Enhancer of zeste
E(z) acts to repress knirps.
Anteroposterior polarity of the Drosophila embryo is initiated by the localized activities of maternal genes bicoid and nanos. They establish a gradient of the Hunchback (HB) morphogen. nanos determines the distribution of the maternal HB protein by inhibiting its translation. To identify further components of this pathway, suppressors of nanos mutations have been isolated. In the absence of nanos, high levels of HB protein repress the abdomen-specific genes knirps and giant. In suppressor-of-nanos mutants, knirps and giant are expressed in spite of high HB levels. The suppressors are alleles of Enhancer of zeste . A small region of the knirps
promoter mediates the regulation by E(Z) and HB (Pelegri, 1994).
The requirements for the multi sex combs (mxc) gene during development have been examined to gain further insight into the mechanisms and developmental processes that depend on the important trans-regulators forming the Polycomb group (PcG) in Drosophila. Although mxc has not yet been cloned, it is known to be allelic with the tumor suppressor locus lethal (1) malignant blood neoplasm [l(1)mbn]. The mxc product is dramatically needed in most tissues because its loss leads to cell death after a few divisions. mxc also has a strong maternal effect. Hypomorphic mxc mutations are found to enhance other PcG gene mutant phenotypes and cause ectopic expression of homeotic genes, confirming that PcG products are cooperatively involved in repression of selector genes outside their normal expression domains. The mxc product is needed for imaginal head specification, through regulation of the ANT-C gene Deformed. This analysis reveals that mxc is involved in the maternal control of early zygotic gap gene expression known to involve some other PcG genes and suggests that the mechanism of this early PcG function could be different from the PcG-mediated regulation of homeotic selector genes later in development (Saget, 1998).
Induction of uncontrolled growth and deregulation of Hox genes are linked in mammals, where Hox products can induce leukemia. In Drosophila, modification of homeotic gene expression causes homeosis, sometimes associated with increased proliferation but not with uncontrolled tumorous growth, possibly because
the identity of each segment is specified by a combination of HOM products. Loss or gain of one HOM gene will likely lead to a new combination that is found elsewhere in wild type, and cells expressing this combination could be expected to follow the corresponding developmental pathway and give rise to homeotic transformations. However, because each cellular identity apparently corresponds to a given proliferation rate, loss or ambiguity of identity due to deregulation of several selector genes in a single cell, such as mxc mutations apparently induce, could lead to loss of proliferation control. Identification of mxc partners and targets, as well as of the molecular nature of the mxc product, may help throw light on the genes and mechanisms involved in this process (Saget, 1998).
It has been proposed that certain PcG genes are required for the maintenance of the expression domains of knirps and giant, through a mechanism similar to the regulation of homeotic genes. The regionalization of the Drosophila embryo depends on the maternally supplied products of bicoid (bcd), hunchback (hb), and nanos (nos). Nos represses the translation of the maternal HB mRNA in the posterior embryonic region. This permits the expression of the zygotic gap genes knirps (kni) and giant (gt), which specify posterior identities. These genes would otherwise be repressed by Hb. Embryos from nos/nos mothers form no abdominal segments, but this phenotype can be rescued by a total lack of hb in the maternal germline. It can also be dominantly rescued by the mutation of maternally supplied regulator molecules that normally repress kni and gt in the zygote. Pelegri and Lehmann (1994) have shown that certain mutant products of the PcG genes E(z), Psc, and pleiohomeotic can partially rescue nos by such a maternal effect. To determine if mutation of mxc also affects this regulation, the cuticles of embryos were examined from mxc/+;hb nos/nos mothers that were heterozygous for different mxc mutations. This genetic background was used because a decrease in the amount of maternal hb product can partially rescue the nos phenotype in F1 embryos. Such embryos can differentiate a few abdominal denticle belts and form an adequate background to evaluate increased rescue of nos. Thus loss-of-function PcG mutations should have a strong effect on rescue, and the embryos from hb nos/nos mothers that have two PcG mutations in their genetic background should permit increased rescue of the nos phenotype (Saget, 1998).
Any of three E(z)son (suppressor of nanos) alleles or a hypomorphic pleiohomeotic allele partially rescue the phenotypes of hb nos/nos progeny by a maternal effect; deficiencies covering E(z) or the Psc/Su(z)2 complex also allow some maternal rescue of hb nos/nos progeny, yet the strongest effect is observed with the gain-of-function E(z)son alleles. The EMS-induced allele mxcG48 rescues the hb nos/nos progeny phenotype, whereas a deficiency of mxc does not. Some rescue with the Psc/Su(z)2 complex deletion Df(2)vgB is also observed and strong rescue (consistently >50%) is observed with an EMS-induced pleiohomeotic allele phob, described as amorphic. This suggests that phob and mxcG48 are probably not amorphic alleles, and that maternal rescue of hb nos/nos progeny by a PcG gene is most efficient with a non-null mutation (Saget, 1998).
Segmentation of embryos from transheterozygous mothers was also examined. Because neither a reduction of wild-type PcG product nor two PcG mutations in trans in the hb nos/nos mothers increases nos rescue, these data strongly suggest that, whatever the mechanism of gap gene regulation by these PcG mutations may be, it does not function like the PcG-mediated maintenance of homeotic gene expression in embryos and in imaginal discs. The strong rescue provided by several non-null EMS-induced mutations, which may produce mutant proteins, leads to a proposal that modified PcG proteins are poisoning a normal process. How this process depends on wild-type regulation by PcG products has yet to be established (Saget, 1998).
Polycomb group (PcG) proteins maintain the transcriptional silence of target genes through many cycles of cell division. This study provides evidence for the sequential binding of PcG proteins at a Polycomb response element (PRE) in proliferating cells in which the sequence-specific DNA binding Pho and Phol proteins directly recruit E(z)-containing complexes, which in turn methylate histone H3 at lysine 27 (H3mK27). This provides a tag that facilitates binding by a Pc-containing complex. In wing imaginal discs, these PcG proteins also are present at discrete locations at or downstream of the promoter of a silenced target gene, Ubx. E(z)-dependent H3mK27 is also present near the Ubx promoter and is needed for Pc binding. The location of E(z)- and Pc-containing complexes downstream of the Ubx transcription start site suggests that they may inhibit transcription by interfering with assembly of the preinitiation complex or by blocking transcription initiation or elongation (Wang, 2004; full text of article).
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).
Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? This study investigate how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell (GSC) lineage. It was found that the silenced state, set up in precursor cells, is relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global downregulation of Polycomb repressive complex 2 (PRC2) components, recruitment of hypophosphorylated RNA polymerase II (Pol II) to promoters, as well as the expression and action of testis-specific homologs of TATA-binding protein-associated factors (tTAFs). In addition, action of the testis-specific meiotic arrest complex (tMAC; Drosophila RB, E2F and Myb), a tissue-specific version of the mammalian MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell type-specific localization of PRC1 components and tTAFs within the spermatocyte nucleolus. Together, the action of the tMAC and tTAF cell type-specific chromatin and transcription machinery leads to loss of Polycomb and release of stalled Pol II from the terminal differentiation gene promoters, allowing robust transcription (Chen, 2011).
The results suggest a stepwise series of developmentally programmed events as terminal differentiation genes convert from a transcriptionally silent state in precursor cells to full expression in differentiating spermatocytes. In precursor cells, differentiation genes are repressed and associated with background levels of hypophosphorylated Pol II and H3K4me3. These genes also display elevated levels of H3K27me3 and Polycomb at the promoter region, suggesting that they are acted upon by the PcG transcriptional silencing machinery. Notably, the differentiation genes studied in precursor cells in this study did not show the hallmark bivalent chromatin domains enriched for both the repressive H3K27me3 mark and the active H3K4me3 mark that have been characterized for a cohort of differentiation genes in mammalian ESCs (Chen, 2011).
The cell fate switch from proliferating spermatogonia to the spermatocyte differentiation program initiates both global and local changes in the transcriptional regulatory landscape, starting a cell type-specific gene expression cascade that eventually leads to robust transcription of the terminal differentiation genes. Globally, soon after the switch from spermatogonia to spermatocytes, core subunits of the PRC2 complex are downregulated, including E(z), the enzyme that generates the H3K27me3 mark. Locally, after male germ cells become spermatocytes, Pol II accumulates at the terminal differentiation gene promoters, although these genes still remain transcriptionally silent, with low H3K4me3 and high Polycomb protein levels near their promoters (Chen, 2011).
The next step awaits the expression of spermatocyte-specific forms of core transcription machinery and chromatin-associated regulators, including homologs of subunits of both the general transcription factor TFIID (tTAFs) and the MIP/dREAM complex (Aly and other testis-specific components of tMAC). The tMAC complex acts either locally or globally, perhaps at the level of chromatin or directly through interaction with tTAFs, to allow recruitment of tTAFs to promoters of target terminal differentiation genes. The action of tTAFs then allows full and robust transcription of the terminal differentiation genes, partly by displacing Polycomb from their promoters (Chen, 2011).
Strikingly, the two major PcG protein complexes appear to be regulated differently by the germ cell developmental program: whereas the PRC2 components E(z) and Su(z)12 are downregulated, the PRC1 components Polycomb, Polyhomeotic and dRing continue to be expressed in spermatocytes. The global downregulation of the epigenetic 'writer' E(z) in spermatocytes might facilitate displacement of the epigenetic 'reader', the PRC1 complex, from the differentiation genes, with the local action of tTAFs at promoters serving to select which genes are relieved of PRC1. In addition, the tTAFs act at a second level to regulate Polycomb by recruiting and accompanying Polycomb and several other PRC1 components to a particular subnucleolar domain in spermatocytes. It is not yet known whether sequestering of PRC1 to the nucleolus by tTAFs plays a role in the activation of terminal differentiation genes, perhaps by lowering the level of PRC1 that is available to exchange back on to differentiation gene promoters. Conversely, recruitment of PRC1 to the nucleolar region might have a separate function, such as in chromatin silencing in the XY body as observed in mammalian spermatocytes
(Chen, 2011).
The findings indicate that, upon the switch from spermatogonia to spermatocytes, the terminal differentiation genes go through a poised state, marked by presence of both active Pol II and repressive Polycomb, before the genes are actively transcribed. Stalled Pol II and abortive transcript initiation are emerging as a common feature in stem/progenitor cells. This mechanism may prime genes to rapidly respond to developmental cues or environmental stimuli. Stalled Pol II could represent transcription events that have initiated elongation but then pause and await further signals, as in the regulation of gene expression by the androgen receptor or by heat shock. Alternatively, Pol II might be trapped at a nascent preinitiation complex, without melting open the DNA, as found in some instances of transcriptional repression by Polycomb. Although ChIP analyses did not have the resolution to distinguish whether Pol II was stalled at the promoter or had already initiated a short transcript, the results with antibodies specific for unphosphorylated Pol II suggest that Pol II is trapped in a nascent preinitiation complex. The PRC1 component dRing has been shown to monoubiquitylate histone H2A on Lys119 near or just downstream of the transcription start site. It is proposed that in early spermatocytes, before expression of the tTAFs and tMAC, the local action of PRC1 in causing H2AK119ub at the terminal differentiation gene promoters might block efficient clearing of Pol II from the preinitiation complex and prevent transcription elongation (Chen, 2011).
Removal of PRC1 from the promoter and full expression of the terminal differentiation genes in spermatocytes require the expression and action of tMAC and tTAFs. Cell type-specific homologs of TFIID subunits have been shown to act gene-selectively to control developmentally programmed gene expression. For example, incorporation of one subunit of the mammalian TAF4b variant into TFIID strongly influences transcriptional activation at selected promoters, directing a generally expressed transcriptional activator to turn on tissue-specific gene expression (Chen, 2011).
The local action of the tTAFs to relieve repression by Polycomb at target gene promoters provides a mechanism that is both cell type specific and gene selective, allowing expression of some Polycomb-repressed genes while keeping others silent. Similar developmentally programmed mechanisms may also reverse PcG-mediated epigenetic silencing in other stem cell systems. Indeed, striking parallels between the current findings and recent results from mammalian epidermis suggest that molecular strategies are conserved from flies to mammals. In mouse epidermis, the mammalian E(z) homolog Ezh2 is expressed in stem/precursor cells at the basal layer of the skin. Strikingly, as was observed for E(z) and Su(z)12 in the Drosophila male GSC lineage, the Ezh2 level declines sharply as cells cease DNA replication and the epidermal differentiation program is turned on. Overexpression of Ezh2 in epidermal precursor cells delays the onset of terminal differentiation gene expression, and removal of the Ezh2-generated H3K27me3 mark by the Jmjd3 (Kdm6b) demethylase is required for epidermal differentiation (Chen, 2011 and references therein).
In particular, the results suggest a possible explanation for the conundrum that, although PcG components are bound at many transcriptionally silent differentiation genes in mammalian ESCs, loss of function of PcG components does not cause loss of pluripotency but instead causes defects during early embryonic differentiation. In Drosophila male germ cells, events during the switch from precursor cell proliferation to differentiation are required to recruit Pol II to the promoters of differentiation genes. Without this differentiation-dependent recruitment of Pol II, loss of Polycomb is not sufficient to precociously turn on terminal differentiation genes in precursor cells. Rather, Polycomb that is pre-bound at the differentiation gene promoters might serve to delay the onset of their transcription after the mitosis-to-differentiation switch. Robust transcription must await the expression of cell type- and stage-specific components of the transcription machinery. These might in turn guide gene-selective reversal of Polycomb repression to facilitate appropriate differentiation gene expression in specific cell types (Chen, 2011).
The ability of a chimeric HP1-Polycomb (PC) protein to bind both to heterochromatin and to euchromatic sites of PC protein binding was exploited to detect stable protein-protein interactions in vivo. Endogenous PC protein
is recruited to ectopic heterochromatic binding sites by the chimeric protein. Posterior sex combs (PSC) protein also is recruited to heterochromatin by the chimeric protein, demonstrating that PSC protein participates in direct protein-protein interaction with PC protein or PC-associated proteins. In flies carrying temperature-sensitive alleles of Enhancer of zeste[E(z)] the general decondensation of polytene chromosomes that occurs at the restrictive temperature is associated with loss of binding of endogenous PC and chimeric HP1-Polycomb protein to euchromatin, but binding of HP1 and chimeric HP1-Polycomb protein to the heterochromatin is maintained. The E(z) mutation also results in the loss of chimera-dependent binding to heterochromatin by endogenous PC and PSC proteins at the restrictive temperature, suggesting that interaction of these proteins is mediated by E(Z) protein. A myc-tagged full-length Suppressor 2 of zeste [SU(Z)2] protein interacts poorly or not at all with ectopic Pc-G complexes, but a truncated SU(Z)2 protein is strongly recruited to all sites of chimeric protein binding. Trithorax protein is not recruited to the heterochromatin by the chimeric HP1-Polycomb protein, suggesting either that this protein does not interact directly with Pc-G complexes or that such interactions are regulated. Ectopic binding of chimeric chromosomal proteins provides a useful tool for distinguishing specific protein-protein interactions from specific protein-DNA interactions important for complex assembly in vivo (Platero, 1996).
The in vivo distribution of the E(Z) protein shows it to be ubiquitously present in embryonic and larval nuclei. In salivary gland polytenized nuclei, the identifiable E(Z) chromosome binding sites are a subset of those described for other Polycomb-group proteins, suggesting that E(Z) may also participate in Polycomb-group complexes. E(Z) binds to chromosomes in a DNA sequence-dependent manner, as illustrated by the creation of a new E(Z)-binding site at the location of a P element reporter construct that contains a Polycomb response element (PRE). This P element contains a 14.5 kb segment from the bxd/pbx Ubx regulatory region. The sequences of one null and three temperature-sensitive E(z) alleles are presented. These mutations diminish all chromosome binding of E(Z) protein. It is suggested that a Cys-rich region altered in these mutations functions as a DNA binding domain. E(Z) binding is noticibly weaker in trx mutants. A reduced level of E(Z) chromosome binding may be due to alteration in the expression of one or more other proteins that are involved in E(Z) binding and does not necessarily imply a direct interaction between E(Z) and TRX (Carrington, 1997).
The Extra sex combs (Esc) and Enhancer of zeste [E(z)] proteins, members of the Polycomb group (PcG) of transcriptional
repressors, interact directly and are coassociated in fly embryos. These two proteins are components of a 600-kDa
complex in embryos. Using gel filtration and affinity chromatography, it has been shown that this complex is biochemically distinct from previously
described complexes containing the PcG proteins Polyhomeotic, Polycomb, and Sex comb on midleg. In addition, evidence is presented
that Esc is phosphorylated in vivo and that this modified Esc is preferentially associated in the complex with E(z). Modified Esc
accumulates between 2 and 6 h of embryogenesis, which is the developmental time when esc function is first required. Mutations in E(z) reduce the ratio
of modified to unmodified Esc in vivo. Germ line transformants were generated that express Esc proteins bearing site-directed mutations that disrupt
Esc-E(z) binding in vitro. These mutant Esc proteins fail to provide esc function, show reduced levels of modification in vivo, and are still assembled into
complexes. Taken together, these results suggest that Esc phosphorylation normally occurs after assembly into Esc-E(z) complexes and that Esc contributes to the
function or regulation of these complexes (Ng, 2000).
ESC mRNA is expressed primarily during early development, with the highest levels
being found before 4 h of embryogenesis. This early expression has prompted the hypothesis that esc functions in the transition
between initiation of homeotic gene repression by gap proteins, such as hunchback, and maintenance of this repression by PcG proteins. This transition occurs at about 4 h, when gap gene products decay. Esc protein is
expressed at peak levels at 6 to 12 h, after ESC mRNA has decayed to low levels. In addition, Esc is detected until the end of
embryogenesis. The presence of substantial levels of Esc in mid- to late-stage embryos suggests that Esc may play a greater role than simply in the transition
between gap protein and PcG protein repression. In addition, a second peak of Esc protein is detected during larval and pupal stages, consistent with its
nonessential function in imaginal discs (Ng, 2000).
The predicted Esc structure identifies two surface-accessible regions likely to contain phosphorylation sites: the highly charged N terminus and the surface
loops of the ß-propeller. The Esc phosphorylation sites have not been mapped.
However, it is predicted that Esc is serine/threonine phosphorylated, because many of the Ser and Thr residues are surface accessible. In particular, the N-terminal tail
is very rich in Ser and Thr residues (35%), a feature which has been conserved in Esc during evolution. A scan of the accessible Esc regions for
consensus kinase recognition motifs identifies numerous possible modification sites and is therefore not particularly instructive (Ng, 2000).
Gel filtration experiments also show that modified Esc is found preferentially in the Esc-E(Z) complex while unmodified Esc behaves predominantly as
unassociated monomer. Interestingly, mutant Esc proteins with reduced levels of modification also associate in complexes with the same apparent molecular mass as
the wild-type complex. This suggests that Esc modification is not required for its stable association in complexes. Consistent with this idea,
low levels of unmodified wild-type Esc are reproducibly detected in the 600-kDa complex. Based on these data, a model is favored in which Esc modification contributes to function
rather than to assembly of the complex. The finding that E(z) function is required for wild-type levels of Esc modification further suggests that this
modification occurs after Esc has complexed with its partners (Ng, 2000).
The mutant Esc proteins described in this study show reduced Esc-E(z) binding in vitro. Therefore, it was surprising to find that these mutants assemble into
complexes of apparently wild-type size. It is suggested that Esc may bind to multiple protein partners in the Esc-E(z) complex, such that specific disruption of
Esc-E(z) interaction still allows complex assembly. In support of this idea, ß-propeller proteins have been shown to make simultaneous contacts with
multiple partners (Ng, 2000).
The PcG proteins Pc and Ph are associated in a complex estimated to be 2 MDa. In addition, Ph and Ph
coimmunoprecipitate and interact with another PcG protein, Psc. The Esc-E(Z) complex is biochemically distinct from
complexes containing Ph. In agreement with this, a Ph-Pc-Psc complex does not contain E(z). Taken
together, these results support a model in which there are at least two distinct PcG complexes in vivo, one containing Esc and E(z) and the other containing
Ph, Pc, and Psc. Consistent with this idea, the mammalian Esc and E(z) homologs, EED and EZH2, fail to coimmunoprecipitate with the mammalian Ph, Psc,
and Pc homologs. In addition, EED and EZH2 do not colocalize with mammalian PH, PSC, and PC within nuclei of osteosarcoma cells.
Furthermore, the patterns of pairwise interactions among Drosophila PcG proteins are reiterated among their mammalian counterparts, which suggests that this division of labor in the PcG has been conserved in evolution (Ng, 2000 and references therein).
Although the existence of at least two different PcG complexes has been established, the complete spectrum of PcG protein interactions has not yet been elucidated.
There appears to be further division among Ph-Pc-Psc complexes, which have different compositions at different target genes. In addition, multiple complexes
containing the mammalian Ph, Pc, and Psc proteins have been detected. Moreover, there are additional PcG proteins, such as Asx, Pcl, and Pho, whose
in vivo associations have yet to be described. Some of these proteins may correspond to as yet unidentified components of Esc-E(z) or Ph-Pc-Psc complexes, or they may sort into additional distinct complexes. In particular, complexes containing Pho, the only known DNA-binding member of the PcG, may be
important for targeting other PcG complexes to sites of action. It is noted that Pho is not detected as a stable member of either the Esc-E(z) or
Ph-Pc-Psc complexes (Ng, 2000).
Despite the presence of biochemically separable PcG complexes, the similar mutant phenotypes and genetic interactions of PcG genes indicate that they work
together at some level. Any model for PcG repression must therefore accommodate both the biochemical separability and functional synergy of PcG complexes. One
possibility is that repression requires multiple chromatin-modifying events by the different PcG complexes. This would be similar to the in vivo synergy between the
chromatin-modifying SWI-SNF and SAGA complexes, which are both required for maintenance of HO expression in yeast. An alternative possibility is that
one PcG complex directly modifies chromatin while the other complex counteracts trithorax group activation by inhibiting the chromatin-remodeling activity of the
brahma complex. Indeed, the first evidence that a PcG complex may covalently modify chromatin is provided by the recent report of histone deacetylase
activity associated with mammalian homologs of Esc and E(z) (Ng, 2000 and references therein).
These mechanisms are inconsistent with an esc role limited to the transition from gap repressors to PcG repressors. Instead, it is suggested that Esc is
more globally involved in chromatin regulation and that this involvement is most critical early in fly development. Consistent with a global role, EED mRNA is
expressed in many tissues during mouse development. Furthermore, the C. elegans homolog of Esc, MES-6, is a transcriptional repressor that functions in
germ line development. MES-6 in worms therefore plays a distinct developmental role from Esc in flies. This suggests that Esc participates in a general
repression mechanism that has been adapted for use in different cell lineages, rather than in the specific transition between gap protein and PcG protein repression (Ng, 2000 and references therein).
If Esc-E(z) complexes function as general chromatin regulators, the early requirement for Esc in Drosophila must be reconciled with the need for long-term PcG
repression during development. One possibility is that another protein replaces Esc in the Esc-E(z) complex at late developmental stages, when Esc is no longer
critically required. Alternatively, E(z) may associate with a completely different set of PcG proteins to supply the biochemical function provided by Esc-E(z)
complexes during embryogenesis. To address these possibilities, the nature of E(z) complexes at postembryonic stages will have to be investigated (Ng, 2000).
The products of Polycomb group (PcG) genes are required for the epigenetic repression of a number of important developmental regulatory genes, including homeotic genes. Enhancer of zeste [E(Z)] is a Drosophila PcG protein that binds directly to another PcG protein, Extra Sex Combs (ESC), and is present along with ESC in a 600-kDa complex in Drosophila embryos. Using yeast two-hybrid and in vitro binding assays, it was shown that E(Z) binds directly to another PcG protein, Polycomblike (PCL). PCL.E(Z) interaction is shown to be mediated by the plant homeodomain (PHD) fingers domain of PCL, providing evidence that this motif can act as an independent protein interaction domain. An association was also observed between PHF1 and EZH2, human homologs of PCL and E(Z), respectively, demonstrating the evolutionary conservation of this interaction. E(Z) was found to not interact with the PHD domains of three Drosophila trithorax group (trxG) proteins, which function to maintain the transcriptional activity of homeotic genes, providing evidence for the specificity of the interaction of E(Z) with the PCL PHD domain. Coimmunoprecipitation and gel filtration experiments demonstrate in vivo association of PCL with E(Z) and ESC in Drosophila embryos. The implications of PCL association with ESC.E(Z) complexes is discussed and the possibility that PCL may either be a subunit of a subset of ESC.E(Z) complexes or a subunit of a separate complex that interacts with ESC.E(Z) complexes (O'Connell, 2001; full text of article).
The Drosophila Polycomb Group (PcG) proteins are
required for stable long term transcriptional silencing of
the homeotic genes. Among PcG genes, esc is unique
in being critically required for establishment of PcG-mediated
silencing during early embryogenesis, but not for
its subsequent maintenance throughout development. Esc has been shown to be physically associated
with the PcG protein E(Z). Esc,
together with E(z), is present in a 600 kDa complex that is
distinct from complexes containing other PcG proteins. This Esc complex has been purified and it also
contains the histone deacetylase Rpd3 and the histone-binding
protein p55 (Chromatin assembly factor 1 subunit), which is also a component of the
chromatin remodeling complex NURF and the chromatin
assembly complex CAF-1. The association of Esc and
E(z) with p55 and Rpd3 is conserved in mammals. Rpd3 is required for silencing mediated by a
Polycomb response element (PRE) in vivo and E(z)
and Rpd3 are bound to the Ubx PRE in vivo, suggesting
that they act directly at the PRE. It is proposed that histone
deacetylation by this complex is a prerequisite for
establishment of stable long-term silencing by other
continuously required PcG complexes (Tie, 2001).
To test whether the association of Esc and E(z) with p55/Caf1 and
Rpd3 has been conserved in mammals, the
human complex containing the Esc homolog (EED) was examined for the
presence of Rpd3 and p55 homologs. Database searches
reveal that Drosophila Rpd3 is most closely related to
two human histone deacetylases, HDAC1 and HDAC2
(77% and 75% identical to Rpd3). Similarly, there are
two closely related p55 homologs in mammals,
RbAp48 and RbAp46 (91% and 86% identical to p55: Drosophila homolog Caf1).
RbAp48 and RbAp46 have also been found together in
the SIN3 and Mi-2 deacetylase complexes, as have
HDAC1 and HDAC2. A test was performed to see
whether all four proteins are associated with the
human EED complex. A GST-ESC fusion
protein encoding full-length Esc can pull down full-length
in vitro translated Esc and a GST-ESC1-60 fusion
protein encoding just the N-terminal 60 residues of Esc
is sufficient to pull down full-length in vitro translated
Esc. Similarly, GST-EED1-81, which
contains the corresponding N-terminal region of EED, binds directly to in vitro
translated EED. In addition to FLAG-Esc, GST-ESC1-60 also
pulls down p55 and Rpd3 from Drosophila embryo
nuclear extract. This strongly suggests that GST-ESC1-
60 specifically pulls down the Esc complex. GST-EED1-81 pulls down HDAC1, HDAC2 and RbAp48
from HeLa cell nuclear extract.
RbAp46 has also been detected. Thus, the association of ESC with
p55 and Rpd3 is mirrored in the conserved association of
mammalian EED with RbAp48, RbAp46 and HDAC1 and
HDAC2. These results confirm the previously reported
association of EED with HDAC1 and HDAC2 (Tie, 2001).
The presence of p55 in the ESC complex provides a direct
molecular link to chromatin. The highly conserved mammalian
p55 homologs, RbAp48 and RbAp46, have been shown to bind
directly to histone H4 and possibly H2A, but not H2B or H3. The N- and C-terminal regions of RbAp48 that mediate
binding to histone H4 are virtually identical to the
corresponding regions of Drosophila p55,
strongly suggesting that p55 has the same histone-binding
specificity (Tie, 2001).
What, then, is the role of p55 in the Esc complex? It is
unlikely that p55 is responsible for the selective recruitment or
targeting of Esc and E(Z) to the ~100 specific chromosomal sites
at which they co-localize. The histone-binding
activity of p55 does not, by itself, suggest a mechanism for such
specificity and p55 binds to many more sites on the polytene
chromosomes than Esc and E(Z),
presumably reflecting its distribution in other complexes, such as
CAF1 and NURF. It seems more likely that p55 acts after the
Esc complex is recruited and serves to direct the deacetylase
activity of Rpd3 to local histone substrates. This is analogous to
the role proposed for RbAp46 in the heterodimeric HAT1
complex. RbAp46 greatly stimulates the acetyltransferase
activity of the non-histone-binding HAT1 catalytic subunit,
presumably by tethering it to its substrate via its histone-binding
activity. Similarly,
although recombinant Rpd3 can deacetylate histone H4 in vitro,
free Rpd3 does not bind to H4 when the two are co-expressed
in vivo and is unlikely to be able to
deacetylate nucleosomal histones. This suggests that p55 may
play a similar essential role in the Esc complex by targeting
Rpd3 to histone substrates for deacetylation (Tie, 2001).
The presence of Rpd3 in the Esc complex suggests that
histone deacetylation is an intrinsic activity of the Esc
complex and that Rpd3 is required for PRE-mediated
silencing. The related mammalian EED complex has been
shown to contain the Rpd3 homologs HDAC1 and HDAC2,
and immunoprecipitates containing this complex can
deacetylate a histone H4 tail-peptide in vitro. In yeast, Rpd3-dependent repression in vivo has
been shown to be associated with deacetylation of histones H4
and H3.
Which nucleosomes would be deacetylated by the Esc
complex? Histone deacetylation by yeast Rpd3 appears to be
highly localized, extending only one or two nucleosomes from
a site to which it is recruited. Since components of the Esc complex
are physically associated with the Ubx PRE in vivo, Esc-mediated
deacetylation may be restricted to nucleosomes
comprising and immediately adjacent to PREs. Nucleosomes
outside the PRE might also be targeted if the PRE has long-range
interactions with the promoter or if the Esc complex
itself also binds to the promoter or other regions outside the Ubx
PRE, a possibility that the data presented here do not rule out.
Although an effect is observed of several Rpd3 mutations on
silencing of a PRE-mini-white reporter, which is an extremely
sensitive assay, PcG phenotypes have not been reported for
Rpd3 mutants. A hypomorphic Rpd3 allele associated with the
insertion of a P-element transposon in the noncoding 5'
untranslated region has been analyzed in the most detail.
Homozygous mutant embryos derived from germline clones of
this allele do not exhibit PcG phenotypes, but have a pair-rule
phenotype similar to that of ftz mutants. Abundant ubiquitously
distributed Rpd3 RNA and protein of maternal origin are
detectable in early (0-2 hour) wild-type embryos, but are
reduced no more than fivefold in these Rpd3 mutant embryos
derived from germline clones. By stage 9-10, the level of
maternally derived Rpd3 RNA and protein is greatly
diminished. Localized zygotic expression of Rpd3 becomes
detectable in the brain and ventral nervous system of wild-type
embryos, but is not detectable in these mutant embryos, suggesting that this
Rpd3 allele may have a stronger effect on zygotic expression
than maternal expression. If Rpd3 protein derived from
maternally synthesized RNA is sufficient to promote
development of a normal cuticular phenotype, then it remains
possible these mutant embryos may contain sufficient
maternally derived protein to do so and that germline clones of
a true null Rpd3 allele would display PcG phenotypes.
Alternatively, it is possible that the function of Rpd3 in the
Esc complex is not absolutely essential for Esc-dependent
silencing or is redundant, i.e. when eliminated, it can be
compensated by another histone deacetylase, either one
normally associated with the Esc complex or a related one that
can associate with the complex in the absence of Rpd3. A
number of other histone deacetylases have been identified in
Drosophila and at least two are reported to be ubiquitously
distributed in the early embryo (Tie, 2001).
However, unlike mammals, which have two very closely
related Rpd3 orthologs (HDAC1 and HDAC2), both of which
are associated with mouse EED, the Drosophila genome
contains no equally closely related homolog of Rpd3. The next
most closely related Drosophila HDAC is an unequivocal
ortholog of mammalian HDAC3, which
is a class I HDAC like Rpd3. Interestingly, mouse HDAC3 has
been reported to interact with the mouse Esc homolog EED
in a yeast two-hybrid assay,
consistent with the possibility that Rpd3 function in the Esc
complex might be at least partially redundant. Further genetic
analysis of Rpd3 should help to clarify its role in the Esc
complex (Tie, 2001).
The 600 kDa Esc complex is distinct from complexes
containing PC and other PcG proteins. This suggests that the
Esc complex and other PcG complexes are likely to have
separate functions. Furthermore, in embryos lacking any
functional Esc protein, some weak residual Pc-dependent
silencing activity is still detected, also
supporting separate, if interdependent, functions. Similar
conclusions have been drawn for the homologous mammalian
PcG complexes, which have been reported to be expressed in temporally distinct stages of B cell
differentiation, further suggesting they have distinct functions. In Drosophila, derepression of
homeotic genes is detected slightly earlier in Esc mutants than
in other PcG mutants, raising the possibility
that Esc complex function might be required earlier than other
PcG complexes. However, unlike the apparent temporal
separation of the homologous complexes during mammalian B
cell development, both Esc- and PC-containing complexes are
present together throughout most of embryogenesis, before
Esc disappears, and E(z), like other PcG proteins, is required
continuously throughout development. The phenotypic
similarities between Esc, E(z) and other PcG mutants, the
genetic interactions among them and their common association
with PREs, suggests that their functions, however distinct at
the biochemical level, are interdependent (Tie, 2001).
What role might Esc-mediated histone deacetylation play
in PcG silencing? Given the critical early requirement for Esc,
Esc-mediated deacetylation of PRE-associated nucleosomes
might be an essential prerequisite for the initial binding of one
or more components of PRC1 or other PcG complexes to
PREs. A schematic model is presented for such a function
of the Esc complex in which Esc complex-mediated
deacetylation of PRE associated histones is a critical step in
establishing stable long-term PcG silencing. Alternatively, the
Esc complex may be required for events subsequent to the
initial binding of other PcG proteins to a PRE, perhaps for their
assembly into active silencing complexes or for interaction of
PRE-bound PcG complexes with the promoter. Indeed,
repression of a reporter gene by a tethered GAL4-Pc fusion
protein remains dependent on endogenous Esc and E(z) as
well as other PcG proteins. This indicates that,
at least for PC, constitutive binding to DNA does not bypass
the requirement for Esc and E(z). This also suggests that
while the biochemical evidence reveals no stable direct association of the
Esc complex with other PcG complexes, it remains possible
that there is a transient or less stable association in vivo that is
essential for establishing PcG silencing (Tie, 2001).
The association of mammalian EED with the two closely
related HDACs and two histone-binding proteins could reflect
the existence of two separate EED complexes or some different
functionality of the EED complex compared with the Esc
complex. Consistent with this latter possibility, EED has
recently been shown to be required after embryogenesis for
aspects of adult hematopoietic development. Interestingly, analysis of the
complete Drosophila genome sequence
using the BLASTP and TBLASTN algorithms reveals that p55 has no other
closely related Drosophila homologs, strongly suggesting that
it is the functional counterpart of both RbAp48 and RbAp46
in Drosophila. Likewise, Rpd3 is the only Drosophila
counterpart of mammalian HDAC1 and HDAC2. Given the
remarkably high degree of similarity between RbAp48 and
RbAp46 and HDAC1 and HDAC2, it is not yet clear whether
each of these proteins has a distinct or redundant role in the
EED complex. Perhaps this situation reflects a greater degree
of functional specialization or versatility within the
mammalian EED complexes. Since HDAC1 and HDAC2 have
also been found together with RbAp48 and RbAp46 in other
co-repressor complexes, it is also possible that the EED and
Esc complexes represent specialized relatives of these
complexes, perhaps more dedicated to a specific subset of
genes (Tie, 2001).
The Drosophila Enhancer of zeste [E(z)] gene encodes a member of the Polycomb group of transcriptional repressors. This study provides evidence for direct physical interaction between E(Z) and dSAP18, which previously has been shown to interact with Drosophila GAGA factor and Bicoid proteins. dSAP18 shares extensive sequence similarity with a human polypeptide originally identified as a subunit of the SIN3A-HDAC (switch-independent 3-histone deacetylase) co-repressor complex. Yeast two-hybrid and in vitro binding assays demonstrate direct E(Z)-dSAP18 interaction and show that dSAP18 is capable of interacting with itself. Co-immunoprecipitation experiments provide evidence for in vivo association of E(Z) and dSAP18. Gel filtration analysis of embryo nuclear extracts shows that dSAP18 is present in native protein complexes ranging from approximately 1100 to approximately 450 kDa in molecular mass. These studies provide support for a model in which dSAP18 contributes to the activities of multiple protein complexes, and potentially may mediate interactions between distinct proteins and/or protein complexes (Wang, 2002).
In Drosophila, PcG complexes provide heritable transcriptional silencing of target genes. Among them, the ESC/E(Z) complex is thought to play a role in the initiation of silencing whereas other complexes such as the PRC1 complex are thought to maintain it. PcG complexes are thought to be recruited to DNA through interaction with DNA binding proteins such as the GAGA factor, but no direct interactions between the constituents of PcG complexes and the GAGA factor have been reported so far. The Drosophila corto gene interacts with E(z) as well as with genes encoding members of maintenance complexes, suggesting that it could play a role in the transition between the initiation and maintenance of PcG silencing. Moreover, corto also interacts genetically with Trl, which encodes the GAGA factor, suggesting that it may serve as a mediator in recruiting PcG complexes. Corto bears a chromo domain, and evidence is provided for in vivo association of Corto with ESC and with PC in embryos. Moreover, GST pull-down and two-hybrid experiments show that that Corto binds to E(Z), ESC, PH, SCM and GAGA and co-localizes with these proteins on a few sites on polytene chromosomes. These results reinforce the idea that Corto plays a role in PcG silencing, perhaps by confering target specificity (Salvaing, 2003).
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).
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).
The Polycomb Group proteins are required for stable long-term maintenance of transcriptionally repressed states. Two distinct Polycomb Group complexes have been identified, a 2-MDa PRC1 complex and a 600-kDa complex containing the ESC and E(Z) proteins together with the histone deacetylase RPD3 and the histone-binding protein p55. There are at least two embryonic ESC/E(Z) complexes that undergo dynamic changes during development and a third larval E(Z) complex that forms after disappearance of ESC. A larger embryonic ESC complex has been identified containing RPD3 and p55, along with E(Z), that is present only until mid-embryogenesis, while the previously identified 600-kDa ESC/E(Z) complex persists until the end of embryogenesis. Constitutive overexpression of ESC does not promote abnormal persistence of the larger or smaller embryonic complexes and does not delay a dissociation of E(Z) from the smaller ESC complex or delay appearance of the larval E(Z) complex, indicating that these changes are developmentally programmed and not regulated by the temporal profile of ESC itself. Genetic removal of ESC prevents appearance of E(Z) in the smaller embryonic complex, but does not appear to affect formation of the large embryonic ESC complex or the PRC1 complex. The ESC complex is already bound to chromosomes in preblastoderm embryos and genetic evidence is presented that ESC is required during this very early period (Furuyama, 2003).
Polycomb group (PcG) proteins are required to maintain stable repression of the homeotic genes and others throughout development. The PcG proteins ESC and E(Z) are present in a prominent 600-kDa complex as well as in a number of higher-molecular-mass complexes. A 1-MDa ESC/E(Z) complex has been identified and characterized that is distinguished from the 600-kDa complex by the presence of the PcG protein Polycomblike (PCL) and the histone deacetylase RPD3. In addition, the 1-MDa complex shares with the 600-kDa complex the histone binding protein p55 and the PcG protein SU(Z)12. Coimmunoprecipitation assays performed on embryo extracts and gel filtration column fractions indicate that, during embryogenesis E(Z), SU(Z)12, and p55 are present in all ESC complexes, while PCL and RPD3 are associated with ESC, E(Z), SU(Z)12, and p55 only in the 1-MDa complex. Glutathione transferase pulldown assays demonstrate that RPD3 binds directly to PCL via the conserved PHD fingers of PCL and the N terminus of RPD3. PCL and E(Z) colocalize virtually completely on polytene chromosomes and are associated with a subset of RPD3 sites. As shown for E(Z) and RPD3, PCL and SU(Z)12 are also recruited to the insertion site of a minimal Ubx Polycomb response element transgene in vivo. Consistent with these biochemical and cytological results, Rpd3 mutations enhance the phenotypes of Pcl mutants, further indicating that RPD3 is required for PcG silencing and possibly for PCL function. These results suggest that there may be multiple ESC/E(Z) complexes with distinct functions in vivo (Tie, 2003).
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 (PRED), 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).
SIR2 was originally identified in S. cerevisiae for its role in epigenetic silencing through the creation of specialized chromatin domains. It is the most evolutionarily conserved protein deacetylase, with homologs in all kingdoms. SIR2 orthologs in multicellular eukaryotes have been implicated in lifespan determination and regulation of the activities of transcription factors and other proteins. Although SIR2 has not been widely implicated in epigenetic silencing outside yeast, Drosophila SIR2 mutations were recently shown to perturb position effect variegation, suggesting that the role of SIR2 in epigenetic silencing may not be restricted to yeast. Evidence is presented that Drosophila SIR2 is also involved in epigenetic silencing by the Polycomb group proteins. Sir2 mutations enhance the phenotypes of Polycomb group mutants and disrupt silencing of a mini-white reporter transgene mediated by a Polycomb response element. Consistent with this, SIR2 is physically associated with components of an E(Z) histone methyltransferase complex. SIR2 binds to many euchromatic sites on polytene chromosomes and colocalizes with E(Z) at most sites. It is concluded that SIR2 is involved in the epigenetic inheritance of silent chromatin states mediated by the Drosophila Polycomb group proteins and is physically associated with a complex containing the E(Z) histone methyltransferase (Furuyama, 2004).
The ability of Sir2 mutations to enhance PcG mutant phenotypes and perturb PRE-mediated silencing indicates that SIR2 plays a role in Polycomb silencing. However, like their yeast and C. elegans counterparts, Drosophila Sir2 mutants are viable under standard laboratory conditions, and they do not exhibit obvious PcG phenotypes. Mutations in several other genes that play a role in Polycomb silencing enhance the phenotypes of PcG mutants but do not themselves exhibit Polycomb phenotypes. These include E(Pc), Su(z)2, and the histone deacetylase Rpd3/HDAC1. Uncovering the role of Sir2 in Polycomb silencing required sensitive genetic assays. This could be due to functional redundancy; four other Drosophila genes encode conserved SIR2 paralogs, corresponding respectively to the mammalian SIRT2 (similar to yeast HST2), SIRT4, and the closely related SIRT6 and SIRT7. Although these SIR2 paralogs are likely to have physiological roles distinct from that of SIR2, in the absence of SIR2, one or more of them might at least partially compensate for the function of SIR2 in Polycomb silencing. In S. cerevisiae, the Sir2p paralog Hst1p, which normally functions as a gene-specific repressor, can rescue the silencing defects of Sir2 mutants when it is overexpressed or targeted to the mating-type locus. Another yeast Sir2p paralog, Hst2p, although not required for silencing, improves rDNA silencing when it is overexpressed almost as efficiently as overexpressed Sir2p itself, even though Hst2p remains exclusively cytoplasmic (Furuyama, 2004).
It is also possible that the another deacetylase, e.g., RPD3/HDAC1, which is also present in E(Z) complexes, may be able to at least partially substitute for the SIR2 function in these complexes. Indeed, Drosophila RPD3 and SIR2 appear to have similarly broad substrate specificities, at least in vitro. Alternatively, SIR2 may be more critically required for Polycomb silencing under particular environmental or nutritional conditions that differ from standard laboratory conditions. It was originally suggested that the NAD+ dependence of SIR2 deacetylase activity (or its inhibition by nicotinamide could serve to link SIR2 activity to environmental or nutritional conditions. Indeed, the yeast Sir2p paralog Hst1p has been shown to regulate genes involved in de novo NAD+ biosynthesis by functioning as a direct sensor of cellular NAD+ levels. Various stresses and nutritional conditions appear to regulate the expression or activity of mammalian SIRT1 as well as the association of SIRT1 with its protein substrates. By analogy, the requirement for SIR2 or its activity in Polycomb silencing may be modulated by environmental or nutritional conditions, perhaps so that the fidelity of Polycomb silencing and its epigenetic inheritance is maintained under unfavorable or stressful culture conditions during larval life (Furuyama, 2004).
The physical association of Drosophila SIR2 with E(Z), RPD3, and p55 is the first evidence that SIR2 is associated with proteins known to be involved in epigenetic silencing in multicellular eukaryotes. The association of SIR2 with E(Z) was only detected in post-embryonic extracts, despite the presence of SIR2 and E(Z) in embryos. This suggests that E(Z) complex(es) differ in their composition and possibly their physiological functions at different developmental stages. It also suggests that the role of Drosophila SIR2 in Polycomb silencing may be restricted to post-embryonic stages. The transition from embryonic to larval period upon hatching from the egg marks the onset of active feeding and concomitant exposure to fluctuations in nutrient sources and other environmental variables from which embryonic development may be relatively more insulated. The differential association of SIR2 with E(Z) complexes during the larval stages may serve to increase the fidelity of PcG silencing under stressful conditions, a function that might not be expected to be uncovered without sensitive genetic assays or knowledge of the conditions that would render SIR2 more critical for maintenance of PcG silencing (Furuyama, 2004).
The high degree of protein sequence conservation among SIR2 orthologs from divergent species suggests that their biological functions, including their roles in epigenetic silencing, are also likely to be generally conserved. The conserved NAD+-dependent histone deacetylase activity and chromosomal localization of the Drosophila SIR2 protein is further consistent with this. However, although other components of E(Z) complexes, including RPD3 and the histone binding protein p55, have been highly conserved among all eukaryotes during evolution, an unequivocal E(Z) ortholog is not identifiable in S. cerevisiae or S. pombe, despite the presence of E(Z) orthologs in plants and animals and the presence of SET domain-containing histone methyltransferases in yeast. Conversely, Drosophila and mammals contain no identifiable homologs of S. cerevisiae SIR3 and SIR4, two key proteins that collaborate with SIR2 in the creation of silent chromatin domains at the mating-type loci and telomeres. This suggests that the mechanisms underlying SIR2-dependent silencing in yeast and multicellular eukaryotes, although broadly similar, are likely to differ in additional mechanistic details. On the other hand, the conservation of PcG proteins between Drosophila and mammals suggests that the association of SIR2 with E(Z) complex(es) is also likely to be conserved in mammals (Furuyama, 2004).
At present it is not evident why both NAD+-dependent (SIR2) and NAD+-independent (RPD3) HDACs are associated with E(Z) in larval extracts, but this arrangement is not unique. The S. cerevisiae SET domain protein SET3 is also found in a complex that contains two HDACs, including Hos2p, an RPD3-related class I HDAC, and Hst1p, which is closely related to yeast and Drosophila SIR2. Drosophila Hairy also interacts with both Rpd3 and Sir2. Perhaps in such situations each HDAC functions in different contexts or deacetylates different substrates. Drosophila RPD3 is found in a complex with the SET domain protein SU(VAR)3-9 and appears to be required for SU(VAR)3-9 histone methyltransferase function in vivo. It remains to be determined whether SIR2 is required for or modulates the histone methyltransferase function of E(Z) in vivo. The developmentally regulated association of SIR2 with E(Z) raises the interesting possibility that SIR2 may alter the activity or substrate specificity of E(Z). Although the chromosomal association of Drosophila SIR2 suggests it could target histones, the identification of multiple transcription factors and other proteins as substrates of mammalian SIRT1 suggests that the SIR2 associated with E(Z) may also have other non-histone substrates that regulate transcriptional silencing, perhaps including proteins in the E(Z) complex itself (Furuyama, 2004).
The ESC-E(Z) complex of Drosophila Polycomb group (PcG) repressors is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes, and related HMTases control germ line development in worms, flowering in plants, and X inactivation in mammals. The fly complex contains a catalytic SET domain subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55/CAF-1. The four-subunit complex is >1,000-fold more active than E(Z) alone. ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. Loss of ESC disrupts global methylation of histone H3-lysine 27 in fly embryos. Subunit mutations identify domains required for catalytic activity and/or binding to specific partners. Missense mutations are described in surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS domain of SU(Z)12, which each disrupt HMTase activity but preserve complex assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce active HMTase. A recombinant worm complex containing the E(Z) homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase activity from a distinct partner set (Ketel, 2005).
In vitro and in vivo data indicate that the noncatalytic ESC subunit makes a critical contribution to HMTase function of the ESC-E(Z) complex. In particular, since global levels of H3-K27 methylation are similarly reduced by genetic loss of ESC or E(Z), ESC appears to be an obligate functional partner for E(Z) HMTase activity (Ketel, 2005).
Two main molecular explanations are envisioned for the ESC requirement. (1) ESC could potentiate HMTase activity through direct interaction with E(Z). ESC binding could trigger a conformational change in E(Z) that improves catalytic efficiency, and/or ESC residues could directly interact with and influence the E(Z) active site. (2) Alternatively, the main role of ESC could be to bind nucleosomes. In this scenario, ESC would boost HMTase activity by facilitating interaction of the enzyme complex with its substrate. Based on several lines of evidence, a mechanism is favored that works through direct ESC-E(Z) contact. (1) The ESC M236K and V289M mutations, which significantly reduce HMTase activity, are located in surface loops previously shown to mediate direct ESC contact with E(Z). Furthermore, M236K displays dominant-negative properties in vivo. This genetic behavior is consistent with an enzyme complex that assembles normally but is compromised in catalytic function. (2) A recent report documents that ESC lacks nucleosome-binding activity on its own and that addition of ESC to a trimeric NURF-55/SU(Z)12/E(Z) complex has little additive effect on ability to bind nucleosomes. (3) ESC potentiation through direct E(Z) binding is supported by evolutionary considerations. Every organism examined that has an E(Z) homolog, ranging from plants to worms, flies, and humans, has at least one ESC homolog. In addition, 28 residues within the ESC surface loops implicated in E(Z) binding are identical from flies to humans. This conservation may reflect a tight functional requirement wherein direct ESC-E(Z) partnership, combining to produce HMTase activity, is maintained by evolutionary pressure. Future studies will be needed to define the precise biochemical mechanism by which ESC potentiates HMTase activity, including tests for binding-induced conformational changes in E(Z) (Ketel, 2005).
Studies on the mammalian homolog of ESC, called EED, have also highlighted its important role as a regulatory subunit. In human cells, multiple EED isoforms are expressed, which differ in the extents of their N-terminal tails. These isoforms are generated by alternative start codon usage of the same EED mRNA. Intriguingly, 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. Thus, it appears that the ESC/EED subunit can influence both catalytic efficiency and lysine substrate preference. In the fly system, ESC isoforms produced from the same mRNA have not been detected. Instead, alternative ESC isoforms could be supplied by an esc-related gene (CG5202) located about 150 kb proximal to esc. Since mutations in this second esc gene, called esc-like (escl), have not yet been reported, its in vivo contributions remain to be assessed. However, since genetic loss of ESC alone dramatically reduces global methylation of H3-K27 in fly embryos, it is concluded that ESC is the predominant functional E(Z) partner during embryonic stages (Ketel, 2005).
Studies on recombinant complexes show that fly SU(Z)12 is absolutely required for HMTase activity of the ESC-E(Z) complex. A key requirement for SU(Z)12 in mammalian EZH2 complexes has also been established based upon in vitro tests and loss-of-function studies in vivo. How does SU(Z)12 contribute molecularly to HMTase activity? Again, two main possibilities are envisioned: influence through direct contact with E(Z) or by mediating nucleosome binding. To address this, it is instructive to consider the SU(Z)12 mutants affecting the conserved VEFS domain. Deletion of the entire VEFS domain eliminates assembly of the fly complex by disrupting SU(Z)12-E(Z) binding. Pairwise binding assays with mammalian SU(Z)12 have similarly shown that the VEFS domain is needed for binding to EZH2 in vitro. Thus, a conserved function of this domain is to contact E(Z). However, missense mutations within the VEFS domain, D546A and E550A, preserve full complex assembly yet have reduced levels of HMTase. Taken together, these results implicate the VEFS domain in both binding to E(Z) and potentiating its enzyme activity, which suggests a connection between these two functions. In contrast, a recent report provides evidence that SU(Z)12 contributes to affinity for nucleosomes. Although SU(Z)12 cannot bind to nucleosomes by itself, the SU(Z)12/NURF-55 dimer has nucleosome-binding properties that are similar to those of the four-subunit complex. Thus, as also suggested for the human PRC2 complex, at least one role of SU(Z)12 is to mediate nucleosome binding. Further work will be needed to define the SU(Z)12 functional domains required for interactions with NURF-55 and with nucleosomes. Based on the available data, SU(Z)12 potentiation of E(Z) HMTase activity may involve both direct E(Z) contact and facilitated binding to nucleosome substrate (Ketel, 2005).
Although the SET domain is the most well-characterized functional domain of E(Z), the adjacent cysteine-rich CXC domain is also remarkably conserved from flies to humans. To address CXC function, in vitro properties of two missense mutants, C545Y and C603Y were analyzed. Both mutations correspond to E(z) loss-of-function alleles; in particular, in vivo effects of the E(z)61 mutation (C603Y) have been well documented. This mutation disrupts global H3-K27 methylation in embryos and causes loss of methyl-H3-K27 from a Hox target gene in imaginal discs. Mutant complexes bearing E(Z)-C603Y can assemble normally but show an approximately 10-fold reduction in HMTase levels. C545Y causes a more modest HMTase reduction, which parallels results obtained with the analogous substitution (C588Y) in human EZH2. These results suggest that the CXC domain interfaces with the SET domain to produce robust HMTase activity. In this regard, the CXC domain could be considered similar to cysteine-rich "preSET" domains required for robust HMTase activity in other SET domain proteins. Another effect of these CXC mutations in vivo is that they dislodge E(Z) from target sites in chromatin. Although the molecular basis for this dissociation is not known, in vitro assembly results suggest that it is not due to wholesale destabilization of the ESC-E(Z) complex. The dissociation may reflect another proposed role for the CXC domain, which is to interact with the PcG targeting factor PHO (Ketel, 2005).
In vitro tests were performed to investigate the role of E(Z) domain II. Both the complete domain deletion and the C363Y missense mutation show that domain II is required for stable association of E(Z) with SU(Z)12. Thus, the composite domain organization of E(Z) reflects division of labor among catalytic functions and requirements for complex assembly. In addition, it appears that none of the E(Z) domains are specifically built for nucleosome interactions; E(Z) plays little or no role itself in stable binding of the complex to nucleosomes (Ketel, 2005).
The NURF-55 subunit is distinct from the other three subunits in several ways. (1) It makes only minimal contributions to in vitro HMTase activity in both the fly and mammalian complexes. (2) Whereas the other three subunits appear dedicated to PcG function, NURF-55 is present in diverse chromatin-modifying complexes, including NURF, chromatin assembly factor 1 (CAF-1), and histone deacetylase complexes. The ability of the mammalian NURF-55 homologs RbAp46 and RbAp48 to bind to free histone H4 has led to the suggestion that NURF-55 may help chromatin complexes interact with substrate. Indeed, the absence of a NURF-55-related protein from the trimeric worm MES complex could help explain its inability to methylate free histones. The free histone-binding property of NURF-55 has also prompted the intriguing suggestion that silencing by the ESC-E(Z) complex in vivo could involve methylation of histones prior to nucleosome assembly. Since NURF-55 loss-of-function alleles have not been described in flies, many questions about roles of NURF-55 remain to be addressed. Even with alleles available, the multiplicity of NURF-55-containing complexes will likely complicate in vivo dissection of its PcG functions (Ketel, 2005).
The basic enzymatic function of the ESC-E(Z) complex, to methylate H3-K27, is shared between the worm, fly, and mammalian versions. Another similarity revealed from this study of recombinant worm complexes is that robust HMTase activity depends critically upon the two noncatalytic subunits. Although MES-6 and MES-3 can each individually bind to the catalytic subunit, MES-2, all three subunits are required together to produce enzyme activity. Since worm MES-6 is a WD repeat protein related to fly ESC, it seems likely that MES-6 and ESC potentiate HMTase activity through similar mechanisms. It is suggested that this mechanism entails direct subunit interactions rather than an influence upon affinity for nucleosomes. However, a major puzzle is presented by the dissimilarity between worm MES-3 and fly SU(Z)12. Though each is required for HMTase activity in their respective complexes, no relatedness was recognized between these two proteins in primary sequence or predicted secondary structure arrangement. From an evolutionary standpoint, it appears that SU(Z)12 represents the more ancient partner, since it is functionally conserved across plant and animal kingdoms. MES-3, a novel protein, may have evolved more recently to replace SU(Z)12 in the worm complex. Since a molecular role attributed to SU(Z)12 in the fly complex is nucleosome binding, it is speculated that MES-3 may supply this function for the worm complex. There are many strategies for building nucleosome contact domains, as represented among divergent chromatin proteins, so MES-3 could have acquired functional similarity without overt sequence similarity to SU(Z)12. In this view, MES-3 function in the worm complex would require, at minimum, affinity for nucleosomes and ability to bind MES-2. In this regard, it is interesting that MES-2 appears to lack domain II, which is needed in E(Z) for stable binding to SU(Z)12. Presumably, MES-2 has instead acquired a site for stable MES-3 interaction. In summary, it is suggested that the E(Z)/ESC and MES-2/MES-6 dimers have been conserved as core subunits of the HMTase complex, whereas the additional required partners in each complex, SU(Z)12 and MES-3, have been allowed to diverge. Future studies will be needed, including functional tests of chimeric worm and fly proteins, to address such a model (Ketel, 2005).
The Drosophila Polycomb group protein E(z) is a histone methyltransferase (HMTase) that is essential for maintaining HOX gene silencing during development. E(z) exists in a multiprotein complex called Polycomb repressive complex 2 (PRC2) that also contains Su(z)12, Esc and Nurf55 (Caf1). Reconstituted recombinant PRC2 methylates nucleosomes in vitro, but recombinant E(z) on its own shows only poor HMTase activity on nucleosomes. This study investigated the function of the PRC2 subunits. It was shown that PRC2 binds to nucleosomes in vitro but that individual PRC2 subunits alone do not bind to nucleosomes. By analysing PRC2 subcomplexes, it was shown that Su(z)12-Nurf55 is the minimal nucleosome-binding module of PRC2 and that Esc contributes to high-affinity binding of PRC2 nucleosomes. Nucleosome binding of PRC2 is not sufficient for histone methylation and only complexes that contain Esc protein show robust HMTase activity. These observations suggest that different subunits provide mechanistically distinct functions within the PRC2 HMTase: the nucleosome-binding subunits Su(z)12 and Nurf55 anchor the E(z) enzyme on chromatin substrates, whereas Esc is needed to boost enzymatic activity (Nekrasov, 2005).
Baculovirus expression vectors have been used to reconstitute and purify recombinant tetrameric PRC2 from Sf9 cells. To identify intermolecular interactions within this complex, tests were performed for reconstitution of PRC2 subcomplexes on coexpression of two or more subunits in Sf9 cells. In each case, the Flag-epitope tag present on one of the subunits was used for affinity purification from Sf9 cell extracts. The following stable dimeric complexes could be purfied: Esc-E(z), E(z)-Su(z)12, E(z)-Nurf55 and Su(z)12-Nurf55. In contrast, purification either from cells expressing Flag-Esc and Nurf55 or from cells expressing Flag-Esc and Su(z)12 resulted in the isolation of Flag-Esc protein only, suggesting that these proteins do not bind directly to each other. Purification from Sf9 cells expressing three subunits allowed the isolation of trimeric Esc-E(z)-Su(z)12 and E(z)-Su(z)12-Nurf55 complexes. Finally, tetrameric PRC2 was isolated, using either Flag-E(z) or Flag-Su(z)12 for affinity purification. Importantly, each of these complexes was stable in buffers containing up to 1 M KCl. Taken together, these data suggest that E(z) binds tightly to Su(z)12, Esc and Nurf55 and that Su(z)12 also binds to Nurf55. The failure to isolate dimeric complexes that contain Esc and Su(z)12 or those that contain Esc and Nurf55 indicates that E(z), Su(z)12 and Nurf55 form a trimeric core complex to which Esc binds through interaction with E(z). The observation that E(z) forms stable dimeric complexes with either Esc or Nurf55 in this reconstitution assay is consistent with earlier studies that reported physical interactions between these proteins in glutathione-S-transferase (GST) pull-down assays (Nekrasov, 2005).
It is noted that the molecular architecture of mammalian PRC2 is unclear at present; conflicting data on intermolecular interactions between subunits have been reported. Specifically, it has been reported that human EZH2, SU(Z)12 and RbAp48 all bind to EED, the Esc homologue, and that EZH2 does not interact with SU(Z)12 or RbAp48 in GST pull-down assays; it has been proposed that EED is the core component of the complex and EZH2 associates with other components through EED. It has also been reported that EZH2 binds to SUZ12 in GST pull-down assays, consistent with the finding that Drosophila E(z) and Su(z)12 form a stable complex (Nekrasov, 2005).
The HMTase activity of recombinant E(z) protein is significantly lower than the activity observed with recombinant tetrameric PRC2. A simple mechanistic explanation would be that one or several PRC2 subunits are needed for nucleosome binding to facilitate interaction of the E(z) HMTase with its substrate, the histone H3 tail. Since it is not known whether any of the PRC2 subunits binds to nucleosomes, tests were performed to see whether complex components alone or in combination could form stable complexes with mononucleosomes, in a bandshift assay. To this end, mononucleosomes were reconstituted with recombinant core histones that were expressed in E. coli and a 201 base pairs (bp) long radioactively labelled DNA template that contained a strong nucleosome-binding sequence called '601'. When recombinant tetrameric PRC2 was incubated with such mononucleosomes and the reaction mixture was resolved on a polyacrylamide gel, distinct, slowly migrating complexes were observed that appeared in a concentration-dependent manner. In contrast, when PRC2 was incubated with naked 601 DNA template, it was not possible to resolve specific protein-DNA complexes. Together, these observations suggest that PRC2 binds to mononucleosomes and that these protein-nucleosome complexes remain stably associated under electrophoretic conditions. Individual PRC2 subunits were tested for nucleosome binding, but no formation of protein-nucleosome complexes was detected with any of the four proteins. This suggests that more than one subunit is needed for nucleosome binding and therefore the different di- and trimeric PRC2 subcomplexes were tested. Among the different subcomplexes, only incubation with the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complexes results in the appearance of distinct, slowly migrating protein-nucleosome complexes. These protein-nucleosome complexes migrate similarly to the complexes observed with tetrameric PRC2, but two- to threefold higher concentrations of the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complex are needed to shift all of the nucleosome probe. Thus, the presence of Esc in PRC2 increases the affinity of the complex for nucleosomes or allows the complex to bind more stably under the experimental conditions compared with PRC2 subcomplexes that lack Esc. In contrast to the distinct protein-nucleosome complexes observed with the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complex, no specific protein-nucleosome complexes are formed if nucleosomes are incubated with the E(z)-Su(z)12, E(z)-Nurf55, Esc-E(z) or Esc-E(z)-Su(z)12 complexes. However, incubation with high concentrations of the trimeric Esc-E(z)-Su(z)12 complex also shifts almost all of the nucleosome probe, and much of the probe is retained in the well of the gel. Thus, it seems that the Esc-E(z)-Su(z)12 complex also binds to nucleosomes but that it binds in a manner distinct from the other PRC2 (sub)complexes. Taken together, these binding assays suggest that several subunits need to cooperate for nucleosome binding of PRC2, as follows: (1) Su(z)12 is essential for nucleosome binding because only complexes containing Su(z)12 bind; (2) the minimal nucleosome-binding complex contains Su(z)12 and Nurf55, and only complexes that contain these two proteins give rise to distinct, slowly migrating protein-nucleosome complexes; Su(z)12 and Nurf55 together thus form the minimal nucleosome-binding module of PRC2; (3) as discussed above, Esc also contributes to nucleosome binding because (a.) tetrameric PRC2 binds more strongly than the E(z)-Su(z)12-Nurf55 complex and (b.) in the absence of Nurf55, that is, in the Esc-E(z)-Su(z)12 complex, Esc seems to cooperate with Su(z)12 to cause retention of the nucleosome probe. E(z) is thus the only subunit for which no contribution to nucleosome binding was detected. Note that comparable concentrations of the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complex are required to shift 50% of the nucleosome probe (Nekrasov, 2005).
Nurf55 and Esc are both WD40 repeat proteins. RbAp46 and RbAp48, the mammalian homologues of Nurf55, have been reported to bind directly to helix 1 of histone H4, a portion of H4 that is thought to be inaccessible within the nucleosome, and, consistent with this, RbAp46 and RbAp48 are unable to bind to H4 in nucleosomal templates. As shown in this study, Drosophila Nurf55 or Esc alone are not able to bind to mononucleosomes but they bind in combination with Su(z)12, which, by itself, also does not bind to nucleosomes. It is possible that the combination of Su(z)12 and Nurf55 or Esc is needed to create the necessary surface for stable nucleosome binding. Alternatively, it could be that these proteins act in a cooperative manner to disrupt histone-DNA contacts locally to expose the histone core (i.e. H4) for binding by Nurf55 or Esc (Nekrasov, 2005).
These results suggest that Su(z)12 together with Nurf55 or Esc tethers the complex to nucleosomes, whereas E(z), the catalytic subunit of the complex, contributes little to nucleosome binding. Next the HMTase activity of the different PRC2 (sub)complexes were analyzed. As substrates for these reactions, non-radiolabelled mononucleosomes were used identical to those used in the bandshift assay. Recombinant tetrameric PRC2 methylates H3 in mononucleosomes. In contrast, E(z) protein alone, the dimeric Esc-E(z), and the Su(z)12-E(z) or E(z)-Nurf55 complexes do not detectably methylate mononucleosomes. Strikingly, the Su(z)12-E(z)-Nurf55 complex also shows no detectable HMTase activity, whereas the Esc-E(z)-Su(z)12 complex methylates H3 in mononucleosomes with efficacy similar to tetrameric PRC2. Thus, no straightforward correlation is observed between nucleosome binding in bandshift assays and HMTase activity. In particular, the Su(z)12-E(z)-Nurf55 complex seems to bind to nucleosomes with only two- to threefold lower affinity than tetrameric PRC2, but shows markedly reduced HMTase activity. In contrast, the Esc-E(z)-Su(z)12 complex, which shows almost no nucleosome binding at the concentration used in the HMTase assay, shows HMTase activity comparable with PRC2. Together, these data suggest that nucleosome binding is not sufficient for HMTase activity and that Esc has a crucial role in boosting the enzymatic activity of E(z). It is possible that Esc is required to dock the complex in a specific orientation on the nucleosome that presents the H3 tail in a particularly favourable position to the E(z) enzyme. Alternatively, Esc could directly increase the catalytic activity of E(z) by inducing a conformational change in the enzyme. However, it is important to note that the Esc-E(z) complex shows no detectable HMTase activity on mononucleosomes and the presence of Su(z)12 is thus essential for HMTase activity of the Esc-E(z)-Su(z)12 complex. The bandshift data show that Su(z)12 is strictly needed for nucleosome binding of PRC2. At the low complex concentrations used in the HMTase assay, there is probably very little nucleosome binding of the Esc-E(z)-Su(z)12 complex; nevertheless, it seems probable that the nucleosome interactions that Su(z)12 shows in cooperation with Esc contribute to the HMTase activity of the Esc-E(z)-Su(z)12 complex. Finally, it is puzzling that the absence of Nurf55 from the complex (i.e. in the Esc-E(z)-Su(z)12 complex) does not seem to diminish HMTase activity because Nurf55 is important for the formation of stable PRC2-nucleosome complexes in bandshift assays. It is possible that Nurf55 is not needed for HMTase activity under the assay conditions that were used but that it is important for methylation of chromatin in vivo (Nekrasov, 2005).
Recent studies have shown that SU(Z)12 is crucial for HMTase activity of mammalian PRC2 in vitro, but the molecular basis for this requirement has remained unclear. This study reports two main findings: (1) Su(z)12 has a crucial role in nucleosome binding of Drosophila PRC2; (2) PRC2 subcomplexes that bind to nucleosomes but lack Esc are poorly active; Esc thus has an important role in boosting HMTase activity of the complex (Nekrasov, 2005).
The requirement of Su(z)12 for HMTase activity can for the most part be explained by its ability to tether PRC2 to nucleosomes, and the data suggest that nucleosome binding requires Su(z)12 in combination with either Nurf55 or Esc. Although both Nurf55 and Esc contribute to nucleosome binding of the complex, only inclusion of Esc leads to an increase in HMTase activity. The contribution of Esc to HMTase activity thus goes beyond the activity that one would expect if Esc were required only for nucleosome binding. In summary, these data suggest that the Su(z)12, Nurf55 and Esc subunits all contribute to nucleosome binding of PRC2 but that these three subunits make distinct contributions to the activation of the E(z) HMTase (Nekrasov, 2005).
The findings reported here imply that E(z) HMTase activity in vivo could be regulated at the level of chromatin binding and/or enzyme activity by modulating the abundance or activity of different PRC2 subunits. It is important to discuss the results reported here in the context of the in vivo requirement for different PRC2 subunits. Genetic studies have shown that Su(z)12 and E(z) are required throughout development to maintain silencing of HOX genes in Drosophila and that this process requires the enzymatic activity of E(z). In contrast, Esc protein is required early in development and then becomes to a large extent, although not completely, dispensable for maintenance of HOX gene silencing during postembryonic development. There are two possible explanations for the paradoxical observation that Esc is required for strong HMTase activity in vitro but that the protein seems to be largely dispensable for the HMTase activity of E(z) that is needed to maintain HOX gene silencing during larval development. (1) It is possible that strong HMTase activity of PRC2 is required primarily early in embryogenesis and that, once it is established, H3-K27 methylation can be maintained with a catalytically less active form of the complex (i.e., lacking Esc). (2) It is possible that another protein substitutes for Esc at later developmental stages. It is noted that the Drosophila genome encodes a second Esc-like protein (CG5202) but, at present, it is not known whether this protein is required for HOX gene silencing (Nekrasov, 2005).
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).
PcG protein complex PRC2 is thought to be the histone methyltransferase (HMTase) responsible for H3-K27 trimethylation at Polycomb target genes. This study reports the biochemical purification and characterization of a distinct form of Drosophila PRC2 that contains the Polycomb group protein Polycomblike (Pcl). Like PRC2, Pcl-PRC2 is an H3-K27-specific HMTase that mono-, di- and trimethylates H3-K27 in nucleosomes in vitro. Analysis of Drosophila mutants that lack Pcl unexpectedly reveals that Pcl-PRC2 is required to generate high levels of H3-K27 trimethylation at Polycomb target genes but is dispensable for the genome-wide H3-K27 mono- and dimethylation that is generated by PRC2. In Pcl mutants, Polycomb target genes become derepressed even though H3-K27 trimethylation at these genes is only reduced and not abolished, and even though targeting of the Polycomb protein complexes PhoRC and PRC1 to Polycomb response elements is not affected. Pcl-PRC2 is thus the HMTase that generates the high levels of H3-K27 trimethylation in Polycomb target genes that are needed to maintain a Polycomb-repressed chromatin state (Nekrasov, 2007).
Genetic studies using Drosophila first identified Polycomb group (PcG) genes as regulators that are required for the long-term repression of HOX genes during development. To date, 17 different genes in Drosophila are classified as PcG members because mutations in these genes cause misexpression of HOX genes. All Drosophila PcG genes are also conserved in mammals and at least some of them are also conserved in plants. In all these organisms, PcG gene products function as repressors of HOX and/or other regulatory genes that control specific developmental programs. Moreover, recent studies that analyzed genome-wide binding of PcG proteins in Drosophila and in mammalian cells have identified a large number of target sites, and thus a whole new set of genes that potentially is subject to PcG repression (Nekrasov, 2007).
Biochemical purification and characterization of PcG protein complexes has advanced understanding of the PcG system. To date, three distinct PcG protein complexes have been isolated from Drosophila: PhoRC, PRC1 and PRC2. Biochemically purified Drosophila PRC2 contains the three PcG proteins Enhancer of zeste [E(z)], Suppressor of zeste 12 [Su(z)12] and Extra sex combs (Esc) and, in addition, Nurf55 (Caf1), a protein that is present in many different chromatin complexes. Drosophila PRC2 and the homologue mammalian complex are histone methyltransferases (HMTases) that specifically methylate H3-K27 in nucleosomes. Chromatin immunoprecipitation (X-ChIP) analyses in Drosophila showed that PRC2 binds in a localized manner at Polycomb response elements (PREs) of target genes, but that H3-K27 trimethylation is present across the whole upstream control, promoter and coding region of these genes. Studies that compared the inactive and active state of the HOX gene Ubx in developing Drosophila have found that PRC2 is constitutively bound at PREs and, surprisingly, that the whole upstream control region is constitutively trimethylated at H3-K27. However, presence or absence of H3-K27 trimethylation in the Ubx promoter and coding region correlates tightly with the gene being repressed or active, respectively. H3-K27 trimethylation is thus a distinctive mark of PcG-repressed chromatin (Nekrasov, 2007).
Analysis of E(z) mutants suggests that E(z) is also responsible for the genome-wide H3-K27 mono- and dimethylation that has been reported to be present on more than 50% of H3 in Drosophila. However, biochemical analyses showed that E(z) protein alone does not bind to nucleosomes and is virtually inactive as an enzyme; E(z) needs to associate with Su(z)12 and Nurf55 for nucleosome binding and with Esc for enzymatic activity. This implies that the genome-wide H3-K27 mono- and dimethylation is generated by PRC2 or another E(z)-containing complex that is able to interact in a non-targeted manner with nucleosomes across the whole genome. Conversely, this raises the question whether H3-K27 trimethylation at PcG target genes is simply a consequence of PRC2 being targeted to PREs or whether additional features such as post-translational modifications or associated factors are required (Nekrasov, 2007).
Previous studies reported that the PcG protein Polycomblike (Pcl) interacts with E(z) in GST pull-down, yeast two-hybrid and co-immunoprecipitation assays. Like most other PcG proteins, Pcl has also been found to be bound at PREs in Drosophila. However, to date, no Pcl-containing complexes have been purified and the role of Pcl in PcG repression has remained enigmatic. This study reports the biochemical purification of Pcl complexes. Pcl is shown to exist in a stable complex with PRC2. This analyses demonstrate that this Pcl complex plays a critical role in generating high levels of repressive H3-K27 trimethylation at PcG target genes (Nekrasov, 2007).
Biochemically purified Pcl complexes contain Pcl together with the four core subunits of PRC2. In contrast, biochemically purified E(z) complexes contain only substoichiometric amounts of Pcl and the previously described purifications of PRC2 failed to reveal Pcl in the purified material. Moreover, fractionation of crude nuclear extracts by gel filtration indicated that Pcl and PRC2 components Esc, E(z) and Su(z)12 co-fractionate in high-molecular-weight assemblies, but that the bulk of these other PRC2 components is present in lower-molecular-weight fractions that do not contain Pcl. Taken together, these observations suggest that only a fraction of PRC2 is associated with Pcl and that Pcl-PRC2 is a distinct complex (Nekrasov, 2007).
Previous X-ChIP studies showed that Pcl and Su(z)12 colocalize at Ubx and Abd-B PREs. This suggests that Pcl-PRC2 is bound at these PREs. In this study, the analysis of Drosophila mutants that lack Pcl protein and therefore lack Pcl-PRC2, provided insight into the function of this complex. The results provide strong evidence that Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation in the chromatin of PcG target genes. Unlike in E(z) or Su(z)12 mutants, removal of Pcl in embryos or in imaginal discs only reduces but does not eliminate H3-K27 trimethylation. Nevertheless, repression of several PcG target genes is abolished in Pcl mutants. This suggests that not only the mere presence of H3-K27me3, but presence of high levels of H3-K27me3 is crucial for maintaining these PcG target genes in the repressed state. Previous studies on the Ubx gene suggested that presence of H3-K27 trimethylation in the promoter and coding region is critical for PcG repression. One possibility would be that it is the overall density of H3-K27me3-marked nucleosomes across the promoter and coding region that determines whether a PcG target gene is repressed. Another possibility would be that even though a whole chromatin domain becomes trimethylated at H3-K27, only a few H3-K27me3-marked nucleosomes at a particular position (e.g., around the transcription start site) are actually required for repression, and failure to maintain this trimethylation results in loss of repression (Nekrasov, 2007).
The observation that Su(z)12 binding and H3-K27 trimethylation are reduced but not lost in the absence of Pcl is consistent with the idea that Pcl might help anchoring PRC2 to PREs, but it also suggests that at least some PRC2 must be targeted to PREs independently of Pcl. It seems likely that the residual H3-K27 trimethylation present in Pcl mutant embryos and in Pcl mutant clones in imaginal discs is generated by PRC2 that is bound at PREs independently of Pcl. In this context it is important to note that not only Pcl-PRC2 but also PRC2 is able to trimethylate H3-K27 in recombinant nucleosomes in vitro. Apart from the suggested role in tethering of PRC2 to PREs, it is possible that Pcl also functions in a post-recruitment step to help PRC2 generate high levels of H3-K27 trimethylation at target genes. For example, the tudor domain and PHD fingers of PRE-bound Pcl might interact with modified nucleosomes in the promoter and coding region of target genes to ensure that they become trimethylated at H3-K27 by the associated PRE-tethered PRC2 (Nekrasov, 2007).
Finally, no evidence was found that Pcl-PRC2 would be required for the genome-wide H3-K27 mono- and di-methylation. X-ChIP analyses suggest that H3-K27 mono- and dimethylation across the genome might even slightly increase in the absence of Pcl. In contrast, there is a loss of all H3-K27 methylation in either E(z) or Suz)12 mutants. This suggests that PRC2 or another E(z)-containing complex generates the genome-wide H3-K27 mono- and dimethylation. The experiments in Pcl mutants thus allowed dissection of the role of different H3-K27 methylation states in Drosophila. The selective reduction of H3-K27me3 levels, and the concomitant loss of repression of PcG target genes in Pcl mutants, provides compelling evidence that only the trimethylated state of H3-K27 is functional in PcG repression in Drosophila. Pcl-PRC2 is evidently critically needed to generate the high levels of H3-K27 trimethylation that are required to maintain a Polycomb-repressed chromatin state (Nekrasov, 2007).
Polycomb-group (PcG) proteins are highly conserved epigenetic transcriptional repressors that play central roles in numerous examples of developmental gene regulation. Four PcG repressor complexes have been purified from Drosophila embryos: PRC1, PRC2, Pcl-PRC2 and PhoRC. Previous studies described a hierarchical recruitment pathway of PcG proteins at the bxd Polycomb Response Element (PRE) of the Ultrabithorax (Ubx) gene in larval wing imaginal discs. The DNA-binding proteins Pho and/or Phol are required for target site binding by PRC2, which in turn is required for chromosome binding by PRC1. This study identified a novel larval complex that contains the PcG protein Polycomblike (Pcl) that is distinct from PRC1 and PRC2 and which is also dependent on Pho and/or Phol for binding to the bxd PRE in wing imaginal discs. RNAi-mediated depletion of Pcl in larvae disrupts chromosome binding by E(z), a core component of PRC2, but Pcl does not require E(z) for chromosome binding. These results place the Pcl complex (PCLC) downstream of Pho and/or Phol and upstream of PRC2 and PRC1 in the recruitment hierarchy (Savla, 2008).
Drosophila Polycomb-group (PcG) genes were originally identified as negative regulators of Hox genes. PcG-mediated silencing in Drosophila occurs in essentially two broadly defined stages: assumption of transcriptional repression responsibilities from gene-specific transcription factors in early embryos, followed by maintenance of the silenced state through many cycles of cell division beginning in mid-late-stage embryos and continuing throughout the remainder of development (Savla, 2008).
Although much of the genetic analysis of PcG functions and studies of the mechanisms by which PcG proteins are targeted to specific genomic sites have focused on their activities in larval tissues, in vitro biochemical analyses have focused on PcG complexes isolated from embryos: PRC1, PRC2 and PhoRC. PRC1 possesses multiple chromatin modifying activities in vitro suggesting that it, among PcG complexes, might be most directly responsible for preventing transcription. The primary functions of PhoRC and PRC2 appear to be to recruit and/or stabilize target site binding by PRC1, and potentially other PcG proteins. PhoRC includes the DNA-binding PcG protein Pleiohomeotic (Pho), which binds to sites within Polycomb Response Elements (PREs) that serve as docking platforms for PcG proteins. Pho directly interacts with components of both PRC1 and PRC2, and is required for recruitment of both complexes. The E(z) subunit of PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), facilitating recruitment of PRC1 (Savla, 2008).
A variant of PRC2 has recently been described that includes the PcG protein Polycomblike (Pcl). On the basis of gel filtration analysis of native complexes in embryo nuclear extracts and the stoichiometry of the purified Pcl-PRC2 complex, it appears that the majority of embryonic Pcl is present in Pcl-PRC2, but that the other PRC2 core subunits, E(z), Su(z)12, Esc and NURF55 (also known as Caf1 - FlyBase), predominantly are in a complex(es) lacking Pcl. It has been proposed that inclusion of Pcl in PRC2 is required for high levels of H3K27me3 in vivo, although the in vitro histone methyltransferase activity of Pcl-PRC2 is indistinguishable from that of PRC2 lacking Pcl. In this study, a larval Pcl-containing complex is identified that is distinct from PRC2 and PRC1 and shown to be required for chromosome binding by these PcG complexes (Savla, 2008).
In order to examine potential differences between embryonic and larval
stage PcG complexes, larval nuclear extracts were fractionated over a Superose 6 gel filtration column, and western blots of the fractions were probed with anti-E(z) and anti-Pcl antibodies. Larval E(z)-containing complexes have a relative mass of ~500 to 600 kDa, similar to that of embryonic PRC2 complexes that lack Pcl. However, Pcl was undetectable in E(z)-containing fractions and appeared to be in a complex with a relative mass of ~1500 kDa. This is different from the fractionation profile of Pcl from embryo extracts, in which it co-fractionates with E(z) in native complexes with relative mass estimates in the range of ~650 kDa to 1000 kDa, suggesting that, unlike its association with a subset of PRC2 complexes in embryos, Pcl functions as a component of a distinct complex in larvae, which will be referred to as the Pcl-Complex (PCLC) (Savla, 2008).
In order to further investigate the relationship of Pcl with other PcG
proteins and its role in PcG-mediated silencing in larvae, chromatin
immunoprecipitation (ChIP) assays were performed on wing imaginal discs. The
PcG maintains the transcriptional silence of the Hox gene
Ultrabithorax (Ubx) in the epithelial cells of wing discs. Other
PcG proteins, including the DNA-binding proteins Pho and Phol and components
of the PRC1 and PRC2 complexes, have previously been shown to be present at
the major PRE in the Ubx cis-regulatory bxd region in this tissue.
Consistent with a previous report, Pcl also was detected at the bxd PRE, and appears to largely colocalize with E(z) and Phol (Savla, 2008).
A hierarchical relationship among PcG proteins at
the bxd PRE in which Pho and/or Phol are required, but are not
necessarily sufficient, for recruitment of PRC2, which in turn facilitates
recruitment of PRC1. In order to determine how Pcl might fit into this recruitment pathway, ChIP assays were performed on E(z) mutant wing
imaginal discs. E(z)61 is a temperature-sensitive allele
that displays nearly wild-type activity at 18°C, but strongly reduced
activity at 29°C. Following shift from 18°C to 29°C, bxd PRE
binding by E(z)61 protein is rapidly lost and along with it the
detection of H3K27me3 and Pc in this region. ChIP assays of wing discs dissected from E(z)61 larvae 24 hours following shift from 18° to 29°C confirmed loss of E(z) from the PRE, but revealed no effect on Pcl and Phol binding to PRE fragments 3 and 4, but a slight decrease of both proteins at the PRE 2 fragment. It is speculated that Pcl and Phol signals at this proximal edge of the PRE are partly due to protein-protein cross-links, which might be reduced in the absence of PRC2. Retention of Pcl at the PRE in the absence of E(z) and by extension absence of PRC1, which requires PRC2 for binding to this region, confirms that Pcl is not a stable subunit of larval versions of either PRC1 or PRC2 and is consistent with its inclusion in a distinct complex (Savla, 2008).
Flies that are homozygous for null Pcl alleles die as embryos and
no conditional Pcl alleles exist, precluding reciprocal experiments
on Pcl mutant larvae. Therefore, transgenic fly lines were generated
that contain inserts of a pWIZ-Pcl construct, which expresses Pcl shRNA under
the control of Gal4, permitting inducible RNAi-mediated knockdown of Pcl in
combination with Gal4 drivers. Individuals that contain both pWIZ-Pcl
and P{GAL4-da.G32}, which constitutively expresses Gal4, died as early pupae and exhibited dramatically reduced levels of Pcl in wing
imaginal discs. E(z) levels were not affected. ChIP assays of these Pcl-depleted wing discs confirmed reduced Pcl levels at the bxd PRE and revealed commensurate loss of E(z). Thus, although Pcl does not require PRC2 for PRE binding, Pcl, presumably functioning as a subunit of PCLC, is needed for stable binding of PRC2 to the bxd PRE. Phol remains at the PRE in the absence of Pcl (Savla, 2008).
In order to determine whether Pcl (like components of PRC1 and PRC2)
requires Pho and/or Phol for PRE binding, ChIP assays were performed using
wing imaginal discs from phol81A; pho1 larvae.
Consistent with their role in recruiting other PcG proteins, Pcl
was lost from the bxd PRE in the absence of Pho and Phol. These
observations at the bxd PRE also appear to generally apply to PcG-binding sites
throughout the genome (Savla, 2008).
These results demonstrate the existence of a distinct Pcl protein complex
in larvae that is required for recruitment of PRC2 to chromosomal target sites
and/or to stabilize its binding. As previously described, E(z), as a core
subunit of PRC2, is required for target site binding by PRC1. Therefore, Pcl is indirectly required for chromosome binding by PRC1 as well, although direct interaction with PRC1 cannot be ruled out, similar to the way in which Pho may contribute to target site binding by PRC1 by interacting both
with PRC2 subunits and with Pc, a core subunit of PRC1
(Savla, 2008 and references therein).
In vitro histone methyltransferase assays of Pcl-PRC2 show that its
activity and specificity for methylation of H3K27 are essentially
indistinguishable from that of PRC2 complexes lacking Pcl. ChIP analysis of
Pcl mutant embryos has shown that Pcl does not seem to be required
for target site binding by other PRC2 subunits, but that it may be needed for
high levels of trimethylation of H3K27. One
explanation for these observations is that the contribution of Pcl to Pcl-PRC2
in embryos might be to mediate interaction with other proteins that are yet to
be identified. In larvae, Pcl exists as a subunit of a distinct complex. Given
the ability of Pcl to directly interact with several PRC2 subunits, colocalization of Pcl and E(z) at the PRE, and dependence of E(z) on Pcl for binding to the bxd PRE and other genomic sites, it is likely that
PCLC is closely associated with PRC2 at target sites in larvae. In both
embryos and larvae, some of the activities attributed to Pcl might, upon
further inspection, be due to the activities of other Pcl-associated proteins,
the close apposition of which with PRC2 and other PcG complexes may be
mediated by Pcl. The differential deployment of Pcl as a subunit of PRC2 and
as a subunit of PCLC at distinct developmental stages is intriguing and might
reflect the different molecular activities needed for establishment of
silencing in embryos and maintenance of the silenced state in larval tissues. A more detailed understanding of the mechanisms by which Pcl contributes to PcG silencing will require identification of the other proteins contained within the larval PCLC complex and the potential biochemical activities of the complex (Savla, 2008).
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).
Jarid2 was recently identified as an important component of the mammalian Polycomb repressive complex 2 (PRC2), where it has a major effect on PRC2 recruitment in mouse embryonic stem cells. Although Jarid2 is conserved in Drosophila, it has not previously been implicated in Polycomb (Pc) regulation. Therefore, Drosophila Jarid2 and its associated proteins were purified, and it was found that Jarid2 associates with all of the known canonical PRC2 components, demonstrating a conserved physical interaction with PRC2 in flies and mammals. Furthermore, in vivo studies with Jarid2 mutants in flies demonstrate that among several histone modifications tested, only methylation of histone 3 at K27 (H3K27), the mark implemented by PRC2, was affected. Genome-wide profiling of Jarid2, Su(z)12 (Suppressor of zeste 12), and H3K27me3 occupancy by chromatin immunoprecipitation with sequencing (ChIP-seq) indicates that Jarid2 and Su(z)12 have very similar distribution patterns on chromatin. However, Jarid2 and Su(z)12 occupancy levels at some genes are significantly different, with Jarid2 being present at relatively low levels at many Pc response elements (PREs) of certain Homeobox (Hox) genes, providing a rationale for why Jarid2 was never identified in Pc screens. Gene expression analyses show that Jarid2 and E(z) (Enhancer of zeste, a canonical PRC2 component) are not only required for transcriptional repression but might also function in active transcription. Identification of Jarid2 as a conserved PRC2 interactor in flies provides an opportunity to begin to probe some of its novel functions in Drosophila development (Herz, 2012).
Different and distinct gene expression patterns are established during development, which need to be maintained and regulated. This is important to allow for the integrity of cell identity and thus the functional preservation of tissues and organs. However, at the same time, transcribed loci must be equipped with an intrinsic flexibility to regulate these expression patterns and initiate changes if necessary. The core components that are required for the maintenance of gene expression or gene repression have been characterized quite extensively to date . Trithorax and Polycomb group genes play antagonistic roles in determining whether a gene is transcriptionally turned on or off, respectively. In Drosophila, so far four distinct complexes, pleiohomeotic repressive complex (PhoRC), Polycomb repressive complex 2 (PRC2), Polycomb repressive complex 1 (PRC1), and recently Polycomb repressive deubiquitinase (PR-DUB) have been described to play a role in Polycomb group-mediated gene repression. However, little is known about the factors involved in controlling recruitment and activity of these complexes on chromatin or about the mechanisms that drive such changes. It should be expected that quite a significant number of proteins would convey Polycomb group-mediated transcriptional changes in order to allow an uncoupling of individual gene activity from that of a group of Polycomb group-controlled genes. Functional redundancy might account for part of the problem to discover such candidates. Furthermore, biochemical approaches might be hindered by the fact that such context-specific and more gene-specific recruiters are contained in only a minor fraction of Polycomb repressive complexes (Herz, 2012).
Recently, Jarid2 the founding member of the JmjC domain-containing protein family, which plays important developmental roles in mice and Drosophila, has been characterized as a component of PRC2 in embryonic stem (ES) cells. The consensus indicates that in ES cells, PRC2 recruitment to many of its targets requires Jarid2. However, levels of bulk histone 3 trimethylated at K27 (H3K27me3) in ES cells depleted of Jarid2 were reported to be only slightly changed at best. This also holds true when individual PRC2 target genes are analyzed. Even though core components of the PRC2 complex were lost from chromatin in the absence of Jarid2, H3K27me3 was not reproducibly affected to a similar degree. Additionally, gene expression analyses in Jarid2-/- ES cells did not confirm a genome-wide derepression of PRC2 target genes as would be expected for any core component of PRC2 (Landeira, 2010; Herz, 2012 and references therein).
To further address whether Jarid2 constitutes a core PRC2 component, is involved in recruitment of PRC2 to chromatin, and regulates H3K27 methylation in Drosophila, a Jarid2 complex was purified from flies and a global in vivo analysis of was performed of Suppressor of zeste 12 [Su(z)12] and H3K27me3 occupancy in Jarid2 mutant animals. The data confirm that Drosophila Jarid2 purifies with the core members of the PRC2 complex. In imaginal discs, global H3K27me3 levels are only weakly but reproducibly affected under Jarid2 mutant and Jarid2-overexpressing conditions. These genome-wide studies suggest that in Drosophila, under physiological conditions, Jarid2 does not appear to be a canonical component of the PRC2 complex as PRC2 recruitment is not altered on most target genes in Jarid2 mutant animals. Interestingly, overexpression of Jarid2 results in reduced Su(z)12 binding and changed chromatin compaction on polytene chromosomes, highlighting a possible role for Jarid2 in altering chromatin architecture. Genome-wide, Jarid2 and Su(z)12 binding correlate very well. However, certain loci, such as Homeobox (Hox) genes, differ significantly from this pattern. Here, Jarid2 occupancy on Polycomb response elements (PREs) is often very low where usually the highest enrichment for Su(z)12 can be observed. Gene expression analyses suggest a PRC2-dependent and -independent role for Jarid2 in transcriptional regulation. Jarid2 appears to be involved in the regulation of a certain number of PRC2 target genes and also transcriptionally controls a subset of genes independently of PRC2. These data not only imply a function for Jarid2 and PRC2 in transcriptional repression but also support a possible role for both Jarid2 and PRC2 in active transcription on genes that are occupied by these factors (Herz, 2012).
This study describes the purification of a Jarid2 complex in Drosophila. Consistent with previous results in mammalian systems, Jarid2 was found to be a component of PRC2. Evidence is provided that in imaginal discs and on polytene chromosomes, Jarid2 is required to fine-tune global H3K27me3 levels. Jarid2 might accomplish this by modulating the activity of the core complex [E(z), Su(z)12, Esc, and Caf1]. The data indicate that Jarid2 could play an inhibitory role in the implementation of H3K27me3 as Jarid2 mutant imaginal disc clones display a global increase and as overexpression of Jarid2 results in a reduction in H3K27me3. Despite having a JmjC domain, Jarid2 has been predicted and reported to be catalytically inactive as a histone demethylase. Therefore, it is unlikely but not impossible that it could function in this manner toward H3K27me3, thereby counteracting PRC2 activity. Even if Jarid2 would be inactive as a histone demethylase, it might still be able to bind to chromatin and prevent spreading of the H3K27me3 mark, such as opposing a possible positive spreading effect of Esc (EED in mammals) (Herz, 2012).
Furthermore, even though Jarid2 could be purified with the PRC2 core members and its occupancy generally correlates very well with canonical PRC2 components such as Su(z)12, it does not appear to play a significant role in regulating PRC2 recruitment in a physiological context, as assessed by Jarid2 mutant animal studies. Apparent differences with published mammalian studies, which imply a major role for Jarid2 in recruitment of PRC2, could be explained by variation in the mechanisms employed or by the fact that the recruitment of PRC2 in ES cells generally differs from that in differentiated tissues. For example, PREs have been known to be highly effective in recruiting PRC2 to target sites in Drosophila. In mammals, attempts have been made to identify functionally analogous sequences but with only limited success. Indeed, it seems more likely that the recruitment of PRC2 in mammals not only requires specific sequences but is also more dependent on additional factors (proteins and RNA), which might explain why PRC2 recruitment is more strongly affected in Jarid2-depleted cells and why PRC1 recruitment in some instances appears to be dependent on PRC2 (H3K27me3). However, the data in Drosophila salivary glands suggest that recruitment of PRC2 (and methylation of H3K27) is not a prerequisite for targeting of PRC1, and the generality of this mechanism is also increasingly questioned in the mammalian system. Nonetheless, when Jarid2 is overexpressed in Drosophila, changes in chromosome compaction can be observed. Under these conditions, Jarid2 extensively occupies the chromosomes, and Su(z)12 localization and H3K27me3 are negatively affected). It is possible that increasing Jarid2 levels beyond a certain physiological level might interfere with PRC2 integrity. Larger amounts of Jarid2 might alter the stoichiometry of the PRC2 subunits, resulting in destabilization of the PRC2 complex on chromatin (Herz, 2012).
Jarid2 also behaves differently from other canonical PRC2 members in Drosophila, as is evident from its binding pattern on certain Hox genes. At Hox genes, occupancy of PRE sites by canonical PRC2 members is one of the highest in the whole genome. In contrast, Jarid2 displays relatively low occupancy on many of these loci, implying a minor or different function for Jarid2 in controlling transcription of these well-described PRC2 targets. It is also possible that at these loci Jarid2 has a more transient association or even that it is less accessible to interact with the antibodies that were have generated. However, these findings are also in agreement with modifier screens that have been performed in Drosophila to identify major regulators of Polycomb group-mediated phenotypes but that were unable to capture Jarid2 (Herz, 2012).
Additionally, the data suggest that Jarid2 appears to control PRC2-dependent transcription, although not necessarily in the same way as expected for canonical PRC2 members. For example, in contrast to the mammalian findings, this study observed that PRC2-mediated transcriptional regulation by Jarid2 in Drosophila is generally independent of changes in Su(z)12 occupancy and does not correlate with changes in H3K27me3 enrichment. However, it needs to be stressed that most Jarid2/PRC2 cobound genes with altered expression patterns in Jarid2 mutants and E(z)-RNAi larvae contain no or low levels of H3K27me3, which is in contrast to the mammalian system where PRC2 components are usually found only at genes with high H3K27me3 enrichment. Nonetheless, in Drosophila, genes with high H3K27me3 enrichment exist that change in transcription in Jarid2 mutants and E(z)-RNAi animals, demonstrating that H3K27me3 is not necessarily instructive for transcriptional repression per se. To date most of the evidence ascribing to H3K27me3 the role of a repressive mark is based on correlation from the observation that PRC2 components colocalize with H3K27me3 and that the respective genes seem to be transcriptionally silenced. The data imply that this might generally be the case but that there are also exceptions to the rule. That certain H3K27me3 patterns can also be connected to transcriptionally active genes in mammals has just recently been reported (Young, 2011; Herz, 2012 and references therein).
Finally, the results imply that Jarid2 and PRC2 are not only involved in maintenance of gene repression but could also function in active transcriptional processes such as transcriptional activation of elongation. This is in agreement with previous reports and demonstrates that PRC2 has cellular functions that extend beyond what was learned from its role at Hox genes. Importantly, the current studies also suggest that despite a very good correlation of Jarid2 and Su(z)12 occupancies, Jarid2 might function in transcriptional repression and activation independently of the canonical PRC2 complex [E(z)] and vice versa. This distinction in target genes between Jarid2 and canonical PRC2 components [E(z)] provides additional confirmation that Jarid2 in some respects behaves fundamentally differently than the canonical PRC2 complex. Together with the varied functions proposed for Jarid2 in mammals, these studies highlight the diverse aspects of Jarid2 function in PRC2-mediated gene regulation (Herz, 2012).
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).
Polycomb group proteins (PcG) exert conserved epigenetic functions that convey maintenance of repressed transcriptional states, via post-translational histone modifications and high order structure formation. During S-phase, in order to preserve cell identity, in addition to DNA information, PcG-chromatin-mediated epigenetic signatures need to be duplicated requiring a tight coordination between PcG proteins and replication programs. However, the interconnection between replication timing control and PcG functions remains unknown. Using Drosophila embryonic cell lines, this study found that, while presence of specific PcG complexes and underlying transcription state are not the sole determinants of cellular replication timing, PcG-mediated higher-order structures appear to dictate the timing of replication and maintenance of the silenced state. Using published datasets it was shown that PRC1, PRC2, and PhoRC complexes differently correlate with replication timing of their targets. In the fully repressed BX-C, loss of function experiments revealed a synergistic role for PcG proteins in the maintenance of replication programs through the mediation of higher-order structures. Accordingly, replication timing analysis performed on two Drosophila cell lines differing for BX-C gene expression states, PcG distribution, and chromatin domain conformation revealed a cell-type-specific replication program that mirrors lineage-specific BX-C higher-order structures. This work suggests that PcG complexes, by regulating higher-order chromatin structure at their target sites, contribute to the definition and the maintenance of genomic structural domains where genes showing the same epigenetic state replicate at the same time (Lo Sardo, 2013).
The epigenome in its overall complexity, including covalent modifications of DNA and histones, higher-order chromatin structures and nuclear positioning, influences transcription and replication programs of the cell. It is well known that timing of DNA replication is correlated with relative transcription state, in particular transcriptionally active genes tend to replicate early and inactive genes tend to replicate late. However, in recent years, genome-wide analyses revealed several exceptions to this rule. These and other evidence suggested that the transcriptional potential of chromatin, expressed as histone modifications and transcription factors binding (rather than the process of transcription per se) is most closely related to replication timing. A recent work in Drosophila has shown that the selection and the timing of firing of replication origins are associated with distinct sets of chromatin marks and DNA binding proteins (Eaton, 2011). This reinforces previous works showing that mutation, overexpression, depletion or tethering of chromatin modifying proteins to specific loci in yeast, Drosophila and vertebrates determines changes in replication timing locally or/and at a global level. In mammals, it has been suggested that higher-order chromatin structures more than basal epigenome modifications better correlate with replication timing profiles. Although several proteins have been reported to control higher-order chromatin structure formation, their role in replicon structure and replication timing regulation remains to be elucidated. Among these, cohesins have been shown to co-localize with ORC binding sites and to influence replication origin choice and density through the regulation of specific chromatin loops. Previously, it has been reported that PcG proteins are key regulators of higher-order chromatin structures and that condensins complex components and Topoisomerase II take part in PRE and BX-C silencing function. Moreover, depletion of the mammalian PC homologue M33 determines a switch of the INK4a/ARF locus replication timing, suggesting a role for PcG proteins in the regulation of replication programs at their targets (Lo Sardo, 2013).
However, the interplay between PcG-mediated silencing, higher-order structures and control of replication timing in Drosophila has not been elucidated. This issue has been addressed on a genome-wide level; H3K27me3 enriched and PRC2 bound sequences were found to replicate later than their unbound counterparts. Surprisingly, the same is not true for PRC1 or PhoRC target sites, where the binding of PcG proteins does not significantly correlate with genome-wide replication timing distributions, highlighting a difference between PRC1, PhoRC and PRC2 complexes at a genome-wide scale. Notably, replication timing is more correlated to PRC1 binding at transcribed TSSs than at silenced TSSs (Lo Sardo, 2013).
To investigate the possible contribution of PRC1 and other PcG complexes at repressed genes, the functional interplay between PcG-dependent epigenetic signatures and maintenance of replication programs were analyzed at one of the major PcG targets: the Drosophila BX-C. After depletion of single PcG proteins in S2 cells, reactivation was found of BX-C genes and their related PREs. Interestingly, depletion of PHO protein causes only a mild effect on homeotic genes transcription, although this protein has been reported to be required for recruitment of PRC1 and 2. This suggests that multiple additional mechanisms of recruitment, such as ncRNAs or other protein partners, may act simultaneously at PcG target loci, as described particularly in mammalian cells. Interestingly, also 3C analysis in single PcG depleted cells reveals a different response to E(z) depletion with respect to PC and PHO depletions. In particular, in PC and PHO depleted cells no change or the small reduction of some PRE-PRE and PRE-promoter BX-C interactions were seen, while in E(z) depleted cells even an increase in specific crosslinking frequencies for some interactions was observed. Moreover, both 3C and replication timing analysis in single PcG depleted cells show that transcription per se cannot dramatically perturb the BX-C higher-order structures neither change the timing of replication. This result is in agreement with recent findings in mammalian cells showing that spatial chromatin organization and replication timing are not a direct consequence of transcription (Lo Sardo, 2013).
Conversely, simultaneous depletion of components of the three major PcG complexes (PhoRC, PRC2 and PRC1) determines major changes in BX-C transcription as well as in higher-order structure and an anticipation in replication timing, suggesting that PcG proteins act synergistically on three-dimensional structures and on replication program maintenance. In line with these findings, in recovered cells, BX-C topological structure and PRE replication timing are indistinguishable from controls, suggesting that the observed variations are not sufficient to determine a stable epigenetic switch. In this context the more stable contacts might hamper an irreversible disruption of the three-dimensional BX-C structure (Lo Sardo, 2013).
These findings were further confirmed by the comparison of two different cell lines: S2 and S3 that differ for their embryonic origin. Previous studies have shown that in S3 cells, active transcription of AbdB is associated with different topological conformation of the locus, where AbdB gene and its regulative PREs are topologically separated from the other repressed and clustered epigenetic elements of the locus. This study found that distinct chromatin structures in S2 and S3 are associated with different replication timings, thus confirming that these epigenetic parameters vary in parallel (Lo Sardo, 2013).
This analysis, in line with recent observations, indicates that the genome may be organized into distinct structural and functional domains in which DNA regions that stay together replicate together as a stable unit for many cell generations irrespective of single gene transcription state. It was shown that major adjustments of chromatin higher-order structure and replication program are necessary for a correct differentiation and are required for reprogramming of cell identity. The high stability of higher-order chromatin structures and replication programs can explain one of the underlying molecular basis counteracting cellular reprogramming and representing an epigenetic barrier and PcG complexes may play an important role in the maintenance of this barrier. The data show that correct levels of PcG components can fully restore silencing, higher-order structures and late replication timing at derepressed BX-C gene loci. Of course, additional functions may be involved in the maintenance of these epigenetic parameters either at the BX-C and in the rest of the genome. For example, other factors involved in the regulation of higher-order chromatin structure, including the insulator CTCF protein, condensin complex subunits and Topoisomerase II, were shown to have a role in PcG-mediated gene silencing function. Interestingly, Topoisomerase II has been shown to be required for a global resetting of replicon organization in the context of somatic cell reprogramming. Hence, a deeper understanding of the functional interplay between epigenetic mechanisms modulating the stability of higher-order chromatin structure and replication program will be crucial to unravel the molecular basis (Lo Sardo, 2013).
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).
Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28) whose function is unclear. To assess the importance of H3S28, a Drosophila H3 histone mutant was generated with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2 (containing Esc, Su(z)12, E(z) and Nurf55), suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, these data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis (Yung, 2015).
This report has established a H3S28A histone mutant in Drosophila. In theory, this mutation could have two different effects on the polycomb system. (1) It could be that PcG proteins are not evicted from H3K27me3-binding sites in the absence of H3S28ph, and thus, PcG target genes might become ectopically repressed or (2) the mutation at H3S28 or the absence of H3S28ph could compromise PcG functions, resulting in derepression of PcG target genes. No evidence was found for the first possibility, although it is formally possible that H3S28 is phosphorylated under certain developmental conditions or in response to particular stimuli to counteract polycomb silencing. Instead, the data point to an inhibition of PRC2 activity by the H3S28A mutation. This inhibition is independent of active H3K27 demethylation by dUtx. Besides, RNAi against Aurora B kinase and hence depletion of H3S28ph did not hamper polycomb silencing. On the other hand, H3S28A nucleosomes proved to be a suboptimal substrate for in vitro PRC2 HMT activity. Although a 3D structure of the human Ezh2 SET domain is available, the exact contribution of the hydroxyl group of H3S28 for H3K27 methylation is difficult to deduce from the available data. vSET, the only other protein capable of H3K27 methylation in the absence of PRC2 subunits, does not require H3S28 for catalysis, whereas it does use H3A29 to define substrate specificity. Clearly, more work will be required to determine the exact structural and biochemical role of H3S28 in PRC2 catalysis. Consistent with the in vitro HMT assays, in vivo the H3S28A mutant exhibits defects in H3K27 methylation and shows similar, though milder, Hox derepression profiles and transformation phenotypes to those observed in H3K27R mutant flies (Yung, 2015).
Interestingly, the 'KS' module is frequently found in Ezh2 substrates other than K27S28 of histone H3. These include K26S27 of human histone H1 variant H1b (H1.4), K38S39 of the nuclear orphan receptor RORα, and K180S181 of STAT3. Whether these serine residues act similarly to H3S28 to support methylation of the adjacent lysine residue remains unknown. Of note, some other Ezh2 substrates can be methylated despite the lack of a 'KS' module. These include K26 of mouse histone H1 variant H1e, K49 of STAT3, and K116 of Jarid2, where the lysine residue is followed by an alanine, glutamate, and phenylalanine, respectively. Moreover, the link between peptide sequence and enzymology of Ezh2 was shown to differ in non-histone substrates. Hence, the role of serine following the Ezh2 methylation target amino acid might not be extrapolated to all other Ezh2 substrates and should be tested individually (Yung, 2015).
Previous reports revealed discrepancies in Drosophila PcG protein localization on mitotic chromosomes depending on staining protocols and tissue types. Nonetheless, live imaging of Pc-GFP, Ph-GFP, and E(z)-GFP in early Drosophila embryos has suggested that the majority of these PcG components are dissociated from mitotic chromosomes. Because stress-induced H3S28ph evicts PcG complexes during interphase, one might expect rebinding of PcG proteins on mitotic chromosomes depleted of H3S28ph. Whereas loss of Ph from mitotic chromosomes was observed in WT background, significant Ph association was not observed in H3S28A mutant condition. The reduced levels of H3K27me3 in the H3S28A mutant could contribute to this observation. Alternatively, other mechanisms might operate to dissociate the majority of PcG proteins during mitosis (Yung, 2015).
The establishment of the histone replacement system in Drosophila has proven to be an important tool to complement functional characterization of chromatin modifiers. Whereas depletion of H3K27 methylation, either by mutation of the histone mark writer E(z) or by mutation of the histone itself in the H3K27R mutant, leads to similar loss of polycomb-dependent silencing, other histone mutations revealed different phenotypes than the loss of their corresponding histone mark writers. For example, H3K4R mutations in both H3.2 and H3.3, hence a complete loss of H3K4 methylation, did not hamper active transcription. Also, the loss of H4K20 methylation upon H4K20R mutation unexpectedly supports development and does not phenocopy cell cycle and gene silencing defects reported upon the loss of the H4K20 methylase PR-Set7. In this study, by comparing the phenotype of Aurora B knockdown and H3S28A mutation in vivo, together with in vitro HMT assay, the requirement of the unmodified H3S28 residue is specifically attributed to supporting PRC2 deposition of H3K27 methylation (Yung, 2015).
Whereas the published data suggest that H3S28 phosphorylation might be important for eviction of PcG components for derepression of PcG target genes upon stimulatory cues, the data reveal a so far unacknowledged function of the unphosphorylated state of H3S28. This study shows that serine 28 is required to enable proper methylation of H3K27 by PRC2 and thus to establish polycomb-dependent gene silencing. Serine 28 of histone H3 is universally conserved in species that display canonical PRC2-dependent silencing mechanisms. Given the fact that no major mitotic defects are found upon its mutation, it is proposed that the major role of this residue is to ensure optimal PRC2 function while facilitating the removal of polycomb proteins in response to signals that induce phosphorylation (Yung, 2015).
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