Histone H3.3A

Germline chromatin undergoes dramatic remodeling events involving histone variants during the life cycle of an organism. A universal histone variant, H3.3, is incorporated at sites of active transcription throughout the cell cycle. The presence of H3.3 in chromatin indicates histone turnover, which is the energy-dependent removal of preexisting histones and replacement with new histones. H3.3 is also incorporated during decondensation of the Drosophila sperm pronucleus, indicating a direct role in chromatin remodeling upon fertilization. This study presents a system to monitor histone turnover and chromatin remodeling during C. elegans development by following the developmental dynamics of H3.3. Worm strains were generated expressing green fluorescent protein- or yellow fluorescent protein-fused histone H3.3 proteins, HIS-71 and HIS-72. It was found that H3.3 is retained in mature sperm chromatin, raising the possibility that H3.3 transmits epigenetic information via the male germline. Upon fertilization, maternal H3.3 enters both male and female pronuclei and is incorporated into paternal chromatin, apparently before the onset of embryonic transcription, suggesting that H3.3 can be incorporated independent of transcription. In early embryos, H3.3 becomes specifically depleted from primordial germ cells. Strikingly, the X chromosome becomes deficient in H3.3 during gametogenesis, indicating a low level of histone turnover. These results raise the possibility that the asymmetry in histone turnover between the X chromosome and autosomes is established during gametogenesis. H3.3 patterns are similar to patterns of H3K4 methylation in the primordial germ cells and on the X chromosome during gametogenesis, suggesting that histone turnover and modification are coupled processes. This demonstration of dynamic H3.3 incorporation in nondividing cells provides a mechanistic basis for chromatin changes during germ cell development (Ooi, 2006: full text of article).

Deposition of the major histone H3 (H3.1) is coupled to DNA synthesis during DNA replication and possibly DNA repair, whereas histone variant H3.3 serves as the replacement variant for the DNA-synthesis-independent deposition pathway. To address how histones H3.1 and H3.3 are deposited into chromatin through distinct pathways, deposition machineries for these histones were purified. The H3.1 and H3.3 complexes contain distinct histone chaperones, CAF-1 and HIRA, that are shown to be necessary to mediate DNA-synthesis-dependent and -independent nucleosome assembly, respectively. Notably, these complexes possess one molecule each of H3.1/H3.3 and H4, suggesting that histones H3 and H4 exist as dimeric units that are important intermediates in nucleosome formation. This finding provides new insights into possible mechanisms for maintenance of epigenetic information after chromatin duplication (Tagami, 2004).

An inducible system has been developed to visualize gene expression at the levels of DNA, RNA and protein in living cells. The system is composed of a 200 copy transgene array integrated into a euchromatic region of chromosome 1 in human U2OS cells. The condensed array is heterochromatic as evidenced by its association with HP1, histone H3 methylated at lysine 9, and several histone methyltransferases. Upon transcriptional induction, HP1alpha is depleted from the locus and the histone variant H3.3 is deposited, suggesting that histone exchange is a mechanism through which heterochromatin is transformed into a transcriptionally active state. RNA levels at the transcription site increase immediately after the induction of transcription and the rate of synthesis slows over time. Using this system, changes in chromatin structure were corrolated with the progression of transcriptional activation allowing a real-time integrative view of gene expression (Janicki, 2004).

Variant histone H3.3 is incorporated into nucleosomes by a mechanism that does not require DNA replication and has also been implicated as a potential mediator of epigenetic memory of active transcriptional states. This study has used chromatin immunoprecipitation analysis to show that H3.3 is found mainly at the promoters of transcriptionally active genes. H3.3 combines with H3 acetylation and K4 methylation to form a stable mark that persists during mitosis. These results suggest that H3.3 is deposited principally through the action of chromatin-remodelling complexes associated with transcriptional initiation, with deposition mediated by RNA polymerase II elongation having only a minor role (Chow, 2005).

Histones are the fundamental components of the nucleosome. Physiologically relevant variation is introduced into this structure through chromatin remodeling, addition of covalent modifications, or replacement with specialized histone variants. The histone H3 family contains an evolutionary conserved variant, H3.3, which differs in sequence in only five amino acids from the canonical H3, H3.1, and was shown to play a role in the transcriptional activation of genes. Histone H3.3 contains a serine (S) to alanine (A) replacement at amino acid position 31 (S31). Both MS and biochemical methods demonstrate that this serine is phosphorylated (S31P) during mitosis in mammalian cells. In contrast to H3 S10 and H3 S28, which first become phosphorylated in prophase, H3.3 S31 phosphorylation is observed only in late prometaphase and metaphase and is absent in anaphase. Additionally, H3.3 S31P forms a speckled staining pattern on the metaphase plate, whereas H3 S10 and H3 S28 phosphorylation localizes to the outer regions of condensed DNA. Furthermore, in contrast to phosphorylated general H3, H3.3 S31P is localized in distinct chromosomal regions immediately adjacent to centromeres. These findings argue for a unique function for the phosphorylated isoform of H3.3 that is distinct from its suspected role in gene activation (Hake, 2005).

Deposition of variant histones provides a mechanism to reset and to potentially specify chromatin states. The distribution was determined of H3 and its variant H3.3 relative to chromatin structure and elongating polymerase. H3.3 is enriched throughout active genes similar to polymerase, yet its distribution is very distinct from that of several euchromatic histone modifications, which are highly biased toward the 5' part of active genes. Upon gene induction displacement of both H3 and H3.3 was observed, followed by selective deposition of H3.3. These results support a model in which H3.3 deposition compensates for transcription-coupled nucleosomal displacement yet does not predetermine tail modifications (Wirbelauer, 2005).

In mammalian fertilization, the paternal genome is delivered to the secondary oocyte by sperm with protamine compacted DNA, while the maternal genome is arrested in meiotic metaphase II. Thus, at the beginning of fertilization, the two gametic chromatin sets are strikingly different. This study elaborates on this contrast by reporting asymmetry for histone H3 type in the pre-S-phase zygote when male chromatin is virtually devoid of histone H3.1/H3.2. Localization of the histone H3.3/H4 assembly factor Hira with the paternal chromatin indicates the presence of histone H3.3. In conjunction with this, a systematic immunofluorescence analysis of histone N-tail methylations at position H3K4, H3K9, H3K27 and H4K20 up to the young pronucleus stage was performed; asymmetries were found to be systematic for virtually all di- and tri-methylations but not for mono-methylation of H3K4 and H4K20, the only marks studied present in the early male pronucleus. For H4K20 the expanding male chromatin is rapidly mono-methylated. This coincides with the formation of maternally derived nucleosomes, a process that is observed as early as sperm chromatin decondensation occurs. Absence of tri-methylated H3K9, tri-methylated H4K20 and the presence of loosely anchored HP1-ß combined with the homogenous presence of mono-methylated H4K20 suggests the absence of a division of the paternal chromatin in eu- and heterochromatin. In summary, the male (in contrast to female) G1 chromatin, is uniform and contains predominantly histone H3.3 as histone H3 variant (van der Heijden, 2005).

Until now it has not been shown definitively at which phase of decondensation of the sperm nucleus the actual removal of protamines takes place and maternal nucleosome deposition commences. This study correlates the change of chromatin morphology with the presence or absence of protamines. Extrusion of protamines starts before decondensation and within 30 min post-gamete fusion the male chromatin does not co-localize with the protamines any more. A steep increase in nucleosome density is observed after the start of decondensation. Hira, a histone chaperone that deposits H3.3-H4 dimers onto DNA, co-localizes with the expanding sperm chromatin in the zygote. Apparently, the onset of deposition of maternally derived nucleosomes takes place at the moment of paternal chromatin expansion. The only indication that DNA might be completely devoid of nucleosomes and protamines during expansion of the sperm chromatin was the brighter staining by the antibody specific for dsDNA at the interphase of condensed and decondensed chromatin. This finding also indicates an absolute coincidence between decondensation and nucleosome formation (van der Heijden, 2005).

This study has obtained a clear picture of paternal chromatin remodeling; protamines are removed from expanding chromatin, which is completed within 30 min after gamete fusion. During this period nucleosome formation starts, continuing over a period of roughly 4 h in the zygote as deduced by absence of Hira in 52% of the paternal PNs at around that time point (van der Heijden, 2005).

The nucleosome core particle consists of two copies of Histone H2A, H2B, H3 and H4. For Histone H2A, H2B and H3 variants have been described. Histone H3 has three functionally different subtypes: CenH3, which is strictly localized in the centromeres and highly diverged in sequence from the canonical H3 of which two functional variants exists: the replication variants (H3.1 and H3.2), and the replacement variant (H3.3). The utilisation of the H3 replication variants, which differ from each other in only one amino acid, is strictly limited to DNA synthesis linked nucleosome assembly. When nucleosomes are formed without occurrence of DNA synthesis, H3.3 is incorporated. This specialisation of H3 subtypes is reflected in their chaperones. H3.1 (and H3.2) is deposited as a H3-H4 dimer by Caf-1 while H3.3 is deposited, also as a H3-H4 dimer, by Hira (van der Heijden, 2005).

The deposition of maternal nucleosomes onto paternal DNA directly after gamete fusion is not accompanied by DNA synthesis. These nucleosomes will contain H3.3 as histone H3 variant. Characteristic for mouse sperm chromatin is the near absence of nucleosomes. Therefore virtually all the paternal chromatin will be wrapped around newly deposited H3.3 containing maternal nucleosomes (van der Heijden, 2005).

Upon fertilization, the gametes undergo a drastic reprogramming that includes changes in DNA methylation and histone modifications. Currently, it is not known whether replacement of the major histones by histone variants is also involved in these processes. This study examined the expression and localization of the histone variant H3.3 in early mouse embryogenesis. H3.3 was shown to be present in the oocyte as a maternal factor. It is then incorporated preferentially into the male pronucleus before genome activation, pointing towards an asymmetry in histone composition between the two pronuclei. This is in line with the male pronucleus bearing transcriptional activation first. The same distribution was observed when the localisation of a tagged version of H3.3 was followed. H3.3 was detected in the nuclei of mouse embryos in all of the stages analysed, from the zygote to the blastocyst stage, suggesting that the epigenetic mechanisms in the early embryo not only involve changes in histone modifications but may also include histone replacement (Torres-Padilla, 2006).

Nucleosomes containing the histone variant H3.3 tend to be clustered in vivo in the neighborhood of transcriptionally active genes and over regulatory elements. It has not been clear, however, whether H3.3-containing nucleosomes possess unique properties that would affect transcription. This study reports that H3.3 nucleosomes isolated from vertebrates, regardless of whether they are partnered with H2A or H2A.Z, are unusually sensitive to salt-dependent disruption, losing H2A/H2B or H2A.Z/H2B dimers. Immunoprecipitation studies of nucleosome core particles (NCPs) show that NCPs that contain both H3.3 and H2A.Z are even less stable than NCPs containing H3.3 and H2A. Intriguingly, NCPs containing H3 and H2A.Z are at least as stable as H3/H2A NCPs. These results establish an hierarchy of stabilities for native nucleosomes carrying different complements of variants, and suggest how H2A.Z could play different roles depending on its partners within the NCP. They also are consistent with the idea that H3.3 plays an active role in maintaining accessible chromatin structures in enhancer regions and transcribed regions. Consistent with this idea, promoters and enhancers at transcriptionally active genes and coding regions at highly expressed genes have nucleosomes that simultaneously carry both H3.3 and H2A.Z, and should therefore be extremely sensitive to disruption (Jin, 2007).

The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3

The histone variant H3.3 marks active chromatin by replacing the conventional histone H3.1. This study investigated the detailed mechanism of H3.3 replication-independent deposition. The death domain-associated protein DAXX and the chromatin remodeling factor ATRX (alpha-thalassemia/mental retardation syndrome protein) are specifically associated with the H3.3 deposition machinery. Bacterially expressed DAXX has a marked binding preference for H3.3 and assists the deposition of (H3.3-H4)2 tetramers on naked DNA, thus showing that DAXX is a H3.3 histone chaperone. In DAXX-depleted cells, a fraction of H3.3 was found associated with the replication-dependent machinery of deposition, suggesting that cells adapt to the depletion. The reintroduced DAXX in these cells colocalizes with H3.3 into the promyelocytic leukemia protein (PML) bodies. Moreover, DAXX associates with pericentric DNA repeats, and modulates the transcription from these repeats through assembly of H3.3 nucleosomes. These findings establish a new link between the PML bodies and the regulation of pericentric DNA repeat chromatin structure. Taken together, these data demonstrate that DAXX functions as a bona fide histone chaperone involved in the replication-independent deposition of H3.3 (Drané 2010).

Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity

Establishment of a proper chromatin landscape is central to genome function. H3 variant distribution can be explained by specific targeting and dynamics of deposition involving the CAF-1 and HIRA histone chaperones. Experiments with human cells reveal that impairing replicative H3.1 incorporation via CAF-1 enables an alternative H3.3 deposition at replication sites via HIRA. Conversely, the H3.3 incorporation throughout the cell cycle via HIRA cannot be replaced by H3.1. ChIP-seq analyses reveal correlation between HIRA-dependent H3.3 accumulation and RNA pol II at transcription sites and specific regulatory elements, further supported by their biochemical association. The HIRA complex shows unique DNA binding properties, and depletion of HIRA increases DNA sensitivity to nucleases. It is proposed that protective nucleosome gap filling of naked DNA by HIRA leads to a broad distribution of H3.3, and HIRA association with Pol II ensures local H3.3 enrichment at specific sites. The importance of this H3.3 deposition as a salvage pathway to maintain chromatin integrity is discussed (Ray-Gallet, 2011).

Hira-dependent Histone H3.3 deposition facilitates PRC2 recruitment at developmental loci in ES cells

Polycomb repressive complex 2 (PRC2) regulates gene expression during lineage specification through trimethylation of lysine 27 on histone H3. In Drosophila, polycomb binding sites are dynamic chromatin regions enriched with the histone variant H3.3. This study shows that, in mouse embryonic stem cells (ESCs), H3.3 is required for proper establishment of H3K27me3 at the promoters of developmentally regulated genes. Upon H3.3 depletion, these promoters show reduced nucleosome turnover measured by deposition of de novo synthesized histones and reduced PRC2 occupancy. Further, this study shows H3.3-dependent interaction of PRC2 with the histone chaperone, Hira, and that Hira localization to chromatin requires H3.3. The data demonstrate the importance of H3.3 in maintaining a chromatin landscape in ESCs that is important for proper gene regulation during differentiation. Moreover, these findings support the emerging notion that H3.3 has multiple functions in distinct genomic locations that are not always correlated with an 'active' chromatin state (Banaszynski, 2013).

H3.3 actively marks enhancers and primes gene transcription via opening higher-ordered chromatin

The histone variants H3.3 and H2A.Z have recently emerged as two of the most important features in transcriptional regulation, the molecular mechanism of which still remains poorly understood. This study investigated the regulation of H3.3 and H2A.Z on chromatin dynamics during transcriptional activation. In vitro biophysical and biochemical investigation showed that H2A.Z promotes chromatin compaction and represses transcriptional activity. Surprisingly, with only four to five amino acid differences from the canonical H3, H3.3 greatly impaires higher-ordered chromatin folding and promotes gene activation, although it has no significant effect on the stability of mononucleosomes. It was further demonstrated that H3.3 actively marks enhancers and determines the transcriptional potential of retinoid acid (RA)-regulated genes via creating an open chromatin signature that enables the binding of RAR/RXR. Additionally, the H3.3-dependent recruitment of H2A.Z on promoter regions results in compaction of chromatin to poise transcription, while RA induction results in the incorporation of H3.3 on promoter regions to activate transcription via counteracting H2A.Z-mediated chromatin compaction. These results provide key insights into the mechanism of how histone variants H3.3 and H2A.Z function together to regulate gene transcription via the modulation of chromatin dynamics over the enhancer and promoter regions (Chen, 2013).

Histone H3.3 is required to maintain replication fork progression after UV damage

Unlike histone H3, which is present only in S phase, the variant histone H3.3 is expressed throughout the cell cycle and is incorporated into chromatin independent of replication. Recently, H3.3 has been implicated in the cellular response to ultraviolet (UV) light. Chicken DT40 cells completely lacking H3.3 are hypersensitive to UV light, a defect that epistasis analysis suggests may result from less-effective nucleotide excision repair. Unexpectedly, H3.3-deficient cells also exhibit a substantial defect in maintaining replication fork progression on UV-damaged DNA, which is independent of nucleotide excision repair, demonstrating a clear requirement for H3.3 during S phase. Both the UV hypersensitivity and replication fork slowing are reversed by expression of H3.3 and require the specific residues in the alpha2 helix that are responsible for H3.3 binding its dedicated chaperones. However, expression of an H3.3 mutant in which serine 31 is replaced with alanine, the equivalent residue in H3.2, restores normal fork progression but not UV resistance, suggesting that H3.3[S31A] may be incorporated at UV-damaged forks but is unable to help cells tolerate UV lesions. Similar behavior was observed with expression of H3.3 carrying mutations at K27 and G34, which have been reported in pediatric brain cancers. It is speculated that incorporation of H3.3 during replication may mark sites of lesion bypass and, possibly through an as-yet-unidentified function of the N-terminal tail, facilitate subsequent processing of the damage (Frey, 2014).

Histone H3.3A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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