Histone H3.3A: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Histone H3.3A

Synonyms -

Cytological map position - 25C3

Function - chromatin protein

Keywords - replacement histone, nucleosome assembly associated with transcriptional activation

Symbol - His3.3A

FlyBase ID: FBgn0014857

Genetic map position - 2L

Classification - Histone H3

Cellular location - nuclear

NCBI link: Entrez Gene
His3.3A orthologs: Biolitmine
Recent literature
Paranjape, N. P. and Calvi, B. R. (2016). The histone variant H3.3 is enriched at Drosophila amplicon origins but does not mark them for activation. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27172191
Eukaryotic DNA replication begins from multiple origins. The origin recognition complex (ORC) binds origin DNA and scaffolds assembly of a pre-Replicative Complex (pre-RC), which is subsequently activated to initiate DNA replication. In multicellular eukaryotes, origins do not share a strict DNA consensus sequence and their activity changes in concert with chromatin status during development, but mechanisms are ill-defined. Previous genome-wide analyses in Drosophila and other organisms have revealed a correlation between ORC binding sites and the histone variant H3.3. This correlation suggests that H3.3 may designate origin sites, but this idea has remained untested. To address this question, this study examined the enrichment and function of H3.3 at the origins responsible for developmental gene amplification in the somatic follicle cells of the Drosophila ovary. H3.3 was found to be abundant at these amplicon origins. H3.3 levels remained high when replication initiation was blocked, indicating that H3.3 is abundant at the origins before activation of the pre-RC. H3.3 was also enriched at the origins during early oogenesis, raising the possibility that H3.3 bookmarks sites for later amplification. However, flies null mutant for both of the H3.3 genes in Drosophila did not have overt defects in developmental gene amplification or genomic replication, suggesting that H3.3 is not essential for the assembly or activation of the pre-RC at origins. Instead, the results imply that the correlation between H3.3 and ORC sites reflects other chromatin attributes that are important for origin function.
Penke, T. J. R., McKay, D. J., Strahl, B. D., Matera, A. G. and Duronio, R. J. (2018). Functional redundancy of variant and canonical histone H3 Lysine 9 modification in Drosophila. Genetics 208(1):229-244. PubMed ID: 29133298
Histone post-translational modifications (PTMs) and differential incorporation of variant and canonical histones into chromatin are central modes of epigenetic regulation. Despite similar protein sequences, histone variants are enriched for different suites of PTMs compared to their canonical counterparts. For example, variant histone H3.3 occurs primarily in transcribed regions and is enriched for "active" histone PTMs like Lys9 acetylation (H3.3K9ac), whereas the canonical histone H3 is enriched for Lys9 methylation (H3K9me), which is found in transcriptionally silent heterochromatin. To determine the functions of K9 modification on variant versus canonical H3, the phenotypes caused by engineering H3.3(K9R) and H3(K9R) mutant genotypes in Drosophila melanogaster were compared. Whereas most H3.3(K9R) and a small number of H3(K9R) mutant animals are capable of completing development and do not have substantially altered protein coding transcriptomes, all H3.3(K9R) H3(K9R) combined mutants die soon after embryogenesis and display decreased expression of genes enriched for K9ac. These data suggest that the role of K9ac in gene activation during development can be provided by either H3 or H3.3. Conversely, it was found that H3.3K9 is methylated at telomeric transposons, and this mark contributes to repressive chromatin architecture, supporting a role for H3.3 in heterochromatin that is distinct from that of H3. Thus, these genetic and molecular analyses demonstrate that K9 modification of variant and canonical H3 have overlapping roles in development and transcriptional regulation, though to differing extents in euchromatin and heterochromatin.
Berlandi, J., Chaouch, A., De Jay, N., Tegeder, I., Thiel, K., Shirinian, M., Kleinman, C. L., Jeibmann, A., Lasko, P., Jabado, N. and Hasselblatt, M. (2019). Identification of genes functionally involved in the detrimental effects of mutant histone H3.3-K27M in Drosophila melanogaster. Neuro Oncol. PubMed ID: 30715493
Recurrent specific mutations in evolutionarily conserved Histone 3 (H3) variants drive pediatric high-grade gliomas (HGG), but little is known about their downstream effects. The aim of this study was to identify genes involved in the detrimental effects of mutant H3.3-K27M, the main genetic driver in lethal midline HGG, in a transgenic Drosophila model. Mutant and wild-type histone H3.3 expressing flies were generated using a phiC31-based integration system. Genetic modifier screens were performed by crossing H3.3-K27M expressing driver strains and 194 fly lines expressing shRNA targeting genes selected based on their potential role in the detrimental effects of mutant H3. Expression of the human orthologues of genes with functional relevance in the fly model was validated in H3-K27M mutant HGG. Ubiquitous and midline glia-specific expression of H3.3-K27M but not wild-type H3.3 caused pupal lethality, morphological alterations and decreased H3K27me3. Knockdown of 17 candidate genes shifted the lethal phenotype to later stages of development. These included histone modifying and chromatin remodeling genes as well as genes regulating cell differentiation and proliferation. Notably, several of these genes were over-expressed in mutant H3-K27M mutated HGG. It is concluded that rapid screening, identification and validation of relevant targets in "oncohistone" mediated pathogenesis has proven a challenge and a barrier to providing novel therapies. These results provide further evidence on the role of chromatin modifiers in the genesis of H3.3-K27M. Notably, they validate Drosophila as a model system for rapid identification of relevant genes functionally involved in the detrimental effects of H3.3-K27M mutagenesis.
Ahmad, K. and Henikoff, S. (2021). The H3.3K27M oncohistone antagonizes reprogramming in Drosophila. PLoS Genet 17(7): e1009225. PubMed ID: 34280185
Development proceeds by the activation of genes by transcription factors and the inactivation of others by chromatin-mediated gene silencing. In certain cases development can be reversed or redirected by mis-expression of master regulator transcription factors. This must involve the activation of previously silenced genes, and such developmental aberrations are thought to underlie a variety of cancers. This study expressed the wing-specific Vestigial master regulator to reprogram the developing eye, and test the role of silencing in reprogramming using an H3.3K27M oncohistone mutation that dominantly inhibits histone H3K27 trimethylation. Production of the oncohistone was found to block eye-to-wing reprogramming. CUT&Tag chromatin profiling of mutant tissues shows that H3K27me3 of domains is generally reduced upon oncohistone production, suggesting that a previous developmental program must be silenced for effective transformation. Strikingly, Vg and H3.3K27M synergize to stimulate overgrowth of eye tissue, a phenotype that resembles that of mutations in Polycomb silencing components. Transcriptome profiling of elongating RNA Polymerase II implicates the mis-regulation of signaling factors in overgrowth. These results demonstrate that growth dysregulation can result from the simple combination of crippled silencing and transcription factor mis-expression, an effect that may explain the origins of oncohistone-bearing cancers.
Chaouch, A., Berlandi, J., Chen, C. C. L., Frey, F., Badini, S., Harutyunyan, A. S., Chen, X., Krug, B., Hebert, S., Jeibmann, A., Lu, C., Kleinman, C. L., Hasselblatt, M., Lasko, P., Shirinian, M. and Jabado, N. (2021). Histone H3.3 K27M and K36M mutations de-repress transposable elements through perturbation of antagonistic chromatin marks. Mol Cell. PubMed ID: 34739871
Histone H3.3 lysine-to-methionine substitutions K27M and K36M impair the deposition of opposing chromatin marks, H3K27me3/me2 and H3K36me3/me2. This study shows that these mutations induce hypotrophic and disorganized eyes in Drosophila eye primordia. Restriction of H3K27me3 spread in H3.3K27M and its redistribution in H3.3K36M result in transcriptional deregulation of PRC2-targeted eye development and of piRNA biogenesis genes, including krimp. Notably, both mutants promote redistribution of H3K36me2 away from repetitive regions into active genes, which associate with retrotransposon derepression in eye discs. Aberrant expression of krimp represses LINE retrotransposons but does not contribute to the eye phenotype. Depletion of H3K36me2 methyltransferase ash1 in H3.3K27M, and of PRC2 component E(z) in H3.3K36M, restores the expression of eye developmental genes and normal eye growth, showing that redistribution of antagonistic marks contributes to K-to-M pathogenesis. These results implicate a novel function for H3K36me2 and showcase convergent downstream effects of oncohistones that target opposing epigenetic marks (Chaouch, 2021).
Farago, A., Zsindely, N., Farkas, A., Neller, A., Siagi, F., Szabo, M. R., Csont, T. and Bodai, L. (2022). Acetylation State of Lysine 14 of Histone H3.3 Affects Mutant Huntingtin Induced Pathogenesis. Int J Mol Sci 23(23). PubMed ID: 36499499
Huntington's Disease (HD) is a fatal neurodegenerative disorder caused by the expansion of a polyglutamine-coding CAG repeat in the Huntingtin gene. One of the main causes of neurodegeneration in HD is transcriptional dysregulation that, in part, is caused by the inhibition of histone acetyltransferase (HAT) enzymes. HD pathology can be alleviated by increasing the activity of specific HATs or by inhibiting histone deacetylase (HDAC) enzymes. To determine which histone's post-translational modifications (PTMs) might play crucial roles in HD pathology, this study investigated the phenotype-modifying effects of PTM mimetic mutations of variant histone H3.3 in a Drosophila model of HD. Specifically, the mutations (K→Q: acetylated; K→R: non-modified; and K→M: methylated) of lysine residues K9, K14, and K27 of transgenic H3.3 was studied. In the case of H3.3K14Q modification, the amelioration was observed of all tested phenotypes (viability, longevity, neurodegeneration, motor activity, and circadian rhythm defects), while H3.3K14R had the opposite effect. H3.3K14Q expression prevented the negative effects of reduced Gcn5 (a HAT acting on H3K14) on HD pathology, while it only partially hindered the positive effects of heterozygous Sirt1 (an HDAC acting on H3K14). Thus, it is concluded that the Gcn5-dependent acetylation of H3.3K14 might be an important epigenetic contributor to HD pathology.
Salzler, H. R., Vandadi, V., McMichael, B. D., Brown, J. C., Boerma, S. A., Leatham-Jensen, M. P., Adams, K. M., Meers, M. P., Simon, J. M., Duronio, R. J., McKay, D. J. and Matera, A. G. (2023). Distinct roles for canonical and variant histone H3 lysine-36 in Polycomb silencing. Sci Adv 9(9): eadf2451. PubMed ID: 36857457
Polycomb complexes regulate cell type-specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. This study showa that mutants of Drosophila replication-dependent (H3.2(K36R)) or replication-independent (H3.3(K36R)) histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined (H3.3(K36R)H3.2(K36R)) mutants display widespread Hox gene misexpression and fail to develop past the first larval stage. Chromatin profiling revealed that the H3.2(K36R) mutation disrupts H3K27me3 levels broadly throughout silenced domains, whereas these regions are mostly unaffected in H3.3(K36R) animals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive Polycomb response elements located outside of silenced domains but relatively depleted from those inside. It is concluded that H3.2 and H3.3 K36 residues collaborate to repress Hox genes using different mechanisms.
McPherson, J. E., Grossmann, L. C., Salzler, H. R., Armstrong, R. L., Kwon, E., Matera, A. G., McKay, D. J. and Duronio, R. J. (2023). Reduced histone gene copy number disrupts Drosophila Polycomb function. Genetics 224(4). PubMed ID: 37279945
The chromatin of animal cells contains two types of histones: canonical histones that are expressed during S phase of the cell cycle to package the newly replicated genome, and variant histones with specialized functions that are expressed throughout the cell cycle and in non-proliferating cells. This study demonstrates that variant histone H3.3 is essential for Drosophila development only when canonical histone gene copy number is reduced, suggesting that coordination between canonical H3.2 and variant H3.3 expression is necessary to provide sufficient H3 protein for normal genome function. To identify genes that depend upon, or are involved in, this coordinate regulation a screen was performed for heterozygous chromosome 3 deficiencies that impair development of flies bearing reduced H3.2 and H3.3 gene copy number. We identified two regions of chromosome 3 that conferred this phenotype, one of which contains the Polycomb gene, which is necessary for establishing domains of facultative chromatin that repress master regulator genes during development. It was further found that reduction in Polycomb dosage decreases viability of animals with no H3.3 gene copies. Moreover, heterozygous Polycomb mutations result in de-repression of the Polycomb target gene Ubx and cause ectopic sex combs when either canonical or variant H3 gene copy number is reduced. It is concluded that Polycomb-mediated facultative heterochromatin function is compromised when canonical and variant H3 gene copy number falls below a critical threshold.

DNA in eukaryotic cells is packaged into nucleosomes, the structural unit of chromatin. Both DNA and bulk histones are extremely long-lived, because old DNA strands and histones are retained when chromatin duplicates. In contrast, the Drosophila HSP70 genes rapidly lose histone H3 and acquire variant H3.3 histones as these genes are induced. Histone replacement does not occur at artificial HSP70 promoter arrays, demonstrating that transcription is required for H3.3 deposition. The H3.3 histone is enriched in all active chromatin and throughout large transcription units, implying that deposition occurs during transcription elongation. Strikingly, the stability of chromatin-bound H3.3 differs between loci: H3.3 turns over at continually active rDNA genes, but becomes stable at induced HSP70 genes that have shut down. It is concluded that H3.3 deposition is coupled to transcription, and continues while a gene is active. Repeated histone replacement suggests a mechanism to both maintain the structure of chromatin and access to DNA at active genes (Schwartz, 2005).

Transcriptional regulation in eukaryotes requires the coordinated recruitment of RNA polymerase II along with a variety of activators, repressors, chromatin-remodeling factors, and other chromatin-associated proteins. Histone variants have also been implicated in transcriptional regulation. All eukaryotic genomes encode four conserved core histones that package bulk chromatin, but a fraction of chromatin is packaged by alternate histones. Three lines of evidence support the argument that an alternate H3 histone subtype in animals -- the variant H3.3 -- plays a role in transcriptionally active chromatin: (1) a pulse of epitope-tagged H3.3 protein localized to gene-rich chromatin in Drosophila cells (Ahmad 2002); (2) H3.3 became enriched at a transgene array after its induction (Janicki, 2004); (3) H3.3 is enriched for covalent histone modifications associated with active chromatin, while repressive modifications are enriched on the H3 histone (McKittrick, 2004). These observations suggest that H3.3 is a common component of active chromatin (Schwartz, 2005).

Studies of the H3.3 variant have also suggested that distinctive nucleosome assembly occurs in active chromatin. Recent biochemical and in vivo assays demonstrate that there are both DNA replication-coupled and replication-independent (RI) nucleosome assembly pathways in eukaryotic nuclei. DNA replication-coupled assembly serves to package newly synthesized DNA into nucleosomes, but the existence of RI assembly pathways implies that previously chromatinized templates are also loading new histones. One RI pathway exclusively deposits newly synthesized histone H4 and the H3.3 variant and operates specifically at active chromatin in interphase nuclei (Ahmad, 2002; Tagami, 2004). However, the relationship between transcriptional activity and H3.3 has not been defined. It is unknown if H3.3 deposits during or after transcription of a gene, or how frequently RI assembly occurs. Conceivably, occasional RI assembly would have little effect on the physical properties of chromatin and the stability of histone modifications. Alternatively, the structure of active chromatin might be disrupted if RI assembly occurs frequently (Schwartz, 2005).

This study demonstrates that H3.3 deposition is triggered by gene induction, and deposition occurs while genes are transcribed. H3.3 deposition is also associated with histone removal and degradation that depends on the activity of a locus. Repeated rounds of replacement may function to destabilize active chromatin. The progressive degradation of H3.3 histone subtypes is distinct from the conservative exchange of H2A/H2B subtypes, and explains constitutive synthesis of new H3.3 histone (Schwartz, 2005).

Cells use two pathways for the assembly of nucleosomes. The replication-coupled pathway suffices to package the bulk genome as DNA is replicated, using histones synthesized from the major canonical genes. The RI pathway acts on transcriptionally active loci and specifically uses the H3.3 histone variant (Ahmad, 2002; Janicki 2004; Tagami 2004). This study shows that RI assembly begins within minutes after gene induction and operates while genes are active. The binding of transcription factors to artificial promoter arrays is insufficient to trigger H3.3 deposition, implying that RI assembly is tied to a late step in gene induction. The findings that H3.3 becomes distributed throughout long transcribed genes suggest that RNA polymerase progression itself is involved in histone displacement and replacement. Finally, the stability of H3.3 in chromatin depends on transcriptional status: At continually active loci, H3.3 deposits and then leaves, but when genes become repressed, incorporated H3.3 becomes stable. This dependence is explained if transcription causes the displacement of chromatin-bound histones (Schwartz, 2005).

In vitro experiments show that RNA polymerases often pause when they run into a nucleosome. Transcription through a nucleosome requires the transient disruption of histone-DNA contacts so that the polymerase can progress. A key question is whether the loss of these contacts releases histones from DNA as the polymerase passes, or if the histones remain associated. The current view is that accessory factors assist polymerases by moving histone octamers along the template, or by returning any histones that are displaced. However, the rapid stimulation of H3.3 replacement by gene induction implies that, at least sometimes, displaced histones are not restored. The enrichment of H3.3 at all active chromatin in Drosophila indicates that the loss of displaced histones is a general feature of transcription from chromatin templates (Schwartz, 2005).

There are two processes that might link histone variant replacement to transcription. (1) The polymerase may displace histones, thus producing a naked template suitable for RI assembly machinery. Such a dependence on prior nucleosome disassembly would effectively restrict H3.3 replacement to active genes. (2) Factors that accompany elongating RNA polymerase may catalyze histone replacement. Support for this idea comes from the detection of transcriptional regulators in isolated H3.3 predeposition complexes (Tagami, 2004). Tagami suggested that these regulators might serve as protein interfaces to recruit H3.3 to sites of active transcription. Indeed, a protein interaction between the histone chaperone CAF large subunit and the DNA replication fork protein PCNA serves to enhance replication-coupled deposition of histone H3 (Shibahara, 1999). An analogous role might be performed by transcription factors that interact with the HIRA chaperone in H3.3 predeposition complexes. Additionally, the histone-binding proteins Spt5 and SPT6 are known to localize to transiently induced HSP70 genes, and are thought to manipulate nucleosomes for transcription (Andrulis, 2000). Such factors might drive histone replacement to clear DNA for polymerase passage. SPT5 and SPT6 are not known to localize to sites of RNA Pol I transcription, so there may also be analogous polymerase-specific factors that clear DNA. Since SPT5 and SPT6 leave HSP genes after transient induction, replacement would also cease and the genes would retain high levels of H3.3. Thus, H3.3 replacement leaves behind an extremely stable marker of past transcriptional activity (Schwartz, 2005).

The observation that chromatin-bound H3.3 has a short half-life (~24 h) is in stark contrast to the half-life of histones from bulk chromatin. Measurements of in vivo histone exchange have demonstrated recycling of H2A and H2B between sites within a nucleus, and at least one complex is capable of exchanging these histones between chromatin and histone chaperones. However, recycling of H3 and H4 was not detected, and in current experiments bulk H2B-GFP does not degrade. The fate of H3.3 when it is displaced from chromatin distinguishes H3.3 replacement from other kinds of chromatin exchange and remodeling, and suggests that replication-independent nucleosome assembly and disassembly is not an exchange reaction. This appears to be a general property of replacement H3 histones, because alfalfa replacement H3 also has a fast rate of turnover (Waterborg 1993). The rapid turnover of replacement H3 histones explains why all eukaryotes have constitutively expressed H3.3-type genes (Thatcher, 1994; Malik, 2003), because a continuous supply will be required to replenish active chromatin. Perhaps the purpose of specific histone degradation is to prevent exchange between chromatin sites. Chromatin-bound H3 and H4 are heavily modified, and it is suggested that H3/H4 subparticles are degraded to prevent moving modified histones to new sites. In contrast, the relative paucity of modifications on the H2A and H2B histones means that their exchange between sites has no detrimental effect (Schwartz, 2005).

Two other instances of histone degradation have been well-characterized. Chromatin-bound centromeric histones in budding yeast (CSE4) are subject to proteasome-mediated degradation that may limit this histone variant to centromeres (Collins 200). A separate system that degrades excess soluble histones has also been described (Gunjan, 2003). The current experiments do not distinguish whether H3.3 is degraded as it is displaced from chromatin or once it is soluble. Degradation of the histone may occur by a proteosome-catalyzed mechanism, or by a general response to damaged proteins. However, histone degradation in differentiated cells appears to discriminate between histone subtypes, because H2B-GFP is not similarly degraded. Since replication-independent histone deposition uses specialized chromatin assembly factors, the results raise the possibility that there are histone disassembly factors that specifically degrade H3.3. This could be tested with drugs that block known degradation pathways (Schwartz, 2005).

The H3 and H3.3 histones are extremely similar, differing by only four amino acid residues in Drosophila. Three of these specify whether the histone can be used for replication-coupled or RI nucleosome assembly, but these residues are not accessible in the complete nucleosome (Ahmad, 2002). This extreme similarity raises a paradox: What can be the function of replication-independent assembly, since new assembly results in a virtually identical nucleosome? In fact, the similarity between H3 and H3.3 distinguishes it from other histone variants like H2A.Z, which do introduce structural differences into the nucleosome. A possible resolution to this paradox comes from observations of the dynamics of RI assembly. Replication-coupled histone deposition only occurs once every cell cycle at a site, but RI deposition is a continual process at active genes. Thus, inactive chromatin retains the same histones for long periods of time, but active chromatin repeatedly shed its histones. Rapid turnover of histones at active genes has three implications for chromatin structure (Schwartz, 2005).

(1) The maintenance of any modification state in active chromatin will require ongoing activity of histone-modifying enzymes to modify newly arriving histones. Acetyl modifications do rapidly turn over in vivo, and can be removed enzymatically, but histone replacement will also contribute to turnover rates. Replacement may enhance the specificity of histone-modifying enzymes by continually removing modifications from active chromatin. Histone replacement also provides a mechanism to remove histone modifications that may not be metabolized. One example of this is the activation of heterochromatic gene arrays (Ahmad, 2002; Janicki 2004), where chromatin is marked by methylation at Lys 9 of the H3 tail (H3K9me). Activation of these gene arrays results in the coincident disappearance of H3K9me and deposition of H3.3, as if removal of the methylation mark occurs by histone replacement. In contrast, some histone modifications that are set during gene expression remain present after transcription and histone replacement ceases. Indeed, the retention of H3.3 at previously active chromatin may require that the variant maintain its extreme similarity with H3 so that they both can be repressed by the same modification systems (Schwartz, 2005).

(2) Continual histone replacement means that active regions have little time to 'mature' their chromatin between rounds of histone deposition. Nascent chromatin produced by replication-coupled assembly matures into a nuclease-resistant form within 1 h, and this delay is thought to reflect the rates for removing predeposition modifications, establishing new modifications, and condensing chromatin. If the rates for modification and condensation after RI assembly are similar to replicated chromatin, rapid histone turnover will continually reset chromatin to an 'immature' uncondensed state (Schwartz, 2005).

(3) High DNA accessibility is a characteristic feature of active chromatin, and this feature may simply result from ongoing histone replacement. This idea is supported by recent experiments in budding yeast demonstrating that the inducible accessibility of the PHO5 promoter is due to loss of nucleosomes. Earlier studies observed a slight decrease in the density of histone H4 at activated HSP70 genes, and concluded that transcription occurs without displacing histones from chromatin. However, a small decrease could result if displaced histones are rapidly replaced with new ones. Indeed, in budding yeast the density of histones appears to drop at very high transcription rates. Such transient structural instability of active chromatin would allow efficient polymerase elongation (Schwartz, 2005).

Nucleosome-depleted chromatin gaps recruit assembly factors for the H3.3 histone variant

Most nucleosomes that package eukaryotic DNA are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions of the genome. Replication-independent nucleosome replacement using the H3.3 histone variant efficiently repackages these regions, but how histones are recruited to these sites is unknown. This study used an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to define steps in the mechanism of nucleosome replacement. Xnp chromatin remodeler and the Hira histone chaperone were found to independently bind nucleosome-depleted chromatin. Surprisingly, these two factors are only displaced when new nucleosomes are assembled. H3.3 deposition assays reveal that Xnp and Hira are required for efficient nucleosome replacement, and double-mutants are lethal. It is proposed that Xnp and Hira recognize exposed DNA and serve as a binding platform for the efficient recruitment of H3.3 predeposition complexes to chromatin gaps. These results uncover the mechanisms by which eukaryotic cells actively prevent the exposure of DNA in the nucleus (Schneiderman, 2012).

DNA in the eukaryotic nucleus is associated with histone proteins to form nucleosomes, the fundamental units of chromatin. Most nucleosomes are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions of the genome. These regions are repackaged by replication-independent (RI) nucleosome replacement using the H3.3 histone variant. This process results in the enrichment of the H3.3 histone variant at all sites where nucleosomes are unstable or disrupted (Schneiderman, 2012 and references therein).

How H3.3 is delivered to dynamic chromatin sites is unknown. However, biochemical isolation of predeposition complexes has identified shared and distinctive assembly factors that associate with the H3 and H3.3 histones and mediate the replication-coupled or RI assembly of nucleosomes, respectively. These factors include histone chaperones and chromatin remodelers that are important for new nucleosome assembly, and might potentially target histones to active chromatin regions. However, mutants in some of these factors have surprisingly limited phenotypes. The Hira chaperone promotes H3.3 deposition at genes but is only essential for H3.3 deposition on sperm chromatin during fertilization. In Drosophila the ATRX/XNP remodeler homolog Xnp colocalizes with H3.3 in somatic cells, but is not essential (Schneiderman, 2009). In mammals, ATRX/XNP promotes H3.3 deposition only at telomeres and some heterochromatic sequences. These results have raised the possibilities that H3.3 assembly factors are redundant or that additional factors involved in the deposition of this histone variant exist. Loss of H3.3 itself can be compensated in somatic cells by the major H3 histone, suggesting that assembly of any nucleosome suffices (Schneiderman, 2012 and references therein).

This work used an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to study the mechanism of nucleosome replacement. Evidence is provided that H3.3 predeposition factors mediate two separable steps in RI nucleosome assembly. The Xnp and Hira factors bind genomic sites when nucleosomes are disassembled, thereby marking sites for RI assembly. Strikingly, it was also demonstrated that Hira and Xnp are redundant for RI nucleosome assembly in somatic nuclei. The results further reveal that RI nucleosome replacement is essential for chromatin structure and viability, and uncover a cellular system that surveys chromatin for defects and promotes its repair (Schneiderman, 2012).

This study used the inducible Hsp70 genes as a controlled in vivo system to deplete nucleosomes from chromatin. Heat-shock activates transcription of Hsp70, displacing nucleosomes and increasing the sensitivity of the locus to digestion by micrococcal nuclease (MNase). After heat-shock, H3.3-containing nucleosomes repackage the locus. To generate persistently nucleosome-depleted chromatin, the Hsp70 genes in H3.3-deficient cells. ChIP experiments using an anti-H3 antibody show similar amounts of histone H3 at the Hsp70 genes in wild-type and H3.3-deficient salivary glands, and the genes show similar protection from MNase digestion. These data demonstrate that Hsp70 sequences are fully protected by nucleosomes before induction in wild-type and H3.3-deficient salivary glands. In both genotypes induced Hsp70 genes become hypersensitive to MNase as nucleosomes are lost. In wild-type cells after heat-shock nuclease protection is restored, but is not restored in H3.3-deficient glands. This finding demonstrates that nucleosomes are not replaced after Hsp70 induction in the absence of H3.3. This system allows the teasing apart of cause-and-effect in analyzing the effects of nucleosome assembly factors (Schneiderman, 2012).

Previous work has described that the Xnp chromatin remodeler colocalizes with H3.3 in chromatin (Schneiderman, 2009). Mammalian data has suggested that the Xnp homolog ATRX mediates H3.3 deposition at telomeres and at transcribed heterochromatic repeat sequences, although its nuclear distribution is much broader. The Hira histone chaperone has been implicated in global nucleosome replacement after the removal of protamines from the sperm nucleus during fertilization and near genes in mammalian cells. Purification of nuclear soluble H3.3-containing complexes showed that ATRX and Hira are in two separate complexes that mediate H3.3 deposition at distinct target sites. ASF1 is a general histone chaperone that complexes with new histone dimers in the cytoplasm and escorts them into the nucleu. This study used antibodies to Drosophila Xnp, Hira, and ASF1 to track their localization during Hsp70 induction and subsequent RI nucleosome assembly in polytene chromosomes of larval salivary glands (Schneiderman, 2012).

Heat-shock rapidly activates transcription of Hsp70, and elongating RNA polymerase II becomes strongly localized in puffs at the transcribing genes. After cessation of a heat-shock, the puffs regress and RNA polymerase II leaves the locus within 30 min. Neither Xnp, Hira, nor ASF1 were found to be enriched at the Hsp70 loci before induction. However, upon induction all three proteins are rapidly recruited to the Hsp70 loci. Xnp, Hira, and ASF1 remain associated with chromatin and the genes are transcribed, but then leave the Hsp70 genes after heat-shock. Their dynamic recruitment implicates these assembly factors in cotranscriptional nucleosome dynamics (Schneiderman, 2012).

The induction of Hsp70 was followed in H3.3-deficient salivary glands, where transcription produces nucleosome-depleted chromatin. Both Northern analysis and cytological observations of RNA polymerase II showed that Hsp70 was induced in wild-type and H3.3-deficient salivary glands, although H3.3-deficient glands produce ~50% less mRNA. Two effects on the recruitment of RI assembly factors were observed . First, Xnp, Hira, and ASF1 are all rapidly recruited to the induced Hsp70 genes at moderately reduced levels, indicating that the Hsp70 genes are less efficiently induced in this genotype. Second, both Xnp and Hira - but not ASF1 - accumulate and persist at the Hsp70 genes after heat-shock. The correspondence between persistent nucleosome depletion and the persistent binding of these factors is striking. This correspondence is a distinctive property of these two RI assembly factors, because the histone chaperone ASF1 is also cotranscriptionally recruited but dissociates both in wild-type and in H3.3-deficient cells. Finally, retention of Xnp and Hira is not a result of ongoing transcription, because RNA polymerase II rapidly leaves the Hsp70 loci after heat-shock and transcript production ceases. It is concluded that once Xnp and Hira bind nucleosome-depleted chromatin, they are only displaced when new nucleosomes assemble (Schneiderman, 2012).

How are Xnp and Hira recruited to nucleosome-depleted chromatin? These factors may be directly recruited by transcriptional machinery to active genes. Alternatively, Xnp and Hira may bind a structural feature common to chromatin gaps, or may simply bind exposed DNA. These factors might be complexed with DNA-binding factors or may bind DNA themselves. The homologous ATRX remodeler contains an ADD (ATRX-DNMT3-DNMT3L) domain that can bind DNA or histone tails. Indeed, ATRX is recruited to the genomes of DNA viruses as they enter the nucleus, suggesting that it may directly bind exposed DNA. A recent study has shown that the mammalian Hira chaperone may also directly bind exposed DNA at chromatin gaps. This study found that Xnp and Hira bind independently at induced Hsp70 genes in null mutants of the other factor, implying that there may be multiple ways that RI assembly factors recognize exposed DNA (Schneiderman, 2012).

Although both Xnp and Hira have been implicated in RI nucleosome assembly, mutants in these factors have surprisingly limited phenotypes. These results have raised the possibility that H3.3 assembly factors are redundant. To test if these factors promote nucleosome replacement at these sites, deposition of GFP-tagged truncated H3.3 histone into chromatin was assayed in wild-type and mutant genotypes. H3.3core-GFP can only be incorporated by RI nucleosome assembly, and the histone labels active genes.A pulse of H3.3core-GFP was produced in salivary glands and chromosome spreads were prepared 2 h later to assess the efficiency of RI nucleosome assembly. In wild-type cells, H3.3core-GFP strongly labels chromosome arms and active genes. In contrast, the H3.3core-GFP protein is efficiently produced in xnp-null mutant cells, but only a fraction of the protein deposits onto chromosomes; instead, most of the protein accumulates within the nucleolus. This protein does not coincide with DNA in the nucleolus, and may be predeposition or aggregated histones. Hira mutants have a similar reduction in H3.3 deposition: H3.3core-GFP protein is produced, but most protein accumulates in the nucleolus. These results demonstrate that the rate of RI nucleosome assembly is reduced in both xnp and Hira mutants, although some assembly can still occur. Indeed, longer expression of tagged histones in xnp or Hira mutants does achieve apparently normal levels (Schneiderman, 2012).

To test if Xnp and Hira have redundant roles in nucleosome replacement, Hira;xnp double-mutant animals were generated. Single-mutants are fully viable, but double-mutant larvae grow slower than wild-type siblings and die during larval development. Therefore H3.3core-GFP deposition was measured in these double-mutants. Strikingly, high levels of H3.3core-GFP were produced in Hira;xnp animals, but all of the protein accumulates in the nucleolus, with no detectable staining of chromosomes. It is concluded that both Hira and Xnp contribute to the efficiency of H3.3 RI nucleosome assembly, but this fails when both factors are eliminated (Schneiderman, 2012).

The results lead to the suggestion that Xnp and Hira identify nucleosome-depleted chromatin and promote new nucleosome assembly through a stepwise process (see Nucleosome-depleted chromatin gaps recruit assembly factors for the H3.3 histone variant). In the first step, Xnp and Hira bind exposed DNA at chromatin gaps, thereby marking sites where a nucleosome has been displaced. In the second step, delivery factors carrying new histones are recruited by binding to Xnp and Hira at chromatin gaps. In the final step, these factors assist in the transfer of histones from delivery chaperones to DNA, and Xnp and Hira are released when nucleosome assembly is complete (Schneiderman, 2012).

A previous study showned that the Xnp remodeler is found at all sites where H3.3 is enriched, including active genes (Schneiderman, 2009). The Hira chaperone also localizes at active genes. However, there are additional sites in the genome where the two assembly factors do not coincide. First, the major site for Xnp binding is at a nucleosome-depleted satellite block, where Hira is not found. Second, most Hira is localized to the repeated ribosomal DNA (rDNA) within the nucleolus, where Xnp is not found. This finding implies that the two factors are not redundant at all sites where H3.3 is deposited (Schneiderman, 2012).

The rDNA genes are repressed in late-stage salivary glands. Therefore, deposition of H3.3 was assayed in the somatic follicle cells of ovaries, where rDNA is highly transcribed. In this cell type, Xnp localizes broadly in the nucleus but not within the nucleolus, but most Hira protein forms foci within the nucleolus. A pulse of epitope-tagged H3.3 produced in follicle cells broadly labels the nucleus and foci within the nucleolus, corresponding to transcriptionally active sites in this cell type. Follicle cells from xnp mutant ovaries also show nucleolar labeling with H3.3-GFP, demonstrating that Xnp is not required for nucleolar RI assembly. In contrast, a pulse of epitope-tagged H3.3 in Hira mutant follicle cells does not deposit in the nucleolus. Thus, rDNA chromatin must rely on the Hira chaperone for H3.3 deposition. It is concluded that Xnp and Hira are redundant at many sites within the genome, but some sites rely on individual factors for replacement nucleosome assembly. Although neither Hira nor Xnp are individually required for viability, there may be more subtle phenotypes that occur in repetitive sites in the genome (Schneiderman, 2012).

If Xnp and Hira are generally involved in recognizing chromatin gaps, the localization of these factors should be affected in H3.3-deficient cells. Indeed, new binding sites for Xnp appear in rDNA chromatin after H3.3 knock-down. Hira is also recruited to the nucleosome-depleted satellite block after H3.3 knock-down. Strikingly, the area of the nucleosome-depleted satellite block is ∼2.5-times larger after H3.3 knock-down, and the Xnp signal at this site is elevated. This finding implies that RI assembly is normally required for compaction of the satellite. The redundancy of Hira and Xnp implies that their importance for H3.3 deposition is underestimated in single-mutants. Both the relocalization and increased binding of Xnp and Hira after H3.3 knock-down support the idea that these assembly factors are recruited to aberrant, nucleosome-depleted chromatin. As persistent exposure of DNA may disrupt transcriptional regulation or allow DNA damage, surveying chromatin for gaps with RI assembly factors may be critical for genome stability and function (Schneiderman, 2012).

The Drosophila DAXX like protein (DLP) cooperates with ASF1 for H3.3 deposition and heterochromatin formation

Histone variants are non-allelic isoforms of canonical histones and they are deposited, in contrast to canonical histones, in a replication-independent (RI) manner. RI deposition of H3.3, a histone variant from the H3.3 family, is mediated in mammals by distinct pathways involving either the histone regulator A (HIRA) complex or the death-associated protein (DAXX)/alpha-thalassemia X-linked mental retardation protein (ATRX) complex. This study investigated the function of Drosophila DAXX Like Protein (DLP) by using both fly genetics approaches and protein biochemistry. DLP specifically interacts with H3.3 and shows a prominent localization on the base of the X chromosome, where it appears to act in concert with XNP the Drosophila homolog of ATRX, in heterochromatin assembly and maintenance. The functional association between DLP and XNP is further supported by a series of experiments, which illustrate genetic interactions and DLP-XNP-dependent localization of specific chromosomal proteins. In addition, DLP both participates in RI deposition of H3.3 and associates with the anti-silencing factor-1 (ASF1). It is suggested, in agreement with a recently proposed model, that DLP and ASF1 are part of a pre-deposition complex, which is recruited by XNP and is necessary to prevent DNA exposure in the nucleus (Fromental-Ramain, 2017).

This study has identified DLP as the Drosophila homolog of DAXX. DLP is involved, likely in concert with XNP/dATRX, in the formation of pericentric heterochomatin of the X chromosome. Moreover, DLP is implicated in RI deposition of the histone variant H3.3 and may constitute with ASF1 the central core of a pre-deposition complex, recruited to chromatin gaps by XNP. The existence of such complex was recently suggested (Fromental-Ramain, 2017 and references therein).

In spite of the fact that both proteins do not molecularly associate as their mammal homologs do, this study provides evidence that DLP and XNP functions are closely linked. DLP and XNP are located on the base of the X chromosome and analysis of animals simultaneously mutant for both dlp and xnp revealed that DLP and XNP likely act together during heterochromatin formation. In addition, both DLP and XNP are located next to distal heterochromatic marker HP1 on the X chromosome of larvae carrying the ln(1)wm4h rearrangement. Functional interactions between DLP and XNP 55 were also supported by the similar behavior of DLP and XNP in H3.3 deficient cells. In wild-type cells, in addition to the base of the X chromosome, expression of XNP is detected at many sites across the chromosome arms where DLP is not observed. In H3.3 knock-down-cells, DLP and XNP are present at many euchromatic sites of the chromosomes and are simultaneously associated with nucleolar chromatin of the rDNA. Finally, overexpressed DLP binds to many interbands on the polytene chromosomes, suggesting that DLP may also be involved in chromatin organization at euchromatic sites in addition to the pericentric heterochromatin. However, this latter observation should be viewed with caution since it cannot be ruled out that over-expressed DLP is not present in its usual complex and is consequently mis-targeted. Additional support for functional interactions between XNP and DLP is provided by genetic interactions between xnp and dlp. Indeed, loss of xnp function is characterized by reduced viability, which is further aggravated when dlp function is simultaneously reduced, strongly indicating that xnp and dlp may functionally cooperate during regulation of common targets. How XNP is recruited to nucleosome-depleted chromatin remains an important issue. XNP may be recruited by transcriptional machinery to active genes. Alternatively, XNP may bind structural motifs common to chromatin gaps, or may simply bind exposed DNA. The homologous ATRX contains a PHD domain that can bind DNA or histones tails. Recent work demonstrates that mammalian Hira may bind exposed DNA at chromatin gaps. Moreover, Hira and XNP bind active regions independently of one another. Hence, there may be multiple ways that RI assembly factors recognize exposed DNA (Fromental-Ramain, 2017).

In Drosophila, loss of H3.3 has a large impact on viability and fertility of both males and females. The Drosophila genome encompasses two single copy genes, H3.3A and H3.3B, which code for the same protein. H3.3 is highly expressed in mitotic, meiotic and post-meiotic male germ cells, probably reflecting high transcriptional activity. Interestingly, high dlp expression is observed in primary spermatocytes, in meiotic spermatocytes and also in the germinal vesicle, suggesting that it may have important functions during development of germ cells. In Drosophila testis, H3.3 disappears with the bulk of histones, prior to accumulation of protamine and other sperm-specific nuclear basic proteins, leading to sperm DNA compaction at late stages of spermiogenesis. At fertilization, assembly of nucleosomes on paternal DNA immediately follows the rapid loss of protamines from the decondensing male nucleus and is dependent on maternally provided factors like Hira and YEM. HIRA and YEM are crucial since male pronuclei fail to decondense at the pronuclear stage in eggs derived from female mutants for HIRA and YEM. Hence, function of HIRA/YEM at fertilization represents a unique example where deficient chromatin activity cannot be compensated by other redundant factors (Fromental-Ramain, 2017).

In contrast, many examples suggest that H3.3 chaperones/chromatin remodeling complexes may display functional redundancy as mutants in these factors have limited phenotypes. In this context, DLP may be viewed as a typical example. The null allele dlpG and the dlp45 allele encoding a truncated protein lacking the C-terminal DHR necessary for H3.3 binding are viable and fertile, indicating that DLP and other chromatin factors may share common functions. Alternatively, DLP may display accessory functions during development of germ cells. Characterization of the phenotypes of double-mutant animals during germ cell development would help to resolve this important issue (Fromental-Ramain, 2017).

H3.3 was initially seen as a characteristic of active genes with histone turnover occurring as a consequence of transcription. More recent studies revealed that H3.3 is widespread within the genome. In particular, H3.3 is deposited by ATRX/DAXX at telomeres and pericentric repeats. Interestingly, ATRX and DAXX are components of the same chromatin-remodeling complex and physically interact. Recently, Schneiderman (2012) proposed a model on how XNP, the Drosophila homolog of ATRX, and HIRA identify nucleosome-depleted DNA following gene activation, and promote nucleosome assembly through a three steps process. Initially, XNP and HIRA bind exposed DNA at chromatin gaps where nucleosomes have been displaced. Subsequently they co-operate to recruit a predeposition complex including ASF1 and histones. In the final step, XNP and HIRA assist the transfer of histones from delivery factors to DNA and are released when nucleosome assembly is complete. Even if XNP and DLP do not physically interact, this study provides several evidences suggesting that DLP could be a component of the predeposition complex recruited by XNP/HIRA (Fromental-Ramain, 2017).

Both HIRA and XNP have been implicated in RI nucleosome assembly, but mutants of these factors have only limited phenotypes, revealing that they have redundant functions. Thus, single mutants of either xnp or hira weakly affect H3.3 deposition, which is abolished in a double-mutant of xnp and hira. This observation highlights the need for two distinct pathways during RI nucleosome assembly, one mediated by HIRA/YEM and the other by XNP. This study assigns a role to DLP during RI H3.3 deposition since H3.3 incorporation is affected in animals lacking DLP. DLP is thought to co-operate with XNP and it was surprising to observe H3.3 deposition in animals lacking HIRA and DLP. Hence, it is speculated that the pathway mediated by XNP is always functional, although less efficient. It has been recently proposed that XNP recognizes exposed DNA when a nucleosome has been displaced, and serves as a binding platform for the recruitment of H3.3 predeposition complexes to chromatin gaps. Such complexes are believed to contain (H3.3-H4) heterodimers, ASF1 and additional factors. In line with this the data revealed physical interactions between ASF1 and DLP in protein extracts made from baculovirus-infected Sf9 cells co-expressing DLP and ASF1, suggesting that DLP may be one of these additional factors (Fromental-Ramain, 2017).

These data provides additional links between HIRA, DLP, ASF1 and XNP during H3.3 incorporation but how they functionally interact during development remains an open question. Identification of their genomic targets and characterization of their activities during H3.3 deposition would obviously help to resolve this important issue (Fromental-Ramain, 2017).

cDNA clone length - 772

Bases in 5' UTR - 97

Exons - 2

Bases in 3' UTR - 264


Amino Acids - 136

Structural Domains

The histone H3 family contains an evolutionary conserved variant, H3.3, that differs in sequence in only five amino acids from the canonical H3. There are four differences between Drosophila H3 and H3.3.

Histone H3.3A: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 15 December 2023

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