Histone H3: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Histone H3

Synonyms - H3

Cytological map position -

Function - core histone

Keywords - chromatin

Symbol - His3

FlyBase ID: FBgn0001199

Genetic map position -

Classification - histone-fold/TFIID-TAF/NF-Y domain

Cellular location - nuclear



NCBI links: UniGene | Entrez Gene
Recent literature
Laprell, F., Finkl, K. and Muller, J. (2017). Propagation of Polycomb-repressed chromatin requires sequence-specific recruitment to DNA. Science [Epub ahead of print]. PubMed ID: 28302792
Summary:
Epigenetic inheritance models posit that during Polycomb repression, Polycomb Repressive Complex 2 (PRC2) propagates histone H3K27 tri-methylation (H3K27me3) independently of DNA sequence. This study shows that insertion of Polycomb Response Element (PRE) DNA into the Drosophila genome creates extended domains of H3K27me3-modified nucleosomes in the flanking chromatin and causes repression of a linked reporter gene. After excision of PRE DNA, H3K27me3 nucleosomes become diluted with each round of DNA replication and reporter gene repression is lost, whereas in replication-stalled cells, H3K27me3 levels stay high and repression persists. Hence, H3K27me3-marked nucleosomes provide a memory of repression that is transmitted in a sequence-independent manner to daughter strand DNA during replication. In contrast, propagation of H3K27 tri-methylation to newly incorporated nucleosomes requires sequence-specific targeting of PRC2 to PRE DNA.
Meers, M. P., Henriques, T., Lavender, C. A., McKay, D. J., Strahl, B. D., Duronio, R. J., Adelman, K. and Matera, A. G. (2017). Histone gene replacement reveals a post-transcriptional role for H3K36 in maintaining metazoan transcriptome fidelity. Elife 6. PubMed ID: 28346137
Summary:
Histone H3 lysine 36 methylation (H3K36me) is thought to participate in a host of co-transcriptional regulatory events. To study the function of this residue independent from the enzymes that modify it, a 'histone replacement' system was used in Drosophila to generate a non-modifiable H3K36 lysine-to-arginine (H3K36R) mutant. Global dysregulation of mRNA levels was observed in H3K36R animals that correlates with the incidence of H3K36me3. Similar to previous studies, it was found that mutation of H3K36 also resulted in H4 hyperacetylation. However, neither cryptic transcription initiation, nor alternative pre-mRNA splicing, contributed to the observed changes in expression, in contrast with previously reported roles for H3K36me. Interestingly, knockdown of the RNA surveillance nuclease, Xrn1, and members of the CCR4-Not deadenylase complex, restored mRNA levels for a class of downregulated, H3K36me3-rich genes. A post-transcriptional role is proposed for modification of replication-dependent H3K36 in the control of metazoan gene expression.
Colmenares, S. U., Swenson, J. M., Langley, S. A., Kennedy, C., Costes, S. V. and Karpen, G. H. (2017). Drosophila histone demethylase KDM4A has enzymatic and non-enzymatic roles in controlling heterochromatin integrity. Dev Cell 42(2): 156-169.e155. PubMed ID: 28743002
Summary:
Eukaryotic genomes are broadly divided between gene-rich euchromatin and the highly repetitive heterochromatin domain, which is enriched for proteins critical for genome stability and transcriptional silencing. This study shows that Drosophila KDM4A (dKDM4A), previously characterized as a euchromatic histone H3 K36 demethylase and transcriptional regulator, predominantly localizes to heterochromatin and regulates heterochromatin position-effect variegation (PEV), organization of repetitive DNAs, and DNA repair. dKDM4A demethylase activity is dispensable for PEV. In contrast, dKDM4A enzymatic activity is required to relocate heterochromatic double-strand breaks outside the domain, as well as for organismal survival when DNA repair is compromised. Finally, DNA damage triggers dKDM4A-dependent changes in the levels of H3K56me3, suggesting that dKDM4A demethylates this heterochromatic mark to facilitate repair. It is concluded that dKDM4A, in addition to its previously characterized role in euchromatin, utilizes both enzymatic and structural mechanisms to regulate heterochromatin organization and functions.
Zenk, F., Loeser, E., Schiavo, R., Kilpert, F., Bogdanovic, O. and Iovino, N. (2017). Germ line-inherited H3K27me3 restricts enhancer function during maternal-to-zygotic transition. Science 357(6347): 212-216. PubMed ID: 28706074
Summary:
Gametes carry parental genetic material to the next generation. Stress-induced epigenetic changes in the germ line can be inherited and can have a profound impact on offspring development. However, the molecular mechanisms and consequences of transgenerational epigenetic inheritance are poorly understood. This study found that Drosophila oocytes transmit the repressive histone mark H3K27me3 to their offspring. Maternal contribution of the histone methyltransferase Enhancer of zeste, the enzymatic component of Polycomb repressive complex 2, is required for active propagation of H3K27me3 during early embryogenesis. H3K27me3 in the early embryo prevents aberrant accumulation of the active histone mark H3K27ac at regulatory regions and precocious activation of lineage-specific genes at zygotic genome activation. Disruption of the germ line-inherited Polycomb epigenetic memory causes embryonic lethality that cannot be rescued by late zygotic reestablishment of H3K27me3. Thus, maternally inherited H3K27me3, propagated in the early embryo, regulates the activation of enhancers and lineage-specific genes during development.
Colmenares, S. U., Swenson, J. M., Langley, S. A., Kennedy, C., Costes, S. V. and Karpen, G. H. (2017). Drosophila histone demethylase KDM4A has enzymatic and non-enzymatic roles in controlling heterochromatin integrity. Dev Cell 42(2): 156-169.e155. PubMed ID: 28743002
Summary:
Eukaryotic genomes are broadly divided between gene-rich euchromatin and the highly repetitive heterochromatin domain, which is enriched for proteins critical for genome stability and transcriptional silencing. This study shows that Drosophila KDM4A (dKDM4A), previously characterized as a euchromatic histone H3 K36 demethylase and transcriptional regulator, predominantly localizes to heterochromatin and regulates heterochromatin position-effect variegation (PEV), organization of repetitive DNAs, and DNA repair. dKDM4A demethylase activity is dispensable for PEV. In contrast, dKDM4A enzymatic activity is required to relocate heterochromatic double-strand breaks outside the domain, as well as for organismal survival when DNA repair is compromised. Finally, DNA damage triggers dKDM4A-dependent changes in the levels of H3K56me3, suggesting that dKDM4A demethylates this heterochromatic mark to facilitate repair. It is concluded that dKDM4A, in addition to its previously characterized role in euchromatin, utilizes both enzymatic and structural mechanisms to regulate heterochromatin organization and functions.
Morgan, M. A. J., Rickels, R. A., Collings, C. K., He, X., Cao, K., Herz, H. M., Cozzolino, K. A., Abshiru, N. A., Marshall, S. A., Rendleman, E. J., Sze, C. C., Piunti, A., Kelleher, N. L., Savas, J. N. and Shilatifard, A. (2017). A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation. Genes Dev 31(19): 2003-2014. PubMed ID: 29089422
Summary:
Histone H3 Lys4 (H3K4) methylation is a chromatin feature enriched at gene cis-regulatory sequences such as promoters and enhancers. This study identified an evolutionarily conserved factor, BRWD2/PHIP, which colocalizes with histone H3K4 methylation genome-wide in human cells, mouse embryonic stem cells, and Drosophila. Biochemical analysis of BRWD2 demonstrated an association with the Cullin-4-RING ubiquitin E3 ligase-4 (CRL4) complex, nucleosomes, and chromatin remodelers. BRWD2/PHIP binds directly to H3K4 methylation through a previously unidentified chromatin-binding module related to Royal Family Tudor domains, which has been named the CryptoTudor domain. Using CRISPR-Cas9 genetic knockouts, it was demonstrated that COMPASS H3K4 methyltransferase family members differentially regulate BRWD2/PHIP chromatin occupancy. Finally, it was demonstrated that depletion of the single Drosophila homolog dBRWD3 results in altered gene expression and aberrant patterns of histone H3 Lys27 acetylation at enhancers and promoters, suggesting a cross-talk between these chromatin modifications and transcription through the BRWD protein family.
Joos, J. P., Saadatmand, A. R., Schnabel, C., Viktorinova, I., Brand, T., Kramer, M., Nattel, S., Dobrev, D., Tomancak, P., Backs, J., Kleinbongard, P., Heusch, G., Lorenz, K., Koch, E., Weber, S. and El-Armouche, A. (2018). Ectopic expression of S28A-mutated Histone H3 modulates longevity, stress resistance and cardiac function in Drosophila. Sci Rep 8(1): 2940. PubMed ID: 29440697
Summary:
Histone H3 serine 28 (H3S28) phosphorylation and de-repression of polycomb repressive complex (PRC)-mediated gene regulation is linked to stress conditions in mitotic and post-mitotic cells. To better understand the role of H3S28 phosphorylation in vivo, a Drosophila strain was studied with ectopic expression of constitutively-activated H3S28A, which prevents PRC2 binding at H3S28, thus mimicking H3S28 phosphorylation. H3S28A mutants showed prolonged life span and improved resistance against starvation and paraquat-induced oxidative stress. Morphological and functional analysis of heart tubes revealed smaller luminal areas and thicker walls accompanied by moderately improved cardiac function after acute stress induction. Whole-exome deep gene-sequencing from isolated heart tubes revealed phenotype-corresponding changes in longevity-promoting and myotropic genes. Changes were also found in genes controlling mitochondrial biogenesis and respiration. Analysis of mitochondrial respiration from whole flies revealed improved efficacy of ATP production with reduced electron transport-chain activity. Finally, posttranslational modification of H3S28 was examined in an experimental heart failure model, and increased H3S28 phosphorylation levels were observed in HF hearts. These data establish a critical role of H3S28 phosphorylation in vivo for life span, stress resistance, cardiac and mitochondrial function in Drosophila. These findings may pave the way for H3S28 phosphorylation as a putative target to treat stress-related disorders such as heart failure.
Ma, Z., Wang, H., Cai, Y., Wang, H., Niu, K., Wu, X., Ma, H., Yang, Y., Tong, W., Liu, F., Liu, Z., Zhang, Y., Liu, R., Zhu, Z. J. and Liu, N. (2018). Epigenetic drift of H3K27me3 in aging links glycolysis to healthy longevity in Drosophila. Elife 7. PubMed ID: 29809154
Summary:
Epigenetic alteration has been implicated in aging. However, the mechanism by which epigenetic change impacts aging remains to be understood. H3K27me3, a highly conserved histone modification signifying transcriptional repression, is marked and maintained by Polycomb Repressive Complexes (PRCs). This study explores the mechanism by which age-modulated increase of H3K27me3 impacts adult lifespan. Using Drosophila, it was revealed that aging leads to loss of fidelity in epigenetic marking and drift of H3K27me3 and consequential reduction in the expression of glycolytic genes with negative effects on energy production and redox state. This study shows that a reduction of H3K27me3 by PRCs-deficiency promotes glycolysis and healthy lifespan. While perturbing glycolysis diminishes the pro-lifespan benefits mediated by PRCs-deficiency, transgenic increase of glycolytic genes in wild-type animals extends longevity. Together, it is proposed that epigenetic drift of H3K27me3 is one of the molecular mechanisms that contribute to aging and that stimulation of glycolysis promotes metabolic health and longevity.
Hundertmark, T., Gartner, S. M. K., Rathke, C. and Renkawitz-Pohl, R. (2018). Nejire/dCBP-mediated histone H3 acetylation during spermatogenesis is essential for male fertility in Drosophila melanogaster. PLoS One 13(9): e0203622. PubMed ID: 30192860
Summary:
Spermatogenesis in many species including Drosophila melanogaster is accompanied by major reorganisation of chromatin in post-meiotic stages, involving a nearly genome-wide displacement of histones by protamines, Mst77F and Protamine-like 99C. A proposed prerequisite for the histone-to-protamine transition is massive histone H4 hyper-acetylation prior to the switch. This study investigated the pattern of histone H3 lysine acetylation and general lysine crotonylation in D. melanogaster spermiogenesis to elucidate a possible role of these marks in chromatin reorganisation. Lysine crotonylation was strongest prior to remodelling and the deposition of this mark depended on the acetylation status of the spermatid chromatin. In contrast to H4 acetylation, individual H3 acetylation marks displayed surprisingly distinct patterns during the histone-to-protamine transition. Nejire, a histone acetyl transferase, is expressed during the time of histone-to-protamine transition. Nejire knock down led to strongly reduced fertility, which correlated with misshaped spermatid nuclei and a lack of mature sperm. protA and prtl99C transcript levels were reduced after knocking down Nejire. ProtB-eGFP, Mst77F-eGFP and Prtl99C-eGFP were synthesized at the late canoe stage, while histones were often not detectable. However, in some cysts histones persist in parallel to protamines. Therefore, it was hypothesized that complete histone removal requires multiple histone modifications besides H3K18ac and H3K27ac. In summary, H3K18 and H3K27 acetylation during Drosophila spermatogenesis is dependent on Nejire or a yet uncharacterized acetyl transferase. Nejire is required for male fertility since Nejire contributes to efficient transcription of protA and prtl99C, but not Mst77F, in spermatocytes, and to maturation of sperm.
Guio, L., Vieira, C. and Gonzalez, J. (2018). Stress affects the epigenetic marks added by natural transposable element insertions in Drosophila melanogaster. Sci Rep 8(1): 12197. PubMed ID: 30111890
Summary:
Transposable elements are emerging as an important source of cis-acting regulatory sequences and epigenetic marks that could influence gene expression. However, few studies have dissected the role of specific transposable element insertions on epigenetic gene regulation. Bari-Jheh is a natural transposon that mediates resistance to oxidative stress by adding cis-regulatory sequences that affect expression of nearby genes. This work, integrated publicly available ChIP-seq and piRNA data with chromatin immunoprecipitation experiments to get a more comprehensive picture of Bari-Jheh molecular effects. Bari-Jheh was shown to be enriched for H3K9me3 in nonstress conditions, and for H3K9me3, H3K4me3 and H3K27me3 in oxidative stress conditions, which is consistent with expression changes in adjacent genes. It was further shown that under oxidative stress conditions, H3K4me3 and H3K9me3 spread to the promoter region of Jheh1 gene. Finally, another insertion of the Bari1 family was associated with increased H3K27me3 in oxidative stress conditions suggesting that Bari1 histone marks are copy-specific. It is concluded that besides adding cis-regulatory sequences, Bari-Jheh influences gene expression by affecting the local chromatin state.
Armstrong, R. L., Penke, T. J. R., Chao, S. K., Gentile, G. M., Strahl, B. D., Matera, A. G., McKay, D. J. and Duronio, R. J. (2019). H3K9 Promotes under-replication of pericentromeric heterochromatin in Drosophila salivary gland Polytene Chromosomes. Genes (Basel) 10(2). PubMed ID: 30700014
Summary:
Chromatin structure and its organization contributes to the proper regulation and timing of DNA replication. Yet, the precise mechanism by which chromatin contributes to DNA replication remains incompletely understood. This is particularly true for cell types that rely on polyploidization as a developmental strategy for growth and high biosynthetic capacity. During Drosophila larval development, cells of the salivary gland undergo endoreplication, repetitive rounds of DNA synthesis without intervening cell division, resulting in ploidy values of ~1350C. S phase of these endocycles displays a reproducible pattern of early and late replicating regions of the genome resulting from the activity of the same replication initiation factors that are used in diploid cells. However, unlike diploid cells, the latest replicating regions of polyploid salivary gland genomes, composed primarily of pericentric heterochromatic enriched in H3K9 methylation, are not replicated each endocycle, resulting in under-replicated domains with reduced ploidy. This study employed a histone gene replacement strategy in Drosophila to demonstrate that mutation of a histone residue important for heterochromatin organization and function (H3K9), but not mutation of a histone residue important for euchromatin function (H4K16), disrupts proper endoreplication in Drosophila salivary gland polyploid genomes thereby leading to DNA copy gain in pericentric heterochromatin. These findings reveal that H3K9 is necessary for normal levels of under-replication of pericentric heterochromatin and suggest that under-replication at pericentric heterochromatin is mediated through H3K9 methylation.
Shindo, Y. and Amodeo, A. A. (2019). Dynamics of free and chromatin-bound Histone H3 during early embryogenesis. Curr Biol. PubMed ID: 30639105
Summary:
During zygotic genome activation (ZGA), the chromatin environment undergoes profound changes, including the formation of topologically associated domains, refinements in nucleosome positioning on promoters, and the emergence of heterochromatin. In many organisms, including Drosophila, ZGA is associated with the end of a period of extremely rapid, exponential cleavage divisions that are facilitated by large maternally provided pools of nuclear components. It is therefore imperative to understand how the supply of chromatin components relative to the exponentially increasing demand affects nuclear and chromatin composition during early embryogenesis. This study examined the nuclear trafficking and chromatin dynamics of histones during the cleavage divisions in Drosophila using a photo-switchable H3-Dendra2 reporter. Total H3-Dendra2 in the nucleus decreases with each cleavage cycle. This change in nuclear composition is due to depletion of large pools (>50%) of free protein that are present in the early cycles. The per nucleus import rate halves with each cycle, and a mathematical model was constructed in which increasing histone demand determines the dynamics of nuclear H3 supply. Finally, it was shown that these changes in H3 availability correspond to a large (~ 40%) reduction in global H3 occupancy on the chromatin, which is compensated by the increased incorporation of H3.3. The observed changes in free nuclear H3 and chromatin composition may contribute to the cell-cycle slowing, changes in chromatin structure, and the onset of transcription associated with this developmental stage.
BIOLOGICAL OVERVIEW

Distinct modifications of histone amino termini, such as acetylation, phosphorylation and methylation, have been proposed to underlie a chromatin-based regulatory mechanism that modulates the accessibility of genetic information. Histone lysine methylation occurs on lysines 4, 9, 27, 36, and 79 of Histone H3 and on lysine 20 of Histone H4. Biochemical and genetic studies indicate that methylation of different lysine residues, with the exception of H3-K79, is catalyzed by different SET domain-containing proteins. Histone H3 methylated at lysine 9 (H3-mLys9) is characteristic of the heterochromatic (genetically silent) state. Immunofluorescent staining of Drosophila polytene chromosomes shows that the bulk of the H3-mLys9 is present in the pericentric heterochromatin and in a banded pattern on the fourth chromosome, known sites of repetitive DNA. Similarly, chromatin immunoprecipitation experiments demonstrate that H3-mLys9 is a prominent component of the silent mating type locus in fission yeast (Schizosaccharomyces pombe), while essentially absent from flanking regions containing inducible genes. Methylation of histone H3-Lys9 has also been associated with the silencing of euchromatic genes (Richards, 2002 and references therein).

A key role for gene silencing and specification of heterochromatin is shown by the demonstration that mammalian homologs of Drosophila Su(var)3-9, including human SUV39H1 and murine Suv39h1, encode enzymes that specifically methylate histone H3 on lysine 9. Su(var)3-9 was originally identified as a suppressor of PEV in Drosophila, indicating that the wild-type gene product is involved in heterochromatin formation. A homolog in S. pombe, Clr4, is also a specific histone H3-Lys9 methyltransferase, suggesting that this activity is widely distributed and well conserved. clr4 mutants exhibit reduced heterochromatin formation at centromeres, with elevated mitotic chromosome loss and reduced silencing within both pericentromeric heterochromatin and the silent mating type locus. Similarly, mammalian Su(var)3-9-like proteins have been implicated in both centromere activity and gene silencing. Disruption of the murine Suv39h1 and Suv39h2 paralogs causes genome instability, chromosome missegregation, and male meiotic defects (Richards, 2002 and references therein).

Drosophila Polycomb Group (PcG) complexes are responsible for the maintenance of the repressed state of genes subject to their control. The best-known PcG targets are the homeotic genes, which are activated in the early embryo by the products of segmentation genes. At this stage, transient, localized activators and repressors determine the segmental domains of expression of each homeotic gene but, after gastrulation, epigenetic (genetically based non-heritable) mechanisms take over to maintain the segmental pattern of expression for the rest of development. These mechanisms are mediated by the Polycomb Response Elements (PREs), regulatory regions of several hundred base pairs, where two kinds of chromatin complexes are assembled. One kind, the PcG complexes, is repressive and can maintain a silent state. The other kind involves the Trithorax protein (Trx) and mediates the persistence of the active state. Which of the two predominates depends on the state of activity of the target promoter at the blastoderm stage of development. If the genes had been repressed by early regulators, the PcG silencing mechanisms maintain the repressed state throughout the rest of development. If the gene was active in the early embryo, the Trx function stimulates its expression and prevents later silencing by the PcG complexes. The gene remains then unrepressed and potentially active for the rest of development. Thus, early transcriptional activity of a target gene sets a mark that maintains transcriptional competence and prevents the establishment of PcG silencing, while early repression results in an antagonistic mark that ensures the maintenance of the silenced state in subsequent cell cycles (Czermin, 2002 and references therein).

Formally, therefore, the PRE mediates both the memory of the silent state and the memory of the derepressed state. The repressive memory is illustrated by the fact that, although PcG proteins are nearly ubiquitous, at every cell division they restore the repressed chromatin state only in cells in which their target genes had been previously repressed. The memory of the derepressed state is shown by the fact that if the target gene is active in the early embryo, or if derepression is forced by massive doses of activator, the derepressed state is inherited by the progeny cells. This memory is affected by trx mutations. In the absence of trx function, cells in which a PRE-containing construct had been activated in the early embryo may lose the derepressed state and become silenced again (Czermin, 2002 and references therein).

In the preblastoderm embryo, Polycomb complexes are assembled at the PREs, which contain consensus sequences for DNA binding proteins such as GAGA factor and Pleiohomeotic (Pho), the fly homolog of the mammalian YY1 factor. These, together with other, unidentified DNA binding proteins, recruit cooperatively a PcG complex that includes Pc, Ph, and GAGA factor but also Esc, E(z), Pho, and Rpd3. The E(z)/Esc/Pho complex dissociates from the Pc-containing complex after the blastoderm stage, and Esc ceases to be produced by the end of embryogenesis. However, the E(z) protein continues to be needed, at least intermittently, to maintain the silent state and is most likely recruited to the PRE by the Pho DNA binding protein. Experiments with Pc targeted to a reporter gene by the LexA DNA binding domain show that, while it can recruit the Esc/E(z) component to establish silencing in the early embryo, it can no longer recruit E(z) at later stages, when the early complex has dissociated. LexA-Pc repression continues in the embryo but the memory of the repressed state is then lost and the reporter gene becomes derepressed during larval stages. These results suggest that E(z) might mediate the creation of a chromatin mark necessary for repression and responsible for maintaining the memory of the silent state (Czermin, 2002 and references therein).

Trx and E(z) are therefore good candidates for the functions required for the positive and negative memories, respectively. Structurally, these two proteins share with Su(var)3-9 the SET domain, named after the three founding members Su(var)3-9, E(z), and Trx. Advances in the past years have shown that the SET domain in many proteins is responsible for a histone H3 methyltransferase (MTase) activity. With one exception, all reported histone MTases that methylate lysine residues contain a SET domain, which harbors the amino acids important for MTase function. Su(var)3-9 and its homologs, in particular, are necessary for the formation of heterochromatic complexes in mammals, flies, and fission yeast. Their activities methylate lysine 9 of histone H3, which becomes a binding site for the chromodomain of heterochromatin proteins such as Hp1 (Czermin, 2002 and references therein).

It was reasoned that Trithorax and Enhancer of zeste might reside in complexes that possess MTase activities. Pc contains a chromodomain whose structure and essential residues are homologous to those found in Hp1 and related methyl lysine binding proteins and might therefore recognize a nucleosomal methylation mark. Both Trx and E(z) complexes have now been shown to contain an H3 MTase activity. To study the Esc/E(z) complex, Drosophila nuclear extract was fractionated and it was asked if a MTase activity copurifies with E(z) and Esc. A complex containing E(z) and Esc trimethylates lysine 9 and methylates lysine 27 of histone H3 and the trimethylated lysine 9 mark is closely correlated with PcG binding sites on polytene chromosomes. The conjecture that the Esc/E(z) complex contains a histone MTase activity has been confirmed by the finding that the complex immunoprecipitated by anti-Esc or purified biochemically methylates in vitro histone H3 whether assembled in a nucleosome, as a free histone, or in the form of oligopeptides. The purified complex contains several components as predicted from previous studies: in addition to E(z), Su(z)12, Esc, p55, and Rpd3 are found. An additional component of approximately 168 kDa remains to be identified (Czermin, 2002).

The activity of the Esc/E(z) complex leads to trimethylation of lysine 9 and probably also of lysine 27 of H3. In vivo, an antibody directed against dimethylated H3 lysine (9me2K9 antibody) does not detectably stain chromosomal PcG sites while a me3K9 antibody directed against trimethylated H3 lysine decorates all chromosomal PcG sites. Whether three methyl groups are added processively or by independent events and whether lysine 9 and lysine 27 are targeted simultaneously remains to be elucidated. The partial ability of the complex to methylate the peptide acetylated at lysine 9 is accounted for by the presence of the Rpd3 deacetylase (Czermin, 2002).

The Su(var)3-9 product has been reported to dimethylate H3 lysine 9, constituting a mark for heterochromatic complexes (Rea, 2000; Lachner, 2001). However, antibody staining of polytene chromosomes detects abundant trimethyl lysine 9 in heterochromatin as well as most of chromosome 4, sites that do not contain PcG complexes. At least some of the heterochromatic me3K9 is lost in Su(var)3-9 mutants. A participation of E(z) in heterochromatic H3 methylation cannot be excluded. This function would be consistent with the report that E(z) mutations are also suppressors of heterochromatic position-effect variegation. However, a more distinct difference between the Su(var)3-9 mark and the E(z) mark is the methylation of H3 K27. In vitro, Su(var)3-9 does not methylate the K27 peptide. It will be important to test whether K27 methylation is present in heterochromatin, but it is likely that K27 methylation differentiates heterochromatic from PcG sites (Czermin, 2002).

Suvar3-9 methylation of lysine 9 of histone H3 is thought to stabilize or even target Hp1-containing heterochromatic complexes through binding of the Hp1 chromodomain to the methylated lysine 9 (Bannister, 2001; Lachner, 2001). The structure of the Hp1 chromodomain bound to histone H3 di- or tri-methylated at lysine 9 shows that either peptide fits in a groove and lodges the methyllysine in a hydrophobic pocket (Nielsen, 2002; Jacobs, 2002). This was confirmed by peptide binding experiments and makes it unlikely that H3 K9 methylation would be sufficient to discriminate between the heterochromatic methylation imprint and the PcG methylation imprint. The parallelism suggests that the chromodomain of Pc would recognize trimethyl lysine 9 H3. Pc has been shown to bind to histone H3 and to nucleosomes in vitro; however, the domain involved does not appear to be the chromodomain but the C-terminal region (Breiling, 1999). Binding experiments detected little increased affinity of Pc for the trimethyl K9 peptide compared to the unmethylated peptide. Instead, methyl K27 appears to make the major contribution to Pc affinity for methylated H3. The amino acid context of K27 (KAARKS) resembles that of K9 (QTARKS) but Hp1 binds weakly to a methylated K27 peptide (Nielsen, 2002; Jacobs, 2002). It remains to be seen which domain of Pc interacts with methylated H3 and whether Hp1 can bind to meK9 meK27 H3. Nevertheless, the presence of me3K9 at PcG sites, as well as in heterochromatin and chromosome 4, suggests that the meK27 or other factors must contribute to discriminate between heterochromatic and PcG sites (Czermin, 2002).

Specific recognition of the methylated histone H3 by Polycomb complexes might be facilitated by other PcG components or by other modifications of the histones. E(z)-dependent methylation might contribute to the stability of the PcG complex, particularly in the early stages of assembly at the PRE, for example, by permitting complex formation to spread to neighboring sequences for a distance of 2- 3 kb. However, the fact that in the E(z)S2 mutant chromosomes the trimethylation mark is lost well before the binding of PcG proteins indicates that methylation is not essential for the binding of the complex. Alternatively, the trimethyl mark might signify the difference between the mere recruitment of a PcG complex and its repressive function. Chromatin immunoprecipitation shows that PcG complexes are present at some PREs whether or not the corresponding gene is repressed, implying that, while recruitment of the complexes may be constitutive, the decision to repress or not depends on other features transmitted epigenetically. The methylation might then constitute the epigenetic mark triggering the silent state. It is interesting to note, therefore, that two polytenic sites that are strongly stained with anti-Psc antibody but very weakly with anti-me3K9 antibody are 2D and 49F, respectively, the sites of the PcG genes ph and Psc. These PcG genes are downregulated but not silenced by PcG mechanisms. If trimethylation of H3 lysine 9 signals strong silencing, these sites might be expected to be occupied by PcG complexes but only partly repressed (Czermin, 2002).

A role of H3 methylation in the assembly of stable PcG complexes is suggested by another observation. When the chromodomain in Hp1 is substituted by the chromodomain of Pc, the chimeric Hp1 is recruited to PcG binding sites on polytene chromosomes. This implies either that the chromodomain is sufficient to recognize and bind to the meK9 meK27 H3 or that the Pc chromodomain specifies critical interactions with other PcG components that are recruited to PcG sites. That the latter interpretation is correct is shown by the fact that the chimeric Hp1 also recruits PcG proteins to heterochromatin, where they are not normally found, and that this recruitment is dependent on E(z) function. Strong staining is observed with the anti-me3K9 antibody in the chromocenter and chromosome 4, implying that me3K9 H3 is widespread in heterochromatin. If this is, in fact, due to E(z) activity, it would explain the intervention of E(z) in the heterochromatic recruitment of PcG proteins mediated by the chimeric Hp1 (Czermin, 2002).

It would be important, therefore, to determine whether E(z) contributes to heterochromatic me3K9. Anti-E(z) antibody does not stain the chromocenter of salivary polytenic chromosomes. In E(z)S2 mutant larvae raised at nonpermissive temperature, me3K9 staining is still seen in heterochromatin although with very variable intensity. One possible explanation for these two observations is that since heterochromatin is very little replicated in polytene chromosomes, the methylated H3 produced before the temperature shift might perdure a long time at nonpermissive temperature (Czermin, 2002).

It is interesting to note that anti-me3K9 staining at telomeres is not affected in Su(var)3-9 null polytene chromosomes but is lost in the absence of E(z) function. Telomeres, particularly those of chromosome 2R and 2L, stain prominently with antibodies against PcG proteins but also against Hp1. A critical role has been attributed to telomeric Hp1 in 'capping' chromosome termini and preventing telomeric fusions, failure to segregate chromosomes, and chromosome breakage. The fact that Drosophila Su(var)3-9 null mutants are perfectly viable implies that this role of Hp1 is not dependent on Su(var)3-9. In contrast, embryos lacking both maternal and zygotic E(z) function are reported to have mitotic phenotypes similar to those attributed to Hp1 mutants. It is likely, therefore, that the recruitment of Hp1 to telomeric sites depends at least in part on E(z) and not on Su(var)3-9. Unfortunately, the evidence obtained with the E(z)S2 mutant is inconclusive (Czermin, 2002).

It is concluded that a combination of histone methylation marks could be a major factor in the establishment of stable patterns of homeotic gene expression and constitutes the molecular basis of a cellular memory system. The analysis of the existing methylation patterns in cells expressing different homeotic genes by chromatin immunoprecipitation will give further insight into how the MTases such as E(z) are targeted to different sites and how they are regulated at different developmental stages (Czermin, 2002).

CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing

Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. This study shows that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. It is proposed that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing (Tie, 2009).

The major findings of this work are: (1) that Drosophila CBP acetylates H3K27; (2) that this acetylation requires TRX; and (3) that it prevents H3K27 trimethylation by E(Z) at Polycomb target genes and antagonizes Polycomb silencing. The remarkably complementary developmental profiles of H3K27ac and H3K27me3 (but not H3K27me2) during embryogenesis suggest that the deposition of H3K27me3, which increases steadily after ~4 hours with the onset of Polycomb silencing, occurs at the expense of a substantial fraction of the H3K27ac already present. This suggests that the establishment of Polycomb silencing might require active deacetylation of this pre-existing H3K27ac. The reciprocal effects of knockdown and overexpression of CBP and E(Z) on H3K27 trimethylation and acetylation in bulk chromatin further suggest that the two modifications constitute alternative chromatin states associated with active and inactive genes. Consistent with this, ChIP-chip experiments revealed that H3K27me3 and H3K27ac are mutually exclusive genome wide. Moreover, in S2 cells, the inactive abd-A gene does not have the H3K27ac modification in its promoter region, but acquires it upon RNAi knockdown of E(Z). It will be important to determine whether such a modification switch occurs genome wide after loss of E(Z) (Tie, 2009).

The ability of E(Z) overexpression to suppress the small rough eye phenotype of CBP overexpressers further supports the conclusion that H3K27 trimethylation by E(Z) antagonizes H3K27 acetylation by CBP and suggests that deacetylation of H3K27 by RPD3, and possibly other deacetylases, might be a prerequisite for subsequent methylation by E(Z) and therefore important for reversal of an active state. Conversely, the ability of CBP and TRX overexpression to increase the global H3K27ac level at the expense of H3K27me3 suggests that either active demethylation of H3K27me3 by the H3K27-specific demethylase UTX (Agge, 2007; Lee, 2007b; Smith, 2008), or histone replacement (Ahmad, 2002), might be a prerequisite to acetylation by CBP. Indeed, depletion of Drosophila UTX in vivo using a GAL4-inducible UTX RNAi transgene line results in an increase in H3K27me3, as previously reported (Smith, 2008), and in a marked decrease in H3K27ac. These data, together with the evidence of developmentally programmed reversal of Polycomb silencing, now suggest that the widely accepted stability of Polycomb silencing during development might be more dynamically regulated than previously appreciated (Tie, 2009).

This is the first report that CBP/p300 acetylates H3K27. Recombinant Drosophila CBP acetylates H3K27 and K18 in vivo and in vitro. The greatly reduced H3K27ac levels in CBP-depleted S2 cells also strongly suggest that CBP is the major H3K27 acetylase in Drosophila. The conservation of H3K27 acetylation by human p300, together with the reported association of CBP with the TRX homolog MLL in humans (Ernst, 2001), suggest that it is likely to play a similar role in antagonizing Polycomb silencing in mammals (Tie, 2009).

The genome-wide distribution of H3K27ac, as estimated from human ChIP-chip experiments, appears very similar to that of H3K4me3. This suggests that H3K27ac is much more widely distributed than just at Polycomb target genes, which are estimated to number several thousand in mammalian cells and hundreds in Drosophila. Although these numbers could grow with the identification of additional Polycomb-silenced genes in additional cell types, the recently reported strong correlation of H3K27ac with active genes suggests that it plays an additional role(s) in promoting the transcription of active genes, including those that are never targets of Polycomb silencing. (Note that the H3K27ac at non-Polycomb target genes will not be directly affected by global changes in H3K27me3.) Interestingly, like H3K27me3, H3K27ac appears on the transcribed regions of Polycomb target genes, which might reflect a role for H3K27ac in facilitating transcriptional elongation, and, conversely, a role for H3K27me3 in inhibiting elongation. In addition to its anti-silencing role in preventing H3K27 trimethylation, H3K27ac may also serve as a signal for recruitment of other proteins with additional enzyme activities that alter local chromatin structure further to facilitate or promote transcription. Prime candidates are those containing a bromodomain, a conserved acetyl-lysine-binding module present in several dozen chromatin-associated proteins, including a number of TrxG proteins that also antagonize Polycomb silencing (Tie, 2009).

The results presented in this study provide new insight into how TRX and CBP function together to antagonize Polycomb silencing. Robust H3K27 acetylation by CBP is dependent on TRX, suggesting that H3K27ac plays a crucial role in the anti-silencing activity of TRX. Consistent with this, preliminary genetic evidence suggests that the Polycomb phenotypes caused by TRX overexpression are dependent on CBP, as they are suppressed by RNAi knockdown of CBP. The nature of this dependence is currently unknown, but could involve targeting of CBP by TRX or regulation of the H3K27 acetylation activity of CBP by TRX (Tie, 2009).

The physical association of TRX and CBP and the widespread coincidence of H3K27ac and H3K4me3 sites in the human ChIP-chip data further suggest that the two modifications might be coordinately executed by TRX and CBP. However, the results also raise the possibility that H3K4 trimethylation by TRX itself might be less important for antagonizing Polycomb silencing than H3K27 acetylation. This possibility is also suggested by the discovery of Polycomb-silenced genes in ES and human T cells that contain 'bivalent' marks (both H3K4me3 and H3K27me3) in their promoter regions (although the H3K4me3 levels at these inactive genes are typically lower, on average, than they are at active genes, hinting at the possible importance of quantitative effects of the two marks) (Tie, 2009).

A speculative model is proposed for the regulation of Polycomb silencing that incorporates the activities of TRX, CBP, E(Z), RPD3 and UTX. Repressed genes are marked with H3K27me3. H3K27 trimethylation by PRC2 (which can also control DNA methylation in mammals) requires RPD3 (and possibly other histone deacetylases) to deacetylate any pre-existing H3K27ac. H3K27me3 promotes binding of PC-containing PRC1 complexes, which may inhibit H3K27 acetylation and maintain silencing through 'downstream' events, including those promoted by the H2AK119 mono-ubiquitylation mediated by its RING subunit. Conversely, active genes are marked with H3K4me3 and H3K27ac. H3K27 acetylation by CBP is dependent on TRX and possibly other TrxG proteins, as suggested by the observation that H3K27me3 levels are significantly increased on salivary gland polytene chromosomes from trx, ash1 and kis mutants. The current results predict that this increase will be accompanied by a decrease in H3K27ac. Interestingly, ash1 encodes another HMTase that also interacts with CBP and antagonizes Polycomb silencing. Acetylation of H3K27 is likely to also require the K27-specific demethylase UTX when removal of pre-existing H3K27me3 is a prerequisite for acetylation, e.g. for developmentally regulated reversal of Polycomb silencing at the onset of differentiation. H3K27ac prevents H3K27 trimethylation and might also serve as a signal for recruitment of other TrxG proteins with additional chromatin-modifying activities that may protect the H3K27ac modification and also alter local chromatin structure to promote transcription and further inhibit Polycomb silencing (Tie, 2009).

Histone H3 Serine 28 is essential for efficient Polycomb-mediated gene repression in Drosophila

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).

The H3K9 methyltransferase SETDB1 maintains female identity in Drosophila germ cells

The preservation of germ cell sexual identity is essential for gametogenesis. This study shows that H3K9me3-mediated gene silencing is integral to female fate maintenance in Drosophila germ cells. Germ cell specific loss of the H3K9me3 pathway members, the H3K9 methyltransferase SETDB1, WDE, and HP1a, leads to ectopic expression of genes, many of which are normally expressed in testis. SETDB1 controls the accumulation of H3K9me3 over a subset of these genes without spreading into neighboring loci. At phf7, a regulator of male germ cell sexual fate, the H3K9me3 peak falls over the silenced testis-specific transcription start site. Furthermore, H3K9me3 recruitment to phf7 and repression of testis-specific transcription is dependent on the female sex determination gene Sxl. Thus, female identity is secured by an H3K9me3 epigenetic pathway in which Sxl is the upstream female-specific regulator, SETDB1 is the required chromatin writer, and phf7 is one of the critical SETDB1 target genes (Smolko, 2018).

In metazoans, germ cell development begins early in embryogenesis when the primordial germ cells are specified as distinct from somatic cells. Specified primordial germ cells then migrate into the embryonic gonad, where they begin to exhibit sex-specific division rates and gene expression programs, ultimately leading to meiosis and differentiation into either eggs or sperm. Defects in sex-specific programming interferes with germ cell differentiation leading to infertility and germ cell tumors. Successful reproduction, therefore, depends on the capacity of germ cells to maintain their sexual identity in the form of sex-specific regulation of gene expression (Smolko, 2018).

In Drosophila melanogaster, germ cell sexual identity is specified in embryogenesis by the sex of the developing somatic gonad. However, extrinsic control is lost after embryogenesis and sexual identity is preserved by a cell-intrinsic mechanism. The Sex-lethal (Sxl) female-specific RNA binding protein is an integral component of the cell-intrinsic mechanism, as loss of Sxl specifically in germ cells leads to a global upregulation of spermatogenesis genes and a germ cell tumor phenotype. Remarkably, sex-inappropriate transcription of a single gene, PHD finger protein 7 (phf7), a key regulator of male identity, is largely responsible for the tumor phenotype. Depletion of phf7 in mutants lacking germline Sxl suppresses the tumor phenotype and restores oogenesis. Moreover, forcing PHF7 protein expression in ovarian germ cells is sufficient to disrupt female fate and give rise to a germ cell tumor. Interestingly, sex-specific regulation of phf7 is achieved by a mechanism that relies primarily on alternative promoter choice and transcription start site (TSS) selection. Sex-specific transcription produces mRNA isoforms with different 5' untranslated regions that affect translation efficiency, such that PHF7 protein is only detectable in the male germline. Although the Sxl protein is known to control expression post-transcriptionally in other contexts the observation that germ cells lacking Sxl protein show defects in phf7 transcription argues that Sxl is likely to indirectly control phf7 promoter choice. Thus, how this sex-specific gene expression program is stably maintained remains to be determined (Smolko, 2018).

This study reports the discovery that female germ cell fate is maintained by an epigenetic regulatory pathway in which SETDB1 (aka EGGLESS, KMT1E, and ESET) is the required chromatin writer and phf7 is one of the critical SETDB1 target genes. SETDB1 trimethylates H3K9 (H3K9me3), a feature of heterochromatin. Using tissue-specific knockdown approaches this study established that germ cell specific loss of SETDB1, its protein partner WINDEI [WDE, aka ATF7IP, MCAF1 and hAM10], and the H3K9me3 reader, HP1a, encoded by the Su(var)205 locus, leads to ectopic expression of euchromatic protein-encoding genes, many of which are normally expressed only in testis. It was further found that H3K9me3 repressive marks accumulate in a SETDB1 dependent manner at 21 of these ectopically expressed genes, including phf7. Remarkably, SETDB1 dependent H3K9me3 deposition is highly localized and does not spread into neighboring loci. Regional deposition is especially striking at the phf7 locus, where H3K9me3 accumulation is restricted to the region surrounding the silent testis-specific TSS. Lastly, this study found that H3K9me3 accumulation at many of these genes, including phf7, is dependent on Sxl. Collectively these findings support a model in which female fate is preserved by deposition of H3K9me3 repressive marks on key spermatogenesis genes (Smolko, 2018).

This study reveals a role for H3K9me3 chromatin, operationally defined as facultative heterochromatin, in securing female identity by silencing lineage-inappropriate transcription. H3K9me3 pathway members, the H3K9 methyltransferase SETDB1, its binding partner WDE, and the H3K9 binding protein HP1a, are required for silencing testis gene transcription in female germ cells. These studies further suggest a mechanism in which SETDB1, in conjunction with the female fate determinant Sxl, controls transcription through deposition of highly localized H3K9me3 islands on a select subset of these genes. The male germ cell sexual identity gene phf7 is one of the key downstream SETDB1 target genes. H3K9me3 deposition on the region surrounding the testis-specific TSS guaranties that no PHF7 protein is produced in female germ cells. In this model, failure to establish silencing leads to ectopic PHF7 protein expression, which in turn drives aberrant expression of testis genes and a tumor phenotype (Smolko, 2018).

Prior studies have established a role for SETDB1 in germline Piwi-interacting small RNA (piRNA) biogenesis and TE silencing. However, piRNAs are unlikely to contribute to sexual identity maintenance as mutations that specifically interfere with piRNA production, such as rhino, do not cause defects in germ cell differentiation. These findings, together with the observation that rhino does not control sex-specific phf7 transcription, suggests that the means by which SETDB1 methylates chromatin at testis genes is likely to be mechanistically different from what has been described for piRNA-guided H3K9me3 deposition on TEs.

The accumulation of H3K9me3 at many of these genes, including phf7, is dependent on the presence of Sxl protein. Thus, these studies suggest that Sxl is required for female-specific SETDB1 function. Sxl encodes an RNA binding protein known to regulate its target genes at the posttranscriptional levels. Sxl control may therefore be indirect. However, studies in mammalian cells suggest that proteins with RNA binding motifs are important for H3K9me3 repression, raising the tantalizing possibility that Sxl might play a more direct role in governing testis gene silencing. Further studies will be necessary to clarify how the sex determination pathway feeds into the heterochromatin pathway (Smolko, 2018).

phf7 stands out among the cohort of genes regulated by facultative heterochromatin because of its pivotal role in controlling germ cell sexual identity. Because ectopic protein expression is sufficient to disrupt female fate, tight control of phf7 expression is essential. phf7 regulation is complex, employing a mechanism that includes alternative promoter usage and TSS selection. This study reports that SETDB1/H3K9me3 plays a critical role in controlling phf7 transcription. In female germ cells, H3K9me3 accumulation is restricted to the region surrounding the silent testis-specific transcription start site. Dissolution of the H3K9me3 marks via loss of Sxl or SETDB1 protein is correlated with transcription from the upstream testis-specific site and ectopic protein expression, demonstrating the functional importance of this histone modification. Together, these studies suggest that maintaining the testis phf7 promoter region in an inaccessible state is integral to securing female germ cell fate (Smolko, 2018).

Although the loss of H3K9me3 pathway members in female germ cells leads to the ectopic, lineage-inappropriate transcription of hundreds of genes, integrative analysis identified only 21 SETDB1/H3K9me3 regulated genes. Given that one of these genes is phf7 and that ectopic PHF7 is sufficient to destabilize female fate, it is likely that inappropriate activation of a substantial number of testis genes is a direct consequence of ectopic PHF7 protein expression. How PHF7 is able to promote testis gene transcription is not yet clear. PHF7 is a PHD-finger protein that preferentially binds to H3K4me2, a mark associated with poised, but inactive genes and linked to epigenetic memory. Thus, one simple model is that ectopic PHF7 binds to H3K4me2 marked testis genes to tag them for transcriptional activation (Smolko, 2018).

It will be interesting to explore whether any of the other 20 SETDB1/H3K9me3 regulated genes also have reprogramming activity. In fact, ectopic fate-changing activity has already been described for the homeobox transcription factor Lim1 in the eye-antenna imaginal disc. However, whether Lim1 has a similar function in germ cells is not yet known. Intriguingly, protein prediction programs identify three of the uncharacterized testis-specific genes as E3 ligases. SkpE is a member of the SKP1 gene family, which are components of the Skp1-Cullin-F-box type ubiquitin ligase. CG12477 is a RING finger domain protein, most of which are believed to have ubiquitin E3 ligases activity. CG42299 is closely related to the human small ubiquitin-like modifier (SUMO) E3 ligase NSMCE2. Given studies that have linked E3 ligases to the regulation of chromatin remodeling, it is tempting to speculate that ectopic expression of one or more of these E3 ligases will be sufficient to alter cell fate. Future studies focused on this diverse group of SETDB1/H3K9me3 regulated genes and their role in reprogramming may reveal the multiple layers of regulation required to secure cell fate (Smolko, 2018).

The SETDB1-mediated mechanism for maintaining sexual identity uncovered in this study may not be restricted to germ cells. Recent studies have established that the preservation of sexual identity is essential in the adult somatic gut and gonadal cells for tissue homeostasis. Furthermore, the finding that loss of HP1a in adult neurons leads to masculinization of the neural circuitry and male specific behaviors suggests a connection between female identity maintenance and H3K9me3 chromatin. Thus, it is speculated that SETDB1 is more broadly involved in maintaining female identity (Smolko, 2018).

These studies highlight an emerging role for H3K9me3 chromatin in cell fate maintenance. In the fission yeast S. pombe, discrete facultative heterochromatin islands assemble at meiotic genes that are maintained in a silent state during vegetative growth. Although less well understood, examples in mammalian cells indicate a role for SETDB1 in lineage-specific gene silencing. Thus, silencing by SETDB1 controlled H3K9 methylation may be a widespread strategy for securing cell fate. Interestingly, H3K9me3 chromatin impedes the reprogramming of somatic cells into pluripotent stem cells (iPSCs). Conversion efficiency is improved by depletion of SETDB1. If erasure of H3K9me3 via depletion of SETDB1 alters the sexually dimorphic gene expression profile in reprogrammed cells, as it does in Drosophila germ cells, the resulting gene expression differences may cause stem cell dysfunction, limiting their therapeutic utility (Smolko, 2018).


GENE STRUCTURE

4.8kb and 5.0kb repeats containing the histone genes His1, His2A, His2B, His3 and His4 were present in all of the more than 20 D. melanogaster strains studied. The strains differ in the relative amounts of the two repeat types, with the 5.0kb repeat always present in equal or greater amounts than the 4.8kb repeat. The strains also differ in a number of far less abundant fragments containing histone gene sequences. The expression of HIS-C genes, including His3, during oogenesis has been studied, and compared to periods of DNA synthesis and actin expression during this developmental stage. The D. virilis core histone genes (Dvir\His2B, Dvir\His3, Dvir\His4 and Dvir\His2A), are arranged in the same order and orientation as the D. melanogaster core histone genes (His2B, His3, His4 and His2A). However, the His1 gene that is located between His2B and His3 in D. melanogaster is not found between Dvir\His2B and Dvir\His3 in D. virilis. The genomic organization of the histone genes in D. hydei closely resembles that of D. melanogaster. The position of the homologous histone gene repeats within the nuclei of early embryo cells has been investigated. The two homologous histone gene clusters are distinct and separate through all stages of the cell cycle up to nuclear cycle 13. During interphase of cycle 14, the two clusters colocalize with high frequency, and move from near the midline of the nucleus towards the apical side. The codon bias of the histone genes from D. melanogaster and D. hydei illustrates that the generalization -- that abundantly expressed genes have a high codon bias and low rates of silent substitution -- does not hold for the histone genes. DNA replication of the 5kb histone gene repeating unit in tissue culture cells (Drosophila Kc cells) initiates at multiple sites located within the repeating unit. Several replication pause sites are located at 5' upstream regions of some histone genes. The TFIID complex interacts with the promoter of His3 making contacts at the TATA element, initiator, +18 and +28 regions. Distinct specific subsets of lysines are utilized during deposition-related His4 diacetylation (FlyBase record for His3 and references therein).


PROTEIN STRUCTURE

Amino Acids - 135

For information on H3 structure see Structures of Histone Proteins and Proteins Containing the Histone Fold Motif and associated links.


Histone H3: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 December 2018

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