InteractiveFly: GeneBrief

Histone H2A: Biological Overview | References

Gene name - Histone H2A

Synonyms -

Cytological map position - 39D3-39E1

Function - chromatin constituent

Keywords - nucleosome, polycomb silencing, ubiquitination, histone crosstalk

Symbol - His2A

FlyBase ID: FBgn0001196

Genetic map position - His2A represents a gene family, individual members of the family are: CG31618, CG33808, CG33814, CG33817, CG33820, CG33823, CG33826, CG33829, CG33832, CG33835, CG33838, CG33841, CG33844, CG33847, CG33850, CG33853, CG33856, CG33859, CG33862, CG33865.

Classification - Histone 2A

Cellular location - nuclear

NCBI link: EntrezGene
His2A orthologs: Biolitmine
Recent literature
Doiguchi, M., et al. (2016). SMARCAD1 is an ATP-dependent stimulator of nucleosomal H2A acetylation via CBP, resulting in transcriptional regulation. Sci Rep 6: 20179. PubMed ID: 26888216
This study discovered an ATP-dependent histone H2A acetylation activity in Drosophila nuclear extracts. This activity was column purified and demonstrated to be composed of the enzymatic activities of CREB-binding protein (CBP) and SMARCAD1, which belongs to the Etl1 subfamily of the Snf2 family of helicase-related proteins. SMARCAD1 enhanced acetylation by CBP of H2A K5 and K8 in nucleosomes in an ATP-dependent fashion. Expression array analysis of S2 cells having ectopically expressed SMARCAD1 revealed up-regulated genes. Using native genome templates of these up-regulated genes, it was found that SMARCAD1 activates their transcription in vitro. Knockdown analysis of SMARCAD1 and CBP indicated overlapping gene control, and ChIP-seq analysis of these commonly controlled genes showed that CBP is recruited to the promoter prior to SMARCAD1. Moreover, Drosophila genetic experiments demonstrated interaction between SMARCAD1/Etl1 and CBP/nej during development. The interplay between the remodeling activity of SMARCAD1 and histone acetylation by CBP sheds light on the function of chromatin and the genome-integrity network.
Kahn, T.G., Dorafshan, E., Schultheis, D., Zare, A., Stenberg, P., Reim, I., Pirrotta, V. and Schwartz, Y.B. (2016). Interdependence of PRC1 and PRC2 for recruitment to Polycomb Response Elements. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 27557709
Polycomb Group (PcG) proteins are epigenetic repressors essential for control of development and cell differentiation. They form multiple complexes of which PRC1 and PRC2 are evolutionary conserved and obligatory for repression. The targeting of PRC1 and PRC2 is poorly understood and has been proposed to be hierarchical and involve tri-methylation of histone H3 (H3K27me3) and/or monoubiquitylation of histone H2A (H2AK118ub). This study tested this hypothesis using the Drosophila model. It was discovered that neither H3K27me3 nor H2AK118ub is required for targeting PRC complexes to Polycomb Response Elements (PREs). PRC1 can bind PREs in the absence of PRC2 but at many PREs PRC2 requires PRC1 to be targeted. It was shown that one role of H3K27me3 is to allow PcG complexes anchored at PREs to interact with surrounding chromatin. In contrast, the bulk of H2AK118ub is unrelated to PcG repression. These findings radically change the view of how PcG repression is targeted and suggest that PRC1 and PRC2 can communicate independently of histone modifications.

Yang, D. and Ioshikhes, I. (2016). Drosophila H2A and H2A.Z nucleosome sequences reveal different nucleosome positioning sequence patterns. J Comput Biol [Epub ahead of print]. PubMed ID: 27992255
Nucleosomes are implicated in transcriptional regulation as well as in packing and stabilizing the DNA. Nucleosome positions affect the transcription by impeding or facilitating the binding of transcription factors. The DNA sequence, especially the periodic occurrences of dinucleotides, is a major factor that affects the nucleosome positioning. This study analyzed the Drosophila DNA sequences bound by H2A and H2A.Z nucleosomes. Periodic patterns of dinucleotides (nweak-weak/strong-strong or purine-purine/pyrimidine-pyrimidine) were identified as WW/SS and RR/YY nucleosome positioning sequence (NPS) patterns. The WW/SS NPS pattern of the H2A nucleosome has a 10-bp period of weak-weak/strong-strong (W = A or T; S = G or C) dinucleotides. The 10-bp periodicity, however, is disrupted in the middle of the sequence. At the dyad, the SS dinucleotide is preferred. On the other hand, the RR/YY NPS pattern has an 18-bp periodicity of purine-purine/pyrimidine-pyrimidine (R = A or G; Y = T or C) dinucleotides. The NPS patterns from H2A.Z nucleosomes differ from the NPS patterns from H2A nucleosomes. The RR/YY pattern of H2A.Z nucleosomes has major peaks shifted by 10 bp deviated from the H2A nucleosome pattern. The H2A and H2A.Z nucleosomes have different sequece preferences. The shifted peaks coincide with DNA regions interacting with the histone loops.
Foglizzo, M., Middleton, A. J., Burgess, A. E., Crowther, J. M., Dobson, R. C. J., Murphy, J. M., Day, C. L. and Mace, P. D. (2018). A bidentate Polycomb Repressive-Deubiquitinase complex is required for efficient activity on nucleosomes. Nat Commun 9(1): 3932. PubMed ID: 30258054
Attachment of ubiquitin to lysine 119 of Histone 2A (H2AK119Ub) is an epigenetic mark characteristic of repressed developmental genes, which is removed by the Polycomb Repressive-Deubiquitinase (PR-DUB) complex. Here we report the crystal structure of the Drosophila PR-DUB, revealing that the deubiquitinase Calypso and its activating partner ASX form a 2:2 complex. The bidentate Calypso-ASX complex is generated by dimerisation of two activated Calypso proteins through their coiled-coil regions. Disrupting the Calypso dimer interface does not affect inherent catalytic activity, but inhibits removal of H2AK119Ub as a consequence of impaired recruitment to nucleosomes. Mutating the equivalent surface on the human counterpart, BAP1, also compromises activity on nucleosomes. Together, this suggests that high local concentrations drive assembly of bidentate PR-DUB complexes on chromatin-providing a mechanistic basis for enhanced PR-DUB activity at specific genomic foci, and the impact of distinct classes of PR-DUB mutations in tumorigenesis.
Yang, L., Ma, Z., Wang, H., Niu, K., Cao, Y., Sun, L., Geng, Y., Yang, B., Gao, F., Chen, Z., Wu, Z., Li, Q., Shen, Y., Zhang, X., Jiang, H., Chen, Y., Liu, R., Liu, N. and Zhang, Y. (2019). Ubiquitylome study identifies increased histone 2A ubiquitylation as an evolutionarily conserved aging biomarker. Nat Commun 10(1): 2191. PubMed ID: 31113955
he long-lived proteome constitutes a pool of exceptionally stable proteins with limited turnover. Previous studies on ubiquitin-mediated protein degradation primarily focused on relatively short-lived proteins; how ubiquitylation modifies the long-lived proteome and its regulatory effect on adult lifespan is unclear. This study profiles the age-dependent dynamics of long-lived proteomes in Drosophila by mass spectrometry using stable isotope switching coupled with antibody-enriched ubiquitylome analysis. The data describe landscapes of long-lived proteins in somatic and reproductive tissues of Drosophila during adult lifespan, and reveal a preferential ubiquitylation of older long-lived proteins. An age-modulated increase of ubiquitylation was found on long-lived histone 2A protein in Drosophila, which is evolutionarily conserved in mouse, monkey, and human. A reduction of ubiquitylated histone 2A in mutant flies is associated with longevity and healthy lifespan. Together, these data reveal an evolutionarily conserved biomarker of aging that links epigenetic modulation of the long-lived histone protein to lifespan.
Fursova, N. A., Turberfield, A. H., Blackledge, N. P., Findlater, E. L., Lastuvkova, A., Huseyin, M. K., Dobrinic, P. and Klose, R. J. (2021). BAP1 constrains pervasive H2AK119ub1 to control the transcriptional potential of the genome. Genes Dev 35(9-10): 749-770. PubMed ID: 33888563
Histone-modifying systems play fundamental roles in gene regulation and the development of multicellular organisms. Histone modifications that are enriched at gene regulatory elements have been heavily studied, but the function of modifications found more broadly throughout the genome remains poorly understood. This is exemplified by histone H2A monoubiquitylation (H2AK119ub1), which is enriched at Polycomb-repressed gene promoters but also covers the genome at lower levels. Using inducible genetic perturbations and quantitative genomics, this study found that the BAP1 deubiquitylase plays an essential role in constraining H2AK119ub1 throughout the genome. Removal of BAP1 leads to pervasive genome-wide accumulation of H2AK119ub1, which causes widespread reductions in gene expression. Elevated H2AK119ub1 preferentially counteracts Ser5 phosphorylation on the C-terminal domain of RNA polymerase II at gene regulatory elements and causes reductions in transcription and transcription-associated histone modifications. Furthermore, failure to constrain pervasive H2AK119ub1 compromises Polycomb complex occupancy at a subset of Polycomb target genes, which leads to their derepression, providing a potential molecular rationale for why the BAP1 ortholog in Drosophila has been characterized as a Polycomb group gene. Together, these observations reveal that the transcriptional potential of the genome can be modulated by regulating the levels of a pervasive histone modification.
Zhang, H., Eerland, J., Horn, V., Schellevis, R. and van Ingen, H. (2021). Mapping the electrostatic potential of the nucleosome acidic patch. Sci Rep 11(1): 23013. PubMed ID: 34837025
he nucleosome surface contains an area with negative electrostatic potential known as the acidic patch, which functions as a binding platform for various proteins to regulate chromatin biology. The dense clustering of acidic residues may impact their effective pKa and thus the electronegativity of the acidic patch, which in turn could influence nucleosome-protein interactions. This study set out to determine the pKa values of residues in and around the acidic patch in the free H2A-H2B dimer using NMR spectroscopy. A refined solution structure is presented of the H2A-H2B dimer based on intermolecular distance restraints, displaying a well-defined histone-fold core. The conserved histidines H2B H46 and H106 that line the acidic patch have pKa of 5.9 and 6.5, respectively, and most acidic patch carboxyl groups have pKa values well below 5.0. For H2A D89, strong evidence was found for an elevated pKa of 5.3. These data establish that the acidic patch is highly negatively charged at physiological pH, while protonation of H2B H106 and H2B H46 at slightly acidic pH will reduce electronegativity. These results will be valuable to understand the impact of pH changes on nucleosome-protein interactions in vitro, in silico or in vivo.


Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants. In Drosophila, genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other developmental regulator genes in cells where they must stay inactive. Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes. Among those, Polycomb repressive complex 1 (PRC1) and the related dRing-associated factors (dRAF) complex contain an E3 ligase activity for monoubiquitination of histone H2A. This study shows that the uncharacterized Drosophila PcG gene calypso encodes the ubiquitin carboxy-terminal hydrolase BAP1. Biochemically purified Calypso exists in a complex with the PcG protein ASX, and this complex, named Polycomb repressive deubiquitinase (PR-DUB), is bound at PcG target genes in Drosophila. Reconstituted recombinant Drosophila and human PR-DUB complexes remove monoubiquitin from H2A but not from H2B in nucleosomes. Drosophila mutants lacking PR-DUB show a strong increase in the levels of monoubiquitinated H2A. A mutation that disrupts the catalytic activity of Calypso, or absence of the ASX subunit abolishes H2A deubiquitination in vitro and HOX gene repression in vivo. Polycomb gene silencing may thus entail a dynamic balance between H2A ubiquitination by PRC1 and dRAF, and H2A deubiquitination by PR-DUB (Scheuermann, 2010; full text of article).

A genetic screen for Drosophila mutants with PcG phenotypes recently identified calypso as a complementation group with two lethal alleles that complemented mutations in any other PcG gene (Gaytán de Ayala Alonso, 2007). The calypso mutation was mapped, and the following findings established that calypso corresponds to the uncharacterized gene CG8445. First, calypso1 and calypso2, two independently isolated lethal calypso alleles, both contained a cytosine to thymine mutation in CG8445 that creates a premature termination codon, whereas the parental chromosome on which these mutations had been induced contained a wild-type cytosine. Second, a transgene expressing a tandem affinity purification (TAP)-tagged form of the CG8445 protein under control of the α-tubulin1 promoter rescued calypso mutant animals into viable and fertile adults. Third, calypso1 and calypso2 mutants did not express detectable levels of CG8445 protein. Therefore the CG8445 gene was named calypso (Scheuermann, 2010).

calypso encodes a polypeptide of 471 amino acids that is a member of the ubiquitin C-terminal hydrolase (UCH) subclass of deubiquitinating enzymes. UCH domains are cysteine proteases that hydrolyse the isopeptide bond between the C-terminal glycine of ubiquitin and the lysine side chain in the conjugated protein. The closest human homologue of Calypso is BAP1, a nuclear protein that possesses tumour suppressor activity. The Calypso protein thus represents Drosophila BAP1 (Scheuermann, 2010).

Western blot analyses of Drosophila nuclear extracts and staining of imaginal discs with anti-Calypso antibodies showed that the Calypso protein is localized in nuclei. To identify interaction partners of Calypso, proteins associated with a TAP-Calypso fusion protein were purified from nuclear extracts of embryos that carried the α-tubulin1-TAP-calypso transgene. The purified material was separated on SDS-polyacrylamide gels and four major protein bands were identified. Sequencing of peptides from these bands by nanoelectrospray tandem mass spectrometry identified the 55-kDa band as the Calypso bait protein, whereas the other three bands all represented fragments of the PcG protein Additional sex combs (ASX). Analysis of other gel regions and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of total purified material confirmed that ASX was the main protein co-purifying with TAP-Calypso. ASX is a PcG protein required for long-term repression of HOX genes during Drosophila development, but it had not been identified in previously characterized PcG protein complexes and its molecular function has remained largely elusive. Calypso and ASX are thus components of a new, bona fide PcG protein complex that was named Polycomb repressive deubiquitinase (PR-DUB) (Scheuermann, 2010).

Testa were performed to see whether Drosophila PR-DUB complexes could be reconstituted from recombinant Calypso and ASX proteins. Using baculovirus vectors, Flag-Calypso and haemagglutinin (HA)-ASX(1-1668) or HA-ASX(2-337) were expressed as individual proteins in Sf21 cells, the cell lysates were mixed and Flag affinity purification was performed. This strategy resulted in the isolation of stable Calypso-ASX complexes and showed that Calypso interacts with the amino-terminal 337 amino acids of ASX. ASX also formed stable complexes with Calypso(C131S), a mutant Calypso protein in which the predicted catalytic cysteine in the UCH domain had been substituted by serine. The interaction between Calypso and ASX(2-337) was specific because ASX(2-337) did not bind to the PcG proteins Flag-ESC or Flag-Sce under the same assay conditions. Using the same strategy, it was found that human BAP1 also forms a stable complex with the N-terminal domain of human ASXL1 (ASXL1(2-365)) but not with the human PcG proteins BMI1 or RING1A). Like Drosophila Calypso and ASX, human BAP1 and ASXL1 proteins could thus also be assembled into a stable PR-DUB complex, demonstrating the evolutionary conservation of this interaction (Scheuermann, 2010).

On polytene chromosomes, ASX protein binds at chromosome intervals encompassing the HOX genes and at many other chromosomal sites that co-map with binding sites for other PcG proteins (Sinclair, 1998). The genome-wide PR-DUB binding profile in the chromatin of Drosophila larvae was determined by performing chromatin immunoprecipitation (ChIP) assays with antibodies against Calypso and ASX proteins. The precipitated material was hybridized to high-density whole-genome tiling arrays and analysed with TileMap, using a stringent cutoff. Genomic regions were considered that were only significantly enriched by both anti-Calypso and anti-ASX antibodies, and thus obtained a high-confidence set of 879 genomic sites bound by PR-DUB. The PR-DUB binding profile were considered along with the profiles of the PRC1 subunit Ph and the PhoRC subunit Pho in imaginal disc cells. PR-DUB is co-bound together with Ph and Pho at Polycomb response elements (PREs) of a large set of PcG target genes, such as the HOX gene. PR-DUB is thus a core PRE-binding complex, like PhoRC, PRC1 and PRC2. To extend these analyses, the binding of Calypso and ASX was considered in wing imaginal disc cells in which the HOX gene Ultrabithorax (Ubx) is inactive, and in haltere/third leg imaginal disc cells in which Ubx is expressed, at the same 16 locations across the Ubx transcription unit where binding of the PcG protein complexes PhoRC, PRC1 and PRC2 was previously analysed (Papp, 2006). Like these other PcG protein complexes, PR-DUB was bound at Ubx PREs both in cells where Ubx is repressed and in cells where it is active (Scheuermann, 2010).

To characterize the deubiquitinase activity of PR-DUB, whether the Drosophila complex could cleave the fluorogenic substrate ubiquitin-amidomethylcoumarin (Ub-AMC) was tested. Calypso alone hydrolysed the Ub-AMC bond, but the Calypso-ASX(2-337) complex was substantially more active in catalysing this reaction. PR-DUB thus functions as a deubiquitinase in vitro and the catalytic activity of Calypso is strongly enhanced by association with the N-terminal domain of ASX. Because Drosophila PR-DUB associated with the chromatin of target genes, it was then asked whether PR-DUB deubiquitinates histone H2A or H2B. Monoubiquitination of H2A (H2Aub1) at Lys 119 in vertebrates and Lys 118 in Drosophila by PRC1-like and dRAF, respectively, is thought to be critical for PcG repression. Monoubiquitination of H2B (H2Bub1) at Lys 120 in vertebrates (corresponding to Lys 117 in Drosophila) is catalysed by a different E3 ligase, RNF20 (also known as BRE1), and has been implicated in transcriptional elongation. Recombinant mononucleosomes that contained either H2Aub1 or H2Bub1 were reconstituted and used as substrates in deubiquitination assays. Notably, the Drosophila Calypso-ASX(2-337) and Calypso-ASX(1-1668) complexes and the human BAP1-ASXL1(2-365) complex all deubiquitinated H2Aub1 but not H2Bub1 in nucleosomes. Deubiquitination of H2Aub1 required both the presence of the catalytic cysteine in Calypso and the association of ASX with Calypso or of ASXL1 with BAP1, respectively. Moreover, PR-DUB showed only very poor activity for cleaving polyubiquitin chains that were linked through either Lys 63 or Lys 48. PR-DUB thus specifically deubiquitinated H2Aub1 in nucleosomes in these assays (Scheuermann, 2010).

How the lack of PR-DUB affects H2Aub1 levels was investigated in developing Drosophila. In embryos that are homozygous for Asx22P4, ASX protein is undetectable and Calypso protein levels are very drastically diminished. Asx22P4 mutant embryos thus have severely reduced levels of PR-DUB. Bulk histones were isolsted from wild-type and Asx22P4 homozygous embryos by acid extraction, and compared the levels of H2Aub1, H2Bub1, H3K27me3 and H4K4me3 in the two genotypes. Bulk H2Aub levels were almost tenfold increased in Asx22P4 mutant embryos. In contrast, the level of the PcG-specific histone tri-methylation mark H3K27me3 was comparable in Asx22P4 mutant and wild-type embryos. Unexpectedly, a weak increase in H2Bub levels was found and a very slight concomitant increase in H3K4me3 levels. The higher H2Bub levels could be an indirect consequence of widespread global H2A ubiquitination, but it is also possible that, in vivo, PR-DUB deubiquitinates both H2A and H2B. Previous studies reported that the monoclonal antibody E6C5 specifically recognizes H2Aub1 in mammalian cells, but it has not been possible to specifically monitor H2Aub1 levels by ChIP in Drosophila using the commercially available E6C5 antibody (Scheuermann, 2010).

Finally, whether the deubiquitinase activity of PR-DUB is required for PcG repression was tested. To this end a transgene rescue assay was used and it was asked whether the catalytically inactive Calypso(C131S) protein can repress the PcG target gene Ubx in Drosophila larvae, as follows. Clones of calypso2 mutant cells in larval imaginal discs lack detectable Calypso protein and fail to repress Ubx. However, a regular supply of wild-type Calypso protein from a heat-inducible hsp70-calypso transgene fully rescues repression of Ubx in such clones. In contrast, the catalytically inactive Calypso(C131S) protein expressed from a hsp70-calypso(C131S) transgene failed to rescue repression, and Ubx was misexpressed as in control animals lacking any hsp70-calypso transgene. PR-DUB deubiquitinase activity is thus critically required for repression of PcG target genes in Drosophila (Scheuermann, 2010).

The following conclusions can be drawn from the work reported in this study: PR-DUB is a new PcG protein complex that comprises the Calypso and ASX proteins; PR-DUB is bound at the PREs of PcG target genes in Drosophila; reconstituted recombinant Drosophila or human PR-DUB deubiquitinate H2A in nucleosomes in vitro; Drosophila mutants lacking PR-DUB show an increase in global H2Aub1 levels; and a mutation in Calypso that disrupts H2A deubiquitinase activity in vitro impairs repression of HOX genes in Drosophila. This analyses identified nucleosomal H2Aub1 as the preferred PR-DUB substrate; the complex failed to deubiquitinate nucleosomal H2Bub1 and showed only very poor activity for cleaving polyubiquitin chains. It is possible that PR-DUB also deubiquitinates other proteins, but this paper deals with its possible role in H2A deubiquitination. The observation that repression of PcG target genes in Drosophila requires not only the H2A ubiquitinase activity of PRC1 and dRAF but also PR-DUB may seem surprising. However, simultaneous depletion of Sce (that is, the H2A ubiquitinase subunit of PRC1 and dRAF and PR-DUB in embryos results in a more rapid loss of HOX gene repression and consequently more severe transformation of body segments than the depletion of Sce or PR-DUB alone. This suggests that appropriately balanced H2Aub1 levels in target gene chromatin may be critical for maintaining a Polycomb-repressed state. One possibility would be that PRC1/dRAF and PR-DUB act locally within target gene chromatin; the presence of H2Aub1 in some regions of a gene may be critical for repression but may be detrimental to it in others. Alternatively, H2A ubiquitination and deubiquitination may have to occur in a temporally regulated cycle to maintain repression, similar to what has been proposed for H2B ubiquitination and deubiquitination during transcriptional elongation. Interestingly, calypso and Asx mutant embryos show not only derepression of HOX genes but also a partial loss of HOX gene expression in the central nervous system. This loss of HOX gene expression seems to be restricted to the nervous system -- no reduction of HOX gene expression in the embryonic epidermis or in imaginal disc cells has been detected. Thus, even though PR-DUB is primarily required for repressing PcG target genes outside their expression domains, it might also be needed to fine-tune expression levels within these domains in certain tissues, perhaps by preventing repressive hyper-ubiquitination of H2A by PRC1 or dRAF complexes. It will be interesting to determine whether the mammalian complex has a similar prominent role in PcG repression during development and for maintenance of stem cell pluripotency, and to explore how the tumour suppressor activity of BAP1 relates to the H2A deubiquitinase activity of human PR-DUB (Scheuermann, 2010).

dRYBP counteracts chromatin-dependent activation and repression of transcription

Chromatin dependent activation and repression of transcription is regulated by the histone modifying enzymatic activities of the trithorax (trxG) and Polycomb (PcG) proteins. To investigate the mechanisms underlying their mutual antagonistic activities this study analyzed the function of Drosophila Ring and YY1 Binding Protein (dRYBP), a conserved PcG- and trxG-associated protein. dRYBP is ubiquitylated and binds ubiquitylated proteins. Additionally dRYBP was shown to maintain H2A monoubiquitylation, H3K4 monomethylation and H3K36 dimethylation levels and does not affect H3K27 trimethylation levels. Further it was shown that dRYBP interacts with the repressive SCE (Ring) and dKDM2 (Lysine (K)-specific demethylase 2) proteins as well as the activating dBRE1 protein. Analysis of homeotic phenotypes and post-translationally modified histones levels show that dRYBP antagonizes dKDM2 and dBRE1 functions by respectively preventing H3K36me2 demethylation and H2B monoubiquitylation. Interestingly, the results show that inactivation of dBRE1 produces trithorax-like related homeotic transformations, suggesting that dBRE1 functions in the regulation of homeotic genes expression. These findings indicate that dRYBP regulates morphogenesis by counteracting transcriptional repression and activation. Thus, they suggest that dRYBP may participate in the epigenetic plasticity important during normal and pathological development (Fereres, 2014).

PR-DUB preserves Polycomb repression by preventing excessive accumulation of H2Aub1, an antagonist of chromatin compaction

The Polycomb repressive complexes PRC1, PRC2, and PR-DUB repress target genes by modifying their chromatin. In Drosophila, PRC1 compacts chromatin and monoubiquitinates histone H2A at lysine 118 (H2Aub1), whereas PR-DUB is a major H2Aub1 deubiquitinase, but how H2Aub1 levels must be balanced for Polycomb repression remains unclear. This study shows that in early embryos, H2Aub1 is enriched at Polycomb target genes, where it facilitates H3K27me3 deposition by PRC2 to mark genes for repression. During subsequent stages of development, H2Aub1 becomes depleted from these genes and is no longer enriched when Polycomb maintains them repressed. Accordingly, Polycomb targets remain repressed in H2Aub1-deficient animals. In PR-DUB catalytic mutants, high levels of H2Aub1 accumulate at Polycomb target genes, and Polycomb repression breaks down. These high H2Aub1 levels do not diminish Polycomb protein complex binding or H3K27 trimethylation but increase DNA accessibility. H2Aub1 interferes with nucleosome stacking and chromatin fiber folding in vitro. Consistent with this, Polycomb repression defects in PR-DUB mutants are exacerbated by reducing PRC1 chromatin compaction activity, but Polycomb repression is restored if PRC1 E3 ligase activity is removed. PR-DUB therefore acts as a rheostat that removes excessive H2Aub1 that, although deposited by PRC1, antagonizes PRC1-mediated chromatin compaction (Bonne, 2022).

This study investigated the molecular function of H2Aub1 in developing Drosophila with a focus on elucidating why and how H2Aub1 deubiquitinase activity of PR-DUB is needed for Polycomb repression (Bonne, 2022).

The following main conclusions can be drawn from the work reported in this study. First, the genomic H2Aub1 profile shaped by the antagonistic actions of PRC1 and PR-DUB is unexpectedly dynamic during development. This balance permits PRC1 to generate H2Aub1 domains at Polycomb target genes in early embryos, but subsequently, it is H2Aub1 deubiquitination by PR-DUB that dominates and shapes a near-uniform, low-level H2Aub1 landscape in late stage embryos and larvae. Second, the early H2Aub1 domains are required for the rapid establishment of H3K27me3 domains; however, at HOX genes, this pathway does not appear to be critical because, with a delay, PRC2 generates H3K27me3 domains at these loci also in animals lacking H2Aub1. In contrast, the formation of noncanonical H3K27me3 domains strictly depends on H2Aub1. Third, in wild-type Drosophila, H2Aub1 is no longer enriched at Polycomb target genes during developmental stages when the Polycomb machinery acts to repress transcription of these genes. Together with the finding that Polycomb repression appears intact in animals lacking H2Aub1, this corroborates that chromatin compaction but not H2Aub1 is central to the PRC1 repression mechanism. Fourth, H2Aub1 inhibits chromatin fiber folding of nucleosome arrays in vitro. H2Aub1 therefore directly impacts the structural organization of chromatin. Fifth, in PR-DUB mutants, excessive H2Aub1 accumulation at HOX genes increases DNA accessibility and disrupts Polycomb repression at these loci. High H2Aub1 levels at these target genes are therefore detrimental to Polycomb repression. Sixth, removal of PRC1 E3 ligase activity restores Polycomb repression in PR-DUB mutants, whereas reduction of PRC1 chromatin compaction activity exacerbates the repression defects. This suggests that PR-DUB preserves Polycomb repression by antagonizing persistent H2Aub1 deposition by PRC1 in order to allow effective chromatin compaction by PRC1 (Bonne, 2022).

Atomic structures of diverse nucleosome arrays revealed that stacking interactions between the solvent-exposed surfaces of the histone octamers in n and n+2 nucleosomes are a common feature. In vivo, chromatin does not exist as long regularly folded fibers, but direct contacts between n and n+2 nucleosomes are also widely observed using sequencing-based approaches. Moreover, tomographic studies suggest that nucleosome stacking is a prevalent feature of chromatin organization in vertebrate nuclei. FRET measurements in this study show that these stacking interactions are critical for folding of unmodified nucleosome arrays in solution and that the presence of ubiquitin at H2AK119 sterically prevents nucleosome-nucleosome stacking. In conclusion, the results show that in addition to providing a binding site for PRC2.2 and variant PRC1 complexes, H2Aub1 can also directly impact the structural organization of chromatin (Bonne, 2022).

To relate the observations from these in vitro studies to the effects of H2Aub1 on chromatin compaction in vivo, it is important to consider the density of H2Aub1-modified nucleosomes within a given stretch of chromatin in cells. In late stage wild-type embryos, only a few percent of total H2A carry the ubiquitin modification. Considering that H2Aub1 is globally distributed across the entire genome in these embryos, one would expect that only a small fraction of nucleosomes across any given stretch of chromatin contains H2Aub1. During the stages when Polycomb repression acts, nucleosome stacking in Polycomb target gene chromatin is therefore unlikely to be grossly impaired. In contrast, the 20-fold to 30-fold increase in H2Aub1 nucleosome density in HOX gene chromatin in late stage PR-DUB mutant embryos could interfere with regular nucleosome stacking. The increased DNA accessibility at several Polycomb target genes in PR-DUB mutants is consistent with a less compact chromatin organization at these loci (Bonne, 2022).

The data argue that the high H2Aub1 accumulation in PR-DUB mutants does not interfere with Polycomb complex binding to PREs or H3K27me3 deposition in the flanking chromatin. The enhancement of the Polycomb repression defects in PR-DUB mutants with reduced PRC1 dosage (i.e., in Pc/+; Asx0 animals) is consistent with a scenario in which the excess of H2Aub1 directly impacts the ability of target gene-associated PRC1 to compact chromatin. This conclusion is in contrast to the recent suggestion that defective Polycomb repression in PR-DUB mutant mouse ESCs might be caused by reduced H3K27me3 deposition at Polycomb target genes. Specifically, these studies proposed that the genome-wide low increase in H2Aub1 levels in PR-DUB mutants would sequester PRC2.2 and thereby titrate it away from Polycomb target genes. The currenr data provide no support for such a scenario in Drosophila (Bonne, 2022). s

The morphological defects in PR-DUB mutants suggest that Polycomb repression is primarily disrupted at HOX genes. Moreover, the repression defects at HOX genes are remarkably tissue- and stage-specific and not as widespread as in the case of mutants lacking PRC1 or PRC2. Even at HOX genes, the high H2Aub1 levels therefore still permit the Polycomb machinery to sustain repression in a fraction of cells. A possible explanation could be that the high H2Aub1 nucleosome density antagonizes chromatin compaction by PRC1 but does not fully abrogate it, and consequently, only some but not all tissue-specific enhancers in HOX genes are able to overcome this residual repression (Bonne, 2022).

What is the molecular basis for the formation of very different genomic H2Aub1 profiles in early and late embryos? Absolute quantification of proteins revealed that the copy number of Sce molecules per nucleus is comparable in early and late stage embryos. In contrast, the copy numbers of Calypso and Asx proteins in nuclei from early stage embryos are very low, and both proteins are fivefold to sevenfold more abundant in nuclei from late stage embryos. The lower abundance of PR-DUB relative to PRC1 in 0- to 6-h-old embryos could therefore provide a simple mechanistic explanation why PRC1 is able to generate H2Aub1 domains at Polycomb target genes early but not late in embryogenesis. Alternative scenarios to explain the different H2Aub1 landscapes in early and late embryos could be modulation of PRC1 or PR-DUB activity or differences in genomic targeting of these complexes during the different stages of embryogenesis (Bonne, 2022).

The formation of the H3K27me3 landscape is thought to rely on the combined action of PRC2.1 and PRC2.2, whereby the relative contributions by these two complexes vary at different genomic locations. This study used H2Aub1-deficient embryos that lack the nucleosomal binding site specific for PRC2.2. H2Aub1-deficient embryos show a genome-wide reduction of the H3K27me3 profile that is particularly pronounced in early embryos. During this stage of development, H2Aub1 and PRC2.2 therefore contribute significantly to H3K27me3 domain formation, analogously to what has been reported in mouse ESCs. Intriguingly, in later stage H2Aub1-deficient embryos, regular domains with near-normal coverage of H3K27me3 appear at HOX and many other canonical Polycomb target genes. This recovery of H3K27me3 domains explains why H2Aub1-deficient animals do not show the repression defects associated with loss of PRC2 activity. The recovery of near-normal levels of H3K27me3 at canonical H3K27me3 domains in late stage H2Aub1-deficient Drosophila embryos argues that the system, at least in flies, is considerably more plastic than anticipated (Bonne, 2022).

A simple straightforward finding of this study was that H2Aub1 is not enriched at Polycomb target genes during the embryonic and larval stages when PRC1 is critically required to repress these genes. This is consistent with the finding that SceI48A mutants show no Polycomb mutant phenotypes and no general transcriptional deregulation of Polycomb target genes in late stage embryos. Together, these observations all argue against a critical role of H2Aub1 in the actual repression mechanism by which PRC1 blocks transcription. Identifying the reason why SceI48A or H2AK117R/K118R/K121R/K122R mutants die as late stage embryos will require further studies, including experiments to investigate whether the low uniform levels of H2Aub1 across the genome might have an essential function in a process other than transcriptional regulation (Bonne, 2022).

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

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

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

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

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

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

Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression

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

The role of the histone H2A ubiquitinase Sce in Polycomb repression

Polycomb group (PcG) proteins exist in multiprotein complexes that modify chromatin to repress transcription. Drosophila PcG proteins Sex combs extra (Sce; dRing) and Posterior sex combs (Psc) are core subunits of PRC1-type complexes. The Sce:Psc module acts as an E3 ligase for monoubiquitylation of histone H2A, an activity thought to be crucial for repression by PRC1-type complexes. This study created an Sce knockout allele and showed that depletion of Sce results in loss of H2A monoubiquitylation in developing Drosophila. Genome-wide profiling identified a set of target genes co-bound by Sce and all other PRC1 subunits. Analyses in mutants lacking individual PRC1 subunits reveals that these target genes comprise two distinct classes. Class I genes are misexpressed in mutants lacking any of the PRC1 subunits. Class II genes are only misexpressed in animals lacking the Psc-Su(z)2 and Polyhomeotic (Ph) subunits but remain stably repressed in the absence of the Sce and Polycomb (Pc) subunits. Repression of class II target genes therefore does not require Sce and H2A monoubiquitylation but might rely on the ability of Psc-Su(z)2 and Ph to inhibit nucleosome remodeling or to compact chromatin. Similarly, Sce does not provide tumor suppressor activity in larval tissues under conditions in which Psc-Su(z)2, Ph and Pc show such activity. Sce and H2A monoubiquitylation are therefore only crucial for repression of a subset of genes and processes regulated by PRC1-type complexes. Sce synergizes with the Polycomb repressive deubiquitinase (PR-DUB) complex to repress transcription at class I genes, suggesting that H2A monoubiquitylation must be appropriately balanced for their transcriptional repression (Gutiérrez, 2012).

This study analyzed how PRC1 regulates target genes in Drosophila to investigate how the distinct chromatin-modifying activities of this complex repress transcription in vivo. Because H2A monoubiquitylation is thought to be central to the repression mechanism of PRC1-type complexes, focus was placed on the role of Sce. The following main conclusions can be drawn from the work reported in this study. First, in the absence of Sce, bulk levels of H2A-K118ub1 are drastically reduced but the levels of the PRC1 subunits Psc and Ph are undiminished. Sce is therefore the major E3 ligase for H2A monoubiquitylation in developing Drosophila but is not required for the stability of other PRC1 subunits. Second, PRC1-bound genes fall into two classes. Class I target genes are misexpressed if any of the PRC1 subunits is removed. Class II target genes are misexpressed in the absence of Ph or Psc-Su(z)2 but remain stably repressed in the absence of Sce or Pc. At class II target genes, Ph and the Psc-Su(z)2 proteins work together to repress transcription by a mechanism that does not require Sce and Pc and is therefore independent of H2A monoubiquitylation. Third, removal of the Ph, Psc-Su(z)2 or Pc proteins results in imaginal disc tumors that are characterized by unrestricted cell proliferation. However, removal of Sce does not cause this phenotype, suggesting that this tumor suppressor activity by the PcG system does not require H2A monoubiquitylation. Finally, these analyses reveal that PRC1 subunits are essential for repressing the elB, noc, dac and pros genes outside of their normal expression domains in developing Drosophila. This expands the inventory of developmental regulator genes in Drosophila for which PcG repression has been demonstrated in a functional assay (Gutiérrez, 2012).

In the Sce33M2 allele Arg65 is mutated to Cys, but this mutant Sce protein is undetectable and therefore does not appear to be stable in vivo. The crystal structure of the Ring1B-Bmi1 complex provides a molecular explanation for this observation: the Arg70 residue in Ring1B that corresponds to Arg65 in Sce is thought to be critical for interaction with Bmi1. A likely scenario therefore is that the SceArg65Cys protein in Drosophila is unstable and is degraded because it is unable to associate with Psc or its paralog Su(z)2. Interestingly, removal of Sce protein has no detectable effect on the levels of the Psc and Ph proteins. Psc is therefore stable in the absence of its binding partner Sce. This is in contrast to the situation in mice in which Ring1B mutant ES cells show a drastic reduction in the levels of the Ring1B partner protein Bmi1 and its paralog Mel18 (Pcgf62) and also a reduction in the levels of Mph2 (Phc2) and Mpc2 (Cbx4) (Leeb, 2007). The interdependence between PRC1 subunits for protein stability is therefore different in mammals and Drosophila (Gutiérrez, 2012).

Reconstitution of the Drosophila PRC1 core complex in a baculovirus expression system suggests that Sce is important for complex stability. At present, it is not know whether the Psc, Ph and Pc proteins still form a complex in vivo in the absence of Sce. It is currently unknown whether Psc, Ph and Pc are still bound to all PRC1 target genes in the absence of Sce. However, the finding that class II genes remain repressed in the absence of Sce, even though their repression depends on Psc-Su(z)2 and Ph, argues against a crucial role of Sce in the targeting of these other PRC1 subunits to these genes. Interestingly, the repression of all class II target genes analyzed in this study always requires both the Ph and the Psc-Su(z)2 proteins. A possible explanation for this observation is that Ph and Psc-Su(2) still form a PRC1 subcomplex in the absence of Sce and that this complex is fully functional to repress class II target genes. Alternatively, it is possible that Ph and Psc-Su(z)2 repress class II target genes as components of as yet uncharacterized complexes that are distinct from PRC1 and Drosophila dRing-associated factors (dRAF) complex (Gutiérrez, 2012).

In vitro, Psc and Su(z)2 proteins compact nucleosome templates, inhibit nucleosome remodeling by SWI/SNF complexes and repress transcription on chromatin templates. The observation that repression of class II target genes requires Psc-Su(z)2 and Ph but not Pc and Sce supports the idea that the chromatin-modifying activities of Psc-Su(z)2 identified in vitro are the main mechanism by which PRC1 represses these genes. Previous structure/function analyses in Drosophila showed that the same domains of the Psc protein responsible for chromatin compaction and remodeling inhibition in vitro are crucial for HOX gene repression in vivo. Chromatin modification by Psc and Su(z)2 is therefore also crucial for repression of class I target genes. Regulation of the class I target gene en further illustrates this point. In some tissues (e.g. in the dorsal hinge region of the wing imaginal disc) repression of en requires all PRC1 core subunits, but in other tissues (e.g. in the pouch of the wing imaginal disc) en remains repressed in the absence of Sce and Pc, and only Psc-Su(z)2 and Ph seem to be crucial to keep the gene inactive. At present, the molecular mechanism of Ph is not well understood. In vitro, Ph protein has the capacity to inhibit chromatin remodeling and transcription but it does so less effectively than Psc. At the target genes analyzed in this study, Ph is required for transcriptional repression wherever Psc-Su(z)2 is required, suggesting that Ph and Psc-Su(z)2 act in concert in this repression. Nevertheless, it is possible that repression of other PRC1 target genes requires a different subset of PRC1 subunits, or that, as in the case of en, the subunit requirement for repression changes depending on the cell type (Gutiérrez, 2012).

In mammals, Ring1B and Ring1A are responsible for the bulk of H2A-K119 monoubiquitylation. Similarly, Sce generates the bulk of H2A-K118 monoubiquitylation in Drosophila, both in tissue culture cells (Lagarou, 2008) and in the developing organism (this study). The requirement for Sce at class I target genes is consistent with the idea that H2A monoubiquitylation of their chromatin is part of the repression mechanism. Repression of a subset of class I genes, namely the HOX genes, also requires the H2A deubiquitinase PR-DUB (Gayt├ín de Ayala Alonso, 2007; Scheuermann, 2010). Moreover, PR-DUB and Sce strongly synergize to repress HOX genes. Specifically, the phenotype of Sce PR-DUB double mutants suggests that H2A monoubiquitylation becomes ineffective for HOX gene repression if PR-DUB is absent. However, embryos that lack PR-DUB alone show a 10-fold increase in the bulk levels of H2A-K118ub1 and it is estimated that ~10% of all H2A molecules become monoubiquitylated in these animals. How could this conundrum be explained? One possibility is that H2A monoubiquitylation and deubiquitylation at HOX gene chromatin need to be regulated in a precisely balanced manner. However, an alternative explanation considers H2A-K118ub1 levels in the context of ubiquitin homeostasis. In particular, the high H2A-K118ub1 levels in PR-DUB mutants suggest that Sce generates widespread H2A monoubiquitylation at most Sce-bound genes and possibly also elsewhere in the genome, but that in wild-type animals PR-DUB continuously deubiquitylates H2A-K118ub1 at these locations and thereby recycles ubiquitin. The observation that PR-DUB is widely co-bound with Sce, not only at HOX but also at many other class I and class II target genes, is consistent with this idea. It is tempting to speculate that the widespread H2A monoubiquitylation in PR-DUB mutants sequesters a substantial fraction of the pool of free ubiquitin. It is therefore possible that removal of PR-DUB effectively depletes the pool of free ubiquitin in the nucleus to an extent that H2A monoubiquitylation at HOX target genes becomes inefficient and, consequently, their repression can no longer be maintained. According to this model, the crucial function of PR-DUB would not be the deubiquitylation of H2A-K118ub1 at HOX genes but rather at class II target genes and elsewhere in the genome where Sce 'wastefully' monoubiquitylates H2A (Gutiérrez, 2012).

Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression

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

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

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

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

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

A histone code in meiosis: the histone kinase, NHK-1, is required for proper chromosomal architecture in Drosophila oocytes

To promote faithful propagation of the genetic material during sexual reproduction, meiotic chromosomes undergo specialized morphological changes that ensure accurate segregation of homologous chromosomes. The molecular mechanisms that establish the meiotic chromosomal structures are largely unknown. This study describes a mutation in a recently identified Histone H2A kinase, nhk-1 (ballchen), in Drosophila that leads to female sterility due to defects in the formation of the meiotic chromosomal structures. The metaphase I arrest and the karyosome, a critical prophase I chromosomal structure, require nucleosomal histone kinase-1 (NHK-1) function. The defects are a result of failure to disassemble the synaptonemal complex and to load condensin onto the mutant chromosomes. Embryos laid by nhk-1-/- mutant females arrest with aberrant polar bodies and mitotic spindles, revealing that mitosis is affected as well. The role of Histone H2A phosphorylation was analyzed with respect to the histone code hypothesis and it was found to be required for acetylation of Histone H3 and Histone H4 in meiosis. These studies reveal a critical role for histone modifications in chromosome dynamics in meiosis and mitosis (Ivanovska, 2005).

This study explored the functional requirements for a newly identified histone kinase in meiosis and found that NHK-1 functions in meiotic progression. The phenotypes of the female-sterile nhk-1Z3-0437 mutant showed that NHK-1 is required for the establishment of several meiosis-specific chromosomal configurations, including the prophase I karyosome, the metaphase I spindle, and the polar body. Histone H2AT119ph, as well as Histone H3K14ac and Histone H4K5ac, were reduced in the mutant oocytes, whereas the other histone modifications examined were unaffected. Strikingly, disassembly of the SC and loading of condensin failed in the mutant. Therefore, it is suggested that NHK-1 and Histone H2AT119ph, the C-terminus of H2A, are required specifically for proper chromosome architecture in meiosis (Ivanovska, 2005).

The histone code hypothesis conjectures functional interactions among histone modifications and between modifications and proteins that bind to them. It has been postulated that histones have signature modification profiles in meiosis to accommodate the meiosis-specific chromosomal events. However, most research in vertebrates has been limited to identifying the modifications and has not provided extensive insight into their function (Ivanovska, 2005).

The finding that NHK-1 is required for meiosis, presumably via its phosphorylation of Histone H2AT119 (Aihara 2004), prompted an examination of the presence of other histone modifications in Drosophila oocytes. It was found that Histone H1 is phosphorylated, HP1 is bound (indicative of histone H3 methylation), and Histone H3 and H4 are acetylated during prophase I of meiosis in Drosophila. The presence of these histone modifications in oocyte nuclei suggests that they play a role in chromosome dynamics during meiosis. It is of interest to note that Histone H4 and H3 were shown to be acetylated in mouse oocytes in prophase I, suggesting an evolutionary conservation. Future analysis of mutations in histone modifying enzymes may shed light on the functions of these modifications in meiosis (Ivanovska, 2005).

One stipulation of the histone code is that histone modifications affect each other. To test this hypothesis with respect to Histone H2AT119ph, the panel of histone modifications was examined in the nhk-1Z3-0437 mutant ovaries. Histone H1ph, HP1 binding, and Histone H4K12ac were unaffected in the mutant oocytes, consistent with Histone H2AT119ph being downstream or independent of them in Drosophila meiosis. In contrast, Histone H3K14ac and Histone H4K5ac were absent specifically from the chromosomes in the nhk-1Z3-0437 mutant oocytes, indicating that Histone H2AT119ph is a prerequisite for acetylation of these residues. Although the significance of these acetylations is unclear at present, it is intriguing to speculate that they play an important role in meiosis (Ivanovska, 2005).

Another stipulation of the histone code is that modifications recruit or exclude proteins from binding to chromatin. One possibility is that Histone H2AT119ph is required for function of the histone acetyltransferases for Histone H3K14 and Histone H4K5, the residues that lack acetylation in the mutant oocytes. There is a precedent for such a cascade of dependencies in transcription: Phosphorylation of H3 Ser 10 leads to acetylation of H3 Lys 14. Histone H2AT119 phosphorylation may also be a prerequisite for proper chromosomal associations of SMC4 and for dissociation of the SC (Ivanovska, 2005).

In conclusion, Histone H2AT119 phosphorylation appears to affect both other histone modifications and the binding of proteins to chromatin as predicted by the histone code hypothesis. Histone H2AT119 phosphorylation by NHK-1 may, therefore, be a key component of a meiotic histone code in oocytes (Ivanovska, 2005).

The Drosophila karyosome is a subnuclear organelle comprised of the prophase I chromosomes. It is the best studied example of a family of similar structures found throughout evolution. Although the exact function of the karyosome is unclear, several Drosophila mutants that disrupt karyosome structure lead to female sterility, suggesting that karyosome formation is required for fertility. In addition, it has been postulated that retaining the oocyte chromosomes in close proximity within the large oocyte nucleus (the germinal vesicle) facilitates proper chromosome segregation during the meiotic divisions. Therefore, understanding the molecular mechanisms that regulate karyosome formation is of great interest (Ivanovska, 2005).

The spindle mutants in Drosophila (spindle-B, spindle-D, and okra) are characterized by defects in karyosome formation, in establishment of the dorsal/ventral polarity of the oocyte and the egg, and defects in repair of the DSBs following recombination. The nhk-1Z3-0437 mutant does not show either of the latter phenotypes, consistent with the NHK-1 histone kinase having a primary role in karyosome formation, whereas the spindle mutants may affect karyosome function as a secondary consequence of other defects. The specific requirement for NHK-1 implicates Histone H2AT119ph in formation of the karyosome. The conservation of the NHK-1 kinase and Thr 119 in Histone H2A among diverse species suggests that this histone modification plays a conserved role in chromosomal dynamics in meiosis (Ivanovska, 2005).

The nhk-1Z3-0437 mutant showed several interesting phenotypes in the oocyte. First, all embryos laid by mutant females showed aberrant polar bodies and an arrest at metaphase of the first mitotic division. Second, a defect was observed in the metaphase I arrest in 50% of late-stage oocytes. Finally, the karyosome failed to form in all early oocytes. It is not clear how the dispersed chromosomes in the mutant karyosomes later form three masses at metaphase I, but it suggests that the chromosomes are not fragmented and that bivalent associations are retained (Ivanovska, 2005).

These results can be interpreted in two ways. First, NHK-1 may be required continuously throughout meiosis for each of the events affected by the Z3-0437 mutation. Continuous requirement may be due to multiple substrates or due to dynamic Histone H2A phosphorylation and dephosphorylation. Second, NHK-1 function may be required specifically for karyosome formation, whereas the metaphase I and the embryonic phenotypes may be a consequence of the karyosome defect. This model suggests that Histone H2AT119ph serves as an epigenetic mark that contributes to proper completion of later events, such as establishment of the metaphase I arrest and progression through the first mitotic division. To test whether the early mitotic defects in the nhk-1 mutant were due to a failure to load condensin in mitosis, the dCAP-D2 condensin subunit was examined and it was found to localize onto chromosomes in embryos from nhk-1 mutant females. Nevertheless, it is thought likely that NHK-1 plays essential roles in mitosis that may be revealed by additional alleles of nhk-1 (Ivanovska, 2005).

Histone H2AT119 is phosphorylated in the follicle cells, the nurse cells, and the oocyte, suggesting that NHK-1 functions in all three ovarian cell types. However, mutant phenotypes were observed specifically in the oocyte. There are two reasons why Histone H2A phosphorylation persists in the mutant nurse and follicle cells and mutant defects were not observed. First, Histone H2A may be phosphorylated by another kinase in the nurse and follicle cells. Second, NHK-1 may phosphorylate Histone H2A in all cell types, but the oocyte may require higher levels of kinase activity for phosphorylation. The levels of Histone H2AT119ph may be reduced in the mutant nurse and follicle cells, but the concentration on these polyploid chromosomes could still permit detection by the antibody. The second idea is favored because partial loss-of-function alleles of essential genes often lead to female sterility. The observation that the nhk-1Z3-0437/Df(3R)Tl-I allelic combination has a stronger mutant phenotype than the nhk-1Z3-0437/Z3-0437 allelic combination is consistent with nhk-1Z3-0437 being a partial loss-of-function allele. Stronger alleles of nhk-1 may, therefore, reveal additional requirements for this kinase in other cell types, including in male meiosis (Ivanovska, 2005).

In conclusion, by using a female-sterile Drosophila mutant, a key step was uncovered in the formation of the karyosome, a conserved structure with an elusive function. Based on the analysis of the mutant phenotype, the following model for karyosome formation is proposed: Following DSB repair and dephosphorylation of Histone H2AvS139, phosphorylation of Histone H2AT119 by NHK-1 leads to disassembly of the SC, loading of condensin onto the chromosomes, and subsequent condensation into a karyosome. In addition to providing insight into karyosome formation, the nhk-1Z3-0437 mutant is an excellent tool for elucidating the interactions of a specific histone modification within the histone code (Ivanovska, 2005).

Nucleosomal histone kinase-1 phosphorylates H2A Thr 119 during mitosis in the early Drosophila embryo

Posttranslational histone modifications are important for the regulation of many biological phenomena. This study shows the purification and characterization of nucleosomal histone kinase-1 (NHK-1). NHK-1 has a high affinity for chromatin and phosphorylates a novel site, Thr 119, at the C terminus of H2A. Notably, NHK-1 specifically phosphorylates nucleosomal H2A, but not free H2A in solution. In Drosophila embryos, phosphorylated H2A Thr 119 is found in chromatin, but not in the soluble core histone pool. Immunostaining of NHK-1 revealed that it goes to chromatin during mitosis and is excluded from chromatin during S phase. Consistent with the shuttling of NHK-1 between chromatin and cytoplasm, H2A Thr 119 is phosphorylated during mitosis but not in S phase. These studies reveal that NHK-1-catalyzed phosphorylation of a conserved serine/threonine residue in H2A is a new component of the histone code that might be related to cell cycle progression (Aihara, 2004: full text of article).

Role of histone H2A ubiquitination in polycomb silencing

Covalent modification of histones is important in regulating chromatin dynamics and transcription. One example of such modification is ubiquitination, which mainly occurs on histones H2A and H2B. Although recent studies have uncovered the enzymes involved in histone H2B ubiquitination and a 'cross-talk' between H2B ubiquitination and histone methylation, the responsible enzymes and the functions of H2A ubiquitination are unknown. This study reports the purification and functional characterization of an E3 ubiquitin ligase complex that is specific for histone H2A. The complex, termed hPRC1L (human Polycomb repressive complex 1-like), is composed of several Polycomb-group proteins including Ring1, Ring2, Bmi1 and HPH2. hPRC1L monoubiquitinates nucleosomal histone H2A at lysine 119. Reducing the expression of Ring2 results in a dramatic decrease in the level of ubiquitinated H2A in HeLa cells. Chromatin immunoprecipitation analysis demonstrated colocalization of dRing with ubiquitinated H2A at the PRE and promoter regions of the Drosophila Ubx gene in wing imaginal discs. Removal of dRing in SL2 tissue culture cells by RNA interference resulted in loss of H2A ubiquitination concomitant with derepression of Ubx. Thus, these studies identify the H2A ubiquitin ligase, and link H2A ubiquitination to Polycomb silencing (Wang, 2004).


Search PubMed for articles about Drosophila Histone H2a

Aihara H., et al. (2004). Nucleosomal histone kinase-1 phosphorylates H2A Thr 119 during mitosis in the early Drosophila embryo. Genes Dev. 18: 877-888. PubMed ID: 15078818

Bonnet, J., Boichenko, I., Kalb, R., Le Jeune, M., Maltseva, S., Pieropan, M., Finkl, K., Fierz, B. and Muller, J. (2022). PR-DUB preserves Polycomb repression by preventing excessive accumulation of H2Aub1, an antagonist of chromatin compaction. Genes Dev. PubMed ID: 36357125

Fereres, S., Simon, R., Mohd-Sarip, A., Verrijzer, C. P. and Busturia, A. (2014). dRYBP counteracts chromatin-dependent activation and repression of transcription. PLoS One 9: e113255. PubMed ID: 25415640

Gaytán de Ayala Alonso A, et al. (2007). A genetic screen identifies novel polycomb group genes in Drosophila. Genetics. 176: 2099-2108. PubMed ID: 17717194

Gutiérrez, L., et al. (2012). The role of the histone H2A ubiquitinase Sce in Polycomb repression. Development 139(1): 117-27. PubMed ID: 22096074

Ivanovska, I., Khandan, T., Ito, T. and Orr-Weaver, T. L. (2005). A histone code in meiosis: the histone kinase, NHK-1, is required for proper chromosomal architecture in Drosophila oocytes. Genes Dev. 19(21): 2571-82. PubMed ID: 16230526

Kalb, R., Latwiel, S., Baymaz, H. I., Jansen, P. W., Muller, C. W., Vermeulen, M., Muller, J. (2014) Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression. Nat Struct Mol Biol 21: 569-571. PubMed ID: 24837194

Lagarou, A., et al. (2008). dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing. Genes Dev. 22: 2799-2810. PubMed ID: 18923078

Leeb, M. and Wutz, A. (2007). Ring1B is crucial for the regulation of developmental control genes and PRC1 proteins but not X inactivation in embryonic cells. J. Cell Biol. 178: 219-229. PubMed ID: 17620408

Papp, B. and Müller, J. (2006). Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins. Genes Dev. 20: 2041-2054. PubMed ID: 16882982

Scheuermann, J. C., et al. (2010). Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB. Nature 465: 243-247. PubMed ID: 20436459

Sinclair, D. A. R., et al. (1998), The Additional sex combs gene of Drosophila encodes a chromatin protein that binds to shared and unique Polycomb group sites on polytene chromosomes. Development 125: 1207-1216. PubMed ID: 9477319

Wang, H., Wang, L., Erdjument-Bromage, H., Vidal, M., Tempst, P., Jones, R.S. and Zhang, Y. (2004). Role of histone H2A ubiquitination in polycomb silencing. Nature 431(7010): 873-878. PubMed ID: 15386022

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date revised: 29 August 2023

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