, it was shown that maternally provided esc⁺ product provides sufficient function during embryogenesis to enable zygotically null esc^– animals to survive to adulthood. These esc^– adults are fertile, healthy, and phenotypically normal except for minor homeotic transformations such as extra sex combs on the meso- and meta-thoracic legs. In contrast, animals that are zygotically null for any other PcG subunit of the ESC-E(Z) complex or PRC1 fail to survive beyond early pupal stages, with most dying by the embryonic/L1 stage (Wang, 2006).

Additional experiments with a conditional esc allele further delimited the main time of ESC function to a period of mid-embryogenesis extending from about the onset of gastrulation (about 3 h at 25°C) until germ band shortening (approximately 9 to 12 h). An independent study that measured phenotypic rescue by a heat-inducible esc⁺ transgene confirmed that the time of ESC action begins at about 3 h of embryogenesis. These genetically determined times of esc⁺ function coincide with the accumulation of ESC protein, which peaks during mid-embryogenesis and declines by the end of embryogenesis (Wang, 2006).

However, full consideration of the genetic evidence also indicates that ESC does contribute to postembryonic PcG repression, particularly in imaginal tissues. Analysis of esc^– larvae showed modest defects in Hox gene repression in imaginal discs as well as in the central nervous system. In particular, this study attributed the extra sex combs phenotype of esc^– larvae to misexpression of the Scr Hox gene in the T2 and T3 leg discs. In addition, production of extra sex combs from patches of esc^– tissue generated by somatic recombination during larval development indicates that the time of ESC action extends into the larval period, at least in leg discs (Wang, 2006).

A postembryonic role is consistent with the detection of ESC on the Ubx gene in wing discs and with the overlapping roles of ESC and ESCL in Ubx repression in disc-derived MCW12 cells. This result might explain why esc^– wing discs did not produce homeotic phenotypes even after sufficient passage to ensure depletion of maternal esc⁺ product; presumably, both ESC and ESCL would need to be disrupted in this tissue to yield robust Hox misexpression. Finally, although it is much less abundant at late developmental times than in embryos, ESC is detected by Western blotting in larval and pupal extracts. Thus, the genetic and molecular data together indicate that ESC does function during postembryonic stages, albeit with a more modest overall contribution than its critical role in embryos (Wang, 2006).

These considerations imply that the developmental division of labor between ESC and ESCL is not simply that ESC functions only in embryos and ESCL takes over for subsequent stages. Rather, although ESC does predominate early, as evidenced by the global loss of H3 K27 methylation in esc^– embryos, postembryonic development appears to involve both ESC and ESCL. It was originally hypothesized that late developmental functions of the esc locus might be executed by an esc⁺ isoform distinct from the embryonic version. The current data confirm that multiple ESC-related proteins do operate during fly development, with a late-acting version supplied by a second copy of the esc gene (Wang, 2006).

The functional context for ESCL during postembryonic development is presumably as a subunit in E(Z)-containing complexes with histone methyltransferase activity. The fact that ESCL can assemble in place of ESC and restore HMTase activity to a reconstituted E(Z) complex indicates that the biochemical roles of ESCL and ESC are similar. ESCL/ESC functional overlap could reflect a mixture of postembryonic E(Z) complexes, with some containing ESCL and others containing ESC. The simplest version of this scenario would entail four-subunit postembryonic HMTase complexes similar to the embryonic core complex of E(Z), SU(Z)12, NURF-55, and ESCL or ESC. However, postembryonic E(Z) complexes have yet to be purified, so their molecular compositions are not yet known. In fact, there is evidence that larval E(Z) complexes may differ from embryonic E(Z) complexes in features besides the ESCL/ESC subunit. For example, the SIR2 histone deacetylase has been reported to associate with larval but not embryonic E(Z) complexes. Much remains to be determined about postembryonic E(Z) complexes, including subunit compositions and characterization of presumed HMTase activity (Wang, 2006).

Although the catalytic subunit E(Z) contains the conserved SET domain, studies on fly, worm, and mammalian homologs reveal that the ESC subunit is also critical for HMTase function. The single loss of ESC from the fly complex or loss of its homolog, MES-6, from the C. elegans complex yields subcomplexes with little or no HMTase activity in vitro. In agreement with this, genetic removal of ESC eliminates most or all methyl-H3 K27 in fly embryos, loss of MES-6 eliminates most or all methyl-H3 K27 in worm germ lines and early embryos, and loss of EED removes most or all methyl-H3 K27 from embryonic mouse cells. The mechanism by which ESC and its relatives potentiate the activity of HMTase complexes is not known. An in vitro study argues against a role for fly ESC in mediating stable contacts with nucleosome substrate. In contrast, loss of ESC by RNA interference in fly S2 cells leads to dissociation of E(Z) from chromatin targets (Wang, 2006).

A biochemical analysis of the human EED-EZH2 complex (also called PRC2) has revealed an intriguing difference in the HMTase depending upon the subtype of EED subunit present in the complex. Multiple isoforms of EED are expressed in HeLa cells that differ in the extents of their N-terminal tails through use of alternative translation start sites. Incorporation of particular EED isoforms into EZH2 complexes can shift the enzyme specificity so that K26 of histone H1 is methylated in addition to H3 K27. Taken together with other studies, this suggests that EED is a regulatory subunit that can influence both substrate specificity and catalytic efficiency of the HMTase (Wang, 2006).

In light of this finding, it seems possible that ESCL-E(Z) complexes might also have HMTase activity with altered lysine specificity. However, both ESCL-E(Z) and ESC-E(Z) recombinant complexes showed similar specificities for H3 K27 in H3/H4 tetramers and no methylation of mammalian histone H1 by either of these recombinant complexes was detected in vitro. It is noted that the human H1 K26 methylation site is embedded in an ARKS sequence, which is also present surrounding H3 K27. This sequence is not conserved in Drosophila histone H1, suggesting that the ability of certain EZH2 complexes to methylate H1 may not be conserved in the fly system. However, there may well be other relevant methylation substrates besides histone H3 and it remains possible that alternative ESC isoforms could alter lysine specificities for these other substrates (Wang, 2006).

Based upon their temporal expression profiles, it seems clear that esc and escl have distinct functions in a developmental context. Their temporal division of labor is most clearly demonstrated by esc^– escl⁺ embryos, which show extreme homeotic transformations accompanied by dramatically reduced levels of methylated H3 K27. This division could be entirely a consequence of differential transcriptional controls built into their divergent promoters. That is, ESC and ESCL could be functionally identical proteins that are just expressed at peak levels at different times. Alternatively, the two proteins may possess intrinsic differences that are also important during development but are not revealed by the assays applied so far. One possibility is that ESC and/or ESCL may play a role in methylation of nonhistone proteins. The only nonhistone proteins yet identified that fly E(Z) complexes can methylate are two subunits of the core complex itself, E(Z) and SU(Z)12. It is not clear if this self-methylation is functionally relevant and, in any case, it occurs at comparable levels with the ESC- and ESCL-containing recombinant complexes (Wang, 2006).

It is also possible that ESC and ESCL could differ in contributions to E(Z) complexes besides HMTase activity. These other functions could include interacting with and recruiting histone deacetylases, mediating physical interactions with PRC1 components, recruiting E(Z) complexes to target loci, and influencing the way E(Z) complexes interact with other (non-K27) histone tail modifications. Indeed, there is evidence for differential association of histone deacetylases with E(Z) complexes at embryonic versus larval stages, which parallels temporal changes in ESC and ESCL abundance. At the same time, ESC and ESCL functions must overlap enough to account for the sufficiency of either one to maintain Ubx repression in at least some postembryonic cells (Wang, 2006).

Definitive answers will require promoter swap experiments in which ESCL is placed under control of the ESC promoter and vice versa, to determine which combinations provide genetic rescue of esc and escl mutations in vivo. Along with this approach, a complete understanding of the developmental role of ESCL will require generation of escl mutant alleles and systematic analysis of the phenotypic consequences of escl loss of function (Wang, 2006).

Bre1 is required for Notch signaling and histone modification

Notch signaling controls numerous cell fate decisions during animal development. These typically involve a Notch-mediated switch in transcription of target genes, although the details of this molecular mechanism are poorly understood. dBre1 has been identified as a nuclear component required cell autonomously for the expression of Notch target genes in Drosophila development. dBre1 affects the levels of Su(H) in imaginal disc cells, and it stimulates the Su(H)-mediated transcription of a Notch-specific reporter in transfected Drosophila cells. Strikingly, dBre1 mutant clones show much reduced levels of methylated lysine 4 on histone 3 (H3K4m), a chromatin mark that has been implicated in transcriptional activation. Thus, dBre1 is the functional homolog of yeast Bre1p, an E3 ubiquitin ligase required for the monoubiquitination of histone H2B and, indirectly, for H3K4 methylation. These results indicate that histone modification is critical for the transcription of Notch target genes (Bray, 2005).

The lethal allele E132 was fortuitously identified among a collection of mutants that modify the wing notching phenotype caused by Armadillo depletion. Genetic mapping of the lethality associated with E132 placed this at 64E8, and it was found to be allelic to an existing mutation, l(3)01640, caused by the P element insertion P1541. Using plasmid rescue of the P element, the site of insertion was localized to the first intron of the open reading frame CG10542, which encodes a predicted protein of 1044 amino acids. The insertion site is 48 nucleotides upstream of the translation initiation codon. Precise excision of P1541 restores viability, confirming that the P element insertion and, by inference, E132 are lethal alleles of CG10542. In support of this, ubiquitous overexpression of the full-length protein encoded by CG10542 rescues the lethality of E132 or P1541 mutant embryos and sustains development to give essentially normal adult flies (with a few minor defects including slightly reduced bristles). CG10542 encodes a conserved protein with close relatives in mammals, C. elegans, plants, and fungi. The Drosophila protein has been named dBre1, after its relative Bre1p in the yeast S. cerevisiae (Bray, 2005).

The hallmarks of the Bre1 proteins are a C-terminal RING finger domain linked to an extensive N-terminal coiled-coil region. The 39 amino acid C3HC4 RING domain is flanked on both sides by ~15 conserved amino acids, suggesting that the fly and mammalian proteins are true orthologs of yeast Bre1p. RING domains are typically found in E3 ubiquitin ligases and frequently mediate the interaction with the E2 ubiquitin-activating enzyme while the other parts of the protein are involved in substrate recognition. The RING domains are therefore critical to catalyze the transfer of ubiquitin from the E2 to the substrate. To confirm the functional importance of the RING domain in dBre1, tests were performed to see whether an N-terminal fragment of dBre1 that lacks the RING domain (ΔRING) could rescue dBre1 mutants. No rescue was observed with any of the 4 transgenic lines (from a total of 814 flies scored), confirming that the RING domain is essential for the function of dBre1 as it is for yeast Bre1p (Bray, 2005).

To examine the subcellular location of full-length dBre1 and the derivative that lacks the RING domain, both forms of the protein were tagged with GFP at the N terminus. Both GFP-dBre1 and GFP-ΔRING are predominantly nuclear in embryonic and imaginal disc cells, although a low level of protein is also detectable in the cytoplasm. This nuclear-cytoplasmic distribution is similar to that of a ΔRING derivative of human Bre1-B when it is overexpressed in mammalian cells. Thus dBre1 appears to be a nuclear protein, like its mammalian counterpart, and deletion of the RING domain does not alter its subcellular distribution even though it abolishes its ability to rescue the mutants (Bray, 2005).

To investigate the role of dBre1 in the fly, homozygous mutant clones were generated in the imaginal disc precursors of the adult structures. Surprisingly, it was found that the majority of defects were similar to those caused by defects in Notch signaling. Thus, adult flies bearing E132 or P1541 mutant clones show notches in the wing margin and aberrant spacing of wing margin bristles, wing blistering and vein defects, fusions of leg segments, and loss of notal bristles and rough eyes. Most of these phenotypes are characteristic of reduced Notch signaling and are distinct from those produced by loss-of-function of other signaling pathways, such as Wingless, Dpp, or Hedgehog signaling that also operate during imaginal disc development. The phenotypic data suggest therefore that dBre1 has a role in promoting Notch signaling (Bray, 2005).

To confirm this, the expression of Notch target genes was examined in dBre1 mutant clones. Since dBre1 mutant clones are considerably smaller than their matched wild-type twin clones, the Minute technique was used to compensate for the growth defect of the mutant clones. In wing imaginal discs, cut and Enhancer of split [E(spl)] are expressed along the prospective wing margin, and their expression depends directly on Notch signaling. Cut expression is absent in large E132 mutant clones, and is lost (3/11) or reduced (6/11) in most P1541 mutant clones. Likewise, E(spl) expression is lost cell autonomously from all E132 mutant clones in the wing. Conversely, expression of spalt, a target of Dpp signaling in the wing, is not reduced in P1541 and E132 mutant cells, indicating that the effects of dBre1 mutation are relatively specific. Similar results are obtained in the eye, where E(spl) expression is also disrupted in E132 clones. Expression in the neurogenic region at the furrow is lost, and elsewhere it is absent or severely reduced, except in the basal layer of undifferentiated cells where expression is independent of Notch. In addition, a derepression of the neuronal cell marker Elav was observed in eye disc clones. The latter indicates excessive neuronal recruitment due to diminished Notch-mediated lateral inhibition (note, however, that the phenotypes are not identical to those produced by complete absence of Notch, which in the eye results in loss of neuronal markers because Notch is needed to promote neural development by alleviating Su(H)-mediated repression. These results demonstrate that dBre1 functions in multiple developmental contexts and, specifically, that it is required for the subset of Notch functions that involve Su(H)-dependent activation of Notch target genes (Bray, 2005).

To further confirm the importance of dBre1 during Notch signaling, it was asked whether any genetic interactions could be detected between overexpressed dBre1 or ΔRING and mutations in Notch (N) or its ligand Delta (Dl). Indeed, overexpression of either protein in the wing disc results in adult phenotypes. In each of 5 ΔRING-expressing lines, mild if consistent mutant phenotypes were observed in both males and females, namely upward-curved wings (due to stronger expression in the dorsal wing compartment), tiny vein deltas, and a significant decrease in wing size. These defects are more severe after overexpression of ΔRING in dBre1 heterozygotes, indicating that ΔRING acts as a weak dominant-negative. Consistent with this, excess ΔRING significantly enhances the phenotypes of N/+ and Dl/+ heterozygotes, resulting in increased vein thickening and additional vein material and, in the case of N/+, also in more frequent wing notching. These genetic interactions support the link between dBre1 and Notch signaling (Bray, 2005).

Excess full-length dBre1 in wing discs causes vein defects whose strength, however, varies considerably between different dBre1-expressing lines, and between males and females (probably because the ms1096.GAL4 driver produces higher expression levels in males). In most lines (4/6), vein thickening and additional vein material were observe only in males, while female wings appear normal. These vein defects in male wings are suppressed to almost normal in dBre1 heterozygotes, suggesting that they are due to increased levels of functional dBre1 protein. The remaining 2 lines produce similar vein defects also in females. Unexpectedly, these defects are enhanced in N/+ and Dl/+ heterozygotes, suggesting that the overexpressed dBre1 interferes with Notch signaling, rather than enhancing it as might have been expected. This anomalous result could be explained if dBre1 is part of a multiprotein complex, in which case its overexpression might interfere with the function of this complex by titrating one of its components. Nevertheless, the genetic interactions between overexpressed dBre1 and Notch and Delta further underscore the link between dBre1 and Notch signaling (Bray, 2005).

To test whether dBre1 directly influences Notch-dependent transcription, Drosophila S2 cells were transfected with Flag-tagged or untagged dBre1, and the activity of a Notch-specific reporter containing 4 Su(H) binding sites (NRE, a luciferase derivative of Gbe+Su(H)_m8) was measured in the presence or absence of low levels of N^ICD. As a control, a reporter was used with mutant Su(H) binding sites [NME, or Gbe+Su(H)_mut]. These experiments reveal a significant stimulation of the NRE reporter by dBre1, especially in the presence of N^ICD. The degree of stimulation is similar to that observed when the ubiquitin ligase Hdm2 is added to transcription assays of Tat activity. dBre1 also elicits a slight stimulation of NME. The fact that overexpressed dBre1 has stimulatory effects on Notch in the transfection assays but not in imaginal discs presumably reflects differences either in the levels of dBre1 or in the amounts of other limiting factors in the two cell contexts. Nevertheless, the transfection assays reveal an intrinsic potential of dBre1 in stimulating the transcription mediated by Su(H) and its coactivator N^ICD (Bray, 2005).

All these results point to a role of dBre1 in promoting Notch signaling. Since other ubiquitin ligases have been shown to influence the levels of specific protein components of the Notch pathway, whether there were any alterations to Notch, Delta, or Su(H) levels in dBre1 mutant clones was investigated. While there are no detectable changes in Notch or Delta staining in dBre1 mutant cells, the levels of Su(H) staining are enhanced slightly but consistently, and cell autonomously, in mutant clones of both dBre1 alleles, regardless of the location of these clones within the disc. This is also obvious in clones induced early in larval development in a non-Minute background in which the mutant dBre1 clones remain small. As an aside, these clones reveal that individual dBre1 mutant cells are enlarged, reminiscent of the yeast bre1p mutant which also shows a 'large cell'phenotype. This phenotype has not been observed in cells lacking Notch signaling, so this aspect of dBre1 function appears distinct from its role in the Notch pathway, and suggests that there are additional molecular targets. Nevertheless, the elevated levels of Su(H) in the dBre1 mutant clones identify Su(H) as one molecular target of dBre1 and suggest that, in the wild-type, dBre1 may expose Su(H) to ubiquitin-mediated degradation. The effects on Su(H) are consistent with the cell-autonomous action of dBre1 on Notch target gene expression, but the fact that removal of dBre1 has a stabilizing effect on Su(H) appears to contradict its stimulating effect on Notch-dependent transcription. Since Su(H) functions as both a repressor and an activator, this may be explained if loss of dBre1 specifically stabilizes the repressor complex. Alternatively, the effect of dBre1 mutations on Su(H) may reflect an indirect bystander activity of dBre1 (Bray, 2005).

Finally, it was asked whether dBre1 has a similar molecular function as its relative yeast Bre1p. The latter is required for the monoubiquitination of histone H2B, which is a prerequisite for the subsequent methylation of histone H3 at K4 by SET1-containing complexes. H3K4 methylation appears to be a chromatin mark for transcriptionally active genes, and yeast bre1p mutants show defects in the transcription of inducible genes that have been ascribed to the lack of H2B ubiquitination and H3K4 methylation at the promoters of these genes. Since there are no in vitro assays for H2B ubiquitination and no antibodies that detect this modified form of H2B, effects of dBre1 mutations on the linked H3K4 methylation were investigated. Wing discs bearing dBre1 mutant clones were stained with an antibody specific for trimethylated H3K4 (H3K4m). This revealed a significant reduction of H3K4m in P1541 mutant clones. More strikingly, in clones of the stronger E132 allele, H3K4m is barely detectable. In contrast, staining of these clones with an antibody against H3K9m does not show any changes in the mutant territory, indicating that the effect in dBre1 mutant clones on the methylation of H3K4 is relatively specific. It is noted that, in wild-type wing discs, there is slight modulation of trimethylated H3K4, with higher levels at the dorsoventral boundary where Notch is activated. However, Notch mutant cells retain robust H3K4m staining, although occasionally show slightly lowered levels compared to adjacent wild-type cells. Thus, the reduced H3K4m staining in dBre1 mutant cells is primarily due to an activity loss of dBre1 rather than due to loss of Notch signaling. Based on its effects on tri-methylated H3K4, it is concluded that dBre1 is indeed the functional homolog of yeast Bre1p. Furthermore, it appears that the activity of dBre1 is essential for the bulk of trimethylated H3K4 in imaginal disc cells (Bray, 2005).

In yeast, H2B ubiquitination and H3K4 methylation are associated with sites of active transcription, but the only identified natural target gene is GAL1. In Drosophila, the target genes of dBre1 evidently include genes regulated by Notch, given the requirement of dBre1 for their transcription. It is therefore conceivable that Su(H) may have a role in targeting dBre1 to their promoters (although it was not possible to detect direct binding or coimmunoprecipitation between dBre1 and Su(H). It is puzzling that dBre1 has a slight destabilizing effect on Su(H), despite being an activating component of Notch signaling. It is believed that this could be a bystander effect of dBre1: evidence suggests that the Bre1p-mediated monoubiquitination of H2B leads to a transient recruitment of proteasome subunits to chromatin, and that the subsequent methylation of H3K4 depends on the activity of these proteasome subunits. Their transient presence at specific target genes may have a destabilizing effect on nearby DNA binding proteins, and the mildly increased levels of Su(H) in dBre1 mutant cells could therefore reflect a failure of proteasome recruitment due to loss of H2B monoubiquitination (Bray, 2005).

Perhaps the most interesting implication of the results is that the dBre1-mediated monoubiquitination of H2B and methylation of H3K4 may be critical steps in the transcription of Notch target genes. Indeed, it appears that the Notch target genes belong to a group of genes whose transcription is particularly susceptible to the much reduced levels of H3K4m in dBre1 mutant cells. Based on the dBre1 mutant phenotypes, there are likely to be other genes in this group, including for example genes controlling cell survival and cell size. Nevertheless, it would appear that the transcription of Notch target genes is particularly reliant on the activity of dBre1. Other examples are emerging where the transcriptional activity of a subset of signal responsive genes is particularly sensitive to the function of a particular chromatin modifying and/or remodelling factor. This sensitivity presumably reflects the molecular mechanisms used by signaling pathways to activate transcription at their responsive enhancers. Understanding why Notch-induced transcription is particularly susceptible to loss of dBre1 function will require knowledge of these underlying molecular mechanisms (Bray, 2005).

Role of the p53 homologue from Drosophila melanogaster in the maintenance of histone H3 acetylation and response to UV-light irradiation

It has been demonstrated that the human tumor suppressor p53 has an important role in modulating histone modifications after UV light irradiation. This work explores if the p53 Drosophila homologue has a similar role. Taking advantage of the existence of polytene chromosomes in the salivary glands of third instar larvae, K9 and K14 H3 acetylation patterns were analyzed in situ after UV irradiation of wild-type and Dmp53 null flies. As in human cells, after UV damage there is an increase in H3 acetylation in wild-type organisms. In Dmp53 mutant flies, this response is significantly affected at the K9 position. These results are similar to those found in human p53 mutant tumor cells with one interesting difference, only the basal H3 acetylation of K14 is reduced in Dmp53 mutant flies, while the basal H3-K9 acetylation is not affected. This work shows, that the presence of Dmp53 is necessary to maintain normal H3-K14 acetylation levels in Drosophila chromatin and that the function of p53 to maintaining histone modifications, is conserved in Drosophila and humans (Rebollar, 2006).

The results presented here show that there are some similarities and differences between fly and human cells. For instance, in wild type third instar larvae there is an increase in the acetylation of K9 and K14 in the histone H3 in response to UV light irradiation. This observation is similar to previous reports in mammalian cells. Other similarity between both systems is that mutations in p53 affect the increase in the K9 acetylation after DNA damage, but not the acetylation of K14 in H3. In contrast, both human p53 and Dm p53 are required to maintain the basal histone H3 acetylation levels. In the case of human cells, the basal K9 acetylation level seems to be preferentially diminished when human p53 is mutated. In the case of Drosophila, K14 basal acetylation is dramatically reduced by the absence of Dm p53. Several scenarios may explain these differences. The first is that since not all p53 functions are conserved between human p53 and Dm p53 and the only region with significant identity between both proteins is the DNA binding domain, it is possible that the interactions with factors involved in histone modifications are different. Another possibility is that cancer cells deficient in human p53 could have other mutations. Usually they are aneuploid and therefore a mutated human p53 may interact with other mutated genes producing a phenotype on histone modifications. It is relevant to mention that in the Dm p53 null fly used in this study only Dm p53 is affected and therefore the effects that were observed in H3 acetylation are due only to this mutation (Rebollar, 2006).

The fact that a deficiency in Dm p53 produces a phenotype in basal H3 acetylation levels and in the increase of histone acetylation after UV light irradiation, indicates there is an important cross-talk between chromatin modifiers, Dm p53 and the nucleotide excision repair machinery in the fly. A similar network has been suggested to exist in human cells and therefore the fly becomes an interesting model to study the mechanisms that operate between DNA damage, p53 and chromatin dynamics. In contrast, the reduction in the basal levels of K14 acetylation in H3, does not have any effect in viability and fertility of the Dm p53 null flies. However, Dm p53 null organisms are very sensitive to UV light irradiation and a short life span. During development, the organism is exposed to genotoxic stress as consequence of the cell metabolism. Dm p53 may participate in the DNA repair during development and it is possible that the reduction in K14 basal acetylation in the Dmp53 null fly is product of a deficient DNA repair mechanism (Rebollar, 2006).

This work opens several interesting avenues that can be explored exploiting Drosophila genetics. For instance different mutant backgrounds in genes involved in genome stability, including Dm p53 can be used for the analyses of different histone modifications after DNA damage. It can also be interesting to find out if these histone modifications are different depending on the chromatin state. Also, since there are two pathways in nucleotide excision repair, transcription coupled repair and global genome repair it will be interesting to know if the increase in histone acetylation after DNA damage is higher in transcribed regions. However it is difficult to determine differences in histone modifications in specific sequences with this kind of analysis. These questions will be eventually answered by doing chromatin immunoprecipitations and genetics (Rebollar, 2006).

Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation

Tri-methylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging and heterochromatin formation. HP1alpha, -beta, and -gamma are released from chromatin during the M phase of the cell cycle, even though tri-methylation levels of histone H3 lysine 9 remain unchanged. However, the additional, transient modification of histone H3 by phosphorylation of serine 10 next to the more stable methyl-lysine 9 mark is sufficient to eject HP1 proteins from their binding sites. Inhibition or depletion of the mitotic kinase Aurora B, which phosphorylates serine 10 on histone H3, causes retention of HP1 proteins on mitotic chromosomes, suggesting that H3 serine 10 phosphorylation is necessary for the dissociation of HP1 from chromatin in M phase. These findings establish a regulatory mechanism of protein-protein interactions, through a combinatorial readout of two adjacent post-translational modifications: a stable methylation and a dynamic phosphorylation mark (Fischle, 2005).

Although histone H3S10ph is widely seen as a hallmark of mitosis, the function of this modification during M phase has been enigmatic. The data suggest that phosphorylation of H3S10 by Aurora B disrupts the chromodomain-H3K9me3 interaction, which is important for HP1 recruitment to chromatin during interphase. This disruption causes a net shift in the dynamic HP1-chromatin binding equilibrium towards the unbound state. In this reaction sequence, dephosphorylation of H3S10 at the end of mitosis re-establishes the overall association of HP1 with chromatin (Fischle, 2005).

It is propose that this binary 'methyl/phos switching' permits dynamic control of the HP1-H3K9me interaction. Intriguingly, the mechanism for HP1 release from M-phase chromatin does not involve a temporary loss of H3K9me3, but instead requires a combination of this unchanging mark and the dynamic H3S10ph modification that is only transiently added to chromatin during mitosis. It is reasoned that stable transmission of the heterochromatin-defining H3K9me3 mark is needed to accurately convey, from one cell generation to the next, which regions of the genome are supposed to be permanently silenced. If removal of HP1 from M-phase chromatin were accomplished by H3K9me3-erasing demethylase activities, the epigenetic information underlying this mark- and effector-system would have to be accurately re-established at the end of every cell cycle (Fischle, 2005).

In addition to H3S10 phosphorylation, other mechanisms might be involved in the mitotic release of HP1 from chromatin. These might include further modifications of the H3-tail, HP1 proteins and/or their interaction partners. Nevertheless, inhibition, knockdown or depletion of Aurora B is sufficient to cause aberrant interaction of all HP1 isoforms with mitotic, condensed chromatin. Although the possibility cannot be excluded that HP1 proteins themselves might be in vivo targets of Aurora B kinase activity (for example, increased association of the xHP1aW57A mutant protein was observed with metaphase chromosomes assembled in DCPC extracts), it is known that the phosphorylation level of HP1b and HP1g does not increase during mitosis. Since phosphorylation of an H3K9me3 peptide is sufficient to dissociate HP1 from this site in vitro, it is concluded that Aurora B-mediated phosphorylation of H3S10 must be the central event in mitotic release of HP1 from chromatin (Fischle, 2005).

Notably, a fraction of HP1a, but not HP1b or HP1g, remains associated with the (peri-)centromeric chromosome region, where it performs important functions for centromere cohesion and kinetochore formation and might be required to identify and define this specialized area of heterochromatin throughout the cell cycle. This mitotic retention of HP1a at centromeres depends on a carboxy-terminal region of the protein, but is independent of the chromodomain8. It is therefore suggested that 'methyl/phos switching' uniformly disrupts HP1-chromatin interaction but that mechanisms other than chromodomain-H3K9me3 interaction are responsible for the lingering HP1a association with pericentromeric regions (Fischle, 2005).

What is the function of HP1 dissociation from chromatin during Mphase? It is tempting to speculate that removal of HP1 is important for allowing access by factors necessary for mediating proper chromatin condensation and faithful chromosome segregation during mitosis. Indeed, inhibition of Aurora B in vertebrate cells results in defects in chromosome alignment, segregation, chromatin-induced spindle assembly and cytokinesis. Furthermore, mutation of H3S10 causes faulty chromosome segregation in Tetrahymena and S. pombe, organisms that rely on HP1 and H3K9me3 for the establishment and maintenance of heterochromatin, but not in Saccharomyces cerevisiae, an organism that lacks this silencing system. Interestingly, most histone phosphorylation sites are rapidly phosphorylated early in M phase. It remains to be seen whether these bursts in histone phosphorylation are directly involved in the release of proteins bound to interphase chromatin, which might need to be removed to ensure faithful progression through mitosis. It is conceivable that similar 'methyl/phos switches' play critical roles in governing other histone-non-histone or even non-histone-nonhistone interactions (Fischle, 2005).

The JIL-1 histone H3S10 kinase regulates dimethyl H3K9 modifications and heterochromatic spreading in Drosophila

A reduction in the levels of the JIL-1 histone H3S10 kinase results in the spreading of the major heterochromatin markers dimethyl H3K9 and HP1 to ectopic locations on the chromosome arms, with the most pronounced increase on the X chromosomes. Genetic interaction assays demonstrated that JIL-1 functions in vivo in a pathway that includes Su(var)3-9, a major catalyst for dimethylation of the histone H3K9 residue, HP1 recruitment, and the formation of silenced heterochromatin. Evidence is provided that JIL-1 activity and localization are not affected by the absence of Su(var)3-9 activity, suggesting that JIL-1 is upstream of Su(var)3-9 in the pathway. Based on these findings, a model is proposed where JIL-1 kinase activity functions to maintain euchromatic regions by antagonizing Su(var)3-9-mediated heterochromatization (Zhang, 2006: full text of article).

According to the histone code hypothesis and the recently proposed binary switch model, phosphorylation of a site adjacent to a methyl mark that engages an effector molecule may regulate its binding. JIL-1 phosphorylates the histone H3S10 residue in euchromatic regions of polytene chromosomes, raising the possibility that this phosphorylation at interphase prevents the recruitment of Su(var)3-9 and/or the dimethylation of the neighboring K9 residue. This, in turn, would affect the binding of HP1, thus antagonizing the formation of silenced heterochromatin at interbands. That different regions of chromatin may have different combinations of posttranslational modifications controlling effector/histone interactions, as predicted by the histone code hypothesis, is underscored by the finding that, in JIL-1 null backgrounds, the level of the dmH3K9 marker and HP1 are preferentially increased on the male and female X chromosomes. It is well documented that the male X chromosome is unique because of the activity of the MSL dosage compensation complex and the MOF histone acetyltransferase, which leads to hyperacetylation of histone H4. However, comparable markers for the female X chromosome have yet to be discovered, and the results are the first indication that markers may exist that distinguish male and female X chromosomes from autosomes, and that this difference may increase the affinity for Su(var)3-9. That the spreading of heterochromatic markers in the absence of JIL-1 occurs on both the male and female X chromosome further indicates that these changes are independent of dosage compensation processes (Zhang, 2006).

Unfortunately, in this study, the possibility that the observed spreading of heterochromatin markers occurred preferentially to specific euchromatic sites could not directly addressed. In JIL-1 null and hypomorphic backgrounds, chromosome morphology is greatly perturbed, and there is an intermixing not only of euchromatin and the compacted chromatin characteristic of banded regions, but also of non-homologous chromatid regions, which become fused and confluent. Thus, an alternative hypothesis that JIL-1 activity may regulate boundary elements that control the spreading of heterochromatic factors cannot be ruled out, or that the two mechanisms may act in concert. However, the spreading of the dmH3K9 marker and HP1 to ectopic locations on the chromosomes is likely to lead to heterochromatization and repression of gene expression at these sites. The results further suggest the possibility that the lethality of JIL-1 null mutants may be due to the repression of essential genes at these ectopic sites as a consequence of the spreading of Su(var)3-9 activity. This hypothesis is supported by genetic interaction assays that demonstrated that the lethality of a severely hypomorphic JIL-1 heteroallelic combination could be almost completely rescued by a reduction in Su(var)3-9 dosage that prevented the ectopic dimethylation of histone H3K9 (Zhang, 2006).

The Su(var)3-1 alleles of JIL-1 consist of dominant gain-of-function alleles that antagonize the expansion of heterochromatin formation. However, evidence is provided that the underlying molecular mechanism of this antagonism is different from that occurring in the loss-of-function null and hypomorphic JIL-1 alleles described in this study. JIL-1^Su(var)3-1 alleles are characterized by deletions of the COOH-terminal domain that do not affect JIL-1 kinase activity or the spreading of heterochromatin markers. Furthermore, the results indicate that the COOH-terminal domain of JIL-1 is required for proper chromosomal localization and that JIL-1^Su(var)3-1 proteins are mislocalized to ectopic chromosome sites. Thus, it is proposed that the dominant gain-of-function effect of the JIL-1^Su(var)3-1 alleles may be attributable to JIL-1 kinase activity at ectopic locations, possibly through the phosphorylation of novel target proteins, or by mis-regulated localization of the phosphorylated histone H3S10 marker. Although the JIL-1^Su(var)3-1 proteins are mislocalized, they still associate with chromosomes and phosphorylate the histone H3S10 residue, suggesting that other regions of the protein have a binding affinity for at least some of the substrates and interaction partners of JIL-1. This is supported by the finding that each of the two kinase domains of JIL-1 can interact with the MSL-complex in vitro (Zhang, 2006).

In summary, evidence is provided that the JIL-1 kinase is a major regulator of histone modifications that affect gene activation, gene silencing and chromatin structure. Thus, it will be informative in future experiments to further explore the interaction of JIL-1 with genes controlling heterochromatin formation, in order to gain a better understanding of the molecular mechanisms of epigenetic gene regulation (Zhang, 2006).

Genome-wide analysis of Polycomb targets in Drosophila

Polycomb group (PcG) complexes are multiprotein assemblages that bind to chromatin and establish chromatin states leading to epigenetic silencing PcG proteins regulate homeotic genes in flies and vertebrates, but little is known about other PcG targets and the role of the PcG in development, differentiation and disease. This study determined the distribution of the PcG proteins PC, E(Z) and PSC and of trimethylation of histone H3 Lys27 (me3K27) in the Drosophila genome using chromatin immunoprecipitation (ChIP) coupled with analysis of immunoprecipitated DNA with a high-density genomic tiling microarray. At more than 200 PcG target genes, binding sites for the three PcG proteins colocalize to presumptive Polycomb response elements (PREs). In contrast, H3 me3K27 forms broad domains including the entire transcription unit and regulatory regions. PcG targets are highly enriched in genes encoding transcription factors, but they also include genes coding for receptors, signaling proteins, morphogens and regulators representing all major developmental pathways (Schwartz, 2006).

The components of PcG complexes are products of PcG genes, first discovered as crucial regulators of homeotic genes in Drosophila. Immunostaining of Drosophila polytene chromosomes, however, showed PcG proteins at about 100 cytological loci, implying a much larger number of target genes. Functional analysis has identified PREs as DNA sequences able to recruit PcG proteins and establish PcG silencing of neighboring genes. Two types of PcG complexes bind to PREs. PRC1-type complexes include a core quartet of proteins: PC, PSC, PH and dRing. PRC2-type complexes include E(Z), which methylates histone H3 Lys27. Mono- and dimethylated Lys27 is widely distributed in the genome, but PcG sites characteristically contain trimethylated Lys27 (me3K27). The activity of the E(Z) complex is essential for stable silencing, and it has been proposed that H3 me3K27 recruits the PRC1 complex through the specific affinity of the PC chromodomain for me3K27. But the relationships between PRC1 and PRC2 complexes, between their binding sites and histone methylation, and between binding, methylation and gene expression are not well understood and remain the subject of debate. The genomic distribution of three PcG proteins [PC, PSC and E(Z)] and of histone H3 me3K27 was examined using using chromatin immunoprecipitation (ChIP). Since PcG target genes may be repressed in some tissues and active in others, a cultured cell line was used to minimize heterogeneity (Schwartz, 2006).

Viewed at the scale of a chromosome arm, the distributions of PC, PSC, E(Z) and me3K27 coincide at a number of distinct binding peaks (which are referred to as 'PcG sites') that correspond to 70% of the bands reported in salivary gland polytene chromosomes stained with the corresponding antibodies. To minimize false positives, the analysis focussed on the PcG sites that showed simultaneous binding of two or more proteins, each above twofold enrichment. Of the 149 PcG sites detected (see the supplemental figure), 95 showed strong binding of all four proteins ('strong' PcG sites), whereas in 54 sites the binding was lower and below threshold for one of the proteins ('weak' PcG sites). At higher resolution, most PcG sites involve two or more genes, often sharing structural or functional similarities. Thus, PcG sites involve the following: engrailed (en) and invected (inv); the PcG genes ph-p and ph-d; the Dorsocross T-box gene cluster; the muscle NK homeobox gene cluster; the wingless cluster; and the two homeotic complexes ANT-C and BX-C (Schwartz, 2006).

The Bithorax complex (BX-C) is a cluster of three homeotic genes (Ubx, abd-A and Abd-B) responsible for segmental identity in the abdomen and posterior thorax. The most prominent features are two sharp binding peaks for all three PcG proteins at the sites of the bx and bxd PREs that control Ubx. No peak was detected over the Ubx proximal promoter, although the entire gene shows a low but significant level of PC. A series of lower peaks emerged in the abd-A region and part of the Abd-B gene. Some of these correspond to the known PREs iab-2. In contrast, the distribution of H3 me3K27 oscillated rapidly above a high plateau that covers Ubx and abd-A but not Abd-B. RT-PCR was used to determine the mRNA levels corresponding to these three genes. Transcription of Ubx and abd-A in these cells was very low but distinctly above background. Abd-B was highly transcribed, at levels 300 times higher than Ubx. This pattern of activity was reflected by the distribution of both PcG proteins and me3K27. It is noted that in the Abd-B regulatory region, the previously characterized Fab-7 and Fab-8 PREs neither bound PcG proteins nor were methylated in these cells. The Abd-B gene has five distinct promoters. A sharp resurgence of both methylation and PcG protein binding in the region of the most upstream Abd-B promoter suggests that, in contrast to the other four promoters, this one might be repressed in the cultured cells. RT-PCR analysis using primers specific for mRNAs initiating from each promoter confirmed that the most upstream promoter is silent and that the other four are active. These results support the view that binding of PcG proteins to PREs is associated with transcriptional quiescence, whereas robust transcriptional activity is accompanied by lack of binding to the PREs and lack of Lys27 methylation over the transcription unit (Schwartz, 2006).

Strong genomic sites bind all three PcG proteins. The PSC and E(Z) peaks generally rise sharply and are contained within less than 2 kb, whereas PC frequently forms a broader peak that may include shoulders or subsidiary peaks absent for E(Z) and PSC and subsides to background more gradually. These peak binding regions are thought of as corresponding to PREs, which they in fact do in the cases where these are known. Additional binding peaks may be found within or downstream of the transcription unit. In contrast, distribution of H3 me3K27 at each site is very broad, forming a domain of tens or even hundreds of kilobases encompassing the transcription unit and regulatory regions of one or more genes but, rather than a level plateau, it consists of a series of deep oscillations (Schwartz, 2006).

The strong binding peaks or putative PREs are often associated with low values or troughs in the methylation profile and at secondary peaks the PC distribution frequently echoes methylation peaks. Overall, their relationship does not support the idea that methylation of Lys27 suffices to recruit binding of PC. It is proposed instead that PC bound to the strong binding peaks, the presumptive PREs, is recruited by proteins that bind specifically to those sequences. The weaker PC binding peaks and tails that mirror the methylation profile near PREs may represent a second mode of PC binding mediated by the interaction of the chromodomain with H3 me3K27 (Schwartz, 2006).

It is supposed that methylation domains initiated by a PRE might spread bidirectionally until they encounter 'active' chromatin, characterized by histone acetylation or methylation of H3 Lys4, marks typical of transcriptionally active genes. Alternatively, specific features might shape the methylation domain either positively, by attracting the methyltransferase complex, or negatively, by blocking productive interactions with the PRE. As in the case of the Abd-B gene or of CG7922 and CG7956 genes, sudden drops in levels of me3K27 are generally associated with transcriptional activity. Are insulators involved in protecting CG7922 and CG7956 from silencing, or is the activity of these two genes simply epigenetically maintained from the time the cell line was originally established? Further work is required to answer this question (Schwartz, 2006).

In many cases, the presumptive PRE lies between divergently transcribed genes such as dco and Sox100B. Which of the two is the PRE target? As PREs can act at distances of 20-30 kb, the proximity of PcG peaks to a promoter is not a reliable guide. It is proposed that the methylation domain is the clue to the target of PcG regulation. A PcG peak is not considered to regulate a promoter if the gene is not included in the methylation domain. When multiple genes are included in the methylation domain, it is likely that they are all affected by PcG regulation. However, this study distinguishes between genes that contain methylation as well as one or more PcG proteins and genes that contain only methylation (Schwartz, 2006).

The 95 'strong' binding sites in the genome encompass a total of 392 genes. Of these 392 genes, 186 contain both PcG binding and methylation, and the remainder are found within broad methylation domains associated with PcG proteins binding but do not bind PcG proteins over their own promoter or transcription unit. They may represent genes not directly targeted but affected by the spread of methylation. An analysis of their ontology indicates that these two classes are in fact very different. Transcription regulators constitute 64.5% of the first set, compared to 4.3% for the full annotation set. Instead they constitute only 4.0% of those genes that contain only me3K27. These comparisons strongly suggest that (1) genes that regulate transcription are preferred PcG targets, and (2) genes that only include the tails of a methylation domain are probably not primary targets of PcG regulation. A similar preference is also seen among the 'weak' binding sites. These include a total of 74 genes containing both PcG proteins and methylation, 28.4% of which encode transcription regulators. Flanking genes containing only methylation include only 5.7% transcription regulators. Although transcription regulators are preferred PcG targets, secreted proteins, growth factors or their receptors, and signaling proteins are also targeted. PcG target genes include components of all the major differentiation and morphogenetic pathways in Drosophila (Schwartz, 2006).

The major features of PcG binding shown by this work are that, although the proteins themselves are highly localized at presumptive PREs, the domain of histone methylation they produce is much broader. If the E(Z) methyltransferase is localized at the PRE, how is the extensive methylation domain produced? A looping mechanism is proposed in which interaction of PRE-bound complexes with flanking chromatin is mediated by the PC chromodomain. The observed broader distribution of PC might result from crosslinking of the chromodomain to methylated H3, reflecting this mechanism (Schwartz, 2006).

Are PREs defined by characteristic sequence motifs? Although the analysis of the sequences underlying the binding peaks will be presented elsewhere, it is noted that Ringrose (2003) devised an algorithm based on GAGA factor, PHO and Zeste binding motifs to identify sequences likely to represent PREs. This algorithm correctly predicts a number of the strong PcG binding sites (27%) and a few of the weaker sites (7%), overall 20%; however, it does not predict the majority of the PcG sites. The reverse is also true: only 22% of the PREs predicted by Ringrose bind PcG proteins in these experiments. Together, these data suggest that additional criteria are necessary to predict most PREs reliably (Schwartz, 2006).

As expected, PcG proteins and me3K27 are associated with transcriptional quiescence, but the data suggest that this is not an absolute condition. Low but significant transcription levels are detected even for the repressed Ubx and abd-A genes. Two target sites, polyhomeotic and the Psc-Su(z)2 site, contain PcG genes, which must be active to ensure the functioning of the PcG mechanism. The polyhomeotic locus is one of two sites in the entire genome that bind PC but lack appreciable levels of E(Z) and of Lys27 methylation. Instead, the Psc-Su(z)2 region is well methylated and binds both PC and E(Z) at multiple peaks. It is concluded that PcG mechanisms do not invariably lead to transcriptional silencing and are compatible with moderate levels of transcription (Schwartz, 2006).

Another point of interest is the number and kind of genes that are PcG targets. Considering the developmental difference between salivary gland cells and the embryo-derived tissue culture cells, the substantial number of shared PcG sites suggests that a majority of target sites are occupied in a large percent of cells. Target genes are in fact predominantly regulatory genes that control major differentiation and morphogenetic pathways. These pathways and their genes are highly conserved, and recent work shows that they are also regulated by PcG in mammals. It might be expected that in a given cell type most alternative genomic programs would be repressed save the subset required in that cell type. The emerging picture from these studies is that PcG regulation is a key mechanism in genomic programming (Schwartz, 2006).

Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins

Polycomb group (PcG) and trithorax group (trxG) proteins act as antagonistic regulators to maintain transcriptional OFF and ON states of HOX and other target genes. To study the molecular basis of PcG/trxG control, the chromatin of the HOX gene Ultrabithorax (Ubx) was analyzed in Ubx^OFF and Ubx^ONcells purified from developing Drosophila. PcG protein complexes PhoRC, PRC1, and PRC2 and the Trx protein are all constitutively bound to Polycomb response elements (PREs) in the OFF and ON state. In contrast, the trxG protein Ash1 is only bound in the ON state; not at PREs but downstream of the transcription start site. In the OFF state, extensive trimethylation was found at H3-K27, H3-K9, and H4-K20 across the entire Ubx gene; i.e., throughout the upstream control, promoter, and coding region. In the ON state, the upstream control region is also trimethylated at H3-K27, H3-K9, and H4-K20, but all three modifications are absent in the promoter and 5' coding region. These analyses of mutants that lack the PcG histone methyltransferase (HMTase) E(z) or the trxG HMTase Ash1 provide strong evidence that differential histone lysine trimethylation at the promoter and in the coding region confers transcriptional ON and OFF states of Ubx. In particular, these results suggest that PRE-tethered PcG protein complexes act over long distances to generate Pc-repressed chromatin that is trimethylated at H3-K27, H3-K9, and H4-K20, but that the trxG HMTase Ash1 selectively prevents this trimethylation in the promoter and coding region in the ON state (Papp, 2006; Full text of article).

Previous studies have shown that PhoRC contains the DNA-binding PcG protein Pho that targets the complex to PREs, and dSfmbt, a novel PcG protein that selectively binds to histone H3 and H4 tail peptides that are mono- or dimethylated at H3-K9 or H4-K20 (H3-K9me1/2 and H4-K20me1/2, respectively) (Klymenko, 2006). PRC1 contains the PcG proteins Ph, Psc, Sce/Ring, and Pc. PRC1 inhibits nucleosome remodeling and transcription in in-vitro assays and its subunit Pc specifically binds to trimethylated K27 in histone H3 (H3-K27me3). PRC2 contains the PcG proteins E(z), Su(z)12, and Esc as well as Nurf55, and this complex functions as a histone methyltransferase (HMTase) that specifically methylates K27 in histone H3 (H3-K27) in nucleosomes (Papp, 2006).

This study used quantitative X-ChIP analysis to examine the chromatin of the HOX gene Ubx in its ON and OFF state in developing Drosophila larvae. Previous genetic studies had established that all of the PcG and trxG proteins analyzed in this study are critically needed to maintain Ubx OFF and ON states in the very same imaginal disc cells in which their binding to Ubx was analyzed in this study. The following conclusions can be drawn from the analyses reported in this study. (1) The PcG protein complexes PhoRC, PRC1, and PRC2 and the Trx protein are all highly localized at PREs, but they are all constitutively bound at comparable levels in the OFF and ON state. (2) The trxG protein Ash1 is bound only in the ON state, where it is specifically localized ~1 kb downstream of the transcription start site. (3) In the OFF state, PRC2 and other unknown HMTases trimethylate H3-K27, H3-K9, and H4-K20 over an extended 100-kb domain that spans the whole Ubx gene. (4) In the ON state, comparable H3-K27, H3-K9, and H4-K20 trimethylation is restricted to the upstream control regions and Ash1 selectively prevents this trimethylation in the promoter and coding region. (5) Repressed Ubx chromatin is extensively tri- but not di- or monomethylated at H3-K27, H3-K9, and H4-K20. (6) Trimethylation of H3-K27, H3-K9, and H4-K20 at imaginal disc enhancers in the upstream control region does not impair the function of these enhancers in the ON state. (7) TBP and Spt5 are bound at the Ubx transcription start site in the ON and OFF state, but Kis is only bound in the ON state. This suggests that in the OFF state, transcription is blocked at a late step of transcriptional initiation, prior to the transition to elongation. A schematic representation of PcG and trxG protein complex binding and histone methylation at the Ubx gene in the OFF and ON state is presented (Papp, 2006).

Unexpectedly, ChIP analysis by qPCR used in this study and in a similar study by the laboratory of Vincent Pirrotta (V. Pirrotta, pers. comm. to Papp, 2006) reveals that the relationship between PcG and trxG proteins and histone methylation is quite different from the currently held views. Specifically, X-ChIP studies have reported that H3-K27 trimethylation is localized at PREs and this led to the model that recruitment of PRC1 to PREs occurs through H3-K27me3 (i.e., via the Pc chromodomain). In contrast, the current study and that by Vincent Pirrotta found H3-K27 trimethylation to be present across the whole inactive Ubx gene, both in wing discs and in S2 cells (V. Pirrotta, pers. comm. to Papp, 2006). No specific enrichment of H3-K27 trimethylation at PREs has been detected; rather, a reduction of H3-K27me3 signals is observed at PREs, consistent with the reduced signals of H3 that are detected at these sites. Consistent with these results, genome-wide analyses of PcG protein binding and H3-K27me3 profiles in S2 cells revealed that, at most PcG-binding sites in the genome, PcG proteins are tightly localized, whereas H3-K27 trimethylation is typically present across an extended domain that often spans the whole coding region. How could the differences between this study and the earlier studies be explained? It should be noted that in contrast to the qPCR analysis used in the current study, previous studies all relied on nonquantitative end-point PCR after 36 or more cycles to assess the X-ChIP results. It is possible that these experimental differences account for the discrepancies (Papp, 2006).

PhoRC, PRC1, and PRC2 are all tightly localized at PREs but they are all constitutively bound at the inactive and active Ubx gene. This suggests that recruitment of PcG complexes to PREs occurs by default. Although all three complexes are bound at comparable levels to the bxd PRE in the inactive and active state and PhoRC is also bound at comparable levels at the bx PRE, it should be pointed out that the levels of PRC1 and PRC2 binding at the bx PRE are about twofold reduced in the active Ubx gene compared with the inactive Ubx gene. Even though there is still high-level binding of PRC1 and PRC2 at the bx PRE, it cannot be excluded that the observed reduction in binding helps to prevent default PcG repression of the active Ubx gene. It is possible that transcription through the bx PRE reduces PRC1 and PRC2 binding at this PRE. Transcription through PREs has been proposed to serve as an 'anti-silencing' mechanism that prevents default silencing of active genes by PREs (Papp, 2006),

The highly localized binding of all three PcG protein complexes at PREs, together with earlier studies on PRE targeting of PcG protein complexes supports the idea that not only PhoRC but also PRC1 and PRC2 are targeted to PRE DNA through interactions with Pho and/or other sequence-specific DNA-binding proteins. In the case of trxG proteins, the binding modes are more diverse. In particular, recruitment of Trx protein to PREs and to the promoter is also constitutive in both states but recruitment of Ash1 to the coding region is clearly observed only at the active Ubx gene. At present, it is not known how Trx or Ash1 are targeted to these sites. It is possible that a transcription-coupled process recruits Ash1 to the position 1 kb downstream of the transcription start site (Papp, 2006).

In contrast to the localized and constitutive binding of PcG protein complexes and the Trx protein, it was found that the patterns of histone trimethylation are very distinct in the active and inactive Ubx gene. The results also suggest that the locally bound PcG and trxG HMTases act across different distances to methylate chromatin. For example, H3-K4 trimethylation is confined to the first kilobase of the Ubx coding region where Ash1 and Trx are bound, whereas H3-K27 trimethylation is present across an extended 100-kb domain of chromatin that spans the whole Ubx gene. This suggests that PRE-tethered PRC2 is able to trimethylate H3-K27 in nucleosomes that are as far as 30 kb away from the bxd or bx PREs. Unexpectedly, it was found that the H3-K9me3 and H4-K20me3 profiles closely match the H3-K27me3 profile. At present it is not known which HMTases are responsible for H3-K9 and H4-K20 trimethylation, but analysis of E(z) mutants indicate that these modifications may be generated in a sequential manner, following H3-K27 trimethylation by PRC2. The molecular mechanisms that permit locally tethered HMTases such as PRE-bound PRC2 to maintain such extended chromatin stretches in a trimethylated state are only poorly understood. However, a recent study showed that the PhoRC subunit dSfmbt selectively binds to mono- and dimethylated H3-K9 and H4-K20 in peptide-binding assays (Klymenko. 2006). One possibility would be that dSfmbt participates in the process that ensures that repressed Ubx chromatin is trimethylated at H3-K27, H3-K9, and H4-K20. For example, dSfmbt, tethered to PREs by Pho, may interact with nucleosomes of lower methylated states (i.e., H3-K9me1/2 or H4-K20me1/2) in the flanking chromatin and thereby bring them into the vicinity of PRE-anchored HMTases that will hypermethylate them to the trimethylated state (Papp, 2006).

These analyses suggest that H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region is critical for Polycomb repression. (1) Although H3-K27, H3-K9, and H4-K20 trimethylation is present at the inactive and active Ubx gene, it is specifically depleted in the promoter and coding region in the active Ubx gene. (2) Misexpression of Ubx in wing discs with impaired E(z) activity correlates well with loss of H3-K27 and H3-K9 trimethylation at the promoter and 5' coding region. It is possible that the persisting H3-K27 and H3-K9 trimethylation in the 3' coding region is responsible for maintenance of repression in those E(z) mutant wing discs cells that do not show misexpression of Ubx. (3) In haltere and third-leg discs of ash1 mutants, the promoter and coding region become extensively trimethylated at H3-K27 and H3-K9, and this correlates with loss of Ubx expression. Previous studies showed that Ubx expression is restored in ash1 mutants cells that also lack E(z) function. Together, these findings therefore provide strong evidence that Ash1 is required to prevent PRC2 and other HMTases from trimethylating the promoter and coding region at H3-K27 and H3-K9. The loss of H3-K4 trimethylation in ash1 mutants is formally consistent with the idea that Ash1 exerts its antirepressor function by trimethylating H3-K4 in nucleosomes in the promoter and 5' coding region, but other explanations are possible (Papp, 2006).

But how might H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region repress transcription? The observation that TBP and Spt5 are also bound to the promoter in the OFF state suggests that these methylation marks do not prevent assembly of the basic transcription apparatus at the promoter. However, the nucleosome remodeling factor Kis is not recruited in the OFF state, and transcription thus appears to be blocked at a late step of transcriptional intiation prior to elongation. It was found that the low-level binding of Pc in the coding region correlates with the presence of H3-K27 trimethylation; i.e., Pc and H3-K27me3 are both present in the OFF state, but are absent in the ON state. One possible scenario would thus be that H3-K27 trimethylation in the promoter and coding region permits direct recruitment of PRC1. According to this view, locally recruited PRC1 would then repress transcription; e.g., by inhibiting nucleosome remodeling in the promoter region. However, several observations are not easily reconciled with such a simple 'recruitment-by-methylation' model. First, peak levels of all PRC1 components are present at PREs and, apart from Pc, very little binding is observed outside of PREs. Second, excision of PRE sequences from a PRE reporter gene during development leads to a rapid loss of silencing, suggesting that transcriptional repression requires the continuous presence of PREs and the proteins that are bound to them. A second, more plausible scenario would therefore be that DNA-binding factors first target PcG protein complexes to PREs, and that these PRE-tethered complexes then interact with trimethylated nucleosomes in the flanking chromatin in order to repress transcription. For example, it is possible that bridging interactions between the Pc chromodomain in PRE-tethered PRC1 and H3-K27me3-marked chromatin in the promoter or coding region permit other PRE-tethered PcG proteins to recognize the chromatin interval across which they should act, e.g., to inhibit nucleosome remodeling in the case of PRC1 or to trimethylate H3-K27 at hypomethylated nucleosomes in the case of PRC2 (Papp, 2006).

The analysis of a HOX gene in developing Drosophila suggests that histone trimethylation at H3-K27, H3-K9, and H4-K20 in the promoter and coding region plays a central role in generating and maintaining of a PcG-repressed state. Contrary to previous reports, the current findings provide no evidence that H3-K27 trimethylation is specifically localized at PREs and could thus recruit PRC1 to PREs; widespread H3-K27 trimethylation is found across the whole transcription unit. The data presented in this study provide evidence that PREs serve as assembly platforms for PcG protein complexes such as PRC2 that act over considerable distances to trimethylate H3-K27 across long stretches of chromatin. The presence of this trimethylation mark in the chromatin that flanks PREs may in turn serve as a signal to define the chromatin interval that is targeted by other PRE-tethered PcG protein complexes such as PRC1. The results reported here also provide a molecular explanation for the previously reported antirepressor function of trxG HMTases; selective binding of Ash1 to the active HOX gene blocks PcG repression by preventing PRC2 from trimethylating the promoter and coding region. It is possible that the extended domain of combined H3-K27, H3-K9, and H4-K20 trimethylation creates not only the necessary stability for transcriptional repression, but that it also provides the molecular marks that permits PcG repression to be heritably maintained through cell division (Papp, 2006).

Polycomb complexes and the propagation of the methylation mark at the Drosophila Ubx gene

Polycomb group proteins are transcriptional repressors that control many developmental genes. The Polycomb group protein Enhancer of Zeste has been shown in vitro to methylate specifically lysine 27 and lysine 9 of histone H3 but the role of this modification in Polycomb silencing is unknown. This study shows that H3 trimethylated at lysine 27 is found on the entire Ubx gene silenced by Polycomb. However, Enhancer of Zeste and other Polycomb group proteins stay primarily localized at their response elements, which appear to be the least methylated parts of the silenced gene. These results suggest that, contrary to the prevailing view, the Polycomb group proteins and methyltransferase complexes are recruited to the Polycomb response elements independently of histone methylation and then loop over to scan the entire region, methylating all accessible nucleosomes. It is proposed that the Polycomb chromodomain is required for the looping mechanism that spreads methylation over a broad domain, which in turn is required for the stability of the Polycomb group protein complex. Both the spread of methylation from the Polycomb response elements, and the silencing effect can be blocked by the gypsy insulator (Kahn, 2006).

The experiments described in this study show clearly that all three PcG proteins tested, Pc, Psc, and E(z), are preferentially located at the PREs. This specificity is clearest and most sharply delineated in the case of Psc and E(z). In the case of PC, the peaks centered at the PREs are much broader, including secondary peaks, and although the binding detected at other Ubx regions decreases to low values, it never reaches the level seen at control sites such as the white gene that possess no PRE. The second basic conclusion from these experiments is that, in contrast to the localization of PcG proteins, the H3 me3K27 profile forms a broad domain that includes the entire Ubx transcription unit and upstream regulatory region. The third important observation is that, contrary to previously published accounts, the PREs themselves appear to contain the lowest levels of me3K27 of the entire domain. This surprising result will be considered first. The lack of apparent methylation at the PREs does not depend on the antibody used or on the level of cross-linking. Comparable results were obtained with two different anti-me3K27 antibodies and with anti-me3K9. Furthermore, the fact that a similar result was obtained with antibody against total histone H3 or histone H2B suggests that nucleosomes are underrepresented at the bxd PRE core. The result is not because of lack of accessibility to the histone or to the epitope: GAGA factor bound to the PRE appears as easily accessible as GAGA factor bound to the Ubx promoter. Salt extraction of the PcG complexes before cross-linking does not qualitatively change the me3K27 binding profile (Kahn, 2006).

In sum, careful quantitative analysis of ChIP indicates that while PcG proteins are principally localized at the PRE, the histone H3 methylation they produce is distributed over the entire Ubx gene. It is evident from this and from the undermethylation of the PRE core that that K27 methylation does not, by itself, recruit PcG complexes. This does not preclude an important role for methylation in PcG binding and silencing but suggests that the relationship between the two requires a more dynamic model (Kahn, 2006).

PREs have been shown to recruit PcG complexes and to produce new binding foci detectable in polytene chromosomes. It is not surprising therefore to find the three PcG proteins tested are associated with the two Ubx PREs. A much smaller peak in microarray profiles for all three proteins can be discerned in the vicinity of the Ubx 3'-exon but its significance is unknown. More surprising was the striking difference between the distributions of Psc and E(z) and that of Pc. E(z) and Psc belong to two different complexes that do not co-precipitate (except in the very early embryo) but are both recruited to the PRE. Pc and Psc are core components of the PRC1-type of PcG complex yet, while Psc is detected almost exclusively at the PREs, Pc has a much broader distribution peaking at the PREs but tailing over considerable distances along the Ubx gene and regulatory regions. The simplest interpretation of this is that a second type of complex containing Pc but not Psc is recruited by a different mechanism to the rest of the Ubx sequences. Alternatively, the same complex, containing both proteins is involved in both cases but the nature of the chromatin contact is different, such that in one case both proteins are well cross-linked to the chromatin but in the second case only Pc is efficiently cross-linked (Kahn, 2006).

Just as striking is the fact that, although the E(z) complex is responsible for the H3 K27 methylation spread over the entire Ubx gene, the E(z) protein is found localized at the PREs. It is concluded that the E(z) complex methylates the Ubx domain by a hit-and-run type of mechanism. Because the methylation is stable, the E(z) complex needs only visit each nucleosome once on the average every cell cycle. It is noted that E(z)-dependent histone H3 K27 dimethylation is highly abundant and widely distributed in the genome but E(z) complexes are not associated with it. Where then does the E(z) that methylates PcG target genes come from? While more complicated scenarios may be imagined, the simplest one involves the E(z) complex bound at the PRE (Kahn, 2006).

It is supposed that the PcG complexes are recruited to PREs by DNA-binding proteins independently of histone methylation. To methylate the entire Ubx domain, the E(z)/ESC histone MTase complex might then detach from the PRE and slide along the chromatin from one nucleosome to the next to survey the entire domain. However, it more likely that both the Pc and the E(z) complexes assembled at the PRE remain associated with the PRE sequences, where they are detected, but that the whole PRE assembly loops over to scan the entire region, methylating all accessible nucleosomes. Such looping models were originally proposed to be mediated by sites of weak PcG complex formation. In a modern version of this type of model, the looping activity would be mediated by the distinct affinity of the Pc protein for histone H3, which is greatly increased by K27 methylation. These affinities would mediate transient interactions of the complexes bound at the PRE with the surrounding chromatin and allow continuous scanning and methylation of unmethylated or hemimethylated nucleosomes (Kahn, 2006).

In such a model, ChIP experiments would always detect a strong PcG presence at the PRE but PcG interactions with the rest of the repressed gene would be distributed over a region, which is very large in the case of the Ubx gene, smaller in the case of the YGPhsW transposon, hence the signal detected at any one site would be weaker in proportion to the extent of the methylated domain. In addition, the contacts between the PcG complex and the rest of the silenced gene would be much more transient than contacts with the PRE. Together, these considerations would explain why ChIP assay gives such low values for PcG proteins over the rest of the methylated domain (Kahn, 2006).

The looping mechanism proposed for the PRE-bound complex strongly resembles that suggested for the interaction between the Locus Control Region and β-globin genes or for enhancer-promoter interactions. Like these interactions, the silencing of a promoter by the PRE is blocked by insulator elements. In transposon constructs, the insertion of a gypsy Su(Hw) insulator between PRE and promoter blocks the spread of methylation. At present, the mechanism of insulator action is not clear and how the block to methylation is achieved is unknown. It is possible that the insulator element produces topological constraints that prevent the PRE-bound complexes from looping beyond the insulator. This would be consistent with the observation that a significant level of Pc presence becomes detectable over the yellow gene when the insulator block is lifted (Kahn, 2006).

Although the data argue against a principal role of histone methylation in the recruitment of Polycomb proteins to their response elements, it seems to be important for both transcriptional repression and stable association of PcG proteins with chromatin. Loss of catalytic E(z) function eventually results in derepression of HOX genes and dissociation of PcG proteins from polytene chromosomes. It is speculated that once the me3K27 domain is established, modified nucleosomes will pave the way for looping interactions of the PRE-bound PcG proteins with the parts of the silenced gene including promoter or enhancer regions. Silencing might then result from hit-and-run interactions with either or both, possibly even resulting in methylation of the associated factors. Alternatively or in addition, trimethylation of K27 and possibly K9 may directly interfere with the signaling cascade of consecutive histone modifications that guide the multistep process of transcription initiation and elongation. Since histone methylation is thought to persist through cell division its immediate presence at the very beginning of the subsequent interphase might win the time necessary for the full assembly of PcG complexes on the PREs before competing transcription has taken over (Kahn, 2006).

Genome-wide chromatin occupancy analysis reveals a role for ASH2 in transcriptional pausing

An important mechanism for gene regulation involves chromatin changes via histone modification. One such modification is histone H3 lysine 4 trimethylation (H3K4me3), which requires histone methyltranferase complexes (HMT) containing the trithorax-group (trxG) protein ASH2. Mutations in ash2 cause a variety of pattern formation defects in the Drosophila wing. This study identified genome-wide binding of ASH2 in wing imaginal discs using chromatin immunoprecipitation combined with sequencing (ChIP-Seq). The results show that genes with functions in development and transcriptional regulation are activated by ASH2 via H3K4 trimethylation in nearby nucleosomes. The occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription was characterized. ASH2 occupancy correlates with phosphorylated forms of RNA Polymerase II and histone activating marks in expressed genes. Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies. Finally, specific motifs were identified associated with ASH2 binding in genes that are differentially expressed in ash2 mutants. The data suggest that recruitment of the ASH2-containing HMT complexes is context specific and points to a function of ASH2 and H3K4me3 in transcriptional pausing control (P´rez-Lluch, 2011).

Work using various model organisms and cultured cells has provided high-resolution profiles of histone modifications and transcription factor binding across different genomes. This study used direct sequencing of ChIP DNA from wing disc to analyse ASH2 function. Because the cell composition of isolated wing disc tissue is rather homogeneous, it has been possible to set apart several attributes. First, ASH2 occupancy correlates with the presence of phosphorylated forms of RNA Polymerase II and activating histone marks in expressed genes. But, a direct role for ASH2 in gene repression as well cannot be dismissed, since ASH2 also targets silenced genes. In support of this, ASH2-interacting proteins HCF-1 and dMyc are involved in both transcriptional activation and repression. Alternatively, silenced ASH2 target genes could be arrested in an intermediate ready-to-go state of transcription, which may be activated by external signals. Second, the results agree with previous observations in Drosophila and Xenopus embryos, where dually marked domains do not seem to be a common feature. It has been reported that bivalently marked chromatin, containing both H3K4 and H3K27 trimethylation, is a hallmark of developmentally regulated silenced promoters in mammalian embryonic stem cells. In contrast, these marks can be coupled to the differential expression pattern of several genes throughout the wing disc, therefore indicating the presence of each individual mark in different cells. A recent report using a similar genome-wide approach in undifferentiated cell-enriched Drosophila testis reveals that differentiation-associated genes are also linked with monovalent modifications. Third, ASH2 binding was used together with activating marks of transcription as a powerful tool to identify previously unannotated genes (P´rez-Lluch, 2011).

The actively transcribed genes in the wing disc are occupied by nucleosomes with histone modifications that are hallmarks of both initiation and elongation. This study has uncovered a positive correlation between activating marks of transcription (both H3K4me3 and H3K36me3) and ASH2 occupancy. This study has also determined that ASH2 contributes to H3K4me3 in nearby nucleosomes. H3K4me3 is associated with the TSS of active genes, whereas H3K27me3 spreads over large regions of chromatin to promote silencing and H3K36me3 is found in actively transcribed regions. Only genes containing H3K36me3 undergo further elongation and produce mature transcripts (P´rez-Lluch, 2011).

Transcriptional regulation is a multistep process controlled by a large complex machinery at the level of recruitment, initiation, pausing and elongation of RNA Polymerase II. A series of recent genome-wide studies indicate that many developmental and inducible genes, prior to their expression, contain RNA Polymerase II bound predominantly in their promoter proximal regions in a stalled state. Nevertheless, not only silenced genes show an enrichment of the RNA Polymerase II density at their TSS as the stalled polymerase is also present at this region in active genes. The presence of ASH2 and H3K4me3 together with PolIIS5P at the TSS of expressed genes is consistent with previous reports proposing that promoter-proximal stalling serves not only to fully repress but also to attenuate transcription of active genes. As recently described, transient stalling of polymerase is a general feature of early elongation, even in highly active genes (P´rez-Lluch, 2011).

The analysis of ash2 mutant flies indicates that ASH2 is performing its canonical function promoting H3K4me3, regardless of the effect on the transcriptional state of its target genes and the context specificity of its recruitment to promoters. In light of the results obtained with RNA Polymerase II modifications in the mutants, it is concluded that ASH2 influences different aspects of transcription. The specific binding motifs identified in differentially regulated genes, together with the co-occupancy of ASH2 and PolIIS5P at the TSS, suggests a role in transcription initiation. Nevertheless, the reduction of PolIIS5P in mutant flies points to a fast escape from stalling in the absence of ASH2 (P´rez-Lluch, 2011).

Distinct sets of accessory factors are associated with polymerase stalling and its escape from this state, acting either by direct interaction with RNA Polymerase II, or by manipulating the chromatin environment. Among these factors, there are proteins associated with polymerase stalling, such as the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF), and others that contribute to escape from stalling, such as the positive transcription-elongation factor-b (P-TEFb) complex and the general transcription factors TFIIS and TFIIF. It remains to be elucidated whether ASH2 interacts directly with some of these factors. However, NELF and GAF have been found linked to promoter-proximal pausing at many genes in Drosophila. A connection between ASH2 and polymerase stalling in developmental genes could, therefore, be envisioned through GAF, since it is known that GAF is a recruiter of PcG and trxG complexes to DNA. In fact, about half of the downregulated genes in ash2 mutants presenting GAGA sites are NELF targets. Furthermore, it has been recently reported that c-Myc regulates RNA Polymerase II pause release by recruiting P-TEFb to its target genes, and it is known that ASH2 interacts with Myc in flies. The enrichment of Ebox and Mnt/Max motifs found in upregulated genes in ash2 mutants (see Characterization of ASH2 binding regions) points to a function of ASH2 through Myc in their transcriptional regulation. A subset of these motifs was characterized in H3K4me3 regions. It has been possible to associate these motifs with downregulated and upregulated genes in ash2 mutants, suggesting differential transcriptional regulation (P´rez-Lluch, 2011).

Several effector proteins that can bind to H3K4me3 determine the functional outcome of this histone modification. The activities of these binding proteins range from activation and repression of transcription, chromatin remodelling or splicing efficiency among others. An additional role for ASH2 during transcript elongation and maturation should not be excluded. Indeed, it has been suggested that methylated H3K4 serves to facilitate the competency of pre-mRNA maturation through the bridging of spliceosomal components. The fact that downregulated and upregulated genes in ash2 mutants display clear differences in size and genomic organization (gene size, alternative isoforms and number of exons) suggests they may be regulated in a different way during transcription and processing of RNA. Finally, recent reports indicate an association of RNA Polymerases II and III at promoter regions of housekeeping genes and a recruitment of RNA Polymerase III through Myc interacting with the cofactor BRF has also been described. However, preliminary experiments discard the implication of other polymerases in the transcription of these housekeeping genes in the absence of ASH2. Taken together, these results support a model in which an ASH2-containing complex would act at different levels of transcriptional regulation (P´rez-Lluch, 2011).

Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture

Genome-wide studies has identified two enhancer classes in Drosophila that interact with different core promoters: housekeeping enhancers (hkCP) and developmental enhancers (dCP). It is hypothesized that the two enhancer classes are occupied by distinct architectural proteins, affecting their enhancer-promoter contacts. It was determined that both enhancer classes are enriched for RNA Polymerase II, CBP, and architectural proteins but there are also distinctions. hkCP enhancers contain H3K4me3 and exclusively bind Cap-H2, Chromator, DREF and Z4, whereas dCP enhancers contain H3K4me1 and are more enriched for Rad21 and Fs(1)h-L. Additionally, the interactions of each enhancer class were mapped utilizing a Hi-C dataset with <1 kb resolution. Results suggest that hkCP enhancers are more likely to form multi-TSS interaction networks and be associated with topologically associating domain (TAD) borders, while dCP enhancers are more often bound to one or two TSSs and are enriched at chromatin loop anchors. The data support a model suggesting that the unique architectural protein occupancy within enhancers is one contributor to enhancer-promoter interaction specificity (Cubenas-Potts, 2017).

This study characterize the protein occupancy, chromatin interactions and architecture profiles for the two enhancer classes found in Drosophila. Each enhancer class has distinct H3K4 methylation states, is bound by both common and distinct architectural proteins, and is involved in distinct types of chromatin interactions. First, it was established that hkCP enhancers exclusively bind CAP-H2, Chromator, DREF and Z4, while dCP enhancers do not and are preferentially enriched for but not exclusively bound by Fs(1)h-L and Rad21. In addition, hkCP enhancers are more likely than dCP enhancers to associate with multiple TSSs, which promotes a higher transcriptional output. Finally, hkCP enhancers preferentially associate with topologically associating domain (TAD) borders, whereas dCP enhancers are enriched at chromatin loop anchors present inside TADs. Interestingly, enhancers activated by both core promoters exhibit more hkCP enhancer like characteristics, indicating that the both CP enhancers may represent an intermediate among the distinctive hkCP and dCP enhancers. Altogether, these results provide strong correlative evidence, supporting a model suggesting that architectural proteins are critical regulators of enhancer-promoter interaction specificity and that the interactions between enhancers and promoters significantly contribute to the generation of 3D chromatin architecture (Cubenas-Potts, 2017).

The importance of architectural proteins in regulating enhancer-promoter interactions in Drosophila is supported by the observation that the vast majority of architectural protein sites present in the genome correspond to enhancers and promoters. Historically, architectural proteins were identified as insulators, which were functionally demonstrated to block enhancer-promoter interactions. The insulator function of architectural proteins correlates with their enrichment at TAD borders. However, several lines of evidence, including ChIA-PET analysis of CTCF- and cohesin-mediated interactions in mammals, suggest that these architectural proteins help mediate long range contacts among regulatory sequences. In Drosophila this study observed that nearly all of the Group 1 and Group 2 architectural protein sites are associated with enhancers or promoters defined by STARR-seq, TSSs or CBP peaks, suggesting that architectural proteins help mediate enhancer-promoter interactions. Notably, Group 3 architectural proteins include the classic insulator proteins CTCF, CP190, Mod(mdg4) and SuHw, and at least 25% of their peaks cannot be explained by enhancers or promoters. It is interesting to speculate that the non-enhancer-promoter sites may be involved in more classical insulator functions or contributing to the chromatin architecture of inactive regions of the genome (Cubenas-Potts, 2017).

The conclusion that architectural proteins are critical regulators of the specificity between enhancers and promoters is supported by two main lines of evidence. First, the current results demonstrate a strong correlation between each enhancer class and distinct architectural protein subcomplexes. Functional evidence supporting this conclusion comes from mutational analyses of the DRE motif in the distinct enhancer classes, which likely recruits DREF and the other hkCP enhancer associated architectural proteins. Zabidi (2015) demonstrated that the tandem DRE motif alone was sufficient to enhance expression of the housekeeping core promoter and that mutation of DRE motifs within an hkCP enhancer reduced its promoter interactions in a luciferase assay. Furthermore, addition of a DRE motif to a dCP enhancer changed its promoter specificity. Because DREF and potentially BEAF-32 bind to the DRE motif, these results strongly support a model suggesting that the differential occupancy of Cap-H2, Chromator, DREF and Z4 in the two enhancer classes is a critical regulator of their specific interactions with the core promoter types. However, the data cannot discount the notion that unique transcription factor binding at proximal TSSs also contribute to the specificity of enhancer-promoter interactions. Although hkCP enhancer identity is most highly correlated with CAP-H2, Chromator, DREF and Z4 localization, these four architectural proteins are not found in isolation within hkCP enhancers. BEAF-32 and CP190 are also strongly enriched in hkCP enhancers, which are also associated with high occupancy APBSs and TAD borders. Thus, the full architectural protein complement at hkCP enhancers is far more complex than the four hkCP-specific architectural proteins. In addition, architectural proteins that are truly unique to dCP enhancers were not detected. Because dCP enhancers exhibit higher cell type specificity, it cannot be discounted that there are additional dCP enhancers present in the Drosophila genome that were not identified by STARR-seq and thus, excluded from this analysis. From these studies, it is unclear if the enrichment of Fs(1)h-L and Rad21, particularly because Fs(1)h-L and Rad21 are present in hkCP enhancers at lower levels, or the absence of BEAF-32, CAP-H2, Chromator, CP190, DREF and Z4 truly distinguishes the architectural protein complexes found at dCP enhancers. In the future, careful biochemical analyses will be required to gain a comprehensive understanding of the complete organization of architectural protein subcomplexes associated with each enhancer class (Cubenas-Potts, 2017).

hkCP enhancers are associated with multi-TSS chromatin interactions and TAD borders. The promoter-clustering by hkCP enhancers results in a dose-dependent increase in transcriptional output for the interacting genes. Thus, one likely molecular mechanism by which hkCP enhancers promote robust transcriptional activation is by increasing the local concentration of RNA Polymerase II and general transcription factors (GTFs) by bringing multiple TSSs into close proximity. It is interesting that the hkCP enhancers, which form promoter clusters, are associated with TAD borders. It is speculated that the hkCP enhancer interactions involve inter-TAD contacts within the A-type compartment, indicative of the formation of transcription factories (70). From this analysis, it is unclear if the hkCP enhancers alone are sufficient for the formation of the 3D interactions or the neighboring TSSs and their associated transcription factors are also contributing to these contacts. It is hypothesized that the genes recruited to the factories contain the housekeeping promoter motifs (DRE, Ohler 1, Ohler 6 and TCT) and that the hkCP enhancer residents Cap-H2, Chromator, DREF and Z4, are critical to the formation of these 3D contacts (Cubenas-Potts, 2017).

dCP enhancers are more likely to be present within TADs and are enriched on the subTAD-like chromatin loop anchors. dCP enhancers do not form promoter clusters, but are more likely to interact with individual TSSs. One possible explanation for this observation is that the genes interacting with dCP enhancers require the binding of sequence-specific transcription factors, and increasing the concentration of GTFs and RNA polymerase II is not an effective mechanism to promote transcriptional output. The chromatin loop association is consistent with dCP enhancers forming a strong contact with a single TSS. However, it is acknowledged that dCP enhancers are likely one of multiple molecular mechanisms contributing to chromatin loop formation. Surprisingly, the chromatin loops that were observed in Drosophila are distinct from the chromatin loops described in humans. A recent study reported approximately 10,000 chromatin loops in the genome of GM12878 lymphoblastoid cells, but this study detected only 458 chromatin loops in Drosophila utilizing a similar method. The reason why there are so few chromatin loops in Drosophila compared to humans is unclear. It is possible that chromatin loops represent a more precise level of architecture within TADs between specific enhancers and promoters in mammals, but because TADs are significantly smaller in flies (median size 32.5 kb compared to 880 kb in mice, the chromatin loops are not as prominent or easily detected in the Drosophila genome. Notably, it appears that the chromatin loops are generated by different architectural proteins in the two species. The chromatin loops in humans are anchored by convergent CTCF motifs, while the results presented in this study demonstrate that the chromatin loop anchors in Drosophila are depleted of CTCF. Because the chromatin loops in Drosophila show a strong enrichment for Fs(1)h-L, a Brd4 homolog, and the architectural proteins Rad21, Nup98, TFIIIC and Mod(mdg4), it is possible that a combination of transcription and architectural proteins is required for chromatin loop formation in flies, which may be different from mammals . Altogether, it is clear that dCP enhancers are involved in individual contacts with TSSs and are likely one mechanism by which chromatin loops form in Drosophila (Cubenas-Potts, 2017).

Surprisingly, only ~20% and ~12.5% of all hkCP enhancer and ~7.5% and ~8.5% of dCP enhancer interactions involve a TSS or enhancer on the opposite anchor, respectively. The biological significance of the enhancer to non-TSS association is unclear. One possible explanation is that current methods for identifying statistically significant interactions are not sufficiently robust and that many of the enhancer to non-TSS interactions are not representative of biologically significant contacts. However, it cannot be discounted that the non-TSS interactions mediated by enhancers are real and the biological significance of these contacts remains to be determined. Throughout this analysis, the patterns of TSS interactions were compared with each enhancer class instead of drawing conclusions about the absolute number of TSSs bound per enhancer, minimizing the impact of any non-specific interactions within the data. Additional molecular studies for the various type of enhancer interactions (enhancer to promoter, enhancer to non-TSS, etc.) will be required to evaluate the various biological contributions of each (Cubenas-Potts, 2017).

This study found that the functional differences between enhancers that activate housekeeping versus developmental genes are reflected in their chromatin and architectural protein composition, and in the type of interactions they mediate. hkCP enhancers are marked by H3K4me3, associate with TAD borders, and mediate large TSS-clustered interactions to promote robust transcription. This class of enhancers contain the architectural proteins CAP-H2, Chromator, DREF and Z4. In contrast, dCP enhancers are marked by H3K4me1, associate with chromatin loop anchors and are more commonly associated with single TSS-contacts. dCP enhancers are depleted of the hkCP-specific architectural proteins and show an enrichment for Fs(1)h-L and Rad21. The results support a model suggesting that the unique occupancy of architectural proteins in the distinct enhancer classes are key contributors to the types of interactions that enhancers can mediate genome-wide, ultimately affecting enhancer-promoter specificity and 3D chromatin organization. In the future, further characterization of the broadly defined housekeeping and developmental enhancers into smaller subclasses may yield additional levels of regulation and formation of unique architectural protein and transcription factor protein complexes as key mediators of long range chromatin contacts (Cubenas-Potts, 2017).

Cdk9 is an essential kinase in Drosophila that is required for heat shock gene expression, histone methylation and elongation factor recruitment

Phosphorylation of the large RNA Polymerase II subunit C-terminal domain (CTD) is believed to be important in promoter clearance and for recruiting protein factors that function in messenger RNA synthesis and processing. P-TEFb is a protein kinase that targets the (CTD). The goal of this study was to identify chromatin modifications and associations that require P-TEFb activity in vivo. The catalytic subunit of P-TEFb, Cdk9, was knocked down in Drosophila using RNA interference. Cdk9 knockdown flies die during metamorphosis. Phosphorylation at serine 2 and serine 5 of the CTD heptad repeat were both dramatically reduced in knockdown larvae. Hsp 70 mRNA induction by heat shock was attenuated in Cdk9 knockdown larvae. Both mono- and trimethylation of histone H3 at lysine 4 were dramatically reduced, suggesting a link between CTD phosphorylation and histone methylation in transcribed chromatin in vivo. Levels of the chromo helicase protein CHD1 were reduced in Cdk9 knockdown chromosomes, suggesting that CHD1 is targeted to chromosomes through P-TEFb-dependent histone methylation. Dimethylation of histone H3 at lysine 36 was significantly reduced in knockdown larvae, implicating CTD phosphorylation in the regulation of this chromatin modification. Binding of the RNA Polymerase II elongation factor ELL was reduced in knockdown chromosomes, suggesting that ELL is recruited to active polymerase via CTD phosphorylation (Eisenberg, 2006).

Cdk9, the catalytic subunit of P-TEFb, is highly conserved among eukaryotes. The yeast kinases Ctk1 and Bur1 are both homologs of Cdk9, and both are CTD kinases in Drosophila, although loss of Bur1 has no effect on CTD phosphorylation yeast. Bur1 is essential but Ctk1 is not (Eisenberg, 2006).

RNAi knockdown of Cdk9 in transgenic flies results in lethality at the pupal stage. This is considerably later than the embryonic lethality reported for C. elegans RNAi knockdown of Cdk9. While this difference could reflect differences in the requirements for Cdk9 in these organisms, it is more likely that differences in timing or efficiency of RNAi, Cdk9 protein turnover and/or maternal Cdk9 loading accounts for the much later lethality in knockdown flies. Nevertheless, these results confirm and extend the finding that P-TEFb is essential in metazoan development (Eisenberg, 2006).

In contrast, Cdk9 homologs in fission yeast and Neurospora are not essential. Since CTD phosphorylation has been linked to promoter clearance, pre-mRNA processing and chromatin modification, it is not possible to say what aspect of P-TEFb activity is essential in metazoa. RNAi knockdown of the Drosophila Cdk9 in cultured cells causes arrest of the cell cycle at the G1-S transition, implicating this kinase in cell cycle control. It is unlikely that cell cycle arrest is causing the lethality in the knockdown flies, since cell cycle mutations in Drosophila generally are associated with reduced or missing imaginal discs, and the discs in Cdk9 knockdown larvae appear overtly normal. The finding that Hsp70 transcripts are reduced in Cdk9 knockdown larvae is consistent with the reduced Hsp70 transcription previously reported in Cdk9 RNAi cultured cells. Hsp 70 is not essential in Drosophila, but the effects on Hsp70 suggest that defects in gene expression could underlie the essential requirement for Cdk9 in Drosophila development (Eisenberg, 2006).

Cdk9 knock-down flies show dramatic reductions in both serine 2 and serine 5 phosphorylation. In contrast, flavopiridol treatment of cultured cells has been found to selectively reduce serine 2 phosphorylation. The significance of this difference is unclear, but could reflect differences in experimental protocol. For example, flavopiridol treatments were limited to 15-20 min, while RNAi knockdown third instar larvae are subject to knockdown conditions for several days before assay. Longer periods of Cdk9 inactivation may be required for reduction in serine 5 phosphorylation. Alternatively, it is possible that knockdown of Cdk9 protein levels results in inhibition of TFIIH, the other known CTD kinase. Regardless of the mechanism, the RNAi knockdown clearly results in reduced phosphorylation of the CTD, enabling a test of the consequences of loss of CTD phosphorylation on chromatin modification and recruitment of RNA Polymerase II-associated factors (Eisenberg, 2006).

Loss of CTD phosphorylation in Cdk9 knockdown larvae is associated with reduced binding of the RNA Polymerase II elongation factor ELL genome-wide. ELL is broadly co-localized with phosphorylated RNA Polymerase II on polytene chromosomes, and is rapidly recruited to heat shock loci after a brief heat shock. These results suggest that the efficient recruitment of ELL to transcribed loci requires CTD phosphorylation. Whether this reflects a direct interaction of ELL with the CTD is unknown (Eisenberg, 2006).

Despite the fact that Elongin A affects the same kinetic parameter in RNA Polymerase II catalysis as ELL, Elongin A binding is not reduced by loss of CTD phosphorylation. As with ELL, the nature of Elongin A binding to RNA Polymerase II is unknown, but these observations suggest their binding can be distinguished by sensitivity to the phosphorylation state of the CTD. Since no increase of Elongin A was observed under conditions of reduced ELL binding, it seems unlikely that ELL and Elongin A compete for RNA Polymerase II binding (Eisenberg, 2006).

Spt4 and Spt5 are subunits of DSIF, which is implicated in the regulation of RNA Polymerase II elongation. Previous work suggested that reduced serine 2 phosphorylation of the RNA Polymerase II CTD has no effect on Spt5 recruitment to a heat shock gene in cultured cells (Ni, 2004). In Cdk9 knockdown flies, in which both serine 2 and 5 phosphorylation are reduced, the chromosomal distribution of Spt5 is unchanged genome-wide. This is consistent with previous reports that Spt5 interacts with both phosphorylated and unphosphorylated RNA Polymerase II (Wen, 1999; Lindstrom, 2001; Lindstrom, 2003; Eisenberg, 2006 and references therein).

The chromo domain motif is a binding site for methylated histone tails. The role of the CHD1 chromo domain in methylated histone binding is controversial. However, recent structural data determined that the double chromo domain of mammalian CHD1 binds methylated H3K4 in vitro (Flanagan, 2005). This study shows that Cdk9 knockdown leads to a loss of chromosomal CHD1. This observation is most easily interpreted as the result of loss of H3K4 methylation that also occurs in Cdk9 knockdown chromosomes. Thus, the finding reported in this study lends support to the in vitro binding data and strongly suggests that the chromo domain-methylated histone interaction plays a dominant role in targeting CHD1 to active chromatin in vivo (Eisenberg, 2006).

The observation that both H3K4 and H3K36 methylation are significantly reduced in Cdk9 knockdown chromosomes suggests a linkage between phosphorylation of the CTD and histone methylation at transcribed genes. In this respect, Cdk9 subsumes activities found in yeast Bur1/Bur2 and yeast Ctk1. Since no significant difference was observed in ASH1 protein levels on Cdk9 knockdown chromosomes, a model is favored in which Cdk9-dependent RNA Polymerase II elongation plays a mechanistic role in H3 tail methylation. In this model, RNA Polymerase II passage destabilizes histone-DNA contacts, making the histones better substrates for efficient methylation. Reduced CTD phosphorylation would lead to reduced rates of RNA Polymerase II transcription genome-wide, resulting in reduced efficiency of histone tail methylation. While the mechanism connecting CTD phosphorylation to RNA Polymerase II elongation rate is likely to be complex in vivo, the observation that reduced CTD phosphorylation is associated with reduced dELL binding suggests that loss of dELL association could be a contributing factor (Eisenberg, 2006).

Mutation in Ash1 in Drosophila results in loss of all detectable H3K4 methylation, but has no effect on H3K36 methylation. This is consistent with independent mechanisms for these two chromatin modifications. A Polymerase II passage model provides a simple mechanism to account for similar effects on both modifications based on substrate availability (Eisenberg, 2006).

A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities

Polycomb response elements (PREs) are specific cis-regulatory sequences needed for transcriptional repression of HOX and other target genes by Polycomb group (PcG) proteins. Among the many PcG proteins known in Drosophila, Pleiohomeotic (Pho) is the only sequence-specific DNA-binding protein. To gain insight into the function of Pho, Pho protein complexes were purified from Drosophila embryos and it was found that Pho exists in two distinct protein assemblies: a Pho-dINO80 complex containing the Drosophila INO80 nucleosome-remodeling complex, and a Pho-repressive complex (PhoRC) containing the uncharacterized gene product Scm-related gene containing four mbt domains (dSfmbt). Analysis of PhoRC reveals that dSfmbt is a novel PcG protein that is essential for HOX gene repression in Drosophila. PhoRC is bound at HOX gene PREs in vivo, and this targeting strictly depends on Pho-binding sites. Characterization of dSfmbt protein shows that its MBT repeats have unique discriminatory binding activity for methylated lysine residues in histones H3 and H4; the MBT repeats bind mono- and di-methylated H3-K9 and H4-K20 but fail to interact with these residues if they are unmodified or tri-methylated. These results establish PhoRC as a novel Drosophila PcG protein complex that combines DNA-targeting activity (Pho) with a unique modified histone-binding activity (dSfmbt). It is proposed that PRE-tethered PhoRC selectively interacts with methylated histones in the chromatin flanking PREs to maintain a Polycomb-repressed chromatin state (Klymenko, 2006).

The regulation of gene expression by Polycomb group (PcG) and trithorax group (trxG) proteins represents a paradigm for understanding the establishment and maintenance of heritable transcriptional states during development. PcG and trxG genes were first genetically identified as regulators that are required for the long-term maintenance of HOX gene expression patterns in Drosophila. PcG proteins keep HOX genes silenced in cells in which they must stay inactive, whereas trxG proteins maintain the active state of these genes in appropriate cells. This regulatory relationship is conserved in vertebrates, where PcG and trxG proteins also regulate HOX gene expression. In addition, mammalian PcG and trxG proteins have also been implicated in X-chromosome inactivation, hematopoietic development, control of cell proliferation, and oncogenic processes (Klymenko, 2006).

Drosophila HOX genes are among the best-studied target genes of the PcG/trxG system. Different studies have led to the identification of specific cis-regulatory sequences in HOX genes that are called Polycomb response elements (PREs) and are required for silencing by PcG proteins. PREs are typically several hundred base pairs in length, and they function as potent transcriptional silencer elements in the context of HOX reporter genes as well as in a variety of other reporter gene assays. This operational definition of PREs is complemented by their classification as DNA sequences to which PcG proteins bind, directly or indirectly. Among the 14 cloned Drosophila PcG genes, only Pleiohomeotic (Pho) and Pho-like (Phol) encode sequence-specific DNA-binding proteins. Pho and Phol bind the same DNA sequence, and while the two proteins act to a large extent redundantly, double mutants show severe loss of HOX gene silencing. DNA-binding sites for Pho and Phol are present in all PREs that have been characterized to date, and mutational analyses of these binding sites have shown that they are essential for silencing by PREs. In contrast, none of the other 12 characterized PcG proteins bind DNA in a sequence-specific manner. However, formaldehyde cross-linking studies showed that several of these proteins specifically associate with the chromatin of PREs in tissue culture cells and in developing embryos and larvae. Biochemical studies revealed that most of these non-DNA-binding PcG proteins are components of either PRC1 or PRC2, two distinct PcG protein complexes that have recently been purified and characterized. Specifically, PRC1 contains the PcG proteins Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph), Sex combs extra/Ring (Sce/Ring), and Sex combs on midleg (Scm), whereas PRC2 contains the three PcG proteins Extra sex combs (Esc), Enhancer of zeste [E(z)], and Suppressor of zeste 12 [Su(z)12] (Klymenko, 2006).

What is the role of Pho and Phol at PREs? Biochemically purified PRC1 and PRC2 do not contain Pho or Phol. Several recent studies investigated possible physical interactions between Pho and PRC1 or PRC2 complex components. Based on coimmunoprecipitation and GST pull-down assays, it was proposed that Pho directly interacts with several different PRC1, PRC2, and SWI/SNF complex components. However, on polytene chromosomes of phol; pho double mutants, the binding of PRC1 and PRC2 to HOX genes and at most other loci is largely unperturbed (Brown, 2003), suggesting that, at least in this tissue, Pho and Phol are not strictly required for keeping PRC1 and PRC2 anchored to HOX genes (Klymenko, 2006).

To gain insight into the biological function of Pho, Pho-containing protein complexes were biochemically purified from Drosophila. The data show that Pho exists in two distinct multiprotein complexes that, contrary to expectation, do not contain any of the previously characterized PcG proteins. The functional analysis of one of these Pho complexes that was named PhoRC provides evidence that its binding to PREs is required for maintaining repressive HOX gene chromatin (Klymenko, 2006).

A tandem affinity purification (TAP) strategy was used to purify Pho protein complexes from Drosophila embryonic nuclear extracts. A transgene that expresses a TAP-tagged Pho fusion protein (Pho-TAP) was expressed under the control of the Drosophila alpha-tubulin promoter, and transgenic flies were generated. To test whether the Pho-TAP protein is functional, the transgene was introduced into the genetic background of animals homozygous for pho¹, a protein-negative allele of pho. pho¹ homozygotes die as pharate adults, but they are rescued into viable and fertile adults that can be maintained as a healthy strain if they carry one copy of the transgene expressing Pho-TAP. The Pho-TAP protein can thus substitute for the endogenous Pho protein, and this shows that the fusion protein is functional (Klymenko, 2006).

Proteins that are associated with the Pho-TAP protein were purified from embryonic nuclear extracts, following the TAP procedure. Seven different polypeptides that consistently copurified with the Pho-TAP bait protein in several independent purifications were identified through sequencing of peptides from individual protein bands by nanoelectrospray tandem mass spectrometry. In addition to Pho, the isolated protein assembly contains the product of CG31212, a protein that is most closely related to yeast INO80, the SWI/SNF2-like nucleosome-remodeling subunit in the yeast INO80 complex. The CG31212 locus as will therefore be referred to as dINO80. Five other subunits of the Pho complex were identified as Reptin (Rept), Pontin (Pon), Actin (Act), and the two actin-related proteins dArp5 and dArp8, which are encoded by CG7940 and CG7846, respectively. These five proteins represent the Drosophila homologs of five core subunits that assemble together with INO80 to form the yeast INO80 complex. Specifically, Rept and Pont are homologs of the yeast Rvb1 and Rvb2 AAA-ATPases that constitute a DNA helicase in the INO80 complex. Act, dArp5, and dArp8 are homologs of the Actin, Arp5, and Arp8 proteins, respectively, that are present in the yeast INO80 complex. Thus, it appears that a Drosophila dINO80 complex copurifies with Pho. In addition, the purified material also contained the product of CG16975, a protein that is not conserved in yeast but is closely related to the product of the murine Scm-related gene containing four mbt domains (Sfmbt); the CG16975 gene is referred to as dSfmbt. The characteristic features of mammalian Sfmbt and the Drosophila dSfmbt protein are four malignant brain tumor (MBT) repeats and a sterile alpha motif (SAM) domain. The Drosophila genome encodes two other proteins that contain MBT repeats and show a similar domain architecture, l(3)mbt and the PcG repressor Scm. Taken together, these findings suggest that Pho exists in multiprotein assemblies that contain a dINO80 complex and dSfmbt but, unexpectedly, none of the previously characterized PcG proteins (Klymenko, 2006).

Since the yeast genome does not contain any dSfmbt-related protein, it was asked whether dSfmbt and dINO80 are part of distinct Pho protein complexes. To this end, crude embryonic nuclear extracts were fractionated by glycerol gradient sedimentation and individual fractions were probed by Western blotting with antibodies against Pho, Pho-like, dINO80, and dSfmbt. The results show that dINO80 and dSfmbt are present in separate fractions of the gradient but that Pho and Pho-like are present in both dINO80- and dSfmbt-containing fractions. dSfmbt and dINO80 thus exist in distinct protein complexes in embryonic nuclear extracts. It should be noted that Pho and Pho-like are also present in fractions that do not contain dINO80 or dSfmbt. This suggests that Pho and Pho-like also exists in soluble protein assemblies that are distinct from the complexes identified in this study, but that these assemblies are not stable enough to be isolated as complexes in the purification scheme (Klymenko, 2006).

It was asked whether components of the purified Pho complexes are associated with PREs in vivo. To this end, chromatin immunoprecipitation (X-ChIP) assays were performed. Drosophila embryos were treated with formaldehyde and DNA that was cross-linked to Pho, dSfmbt, dINO80, Reptin, Pontin, or Ph was immunoprecipitated with antibodies against these proteins. Real-time quantitative PCR was used to measure the abundance of the following endogenous and transgene PREs in the immunoprecipitates. The bxd and iab-7 PREs in the HOX genes Ultrabithorax (Ubx) and Abdominal-B (Abd-B), respectively, are well-characterized, and Pho binds to these PREs in vitro and in vivo. It has been reported that PRE_D, a 572-bp core fragment of the bxd PRE, silences a Ubx-LacZ reporter gene in imaginal discs and in embryos but that point mutations in all six Pho protein-binding sites in this fragment (PRE_{D pho mut}) completely abolish its silencing capacity (Fritsch, 1999). Therefore X-ChIP assays were performed in transformed embryos that carried either the wild-type PRE_D or the mutated PRE_{D pho mut} reporter gene; this allowed direct comparison of protein binding at the transgenic PRE with protein binding at the endogenous bxd and iab-7 PREs in the same preparation of chromatin. Specific PCR primer sets allowed X-ChIP signals at the reporter gene PRE to be distinguished from signals at the endogenous bxd PRE. It was found that Pho, Ph, and, importantly, also dSfmbt are specifically bound at the endogenous bxd and iab-7 PREs but not at sequences flanking those PREs. In contrast, binding of dINO80, Reptin, or Pontin at any of the sequences analyzed (data not shown). Pho, dSfmbt, and Ph are also bound at the PRE_D fragment in the transgene was not detected, but, strikingly, binding signals of Pho, dSfmbt, and Ph are severely reduced at the mutated PRE_{D pho mut} fragment. Taken together, these data show that Pho-dSfmbt complexes are bound at PREs in vivo and that binding of these complexes to PREs requires DNA-binding sites for Pho. Since association of dINO80 complex components with PREs was not detected in this assay, further analysis focused on the characterization of Pho-dSfmbt complexes (Klymenko, 2006).

Therefore, this study shows that the PcG protein Pho exists in two stable protein complexes, a Pho-dINO80 complex and PhoRC. Biochemical and genetic analyses identify PhoRC as a novel PcG protein complex that has a different subunit composition and molecular function than the previously described PcG complexes PRC1 and PRC2. The following conclusions can be drawn from studies of PhoRC: (1) PhoRC contains Pho and dSfmbt, and these two proteins form a very stable complex that can be purified from embryos and reconstituted from recombinant proteins. (2) PhoRC is bound to PREs in vivo, and PRE-targeting of PhoRC requires intact Pho/Pho-like DNA-binding sites. (3) A dSfmbt knockout reveals that dSfmbt is a novel PcG protein that is critically needed for HOX gene silencing. (4) The MBT repeats of dSfmbt are a novel methyl-lysine-recognizing module that selectively binds to the N-terminal tails of histones H3 and H4 if they are mono- or di-methylated at H3-K9 or H4-K20, respectively. PhoRC thus contains sequence-specific DNA-binding activity via the Pho protein and methylated histone-binding activity via dSfmbt (Klymenko, 2006).

Pho and Pho-like are the only PcG proteins with sequence-specific DNA-binding activity. Therefore, it is likely that these factors might tether PRC1 or PRC2 to PREs. Unexpectedly, biochemical purification of Pho complexes revealed that Pho exists in stable assemblies with either the PcG protein dSfmbt or components of the Drosophila INO80 complex. However, native or recombinant Pho complexes that contain PRC1 or PRC2 components were not purified. Similarly, biochemically purified PRC1 and PRC2 also do not contain Pho. PhoRC, PRC1, and PRC2 thus seem to be separate biochemical entities (Klymenko, 2006).

Reconstitution of recombinant PhoRC shows that dSfmbt binds directly to Pho or to Pho-like to form stable dimeric complexes. Coimmunoprecipitation assays indicate that such interactions also take place in Drosophila, and it was found that dSfmbt is associated with Pho or Pho-like in vivo. Moreover, dSfmbt mutants and pho-like; pho double mutants show a comparable loss of HOX gene silencing with similar kinetics. These observations are consistent with dSfmbt being needed for repression by both Pho and Pho-like. Furthermore, the X-ChIP experiments show that Pho/Pho-like DNA-binding sites in PREs are critical for binding of both Pho and dSfmbt at PREs. These data thus suggest that PhoRC is tethered to PREs by Pho or Pho-like (Klymenko, 2006).

Binding of the PRC1 subunit Ph at the bxd PRE also depends on intact Pho protein-binding sites. Could dSfmbt in PRE-bound PhoRC interact with Scm or Ph, for example, through the C-terminal SAM domain and thereby tether PRC1 to PREs? In coimmunoprecipitation experiments, no association of dSfmbt with Ph or Scm was detected. These interactions, if they exist, might be either very weak or exist only transiently. Previous studies reported direct physical interactions between Pho and PRC1 or PRC2 subunits, respectively. A possible scenario could therefore be that multiple weak interactions between Pho and dSfmbt with PRC1 and/or with PRC2 subunits might help to stabilize the binding of these complexes to PREs. It is also possible that the lack of Ph binding to the PRE transgene with mutated Pho sites reflects an indirect role of PhoRC that does not involve direct physical interactions between PhoRC and PRC1. In this context, it is worth noting that, on polytene chromosomes, binding of Ph and other PRC1 components is largely unperturbed in animals that lack both Pho and Pho-like proteins (Klymenko, 2006).

Four consecutive MBT repeats are a key feature of the dSfmbt protein. Fluorescence polarization binding assays suggest that these MBT repeats selectively bind to the N-terminal tail of histones H3 and H4 if these are mono- or di-methylated, but not if the same sites are unmethylated or tri-methylated. This novel discriminatory methyl-lysine-binding activity of MBTs is in stark contrast to the well-documented preference of chromodomains for higher, i.e., tri-methylated, binding sites in histones and could constitute an important general function of chromatin-associated MBT-containing proteins. The dSfmbt methyl-lysine interaction seems to be specific for the H3K9 and H4K20 methylation sites since matched H3 peptides that are methylated at different lysine residues (i.e., H3-K4me instead of H3-K9me) or histone tail peptides in which the methylated lysine residue is embedded in the same amino acid sequence context (i.e., ARKmeS in H3-K27me instead of ARKmeS in H3-K9me) are bound with at least 20-fold lower affinity (Klymenko, 2006).

Since these results suggest that dSfmbt is targeted to HOX gene PREs primarily through interaction with Pho, it was reasoned that binding to methyl-lysine residues in histone tails is not a primary mechanism for targeting dSfmbt to HOX genes. Moreover, recent studies provide evidence that, in the PcG-repressed state, the silenced HOX gene Ubx is tri-methylated at H3-K9, H4-K20, and H3-K27 throughout the gene, whereas lower methylated states of these sites are largely absent. What, then, is the role of Sfmbt in binding histones that are mono- or di-methylated at H3-K9 and H4-K20 in silenced HOX genes? Mono- and di-methylation of H4-K20 are very abundant modifications in Drosophila chromatin, and mass spectroscopic analyses of histones in embryos imply that lower methylated forms of histone H4 (i.e., H4-K20me2) already exist prior to becoming incorporated into chromatin during S phase. It is therefore tempting to speculate that dSfmbt, tethered to PREs by Pho, scans the flanking HOX gene chromatin for nucleosomes that are only mono- or di-methylated at H3-K9 or H4-K20 and docks onto such nucleosomes through its MBT repeats. It is hypothesized that through this bridging interaction, nucleosomes of lower methylated states might be brought into proximity to PRE-bound PRC2 and other currently unknown HMTases that are responsible for local tri-methylation of H3-K9 and H4-K20 in silenced HOX genes. According to this model, PRE-bound PhoRC would act as a 'grappling hook' that tethers mono- and di-methylated histones in silenced HOX gene chromatin to PREs to ensure that they become hypermethylated to the tri-methylated state. Such a chromatin-scanning function might be particularly important during S phase, when newly incorporated histone octamers need to become fully tri-methylated in order to maintain silencing of HOX genes (Klymenko, 2006).

Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3

Histone-tail modifications play a fundamental role in the processes that establish chromatin structure and determine gene expression. One such modification, histone methylation, was considered irreversible until the recent discovery of histone demethylases. Lsd1 was the first histone demethylase to be identified (Shi, 2004). Lsd1 is highly conserved, from yeast to humans, but its function has primarily been studied through biochemical approaches. The mammalian ortholog has been shown to demethylate monomethyl- and dimethyl-K4 and -K9 residues of histone H3. This study, along with a second study by Rudolph (2007) describes the effects of Lsd1 (Suppressor of variegation 3-3) mutation in Drosophila. The inactivation of dLsd1 strongly affects the global level of monomethyl- and dimethyl-H3-K4 methylation and results in elevated expression of a subset of genes. dLsd1 is not an essential gene, but animal viability is strongly reduced in mutant animals in a gender-specific manner. Interestingly, dLsd1 mutants are sterile and possess defects in ovary development, indicating that dLsd1 has tissue-specific functions. Mutant alleles of dLsd1 suppress positional-effect variegation, suggesting a disruption of the balance between euchromatin and heterochromatin. Taken together, these results show that dLsd1-mediated H3-K4 demethylation has a significant and specific role in Drosophila development (Di Stefano, 2007).

Su(var)3-3, the Drosophila homolog of the human LSD1 amine oxidase, demethylates H3K4me2 and H3K4me1 and facilitates subsequent H3K9 methylation by SU(VAR)3-9. Su(var)3-3 dictates the distinction between euchromatic and heterochromatic domains during early embryogenesis. Su(var)3-3 mutations suppress heterochromatic gene silencing, display elevated levels of H3K4me2, and prevent extension of H3K9me2 at pericentric heterochromatin. Su(var)3-3 colocalizes with H3K4me2 in interband regions and is abundant during embryogenesis and in syncytial blastoderm, where it appears concentrated at prospective heterochromatin during cycle 14. In embryos of Su(var)3-3/+ females, H3K4me2 accumulates in primordial germ cells, and the deregulated expansion of H3K4me2 antagonizes heterochromatic H3K9me2 in blastoderm cells. These data indicate an early developmental function for the Su(var)3-3 demethylase in controlling euchromatic and heterochromatic domains and reveal a hierarchy in which Su(var)3-3-mediated removal of activating histone marks is a prerequisite for subsequent heterochromatin formation by H3K9 methylation (Rudolph, 2007).

The homeobox (Hox) gene locus is subject to extensive H3-K4 methylation by trithorax-group proteins. It was therefore asked whether the expression level of the Hox genes Ultrabithorax (Ubx) and abdominal-A (abdA) is affected by dLsd1 depletion. Ubx- and abdA-mRNA levels increased 2-fold in SL2 cells treated with dLsd1 double-stranded RNA (dsRNA). These changes were specific and were not seen with other control genes (dDP and Hid). To verify the relevance of these observations in vivo, the expression of these genes was compared in wild-type and dLsd1^ΔN mutant flies. A significant upregulation of each of these targets was found in dLsd1^ΔN mutant flies, confirming the importance of dLsd1-mediated repression in vivo. Intriguingly, it was observe that this upregulation is age dependent: The difference in gene expression is minimal in larval stages, and, consistent with this, the Hox gene-expression pattern in imaginal discs from dLsd1^ΔN mutant larvae and in embryos is largely unaltered. However, the level of nAcrβ, Ubx, and Abd-B gradually and significantly increases with age after eclosion, suggesting that dLsd1 function is especially important for the regulation of gene expression in adult tissues (Di Stefano, 2007).

The data support a model in which heterochromatin formation and gene silencing in PEV are defined during early embryonic development of Drosophila. A dynamic balance between HMTases and demethylases controls establishment of the functionally antagonistic histone H3K4 and H3K9 methylation marks at the border region of euchromatin and heterochromatin. In transcriptionally silent cleavage nuclei, chromatin is in a naive state with only little H3K9me2 and with H3K4 methylation completely missing. A dramatic transition of chromatin structure occurs during blastoderm formation and cellularization by establishing H3K4 and H3K9 methylation. In contrast to H3K9 acetylation, which is already found in cleavage chromatin, H3K4 methylation at prospective euchromatin appears first at the end of cleavage in cycle 12. In parallel, di- and trimethylation of H3K9 and HP1 binding establish heterochromatin. Pole cells, which are the primordial germ cells of Drosophila, are in a transcriptionally silent state and show extensive H3K9me2 and H3K9me3. During the definition of the euchromatin-heterochromatin boundaries in blastoderm cells and for the establishment of repressive H3K9 methylation marks in primordial germ cells, the SU(VAR)3-3 demethylase plays an early and inductive regulatory role. SU(VAR)3-3 might also be involved in control of early transcriptional activities within Drosophila pericentromeric sequences preceding heterochromatin formation, as suggested by a model of heterochromatin formation that depends on the RNAi pathway (Rudolph, 2007).

Genetic analysis revealed that SU(VAR)3-3 functions upstream of the H3K9 HMTase SU(VAR)3-9 and the heterochromatin-associated proteins HP1 and SU(VAR)3-7 in control of gene silencing in PEV. Combined with earlier studies of epigenetic interactions, heterochromatic gene silencing is established by a sequential action of SU(VAR)3-3, SU(VAR)3-9, the amount of Y heterochromatin, HP1, and SU(VAR)3-7. RPD3 also acts upstream of SU(VAR)3-9, because Rpd3 mutations dominate the dose-dependent PEV enhancer effect of SU(VAR)3-9. Additional genomic copies of Su(var)3-3 are epistatic to a Rpd3 mutation placing the H3K4 demethylase SU(VAR)3-3 together with RPD3 at the top of a mechanistic hierarchy controlling heterochromatic gene silencing in Drosophila. Such a role is in agreement with the enriched association of SU(VAR)3-3 to prospective heterochromatin in early blastoderm nuclei. In Su(var)3-3 null embryos, there is an extension of H3K4me2 and concomitant reduction of H3K9me3 at prospective heterochromatin, suggesting that SU(VAR)3-3 has a protective function at heterochromatic regions to restrict expansion of H3K4 methylation. Similarly, H3K9 acetylation becomes expanded toward heterochromatin. H3K4 methylation precedes H3K9 methylation in blastoderm nuclei, and both SU(VAR)3-3 and SU(VAR)3-9 are abundant proteins within cleavage chromatin. A developmentally regulated silencing complex between SU(VAR)3-3, RPD3, and SU(VAR)3-9 is therefore likely to dictate the distinction between euchromatic and heterochromatic domains during early embryogenesis. A comparable functional crosstalk between human LSD1 and HDAC1/2, which depends on nucleosomal substrates and the CoREST (see Drosophila CoRest) protein, has been demonstrated in vertebrates. The interaction between SU(VAR)3-3 and RPD3 could also explain butyrate sensitivity of Su(var)3-3 mutations. The effect of SU(VAR)3-3 on heterochromatin formation during blastoderm could involve both maternal and zygotic protein. Association of SU(VAR)3-3 with cleavage chromatin is dependent on maternal sources. In contrast, all other effects on gene silencing are zygotically determined, and no maternal effects on PEV were found in any of the Su(var)3-3 mutations. This is also supported by clonal analysis showing early onset and stable maintenance of gene silencing in PEV (Rudolph, 2007).

Phosphorylation of histone H3 at Ser10 by JIL-1 facilitates RNA polymerase II release from promoter-proximal pausing in Drosophila

The Drosophila JIL-1 kinase is known to phosphorylate histone H3 at Ser10 (H3S10) during interphase. This modification is associated with transcriptional activation, but its function is not well understood. Evidence is presented suggesting that JIl-1-mediated H3S10 phosphorylation is dependent on chromatin remodeling by the brahma complex and is required during early transcription elongation to release RNA polymerase II (Pol II) from promoter-proximal pausing. JIL-1 localizes to transcriptionally active regions and is required for activation of the E75A ecdysone-responsive and hsp70 heat-shock genes. The heat-shock transcription factor, the promoter-paused form of Pol II (Pol IIo^ser5), and the pausing factor DSIF (DRB sensitivity-inducing factor) are still present at the hsp70 loci in JIL-1-null mutants, whereas levels of the elongating form of Pol II (Pol IIo^ser2) and the P-TEFb kinase are dramatically reduced. These observations suggest that phosphorylation of H3S10 takes place after transcription initiation but prior to recruitment of P-TEFb and productive elongation. Western analyses of global levels of both forms of Pol II further suggest that JIL-1 plays a general role in early elongation of a broad range of genes. Taken together, the results introduce H3S10 phosphorylation by JIL-1 as a hallmark of early transcription elongation in Drosophila (Ivaldi, 2007).

The eukaryotic cell packages its DNA wrapped around histone proteins to form nucleosomes, the basic units of chromatin. These nucleosomes assemble into higher-order chromatin structures through which the transcription machinery must navigate each time it is signaled to transcribe. Mechanisms have consequently evolved to maintain a flexible chromatin state that can readily respond to intrinsic and extrinsic stimuli and accordingly modulate gene expression. Most prominently, histone-modifying enzymes can methylate, acetylate, and phosphorylate various amino acid residues of histone N termini, thereby changing their affinity for different transcriptional regulators. ATP-dependent chromatin remodeling complexes can also be recruited to alter the position and accessibility of the nucleosome. The binding of specific transcription factors triggers a cascade of events during which these diverse chromatin modulators work in concert to allow the RNA polymerase II (Pol II) machinery to bind target genes, initiate transcription, and elongate the messenger RNA (mRNA). These regulators maintain tight control of transcription throughout the elongation process by continuously communicating with the C-terminal domain (CTD) of the largest subunit of Pol II (Ivaldi, 2007 and references therein).

The CTD of Pol II consists of a heptad repeat (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) that is conserved from yeast to humans. It integrates transcriptional events by interacting with distinct regulatory proteins that recognize different patterns of CTD phosphorylation. When Pol II is first recruited to the promoter as part of the preinitiation complex, its CTD is hypophosphorylated. After Pol II disengages from the promoter, the CTD becomes phosphorylated at Ser5 (Pol IIo^ser5) by TFIIH, a general transcription factor that is part of the Pol II machinery. As part of an early elongation complex, Pol II progresses 20-40 base pairs (bp) downstream from the promoter. It then pauses in a process referred to as promoter-proximal pausing to allow for capping of the nascent mRNA. DRB sensitivity-inducing factor (DSIF, Spt5) and negative elongation factor (NELF) cooperate to repress transcription elongation and maintain this pause. Pol II is released once the P-TEFb kinase is recruited to relieve the negative effects of DSIF and NELF and phosphorylate the CTD at Ser2 (Pol IIo^ser2), marking the onset of productive elongation. The various transcriptional steps are associated with distinct histone modifications and chromatin remodeling complexes. Set1, the enzyme responsible for methylating Lys4 of histone H3 (H3K4) in Saccharomyces cerevisiae, is known to physically associate with the CTD of Pol II when it is phosphorylated at Ser5. At the same time, trimethylation of H3K4 has been found concentrated at the 5' end of transcribed genes. Methylation of Lys36 of H3 (H3K36), on the other hand, is associated with a later step in elongation; this mark accumulates further downstream from the promoter and associates with the CTD when phosphorylated at Ser2. Other modifications, such as lysine acetylation, arginine methylation, and serine phosphorylation, have also been associated with activation of gene expression. Of interest, phosphorylation of histone H3 at the Ser10 residue (H3S10) has been shown to be important for activation of transcription in yeast, Drosophila, and mammalian cells, but its precise role in this process is not well understood (Ivaldi, 2007 and references therein).

Several studies have suggested an important role for H3S10 phosphorylation in specific transcriptional responses to signaling stimuli. The yeast Snf1 kinase phosphorylates H3S10 upon activation of the INO1 gene. In mammalian fibroblasts, rapid phosphorylation of histone H3 concomitant with activation of immediate-early (IE) response genes takes place when cells are treated with growth factors and various stress-inducing agents. Further, Coffin-Lowry syndrome is characterized by impaired transcriptional activation of the c-fos gene and a loss of EGF-induced phosphorylation of histone H3S10. Treatment of immature rat ovarian granulosa cells with follicle-stimulating hormone produces rapid H3S10 phosphorylation in a PKA-dependent manner, suggesting a role for histone phosphorylation in cellular differentiation. Additionally, H3S10 phosphorylation follows the stimulation of the suprachiasmatic nucleus of rats with light and activation of hippocampal neurons. It further appears to play a central role during cytokine-induced gene expression mediated by IkappaB kinase α (IKK-α). What remains unclear from these studies is whether H3S10 phosphorylation is limited to mediating signal transduction events or whether it plays a more general role in the activation of gene expression in vertebrates (Ivaldi, 2007).

Studies in Drosophila suggest that this modification may be required for the transcription of most genes in this organism. Using the heat-shock response as a model system, it has been established that H3S10 phosphorylation patterns parallel those of active genes. Drosophila responds to a rise in temperature by rapidly increasing the transcription of heat-shock genes while repressing genes expressed previously. Before heat shock, phosphorylated H3S10 localizes to euchromatic regions of polytene chromosomes and colocalizes with Pol II. After heat shock, this modification redistributes to the active heat-shock loci and disappears from the rest of the chromosome, where genes are now repressed (Nowak, 2000; Ivaldi, 2007).

Despite these observations, the precise role of H3 phosphorylation in gene activation remains elusive. The mammalian MSK1 and MSK2 kinases, among others, have been shown to be responsible for H3S10 phosphorylation associated with transcription. The Drosophila homolog of MSK1/2, the JIL-1 threonine/serine kinase, has been shown to phosphorylate H3S10 in vitro. H3S10 phosphorylation levels in vivo are dramatically reduced in JIL-1^z2-null mutants. The JIL-1 protein localizes to interband regions of polytene chromosomes and is found up-regulated on the male X chromosome. Furthermore, the JIL-1^z2 allele enhances the phenotype of trx-G mutations. These data indirectly suggest that JIL-1-mediated H3S10 phosphorylation plays an important role in transcriptional activation (Ivaldi, 2007).

This study further characterizes the role of JIL-1-mediated H3S10 phosphorylation in transcription. JIL-1 is required for the transcription of the majority of, if not all, Drosophila genes. Mechanistic analyses place the phosphorylation event subsequent to transcription initiation but prior to productive elongation; JIL-1 plays an integral role in the release of Pol II from promoter-proximal pausing. The data therefore highlight H3S10 phosphorylation as a novel hallmark of early productive elongation in Drosophila (Ivaldi, 2007).

These results establish H3S10 phosphorylation by JIL-1 as a key event during early elongation of transcription in Drosophila. JIL-1 appears to interact with the transcription machinery at most or all actively transcribed regions on Drosophila polytene chromosomes, including active ecdysone and heat-shock genes. At the same time, expression levels of the hsp70 and E75A genes are decreased in JIL-1-null mutants. Importantly, when JIL-1 is mutated, a global decrease in the phosphorylation levels of elongating RNA polymerase II is observed, suggesting that JIL-1 is required for transcription of the majority of genes (Ivaldi, 2007).

The results further elucidate the timing of H3S10 phosphorylation within the framework of the cascade of events that lead to activation of transcription in eukaryotes. Phosphorylation of H3S10 is not required for transcription factor recruitment, since loss of JIL-1 does not affect binding of HSF at the hsp70 genes after heat shock. Also, H3S10 phosphorylation is dependent on BRM chromatin remodeling, which is required genome-wide prior to the recruitment of Pol II. Transcription initiation can take place independently of JIL-1, as shown by the normal levels of Pol IIo^ser5 and H3K4 methylation in JIL-1^z2 mutants, indicating that the chromatin environment in the absence of JIL-1 is still suitable for transcription initiation. However, productive elongation is impaired in these mutants, as is evident by the decrease in Pol IIo^ser2 levels. These findings introduce H3S10 phosphorylation as a new component of an increasingly complex chromatin environment that is required at the onset of transcription elongation in Drosophila, suggesting a role for JIL-1 in the release of Pol II from promoter-proximal pausing and facilitation of early elongation. Specifically, in JIL-1 mutants, P-TEFb is not detected at the induced hsp70 genes while levels of DSIF are maintained. In the absence of P-TEFb, neither DSIF nor Pol II can be phosphorylated, which is sufficient to block productive elongation. It is likely that Pol II arrests in a paused state and cannot elongate in these mutants. It is also possible that Pol II continues to elongate but is unable to communicate with the proper mRNA processing machinery, which is normally contingent on Ser2 phosphorylation of its CTD (Orphanides, 2002). In this case, the mRNA would be produced but quickly degraded, leading to the transcription defects observed in the Northern analyses. Further work is needed to distinguish between these two possibilities (Ivaldi, 2007).

Although JIL-1 is required for transcription, its presence is not sufficient to ensure gene activation, since JIL-1 is present at all previously transcribed genes that are silenced after heat shock, whereas phosphorylated H3S10 is found exclusively at the transcriptionally active heat-shock genes (Nowak, 2000). Nevertheless, recruitment of JIL-1 to the hsp70 gene is transcription dependent. One possibility is that JIL-1 can exist in both active and inactive states. Once recruited to activate a gene, it may eventually be repressed by inactivation rather than disassociation. Alternatively, the net levels of phosphorylated H3S10 could result from a delicate balance between kinase and phosphatase activities. It has been proposed previously that phosphatase 2A (PP2A) plays a major role in transcription-dependent H3S10 phosphorylation (Nowak, 2003). Therefore, even if JIL-1 is actively maintained at silent genes, its action may be counterbalanced by PP2A. Further studies are required to shed light on how JIL-1 activity can be regulated to affect transcription (Ivaldi, 2007).

In vertebrates, phosphorylation of H3S10 seems to be limited to transcription activation of specific genes in the context of particular signal transduction pathways. In fact, activation of the hsp70 genes by different stressors in mammalian cells is associated with distinct signaling pathways that are not always linked to H3S10 phosphorylation. Contrary to the Drosophila response, heat shock elicits histone H4 acetylation instead of H3S10 phosphorylation at the hsp70 loci in mouse fibroblasts. In contrast, both H3S10 phosphorylation and H4 acetylation are detected at the hsp70 genes upon arsenite treatment of the same cells (Thomson. 2004). Therefore, mammals appear to have more diverse mechanisms of transcription activation and may partially rely on H3S10 phosphorylation in a context-dependent manner. In yeast, substituting the H3 Ser10 for an Ala prevents the recruitment of the TATA-binding protein to the INO1 and GAL1 gene promoters, suggesting that H3S10 phosphorylation is required for the assembly of the preinitiation complex. It would be interesting to explore the significance of this apparent diversity across species (Ivaldi, 2007 and references therein).

The results presented in this study shed light on the mechanism of transcription regulation by H3S10 phosphorylation. It has been recently shown that H3S10 phosphorylation antagonizes the binding of the heterochromatin protein HP1 to histone H3 methylated in Lys9 (H3K9) during mitosis in mammalian cells. It was consequently proposed that JIL-1 maintains chromosome structure in Drosophila by counteracting heterochromatin formation and preventing its spreading into euchromatin. This model for JIL-1 activity could explain a lack of transcription in JIL-1^z2 mutants, since any ectopic heterochromatin would make the DNA inaccessible to the Pol II machinery. However, contrary to such a prediction, the current results show that heat-shock puffs are still formed in JIL-1^z2mutants, and transcription factors and the Pol II machinery retain the ability to bind despite the disruption of chromatin structure. Furthermore, transcription can be initiated, as is evident by the phosphorylation of Pol II at Ser5. This requires several components of the core transcription machinery and the procession of Pol II a few bases downstream from the promoter. These results suggest that, rather than contribute to global chromosome structure, JIL-1-mediated H3S10 phosphorylation may be required to maintain a local chromatin environment that serves as a platform for the recruitment of P-TEFb and the consequent release of Pol II from promoter-proximal pausing (Ivaldi, 2007).

It has become increasingly evident that transcription elongation is a rate-limiting step of gene expression that requires tight regulation. It was reported recently that the majority of gene promoters in human embryonic stem cells are occupied by a promoter-proximally paused Pol II, poised for productive elongation (Guenther, 2007). This suggests that the expression of these genes is predominantly regulated at the level of Pol II release rather than during preinitiation. The exact mechanism of P-TEFb recruitment, a key step in this process, remains to be determined. Several transcription regulators have been shown to recruit P-TEFb, but this is the first evidence of a histone modification required precisely at the timing of recruitment (Ivaldi, 2007).

The exact contribution of H3S10 phosphorylation to P-TEFb recruitment remains open to further investigation. Recent reports have shown that the ubiquitous protein 14-3-3 binds to H3 only when phosphorylated at Ser10, and this interaction could provide a mechanistic link between H3S10 phosphorylation and P-TEFb (Macdonald, 2005). It is possible that 14-3-3 interacts with P-TEFb directly or indirectly through other transcription regulators that are known to recruit it. Alternatively, 14-3-3 is known to interact with many chromatin-related proteins, thus providing another avenue to manipulate the local chromatin environment to support P-TEFb recruitment and early elongation. Further analyses will be necessary to test these hypotheses and clarify the role and mechanism of regulation of JIL-1 and H3S10 phosphorylation in gene expression (Ivaldi, 2007).

Histone replacement marks the boundaries of cis-regulatory domains

Cellular memory is maintained at homeotic genes by cis-regulatory elements whose mechanism of action is unknown. Drosophila homeotic gene clusters have been examined by measuring, at high resolution, levels of histone replacement and nucleosome occupancy. Homeotic gene clusters display conspicuous peaks of histone replacement at boundaries of cis-regulatory domains superimposed over broad regions of low replacement. Peaks of histone replacement closely correspond to nuclease-hypersensitive sites, binding sites for Polycomb and trithorax group proteins, and sites of nucleosome depletion. These results suggest the existence of a continuous process that disrupts nucleosomes and maintains accessibility of cis-regulatory elements (Mito, 2007).

Chromatin can be differentiated by the replication-independent replacement of one histone variant with another. For example, histone H3.3 is deposited throughout the cell cycle, replacing H3 that is deposited during replication. Unlike replication-coupled assembly of H3, which occurs in gaps between old nucleosomes on daughter helices, the insertion of H3.3 is preceded by disruption of preexisting histones during transcription and other active processes. H3.3 replacement profiles resemble those for RNA polymerase II, which suggests that gradual replacement of H3.3 occurs in the wake of transiting polymerase to repair disrupted chromatin. This study asked whether histone replacement and nucleosome occupancy are also distinctive at cis-regulatory elements (Mito, 2007).

Log-phase Drosophila S2 cells were induced to produce biotin-tagged H3.3 for two or three cell cycles. DNA was extracted from streptavidin pull-down assay and input material, labeled with Cy3 and Cy5 dyes, and cohybridized to microarrays. To provide a standard, biotin-tagged H3-containing chromatin was profiled in parallel. Analysis of H3.3/H3 levels over the entire 3R chromosome arm revealed that the 350-kb bithorax complex (BX-C) region displays the lowest H3.3/H3 ratio of any region of comparable size on 3R, and the Antennapedia homeotic gene complex (ANTP-C) also displays an unusually low H3.3/H3 ratio. Low H3.3/H3 ratios at the homeotic gene clusters are attributable to infrequent histone replacement, and not to low nucleosome occupancy, because H3.3 levels at the BX-C are far below the median for all of 3R, whereas H3 levels are slightly above the median overall. Even the heterochromatic chromosome 4 includes only shorter (100-kb) stretches that are as depleted in H3.3 as the BX-C (Mito, 2007).

A close-up view of the BX-C iab region reveals the presence of several prominent H3.3 peaks. Notably, the seven highest peaks correspond to the functional boundaries of the seven proximal-to-distal cis-regulatory domains that regulate the abd-A (iab2 to iab4) and Abd-B (iab5 to iab8) homeotic genes successively from anterior to posterior in the abdomen. Conspicuous peaks of H3.3 also correspond to the bxd Polycomb response element (PRE) and to promoters within the Abd-B gene, which is known to be active in S2 cells. Therefore, each of the most prominent H3.3 peaks in the region corresponds to a previously defined cis-regulatory element. These findings are likely to be general, because in budding yeast, promoters and boundaries are also sites of intense histone replacement (Mito, 2007).

A characteristic feature of both boundaries and PREs in the BX-C is that they span deoxyribonuclease I (DNaseI)-hypersensitive sites in a variety of cell types, including S2 cells. To better delineate histone replacement patterns in the vicinity of hypersensitive sites, the entire BX-C was tiled at 20-bp resolution. The bxd, Mcp, Fab-7, and Fab-8 PRE-boundaries each encompass conspicuous peaks of H3.3 abundance that closely correspond to all the known nuclease-hypersensitive sites within the region. Nuclease hypersensitivity identifies sites of relatively accessible DNA, so that their correspondences to peaks of histone replacement suggest that continuous disruption of nucleosomes exposes cis-regulatory DNA relative to surrounding regions (Mito, 2007).

PRE-boundary elements in the BX-C and other regions are binding sites for multiple Polycomb group (PcG) proteins, which have been mapped in an S2 cell line at high resolution. If the process that disrupts nucleosomes also facilitates PcG binding, then a correspondence would be expected between peaks of PcG binding and peaks of H3.3. Indeed, when H3.3 profiles were compared with those for Enhancer-of-zeste (EZ) and Posterior-sex-combs (PSC) PcG proteins, all 10 peaks of PcG binding in the abdominal region were found to be local peaks of H3.3. Likewise at the ANTP-C, all 13 peaks of PcG binding in the Scr-Antp region correspond to high levels of H3.3. H3.3 enrichment at PcG-binding sites is not attributable to higher nucleosome occupancy, because essentially identical results were obtained for H3.3/H3 profiles (Mito, 2007).

Not all PREs in the BX-C are found to be sites of PcG binding; for example, neither Fab-7 nor Fab-8 is detectably bound by EZ or PSC. The fact that all PcG sites are peaks of histone replacement, but not vice versa, suggests that histone replacement at PREs and boundaries is constitutive and independent of the expression of the homeotic genes that they regulate. For example, Abd-B is expressed at high levels in S2 cells and displays the typical H3.3 5' peak for an active gene, whereas Ubx and abd-A are nearly inactive, yet the PREs and boundaries regulating all three genes are sites of conspicuous histone replacement over a low background (Mito, 2007).

Histone replacement averaged over the 175 genomewide EZ+PSC peaks outside of the BX-C and ANTP-C was examined and an H3.3 peak was observed centered over the PcG maximum. Therefore, the strong association between PcG protein binding and histone replacement is not limited to homeotic gene clusters. The genomewide H3.3 peak is higher than that for the BX-C and ANTP-C, presumably because other PcG-binding sites are not superimposed over such deep H3.3 valleys (Mito, 2007).

The colocalization of PcG-binding sites and local peaks of H3.3 suggests that the process that disrupts nucleosomes locally maintains the accessibility of cis-regulatory DNA to PcG proteins. If so, then there should be a lower average occupancy of nucleosomes over sites of PcG protein binding than over their surrounding regions. To test this possibility, nucleosomal DNA and fragmented genomic DNA were hybridized on the same microarrays, and nucleosomal/genomic DNA log ratios were measured. Around peak regions of EZ+PSC binding, nucleosomal DNA was clearly depleted on average, similar to the depletion seen for active gene promoters, and essentially the same results were obtained with different methods for genomic DNA fragmentation. It is concluded that the correspondence between histone replacement and nucleosome depletion is a genomewide feature of PcG-binding sites (Mito, 2007).

In Drosophila, many cis-regulatory elements, including PREs and boundaries, are bound by the trxG proteins, Zeste and GAGA factor (GAF). To test the possibility that histone replacement is enhanced and nucleosome occupancy is reduced where Zeste protein preferentially binds, 390 Zeste-binding sites identified by high-resolution chromatin immunoprecipitation (ChIP) combined with tiling microarrays (ChIP-chip profiling) were aligned, and log ratios of H3.3/H3 and nucleosome occupancy were averaged. A prominent maximum of histone replacement and a sharp minimum of nucleosome occupancy was observed centered over the point of alignment. Similar results were obtained for predicted GAF sites, which suggests that nucleosome disruption is a general feature of trxG protein DNA-binding sites. H3.3 enrichment at PcG- and trxG protein-binding sites results from a replication-independent replacement process, because essentially identical profiles were obtained for H3.3^core, which lacks the N-terminal tail and does not assemble during replication (Mito, 2007).

Like Fab-7 and Fab-8, heat shock gene promoters are prominent sites of GAF binding, nuclease hypersensitivity, and reduced nucleosome occupancy. Heat shock protein Hsp70 genes are constitutively 'poised' for rapid induction, but do not produce detectable mRNAs in the uninduced state. Hsp70 genes were aligned at their 5' ends and H3.3 and H3 profiles were averaged. For comparison, similarly aligned H3.3 and H3 profiles were averaged for all 2165 genes on 3R with known 5' and 3' ends, divided into quintiles based on expression levels. H3.3 patterns were similar to those of highly active genes, with histone replacement levels peaking on either side of heat shock promoters. As do transcriptionally active gene promoters, heat shock genes display prominent H3.3 and H3 dips in abundance that are attributable to partial nucleosome depletion. Constitutive histone replacement also appears to be a feature of poised promoters in vertebrates, because H3.3 is strongly enriched in the upstream region of the chicken folate receptor gene, regardless of whether the gene is active or inactive (Mito, 2007).

What process maintains the chromatin of cis-regulatory elements in a state of flux? Many DNA-binding and chromatin-binding proteins involved in gene regulation display short residence times on DNA, and some mouse transcription factors show dynamic behavior at their functional binding sites. A model for this process has been proposed, involving alternating cycles of nucleosome disruption by a Brahma-related SWI/SNF chromatin-remodeler and transcription factor binding. The binding of PcG and trxG proteins is also dynamic, and it is proposed that a similar cycle of nucleosome disruption and factor binding takes place at boundaries and PREs. Nucleosome disruption by SWI/SNF remodeling complexes would occasionally evict nucleosomes and transiently expose DNA, which would become available to other diffusible factors, including PcG proteins. The continued local presence of nucleosome remodelers would result in another cycle of remodeling, nucleosome depletion, nuclease hypersensitivity, and histone replacement at the site. This model could account for the diversity of trxG proteins, which include DNA-binding proteins (Zeste and GAF), nucleosome remodelers (Brahma and Kismet), and histone methyltransferases (Trithorax and Ash1) that are specific for H3K4, a modification that is highly enriched on H3.3. The resulting dynamic process would allow for proteins that promote opposite epigenetic outcomes to act at common cis-regulatory sites (Mito, 2007).

Nucleosomal core histones mediate dynamic regulation of Poly(ADP-ribose) polymerase 1 protein binding to chromatin and induction of its enzymatic activity

Poly(ADP-ribose) polymerase 1 protein [PARP1; see Poly-(ADP-ribose) polymerase] mediates chromatin loosening and activates the transcription of inducible genes, but the mechanism of PARP1 regulation in chromatin is poorly understood. This study found that Drosophila PARP1 interaction with chromatin is dynamic and that PARP1 is exchanged continuously between chromatin and nucleoplasm, as well as between chromatin domains. Specifically, the PARP1 protein preferentially interacts with nucleosomal particles, and although the nucleosomal linker DNA is not necessary for this interaction, the core histones, H3 and H4, are critical for PARP1 binding. Histones H3 and H4 interact preferentially with the C-terminal portion of PARP1 protein, and the N-terminal domain of PARP1 negatively regulates these interactions. Finally, it was found that interaction with the N-terminal tail of the H4 histone triggers PARP1 enzymatic activity. Therefore, the data collectively suggests a model in which both the regulation of PARP1 protein binding to chromatin and the enzymatic activation of PARP1 protein depend on the dynamics of nucleosomal core histone mediation (Pinnola, 2007).

This paper provides the first insight into the nature of the association of the PARP1 protein with chromatin in vivo and in vitro. The dynamics between free and chromatin-bound PARP1 protein were characterized, and an additional mechanism for these interactions is suggested. It was also demonstrated that PARP1 associates with chromatin on a monucleosomal level in vivo. More specifically, H3 and H4 are preferential binding sites for the C-terminal domain of PARP1 and that DNA is not required for this association in vitro. Histone H4 works as a strong DNA-independent activator of pADPr enzymatic reaction, whereas other histones (especially H2A) inhibit H4-dependent PARP1 activation (Pinnola, 2007).

PARP1 protein is exchanged rapidly between chromatin regions in the nucleus. No difference was detected between the recovery rate of enzymatically inactive PARPe-EGFP protein and active PARP1-DsRed protein isoforms. Therefore, it is proposed that PARP1 enzymatic activity is not required for steady-state dynamics. However, PARP1 inactivation followed by due automodification of PARP1 molecules has been shown to be critical for PARP1 protein removal from chromatin. The existence of two distinct mechanisms controlling PARP1 interaction with chromatin were detected as a result of sucrose gradient purification experiments. That is, unmodified PARP1 molecules co-purifies with nucleosomes, as well as other fractions (Complex I), whereas PARP1 molecules modified with pADPr were segregated to a separate fraction (Complex II). Based on this finding, it is concluded that, indeed, two distinct mechanisms conjoin to control PARP1 molecule interaction with chromatin. One involves a protein-equilibrated binding via association-dissociation, and the other involves irreversible removal of PARP1 from chromatin after automodification. Based on an accepted model, the existence of Complexes I and II was expectedPARP1 protein is associated with chromatin in its inactive state (Complex I), and upon activation it becomes automodified, loses contact with chromatin, and establishes interactions with pADPr-binding proteins (Complex II). Interestingly, the fraction with Complex II also contains a significant amount of unmodified PARP1. This may suggest that there is a nucleoplasmic pool of unmodified PARP1 that can reversibly bind to pADPr (Pinnola, 2007).

A three step model is presented for the regulation of PARP1 protein enzymatic activity in chromatin. Step 1: PARP1 protein is broadly distributed in chromatin because of interaction with core histones in the context of nucleosome. PARP1 is inactive in this state because of inhibitory effect of histone H2A. Step 2: genotoxic stress-dependent PARP1 activation. The N-terminal domain of PARP1 protein serves as a sensor of the double-stranded breaks or nicks in genomic DNA. Upon binding of damaged DNA, it mediates conformational changes, which leads to disruption of interaction with histones and consequently to the activation of PARP1 enzymatic reaction. Step 3: DNA-independent PARP1 activation. Developmental or environmental signals induces local changes in the "histone modification core" and subsequently expose the N-terminal tail of histone H4 and/or hide histone H2A followed by H4-dependent PARP1 activation (Pinnola, 2007).

Similar to H1, PARP1 controls the establishment of silenced chromatin (Tulin, 2002). Recently, it has been shown that PARP1 and H1 work independently. Moreover, they antagonize each other in chromatin. This antagonistic interaction strongly suggests competition for the same binding sites. The site of linker histone binding is known to be the linker DNA in the context of nucleosomal array. Unlike H1, linker DNA is not crucial for PARP protein binding. This, in turn, suggests that if H1 and PARP compete for binding sites, they recognize different but overlapping, epitopes (Pinnola, 2007).

The ability of PARP1 to bind chromatin via nicks in double-stranded DNA, as well as noncanonical DNA structures, has been demonstrated in vitro. Still, the broad PARP1 localization in chromatin in vivo suggests an alternative mechanism for PARP1 protein binding. Histones H2A and H2B have been reported as preferential targets for PARP1 binding in vitro (Buki, 1995) and for enzymatic modification by PARP1. In the current experiments, unmodified PARP1 protein always co-purified with core histones, even after DNA digestion to mononucleosomes. It was also found that the C terminus of PARP1 preferentially binds histones H3 and H4 of histone octamers lacking DNA. The PARP1 C terminus contains the catalytic domain and the sequence required for homodimerization and thus activation. PARP1 C terminus binding to H3/H4 may serve to sequester the domains in PARP1 that are required for activation, and this could account for the broad localization of PARP1 in chromatin. Histone H4 activates, whereas histone H2A completely inhibits, PARP1 protein. These findings support the conclusion that the PARP1 protein is generally silent (enzymatically inactive) in chromatin, although a number of developmental and environmental stimuli could still activate it at specific loci. This activation is required for chromatin decondensation and transcriptional activation in these loci. PARP1 activation always correlates with changes of local histone modification {e.g. phosphorylation of histone H3 co-localized with pADPr in Drosophila puffs}. Therefore, it is hypothesized that changes in histone modification code promote conformational alteration of nucleosomes and therefore expose (or hide) specific domains of histones, which activate (or inhibit) PARP1 (Pinnola, 2007).

Epigenetic blocking of an enhancer region controls irradiation-induced proapoptotic gene expression in Drosophila embryos

Drosophila embryos are highly sensitive to gamma-ray-induced apoptosis at early but not later, more differentiated stages during development. Two proapoptotic genes, reaper and hid, are upregulated rapidly following irradiation. However, in post-stage-12 embryos, in which most cells have begun differentiation, neither proapoptotic gene can be induced by high doses of irradiation. The sensitive-to-resistant transition is due to epigenetic blocking of the irradiation-responsive enhancer region (IRER), which is located upstream of reaper but is also required for the induction of hid in response to irradiation. This IRER, but not the transcribed regions of reaper/hid, becomes enriched for trimethylated H3K27/H3K9 and forms a heterochromatin-like structure during the sensitive-to-resistant transition. The functions of histone-modifying enzymes Hdac1(Rpd3) and Su(var)3-9 and PcG proteins Su(z)12 and Polycomb are required for this process. Thus, direct epigenetic regulation of two proapoptotic genes controls cellular sensitivity to cytotoxic stimuli (Zhang, 2008).

Irradiation responsiveness appears to be a highly conserved feature of reaper-like IAP antagonists. A recently identified functional ortholog of reaper in mosquito genomes, michelob_x (mx), is also responsive to irradiation. These results highlighted that stress responsiveness is an essential aspect of functional regulation of upstream proapoptotic genes such as reaper/hid. It is also worth mentioning that several mammalian BH3 domain-only proteins, the upstream proapoptotic regulators of the Bcl-2/Ced-9 pathway, are also regulated at the transcriptional level (Zhang, 2008).

This study shows that the irradiation responsiveness of reaper and hid is subject to epigenetic regulation during development. The epigenetic regulation of the IRER is fundamentally different from the silencing of homeotic genes in that the change of DNA accessibility is limited to the enhancer region while the promoter of the proapoptotic genes remains open. Thus, it seems more appropriate to refer this as the 'blocking' of the enhancer region instead of the 'silencing' of the gene. This region, containing the putative P53RE and other essential enhancer elements, is required for mediating irradiation responsiveness. ChIP analysis indicates that histones in this enhancer region are quickly trimethylated at both H3K9 and H3K27 at the sensitive-to-resistant transition period, accompanied by a significant decrease in DNA accessibility. DNA accessibility in the putative P53RE locus (18,368k), when measured by the DNase I sensitivity assay, did not show significant decrease until sometime after the transition period. It is possible that other enhancer elements, in the core of IRER_left, are also required for radiation responsiveness. An alternative explanation is that the strong and rapid trimethylation of H3K27 and association of PRC1 at 18,366,000-18, 368,000 are sufficient to disrupt DmP53 binding and/or interaction with the Pol II complex even though the region remains relatively sensitive to DNase I. Eventually, the whole IRER is closed with the exception of an open island around 18,387,000 (Zhang, 2008).

The finding that epigenetic regulation of the enhancer region of proapoptotic genes controls sensitivity to irradiation-induced cell death may have implications in clinical applications involving ionizing irradiation. It suggests that applying drugs that modulate epigenetic silencing may help increase the efficacy of radiation therapy. It also remains to be seen whether the hypersensitivity of some tumors to irradiation is due to the dedifferentiation and reversal of epigenetic blocking in cancer cells. In contrast, loss of proper stress response to cellular damage is implicated in tumorigenesis. The fact that the formation of heterochromatin in the sensitizing enhancer region of proapoptotic genes is sufficient to convey resistance to stress-induced cell death suggests it could contribute to tumorigenesis. In addition, it could also be the underlying mechanism of tumor cells' evading irradiation-induced cell death. This is a likely scenario given that it has been well documented that oncogenes such as Rb and PML-RAR fusion protein cause the formation of heterochromatin through recruiting of a human ortholog of Su(v)3-9. In this regard, the reaper locus, especially the IRER, provides an excellent genetic model system for understanding the cis- and trans-acting mechanisms controlling the formation of heterochromatin associated with cellular differentiation and tumorigenesis (Zhang, 2008).

The developmental consequence of epigenetic regulation of the IRER is the tuning down (off) of the responsiveness of the proapoptotic genes, thus decreasing cellular sensitivity to stresses such as DNA damage. Epigenetic blocking of the IRER corresponds to the end of major mitotic waves when most cells begin to differentiate. Similar transitions were noticed in mammalian systems. For instance, proliferating neural precursor cells are extremely sensitive to irradiation-induced cell death while differentiating/differentiated neurons become resistant to γ-ray irradiation, even though the same level of DNA damage was inflicted by the irradiation. These findings here suggest that such a dramatic transition of radiation sensitivity could be achieved by epigenetic blocking of sensitizing enhancers (Zhang, 2008).

Later in Drosophila development, around the time of pupae formation, the organism becomes sensitive to irradiation again, with LD₅₀ values similar to what was observed for the 4–7 hr AEL embryos. Interestingly, it has also been found that during this period, the highly proliferative imaginal discs are sensitive to irradiation-induced apoptosis, which is mediated by the induction of reaper and hid through P53 and Chk2. However, it remains to be studied whether the reemergence of sensitive tissue is due to reversal of the epigenetic blocking in the IRER or the proliferation of undifferentiated stem cells that have an unblocked IRER (Zhang, 2008).

The blocking of the IRER differs fundamentally from the silencing of homeotic genes in several aspects. (1) The change of DNA accessibility and histone modification is largely limited to the enhancer region. The promoter regions of reaper (and hid) remain open, allowing the gene to be responsive to other stimuli. Indeed, there are a few cells in the central nervous system that could be detected as expressing reaper long after the sensitive-to-resistant transition. Even more cells in the late-stage embryo can be found having hid expression. Yet, the irradiation responsiveness of the two genes is completely suppressed in most if not all cells, transforming the tissues into a radiation-resistant state (Zhang, 2008).

(2) The histone modification of the IRER has a mixture of features associated with pericentromeric heterochromatin formation and canonic PcG-mediated silencing. Both H3K9 and H3K27 are trimethylated in the IRER. Both HP1, the signature binding protein of the pericentromeric heterochromatin, and PRC1 are bound to the IRER. As demonstrated by genetic analysis, the functions of both Su(var)3-9 and Su(z)12/Pc are required for the silencing. Preliminary attempts to verify specific binding of PRC2 proteins to this region were unsuccessful. The fact that none of the mutants tested could completely block the transition seems to suggest that there is a redundancy of the two pathways in modifying/blocking the IRER. It is also possible that the genes tested are not the key regulators of IRER blocking but only have participatory roles in the process (Zhang, 2008).

(3) Within the IRER, there is a small region around 18,386,000 to 18,188,000 that remains relatively open until the end of embryogenesis. Interestingly, this open region is flanked by two putative noncoding RNA transcripts represented by EST sequences. If they are indeed transcribed in the embryo as suggested by the mRNA source of the cDNA library, then the 'open island' within the closed IRER will likely be their shared enhancer/promoter region. Sequences of both cDNAs revealed that there is no intron or reputable open reading frame in either sequence. Despite repeated efforts, their expression was not confirmed via ISH or northern analysis. Overexpression of either cDNA using an expression construct also failed to show any effect on reaper/hid-induced cell death in S2 cells. Yet, sections of the two noncoding RNAs are strongly conserved in divergent Drosophila genomes. The potential role of these two noncoding RNAs in mediating reaper/hid expression and/or blocking of the IRER remains to be studied (Zhang, 2008).

dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing

Transcription regulation involves enzyme-mediated changes in chromatin structure. This study describes a novel mode of histone crosstalk during gene silencing, in which histone H2A monoubiquitylation is coupled to the removal of histone H3 Lys 36 dimethylation (H3K36me2). This pathway was uncovered through the identification of dRING-associated factors (dRAF), a novel Polycomb group (PcG) silencing complex harboring the histone H2A ubiquitin ligase dRING, PSC and the F-box protein, and demethylase Lysine (K)-specific demethylase 2 (dKDM2). In vivo, dKDM2 shares many transcriptional targets with Polycomb and counteracts the histone methyltransferases TRX and ASH1. Importantly, cellular depletion and in vitro reconstitution assays revealed that dKDM2 not only mediates H3K36me2 demethylation but is also required for efficient H2A ubiquitylation by dRING/PSC. Thus, dRAF removes an active mark from histone H3 and adds a repressive one to H2A. These findings reveal coordinate trans-histone regulation by a PcG complex to mediate gene repression (Lagarou, 2008).

This study investigated the molecular mechanisms involved in PcG-mediated gene silencing. The major findings of this work are the following. First, a novel PcG silencing complex was idebtufued tat was named dRAF, harboring core subunits dKDM2, dRING, and PSC. Whereas dRING and PSC are also part of PRC1, the other two PRC1 core subunits, PC and PH, are absent from dRAF. In addition, it was found that significant amounts of PSC and PH are not associated with either PRC1 or dRAF, suggesting they might form part of other assemblages. In short, this work suggests a greater diversity among PcG complexes than previously anticipated. Second, genome-wide expression analysis revealed that dKDM2 and PRC1 share a significant number of target genes. Third, it was found that Pc and dkdm2 interact genetically and cooperate in repression of homeotic genes in vivo. Fourth, dKDM2 counteracts homeotic gene activation by the trxG histone methyltransferases TRX and ASH1. Fifth, a novel trans-histone pathway acting during PcG silencing was uncovered. dKDM2 plays a central role by removal of the active H3K36me2 mark and promoting the establishment of the repressive H2Aub mark by dRING/PSC. Finally, the observation that dKDM2 is required for bulk histone H2A ubiquitylation by dRING/PSC, suggests that dRAF rather than PRC1 is the major histone H2A ubiquitylating complex in cells (Lagarou, 2008).

The term trans-histone pathway was first coined to describe that H2B ubiquitylation is required for H3K4 and H3K79 methylation, whereas the reverse is not the case. Recently, it was found that H2Bub determines the binding of Cps35, a key component of the yeast H3K4 methylase COMPASS complex, providing insight in the molecular mechanism by which two positive marks are coupled. This study describes a different type of trans-histone regulation where the removal of the active H3K36me2 mark is directly linked to repressive monoubiquitylation of H2A. A recent study strongly argued that ASH1 mediates H3K36me2. Significantly, the current genetic and biochemical analysis revealed an in vivo antagonism between dKDM2 and ASH1. Thus, dKDM2 appears to reverse the enzymatic activity of trxG protein ASH1 through H3K36 demethylation, whereas it does not affect H3K4 methylation. The observation that chromatin binding of TRX is ASH1 dependent is likely to be part of the explanation of the strong genetic interaction between dkdm2 and trx. The association of the H3K27me2/3 demethylase UTX with the MLL2/3 H3K4 methylase complexes is an example of coupling removal of a repressive mark to the establishment of an active mark (Lagarou, 2008).

This work revealed that the key H2A E3 ubiquitin ligase dRING is part of two distinct complexes, PRC1 and dRAF. A previous study identified the mammalian BCOR corepressor complex, which harbors RING1, NSPC1, and FBXL10 and other proteins, absent from dRAF. These findings suggest that BCOR and dRAF represent a variety of related but distinct silencing complexes. Reduction of dKDM2 caused a dramatic loss of H2Aub levels, which was comparable with that observed after depletion of dRING or PSC. However, knockdown of PRC1 subunits PC or PH had no effect on H2Aub. These observations suggest that dRAF rather than PRC1 is responsible for the majority of H2A ubiquitylation in cells. This notion was reinforced by in vitro reconstitution experiments, suggesting that dRAF is a more potent H2A ubiquitin ligase than PRC1. An unresolved issue remains the molecular mechanisms that underpin the opposing consequences of either H2A or H2B ubiquitylation. It is intriguing that H2Aub appears to be absent in yeast, present but less prominent than H2Bub in Drosophila, and abundant in mammalian cells. An attractive speculation is that H2Aub becomes more important when genome size increases and noncoding regions and transposons need to be silenced (Lagarou, 2008).

In summary, this study identified the PcG complex dRAF, which employs a novel trans-histone pathway to mediate gene silencing. dKDM2 plays a pivotal role by coupling two distinct enzymatic activities, H3K36me2 demethylation and stimulation of H2A ubiquitylation by dRING/PSC. The results indicate that dRAF is required for the majority of H2Aub in the cell. dKDM2 cooperates with PRC1 but counteracts trxG histone methylase ASH1. These findings uncovered a repressive trans-histone mechanism operating during PcG gene silencing (Lagarou, 2008).

Drosophila HP1c isoform, which efficiently binds H3K9me2,3 in vitro, interacts with the zinc-finger proteins WOC and Relative-of-WOC to regulate gene expression

Heterochromatin protein 1 (HP1) proteins are conserved in eukaryotes, with most species containing several isoforms. Based on the properties of Drosophila HP1a, it has proposed that HP1s bind H3K9me2,3 (Histone 3 methylate on lysine 9) and recruit factors involved in heterochromatin assembly and silencing. Yet, it is unclear whether this general picture applies to all HP1 isoforms and functional contexts. Evidence reported here suggests that Drosophila HP1c regulates gene expression as follows: (1) it localizes to active chromatin domains, where it extensively colocalizes with the poised form of RNApolymerase II (RNApol II), Pol IIo(ser5), and H3K4me3, suggesting a contribution to transcriptional regulation; (2) its targeting to a reporter gene does not induce silencing but, on the contrary, increases its expression, and (3) it interacts with the zinc-finger transcription factors WOC (Without children) and Relative-of-WOC (ROW). Although HP1c efficiently binds H3K9me2,3 in vitro, its binding to chromatin strictly depends on both WOC and ROW. Moreover, expression profiling indicates that HP1c, WOC, and ROW regulate a common gene expression program that, in part, is executed in the context of the nervous system. From this study, which unveils the essential contribution of DNA-binding proteins to HP1c functionality and recruitment, HP1 proteins emerge as an increasingly diverse family of chromatin regulators (Font-Burgada, 2008).

The contribution of chromatin to the regulation of genomic functions is well established. Most frequently, regulation by chromatin involves the establishment of specific patterns of post-translational histone modifications, which result in recruitment of regulatory nonhistone proteins. Heterochromatin protein 1 (HP1) constitutes one of the best-studied examples, where a regulatory nonhistone protein is recruited to chromatin through the recognition of a specific histone modification, di- or trimethylation of Lys 9 on the histone H3 tail (H3K9me2,3). This interaction, which involves the N-terminal chromodomain of HP1, is known to play a fundamental role in the formation and maintenance of heterochromatic domains (Font-Burgada, 2008).

Except in budding yeast, HP1 is widely conserved in eukaryotes, with most species containing several isoforms. HP1 proteins are characterized by a common structural organization consisting of two conserved domains, the N-terminal chromodomain and the C-terminal chromo-shadow domain, which are spaced by a variable nonconserved hinge domain. The existence of multiple isoforms suggests functional specialization, with different isoforms playing different functions. For instance, in Drosophila, three of the five HP1 isoforms (HP1a, HP1b, and HP1c) are ubiquitously expressed, while the other two (HP1d/Rhino and HP1e) are predominantly expressed in the germline. Moreover, ubiquitously expressed HP1 isoforms show differential chromosomal distributions, as HP1a is mainly associated to heterochromatin, while HP1c is excluded from centromeric heterochromatin and HP1b is found both at euchromatic and heterochromatic domains. A similar situation is observed in mammals, where the patterns of localization of the three HP1 isoforms (HP1α, HP1β, and HP1γ) overlap only partially and show differential dynamics during differentiation and cell cycle progression. In contrast, in the nematode C. elegans, two HP1 isoforms exist (HPL1 and HPL2), showing preferential euchromatic association, partially nonoverlapping expression and localization patterns, and distinct mutant phenotypes. Interestingly, the only HP1 protein (TLF2/LHP1) of Arabidopsis thaliana appears to localize exclusively to euchromatin, where it colocalizes with H3K27me3 (Font-Burgada, 2008).

The molecular mechanisms that determine the differential distribution of the various HP1 isoforms, and their differential functional properties, remain largely unknown. Most knowledge about the mechanisms of action of HP1 proteins derives from studies addressing the functional properties of Drosophila HP1a or mammalian HP1α. From these studies, a general picture emerges by which, through the chromodomain, HP1 proteins bind chromatin regions enriched in H3K9me2,3 while, through the chromo-shadow domain, recruiting different factors resulting in various functional outcomes; namely, heterochromatin assembly and gene silencing. It is uncertain whether this general picture applies to all HP1 proteins and possible scenarios. Actually, Drosophila HP1a is known to play a more complex role(s) in the regulation of gene expression, as it is required for the expression of several heterochromatic genes, and certain euchromatic genes. Moreover, Drosophila HP1a is recruited to developmentally regulated genes and heat-shock-induced puffs in an RNA-dependent manner, and in an erythroid cell line, murine HP1α was found associated to actively transcribed genes (Font-Burgada, 2008).

This study reports on the functional characterization of HP1c, a Drosophila HP1 protein of largely unknown properties. The results show that HP1c extensively colocalizes with poised RNA polymerase II (RNApol II) and H3K4me3, a modification that correlates with active chromatin domains. Moreover, targeting HP1c to a reporter construct does not induce silencing but, on the contrary, results in increased expression of the reporter gene. This study also reports on the interaction of HP1c with the zinc-finger proteins WOC (without children), and Relative-of-WOC (ROW), which are putative transcription factors. HP1c efficiently binds H3K9me2,3 in vitro, but its binding to chromatin strictly depends on both WOC and ROW. Moreover, expression profiling indicates that HP1c, WOC, and ROW extensively cooperate to regulate gene expression, especially in the context of the nervous system. These results unveil the essential contribution of sequence-specific DNA-binding proteins to functionality of HP1c and its recruitment to chromatin (Font-Burgada, 2008).

Altogether, these results indicate that HP1c, WOC, and ROW are components of a distinct multiprotein complex. HP1c-WOC interaction is likely to be direct, since WOC contains a canonical PxVxL motif (¹⁵³⁶PHVLL¹⁵⁴⁰), which is known to mediate binding to the chromo-shadow domain of HP1 proteins. This motif is located within the highly conserved C-terminal HC domain of WOC, being also present in the three human homologs. ROW also contains several variant PxVxL motifs, suggesting that it might also bind directly to HP1c. In agreement with these observations, euchromatic localization of HP1c depends on the C-terminal chromo-shadow domain, strongly suggesting that it mediates interaction with WOC and ROW (Font-Burgada, 2008).

This study also shows that binding of HP1c to chromatin depends on WOC and ROW that, in contrast, are reciprocally required for binding to chromatin. Domain structure and organization indicate that WOC and ROW are sequence-specific DNA-binding proteins. These results indicate that chromosomal association of HP1c is largely determined by the recognition of specific DNA sequences, which is in contrast to the situation observed in the case of Drosophila HP1a, or mammalian HP1α, where chromosomal association was found to depend on the recognition of a specific pattern of histone modifications; namely, H3K9 methylation. Several HP1 proteins, including HP1a and HP1α, were reported to interact with a number of transcription factors, replication proteins, and chromatin assembly complexes. Yet, it is unclear whether these interactions mediate recruitment of HP1 to specific sites, and/or in response to particular processes, or they actually take place after recruitment to regulate their functions. What is striking in the case of Drosophila HP1c is that its binding to chromatin is strictly dependent on DNA-binding proteins. The results, however, also show that, through the chromodomain, HP1c efficiently binds H3K9me2,3 in vitro. Actually, overexpression of HP1c-lacI leads to its mislocalization to heterochromatin, likely reflecting binding to H3K9me2,3. In this context, it must be noticed that HP1c shows a partial colocalization with H3K9me3. However, binding of HP1c at these sites is in general weak, being also obliterated in the absence of WOC and ROW. HP1 proteins have been reported to interact with different histone methyltransferases (HMTs), being involved in their recruitment to specific sites. Therefore, it is possible that H3K9 methylation at these sites is actually the consequence of HP1c binding. Altogether, these observations indicate that recognition of H3K9me2,3 is not a major determinant of the association of HP1c with chromatin in vivo. Consistent with this interpretation, replacing the chromodomain of HP1c by that of HP1a does not alter its chromosomal distribution. The interaction with WOC and ROW does not appear to hinder the chromodomain from binding H3K9me2,3 since overexpression of HP1c-lacI brings both HP1c and WOC to heterochromatin. Several other possibilities can account for the inability of HP1c to bind H3K9me2,3 in vivo. The interaction of HP1c with WOC and ROW might be of higher affinity than the interaction with H3K9me2,3. In addition, post-translational modifications could regulate these interactions. It is also possible that HP1c is actively excluded from heterochromatin. Whether binding to H3K9me2,3 plays a role at any stage during development or cell cycle progression remains, however, to be determined (Font-Burgada, 2008).

HP1a and HP1b also localize to euchromatin, yet they show strong binding to heterochromatin. Therefore, WOC and ROW could also play a role in binding of HP1a and HP1b to euchromatin. In fact, euchromatic localization of HP1b is decreased in both woc^RNAi and row^RNAi mutants. This effect, however, is much weaker than that observed in the case of HP1c. In contrast, binding of HP1a to euchromatin is not grossly altered in woc^RNAi and row^RNAi mutants, though its association to some specific loci, such as at the 31C region, appears to be affected. In contrast, binding of HP1a and HP1b to heterochromatin, which depends on H3K9me2,3, is not significantly affected in woc^RNAi and row^RNAi mutants. Similarly, mislocalization of overexpressed HP1c-lacI to heterochromatin, which likely reflects binding to H3K9me2,3, is not affected either in woc^RNAi and row^RNAi mutants (Font-Burgada, 2008).

Altogether, these observations suggest that, in Drosophila, HP1 proteins could be recruited to chromatin by at least two independent mechanisms: (1) recognition of H3K9me2,3, which is instrumental in heterochromatin binding, and (2) interaction with sequence-specific DNA-binding proteins, which mediate euchromatic localization of HP1c and, perhaps, of HP1b and HP1a to some specific loci. Actually, the interaction of HP1 proteins with DNA-binding proteins might be more frequent than anticipated. In fact, in C. elegans, HPL-2 was found to interact with LIN-13, a sequence-specific DNA-binding protein containing multiple zinc-finger domains (Font-Burgada, 2008).

HP1c localizes at multiple active chromatin domains and cooperates with WOC and ROW, which show features characteristic of transcription factors, to regulate gene expression. Moreover, targeting HP1c to a reporter construct promotes expression of the reporter gene. This effect is specific of HP1c, since targeting both HP1a and HP1b induce silencing. These results indicate that, rather than as a silencing factor, HP1c acts as a transcriptional regulator that is recruited to chromatin by sequence-specific DNA-binding proteins (Font-Burgada, 2008).

Other HP1 proteins have also been shown to contribute to the regulation of gene expression, yet their presence is generally associated to silencing. These include Drosophila HP1a, which is required for proper expression of most heterochromatic genes as well as a few euchromatic genes. Mammalian HP1γ has also been shown to localize at active genes in a murine erythroid cell line. In these cases, presence of HP1 appears to be implicated in stabilizing RNA transcripts, or in another RNA-processing event occurring during elongation. In the case of HP1c, however, colocalization with the poised RNApol II form, Pol IIo^ser5, is much stronger than with the elongating form, Pol IIo^ser2, suggesting that HP1c acts at the promoter level rather than during elongation. Consistent with this hypothesis, HP1c shows a much stronger colocalization with H3K4me3, a modification that occurs at promoters, than with H3K36me3, which occurs all through transcribed regions and incorporates during elongation. In full agreement with these results, WOC also shows extensive colocalization with Pol IIo^ser5, which is stronger than with Pol IIo^ser2. These results favor a contribution to the regulation of genes containing poised RNApol II. Actually, recent studies show that the presence of poised RNApol II at promoters is more frequent than anticipated, particularly on developmental control genes. Interestingly, a high proportion of genes coregulated by HP1c, WOC, and ROW act during development and morphogenesis (Font-Burgada, 2008).

The precise molecular mechanism(s) underlying the contribution of HP1c to transcription regulation remains to be determined. However, a contribution to RNApol II recruitment appears unlikely since, in the absence of WOC, RNApol II recruitment is not affected. A contribution to the regulation of poised RNApol II is also uncertain, since no gross changes in the levels of Pol IIo^ser5 and Pol IIo^ser2 are observed in woc-null mutants by either immunostaining or Western analysis. It is possible, however, that HP1c/WOC/ROW act only on a reduced subset of genes containing poised RNApol II. It must also be noticed that HP1c likely participates both in promoting and inhibiting transcription. In fact, among the 158 genes that are differentially expressed in the same direction in hp1c^RNAi, woc^RNAi, and row^RNAi mutants, the number of up-regulated and down-regulated genes is similar, 80 in front of 78. Moreover, consistent with a contribution to repression, out of the 35 genes that are coregulated by HP1c, WOC, and ROW in the context of the nervous system, a significantly higher number of genes are found up-regulated than down-regulated in the mutants, 22 versus 13. In contrast, targeting HP1c to a reporter, though modestly, increases its expression. Altogether, these observations suggest that, depending on the actual functional/promoter context, HP1c can be engaged in either promoting or inhibiting transcription (Font-Burgada, 2008).

The patterns of differentially expressed genes observed in woc^RNAi and row^RNAi mutants show a very strong correlation, indicating that WOC and ROW share a common gene expression program. In addition, these results show that genes regulated by HP1c are also regulated by both WOC and ROW. In contrast, a high proportion (78.3%) of differentially expressed genes change expression both in woc^RNAi and row^RNAi but not in hp1c^RNAi, suggesting that WOC and ROW could also regulate gene expression independently of HP1c. However, the extensive colocalization observed between WOC, ROW, and HP1c argues against this possibility. In contrast, the results show that HP1c protein levels are significantly decreased in woc^RNAi and row^RNAi mutants. In this scenario, stronger synergistic effects would be expected in woc^RNAi and row^RNAi mutants than in hp1c^RNAi, which could result in more genes being differentially expressed. Consistent with this hypothesis, a major proportion of genes that are differentially expressed in all three mutants shows stronger changes in woc^RNAi and row^RNAi than in hp1c^RNAi. Actually, out of the 158 genes that are differentially expressed in the same direction in all three mutants, 77 had smaller changes in hp1c^RNAi than in woc^RNAi or row^RNAi, a statistically significant higher number than the 52.6 genes expected under the assumption that the magnitude of change is the same in all three mutants (Font-Burgada, 2008).

Clustering analysis suggests that the gene expression program coregulated by HP1c, WOC, and ROW is executed, at least in part, in the context of the nervous system. In agreement with this hypothesis, expression of woc and row is high in the nervous system during embryogenesis and larval development, and mutant larvae have reduced brains. Furthermore, one of the human homologs of WOC, DXS6673E/ZNF261, is implicated in a form of X-linked mental retardation (Font-Burgada, 2008).

Others have reported that WOC regulates telomere function, since it is required to prevent chromosomal end-to-end fusions. The physical and functional interactions between WOC, HP1c, and ROW suggest that HP1c and ROW might also regulate telomere function. Actually, at telomeres, colocalization of WOC with HP1c and ROW is also most extensive, with all detectable αWOC bands overlapping with αHP1c and αROW bands and vice versa. However, the incidence of telomere fusions in hp1c^RNAi and row^RNAi mutants is low, being similar to that observed in control flies, carrying an UAS-hairpin construct against an unrelated gene, GFP. This is likely the consequence of both the hypomorph character of the mutations and hyperactivation of the RNAi pathway, which is known to regulate telomere function. Consistent with this hypothesis, the frequency of telomere fusions is also low in woc^RNAi. The contribution of ROW to telomere function was also analyzed in row^l(2)SH2172, which corresponds to a very strong mutation caused by a P-element insertion at the ATG-start codon. The incidence of telomere fusions is significantly higher in homozygous row^l(2)SH2172 flies than in control wild-type flies, confirming its contribution to telomere function. The use of currently unavailable hp1c-null mutations is also likely to confirm the contribution of HP1c to telomere function (Font-Burgada, 2008).

HP1a is also known to regulate telomere function. Several observations, however, indicate that the contribution of WOC/ROW/HP1c is not related to that of HP1a. On one hand, su(var)2-5 mutants show much stronger effects than either woc or row mutants. Furthermore, both telomere length and expression of the telomeric retrotransposons Het-A and TART are increased in su(var)2-5 mutants, but they are not affected in woc-null mutants. In addition, expression profiling data show that, in hp1c^RNAi, woc^RNAi, and row^RNAi mutants, expression of Het-A and TART is not significantly affected (data not shown). Similarly, other genes known to contribute to telomere function do not change expression in hp1c^RNAi, woc^RNAi, and row^RNAi mutants (data not shown). Altogether, these observations suggest that the contribution of WOC/ROW/HP1c to the regulation of telomere function is direct and independent of their contribution to the regulation of gene expression (Font-Burgada, 2008).

The endogenous siRNA pathway is involved in heterochromatin formation in Drosophila

A new class of small RNAs (endo-siRNAs) produced from endogenous double-stranded RNA (dsRNA) precursors was recently shown to mediate transposable element (TE) silencing in the Drosophila soma. These endo-siRNAs might play a role in heterochromatin formation. This has been shown in S. pombe for siRNAs derived from repetitive sequences in chromosome pericentromeres. To address this possibility, the viral suppressors of RNA silencing B2 and P19 were used. These proteins normally counteract the RNAi host defense by blocking the biogenesis or activity of virus-derived siRNAs. It was hypothesized that both proteins would similarly block endo-siRNA processing or function, thereby revealing the contribution of endo-siRNA to heterochromatin formation. Accordingly, P19 as well as a nuclear form of P19 expressed in Drosophila somatic cells were found to sequester TE-derived siRNAs whereas B2 predominantly bound their longer precursors. Strikingly, B2 or the nuclear form of P19, but not P19, suppressed silencing of heterochromatin gene markers in adult flies, and altered histone H3-K9 methylation as well as chromosomal distribution of histone methyl transferase Su(var)3-9 and Heterochromatin Protein 1 in larvae. Similar effects were observed in dcr2, r2d2, and ago2 mutants. These findings provide evidence that a nuclear pool of TE-derived endo-siRNAs is involved in heterochromatin formation in somatic tissues in Drosophila (Fagegaltier, 2009).

This study implicates components of the RNAi pathway in heterochromatin silencing during late Drosophila development. The study also provides correlative evidence supporting a functional link between endo-siRNAs and the formation or maintenance of somatic heterochromatin in flies. The viral proteins NLS-P19 and B2 suppress the silencing of PEV markers and induce aberrant distribution of H3m2K9 and H3m3K9 heterochromatic marks as well as histone H3 methylase Su(var)3-9 in larval tissues. Dcr2 and Ago2 mutations have similar effects. In striking contrast, cytoplasmic P19 has no noticeable effect on chromatin. It is proposed that B2 inhibits Dcr2-mediated processing of double-stranded TE read-through transcripts in the cytoplasm; it is further proposed that NLS-P19 sequesters TE-derived siRNA duplexes. This model implies that part of the cytoplasmic pool of TE-derived endo-siRNA (which might be involved in PTGS events) is translocated back into the nucleus to exert chromatin-based functions. In C. elegans, silencing of nuclear-localized transcripts involves nuclear transport of siRNAs by an NRDE-3 Argonaute protein. A similar siRNA nuclear translocation system, possibly mediated by Ago2, may also exist in flies. Alternatively, an as yet unidentified siRNA duplex transporter may be involved. Deep sequencing analyses show that the fraction of siRNAs sequestered by NLS-P19 is smaller as compared with the one bound by P19 in the cytoplasm. Thus, the poor effects of P19 on nuclear gene silencing may be explained if the cytoplasmic pool of siRNA competes with the pool of siRNA to be translocated in the nucleus (Fagegaltier, 2009).

The Dcr-1 partner Loquacious (Loqs), but not the Dcr-2 partner R2D2, was unexpectedly found to be required for biogenesis of siRNA derived from fold-back genes that form dsRNA hairpins. By contrast, it is noteworthy that loqs mutations had little or no impact on the accumulation of siRNA derived from TE. The finding that r2d2 but not loqs mutation suppresses the silencing of PEV reporters and delocalizes H3m2K9 and H3m3K9 heterochromatic marks agrees with these results and further suggests that siRNA involved in heterochromatin formation and siRNA derived from endogenous hairpins arise from distinct r2d2- and loqs-dependent pathways, respectively. One possible mechanism by which TE- or repeat-derived endo-siRNAs could promote heterochromatin formation is by tethering complementary nascent TE transcripts and guiding Su(var)3-9 recruitment and H3K9 methylation. Identifying which enzymes tether siRNAs to chromatin in animals is a future challenge. In addition, some endo-siRNAs could also impact on heterochromatin formation by posttranscriptionaly regulating the expression of chromatin modifiers, such as Su(var)3-9. In any case, the current results demonstrate the value of viral silencing suppressor proteins in linking siRNAs to heterochromatin silencing in the fly soma, as established in S. pombe and higher plants. Because silencing suppressors are at the core of the viral counterdefensive arsenal against antiviral RNA silencing in fly, whether they also induce epigenetic changes in chromatin states during natural infections by viruses deserves further investigation (Fagegaltier, 2009).

Histone demethylase KDM5A is an integral part of the core Notch-RBP-J repressor complex

Timely acquisition of cell fates and the elaborate control of growth in numerous organs depend on Notch signaling. Upon ligand binding, the core transcription factor RBP-J activates transcription of Notch target genes. In the absence of signaling, RBP-J switches off target gene expression, assuring the tight spatiotemporal control of the response by a mechanism incompletely understood. This study shows that the histone demethylase KDM5A (Little imaginal discs in Drosophila) is an integral, conserved component of Notch/RBP-J gene silencing. Methylation of histone H3 Lys 4 is dynamically erased and re-established at RBP-J sites upon inhibition and reactivation of Notch signaling. KDM5A interacts physically with RBP-J; this interaction is conserved in Drosophila and is crucial for Notch-induced growth and tumorigenesis responses (Liefke, 2010).

Histone lysine demethylases reversibly remove methyl marks, thus facilitating changes in chromatin formation and transcriptional regulation. Histone demethylases have therefore been proposed as promising therapeutic targets of human diseases, including cancer, that are often associated with aberrant histone methylation. This study identified KDM5A as an enzyme responsible for the removal of H3K4me3 at Notch target genes; KDM5A interacts directly with RBP-J via a domain located between PHD2 and PHD3 domain and its C-terminal PHD3 domain. Interestingly, the PHD3 domain was shown recently to bind to H3K4me3 (Wang, 2009). Although it has been suggested that the Arid domain of KDM5A can bind to a short DNA sequence, CCGCCC, the importance of this finding is challenged by the fact that this sequence is very common in CGIs, yet KDM5A is found only at a small number of genes in ChIP-on-chip experiments. Morover, 11 of these putative KDM5A DNA-binding sites can be found at the CGI of Deltex-1, but only one is present in the Deltex-1 enhancer. Therefore, no correlation exists between the position with a high density of putative KDM5A-binding sites and the dynamically regulated H3K4 trimethylation site at the Deltex-1 gene. Furthermore, in EMSA assays no corroborate binding of KDM5A was seen at the proposed sites. Thus, it is hypothesized that the PHD3 domain of KDM5A binds to H3K4m3 at active promoters, and once the H3K4me3 substrate is demethylated, KDM5A is released (Liefke, 2010). The Polycomb group (PcG) proteins play important roles in maintaining gene silencing during development and adult tissue homeostasis, and recent studies have shown that histone demethylase KDM5A is part of a Polycomb complex. Thus, a KDM5A/Polycomb complex, recruited to Notch target genes, could facilitate the removal of active mark H3K4me3 and the subsequent addition of the repressive H3K27me3 marks. Paradoxically, original genetic analysis of Drosophila lid mutations classified Lid as a member of the Trithorax group of genes. Biochemical experiments suggest that Drosophila Lid affects the HDAC activity of Rpd3 (Lee, 2009), and molecular and genetic data show that Lid facilitates activation of dMYC target genes in a demethylase-independent manner, explaining in part the original classification of lid as a positive transcriptional regulator. However, more recent data and this study support a key role for KDM5A demethylase in the dynamics of gene silencing (Liefke, 2010).

Human KDM5A-containing and Drosophila KDM5A/Lid-containing complexes have been analyzed by several groups. KDM5A is found to be part of an MRG15-containing complex comprising multiple subunits, including Sin3B, HDAC1/2, and RbAp46, and two histone acetyltrasferases, TRRAP and Tip60 (Hayakawa, 2007). Interestingly, similar to the current data, these studies unveiled effects of KDM5A/RBP2 on H3K4 trimethylation away from the TSSs in intergenic regions. Moreover, the current findings on dynamic removal of H3K4me3 associated with changes in acetylated H3K9 also point to a combined action of KDM5A and histone deacetylases. Importantly, Drosophila KDM5A/Lid complexes also contain histone deacetylase activity along with histone chaperones ASF1 and NAP1. In agreement with with the current data, several of these Drosophila corepressors affect Notch target gene expression (Moshkin, 2009). However, although all of these data clearly point to a repressive role of KDM5A/Lid, how these enzymes silence specific genes was unknown (Liefke, 2010).

KDM5A is a member of the KDM5 family, which consists of four proteins (KDM5A-D) in mammals. Particularly, KDM5A is highly expressed in the hematopoietic system. KDM5A (RBP2)-deficient mice appear grossly normal but display a mild hematopoietic phenotype, especially in the myeloid compartment. The relatively mild phenotype of KDM5A mouse knockout might be explained in part by some redundancy between the KDM5 paralogs. Yet, one of the up-regulated genes in the KDM5A knockout microarray is Ifi2004, a Notch target gene, indicating that KDM5 paralogs might play redundant as well as specific roles. Loss of KDM5 orthologs in organisms that encode a single KDM5 gene show more severe phenotypes, underscoring the important role of KDM5 demethylases in development. Thus, mutations in Drosophila KDM5A homolog lid often result in lethality before hatching, some animals show a small optic brain lobe and small imaginal discs, and functional inactivation of the C. elegans KDM5A ortholog, Rbr-2, results in undeveloped vulvas or a multivulval phenotype (Liefke, 2010).

Although the discovery of histone demethylases implicates a reversible state of epigenetic gene silencing, it was unanticipated that these chromatin-modifying enzymes exert pathway-specific effects on gene regulation. The dynamic switch-off (and back on) system for Notch target gene expression used in this study allowed revealing of dynamic changes of histone modifications at Notch target genes. It was found that the H3K4me3 is removed with a half-time of about 4 h after inhibition of the Notch pathway by the γ-secretase inhibito GSI. These 4 h cannot be explained by out-dilution through cell cycling; the cell division time here is 24 h. Modulation of H3K4 methylation has also been observed in other biological systems, such as the circadian variation of the transcription of the albumin D-element-binding protein gene in the mouse liver or the X inactivation in early embryonic development, where loss of H3K4me3 is one of the earliest and most characteristic features of chromosome-wide silencing (Liefke, 2010).

Disruption of Notch signaling results in a reduction of H3K4me3 at RBP-J sites, while reactivation re-establishes H3K4me3 levels. This suggests that switch-off and switch-on of Notch target genes depends on a tightly controlled balance of histone H3 methylation and demethylation. This study further identifies the histone demethylase KDM5A as a fundamental element in the switch-off process. For re-establishing H3K4me3 levels, Notch-IC could recruit an H3K4me3 methyltransferase to RBP-J sites of Notch target genes. In contrast to Drosophila genes, ~70% of mammalian genes possess CpG islands (CGIs), including many Notch target genes like Deltex-1, Hes-1, Hes-5, Nrarp, and Hey-1. Genome-wide studies proposed that H3K4 trimethylation remains very constant at CGI-containing promoters in different cell types. This study showed that H3K4me3 stays stable at the CGIs of Deltex-1 and Hes-1 after switching off Notch, but is regulated at the RBP-J-binding site. This finding shows for the first time that H3K4me3 does not necessarily have to be removed from the entire promoter to facilitate gene silencing. Instead, modulation of H3K4me3 at specific regulator elements could be sufficient to regulate gene expression. It will be of interest if the dynamic versus constant H3K4me3 is a more common feature of CGI-containing promoters. Recent technical advances in analyzing histone modifications genome-wide might help to address this question (Liefke, 2010).

In summary, this study unveils that histone methylation is dynamically regulated by Notch signaling: Inhibition of Notch leads to a reduction of H3K4me3 levels at regulatory RBP-J sites, while reactivation of signaling re-establishes high levels of H3K4me3. These biochemical and in vivo data support a role for the histone H3K4me3 demethylase KDM5A/Lid in facilitating the switch from activation to repression state via Su(H)/RBP-J in both Drosophila and mammals. Thus, the histone lysine demethylase KDM5A/Lid is a crucial factor in the silencing process. With the in vivo evidence of Drosophila lid/KDM5A in Notch-induced tumorigenesis, this study suggests a pathway-specific tumor suppressor role of KDM5A in cancer, and provides the basis for studies in novel strategies to manipulate Notch-mediated carcinogenesis (Liefke, 2010).

dKDM5/LID regulates H3K4me3 dynamics at the transcription-start site (TSS) of actively transcribed developmental genes

H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3 was analyzed. ChIP-seq (Little imaginal discs) results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling shows that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol llo^ser5) form. The results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription (Lloret-Llinares, 2012).

This study reports that dKDM5/LID localizes at TSS of developmental genes and regulates H3K4me3. dKDM5/LID target genes are actively transcribed and, though weakly, they are significantly downregulated in lid^RNAi mutant flies. Previous reports already suggested a positive contribution of dKDM5/LID to transcriptio. The current results also show that dKDM5/LID target genes are bound by ASH2, an evolutionarily conserved component of H3K4-KMT2 complexes that localizes at TSS and is required for H3K4me3. In addition, dKDM5/LID target genes are strongly downregulated in ash2 mutant flies. These observations indicate that dKDM5/LID and ASH2 act coordinately to regulate H3K4me3 at TSS of developmental genes for their efficient transcription. dKDM5/LID and ASH2, however, do not bind chromatin simultaneously, indicating that they act at different moments during transcription. These observations strongly favor a model by which ASH2 and dKDM5/LID act sequentially during transcription to facilitate its progression. On this regard, work performed in budding yeast links chromatin modification events to sequential RNApol II activation. At a first step, TFIIH-mediated phosphorylation of CTD^Ser5 recruits scKMT2/SET1 to methylate H3K4, and induces promoter escape. Later, the onset of productive transcription involves phosphorylation of CTD^Ser2, which results in recruitment of H3K36 KMT3/SET2 both in budding yeast and mammals. dKDM5/LID recruitment might also be regulated during transcription cycle progression. In this context, it is possible that, after RNApol II activation and subsequent H3K4-methylation, dKDM5/LID is recruited and transient demethylation resets chromatin to the original 'unmethylated' state, facilitating the next RNApol II molecule to initiate progression through the transcription cycle. Consistent with this model, it was shown that the C-terminal PHD-finger of dKDM5/LID, or the mammalian homolog KDM5A/JARID1A, specifically binds H3K4me2,3 (Wang, 2009) and, furthermore, this study has shown that dKDM5/LID binds chromatin enriched in H3K4me3, whereas chromatin bound by ASH2 is poor in H3K4me3. Finally, the results also show that dKDM5/LID depletion significantly reduces RNApol ll occupancy, in particular by the promoter-proximal Pol IIo^ser5 active form, providing a basis for the positive contribution of dKDM5/LID to transcription. In contrast, occupancy by the elongating Pol IIo^ser2 form is not similarly affected, showing a tendency to be slightly increased. It is possible that, in the absence of dKDM5/LID, constitutive/increased H3K4me3 at TSS affects RNApol II pausing and, hence, transcription efficiency. Actually, it has been shown that depletion of NELF, a factor required for RNApol II pausing, results in a general downregulation of its target genes both in Drosophila and human cells (Lloret-Llinares, 2012).

Several reasons could account for the weakness of the observed effect of dKDM5/LID depletion on gene expression. On one hand, though dKDM5/LID content is strongly reduced in lid^RNAi, depletion is not complete. Note that null lid mutations could not be used, as they are lethal during late embryo/early larvae development. Second, although dKDM5/LID is the only enzyme known to specifically demethylate H3K4me3 in Drosophila, additional KDMs might exist capable of playing a similar function. At this respect, it was reported that dKDM2, which was originally found to demethylate H3K36me2, might also be capable of demethylating H3K4me3. Thus, it is possible that loss of dKDM5/LID is partially compensated by dKDM2. As a matter of fact, a genetic interaction was recently reported between dKDM5/lid and dKDM2 (Lloret-Llinares, 2012).

The proposed function of dKDM5/LID in the regulation of transcription is likely conserved, as it was recently reported that mammalian KDM5B/JARID1B preferentially localizes at TSS of developmental genes and regulates H3K4me3. Mammalian KDM5C/JARID1C has also been shown to bind at TSS. Interestingly, although KDM5B/JARID1B is required to efficiently silence stem and germ cell specific genes during neuronal differentiation, its depletion in ESCs shows also a weak downregulation of target genes. In Drosophila, dKDM5/LID has also been shown to be involved in repression of some developmental genes. In fact, in the wing imaginal disc, ∼20% of dKDM5/LID target genes show no detectable H3K4me3. Altogether, these observations suggest that dKDM5/LID might play a dual function; repressing specific genes during development and, in differentiated cells, regulating H3K4me3 dynamics at TSS during transcription (Lloret-Llinares, 2012).

Chromatin landscape dictates HSF binding to target DNA elements

Sequence-specific transcription factors (TFs) are critical for specifying patterns and levels of gene expression, but target DNA elements are not sufficient to specify TF binding in vivo. In eukaryotes, the binding of a TF is in competition with a constellation of other proteins, including histones, which package DNA into nucleosomes. ChIP-seq assay was used to examine the genome-wide distribution of Drosophila Heat Shock Factor (HSF), a TF whose binding activity is mediated by heat shock-induced trimerization. HSF binds to 464 sites after heat shock, the vast majority of which contain HSF Sequence-binding Elements (HSEs). HSF-bound sequence motifs represent only a small fraction of the total HSEs present in the genome. ModENCODE ChIP-chip datasets, generated during non-heat shock conditions, were used to show that inducibly bound HSE motifs are associated with histone acetylation, H3K4 trimethylation, RNA Polymerase II, and coactivators, compared to HSE motifs that remain HSF-free. Furthermore, directly changing the chromatin landscape, from an inactive to an active state, permits inducible HSF binding. There is a strong correlation of bound HSEs to active chromatin marks present prior to induced HSF binding, indicating that an HSE's residence in 'active' chromatin is a primary determinant of whether HSF can bind following heat shock (Guertin, 2010).

Although bona fide HSF binding sites contain highly specific HSE motifs, only a small fraction of potential HSE motifs are occupied by HSF. To search for HSF-free binding sites, a conservative cut-off was used for conformity to the consensus HSE by using a p-value of 5×10^-6 or less, while ensuring that the flanking region is mappable. There are 708 HSF-free motifs that meet these criteria. Less than 15% (107/815) of the mappable HSE motifs with a p-value of 5×10^-6 or less are detectably bound by HSF after HS. Upon closer inspection, this study found that HSF-free motifs are absolutely HSF-free during non-heat shock (NHS), and these same motifs are either unoccupied or infrequently occupied after HS. In contrast, HSF-bound motifs are either very weakly occupied or unoccupied prior to HS, and show strong inducible binding after HS induction. Therefore, these two categories of motifs, HSF-free and HSF-bound, are distinct from one another (Guertin, 2010).

The distribution of HSF binding sites relative to annotated genes and promoter regions was determined. Annotated genes account for 60.6% of the Drosophila reference genome, however, 72% of the HSF-bound motifs are found within gene boundaries. HSF-bound motifs within promoters [500 bp upstream of a transcription start site (TSS)] were also enriched, accounting for 22% of the total bound motifs, while such promoter regions only account for 3.4% of the total reference genome. In contrast, the classification of the 708 HSF-free motifs is much closer to a background distribution; 63% HSF-free motifs are within genes and 5.5% are within promoters. These results indicate that HSE motifs are not simply enriched within gene and promoter boundaries, but that HSF preferentially interacts with HSEs that are present within genes and promoters (Guertin, 2010).

The ChIP-seq method is used routinely to determine genome-wide factor binding profiles; however, important controls and variations in the ChIP protocol more fully exploit this approach. The implementation of the control RNAi knockdown of HSF allowed elimination of the genome-wide set of false positive signals that were resistant to this knockdown, and prevented the elimination of many true positive binding sites. Another rigorous and complementary control for specificity includes performing independent ChIP experiments with multiple antiserum preparations, each of which is affinity purified with nonoverlapping antigens. The details of ChIP-seq chromatin preparation can also enhance peak detection. Additional crosslinking agents and crosslinkers that target particular types of protein/DNA interactions, such as exclusively probing direct protein/DNA interactions with UV light, can also augment the type and quality of information obtained by the basic ChIP-seq strategy (Guertin, 2010).

The non-sequence dependent specificity observed by TFs can be explained by non-mutually exclusive mechanisms: DNA binding is specifically inhibited by repressive chromatin, aided by active chromatin, or mediated by cooperative interactions with chromatin factors. This study report that repressive marks contribute minimally to restrict HSF binding, as only a small fraction of HSF-free motifs are associated with repressive chromatin. Additionally, it was observed that chromatin containing background levels of active and repressive marks is unfavorable to inducible HSF binding - the default state of an in vivo HSE can be considered inaccessible. In contrast, HSF inducibly binds to sites that contain TFs and marks of active chromatin prior to HS induction. This study has shown that the chromatin landscape can be modified to the permissive state and result in recognition and binding of a previously unbound HSE. This result suggests that HSF does not primarily function to bind DNA cooperatively with other factors, but simply co-occupies the same regions as other TFs, due to the accessible nature of the DNA. These results provide a framework for understanding the binding selectivity of HSF, and mechanistic studies can be anticipated that solidify the rules of in vivo binding specificity (Guertin, 2010).

Activators are generally thought to bind to promoters and recruit either Pol II or coactivators to produce productively elongating Pol II. HSF recruits the acetyltransferase CREB Binding Protein (CBP) and a methyltransferase, Trithorax, directly to HS genes. Paradoxically, this study shows that the chromatin landscape at HSF binding sites contains considerable histone acetylation and methylation prior to detectable HSF binding. HSF recruits these enzymes after HS to broaden the domain or increase the level of histone modifications. Another, non-mutually exclusive, possibility is that cofactors other than histones are the functional targets of recruited transferases. Although this study has describe the landscape at HSF binding sites prior to HS, it still remains unclear which factors are responsible for setting up or maintaining the accessibility of these motifs. Furthermore, many HSF-binding sites are probably passively occupied because they happen to be accessible and HSF binding is non-deleterious, but these sites likely have no function in the HS response. The global chromatin landscape is dynamic throughout development and environmental changes; therefore, it is expected that the HSF binding profile at non-functional sites is dynamic as well. Nonetheless, the HS response is a ubiquitous cellular response, so functional sites are likely to be evolutionarily constrained at the sequence level, and actively maintained in the accessible state at the level of chromatin organization (Guertin, 2010).

The maintenance of functional HSF binding sites may be occurring as a result of a specific class of activators. Non-traditional activators, such as GAF, are known to recruit cofactors that establish an accessible chromatin state, as opposed to directly activating transcription of the local gene. This general mechanism has been characterized at the phaseolin gene in Arabidopsis and at the PHO5 gene in yeast. Taken together, this suggests a step-wise process whereby a repressed site can be potentiated for activator binding and subsequently activated. Additionally, it has been shown that active marks are not simply a product of transcription, as the active marks that are associated with intergenic DNaseI hypersensitive sites and putative enhancers are not correlated with respective gene expression. The results suggest that the landscape may be marked with active histone modifications to allow binding of activators that can stimulate transcription; therefore, the presence of a modification would not be expected to correlate with gene expression if the activator has yet to bind. Further investigation of activator binding sites during non-induced conditions will determine the generality of this observation (Guertin, 2010).

Candidate gene analysis shows that HSF is not sufficient to activate local genes. Although inducibly activated genes are occupied by their cognate transcriptional activator near the TSS, it remains unclear how the majority of activators discriminate between locally bound genes to selectively activate. Strikingly, Caudal exhibits promoter element-specific activation, specifically activating genes that contain the Downstream Promoter Element (DPE). Previously, evidence is presented that the presence of a paused polymerase facilitates activation from an Hsp70 promoter, but it is unclear whether or not this is true for the majority HSF-inducible genes. Combinations of promoter features and gene properties are likely necessary for activation. One certainty, however, is that the recent emergence of genome-wide expression and binding data makes the characterization of complex regulatory mechanisms more exciting and promising than ever (Guertin, 2010).

The enhancer of trithorax and polycomb gene Caf1/p55 is essential for cell survival and patterning in Drosophila development

In vitro data suggest that the human RbAp46 and RbAp48 genes encode proteins involved in multiple chromatin remodeling complexes and are likely to play important roles in development and tumor suppression. However, to date, understanding of the role of RbAp46/RbAp48 and its homologs in metazoan development and disease has been hampered by a lack of insect and mammalian mutant models, as well as redundancy due to multiple orthologs in most organisms studied. This study reports the first mutations in the single Drosophila RbAp46/RbAp48 homolog Caf1, identified as strong suppressors of a senseless overexpression phenotype. Reduced levels of Caf1 expression result in flies with phenotypes reminiscent of Hox gene misregulation. Additionally, analysis of Caf1 mutant tissue suggests that Caf1 plays important roles in cell survival and segment identity, and loss of Caf1 is associated with a reduction in the Polycomb Repressive Complex 2 (PRC2)-specific histone methylation mark H3K27me3. Taken together, these results suggest suppression of senseless overexpression by mutations in Caf1 is mediated by participation of Caf1 in PRC2-mediated silencing. More importantly, the mutant phenotypes confirm that Caf1-mediated silencing is vital to Drosophila development. These studies underscore the importance of Caf1 and its mammalian homologs in development and disease (Anderson, 2011).

Several lines of evidence suggest that the participation of Caf1 in PcG complexes may account for many of the phenotypes observed in flies with altered expression of Caf1. First, Caf1 loss-of-function clones in the eye have phenotypes ranging from slight disorganization and bristle defects to almost complete loss of homozygous tissue in adults, and incomplete rescue of Caf1 results in adult eyes that are small and disorganized. Clones of many PcG genes have similar phenotypes in the eye. Loss of E(z) or Pc causes mild defects in differentiation in the third instar disc, but clones fail to survive in adults. An analogous situation occurs in Caf1^short clones, where expression of Elav, which marks differentiating neurons, is present in Caf1 clones at third instar but Caf1^short tissue is largely missing in the adult. Derepression of Hox genes could account for these phenotypes, as ectopic expression of many Hox genes in the eye field causes small disorganized eyes in adults (Anderson, 2011).

Second, flies with incomplete rescue of Caf1 display a range of homeotic phenotypes, notably transformation of arista to leg. Similar homeotic transformations, including antenna-to-leg transformations, are a hallmark of mutations in PcG genes. Also a genetic interaction was observe between Caf1 and the PRC1 gene Pc, since mutations in Caf1 are able to dominantly suppress the homeotic transformation of second or third leg to first leg in Pc¹⁵/+ males (Anderson, 2011).

Third, the disrupted patterning of Caf1^short mutant heads may also result from PcG dysfunction. It is possible that these patterning defects are non-cell autonomous and may be an indirect result of widespread apoptosis in the eye disc. Normally, when an imaginal disc is injured, remaining cells proliferate and assume correct identities, leading to a perfectly patterned adult structure. This type of regeneration requires that some determined cells must change their fates and involves substantial chromatin remodeling. Under specific circumstances, the disc can regenerate with incorrect patterning, leading to duplication, deletion or transformation of structures, a phenomenon referred to as transdetermination. Levels of many PcG transcripts are increased in transdetermining imaginal discs, and heterozygous mutations in PcG genes can enhance transdetermination in regenerating imaginal discs. Therefore, one interpretation of the patterning defects in Caf1^short mutant discs is that under the stress of widespread apoptosis, the remaining heterozygous tissue is haploinsufficient for the chromatin remodeling activity required to properly regenerate and pattern the injured disc. Consistent with this interpretation, no extra or missing appendages are observed in flies with Caf1^long clones, which show less active Caspase 3 staining at third instar (Anderson, 2011).

Finally, in Caf1 mutant tissue, a reduction is observed in levels of the H3K27me3 mark, which is associated with inactive chromatin and PRC2 activity. These data are consistent with a disruption of PRC2 function as a result of loss of Caf1 and represent the first in vivo evidence that Caf1 is an essential member of this chromatin remodeling complex in an animal model (Anderson, 2011).

One obvious question arises from the current study: why were multiple Caf1 alleles identified in a screen for modifiers of the sens overexpression phenotype? Moreover, it is surprising that mutations in Caf1 were not identified in previous Drosophila modifier screens involving PcG or Rb pathway members. It is proposed that the link between sens and Caf1 is due to the role of Caf1 in PcG-mediated silencing (Anderson, 2011).

Recent evidence suggests that Sens and Hox proteins can compete for binding at overlapping sites at an enhancer of the rhomboid (rho) locus (Li-Kroeger, 2008). When the Hox protein Abdominal-A (Abd-A) binds, transcription of rho is activated, whereas binding by Sens leads to repression of rho. In the embryo, this mechanism acts as a molecular switch to allow differentiation of either chordotonal organs (under control of Sens) or hepatocyte-like cells called oenocytes (by the action of Abd-A). It is proposed that a similar mechanism underlies the suppression of the Sens overexpression phenotype. It is hypothesized that one or more targets of Sens in the eye contain similar overlapping sites that can be bound by either Sens or a Hox protein. During normal development, these loci are bound by neither Sens nor Hox in undifferentiated cells posterior to the furrow, as no Hox genes are known to be widely expressed in the eye field. In the absence of both types of factors, these loci are transcriptionally active, and are necessary to ultimately attain the proper fates of the cells in which they are expressed. When Sens is overexpressed, as in ls, Sens binds to its recognition site in the downstream loci, repressing transcription. Repression of these genes initiates a cascade leading to a change in cell fate; for example, some of the cells that would normally become secondary or tertiary pigment cells now become bristle precursors, giving rise to the extra bristles of ls. However, when one copy of Caf1 is lost, a slight derepression of the Hox genes occurs due to loss of PcG activity. Hox proteins are now able to compete with Sens for the overlapping binding sites, tipping the balance towards activation of downstream genes and attainment of normal cell fate - effectively suppressing ls. The ability of ectopic expression of pb and Antp in the eye to suppress ls is consistent with this hypothesis. Suppression of ls by Hox proteins is particularly significant given that ectopic expression of Antp alone in the eye field leads to a small and disorganized eye. As Sens activity is exquisitely sensitive to Hox proteins, especially in the eye, the screen for modifiers of a sens overexpression phenotype was therefore ideal for identifying mutations in Caf1 (Anderson, 2011).

Previous studies have explored pro-apoptotic roles of Hox proteins and anti-apoptotic roles of Sens. It is therefore possible that one effect of Hox gene derepression in ls eyes suppressed by Caf1 may be restoration of an apoptotic fate in cells that would otherwise form bristle precursors due to ectopic Sens. Abd-A expression in the abdomen during normal third instar larval development leads to apoptosis of proliferating neuroblasts of the central nervous system, and ectopic expression of other Hox genes can also cause neuroblast apoptosis. Accordingly, survival of neuroblasts is dependent on PcG activity to repress Hox gene expression. Furthermore, expression of Sens is necessary in the Drosophila embryonic salivary gland to prevent apoptosis. Thus, one possible mechanism for suppression of ls by Caf1 mutations is that in the ls eye, Sens may promote the ectopic bristle fate partly by repressing apoptotic genes in cells normally fated to die, whereas in the ls eye suppressed by mutations in Caf1, ectopic Hox proteins may promote apoptosis and prevent bristle formation. Increased Ubx expression was not detected by antibody staining; however, a very small increase in one or more Hox proteins may be all that is necessary to change the transcriptional state of downstream loci and prevent the ectopic bristles and other defects in the highly sensitized ls eye - especially in the eye field, where no Hox genes are known to be highly expressed. Furthermore, the fact that multiple Hox proteins can recognize the same DNA binding site offers the possibility that the competitive effect of each Hox protein type on genes with overlapping Sens/Hox binding sites would be additive. Therefore, although loss of one copy of Caf1 may only cause a small derepression of any one Hox gene, mild derepression of many Hox genes collectively can lead to strong repression of the ls phenotype (Anderson, 2011).

Biochemical evidence suggests that Caf1 is a member of multiple complexes that effect gene regulation through chromatin remodeling, suggesting that it is a vital component of the cell's arsenal of chromatin modifying factors. Although the results suggest that disruption of PRC2 function may be the most important consequence of Caf1 gain- or loss-of-function, many phenotypes that were observed in Caf1 mutant tissue are also reminiscent of mutations in members of other complexes previously shown to contain Caf1. It is not surprising that all three alleles of Caf1 in the current study are homozygous lethal, and that Caf1^short cells have poor viability, considering that Caf1 has been found in the NURF and CAF-1 complexes, which have fundamental roles in nucleosome assembly and spacing. The apoptosis observed in eyes with Caf1^short clones is also consistent with a role for Caf1 in the dREAM (Drosophila Rbf, E2F2, and Myb-interacting proteins) complex. Members of the E2f family of transcription factors can complex with Dp proteins and bind short recognition sites to activate transcription. When Rb binds the E2f-Dp complex, transcription is repressed. Like Caf1 homozygotes, homozygous rbf1 null flies die in early larval development. Fully rbf1-deficient embryos display increased apoptosis, a phenotype reminiscent of the increased active Caspase-3 staining seen anterior to the morphogenetic furrow in eye discs with Caf1^short clones (Anderson, 2011).

The mammalian homologs of sens, Growth Factor Independence 1 (Gfi1) and Gfi1b are essential to the development of multiple cell types and have been implicated as oncogenes. Therefore, the possibility that Caf1 links Sens with the activity of PcG complexes through parallel, competing pathways has implications for both Drosophila development and the activity of Gfi1 family members in human development and disease, and warrants additional study beyond the scope of the present work. The results underscore the importance of Caf1 to diverse processes, including cell survival and tissue identity, and highlight the participation of Caf1 in multiple chromatin remodeling complexes. Further studies are needed to fully assess the importance of Caf1 in Drosophila development, as well as its developmental role in other chromatin remodeling complexes (Anderson, 2011).

Gcm/Glide-dependent conversion into glia depends on neural stem cell age, but not on division, triggering a chromatin signature that is conserved in vertebrate glia

Neurons and glia differentiate from multipotent precursors called neural stem cells (NSCs), upon the activation of specific transcription factors. In vitro, it has been shown that NSCs display very plastic features; however, one of the major challenges is to understand the bases of lineage restriction and NSC plasticity in vivo, at the cellular level. This study shows that overexpression of the Gcm transcription factor, which controls the glial versus neuronal fate choice, fully and efficiently converts Drosophila NSCs towards the glial fate via an intermediate state. Gcm acts in a dose-dependent and autonomous manner by concomitantly repressing the endogenous program and inducing the glial program in the NSC. Most NSCs divide several times to build the embryonic nervous system and eventually enter quiescence: strikingly, the gliogenic potential of Gcm decreases with time and quiescent NSCs are resistant to fate conversion. Together with the fact that Gcm is able to convert mutant NSCs that cannot divide, this indicates that plasticity depends on temporal cues rather than on the mitotic potential. Finally, NSC plasticity involves specific chromatin modifications. The endogenous glial cells, as well as those induced by Gcm overexpression display low levels of histone 3 lysine 9 acetylation (H3K9ac) and Drosophila CREB-binding protein (dCBP) Histone Acetyl-Transferase (HAT). Moreover, dCBP targets the H3K9 residue and high levels of dCBP HAT disrupt gliogenesis. Thus, glial differentiation needs low levels of histone acetylation, a feature shared by vertebrate glia, calling for an epigenetic pathway conserved in evolution (Flici, 2011).

Understanding the biology and the potential of stem cells of specific origins is a key issue in basic science and in regenerative medicine. This study shows that NSCs can be fully and stably redirected towards the glial fate in vivo, via a transient, intermediate, state, upon the expression of a single transcription factor. NSC plasticity is temporally controlled and quiescent NSCs cannot be converted; however, plasticity is independent of cell division. Finally, the acquisition of the glial fate involves low histone acetylation, a chromatin modification that is conserved throughout evolution, emphasizing the importance of this mark in glial cells (Flici, 2011).

NSCs produce the different types of neurons and glia that form the nervous system. These precursors can be converted into induced pluripotent cells and even into monocytes, a differentiated fate of an unrelated somatic lineage; however, the in vitro behavior may differ markedly from the in vivo situation. For example, the Achaete-Scute Complex homolog-like 1 transcription factor promotes the expression of oligodendrocyte features upon retroviral injection in the dentate gyrus, but promotes neuronal differentiation from the same progenitors in vitro. The use of NB-specific drivers, markers and conditional overexpression protocols, has led to the demonstration that a single transcription factor can fully convert NSCs into glia in a dose-dependent manner. High Gcm levels probably enable this transcription factor to counteract the endogenous transcriptional program and/or to compensate for the absence of cell-specific co-factors. Quantitative regulation is also required in physiological conditions; for example, the nuclear protein Huckebein enhances the gliogenic potential of Gcm upon triggering its positive autoregulation in a specific lineage. The present study therefore shows for the first time that NSCs can be completely and efficiently redirected in vivo towards a specific fate, also highlighting the importance of quantitative regulation in fate choices (Flici, 2011).

It is widely accepted that NSCs are multipotent precursors; however, their plastic features have not been investigated throughout their life at the cellular level. For example, the existence of a tri-potent NSC with the capacity to generate neurons, astrocytes and oligodendrocytes in the adult brain remains to be demonstrated in vivo. This study demonstrates that NSCs are more plastic at early embryonic stages than at the end of embryogenesis. Furthermore, the intrinsically defined program of quiescence is not compatible with fate conversion, even though quiescent cells are subsequently reactivated. As Drosophila glia are generated at different stages, it is unlikely that a general glial repressor arises late in development and specifically restricts the potential of Gcm. The data rather imply that temporal cues progressively limit NSC plasticity, a feature that may have important consequences in therapeutic applications (Flici, 2011).

It will be of great interest to determine whether such irreversible temporal restriction relies on external cues or whether it reflects an internal clock, as it has been shown for the acquisition of temporal identity, the process by which specific progenies are produced at different developmental stages (Flici, 2011).

Finally, the data show that Gcm does not reprogram neurons. Thus, although other somatic and even germ line cells can be reprogrammed into neurons, these post-mitotic cells seem endowed with an efficient brake to fate conversion. Interestingly, dorsal root ganglia neurons can transdifferentiate from one subtype into another in zebrafish, suggesting that, under some conditions, neurons can adopt a different, but closely related, phenotype. In addition, it cannot be formally excluded that a low percentage of immature neurons adopt a glial or a multipotent phenotype upon Gcm overexpression. Nevertheless, the data indicate that neurons are intrinsically different from other cell types, which may reflect a specific chromatin organization and/or expression of an efficient tumor suppressor molecular network. Transcriptome analyses will help characterizing the molecular signature responsible for the neuronal behavior (Flici, 2011).

Dedifferentiation and transdifferentiation of somatic cells can occur in the absence of mitosis, whereas NSCs plasticity has generally been associated to cell division, as a means to erase transcriptional programs and implement new ones. This study shows that, like terminally differentiated cells, NSCs can be efficiently redirected in the absence of cell division. The concomitant extinction of the endogenous program and activation of the glial program indicate that conversion occurs via an intermediate state, as has been described in B cell to macrophage experimental transdifferentiation. The acquisition of an intermediate state (partial reprogramming) has also been proposed for somatic cell reprogramming. The current findings raise a more general question as to whether intermediate states are common and unstable features of many plastic process including development. These states may reveal competing molecular pathways that in physiological conditions are alternatively consolidated or switched off in response to cell-specific signals. The development of tools enabling tracing these dynamic states will improve understanding of cell plasticity under physiological and experimental conditions (Flici, 2011).

Interestingly, altered tumor suppressor gene expression, which alters the proliferation pathway, leads to ambiguous cell identities, which may reflect the stabilization of intermediate fates. Similarly, Drosophila metastatic cells from brain tumors and several non-dividing NSC cells challenged with Gcm co-express the neuronal and the glial programs. It is proposed that the appropriate activation of the mitotic pathway is necessary for efficient consolidation/extinction of specific fates (Flici, 2011).

The interplay of extrinsic signals, transcription factors and chromatin modifications shape the identity of different cell types. The low and high levels of dCBP as well as H3K9ac truly represent a glial and neuronal signature, respectively. They both depend on gcm, which controls the fate choice, but not on genes downstream to Gcm, which are not sufficient to implement such choice. Thus, full fate conversion is accompanied by a cell-specific chromatin modification (Flici, 2011).

Interestingly, whereas dCBP accumulates at different levels in glia versus neurons and its overexpression or loss affects the levels of H3K9ac, the levels of dGCN5, another HAT that is able to acetylate the H3K9 residue in vivo, are similar in glia and neurons. Moreover, dGCN5 overexpression does not enhance H3K9ac levels nor does it affect the expression of glial genes. These data strongly suggest that the dCBP HAT specifically participates in setting up the H3K9ac signature. It should be noted that dGCN5 is a member of multiprotein complexes, which may explain why its overexpression cannot produce high HAT activity on its own. The balance between HATs and histone deacetylases (HDACs), enzymes with counteracting activities, is thought to be important in the regulation of histone acetylation levels. Although the investigation of histone deacetylation is not in the focus of this paper, the relevance of HDACs in the control of the glia-neuron histone acetylation signature cannot be excluded (Flici, 2011).

The tight regulation of histone acetylation in the nervous system seems to be evolutionarily conserved. Human neuronal disorders are frequently connected to downregulation of histone acetylation and HDAC inhibitors are good candidates as therapeutic tools. Histone acetylation is instrumental for mammalian memory formation and CBP plays an important role in long-term memory processes. Altogether, these data indicate that normal neuronal function requires high levels of histone acetylation (Flici, 2011).

This study shows that low HAT activity is necessary for glial differentiation. The increased levels of histone acetylation by overexpression of dCBP cause downregulation of the majority (but not all) of the tested glial genes, whereas the levels of general nuclear factors remain unchanged. The glial cells do not undergo apoptosis, indicating that high dCBP and histone acetylation levels influence specific pathways rather than generally affecting cell viability. The exact molecular mechanisms are not known, yet the behavior is similar in the mammalian CNS. Oligodendrocyte differentiation requires low levels of histone acetylation, resulting from high amounts of HDACs and low amounts of HATs (CBP and P300). The role of HDACs was further investigated, showing that such enzymes directly repress genes that prevent oligodendrocyte differentiation. Most probably an appropriate balance between HATs and HDACs is the key factor, which produces low levels of histone acetylation and regulates mammalian as well as Drosophila glial differentiation (Flici, 2011).

The broadly accepted model is that histone acetylation weakens the interaction between positively charged histone tails and negatively charged DNA, thereby contributing to transcriptional activation. The current data contradict this simple model. First, the levels of H3K4me3, a histone mark that is connected to actively transcribed genes, are similar in glia and neurons. Second, the total mRNA levels are not different in the two cell populations. Third, and most importantly, dCBP overexpression in glia specifically causes downregulation of a set of glial genes. It seems that the H3K9ac levels reflect specific functional differences between neurons and glia, rather than simply revealing general gene activation. Maybe neurons require more plastic and dynamic regulation of transcription than other cell types and this process requires higher capacity of histone acetylation. Supporting this theory is the finding that a large number of activity-regulated enhancers bind CBP in cortical neuronal cultures. The technological breakthrough will be to analyze the transcriptome and the chromatin landscape of a few cells, which will help understanding the mode of action of dCBP and HDACs in the control of Drosophila glial and neuronal differentiation (Flici, 2011).

Functional antagonism between histone H3K4 demethylases in vivo

Dynamic regulation of histone modifications is critical during development, and aberrant activity of chromatin-modifying enzymes has been associated with diseases such as cancer. Histone demethylases have been shown to play a key role in eukaryotic gene transcription; however, little is known about how their activities are coordinated in vivo to regulate specific biological processes. In Drosophila, two enzymes, dLsd1 [(Suppressor of variegation 3-3), Drosophila ortholog of lysine-specific demethylase 1)] and Lid (little imaginal discs), demethylate histone H3 at Lys 4 (H3K4), a residue whose methylation is associated with actively transcribed genes. These studies show that compound mutation of Lid and dLsd1 results in increased H3K4 methylation levels. However, unexpectedly, Lid mutations strongly suppress dLsd1 mutant phenotypes. Investigation of the basis for this antagonism revealed that Lid opposes the functions of dLsd1 and the histone methyltransferase Su(var)3-9 in promoting heterochromatin spreading at heterochromatin-euchromatin boundaries. Moreover, the data reveal a novel role for dLsd1 in Notch signaling in Drosophila, and a complex network of interactions between dLsd1, Lid, and Notch signaling at euchromatic genes. These findings illustrate the complexity of functional interplay between histone demethylases in vivo, providing insights into the epigenetic regulation of heterochromatin/euchromatin boundaries by Lid and dLsd1 and showing their involvement in Notch pathway-specific control of gene expression in euchromatin (Di Stefano, 2011).

Molecular studies have identified an increasingly large number of histone-modifying enzymes, and biochemical assays readily allow these proteins to be classified, but the more difficult and more important challenge is to understand how these various enzymatic activities are integrated, in vivo, to control biological processes. This study examined the effects of combining mutations in the two H3K4 demethylases Lid and dLsd1 in Drosophila. Thise studies, performed in vivo, show that the interplay between Lid and dLsd1 is dependent on the chromatin context and active signaling pathways. The results show a consistent pattern of genetic interactions between Lid and dLsd1 that is evident in multiple tissues and phenotypes. Unexpectedly, despite their activity as histone H3K4 demethylases, these proteins function antagonistically in a number of functional and developmental contexts. For example, dLsd1 and Lid have opposing functions in the establishment of euchromatin and heterochromatin boundaries. At these locations, the antagonism does not seem to stem from the effects of Lid on H3K4 methylation, but rather from its indirect effects on the spreading of H3K9me2. In addition, while the data show that both Lid and dLsd1 can repress Notch targets within euchromatin when Notch signaling is not active, and that Notch signaling is an important component of the dLsd1 mutant phenotype, genetic evidence supports the hypothesis that Lid and dLsd1 have antagonistic functions in the context of activated Notch signaling. This complex pattern of interactions illustrates that the functional interplay between demethylases, and most likely between other types of chromatin-associated proteins, cannot be rationalized into a single generic model. The evidence that dLsd1 can switch from being a negative regulator of Notch target genes to a positive regulator adds an extra layer of complexity to the interplay between Lid and dLsd1, and strongly supports the concept that the activity of histone demethylases is highly regulated and context-dependent (Di Stefano, 2011).

Genetic and biochemical data support a model for the creation and maintenance of heterochromatin boundaries, proposed by (Rudolph, 2007), in which dLsd1 promotes deacetylation of H3K9 by RPD3 and subsequent methylation of H3K9 by Su(var)3-9, thereby facilitating spreading of heterochromatin. In addition, this study shows an increase in H3K4me1 at the white-rough-est locus in dLsd1 mutant flies, suggesting that active demethylation of H3K4me1 by dLsd1 is an important step in the establishment of heterochromatin. Furthermore, it was found that Lid antagonizes dLsd1 function by promoting euchromatin formation, and that the spreading of heterochromatin seen in Lid mutants is dependent on dLsd1 and Su(var)3-9 activities. Consistent with this notion, H3K9 methylation levels are increased in Lid mutant flies compared with control at the white-rough-est locus and in pericentric heterochromatin. Interestingly, the levels of H3K4me2 and H3K4me3 at the white-rough-est locus are very low and increase only marginally upon Lid mutation, suggesting that Lid function in this context is independent of its histone H3K4 demethylase activity. Previously, Lid had been reported to facilitate activation of Myc target genes in a demethylase-independent manner, and to antagonize Rpd3 histone deacetylase function; moreover, mutation of Lid has been shown to cause a decrease of H3K9 acetylation levels. It is therefore tempting to speculate that Lid opposes the spreading of heterochromatin, independent of its function as a histone H3K4 demethylase, by antagonizing the activity of the dLsd1/Su(var)3-9/Rpd3 complex. This antagonism would explain why, in double mutants for dLsd1 and Lid, the balance between euchromatin and heterochromatin is artificially reset to wild-type levels. Consistently, reorganization of chromatin domains observed in dLsd1 mutant flies affects the expression of genes located at the 2R euchromatin-heterochromatin boundary, an effect that is reversed by mutation of Lid (Di Stefano, 2011).

Given the predominant presence of H3K4 methylation in euchromatin and its important role in determining the transcription status of a gene, it was of interest to establishing the nature of the interplay between Lid and dLsd1 in a euchromatic context. Previous studies had implicated Lid as a crucial factor in the silencing of Notch target genes. The current study shows a cooperative role for Lid and dLsd1 in repressing Notch target gene expression, and suggests that they contribute to repression by maintaining low levels of H3K4 methylation. Repression of Notch target genes is essential for the establishment of Notch-inhibited cell fates, suggesting that Lid and dLsd1 could play a role in proper cell fate specification during Drosophila development. Interestingly, the role of dLsd1 does not seem to be limited to repression of Notch target genes. Indeed, genetic analysis suggests that, in a context in which the Notch signaling pathway is active, dLsd1 switches from a repressor to an activator role. Such a dual role had already been described for Su(H), whose switch from a repressor to an activator has been suggested to be mediated through an exchange of associated proteins. Similarly, in mammalian cells, studies have shown that LSD1 activity can be modulated by changes in composition of the complexes present at the Gh promoter, and, depending on the cell type (somatotroph or lactotroph), LSD1 can act as either an activator or a repressor. Therefore, a possible explanation for the current data is that, depending on the complexes available, dLsd1 can switch from being a repressor to acting as an activator of Notch target genes. Alternatively, dLsd1 mutation could promote derepression of negative regulators of Notch activity, or could directly modulate Notch activity by demethylating crucial components of the Notch-activating complex. Further studies are required to distinguish between these possibilities (Di Stefano, 2011).

These results provide the basis for future studies aimed at investigating whether the dual role of dLsd1 in modulating Notch signaling is conserved in mammals. In mice, LSD1 has been shown to repress the Notch target Hey1 in late stages of pituitary development, suggesting that its ability to regulate Notch target genes is conserved. This pathway-specific function of LSD1 could potentially be exploited to create novel strategies to manipulate Notch-mediated carcinogenesis (Di Stefano, 2011).

Collectively, these results reveal an intricate interplay between the histone demethylases Lid and dLsd1 in the control of higher-order chromatin structure at euchromatin and heterochromatin boundaries affecting developmental gene silencing. They also demonstrate an involvement of dLsd1 and Lid in Notch pathway-specific control of gene expression in euchromatin, and support the idea that, depending on the context, Lid and dLsd1 can favor either transcriptional activation or transcriptional repression (Di Stefano, 2011).

The DEK oncoprotein is a Su(var) that is essential to heterochromatin integrity

Heterochromatin integrity is crucial for genome stability and regulation of gene expression, but the factors involved in mammalian heterochromatin biology are only incompletely understood. This study identified the oncoprotein DEK, an abundant nuclear protein with a previously enigmatic in vivo function, as a Suppressor of Variegation [Su(var)] that is crucial to global heterochromatin integrity. DEK interacts directly with Heterochromatin Protein 1 alpha (HP1alpha) and markedly enhances its binding to trimethylated H3K9 (H3K9me3), which is key for maintaining heterochromatic regions. Loss of Dek in Drosophila leads to a Su(var) phenotype and global reduction in heterochromatin. Thus, these findings show that DEK is a key factor in maintaining the balance between heterochromatin and euchromatin in vivo (Kappes, 2011).

The observation that knocking down DEK expression leads to a marked diminution of the H3K9me3 heterochromatin mark suggested that, as a consequence of the decrease in HP1alpha being brought to histones, there is less efficient recruitment of KMT1 A/B [Su(var)3-9]. Thus, to determine if DEK is indeed a functional member of self-sustaining silencing loops acting through coordinated recruitment of HP1alpha and thus KMT 1 A/B to the four genes (Oct3/4, DHFR, PAX3, and MYOD) examined in HeLa cells, chromatin immunoprecipitation (ChIP) assays were performed. As no KMT 1A/B-specific antibodies were available, stable DEKkd or stable GFP-DEK overexpression was established in HEK293 cell lines that had been engineered previously to stably express low levels of hemagglutinin (HA)-tagged-KMT1 A/B. In the ChIP assays, a significantly reduced abundance of H3K9me3 was found at all genes examined in the HEK293 DEKkd cells, H3K9me3, control, and shDEKB1, confirming results obtained in HeLa cells. Furthermore, reduced gene-specific H3K9me3 levels coincided with a marked reduction in the abundance of KMT1 A/B. In strong support of the knockdown data, significantly increased H3K9me3 levels were identified in GFP-DEK-overexpressing HEK293 cells at the genes investigated, accompanied by increased occupancy of KMT1 A/B at these particular genes. Thus, both knockdown and overexpression studies demonstrate that DEK coordinates the recruitment of KMT1 A/B to specific genes, thus regulating H3K9 trimethylation (Kappes, 2011).

In summary, This study has identified DEK as a novel Su(var) factor with a conserved role in global heterochromatin integrity that functions by augmenting the binding of HP1alpha (and KMT1 A/B) to the H3K9me3 heterochromatic mark. As it has been shown previously that HP1alpha and KMT1 A/B are crucial to self-sustaining silencing loops, disruption of this mechanism can lead to significant changes in the epigenome of a given cell. Furthermore, DEK is the only oncoprotein described that directly affects heterochromatin integrity on a global level. This effect was seen in Drosophila and cell lines derived from patients with cervical cancer (HeLa S3), human embryonic kidney cells, and melanoma cells. The observation that DEK is a key factor in heterochromatin biology suggests that disruption of the balance between euchromatin and heterochromatin could play an important role in the pathogenesis of cancers in which DEK expression is altered. Most notably, the findings indicate that DEK, a nonhistone protein with no known enzymatic activity, plays a vital role in global heterochromatin integrity across species (Kappes, 2011).

Drosophila Set1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription

Histone H3 lysine 4 trimethylation (H3K4me3) is a major hallmark of promoter-proximal histones at transcribed genes. This study reports that a previously uncharacterized Drosophila H3K4 methyltransferase, dSet1, and not the other putative histone H3K4 methyltransferases (Trithorax), is predominantly responsible for histone H3K4 trimethylation. Functional and proteomics studies reveal that dSet1 is a component of a conserved H3K4 trimethyltransferase complex and polytene staining and live cell imaging assays show widespread association of dSet1 with transcriptionally active genes. dSet1 is present at the promoter region of all tested genes, including activated Hsp70 and Hsp26 heat shock genes and is required for optimal mRNA accumulation from the tested genes. In the case of Hsp70, the mRNA production defect in dSet1 RNAi-treated cells is accompanied by retention of Pol II at promoters. These data suggest that dSet1-dependent H3K4me3 is responsible for the generation of a chromatin structure at active promoters that ensures optimal Pol II release into productive elongation (Ardehali, 2011).

Genomic DNA of eukaryotic cells is organized into a nucleoprotein structure called chromatin. The fundamental building block of chromatin is the nucleosome core particle, which consists of 147 base pairs of DNA wrapped around an octamer of the histones H2A, H2B, H3, and H4. Nucleosomes can form an obstacle to processes requiring access to the DNA such as transcription. Covalent modifications of histones at particular residues can alter the properties of nucleosomes by both changing chromatin's compactness and accessibility and by specifying new interactions of histones with transcription factors. One of these modifications, histone H3 lysine 4 trimethylation (H3K4me3), has been shown to be a major conserved mark of chromatin at nucleosomes immediately downstream of promoters of transcribed genes in yeast, Drosophila, and mammals. Although nucleosomes carrying this modification are targeted by a number of transcription and chromatin regulators, its contribution to transcription remains mysterious. In baker's yeast, the mutation in the gene coding for the H3K4 methyltransferase, SET1, does not cause obvious transcription defects during vegetative growth, while in human cells, the functional study of this mark is hampered by the existence of several genes encoding H3K4 trimethyltransferases (Ardehali, 2011 and references therein).

H3K4 methylation is introduced into nucleosomes by the type-2 histone lysine methyltransferases (KMT2s; Allis, 2007). The prototypic KMTs from Drosophila are Suppressor of variegation 3–9, Enhancer of zeste, and Trithorax (Trx), and they share similarity in their catalytic SET domain. Of these, only Trx is H3K4-specific, and has been proposed to be the major KMT2 in flies. Flies possess a second, Trithorax-related protein (Trr), which functions in the regulation of hormone-response gene expression. The two Drosophila Trx relatives are related to the mammalian Mixed lineage leukaemia (Mll)1–4 KMT2s. While some Mll complexes contain subunits found in the yeast Set1 complex (COMPASS), Trx/Mll relatives have also been shown to assemble into complexes that are unrelated to COMPASS and contain histone acetyltransferases. These findings suggest a functional diversification of Trx/Mll complexes. Surprisingly, genome-wide studies in flies and mammals revealed that Trx/Mll relatives do not localize to over 97% of H3K4me3 domains, including those adjacent to transcription start sites (TSSs) of most expressed genes. Recent studies led to the identification of two Set1 orthologues in humans and showed that they assemble into complexes similar to yCOMPASS; however, their contribution to overall H3K4me3 is still obscure. Since a Set1 homologue was not found in Drosophila, the role of Set1 versus Trx/Mll relatives in global, transcription-linked H3K4 trimethylation at promoters is still unclear in multicellular organisms (Ardehali, 2011).

This study reports the identification and characterization of a Set1 homologue from Drosophila, CG40351/dSet1. Proteomics and functional studies revealed that the dSet1 complex is identical in its composition to its human counterpart and has strong H3K4 trimethyltransferase activity towards recombinant nucleosomes. RNAi-mediated knockdown (KD) studies demonstrated that dSet1 is responsible for bulk H3K4 di- and trimethylation, while the KD of Trx or Trr had less pronounced effects on H3K4me2/3. dSet1 co-localizes with H3K4me3 and transcribing Pol II on polytene chromosome, and the loss of the dSet1-complex subunit, dCfp1, diminishes dSet1 and H3K4me3 at transcription puffs. The KD of dSet1 causes reduced mRNA levels at all tested genes, including heat shock (HS) genes. Live cell imaging studies also revealed that EGFP–dSet1 is rapidly recruited to the Hsp70 HS loci upon activation. Photobleaching/recovery assays demonstrated that EGFP–dSet1 is continuously exchanged at the activated hsp70 loci. Moreover, time course HS experiments showed that KD of dSet1 caused increased Pol II levels at the hsp70 promoter during extended HS periods. These data supports a model in which dSet1-dependent H3K4me3 regulates chromatin changes at promoter-proximal nucleosomes that positively influence the release of Pol II into productive elongation, thereby contributing to optimal mRNA levels (Ardehali, 2011).

The Drosophila dSet1 complex is a component of a conserved complex responsible for bulk H3K4me3. dSet1 is required for H3K4me3 at all transcribed genes tested and its loss leads to a reduction of transcription levels accompanied by the accumulation of Pol II at promoters, which occur during phases of maximal transcription up-regulation. These findings support a model in which dSet1-dependent H3K4me3 generates a chromatin architecture facilitating the passage of Pol II through promoter-proximal nucleosomes during multiple rounds of transcription (Ardehali, 2011).

To this date, the role of Trx/Mll versus Set1 orthologues in transcription-linked H3K4 methylation is not fully understood. These studies support that dSet1 and Trx/Mll relatives form distinct subfamilies that most likely have non-overlapping functions in H3K4 methylation. While it has been proposed that Trx functions in complexes similar to Set1 in flies, this study along with other reports support that Set1 homologues have a broader role in transcription-linked H3K4 methylation (Ardehali, 2011 and references therein).

Recent studies showed that Trx might even have a role in development independent of its KMT activity. A C-terminal cleavage product of Trx (Trx-Cter) containing the SET domain was found at sites lacking H3K4me3. Trx-Nter distributed over large domains of active PcG-target genes, which also lacked H3K4me3, but were acetylated at H3K27 (H3K27ac). H3K27ac is introduced by CBP, and its KAT activity depends on its association with Trx-Nter. This acetylation protects H3K27 from methylation by PcG complexes, suggesting that Trx can function in positive gene regulation independent of its KMT activity. Consistent with this model, the loss of the elongation factor, Kismet, abolished the association of Trx with chromatin, while global H3K4me3 levels were unaffected. In these studies, chromosomal H3K4me3 was also unchanged in trx mutants. These findings are consistent with the observations that H3K4me3 nearly fully overlaps with dSet1 and depended at most sites on the dSet1 complex (Ardehali, 2011).

These studies and previous reports support that Trr has a more widespread role in H3K4 methylation compared with Trx. The SET domain of Trr had robust monomethyltransferase activity in vitro, and it was observed that the KD of Trr affected H3K4me1. Since isolated SET domains of KMT2s are only capable of monomethylating H3K4, further functional studies on purified Trr complexes will be necessary to address whether this factor has limited H3K4 KMT activity. In summary, dSet1 complexes are likely to regulate H3K4me3 in most chromatin regions, including the promoters of the majority of active genes, while Trx and Trr might function in diverse complexes with primary roles in developmental gene regulation (Ardehali, 2011).

The characterization of the yeast and human Set1 complexes supported a functional and structural conservation between these complexes (Lee, 2005; Eissenberg, 2010). Proteomics studies indicate that this is also the case for the Drosophila Set1 complex, which is identical in its composition to the human complex. Functional similarities were observed between the Cfp1 subunit of Set1 complexes in metazoans. In dCfp1 mutants, dSet1 dissociated from chromatin and H3K4me3 levels on polytene chromosomes were severely reduced. In humans, Cfp1 is essential for the targeting of hCOMPASS to euchromatin, while yeast yCfp1p/Spp1p is required for the trimethyltransferase activity of yCOMPASS (Ardehali, 2011).

in vitro methyltransferase assays revealed that dSet1 complexes trimethylate H3K4 in recombinant nucleosomes. In vivo, H2B ubiquitination stimulates nucleosomal H3K4 trimethylation by Set1. The data suggests that H2Bub has no direct regulatory role on the dSet1 complex, which is consistent with studies reporting that H2Bub prevents the loss of H2A/H2B heterodimers by Pol II-dependent transcription in vivo. This might explain why the stable association of Set1 complexes with nucleosomes is less dependent on H2Bub in a transcription-independent context. In conclusion, the data supports that dCOMPASS and its counterparts in other eukaryotes are highly conserved at the structural and functional level (Ardehali, 2011).

The role of Set1 in transcription initiation versus elongation is still under debate. It is well established that the 5′ end of the hsp genes are occupied by paused Pol II. The current data indicates that promoter occupancy by Pol II at the HS and many other loci does not depend on dSet1. Pol II levels were also mostly unchanged at transcription puffs in dCfp1 mutants, and the KD of dSet1 did not cause a drop in Pol II levels at the activated Hsp70 gene. Furthermore, dSet1 and H3K4me3 levels increased at hsp loci after their activation, further supporting that dSet1 accumulation positively correlates with transcriptional activity. In fact, dSet1-dependent H3K4me3 appears to directly correlate with gene expression levels, and the KD of dSet1 has stronger impact on highly expressed genes such as α-tubulin (84B), sda, R, or the hsp loci. Interestingly, the hsp26, hsp70, and hsp83 mRNA accumulation defects occurred in dSet1-depleted cells during phases of maximal up-regulation between 10 and 20 min post-HS. Recent genome-wide studies on the role of Ash2, a subunit of the dSet1 complex, revealed that Ash2-dependent H3K4me3 was most critical for the expression of strongly expressed genes (Perez-Lluch, 2011), which is fully consistent with these studies (Ardehali, 2011).

The retention of Pol II at the hsp70 promoter in dSet1-depleted cells is accompanied with a decrease in Pol II density in the body of the gene. This suggests that dSet1 might have a role in the release of Pol II into productive elongation, which has been shown to be the rate-limiting step in transcription. This appears to be different from yeast, in which the promoter occupancy of Pol II was reduced at the MET16 locus in cells lacking SET1. Given the relatively modest effect of dSet1 KD on the Pol II density across the body of Hsp70 gene, it is likely that dSet1-dependent H3K4me3 has other functions in transcription; however, these roles must be restricted to promoter-proximal nucleosomes due to the accumulation of dSet1 in the 5′ regions of active genes. Considering that H3K4me3 is required for the recruitment of pre-mRNA processing and elongation factors to the 5′ regions of genes, it cannot be excluded that the observed hsp mRNA accumulation defects are due in part to the degradation of improperly processed pre-mRNAs. No major changes were observed in the phosphorylation states of Pol IIo along the axis of the activated hsp70 gene. This was not unexpected, since serine 5 phosphorylation and Pol II pausing at the hsp70 promoters precedes H3K4me3, supporting that this process is independent of H3K4me3 (Ardehali, 2011).

The data support a model in which dSet1-dependent H3K4me3 successively leads to the generation of a chromatin structure facilitating the repeated passage of Pol II through promoter-proximal chromatin. Since the release of Pol II into productive elongation is rate limiting, H3K4me3 might form a docking platform for other chromatin modifiers, which generate nucleosomes that are less refractive to Pol II passage. It has been shown that the nucleosomes at promoters of highly expressed genes are enriched for H3.3 and H2A.Z in hyperacetylated form. The candidate H3.3-exchange factor, CHD1, was shown to bind to H3K4-trimethylated nucleosomes, and H3.3 is the main target for H3K4 trimethylation at active genes. It is tempting to speculate that H3K4me3 might contribute to the generation of unstable nucleosomes containing H3.3 and H2A.Z, which would be consistent with the observed continuous exchange of dSet1 at the activated hsp70 puffs. On the other hand, it cannot rule out that histone H3K4me3-independent functions of the dSet1 complex are critical for its role in transcription (Ardehali, 2011).

Finally, dSet1-dependent H3K4me3 is likely to recruit factors that generate a chromatin architecture at promoters that is critical for optimal transcription levels. In fact, a number of transcription and chromatin regulators were confirmed to interact with nucleosomes trimethylated at H3K4; however, repressive complexes like the HDAC1 complexes were also found to be recruited by this mark. Future studies are necessary to identify other key regulators that interact with dSet1 complexes in the regulation of maximal transcription levels (Ardehali, 2011).

Set1 is the main H3K4 di- and tri-methyltransferase throughout Drosophila development

In eukaryotes, the post-translational addition of methyl groups to histone H3 lysine 4 (H3K4) plays key roles in maintenance and establishment of appropriate gene expression patterns and chromatin states. This study describes an essential locus within chromosome 3L centric heterochromatin that encodes the previously uncharacterized Drosophila ortholog (dSet1, CG40351) of the Set1 H3K4 histone methyltransferase (HMT). The results suggest that dSet1 acts as a 'global' or general H3K4 di- and trimethyl HMT in Drosophila. Levels of H3K4 di- and trimethylation are significantly reduced in dSet1 mutants during late larval and post-larval stages, but not in animals carrying mutations in genes encoding other well-characterized H3K4 HMTs such as trr, trx, and ash1. The latter results suggest that Trr, Trx, and Ash1 may play more specific roles in regulating key cellular targets and pathways and/or act as global H3K4 HMTs earlier in development. In yeast and mammalian cells, the HMT activity of Set1 proteins is mediated through an evolutionarily conserved protein complex known as Complex of Proteins Associated with Set1 (COMPASS). Biochemical evidence is presented that dSet1 interacts with members of a putative Drosophila COMPASS complex and genetic evidence is presented that these members are functionally required for H3K4 methylation. Taken together, these results suggest that dSet1 is responsible for the bulk of H3K4 di- and trimethylation throughout Drosophila development, thus providing a model system for better understanding the requirements for and functions of these modifications in metazoans (Hallson, 2012).

Post-translational modification of histones can alter the local chromatin environment and affect the recruitment of transcriptional regulatory machinery. These modifications can play diverse roles in transcriptional activation or silencing, and cross talk between different activating and silencing modifications may fine-tune levels of transcription (Hallson, 2012).

The post-translational addition of up to three methyl groups to histone H3 lysine 4 (H3K4) residues (H3K4me1, H3K4me2, and H3K4me3) correlates with active transcription. H3K4 di- and trimethylation is often enriched at the promoter and 5' coding regions of active genes, whereas H3K4 monomethylation is commonly found near the 3' ends of active genes and within enhancer elements. Although the mechanisms of methyl-H3K4-mediated transcriptional activation are not fully elucidated, trimethyl-H3K4 is thought to act as a docking scaffold for the recruitment of the transcription pre-initiation complex and transcriptionally activating chromatin-remodelling complexes (Hallson, 2012).

In the budding yeast, Saccharomyces cerevisiae, all mono-, di-, and trimethylation of H3K4 is catalyzed by the Set1 enzyme, and the enzymatic activity of Set1 is modulated through a multi-subunit protein complex known as the Complex of Proteins Associated with Set1 (COMPASS). COMPASS is evolutionarily conserved, with functional orthologs of Set1 acting as major H3K4 histone methyltransferases (HMTs) in metazoans (Hallson, 2012 and references therein).

Higher metazoans possess additional H3K4 methylases, the mixed lineage leukemia (MLL) class of proteins, which act through distinct complexes similar to COMPASS. The MLL proteins (MLL1-5) are required at limited but important subsets of gene targets, such as homeotic and hormone response genes. H3K4 methylases identified in Drosophila melanogaster to date include Trx (homologous to MLL1-2), Trr (homologous to MLL3-4), and Ash1. Ash1 and Trx are members of the Trithorax group of proteins that antagonize Polycomb group-mediated gene silencing. In addition, Trx methylates H3K4 at heat-shock loci upon induction and appears to be required for mediating stress responses to heat stimuli. Trr is recruited to and required for H3K4 methylation at gene targets responsive to the insect nuclear hormone ecdysone. Although these HMTs are known to catalyze H3K4 methylation and are widely believed to act as the main global H3K4 methylases in Drosophila, the functional roles of the Drosophila ortholog of Set1 (dSet1) have remained undefined, largely because its location within centric heterochromatin makes genetic and molecular analysis particularly challenging (Hallson, 2012).

In efforts to functionally annotate essential heterochromatic genes in Drosophila, this study has linked dSet1/CG40351, the Drosophila set1 ortholog, to an essential genetic locus previously known as lethal 5 or l(3L)h5, residing in chromosome 3L centric heterochromatin. Surprisingly, dSet1, and not Trx, Trr, or Ash1, was shown to act as the main global H3K4 di- and trimethylase throughout Drosophila development. Genetic and molecular evidence is provided that Drosophila orthologs of other COMPASS members are required for H3K4 methylation and physically interact with dSet1. These findings establish a foundation for examining transcriptional regulatory mechanisms underlying this key post-translational modification (Hallson, 2012).

These results indicate that dSet1 acts as the main global H3K4 methylase throughout Drosophila development and is required for completion of late developmental stages. Although developmental roles of Set1 have been reported in Caenorhabditis elegans, this is the first report that a Set1 ortholog is essential for the somatic development of a multicellular organism (Hallson, 2012).

A persisting maternal contribution of dSet1 mRNA or protein to the catalysis of bulk H3K4me2/me3 during early developmental stages cannot be ruled out. Indeed, RNA sequencing data available on FlyBase indicate that dSet1 is present in embryos aged 0-2 hr post egg lay, suggesting significant maternal loading of dSet1 transcripts. However, knocking down this maternal contribution during embryogenesis by expressing dSet1 RNAi in a dSet1 mutant background only slightly increases lethality at the L3 larval stage relative to dSet1 mutants. It is possible that, in addition to dSet1, other H3K4 methylases are responsible for 'early' bulk H3K4 methylation. Consistent with this, mutations in trr result in major losses of H3K4me2 and H3K4me3 levels during Drosophila embryogenesis. Moreover, bulk H3K4 dimethylation during C. elegans embryogenesis depends mostly on ASH-2, and not on SET-2 (the C. elegans dSet1 ortholog), suggesting that other H3K4 HMT players are involved (Hallson, 2012 and references therein).

Trx and Trr, while apparently not required for bulk H3K4 methylation, may be important for transcriptional regulation of a subset of specific gene targets later in development. This would be consistent with their proposed role in human cells, where the Trr and Trx homologs MLL1-2 and MLL3-4 are thought to methylate H3K4 at a limited number of non-overlapping gene targets. Recent findings suggest that the predominant function of Ash1 is the catalysis of H3K36, and not H3K4 methylation (Hallson, 2012).

This study has shown that dSet1 interacts within the Drosophila COMPASS and has demonstrated the requirement of other Drosophila COMPASS members for H3K4 methylation, facilitating dissection of the functional roles of individual COMPASS members. Upon hcf and dWdr82 RNAi knockdown, only levels of H3K4me3 are reduced, an effect differing from that associated with loss of dSet1 function and suggesting specialized roles for Hcf and dWdr82 within the COMPASS. A nearly complete loss of H3K4me2 and H3K4me3 in ash2 mutants and dRbbp5 and wds RNAi knockdown animals suggests that these members are critical for COMPASS function; a loss of H3K4 monomethylation in these same animals is an effect not observed in dSet1 mutants and suggests distinct roles of dRbbp5, Wds, and Ash2 aside from their roles within the COMPASS (Hallson, 2012).

Although Set1 has been reported to target H3K4me1 in Saccharomyces cerevisiae, there have been no reports indicating that Set1 plays a similar role in metazoans, and no reductions were observed in monomethyl H3K4 in dSet1 mutants. The results also rule out the individual contributions of Ash1, Trr, or Trx to general H3K4 monomethylation. It has been reported that the human MLL/COMPASS complex subunits WDR5, Rbbp5, ASH2L, and DPY-30 form a complex (known as WRAD) that monomethylates recombinant histone H3 at lysine 4 in vitro. The involvement of a Drosophila form of WRAD in H3K4me1 seems plausible as ash2 mutations as well as wds and dRbbp5 RNAi knockdown result in decreased levels of H3K4me1. Alternatively, bulk H3K4 monomethylation may be catalyzed or targeted by an as-yet-uncharacterized H3K4 HMT complex containing these members or by combinatorial effects of Trr- and Trx-containing complexes (Hallson, 2012).

In summary, these results indicate that dSet1 acts through the COMPASS to promote global H3K4 di- and trimethylation and appears to be indispensable during Drosophila development. Another study (Ardehali, 2011) has reported that dSet1 is associated within a COMPASS complex and is responsible for the majority of H3K4 methylation in Drosophila S2 tissue culture cells. Mohan (2011) has also recently reported on the central role of dSet1; moreover, that study has comprehensively characterized all three COMPASS complexes in Drosophila containing dSet1, Trx, or Trr and associated proteins (Mohan, 2011). This work provides a complementary analysis of dSet1 function in the context of whole-organism development and includes data on functional roles for other COMPASS members. These results lay the groundwork for studying mechanisms and functional roles of H3K4 methylation by the COMPASS and other HMTs in metazoans (Hallson, 2012).

The COMPASS family of H3K4 Methylases in Drosophila

Methylation of histone H3 lysine 4 (H3K4) in Saccharomyces cerevisiae is implemented by Set1/COMPASS, which was originally purified based on the similarity of yeast Set1 to human MLL1 and Drosophila Trithorax (Trx). While humans have six COMPASS family members, Drosophila possesses a representative of the three subclasses within COMPASS-like complexes: dSet1 (human SET1A/SET1B), Trx (human MLL1/2), and Trr (human MLL3/4). This study reports the biochemical purification and molecular characterization of the Drosophila COMPASS family. A one-to-one similarity in subunit composition with their mammalian counterparts was observed, with the exception of LPT (lost plant homeodomains [PHDs] of Trr), which copurifies with the Trr complex. LPT is a previously uncharacterized protein that is homologous to the multiple PHD fingers found in the N-terminal regions of mammalian MLL3/4 but not Drosophila Trr, indicating that Trr and LPT constitute a split gene of an MLL3/4 ancestor. This study demonstrates that all three complexes in Drosophila are H3K4 methyltransferases; however, dSet1/COMPASS is the major monoubiquitination-dependent H3K4 di- and trimethylase in Drosophila. Taken together, this study provides a springboard for the functional dissection of the COMPASS family members and their role in the regulation of histone H3K4 methylation throughout development in Drosophila (Mohar, 2011).

Modifications of histones and the protein machinery for the generation and removal of such modifications are highly conserved and are associated with processes such as transcription, replication, recombination, repair, and RNA processing. Histone H3K4 methylation, particularly trimethylation, has been mapped to transcription start sites in all eukaryotes tested and is generally believed to be a hallmark of active transcription. The H3K4 methylation machinery was first identified in yeast and named Set1/COMPASS. Six H3K4 methyltransferase complexes have been identified in humans, including SET1A/B, which are subunits of human COMPASS, and MLL1 to MLL4, which are found in COMPASS-like complexes (Mohar, 2011).

Although Trx and Trr were identified quite some time ago, their relative contributions to different states of overall H3K4 methylation were not known. Studies of human cells and Drosophila cells has shown that SET1 is the major contributor of H3K4 trimethylation levels in cell. During the preparation of the manuscript, a study of Drosophila also showed that dSet1, as a part of COMPASS, is responsible for the majority of H3K4 di- and trimethylation (Ardehali, 2011), which is in line with the findings presented in this study. These findings suggest that dSet1 could be responsible for the deposition of H3K4 trimethylation at the transcription start sites of the most actively transcribed genes as a consequence of postinitiation recruitment via the PAF complex (Smith, 2010: see Recruitment of histone-modifying activities by RNA Pol II). Trx and Trr both show extensive distribution along polytene chromosomes, although neither protein is required for bulk levels of H3K4me3. Perhaps Trx and Trr implement H3K4 methylation in a more gene-specific manner, at distinct stages of transcriptional regulation, or alternatively, have other substrates or functions (Mohar, 2011).

These biochemical studies have demonstrated that the Drosophila complexes are very similar to their mammalian counterparts in subunit composition. These studies have also demonstrated the utility of a baculovirus superinfection system for expressing proteins in Drosophila cells. Large-scale transient transfections offer several potential advantages over generating clonal stable cell lines, one of which is that the overexpression of some proteins could be toxic to cells. This can be a problem even when using inducible promoters, such as the Mtn promoter, due to leaky expression under uninduced conditions. Moreover, the baculovirus infection and expression strategy took about 3 weeks from the cloning of the cDNA into the viral vector, generating the virus, infection of S2 cells, and purification of the complexes from nuclear extracts. In contrast, conventional cloning took 4 months from cloning the cDNA into the vector to generating and characterizing the clonal cell lines. FLAG-HA-dWDR82 was purified from both stably transfected S2 cells and from the superinfection system and both strategies yielded a strikingly similar enrichment of target proteins (Mohar, 2011).

All of the COMPASS family members in Drosophila have several common subunits, namely, Ash2, Rbbp5, Wdr5, and Dpy30, which are homologs of CPS60, CPS50, CPS30, and CPS25, respectively, as well as each having complex-specific subunits. Many of these subunits have established, conserved roles in both the yeast and mammalian complexes: ASH2L is required for proper H3K4 trimethylation, as is CPS60 in yeast; both WDR5 in humans and CPS30 in yeast are required for the mono-, di-, and trimethylation of H3K4, and each is required for proper formation of the COMPASS and MLL complexes. Conservation of this degree in the H3K4 methylation machinery suggests that Drosophila might have similar machinery. However, it had previously been reported that Trx forms a complex with CBP and SBF, but no corresponding complexes have been found in mammals (Mohar, 2011).

The demonstration of the presence of shared components between COMPASS and COMPASS-like complexes in Drosophila supports the findings that these proteins are required for the proper functional architecture critical for the methylation of H3K4. The complex-specific components found in association with the dSet1, Trx, and Trr complexes further demonstrate a one-to-one correspondence of subunits between the Drosophila and human COMPASS family members that will allow the use of Drosophila as a model system for understanding the function of the human complexes. For example, while Set1/COMPASS is conserved from yeast to humans, it is possible that the metazoan complexes have additional functions needed for development. As the subunit compositions of both the SET1A and SET1B complexes are identical, it is likely that their functional analysis would be hindered by redundancy between the two complexes. The presence of a single dSet1 complex in flies may serve as an excellent starting point to dissect the metazoan-specific functions of the SET1 complexes (Mohar, 2011).

MLL-related proteins are multidomain proteins with the capacity to bind to many other proteins that may modulate their function. For example, Menin binds to the extreme N terminus of MLL1/2 and is required for proper targeting of the MLL1/2 complex to chromatin. Owing to its conserved components and interactions, but nonredundant nature, investigation of the Drosophila Trx complex promises to aid in understanding of the MLL1 and MLL2 complexes, specifically in their role in development (Mohar, 2011).

Currently there is very limited understanding of the functions of the various domains within the MLL3/4 proteins. The identification of LPT, which is homologous to the N terminus of MLL3/4, as a component of the Trr complex indicates the importance of PHD fingers residing in the LPT protein for the proper functioning and/or targeting of the Trr complex to chromatin. This separation of the MLL3/4 protein in Drosophila as Trr and LPT could allow dissection of the functions of N and C termini. Various studies have identified mutations in MLL3, MLL4, and UTX in a variety of cancers. Therefore, studies of the LPT-Trr complex could improve understanding of the targeting and regulation of these complexes with relevance to human disease (Mohar, 2011).

Importantly, Drosophila has a single representative of each class of COMPASS family members found in mammals, in which two representatives of each complex exist. In contrast, nematodes, such as the genetically tractable C. elegans, contain only a Set1 and MLL3/4-related protein, but no MLL1/2 representative. Given the power of genetic manipulation, the identification of the COMPASS, Trx, and Trr complexes in Drosophila that share similar subunits with their mammalian counterparts will greatly facilitate an understanding of the biological functions of the H3K4 methylation machinery in development and differentiation (Mohar, 2011).

Drosophila SETDB1 is required for chromosome 4 silencing

Histone H3 lysine 9 (H3K9) methylation is associated with gene repression and heterochromatin formation. In Drosophila, SU(VAR)3–9 is responsible for H3K9 methylation mainly at pericentric heterochromatin. However, the histone methyltransferases responsible for H3K9 methylation at euchromatic sites, telomeres, and at the peculiar Chromosome 4 have not yet been identified. This study shows that DmSETDB1 is involved in nonpericentric H3K9 methylation. Analysis of two DmSetdb1 (eggless) alleles generated by homologous recombination, a deletion, and an allele where the 3HA tag is fused to the endogenous DmSetdb1, reveals that this gene is essential for fly viability and that DmSETDB1 localizes mainly at Chromosome 4. It also shows that DmSETDB1 is responsible for some of the H3K9 mono- and dimethyl marks in euchromatin and for H3K9 dimethylation on Chromosome 4. Moreover, DmSETDB1 is required for variegated repression of transgenes inserted on Chromosome 4. This study defines DmSETDB1 as a H3K9 methyltransferase that specifically targets euchromatin and the autosomal Chromosome 4 and shows that it is an essential factor for Chromosome 4 silencing (Seum, 2007).

Whereas Su(var)3–9 and dG9a are not essential, DmSetdb1 is the first gene described encoding a H3K9 methyltransferase that is required for fly viability. DmSetdb1 transcript can be detected at every stage of development. Analysis by Northern blot confirms that the only transcript is 3.9 kb long, encompassing both CG30422 and CG30426. Early embryos show relative high mRNA levels, suggesting deposition of the transcript in the embryo. Others have reported that DmSetdb1 transcript is not present in 0–3-h embryos when tested by RT-PCR, a result that is not easily reconciled with the observations. DmSetdb1^10.1a homozygotes are rescued with the UAS- DmSetdb1^421–1,261 daGal4 transgene; the males are fertile, while the females are sterile. Thus, the rescue is not complete in females, because of either nonappropriate expression of the transgene or because DmSETDB1^421–1,261 is not full-length. This observation is consistent with the fact that DmSetdb1 (eggless) has been shown to be required for oogenesis. Preliminary data suggest that sterility in rescued females and in eggless mutant alleles is due, at least in part, to defects in germline development. Indeed, using the FLP-ovo^D1 system, no DmSetdb1^10.1a homozygous mutant germline clones could be generated. This suggests that germline-specific expression of DmSetdb1 is required before stage 5 of oogenesis. This does not exclude, however, that a maternal contribution is required for proper oogenesis (Seum, 2007).

The polyclonal antibody directed against a DmSETDB1 peptide that was generated does not recognize DmSETDB1 on polytene chromosomes. Therefore, the DmSetdb1^3HA allele was generated that results in the expression of the endogenous DmSETDB1 protein tagged with 3HA. Such an approach has the advantage that the endogenously expressed protein can be detected with highly specific monocolonal antibodies. This showed that DmSETDB1 localizes at a high level on Chromosome 4 and over the chromosome arms. DmSETDB1 is also present at the chromocenter. It is not known if this feature has any biological significance as DmSETDB1 does not methylate the chromocenter. The association of DmSETDB1 with chromatin is not dependent on its own catalytic activity, since the DmSETDB1^421–1,261(H1195K) mutant protein localizes similarly to DmSETDB1^421–1,261. The mode of DmSETDB1 recruitment thus differs from that of SU(VAR)3–9, since the latter appears to require its HMTase activity for binding to heterochromatin. It is currently not known how DmSETDB1 is recruited to chromatin. Mammalian SETDB1 is recruited to DNA together with HP1, either via the KRAB-zinc-finger protein KAP1 corepressor, or as a component of the MBD1-mAM/MCAF1-SETDB1 complex. It is tempting to speculate that in Drosophila transcriptional repressors also recruit DmSETDB1 onto euchromatin or at Chromosome 4 (Seum, 2007).

Comparative analysis of H3K9 methylation and HP1 profile on polytene chromosomes of wild-type and DmSetdb1^10.1a homozygous mutant larvae shows that DmSETDB1 is involved in some of the H3K9 mono- and dimethyl marks in euchromatin and in dimethyl marks on Chromosome 4. Loss of methylation at Chromosome 4 and euchromatin is coherent with the localization profile of the DmSETDB1 protein itself. Western blot analysis of the H3K9 methylation level in mixed salivary glands, brain, and imaginal discs tissue in DmSetdb1 mutant background shows a decrease in all three H3K9 methyl marks. No change of trimethylation in polytene chromosomes of DmSetdb1^10.1a mutant larvae was evident. This suggests a distinct H3K9 trimethylation profile in the tissues analyzed by Western blot and in polytene chromosomes. This hypothesis is corroborated by the recent finding that DmSETDB1 trimethylates H3K9 in germ and somatic cells of the germarium (Seum, 2007).

The overexpression data provide a mirror image, in that they show the ability of DmSETDB1 to mono-, di-, and trimethylate H3K9. Thus, Drosophila DmSETDB1 and mammalian SETDB1 are conserved with respect to their HMTase activity, as both Drosophila DmSETDB1 and mammalian SETDB1 are H3K9 mono-, di-, and tri-HMTases. Although such a mechanism has not yet been described, it cannot be excluded that DmSETDB1 is exclusively a H3K9 monomethyltransferase providing monomethyl substrates for other enzymes; but in that case, the partner enzyme would not be SU(VAR)3–9, since its absence does not impair Chromosome 4 or euchromatic dimethylation. In mammals, conversion of the H3K9 dimethyl- to the trimethyl-state by SETDB1 is strongly facilitated by the mAM cofactor. Such a mechanism can also be envisaged for DmSETDB1, and CG12340 is a candidate Drosophila homologue of mAM (Seum, 2007).

No HMTase activity of DmSETDB1 could be detected in cell-free conditions. Immunopurified DmSETDB1, regardless of whether expressed in mammalian or in Drosophila S2 embryo cell lines, did not show any activity when tested on GST-H3, GST-H4, core histones, or oligonucleosomes, while mammalian SETDB1 produced under identical conditions showed robust H3 specific activity. It is hypothesized that another protein or a post-translational modification is necessary for HMTase function of DmSETDB1. This activity would not be present in S2 cell line; this is consistent with the fact that overexpression of DmSETDB1 in S2 cells does not induce any increase in H3K9 mono-, di-, or trimethylation (unpublished data) (Seum, 2007).

DmSETDB1 functions in association with HP1; HP1 is recruited when DmSETDB1^421–1,261 is overexpressed and lost from some euchromatic bands and Chromosome 4 in the DmSetdb1^10.1a mutant. In addition, HP1 is required for DmSETDB1-dependent repression of Chromosome 4 variegating transgenes. It is speculated that HP1 is recruited to chromatin by both the DmSETDB1 protein and the H3K9 methyl mark. Indeed, the DmSETDB1 protein is not able to recruit HP1, because the DmSETDB1^421–1,261(H1195K) mutant protein does not influence HP1 localization. On the other hand, the H3K9 methyl mark alone is not sufficient to recruit HP1. Therefore, it is hypothesized that HP1 recognizes the H3K9 methyl mark in association with DmSETDB1, or with another factor. The situation is similar for Suv39H1, where the protein itself does not recruit HP1, despite a direct interaction that is necessary for HP1 binding in collaboration with the H3K9 methyl mark. It is not known if a direct DmSETDB1-HP1 interaction occurs, but two arguments in mammals argue in favor of this. First, KAP1 directly binds HP1 and SETDB1, and in such a complex, contacts between HP1 and SETDB1 are probable. Second, heterochromatin targeted HP1 recruits SETDB1, although an intermediate factor cannot be excluded (Seum, 2007).

Although both DmSETDB1 and SU(VAR)3–9 methylate H3K9, one cannot substitute for the other. Indeed, in a mutant background for one enzyme, the other will not compensate for its absence. In addition, both enzymes function independently; SU(VAR)3–9-mediated H3K9 di- and trimethylation and HP1 deposition at the chromocenter are not affected in the DmSetdb1^10.1a mutant context, and conversely, H3K9 mono- and dimethyl marks at euchromatic arms, dimethyl marks on Chromosome 4, and the associated HP1, are not affected in a Su(var)3–9 mutant background. Surprisingly, SU(VAR)3–9 is present on Chromosome 4; it is most probably recruited by HP1, but it does not induce any H3K9 methylation. Thus, DmSETDB1 and SU(VAR)3–9 exert nonoverlapping and independent functions, suggesting that they accomplish distinct biological roles. It is anticipated that at least one additional HMTase is involved in H3K9 methylation in Drosophila. H3K9 monomethylation at the chromocenter, H3K9 dimethylation at the telomeres, and some of the H3K9 mono- and dimethylation marks at euchromatic bands are not deposited by SU(VAR)3–9 nor DmSETDB1. One candidate, dG9a, was recently shown to methylate H3K9 and to localize to euchromatin (Seum, 2007).

The repressive function of DmSETDB1 demonstrated for Chromosome 4 is consistent with the fact that H3K9 methylation is generally found in association with transcriptional silencing. Indeed, the mammalian SETDB1 homologue fulfills such a function. DmSETDB1 could also be implicated positively in gene expression, since H3K9 di- and trimethylation, as well as HP1γ were recently found in the coding region of active genes. One task will be to identify endogenous genes that are regulated by DmSETDB1 in euchromatin and at Chromosome 4. Genes located in the region 31 are potential candidates, given that the HP1 signal is lost in the DmSetdb1^10.1a mutant. The second set of candidate genes are those physically associated with HP1 but not with SU(VAR)3–9. Large-scale mapping of HP1 and SU(VAR)3–9 targeted loci in embryonic Kc cells has shown that whereas HP1 and SU(VAR)3–9 bind together to transposable elements and pericentric genes, HP1 binds to many genes on Chromosome 4, mostly independently of SU(VAR)3–9. The latter, together with a class of euchromatic genes showing the same protein-factor occupation profile, possibly depend on DmSETDB1 for H3K9 methylation and regulation (Seum, 2007).

DmSETDB1 is the H3K9 HMTase responsible for heterochromatin silencing on Chromosome 4, because variegating transgenes are derepressed in a DmSetdb1^10.1a mutant background. As both alleles have to be mutated in order to obtain an effect, the DmSetdb1 gene is a recessive suppressor of variegation on Chromosome 4. Conversely, loss of a single dose of HP1 or SU(VAR)3–7 results in loss of silencing. This difference could be explained by the fact that DmSETDB1 is an enzyme, whereas HP1 and SU(VAR)3–7 are dosage-sensitive structural components. Alternatively, DmSETDB1 might be present in excess. Heterochromatic variegating reporters are responding to an additional or missing dose of SU(VAR)3–9 when inserted on Chromosomes 2, 3, or X, but not on Chromosome 4. This observation is henceforth explained by the fact that DmSETDB1 mediates H3K9 dimethylation on Chromosome 4. Conversely, and as expected, variegating expression responding to the SU(VAR)3–9 dosage is not under the control of DmSETDB1. This corroborates once again that SU(VAR)3–9 and DmSETDB1 function independently. Mammalian SETDB1 is involved in epigenetic maintenance, since silencing is stably maintained for more than 40 population doublings, once it is established on an integrated reporter by a short transient pulse of the corepressor KAP1 that subsequently recruits SETDB1 and HP1. DmSETDB1 could also be involved in epigenetic maintenance; in that case, transient expression would suffice for long-term repression of Chromosome 4 variegating transgenes (Seum, 2007).

The arm of Chromosome 4 is composed of a minimum of three euchromatic domains interspersed with heterochromatic domains. The variegating P elements that were tested were inserted within the banded region, in or at the edge of heterochromatic domains. Chromosome 4 heterochromatic bands are qualitatively different from centromeric heterochromatin, as they are H3K9 dimethylated and regulated by DmSETDB1, not by SU(VAR)3–9. Two possibilities can be envisaged for the Chromosome 4 domains that are methylated by DmSETDB1. First, they could be representative of equivalent bands at euchromatic arms, which would be smaller and/or more dispersed, and therefore would not yet have been identified functionally. Alternatively, D. melanogaster Chromosome 4 could make use of specific machinery dedicated to gene regulation and/or epigenetic maintenance. The other well-known example of chromosome-specific regulation is the dosage compensation of sex chromosomes. In that case, DmSETDB1 function would depend on partners or DNA sequences specific for Chromosome 4, such as for instance the Chromosome 4-specific factor POF or the Hoppel element, also known as 1360, which is over-represented on the D. melanogaster Chromosome 4, and which could be an initiation site for heterochromatin formation (Seum, 2007).

In conclusion, this study has characterized DmSETDB1 as a major nonheterochromatic H3K9 methyltransferase in Drosophila. It was also demonstrated that DmSetdb1 is an essential gene and that its loss has functional consequences on gene expression on Chromosome 4. This work represents an important step toward the understanding of the differential specificity and mode of action of distinct H3K9 HMTases and underlines a specific mode of regulation of Chromosome 4 in Drosophila (Seum, 2007).

dSETDB1 and SU(VAR)3–9 sequentially function during germline-stem cell differentiation in Drosophila melanogaster

Germline-stem cells (GSCs) produce gametes and are thus true 'immortal stem cells'. In Drosophila ovaries, GSCs divide asymmetrically to produce daughter GSCs and cystoblasts, and the latter differentiate into germline cysts. This study shows that the histone-lysine methyltransferase dSETDB1, located in pericentric heterochromatin, catalyzes H3-K9 trimethylation in GSCs and their immediate descendants. As germline cysts differentiate into egg chambers, the dSETDB1 function is gradually taken over by another H3-K9-specific methyltransferase, SU(VAR)3–9. Loss-of-function mutations in dsetdb1 (eggless) or Su(var)3–9 abolish both H3K9me3 and heterochromatin protein-1 (HP1) signals from the anterior germarium and the developing egg chambers, respectively, and cause localization of H3K9me3 away from DNA-dense regions in most posterior germarium cells. These results indicate that dSETDB1 and SU(VAR)3–9 act together with distinct roles during oogenesis, with dsetdb1 being of particular importance due to its GSC-specific function and more severe mutant phenotype (Yoon, 2008).

This study shows that dSETDB1 is the only HKMTase responsible for the synthesis of H3K9me3 signals in the inner germarium where GSCs and their early descendants are found. When these vasa-positive cells move to region-3 germarium, the H3-K9 trimethylating task is transferred to a combination of dSETDB1 and SU(VAR)3–9, as both enzymes act cooperatively in all other somatic-type cells of the germarium. After the egg chamber buds off from the germarium, the trimethylation activity is now entirely the province of SU(VAR)3–9. The results disclose that the developmental program uses dSETDB1 first and then SU(VAR)3–9 during GSC differentiation, indicating that the two HKMTases perform distinct functions in these germ cells. The role of dSETDB1 in early GSC differentiation is presumably to 'pre-mark' certain regions of chromatin, including the pericentric heterochromatin, with H3K9me3. The biochemical features of these pre-marked regions might be different from those of regions that are substrates of SU(VAR)3–9, and the pre-marked regions may be the platform on which incoming SU(VAR)3–9 further modulates the pre-methylated chromatin regions in later-developing VASA-positive cells. The functional significance of trimethylating, or 'priming', GSC chromatins with dSETDB1 is highlighted by the catastrophic ovarian phenotypes observed in, and the sterility of, the dsetdb1 female homozygote. By contrast, although the egg chambers of the Su(var)3–9¹⁷ mutant completely lacked H3K9me3 signals, which might be expected to result in a phenotype more severe than that of the dsetdb1 mutant, the Su(var)3–9¹⁷ mutant is capable of oogenesis, is able to lay eggs, and is fertile (Yoon, 2008).

Meanwhile, the localization of both dSETDB1 and Su(var)3-9 at DAPI-dense heterochromatin does not necessarily mean that they target the same chromatin loci in early- and late-stage of oogenesis, respectively. The observation that dSETDB1, but not Su(VAR)3-9, is essential for Drosophila oogenesis provides a possibility that the two HKMTases may have different sets of target chromatin regions during oogenesis. It would be interesting to examine whether dsetdb1 phenotypes could be rescued or not if exogenous Su(var)3-9 were expressed at high level in GSCs and their close derivatives (Yoon, 2008).

In germ cells, dSETDB1 locates at DAPI-dense, pericentric heterochromatin. This was unexpected because the mammalian counterpart, SETDB1/Eset, is known to have euchromatin-associated function. Seum (2007) recently reported that, in Drosophila polytene chromosomes, dSETDB1 locates at the fourth chromosome. This fourth chromosome is known to be unusual as it has many characteristics of heterochromatic domains (such as a high-repeat density, no recombination and late replicating) and, at the same time, it shows features of euchromatin (such as being transcriptionally active and having a high gene density); in fact, many of the genes in the fourth chromosome are expressed during development. These characteristics indicate that the banded regions of the fourth chromosome are different from pericentric heterochromatin, which highlights the peculiarity of dSETDB1 localization to DAPI-dense heterochromatin in the germarium (Yoon, 2008).

The location of dSETDB1 at pericentric heterochromatin probably indicates that dSETDB1 participates, at a global level, in regulating chromosome organization and maintaining the chromosome integrity in the germ-lineages. This hypothesis is supported by the observation that the main H3K9me3 spots were displaced and went astray from DNA-dense heterochromatin regions in most region-3 cells of the dsetdb1^G19561 germarium. In addition, the egg chambers of the dsetdb1^G19561 mutant ovary that survived stage six were shown to have disorganized chromosomes in the nurse cell nuclei. The nurse cell chromosomes of the stage-7 egg chambers in both wild-type and Su(var)3–9¹⁷ ovaries were organized into bundles with well-developed, large nucleoli, whereas those in the dsetdb1^G19561 ovaries were simply scattered throughout the nucleoplasm without nucleolar regions; otherwise, all were stained positive in the TUNEL assay. HP1 was diffusely located in these nuclei of dsetdb1^G19561 mutant egg chambers but the HP1 mislocalization is unlikely to be the reason for the scattered chromosomes because the Su(var)3–9¹⁷ egg chambers totally lacked HP1 but had nucleolar regions between bundles of chromosomes. These results suggest that dSETDB1 has a role in coordinating the chromosomal integrity in the germ-cell lineages, and the loss of dSETDB1 function results in a dysregulation of chromosome organization (Yoon, 2008).

In the ovary, the main type of methylation catalyzed by dSETDB1 is H3K9me3. In the dsetdb1^G19561 germarium, the loss of H3K9me3 was limited to the germ cells in the inner germarium. By contrast, in the salivary glands, dSETDB1 primarily synthesizes H3K9me2 at the fourth chromosome at which dSETDB1 itself localizes. Alterations in the H3K9me3 pattern and intensity were not detected in the polytene chromosomes in these studies. This means that dsetdb1 synthesizes either H3K9me2 or H3K9me3, depending on the type of cells in which it functions. By analogy with mammalian SETDB1/Eset, dSETDB1 can produce in vitro all the methylation types such as H3K9me1, H3K9me2 and H3K9me3. Under in vivo conditions, the specificity of SETDB1 activity and the resulting state of methylation depend on regulatory protein(s) associated with SETDB1/Eset. This is shown by the observation that a murine ATFa-associated factor (mAM) tightly associates with SETDB1 and facilitates the SETDB1-dependent conversion of H3K9me2 to H3K9me3. Therefore, the proteins that regulate SETDB1 activity determine the H3-K9 methylation state in certain tissue cells and at particular developmental stages, and this might be true for dSETDB1 in Drosophila (Yoon, 2008).

Relating to likely dSETDB1-associated protein(s), a clue was provided by the observation that a dsetdb1 null mutant, DmSetdb1^10.1a, dies at the late pupal stage, but it can be rescued to progress to the adult stage by expression of a truncated DmSETDB1^421–1,261 transgene, which was constructed by deleting the N-terminal 420 amino acids of the full-length dSETDB1. Of particular interest is the finding that the rescued females are sterile whereas the males are fertile, which is the same phenotype as seen with the dsetdb1^G19561 mutant. This rescue experiment indicates that the truncated DmSETDB1^421–1,261 is enough for the null DmSetdb1^10.1a mutants to survive the pupal stage, but is still insufficient to overcome the female sterility. This provides important clues about the tissue and substrate specificity of dSETDB1. This indicates that the N-terminal region (spanning 1–420 amino acids) of dSETDB1 is instrumental in female fertility. This region likely forms a functional domain that provides a binding site(s) for regulatory protein(s) that positions dSETDB1 at pericentric heterochromatin in the PGCs and GSC-derived cells instead of the fourth chromosomes, and preferentially synthesizes H3K9me3 instead of H3K9me2. Mammalian SETDB1/ESET is known to associate with several transcriptional regulators such as the ERG protein, mAM, KRAB-zinc-finger protein KAP1, and MBD1/MCAF1. It would be interesting to investigate the factor(s) that restricts dSETDB1 to the germ-cell lineages and favors H3K9me3 over H3K9me2 in the ovary (Yoon, 2008).

At present, there is no information on SU(VAR)3-9 function during the Drosophila oogenesis. Because an antibody capable of immunocytochemically detecting SU(VAR)3-9 protein was unavailable, a transgenic line was used that expresses GFP-tagged SU(VAR)3-9 protein as an alternative. It is clear that the ectopic expression pattern shown by the GFP-tagged SU(VAR)3-9 does not always reflect the pattern of endogenous SU(VAR)3-9. Nevertheless, if the SU(VAR)3-9-eGFP were expressed in a cell with endogenous SU(VAR)3-9, the SU(VAR)3-9-eGFP signals would be localized to wherever endogenous SU(VAR)3-9 is located. The results of RISH and RT-PCR analyses showed that Su(var)3-9 is expressed in the ovarioles including the germarium and participates in oogenesis, and the egg chambers were shown to lack H3K9me3 in the Su(var)3–9¹⁷ mutant flies. Therefore, a GFP-tagged SU(VAR)3-9 transgenic fly was used to determine the location of endogenous SU(VAR)3-9 from the ectopically expressed SU(VAR)3-9-eGFP signals in the ovarian cells, and the results showed that the SU(VAR)3-9-eGFP signals were overlapped with H3K9me3/HP1 signals in the egg chambers, indicating that endogenous SU(VAR)3-9 is responsible for H3K9me3 signals in developing egg chambers. The function of SU(VAR)3-9 in the germarium could also be deduced from the localization of SU(VAR)3-9-eGFP signals. In the inner germarium SU(VAR)3-9-eGFP signals were less co-localized with H3K9me3 signals than in the outer germarium. Such a positioning of SU(VAR)3-9-eGFP in the germarium is in agreement with the prediction of endogenous SU(VAR)3-9 function in the outer germarium. Therefore, it is certain that SU(VAR)3-9 also has a role in the oogenesis. Despite its role as an influential epigenetic modifier, the SU(VAR)3-9 function during the oogenesis is likely to be dispensable because Su(var)3-9 null mutant flies are fertile (Yoon, 2008).

HP1 recognizes H3K9me2 and H3K9me3. In the polytene chromosomes of salivary glands, HP1 localizes at the chromocenter and chromosome 4, in agreement with the pattern of H3K9me2, rather than H3K9me3, which is present at the core of the chromocenter. Mutations in the dsetdb1 gene abolish both H3K9me2 and HP1 signals from the fourth chromosome in the salivary glands. By contrast, the HP1 in the nuclei of both the germarium and the developing egg chambers mainly associates with H3K9me3 instead of H3K9me2. The dsetdb1^G19561 germarium and the Su(var)3–9¹⁷ egg chambers have normal-looking H3K9me2 signals but lack H3K9me3, and their nuclei also lack HP1 signals. These observations indicate that in some cells and tissues, HP1 binds either H3K9me2 or H3K9me3, and the preferred substrate depends on the HKMTase(s) itself that recruits and tethers HP1 to their sites of action (Yoon, 2008).

In summary, this study has demonstrated that dsetdb1 is expressed, in a germ cell-specific manner, in the germarium; the germline stem cells and their early descendants reside in the anterior part of the germarium and both H3K9me3 and HP1 signals are abolished with mutations in the dsetdb1 gene. In the GSC-derived cells, dSETDB1 trimethylates H3-K9 residues at pericentric heterochromatin, but this function is performed by SU(VAR)3–9 as germline cysts differentiate into egg chambers. Loss-of-function mutation in Su(var)3–9 abolishes both H3K9me3 and HP1 signals in developing egg chambers. Both dSETDB1 and SU(VAR)3-9 collaborate in the region-3 germarium and a mutation in either of these genes causes localization of H3K9me3 away from DNA-dense regions in the region-3 cells. These findings, therefore, indicate that dsetdb1 and Su(var)3-9 act sequentially to regulate chromosome organization in accordance with the differentiation of the germline-stem cells in Drosophila (Yoon, 2008).

HP1a recruitment to promoters is independent of H3K9 methylation in Drosophila melanogaster

Heterochromatin protein 1 (HP1) proteins, recognized readers of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me), are important regulators of heterochromatin-mediated gene silencing and chromosome structure. In Drosophila three histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9: Su(var)3-9, Setdb1 (Eggless), and G9a. To probe the dependence of HP1a binding on H3K9me, its dependence on these three HKMTs, and the division of labor between the HKMTs, correlations were examined between HP1a binding and H3K9me patterns in wild type and null mutants of these HKMTs. Su(var)3-9 was shown to control H3K9me-dependent binding of HP1a in pericentromeric regions, while Setdb1 controls it in cytological region 2L:31 and (together with POF) in chromosome 4. HP1a binds to the promoters and within bodies of active genes in these three regions. More importantly, however, HP1a binding at promoters of active genes is independent of H3K9me and POF. Rather, it is associated with heterochromatin protein 2 (HP2) and open chromatin. These results support a hypothesis in which HP1a nucleates with high affinity independently of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites (Figueiredo, 2012).

Chromosome 4 is considered to be a repressive environment that is enriched in heterochromatin markers such as HP1a and methylated H3K9. It contains large blocks of repeated sequences and transposable elements interspersed with the genes, and transgenes inserted on the 4^th chromosome often show variegated expression because of partial silencing. Despite its heterochromatic nature genes located on the 4^th chromosome are expressed as strongly on average, or even more strongly, than genes on other chromosomes. Traditionally, the division of genomes into heterochromatic and euchromatic regions was based on cytological characteristics of chromatin in interphase. Today more elaborate definitions of chromatin states are available based on chromatin components, such as the five principal chromatin types defined in (Filion, 2010). According to these definitions, pericentromeric heterochromatin and the 4^th chromosome is highly enriched in 'green-chromatin'. Similar definitions have been constructed by the modENCODE project, distinguishing nine chromatin states (Kharchenko, 2011), one of which (chromatin state 7) corresponds to 'green-chromatin'. HP1a and H3K9me2/me3 are the key components distinguishing green-chromatin and chromatin state 7. Maps of HP1a and H3K9me2/me3 in chromatin from dissected salivary gland tissue correlate with previously reported high-resolution ChIP-chip and DamID maps of chromatin from various cell lines, embryos and fly heads. Thus, the main regional chromosome organization into this chromatin type appears to be stable during development (Figueiredo, 2012).

The results show that the regional enrichment of HP1a depends on region-specificity of the HKMTs. The regional differences observed in whole chromosomes confirm previous results based on chromosome stainings, i.e., loss of Su(var)3-9 causes a reduction of HP1a and H3K9me in pericentromeric regions but not the 4^th chromosome while loss of Setdb1 causes reductions of HP1a and H3K9me on the 4^th chromosome and in region 2L:31. Loss of G9a results in no difference in H3K9me. No clear indications of redundancy were seen between the different HKMTs (Figueiredo, 2012).

The most important observations in this study are the fundamental differences between HP1a enrichment responses in gene bodies and promoters to losses of HKMT and H3K9 methylation. Upon loss of the region-specific HKMT, HP1a is strongly reduced or lost in gene bodies but the HP1a promoter peak is retained. These effects were observed in the pericentromeric region in Su(var)3-9 mutants and both the 4^th chromosome and region 2L:31 in Setdb1 mutants. The observed HP1a binding in promoters is strongly indicative of H3K9me-independent nucleation sites. Interestingly, although the interaction between HP1a and methylated H3K9 is well documented, and was confirmed in these experiments, HP1 proteins have been shown to bind only weakly to reconstituted methylated nucleosomal arrays and purified native chromatin. For example, H3 peptides containing H3K9me3 bind HP1 with relatively weak (μM) affinity. This is in stark contrast to their nM affinity for unmodified histones and the stable interaction of HP1, probably to the histone fold region of H3, that occurs in S phase when DNA replication disrupts the histone octamers. It is concluded that HP1a binds to two distinct targets in chromatin: very stably and methylation-independently to promoters of active genes (probably via interactions within the nucleosomes) and less stably (but with perfect correlation) to methylated H3K9 sites (Figueiredo, 2012).

Considering the methylation-dependent and -independent binding of HP1a it is interesting to note that HP1a is essential for viability>, in contrast to the three studied HKMTs. Su(var)3-9 is not required for viability and homozygous null mutant stocks can be kept. The same is true for G9a. Setdb1 is claimed to be essential in Drosophila and is certainly required for female fertility. Nevertheless, in uncrowded conditions Setdb1^10.1a homozygous flies hatch although they have decreased viability, and pairwise crossings of the HKMT mutants have showed no clear effects in terms of reduced viability. The findings of H3K9me-independent HP1a binding to promoters tempt speculation that HP1a may be essential for survival due to the methylation-independent promoter binding of HP1a. However, HP1a has also been associated with non-chromatin based functions such as linkage to hnRNP particles, suggesting it may also be involved in RNA compaction, although the importance of this function remains elusive (Figueiredo, 2012).

The characterization of the HP1a bound promoter peaks led to two important findings. Firstly, promoters in the 4^th chromosome and region 2L:31 have a significantly higher A/T content compared to promoters at other chromosomal locations. The presence of A/T rich motifs in general HP1a target sites have previously been reported and the results confirm that this is also true for promoter-specific HP1a targets. It has been shown that poly(dA:dT) tracts in promoters disfavour nucleosomes and modulate gene expression levels. In addition, promoters in the 4^th chromosome are more DNase sensitive than promoters at other genomic locations, suggesting that the chromatin structure is more open within these promoters. In fact, it has been proposed that HP1a promotes an open chromatin structure at bound promoters. In contrast, gene bodies in the 4^th chromosome are slightly less accessible to DNase, which again indicates that the HP1a binding to promoters is fundamentally different to the HP1a targeting in gene bodies. The slightly reduced DNase sensitivity in gene bodies is also consistent with the previously observed reduction of transcription elongation efficiency of genes on the 4^th chromosome. Secondly, in a search for chromatin-associated factors that correlate with the HP1a binding promoter peaks, Heterochromatin protein 2 was found to show a close to perfect correlation. HP2 has previously been shown to interact with HP1a , and it has been suggested that HP2 interaction with the HP1a chromo-shadow domain drives HP1a dimerization (Mendez, 2011). The results show that the HP1a-HP2 interactions mainly occur at the HP1a bound promoters. This is consistent with previous observations that all mutations affecting HP1 dimerization abolish the interactions between HP1 and non-modified H3, since these interactions should mainly occur at promoters according to the current data (Figueiredo, 2012).

The results provide strong support for the model proposed by (Dialynas, 2006) that high affinity HP1a binding to the histone fold provides a nucleation site for HP1a targeting to chromatin. It is interesting to note that this incorporation is suggested to occur when the histone fold region of H3 becomes exposed because of active transcription, histone variant exchange or replication. A link between HP1 and replication has also been demonstrated by observed interactions between HP1 and the origin recognition complex (ORC), and the requirement of human ORC association with HP1 for correct targeting to heterochromatin. In addition, HP1a modulates replication timing in Drosophila and reduced levels of HP1a result in delayed replication of chromosome 4. It is speculated that HP1a binding to promoters avoids delay of this heterochromatic region's replication, that it provides an epigenetic nucleation mark for HP1a, and that the resulting nucleation is followed by a low affinity spreading to gene bodies. A transient looping contact model is envisioned in which the low affinity between HP1a and H3K9me provides the means for spreading of HP1a, analogous to the proposed model for the interactions of another Drosophila chromodomain protein, Polycomb (Pc). The chromo-domain of Pc interacts with H3K27me, but the nucleation sites for Pc are the Polycomb Response Elements, which have lower levels of H3K27me. Thus, the nucleation appears to be independent of H3K27me and is followed by spreading caused by transient contacts between Pc and H3K27me. Similarly to HP1a and H3K9me, the affinity of the Pc chromo-domain to H3K27me is relatively weak, with a dissociation constant in the μM range. In the case of HP1a the proposed spreading correlates with (and thus presumably depends on) at least three factors: H3K9me, active transcription and Painting of fourth (POF). The spreading appears to be generally restricted to transcribed genes, although there are two exceptions (onecut and CG1909) on the 4^th chromosome. On the 4^th chromosome, where POF binds to gene bodies, the HP1a enrichment is much higher than in region 2L:31. It should be stressed that HP1a and POF bindings are interdependent and POF also requires Setdb1 to target the 4^th chromosome. Thus, the relationships between these factors remain elusive. Why is the gene body targeting of HP1a on the 4^th chromosome Pof-dependent? This cannot be explained by expression differences, because although expression levels drop in Pof mutants the reductions are minor. It is hypothesized that POF binding to nascent RNA on active chromosome 4 genes may stabilize the interaction between HP1a and H3K9me as an adaptor system linking histone marks to nascent RNA, similar to the chromatin adaptor model for alternative splicing (Figueiredo, 2012).

The enrichment of H3K9me on the 4^th chromosome mainly depends on Setdb1, but in the most proximal region of the 4^th chromosome the H3K9me is Su(var)3-9 dependent. Thus, the proximal region of chromosome 4 is similar to the proximal region of other chromosome arms in this respect. Position-effect variegation studies have shown that although most variegated (partially silenced) transgenic inserts on the 4^th chromosome are suppressed in Setdb1, but not Su(var)3-9 mutants, the reporter insertion 118E-10 is suppressed in Su(var)3-9 mutants. Interestingly, this transgene is inserted in the pericentric region on the 4^th chromosome, i.e., the region that according to this study is dependent on Su(var)3-9 (Figueiredo, 2012).

In summary, this study reports dual binding properties of the HP1a protein: an H3K9me methylation-independent binding at promoters and a methylation-dependent binding within gene bodies suggested to occur by spreading. Like arms of other chromosomes, the proximal region of the 4^th chromosome is enriched in HP1a and Su(var)3-9-dependent H3K9me. However, in contrast to other chromosome arms, the gene-rich portion of the 4^th chromosome is enriched in HP1a and H3K9me, and here the enrichment within gene bodies depends on Setdb1. The methylation-independent HP1a promoter binding correlates with HP2 and with 'open' chromatin structure. It is suggested that the methylation-independent and -dependent binding of HP1a are fundamental steps in the transmission, propagation and spreading of this epigenetic mark, hence the current observations provide important insights and the basis of a novel model of gene regulation in highly heterochromatic regions (Figueiredo, 2012).

Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression

A key step in gene repression by Polycomb is trimethylation of histone H3 K27 by PCR2 to form H3K27me3. H3K27me3 provides a binding surface for PRC1. This study shows that monoubiquitination of histone H2A by PRC1-type complexes to form H2Aub creates a binding site for Jarid2-Aebp2-containing PRC2 and promotes H3K27 trimethylation on H2Aub nucleosomes. Jarid2, Aebp2 and H2Aub thus constitute components of a positive feedback loop establishing H3K27me3 chromatin domains (Kalb, 2014).

Nucleosomes constitute the building blocks of eukaryotic chromosomes. They consist of a core of histone proteins around which DNA is wrapped in two helical turns. The post-translational modification of histones is a key step for the regulation of diverse processes that occur on nucleosomal DNA. Specific histone modifications often decorate arrays of nucleosomes that comprise many kilobases of DNA, but how such extended stretches of chromatin become modified is not well understood. A paradigm for a long-range chromatin-modification mechanism is transcriptional repression by Polycomb protein complexes. The Polycomb system generates two distinct histone modifications: methylation of K27 in histone H3 and monoubiquitination of K119 in histone H2A in vertebrates and of the corresponding K118 in Drosophila H2A. Polycomb repressive complex 2 (PRC2) catalyzes mono-, di- and trimethylation at H3 K27. At inactive Polycomb-target genes, H3 K27 trimethyl marks typically decorate nucleosomes across the entire upstream, promoter and coding region and are essential for repression of these genes. The H3K27me3 modification is recognized by Polycomb, a subunit of the canonical Polycomb repressive complex 1 (PRC1), and is thought to promote PRC1 interaction with chromatin across the entire length of repressed genes. PRC1 has been proposed to repress transcription through chromatin compaction and also through its ubiquitin-ligase activity for H2Amonoubiquitination. To gain insight into the function of H2Aub, this study set out to identify interactors of this modification (Kalb, 2014).

Arrays of four nucleosomes (referred to as oligonucleosomes) were reconstituted with recombinant Drosophila or Xenopus histones and monoubiquitinated H2A in these templates, using appropriate recombinant enzymes. Drosophila monoubiquitinated H2AK118 (H2AK118ub) oligonucleosomes and the corresponding unmodified oligonucleosome control template were used for affinity purification of H2AK118ub-binding proteins from Drosophila embryo nuclear extracts. In parallel, Xenopus monoubiquitinated H2A K119 (H2AK119ub) and unmodified control oligonucleosomes were used to identify vertebrate H2AK119ub interactors in nuclear extracts from mouse embryonic stem cells. In both experiments, quantitative MS analyses identified PRC2 subunits as being among the most highly enriched H2Aub interactors. Jarid2 and Aebp2 were the PRC2 subunits showing highest enrichment in both cases (Kalb, 2014).

The identification of PRC2 as an H2Aub interactor in both flies and vertebrates prompted an analysis of PRC2 histone methyltransferase (HMTase) activity on H2Aub nucleosomes. Recombinant human PRC2 containing EED, EZH2, SUZ12 and RBBP4 (referred to as PRC2) and assemblies of the same complex that in addition contained AEBP2 (AEBP2-PRC2), JARID2 (JARID2-PRC2) or both JARID2 and AEBP2 (JARID2-AEBP2-PRC2) were reconstituted. For substrates, Xenopus mononucleosomes were used that were either unmodified or monoubiquitinated at H2A K119, and in all cases western blot analyses were used with antibodies against either monomethylated H3 K27 (H3K27me1) or H3K27me3 to monitor PRC2 activity. A time-course experiment was performed to compare the activity of PRC2 and JARID2-AEBP2-PRC2 on H2A and H2Aub nucleosomes. It was found that, consistently with earlier reports, the catalytic activity of PRC2 alone is largely unchanged on H2Aub nucleosome templates. As expected, inclusion of JARID2 and AEBP2 in PRC2 resulted in stronger activity for H3 K27 methylation on unmodified nucleosome templates. However, a much stronger increase was used in H3K27me3 formation when JARID2-AEBP2-PRC2 was used for HMTase reactions on H2Aub nucleosomes. It was estimated that JARID2-AEBP2-PRC2 trimethylates H3K27 in H2Aub nucleosomes with an efficiency 25-fold higher than that of PRC2. To assess the contributions of JARID2 and AEBP2 to this stimulation of HMTase activity, the catalytic activity was compared of all four forms of PRC2 on H2A and H2Aub nucleosome substrates. JARID2-PRC2 showed higher H3K27 methyltransferase activity than did PRC2 on unmodified nucleosomes, as previously reported, but this was not further increased on H2Aub nucleosomes. In contrast, AEBP2-PRC2 methylated H3K27 in H2Aub nucleosomes with considerably higher efficiency than in unmodified nucleosomes. This suggests that AEBP2 is critical for the specific activation of PRC2 by H2Aub, whereas JARID2 has a more general function in boosting PRC2 HMTase activity, independently of the H2A modification state (Kalb, 2014).

The work reported in this study reveals that Jarid2-Aebp2-containing PRC2 binds to H2Aub nucleosomes and demonstrates that H3K27 trimethylation by this complex is strongly enhanced on H2Aub nucleosomes. This establishes H2Aub, Aebp2 and Jarid2 as components of a positive feedback loop in which H2Aub promotes PRC2 binding and H3K27 trimethylation, and H3K27me3 in turn promotes binding of the canonical PRC1 via the chromodomain of Polycomb. It is currently not clear whether canonical PRC1 indeed has E3 ligase activity for H2Amonoubiquitination or whether this modification is generated only by forms of PRC1 lacking Polycomb. Intriguingly, in embryonic stem cells, the PRC1-type complexes PRC1.1 and PRC1.6 were also identified as H2Aub interactors, results suggesting an additional feedback loop for H2A ubiquitination in vertebrates. The positive feedback loop for H3K27me3 formation by H2Aub uncovered in this study provides a rationale for how extended domains of Polycomb-repressed chromatin could be generated in both Drosophila and vertebrates. These findings could explain why H3K27me3 levels at Polycomb-target genes are reduced in mouse embryonic stem cells in which H2AK119ub has been depleted. However, it was previously found that bulk H3K27me3 levels were undiminished in late-stage Drosophila larvae in which bulk H2Aub levels had been depleted, thus suggesting that maintenance of H3K27me3-containing chromatin domains does not strictly depend on H2Aub. The H2Aub-mediated feedback loop may thus primarily be required for the initial formation of H3K27me3 chromatin domains when Polycomb repression is first established during the early stages of embryogenesis (Kalb, 2014).

Regulation of H3 continued: Part 2

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