BIOLOGICAL OVERVIEW

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

The basic unit of chromatin is the nucleosome, which is made up of 146 bp of DNA wrapped around a histone octamer composed of two molecules each of the histones H2A, H2B, H3, and H4. Activation of gene expression requires the transcriptional machinery to overcome the compaction of chromatin: work in recent years has uncovered several strategies to accomplish this goal. ATP-dependent chromatin remodeling has been studied extensively as a mechanism to make the DNA accessible to the transcription apparatus. Covalent modification of the unstructured and solvent-exposed N-terminal tails of histones has also been shown to participate in processes such as activation or repression of transcription and chromosome condensation and segregation (Swaminathan, 2005 and references therein).

Replacement of canonical histones with other variants might provide an alternative mechanism for controlling transcription by inducing altered nucleosomal structures; in fact, recent work has implicated ATP-dependent chromatin remodeling complexes in the process of histone variant replacement (Krogan, 2003; Mizuguchi, 2004). The role of histone variants, and specially those of H3 and H2A, in various nuclear processes has been long appreciated. There are at least three different families of H2A variants present in a variety of organisms from yeast to mammals, and the degree of conservation among members of each family is greater than than to the canonical H2A. H2AX is thought to play a role in DNA double-strand break repair; the serine in the SQEY motif of H2AX is phosphorylated at the site of the DNA damage and serves as a signal for the recruitment of repair proteins. Macro H2A1, another H2A variant, has been shown to have a role in X-chromosome inactivation and dosage compensation in mammals, where it is found to localize to the inactive X after silencing has been established (Swaminathan, 2005).

H2A.Z, the Drosophila version of which is the subject of this overview, is a third histone H2A variant highly conserved across species and, therefore, likely to play an important role in chromatin function (van Daal, 1988; Stargell, 1993). H2A.Z is an essential protein in Drosophila and in mice (van Daal, 1992; Faast, 2001), and has been implicated in both activation and repression of transcription (Dhillon, 2000; Santisteban, 2000; Larochelle, 2003). Gene expression analyses using whole-genome microarrays show that H2A.Z (named Htz1 in yeast) is involved in both activation and silencing of transcription in Saccharomyces cerevisiae (Meneghini, 2003). Recent studies have also shown that H2A.Z is enriched in the pericentric heterochromatin (Rangasamy, 2003) during early mammalian development (Swaminathan, 2005).

Heterochromatin consists of highly compacted DNA that is present around the centromeres and telomeres and is also dispersed at certain sites along the chromosomes. Heterochromatin has been found to be essential for proper chromosome segregation and genomic stability and for maintaining dosage compensation by inactivating one of the X chromosomes in female mammals. Recent studies have helped elucidate several steps in the pathway leading to the formation of heterochromatin. The earliest step established so far requires the RNAi machinery for targeting of small RNAs to heterochromatin by the RNA-induced initiation of transcriptional gene silencing (RITS) complex. Subsequent steps require deacetylation of histone H3 Lys 9 followed by methylation of the same residue by Su(var)3-9. This modified histone then recruits HP1, which in turns recruits the Suv4-20 methyltransferase to trimethylate histone H4 at Lys 20 (Swaminathan, 2005 and references therein).

Given the large number of suppressors and enhancers of PEV identified in Drosophila, there must be additional steps involved in the establishment and maintenance of heterochromatin. Although acetylation of histone tails is generally correlated with transcription activation, and establishment of heterochromatin requires deacetylation of H3 Lys 9, acetylation of other residues in H3 or H4 might also play a role in silencing processes. Histone acetyl transferases have been found to be physically associated with Su(var)3-9, and mutations in a MYST domain acetyltransferase behave as suppressors of PEV. These results support the idea that acetylation of specific histone residues might be involved in the formation of heterochromatin. This study demonstrates that the H2A.Z variant of Drosophila, H2Av, plays a role in Pc-mediated silencing and in the establishment of centromeric heterochromatin. In addition, acetylation of H4 Lys 12 is required subsequent to H2Av replacement but before H3 Lys 9 methylation. The results highlight the complexity of the multistep process leading to heterochromatin formation in higher eukaryotes (Swaminathan, 2005).

The establishment of heterochromatin has so far been defined as a four-step process initiated by the RNAi machinery through the production of small RNAs homologous to centromeric DNA repeats that are recruited to prospective heterochromatic regions as part of the RNA-induced initiation of transcriptional gene silencing (RITS) complex. The next step described in this process thus far is the deacetylation and subsequent methylation of histone H3 Lys 9, which serves to recruit HP1. HP1 then recruits the Suv4-20 methyltransferase to trimethylate histone H4 at Lys 20. The work described here suggests that heterochromatin formation is more complex than previously thought, and it involves at least two additional steps. One step requires recruitment of H2Av or replacement of the canonical histone H2A for the H2Av variant. This requirement is highlighted by the observation that mutations in the His2Av gene act as suppressors of position effect variegation by modulating the silencing effect of heterochromatin on the adjacent white gene (Swaminathan, 2005).

The replacement of H2A for H2Av is not specific to heterochromatin, and it may also take place in silenced regions of the euchromatin, since it appears that His2Av behaves genetically as a PcG gene. PcG proteins are responsible for the maintenance of epigenetic silencing of the homeotic genes during Drosophila development. The His2Av gene can be classified as a PcG gene, since mutations in His2Av enhance the phenotype of Pc mutants, suppress the phenotype of mutations in trxG genes, and cause ectopic expression of the Ant gene. The involvement of H2Av in Pc-mediated silencing is not completely unexpected, since H2Av is critical for the establishment of pericentric heterochromatin and both processes share similar strategies. Heterochromatin-induced silencing requires methylation of H3 at Lys 9 by the Su(var)3-9 histone methyltransferase, whereas Pc-induced silencing involves the recruitment of the ESC-E(z) complex to methylate H3 at Lys 27. Although the modified residues are different, in both cases the modification serves as a tag to bind chromo domain-containing proteins, HP1 in the case of pericentric heterochromatin and Pc in euchromatic silencing. Given the parallels between the two processes, it was surprising to find that replacement of H2Av was required for subsequent H3 Lys 9 methylation in heterochromatin but not for H3 Lys 27 methylation in silenced regions of euchromatin. This later conclusion is supported by the observation that neither H3 Lys 27 methylation nor E(z) recruitment is affected by mutations in His2Av (Swaminathan, 2005).

The requirement of H2Av for Pc recruitment but not for H3 Lys 27 methylation points to a slightly different strategy in the establishment of silencing in the euchromatin compared to heterochromatin. Heterochromatic silencing appears to involve a series of events that take place in a linear pathway; each event is dependent on the previous one for proper heterochromatin assembly. In this cascade of events, replacement of H2A by H2Av appears to be an early step in the process, although the results cannot distinguish among the possibilities that H2Av is recruited before, after, or in parallel to the recruitment of the RITS complex by the RNAi machinery. Surprisingly, the observed effects of H2Av on Pc-mediated silencing point to several possible mechanisms, all slightly different from that involved in heterochromatin formation. One formal possibility that would be consistent with the results but that is considered less likely is that H2Av acts downstream of Pc; recruitment of this protein would be required for H2Av replacement, which would then in turn stabilize the binding of the Pc complex. A second possibility is that H2Av replacement is a relatively late event in the process, acting downstream of H3 Lys 27 methylation instead of being required for this modification. In this scenario, H2Av would not be required for the recruitment of the ESC–E(z) complex, but it would be required subsequently to alter chromatin structure and allow Pc recruitment. Alternatively, euchromatic Pc-mediated silencing might be accomplished by two relatively independent parallel pathways that converge at the end to ensure Pc binding to the chromatin. One pathway would alter chromatin structure by recruiting ESC–E(z) and methylating H3 at Lys 27; a second parallel but independent pathway would further alter chromatin structure by replacing H2A for H2Av. Both processes would then be required for the recruitment of the Pc-containing PRC-1 complex (Swaminathan, 2005).

The apparent association of H2Av with silenced regions might appear puzzling in view of findings in other systems. In Tetrahymena, H2A.Z is present in the transcriptionally active macronucleus, but it is not detected in the silenced micronucleus. In S. cerevisiae, the H2A.Z histone variant Htz1 is required for the expression of SWI/SNF-dependent genes such as PHO5 and GAL1; interestingly, although Htz1 is required for activation, it is present at higher levels in the promoters of these genes when they are repressed than when they are transcriptionally active (Santisteban, 2000). Nevertheless, Htz1 has also been shown to be present at silenced loci in yeast, and to be required for HMR and telomere-induced silencing (Dhillon, 2000). A detailed analysis of the role of Htz1 in gene expression in yeast using whole-genome microarrays resulted in the identification of 214 genes that are activated and 107 genes that are repressed by Htz1. Htz1-activated genes are located adjacent to heterochromatin; Htz1 is enriched in the promoters and coding regions of activated genes (Meneghini, 2003), where it appears to block the spreading of heterochromatic silencing (Swaminathan, 2005).

One possible explanation for the functional differences between the yeast and Drosophila H2A.Z homologs is that Drosophila lacks H2A.X, and, therefore, H2Av plays the roles of both histone variants. Nevertheless, the localization of H2A.Z in heterochromatin is not limited to Drosophila. Studies in mice also show an association of this histone variant with pericentric heterochromatin (Rangasamy, 2003). The apparent contradiction between the two conflicting roles for H2A.Z in various organisms could be explained if the role of this histone variant is to assemble a chromatin that is more accessible to other chromatin-remodeling complexes, and that the final result depends on the type of factors recruited to H2A.Z-containing chromatin. This possible role is consistent with the finding that the functionally essential C-terminal domain of H2A.Z (Clarkson, 1999) is exposed on the surface of H2A.Z-containing nucleosomes, and therefore it could serve to recruit other factors (Suto, 2000). Analytical ultracentrifugation experiments suggest that nucleosomes reconstituted with H2A.Z have decreased stability compared to those reconstituted with the canonical H2A histone, and therefore might allow greater accessibility of various factors to H2A.Z-containing chromatin (Abbott, 2001). The decrease in stability is also supported by the crystal structure of H2A.Z-containing nucleosomes, which suggests an altered interface between the H2A.Z-H2B dimer and the H3-H4 tetramer (Suto, 2000). Alternatively, the exposed domain of H2A.Z could mediate internucleosome interactions to give rise to higher-order compacted chromatin structures (Fan, 2002). The H2A.Z-containing chromatin fiber contains regularly spaced nucleosomes (Wallrath, 1995), an arrangement that is characteristic of pericentric heterochromatin (Swaminathan, 2005).

The results discussed here suggest a complex multistep model for heterochromatin assembly. The nature of the first step in the process might involve the RNAi machinery and the recruitment of the RITS complex, although it is also possible that H2Av recruitment takes place prior to this step. The mechanism by which H2Av recruitment is controlled is unclear. Recent studies in yeast have shown that a complex containing swr1, an Snf-2-related ATP-dependent chromatin-remodeling factor, is able to efficiently exchange H2A for H2A.Z (Krogan, 2003; Kobor, 2004; Mizuguchi, 2004). A Drosophila homolog of SWR1 is domino, which has been characterized as a PcG gene and a dominant suppressor of PEV. It is plausible that RITS recruits a Domino-containing chromatin-remodeling complex, which in turn replaces histone H2A for H2Av. This alteration would then facilitate the recruitment of histone-modifying enzymes that would further change chromatin structure. The current results suggest that the next step in the process requires the acetylation of histone H4 Lys 12, but the nature of the enzyme responsible for acetylation of H4 Lys 12 is not known. A candidate for this role could be the product of the chameau gene, which encodes a MYST domain histone acetyltransferase. Mutations in chameau behave as PcG genes and suppress PEV (Grienenberger, 2002), but they fail to affect H4 Lys 12 acetylation, suggesting that the Chameau protein affects a different step in the establishment of heterochromatin. Acetylation of H4 Lys 12 is then followed by methylation of H3 Lys 9 and recruitment of HP1, which in turn tethers Svar4-20 to methylate H4 Lys 20. Additional steps are likely to be required in this complex process. Analysis of the large collection of Su(var) and E(var) mutations identified in Drosophila should make possible the elucidation of additional steps in this pathway, resulting in a comprehensive understanding of the molecular events involved in heterochromatin formation (Swaminathan, 2005).

Nucleosome organization in the Drosophila genome

Comparative genomics of nucleosome positions provides a powerful means for understanding how the organization of chromatin and the transcription machinery co-evolve. This study produced a high-resolution reference map of H2A.Z and bulk nucleosome locations across the genome of the Drosophila and compare it to that from the yeast Saccharomyces cerevisiae. Like Saccharomyces, Drosophila nucleosomes are organized around active transcription start sites in a canonical -1, nucleosome-free region, +1 arrangement. However, Drosophila does not incorporate H2A.Z into the -1 nucleosome and does not bury its transcriptional start site in the +1 nucleosome. At thousands of genes, RNA polymerase II engages the +1 nucleosome and pauses. How the transcription initiation machinery contends with the +1 nucleosome seems to be fundamentally different across major eukaryotic lines (Mavrich, 2008).

Knowledge of the precise location of nucleosomes in a genome is essential to understand the context in which chromosomal processes such as transcription and DNA replication operate. A common theme to emerge from recent genome-wide maps of nucleosome locations is a general deficiency of nucleosomes in promoter regions and an enrichment of certain histone modifications towards the 5' end of genes. A high resolution genomic map of nucleosome locations in the budding yeast S. cerevisiae has further revealed the nucleosomal context of cis-regulatory elements and transcriptional start sites. However, such context has not been established in multicellular eukaryotes, and so fundamental questions remain: Is there a common theme by which genes of multicellular eukaryotes position their nucleosomes with respect to functional chromosomal elements? Are such themes and their underlying rules evolutionarily conserved across eukaryotes? What are the functional implications for those themes that differ across the major eukaryotic lines? To address these questions, a genome-wide high-resolution map of H2A.Z (also known as H2Av) and bulk nucleosome locations was produced in the Drosophila embryo. H2A.Z is widely distributed in Drosophila, but some evidence points to specialized roles. In Saccharomyces, H2A.Z replaces H2A at the 5' end of active genes, and thus provides a focused representation of promoter chromatin architecture (Mavrich, 2008).

Drosophila embryos are composed of a wide variety of cell types in which subsets of genes may elicit distinct gene expression programs. Global gene expression profiles during all stages of Drosophila development from 8-12 h post fertilization to a young adult fly are correlated, which possibly reflects the broad expression pattern of the large repertoire of housekeeping genes in most cell types during development. This general spatial and temporal independence of gene expression provides impetus to use whole embryos to develop a reference nucleosome map. Indeed, a map reveals that nucleosomes are generally well organized, despite cell type heterogeneity (Mavrich, 2008).

Embryos were treated with formaldehyde, and H2A.Z nucleosome core particles were immunopurified. H2A.Z-containing nucleosomes (652,738) were sequenced and mapped to 207,025 consensus locations in the Drosophila r5.2 reference genome, thereby providing >3-fold depth of coverage (see browser at http://drosophila.atlas.bx.psu.edu/). Correction for micrococcal nuclease (MNase) digestion bias was imposed. Those 112,750 nucleosomes detected three or more times were further analysed, although patterns were identical when all nucleosomes were analysed. The internal median error of the data was 4 bp (Mavrich, 2008).

H2A.Z nucleosomes were predominantly distributed at 175-bp intervals from the TSS (compared to 165-bp in Saccharomyces, demonstrating that a predominant organizational pattern exists for H2A.Z nucleosomes in Drosophila embryos that transcends a spatial and temporal context. The H2A.Z pattern was compared to the distribution of bulk nucleosomes (that is, those containing any combination of H2A.Z and H2A), determined using high-density tiling arrays (36-bp probe spacing). Within genic regions, the same organizational pattern was found. For both data sets, a nucleosome-depleted region was evident immediately upstream of the +1 nucleosome, which probably reflects a nucleosome-free core promoter region (NFR), as first detected in Saccharomcyces. Like Saccharomyces, a -1 nucleosome was detected ~180 bp upstream of the TSS. However, in contrast, it lacked H2A.Z (Mavrich, 2008).

Surprisingly, the genic array of Drosophila nucleosomes started ~75 bp further downstream from the equivalent position in Saccharomyces, placing the +1 nucleosome at +135. This shift has important implications in how the TSS is presented to RNA polymerase II (Pol II). In Saccharomyces, the TSS resides within the nucleosome border, potentially allowing the nucleosome to regulate start-site selection and efficiency. In Drosophila, the predominant arrangement of nucleosomes might allow unimpeded access to the TSS, with potential blockage occurring downstream after initiation (Mavrich, 2008).

Drosophila have well-defined core promoter elements, such as TATA, initiator (Inr), downstream promoter element (DPE) and motif ten element (MTE), which bind to the general transcription machinery, although these elements are not found in most genes. For genes lacking these core promoter elements or having a DPE, the canonical nucleosome organization was observed, which was more robust when only H2A.Z-containing nucleosomes were examined. In contrast, genes containing TATA, Inr or MTE had a diminished canonical nucleosome organization and a diminished NFR, indicating that these classes of genes may have a more compact and gene-specific chromatin architecture, including a positioned nucleosome over the TSS. Consequently, they might be more dependent on chromatin remodelling for expression. When genes become transcriptionally competent, resident nucleosomes could adopt a more open and canonical organization, which includes replacing H2A with H2A.Z. Three observations support this hypothesis. First, H2A.Z and bulk nucleosomes at highly expressed genes were more uniformly organized than those at genes with a lower expression. Second, bulk nucleosomes for genes that contained H2A.Z at their 5' end displayed the canonical pattern, whereas those lacking H2A.Z did not. Third, within any class of genes except those having an Inr, H2A.Z nucleosomes adopted a more canonical organization than the bulk set of nucleosomes. These results suggest that transcription and the presence of H2A.Z are linked to an open and uniform chromatin architecture at promoter regions (Mavrich, 2008).

Recent genome sequencing of 12 Drosophila species of differing evolutionary distance has provided an unprecedented opportunity to identify conserved DNA sequence. By comparing the distribution of motifs around the TSS, four recurring patterns were found: 27 motifs were classified as 'nucleosomal', 57 as 'anti-nucleosomal', 12 as 'fixed', and 98 as 'random'. Nucleosomal and anti-nucleosomal patterns matched the general distribution of where nucleosomes were relatively enriched or depleted, respectively, relative to the TSS. Fixed elements were at a defined distance from the TSS, and random elements lacked patterning. The nucleosomal and anti-nucleosomal patterns suggest that certain motifs are organized to be downstream of the TSS in the midst of nucleosomal arrays, whereas others are organized to be upstream of the TSS, where nucleosomes are relatively depleted (Mavrich, 2008).

The organizational relationship of these DNA motifs to individual H2A.Z nucleosomes was examined within the whole genome. Notably, nucleosomal motifs were consistently enriched on the H2A.Z nucleosome surface, whereas anti-nucleosomal motifs were consistently depleted. Individual fixed motifs were mostly depleted of H2A.Z nucleosomes. These findings along with several controls suggest that motifs and nucleosomes adopt a preferred organization around each other, regardless of their genomic location. This organization could be linked to co-evolution of base sequence composition bias in and around nucleosomes. The functional importance of such context remains to be determined (Mavrich, 2008).

Whether the positions of Drosophila H2A.Z nucleosomes are at least partly defined by the underlying DNA sequence pattern was examined, and whether such a pattern might be evolutionarily conserved was investigated. The frequency of dinucleotides across Drosophila H2A.Z nucleosomal DNA was examined because 10-bp periodic patterns of certain dinucleotides enhance the wrapping and positioning of DNA around the histone core. As seen in Saccharomyces, 10-bp periodic patterns of A and/or T (A/T) dinucleotides running counter-phase to G/C dinucleotides was observed. The modest amplitudes of the pattern suggest that such periodicities are infrequent, and are thus used selectively (that is, most nucleosomes lack underlying positioning signals) (Mavrich, 2008).

The rules of nucleosome positioning were further investigated by scanning promoter regions for correlations to nucleosome positioning sequences previously identified for a relatively small number of eukaryotic nucleosomes, in which AA/TT or CC/GG dinucleotides occur in a biased and/or periodic arrangement across nucleosomal DNA. Unlike in yeast, the AA/TT positioning pattern failed to identify nucleosome locations. However, the CC/GG pattern reproduced the exact position of the +1 nucleosomes, indicating that the Drosophila +1 nucleosome may be positioned in part by CC/GG-based positioning sequences that are used preferentially in metazoans. Consistent with this, +1 nucleosomes are highly positioned around the 5' end of genes (Mavrich, 2008).

Despite H2A.Z being enriched at the 5' end of genes, substantial levels were detected throughout the genome, which allowed examination of nucleosome organization at the 3' end of genes. Notably, H2A.Z nucleosome levels spiked near the open reading frame (ORF) end points and then dropped precipitously further downstream into the intergenic regions, where transcripts terminate. The spike occurred ~30 bp upstream from the stop codon and ~160 bp upstream of the transcript poly(A) site. A similar nucleosome drop-off was seen when bulk nucleosomes were examined, but was not evident at genes that lacked H2A.Z. Thus, like the 5' end, the presence of H2A.Z may be linked to a more open chromatin architecture at the 3' end of genes. The change in nucleosome density coincided with alterations in nucleosome positioning sequences. Thus, such '3' NFRs' might be defined in part by the underlying DNA sequence. Conceivably, 3' NFRs might function in transcription termination (Mavrich, 2008).

The location of the +1 nucleosome at the 5' end of genes is notable because its upstream border resides at approximately +62 (relative to the TSS), which is near where Pol II pauses during the transcription cycle. To examine the potential linkage between Pol II pausing and nucleosome positions, the genome-wide location of Pol II was determined in embryos at 1,956 putatively paused genes. Pol II was concentrated in a ~300 bp region that peaked around +90, which overlaps the region bound by the +1 nucleosome; this is consistent with other recent placements. Indeed, the distribution of paused Pol II, as directly measured by permanganate reactivity of thymines on a statistically robust subset of ~50 genes, indicates that pausing occurs between +20 and +50, with the centre at +35. This high-resolution permanganate footprinting data, which represents the most definitive means of assessing Pol II pausing, places the front edge of Pol II (~16 bp downstream of the bubble) within ~10 bp of the +1 nucleosome border (Mavrich, 2008).

The location of the +1 H2A.Z nucleosome was similar (but not identical) whether or not paused Pol II was present, indicating that Pol II was not likely to be the cause of the nucleosome shift compared to Saccharomyces. Instead, the positioned +1 nucleosome might be contributing to Pol II pausing, which is consistent with other studies. Other factors including negative elongation factor (NELF) are likely to make significant contributions to pausing as well (Mavrich, 2008).

Intriguingly, genes that contained a paused Pol II showed a ~10 bp downstream shift of H2A.Z nucleosomes. The same shift was observed if H2A.Z sequencing reads (rather than nucleosomes) or bulk nucleosomes are plotted. The shift suggests that, as part of the pausing process, Pol II collides with the +1 nucleosome, possibly displacing it downstream by one turn of the DNA helix. If the downstream nucleosomes are positioned mainly by the principles of statistical positioning, rather than the underlying DNA sequence, then a shift of the +1 nucleosome is expected to have a ripple effect on downstream nucleosomes (Mavrich, 2008).

To test the prediction that Pol II is engaging the +1 nucleosome, bulk mononucleosomes were prepared from formaldehyde-crosslinked embryos and immunoprecipitated with antibodies directed against Pol II. DNA corresponding to mononucleosomes (~150 bp) was gel-purified and mapped to the entire Drosophila genome with high-resolution tiling arrays. The distribution of nucleosome-Pol II crosslinking at Pol II-paused genes peaked at the +1 nucleosome. This was not seen at genes lacking a paused Pol II or H2A.Z. The selective enrichment at +1 demonstrates that Pol II is predominantly engaged with the +1 nucleosome, and therefore the +1 nucleosome may be instrumental in establishing the paused state (Mavrich, 2008).

The high resolution map of Drosophila nucleosomes reveals evolutionarily conserved and divergent principles of nucleosome organization. Genes that possess H2A.Z nucleosomes are likely to have experienced a transcription event. They tend to have nucleosome-free promoter and termination regions and intervening arrays of uniformly positioned nucleosomes that become less uniform towards the 3' end of the gene. H2A.Z nucleosomes in general might not block assembly of the transcription machinery at transcriptionally 'experienced' promoters. However, repressed promoters or those containing Inr elements do seem to have an H2A nucleosome over the TSS (Mavrich, 2008).

Conserved DNA sequence motifs (and thus any proteins that bind to them) tend to have an organizational relationship with nucleosomes. 'Anti-nucleosomal' motifs including those for proteins such as Engrailed, Even skipped, Fushi tarazu, Giant, Hunchback and Knirps tend to be located upstream of the TSS and might contribute to the exclusion of nucleosomes over the core promoter. Indeed some have anti-nucleosomal activity. 'Nucleosomal' motifs include sites for achaete, antennapedia, dorsal, tramtrack and others. Their preference for locations downstream of the TSS where nucleosomes are well organized raises the possibility that they contribute to nucleosome organization (Mavrich, 2008).

In Saccharomyces, the location of the TSS just inside the +1 nucleosome border allows the nucleosome potentially to exert control over initiation; however, in Drosophila, most genes might position the +1 nucleosome to interact with a transcriptionally engaged paused polymerase. It is not known whether the +1 nucleosome is causative or just participatory in the pausing. It is now becoming clear that metazoans also regulate transcription through Pol II pausing rather than solely through transcription complex assembly. The nucleosome map and its context to DNA regulatory elements, presented here, provides a framework for designing experiments and analysing existing data to understand how metazoans regulate transcription (Mavrich, 2008).

The role of variant histone H2AV in D. melanogaster larval hematopoiesis

Replication-independent histone variants can replace the canonical replication-dependent histones. Vertebrates have multiple H2A variant histones, including H2AZ and H2AX that are present in most eukaryotes. H2AZ regulates transcriptional activation as well as maintenance of gene silencing, while H2AX is important in DNA damage repair. The fruit fly Drosophila melanogaster has only one histone H2A variant (H2AV), which is a chimera of H2AZ and H2AX. This study found that lack of H2AV led to the formation of black melanotic masses in the third instar larvae of Drosophila. The formation of these masses was found in conjunction with a loss of a majority of the primary lymph gland lobes. Interestingly, the cells of the posterior signaling center were preserved in these mutants. Reduction of H2AV levels by RNAi knockdown caused a milder phenotype that preserved the lymph gland structure, but that included precocious differentiation of the prohemocytes located within the medullary zone and secondary lobes of the lymph gland. Mutant rescue experiments suggest that the H2AZ-like rather than the H2AX-like function of H2AV is primarily required for normal hematopoiesis (Grigorian, 2017).

Absence of the variant histone protein H2AV results in the formation of larval melanotic masses containing plasmatocytes and crystal cells. Previous studies have proposed that the formation of melanotic masses can be due either to the response of a normal immune system to abnormal tissue formed during development, or to a developmental defect in the hemocytes of the lymph gland. The current data showing the loss of a majority of the primary lymph gland lobes in the His2Av⁸¹⁰ null mutant, as well as the early differentiation of the medullary zone and secondary lobe prohemocytes when H2AV levels were reduced via RNAi, are consistent with the latter model. The results demonstrate an important role for H2AV during normal hemocyte differentiation and dispersal. Interestingly, studies using a human histiocytic lymphoma cell line or normal macrophages differentiated with macrophage colony stimulating factor (M-CSF; CSF1) have shown an upregulation of the His2Av-related human H2A.Z (H2AFZ) gene during macrophage differentiation. These results imply an evolutionarily conserved role for the closely related H2AV and H2AZ histone variants in blood cell differentiation (Grigorian, 2017).

The presence of black melanotic masses in Drosophila larvae is not restricted to His2Av mutants. This phenotype has previously been observed in mutants of two different ATP-dependent chromatin-remodeling complexes. Dom, which is a catalytic subunit of the dTip60 complex, plays a role in H2A variant exchange in nucleosomes, as well as in DNA damage repair. dom loss-of-function mutants display black melanotic masses that are composed of melanized lymph glands. Mutants have shown that the vertebrate homolog of dom is required for both embryonic and adult hematopoiesis in the laboratory mouse. Loss of a subunit of another ATP-dependent chromatin-remodeling complex, NURF, also causes melanotic masses. In addition, melanotic masses have been observed in mutations that affect various signaling pathways. For example, constitutive activation of the JAK-STAT pathway via the dominant gain-of-function Hop^TUM mutation results in the formation of melanotic masses. Constitutive activation of the Toll pathway via the dominant gain-of-function Tl^10b mutation also causes melanotic masses. These observations raise the question of whether the closely related variant histones H2AV and H2AZ might be required to repress these evolutionarily conserved signaling pathways in hematopoietic cells (Grigorian, 2017).

Although the majority of the cells in the primary lymph gland lobes in His2Av mutants are lost, the Antp-positive cells comprising the PSC are spared and can be seen adjacent to the cardioblasts of the dorsal vessel. In addition, these cells express the Hh ligand that normally prevents premature differentiation of hemocyte precursors. The presence of Antp-positive cells can also be observed in posterior lymph gland lobes, where Antp is not normally expressed. In this regard, previous studies have shown that His2Av can function as a Polycomb Group (PcG) gene, and PcG proteins are known to be important for repressing the transcription of homeotic genes such as Antp. In particular, it has been reported that Antp expression is expanded in the central nervous system of larvae that are mutant for His2Av. Reduction of H2AV levels via RNAi in the prohemocytes of the primary lobes, as well as in the secondary lobes, led to increased differentiation of plasmatocytes and crystal cells. This suggests that H2AV also acts downstream of the signals that originate from the PSC and that maintain the prohemocytes of the medullary zone in an undifferentiated state (Grigorian, 2017).

Reduction of H2AV levels via RNAi causes a less severe phenotype than that of His2Av⁸¹⁰ null mutants, in that the primary lobes of the lymph gland are preserved. However, there is a loss of the undifferentiated prohemocytes found within the medullary zone, as these cells differentiate into mature hemocytes. Previous studies in the testis of Drosophila have shown an important role for H2AV in the maintenance of both the germline and cyst stem cells. Together, these results suggest a possible role for H2AV in the transcriptional control of genes important for stem cell maintenance in general. In this regard, the closely related H2AZ protein of mammals has been reported to be important for the differentiation of embryonic stem cells in culturen (Grigorian, 2017).

H2AV might be exerting its effects on the lymph gland through various signaling pathways that have been shown to orchestrate prohemocyte differentiation. Two pathways that might be affected are the Hh and Wg signaling pathways. Hh has been implicated in maintaining prohemocytes in an undifferentiated state. However, this study observed Hh expression in the PSC of both heterozygous and homozygous mutant His2Av⁸¹⁰ larval lymph glands. Wg has been reported to not only maintain the prohemocyte population in an undifferentiated state, but also to dictate PSC cell number. No significant differences were detected in the staining of prohemocytes and PSC cells with anti-Wg antibodies in homozygous versus heterozygous mutant His2Av⁸¹⁰ larval lymph glands. These results suggest that loss of H2AV might alter the intracellular responses to these ligands rather than their expression (Grigorian, 2017).

Drosophila H2AV is a chimeric protein that plays the roles of two widely conserved variant histones, H2AX and H2AZ. H2AX is important for the DNA damage repair response, while H2AZ is important for both transcriptional activation and gene silencing. Previous studies have shown that H2AV^CT, which lacks H2AX function, is able to rescue the lethal phenotype seen in His2Av⁸¹⁰ null mutants, allowing the organisms to progress to pupation and adulthood. This study found that H2AV^CT was able to partially rescue the His2Av⁸¹⁰ null larval hematopoietic phenotype, arguing that an H2AZ-like function rather than an H2AX-like function of H2AV is required for hematopoiesis. Nevertheless, differentiation within the lymph gland still appeared disrupted and partial loss of the primary lymph gland lobes could be seen. In addition, the expression of Antp was at times seen to expand into the posterior lobes of the lymph gland. This lack of full rescue could be due to a decreased stability of the H2AV^CT protein. However, the presence of H2AV^CT in an otherwise His2Av wild-type background was sufficient to cause abnormalities of the lymph gland lobes. Furthermore, overexpression of wild-type or of phosphorylation mutants of H2AV also caused hematopoietic abnormalities. Together, these results imply that a precise dosage of H2AV protein is essential for normal hematopoiesis in Drosophila. Similar alterations in differentiation might also occur in other organs and tissues. In this regard, care should be taken when using His2Av-GFP and His2Av-RFP transgenes, which are popular markers in live imaging (Grigorian, 2017).

The formation of black melanotic masses in the His2Av⁸¹⁰ null mutant establishes larval hemocytes as a useful tool for further studies of H2AV function. Furthermore, given the role that H2AV plays not only in undifferentiated prohemocytes, but also in the germline and cyst stem cells found in the testis, it will be interesting to test whether H2AV also regulates stem cells found in other tissues (Grigorian, 2017).

Sequestration to lipid droplets promotes histone availability by preventing turnover of excess histones

Because both dearth and overabundance of histones result in cellular defects, histone synthesis and demand are typically tightly coupled. In Drosophila embryos, histones H2B/H2A/H2Av accumulate on lipid droplets (LDs), cytoplasmic fat storage organelles. Without LD-binding, maternally provided H2B/H2A/H2Av are absent, but how LDs ensure histone storage is unclear. Using quantitative imaging, this study uncover when during oogenesis these histones accumulate, and which step of accumulation is LD-dependent. LDs originate in nurse cells (NCs) and are transported to the oocyte. Although H2Av accumulates on LDs in NCs, the majority of the final H2Av pool is synthesized in oocytes. LDs promote intercellular transport of the histone-anchor Jabba and thus its presence in the ooplasm. Ooplasmic Jabba then prevents H2Av degradation, safeguarding the H2Av stockpile. These findings provide insight into the mechanism for establishing histone stores during Drosophila oogenesis and shed light on the function of LDs as protein-sequestration sites (Stephenson, 2021).

Early animal embryogenesis often exhibits rapid cell cycles dominated by DNA replication, mitosis and little to no transcription. Drosophila is a dramatic case where the first 13 nuclear divisions occur every 8-20 min. This speed poses a challenge for histone biology: demand increases exponentially, yet major regulatory mechanisms that control histone expression are unavailable. To meet this demand, many embryos inherit maternally synthesized histones. This study examined the origin of H2Av, H2B and H2A stockpiles in Drosophila (Stephenson, 2021).

Expression of histone messages is increased during S10, but it was unclear when the complementary maternally deposited histone proteins accumulate. A methodological challenge is that enterocytes (ECs) have polyploid nuclei. Each of the nurse cell (NC) nuclei are estimated to contain about 500 times more DNA than diploid nuclei; the 900 follicle cells contain DNA levels about eight times higher than a diploid nucleus. Thus, the histones needed to package NC and follicle cell chromatin are at least tenfold more abundant than the histone stockpile of newly laid embryos, which are estimated to be the equivalent of 1000 diploid nuclei. Detecting accumulation of the oocyte histone stockpile in addition to other histones in the EC is therefore challenging (Stephenson, 2021).

This study followed H2Av accumulation using an imaging approach that specifically quantifies histone signal in the NC and oocyte cytoplasm. H2Av already accumulates in the cytoplasm of S9 NCs and is associated with lipid droplets (LDs). An intriguing possibility is that this LD-associated H2Av pool in the early-stage NCs might support NC endoreplication. This H2Av-LD association likely brings some H2Av into the oocytes, but quantitation indicates that transfer from NCs contributes at most one fifth of the final H2Av pool in mature oocytes. These data are consistent with the possibility that the majority of the ooplasmic H2Av is synthesized in the oocyte; reduced H2Av levels in Jabba^−/− S12 oocytes may represent early loss by degradation rather than defective transfer from NCs (Stephenson, 2021).

After dumping, total H2Av levels continue to rise from S12 through S14. It is proposed that two mechanisms contribute to this rise. First, the translational efficiency of H2Av mRNA is upregulated from S12 to S14. Second, Jabba increases from S12 to S14. As more Jabba protein becomes available, it can presumably recruit more H2Av to LDs and protect it from degradation (Stephenson, 2021).

H2A and H2B levels in embryos are Jabba dependent (Li, 2012), and the pattern of H2B accumulation during oogenesis resembles that of H2Av. Currently, good tools to determine H2A accumulation are lacking, but it is predicted to will follow the same pattern, as canonical histones are typically similarly regulated. It is proposed that increasing H2Av, H2B and H2A accumulation during late oogenesis establishes an LD-bound histone depot for the early embryo (Stephenson, 2021).

It will be interesting to determine whether H3 and H4 accumulation follows a similar pattern. As canonical histones, their transcriptional and translational regulation is likely similar to that of H2A and H2B. However, they are not LD associated nor are their embryonic levels Jabba dependent, so there must be mechanistic differences (Stephenson, 2021).

Newly laid Jabba^−/− eggs have lower H2A, H2B and H2Av levels than wild type. This divergence is established during late oogenesis; H2Av and H2B levels rise in the wild type but drop in Jabba^−/−. Experiments to inhibit proteasome activity reveal that histone degradation plays a major role in bringing about this difference. As high MG132 (proteasome inhibitor) concentrations arrest development, it is infered that under conditions that allow EC development to S14, degradation is only partially inhibited. Yet even with partial inhibition, almost half of the H2Av normally lost in Jabba^−/− is retained. It is concluded that Jabba prevents histone turnover and that a major contributor to turnover is a proteasome-dependent pathway. The remaining turnover not prevented by drug treatment may be due to a proteasome-independent mechanism (Stephenson, 2021).

In the wild type, proteasome inhibition did not increase the H2Av pool beyond levels in untreated ECs. This observation was surprising as 4x Jabba females can accumulate more H2Av than wild type, which presumably indicates that even the wild type produces excess H2Av that is degraded if not protected by Jabba. It was reasoned that the direct effects on proteasome turnover are balanced out by indirect effects that compromise histone or Jabba synthesis. Indeed, high concentrations of the inhibitor abolish any rise in H2Av. Turnover of excess histones by the proteasome is well established in yeast. It is one of numerous mechanisms that prevent accumulation of free histones and the resulting cytotoxicity. It is proposed that H2Av turnover in late oogenesis is due to this general protective machinery; Jabba allows oocytes to deploy this safety feature while also accumulating H2Av needed to provision the embryo. Accordingly, Jabba determines the H2Av pool that is protected and any excess is recognized as a potential hazard. Because the mechanism that targets excess H2Av for proteasomal degradation is not known, it cannot yet be tested what type of damage this mechanism guards against (Stephenson, 2021).

The data suggest that Jabba prevents degradation by physically protecting H2Av. First, H2Av levels scale with Jabba dose, and H2Av levels beyond normal can be achieved by simply doubling Jabba levels. Second, a version of Jabba that is mislocalized to the NC nuclei and not present in oocytes is unable to support high H2Av levels. Finally, a Jabba mutant unable to bind to H2Av resulted in H2Av levels indistinguishable from those in Jabba^−/− (Stephenson, 2021).

To further unravel how Jabba prevents degradation, it will be necessary to identify the machinery that targets excess H2Av to the proteasome. Previous work has identified E3 ligases promoting H3 and H4 turnover, but those for other histones remain uncharacterized. Jabba may physically protect H2Av by shielding a ubiquitylation site. This remains to be tested. An alternate hypothesis is that histone degradation in oocytes occurs independently of ubiquitylation. Intriguingly, during development of the mammalian male germline, histone turnover occurs via ubiquitin-independent proteasome-dependent degradation (Stephenson, 2021).

In embryos, H2Av exchanges between LDs. If similar exchange occurs in oocytes, it is unclear how transient interactions with Jabba are sufficient to prevent H2Av degradation. It is speculated that either the transit time is negligible relative to the time H2Av spends interacting with Jabba or that cytosolic H2Av is accompanied by a chaperone. LD binding promotes availability of Jabba and, consequently, of histones Our analysis provides a first answer to why some histones are stored on LDs, while others are apparently stored in the cytoplasm. Jabba, the protein necessary to stabilize the H2A, H2B and H2Av ooplasmic pool, becomes trapped in NC nuclei if it is not LD bound. Thus, it is absent from the oocyte and cannot perform its protective function. It is proposed that LD binding ensures proper intercellular transport of Jabba, safeguarding its ability to function in the ooplasm (Stephenson, 2021).

H3 and H4 ooplasmic stores are presumably bound to a partner that prevents their degradation, perhaps the histone chaperone NASP. As these histones are apparently cytoplasmic, it is unclear how they and their binding partners avoid mislocalization to NC nuclei (Stephenson, 2021).

It is also unclear why histones are stored on LDs rather than another cytoplasmic structure. A priori, any cytoplasmic organelle that is transferred to oocytes could suppress the NC nuclear import of the histone anchor and promote transport into the oocyte. The large surface area of LDs may provide a readily available and, at these developmental stages, metabolically inert platform for recruitment. Other organelles may not have enough storage capacity or might be functionally impaired by histones on their surface. Alternatively, LD association may be an evolutionary accident and many other organelles might in principle be able to store histones. Identification of a Jabba region sufficient for histone binding will make it possible to address this question in the future (Stephenson, 2021).

But why is JabbaHBR mislocalized to NC nuclei? It is suspected that the histone-binding ability of JabbaHBR leads to its mislocalization, dragged along by histone nuclear import or retained in nuclei via chromatin binding. As histone binding is important to protect against degradation, mislocalization cannot be avoided unless JabbaHBR is anchored outside the nucleus. This idea could not be tested, as JabbaHBR that is unable to interact with histones still accumulates in nuclei, in cultured cells and in NCs. It is hypothesized that a cryptic nuclear localization signal in JabbaHBR promotes nuclear transport even without histone binding (Stephenson, 2021).

This analysis may shed light on how LDs regulate other proteins. LDs can transiently accumulate proteins from other cellular compartments. This has been particularly documented for proteins involved in nuclei acid binding and/or transcriptional regulation: MLX and Perilipin 5 can either be present on LDs or move into the nucleus to regulate transcription. The bacterial transcriptional regulator MLDSR is sequestered to LDs under stress conditions. As for Jabba and H2Av, LD association of such 'refugee proteins' may prevent their premature turnover or may promote their delivery to the correct intra- or intercellular location. A role for LDs in protein delivery to distant cellular compartments might be important in neurons where recent discoveries suggest important, but largely uncharacterized, roles for LDs (Stephenson, 2021).

GENE STRUCTURE

cDNA clone length - 913

Bases in 5' UTR - 149

Exons - 4

Bases in 3' UTR - 338

PROTEIN STRUCTURE

Amino Acids - 141

Structural Domains

The Tetrahymena histone H2A variant designated hv1 is localized exclusively in the transcriptionally active macronucleus and is absent from the quiescent micronucleus. A cDNA clone of the hv1 gene was used to screen a Drosophila cDNA library. A cross-hybridizing clone was recovered and shown by sequence analysis to code for a protein homologous to hv1 as well as to the chicken H2A variant, H2A.F, the sea urchin H2A variant, H2A.F/Z and the mammalian H2A variant H2A.Z. Southern analysis of Drosophila genomic DNA indicates that the H2AvD (H2A variant Drosophila) gene is present in one copy. In situ hybridization places the locus at 97CD on chromosome 3, while the S-phase regulated histone genes are on chromosome 2. Thus the Drosophila H2A variant should be accessible to genetic analysis, which will enable its function to be determined (van Daal, 1988).

The H2AvD sequence contains the nonapeptide, which is conserved in all H2A's, at position 23-31, indicating that this cDNA encodes an H2A-like protein. However, it is clear that the sequence differs markedly from that of the H2A encoded in the Drosophila histone gene cluster. There are 52 amino acid changes in the first 124 amino acids, which indicates a similarity of only 59%. This is approximately the same degree of similarity shown by the H2A variants of chicken (60%), sea urchin (56%-57%), mammals (56%) and Tetrahymena (62%-65%) relative to their respective major S-phase regulated H2A's. The cDNA sequence predicts that H2AvD is a larger molecular weight protein than H2A.1. An antibody to the carboxy terminal portion of H2AvD detects a protein of lower mobility than the major histone H2A on electrophoresis in SDS polyacrylamide gels. An H2A variant (D2) has been previously reported in Drosophila. D2 has a molecular weight of 13,400 daltons and differs from H2A.1 in that it contains no methionine residues and has an increased histidine content. H2AvD shares these properties (van Daal, 1988).

The amino and carboxy terminal ends of the Drosophila and Tetrahymena H2A variants have diverged. However, there is a remarkably high similiarity when comparing the body of the protein in both variants. There are only 16 amino acid changes in the region of amino acids 18-120, which gives a similarity of 84%. The carboxy terminal end of hvl (from amino acid 121) is completely different from that of H2AvD in both amino acid composition and length. The N-terminus shows some sequence similarity (less than 50%), but again the length is different. It should be noted that the major Tetrahymena histone H2A differs significantly from the major H2A's of the other species. The H2A.l's of human, cow, rat and chicken are 95%-99% similar to each other. The Drosophila and sea urchin H2A.l's are 81%-87% similar to those of mammals, chicken and each other. However, the Tetrahymena H2A.1 is only 63-69% similar to those of other species, and so it is perhaps not surprising that H2AvD is quite different from hvl, whereas, it is very similar (97%) to the sea urchin variant (van Daal, 1988).

A comparison of the deduced protein sequences of the H2A variants of chicken (H2A.F), sea urchin (H2A.F/Z) and mammals (H2A.Z) with that of Drosophila (H2AvD) shows clearly that the amino acid sequence of H2AvD is more closely related to the chicken, sea urchin and mammalian H2A variants than it is to the major Drosophila H2A, H2A.l. The conservation in the first 122 amino acids is extremely high. There is 99% similarity between the Drosophila and the sea urchin proteins, with only one amino acid change (ser to asn at position 38). There is 98% similarity between H2AvD and the chicken and mammalian proteins. Both have two amino acid changes (ala to gln at position 21 and ser to thr at position 38 in chicken and ala to thr at position 14 and ala to glu at position 21 in mammals). The carboxyl ends of these four variants all differ. However, the Drosophila variant is the most divergent. The C terminal tail is longer than the others and its sequence is also dissimilar. Southern blot analysis of Drosophila genomic DNA indicates that the H2AvD gene is unique (van Daal, 1988).

date revised: 23 June 2024

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