Dramatic replacement of histone variants during genome remodeling in nuclear-transferred embryos

The genome of differentiated somatic nuclei is remodeled to a totipotent state when they are transplanted into enucleated oocytes. To clarify the mechanism of this genome remodeling, we analyzed changes in the composition of core histone variants in nuclear-transferred embryos, since recent evidence has revealed that chromatin structure can be remodeled as a result of variant histone replacement. We found that the donor cell-derived histone H3 variants H3.1, H3.2, and H3.3, as well as H2A and H2A.Z, were rapidly eliminated from the chromatin of nuclei transplanted into enucleated oocytes. Accompanying this removal, oocyte-stored histone H3 variants and H2A.X were incorporated into the transplanted nuclei, while the incorporation of H2A and H2A.Z was minimal or not detected. The incorporation of these variant histones was DNA replication-independent. These results suggest that most core histone H2A and H3 components are dynamically exchanged between donor nuclei and recipient cytoplasm, which further suggests that replacement of donor cell histones with oocyte-stored histones may play a key role in genome remodeling in nuclear-transferred embryos. In addition, the incorporation patterns of all of the histone variants in the nuclear-transferred embryos were virtually the same as in the fertilized embryos. Only the incorporation pattern of H3.1 differed; it was incorporated into the transplanted donor nuclei, but not in the pronuclei of fertilized embryos. This result suggests that the incorporation of H3.1 has a detrimental effect on the process of genome remodeling and contributes to the low success rate of somatic nuclear cloning.     

Dramatic replacement of histone variants during genome remodeling in nuclear-transferred embryos

The genome of differentiated somatic nuclei is remodeled to a totipotent state when they are transplanted into enucleated oocytes. To clarify the mechanism of this genome remodeling, we analyzed changes in the composition of core histone variants in nuclear-transferred embryos, since recent evidence has revealed that chromatin structure can be remodeled as a result of variant histone replacement. We found that the donor cell-derived histone H3 variants H3.1, H3.2, and H3.3, as well as H2A and H2A.Z, were rapidly eliminated from the chromatin of nuclei transplanted into enucleated oocytes. Accompanying this removal, oocyte-stored histone H3 variants and H2A.X were incorporated into the transplanted nuclei, while the incorporation of H2A and H2A.Z was minimal or not detected. The incorporation of these variant histones was DNA replication-independent. These results suggest that most core histone H2A and H3 components are dynamically exchanged between donor nuclei and recipient cytoplasm, which further suggests that replacement of donor cell histones with oocyte-stored histones may play a key role in genome remodeling in nuclear-transferred embryos. In addition, the incorporation patterns of all of the histone variants in the nuclear-transferred embryos were virtually the same as in the fertilized embryos. Only the incorporation pattern of H3.1 differed; it was incorporated into the transplanted donor nuclei, but not in the pronuclei of fertilized embryos. This result suggests that the incorporation of H3.1 has a detrimental effect on the process of genome remodeling and contributes to the low success rate of somatic nuclear cloning.     

Identification of a replication-independent replacement histone H3 in the basidiomycete Ustilago maydis

Ustilago maydis is a haploid basidiomycete with single genes for two distinct histone H3 variants. The solitary U1 gene codes for H3.1, predicted to be a replication-independent replacement histone. The U2 gene is paired with histone H4 and produces a putative replication-coupled H3.2 variant. These predictions were evaluated experimentally. U2 was confirmed to be highly expressed in the S phase and had reduced expression in hydroxyurea, and H3.2 protein was not incorporated into transcribed chromatin of stationary phase cells. Constitutive expression of U1 during growth produced ~25% of H3 as H3.1 protein, more highly acetylated than H3.2. The level of H3.1 increased when cell proliferation slowed, a hallmark of replacement histones. Half of new H3.1 incorporated into highly acetylated chromatin was lost with a half-life of 2.5 h, the fastest rate of replacement H3 turnover reported to date. This response reflects the characteristic incorporation of replacement H3 into transcribed chromatin, subject to continued nucleosome displacement and a loss of H3 as in animals and plants. Although the two H3 variants are functionally distinct, neither appears to be essential for vegetative growth. KO gene disruption transformants of the U1 and U2 loci produced viable cell lines. The structural and functional similarities of the Ustilago replication-coupled and replication-independent H3 variants with those in animals, in plants, and in ciliates are remarkable because these distinct histone H3 pairs of variants arose independently in each of these clades and in basidiomycetes.     

H3.5 is a novel hominid-specific histone H3 variant that is specifically expressed in the seminiferous tubules of human testes

The incorporation of histone variants into chromatin plays an important role for the establishment of particular chromatin states. Six human histone H3 variants are known to date, not counting CenH3 variants: H3.1, H3.2, H3.3 and the testis-specific H3.1t as well as the recently described variants H3.X and H3.Y. We report the discovery of H3.5, a novel non-CenH3 histone H3 variant. H3.5 is encoded on human chromosome 12p11.21 and probably evolved in a common ancestor of all recent great apes (Hominidae) as a consequence of H3F3B gene duplication by retrotransposition. H3.5 mRNA is specifically expressed in seminiferous tubules of human testis. Interestingly, H3.5 has two exact copies of ARKST motifs adjacent to lysine-9 or lysine-27, and lysine-79 is replaced by asparagine. In the Hek293 cell line, ectopically expressed H3.5 is assembled into chromatin and targeted by PTM. H3.5 preferentially colocalizes with euchromatin, and it is associated with actively transcribed genes and can replace an essential function of RNAi-depleted H3.3 in cell growth.     

The Differential Mobilization of Histones H3.1 and H3.3 by Herpes Simplex Virus 1 Relates Histone Dynamics to the Assembly of Viral Chromatin

During lytic infections, HSV-1 genomes are assembled into unstable nucleosomes. The histones required for HSV-1 chromatin assembly, however, are in the cellular chromatin. We have shown that linker (H1) and core (H2B and H4) histones are mobilized during HSV-1 infection, and proposed that the mobilized histones are available for assembly into viral chromatin. However, the actual relevance of histone mobilization remained unknown. We now show that canonical H3.1 and variant H3.3 are also mobilized during HSV-1 infection. Mobilization required no HSV-1 protein expression, although immediate early or early proteins enhanced it. We used the previously known differential association of H3.3 and H3.1 with HSV-1 DNA to test the relevance of histone mobilization. H3.3 binds to HSV-1 genomes first, whereas H3.1 only binds after HSV-1 DNA replication initiates. Consistently, H3.3 and H3.1 were differentially mobilized. H3.1 mobilization decreased with HSV-1 DNA replication, whereas H3.3 mobilization was largely unaffected by it. These results support a model in which previously mobilized H3.1 is immobilized by assembly into viral chromatin during HSV-1 DNA replication, whereas H3.3 is mobilized and assembled into HSV-1 chromatin throughout infection. The differential mobilizations of H3.3 and H3.1 are consistent with their differential assembly into viral chromatin. These data therefore relate nuclear histone dynamics to the composition of viral chromatin and provide the first evidence that histone mobilization relates to viral chromatin assembly.     

Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis

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, we have purified deposition machineries for these histones. The H3.1 and H3.3 complexes contain distinct histone chaperones, CAF-1 and HIRA, that we show are 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.     

The human histone H3 complement anno 2011

Histones are highly basic, relatively small proteins that complex with DNA to form higher order structures that underlie chromosome topology. Of the four core histones H2A, H2B, H3 and H4, it is H3 that is most heavily modified at the post-translational level. The human genome harbours 16 annotated bona fide histone H3 genes which code for four H3 protein variants. In 2010, two novel histone H3.3 protein variants were reported, carrying over twenty amino acid substitutions. Nevertheless, they appear to be incorporated into chromatin. Interestingly, these new H3 genes are located on human chromosome 5 in a repetitive region that harbours an additional five H3 pseudogenes, but no other core histone ORFs. In addition, a human-specific novel putative histone H3.3 variant located at 12p11.21 was reported in 2011. These developments raised the question as to how many more human histone H3 ORFs there may be. Using homology searches, we detected 41 histone H3 pseudogenes in the current human genome assembly. The large majority are derived from the H3.3 gene H3F3A, and three of those may code for yet more histone H3.3 protein variants. We also identified one extra intact H3.2-type variant ORF in the vicinity of the canonical HIST2 gene cluster at chromosome 1p21.2. RNA polymerase II occupancy data revealed heterogeneity in H3 gene expression in human cell lines. None of the novel H3 genes were significantly occupied by RNA polymerase II in the data sets at hand, however. We discuss the implications of these recent developments.     

DAXX co-folds with H3.3/H4 using high local stability conferred by the H3.3 variant recognition residues

Histone chaperones are a diverse class of proteins that facilitate chromatin assembly. Their ability to stabilize highly abundant histone proteins in the cellular environment prevents non-specific interactions and promotes nucleosome formation, but the various mechanisms for doing so are not well understood. We now focus on the dynamic features of the DAXX histone chaperone that have been elusive from previous structural studies. Using hydrogen/deuterium exchange coupled to mass spectrometry (H/DX-MS), we elucidate the concerted binding-folding of DAXX with histone variants H3.3/H4 and H3.2/H4 and find that high local stability at the variant-specific recognition residues rationalizes its known selectivity for H3.3. We show that the DAXX histone binding domain is largely disordered in solution and that formation of the H3.3/H4/DAXX complex induces folding and dramatic global stabilization of both histone and chaperone. Thus, DAXX uses a novel strategy as a molecular chaperone that paradoxically couples its own folding to substrate recognition and binding. Further, we propose a model for the chromatin assembly reaction it mediates, including a stepwise folding pathway that helps explain the fidelity of DAXX in associating with the H3.3 variant, despite an extensive and nearly identical binding surface on its counterparts, H3.1 and H3.2.     

Differential Histone Acetylation in Alfalfa (Medicago sativa) Due to Growth in NaCl : Responses in Salt Stressed and Salt Tolerant Callus Cultures

The steady state distribution of histone variant proteins and their modifications by acetylation were characterized in wild type and salinity stress adapted alfalfa (Medicago sativa). Isotopic labeling detected dynamic acetylation at four sites in the histone H3 variants and five sites in histones H4 and H2B. Histone variant H3.2 was the most highly acetylated histone with 25% higher steady state acetylation and a two- to threefold higher acetylation labeling than histone H3.1. Histone phosphorylation was limited to histone variants H1.A, H1.B, and H1.C and to histone H2A.3, which was also acetylated. Histone variant composition was unaffected by cellular exposure to NaCl. Histone acetylation was qualitatively similar in salt-tolerant and salt-sensitive cells under normal growth conditions. However, short term salt stress in salt sensitive cells or continued growth at 1% NaCl in salt tolerant cells led to major increases in the multiacetylated forms of histone H4 and the two variants of histone H3. These changes were more pronounced in the diploid than in the tetraploid alfalfa strains. The increase in multiacetylation of core histones serves as an in vivo reporter suggesting an altered intranuclear ionic environment in the presence of salt. It may also represent an adaptive response in chromatin structure to permit chromatin function in a more saline intranuclear environment.     

Nucleosome formation with the testis-specific histone H3 variant, H3t, by human nucleosome assembly proteins in vitro

Five non-allelic histone H3 variants, H3.1, H3.2, H3.3, H3t and CENP-A, have been identified in mammals. H3t is robustly expressed in the testis, and thus was assigned as the testis-specific H3 variant. However, recent proteomics and tissue-specific RT-PCR experiments revealed a small amount of H3t expression in somatic cells. In the present study, we purified human H3t as a recombinant protein, and showed that H3t/H4 forms nucleosomes with H2A/H2B by the salt-dialysis method, like the conventional H3.1/H4. We found that H3t/H4 is not efficiently incorporated into the nucleosome by human Nap1 (hNap1), due to its defective H3t/H4 deposition on DNA. In contrast, human Nap2 (hNap2), a paralog of hNap1, promotes nucleosome assembly with H3t/H4. Mutational analyses revealed that the Ala111 residue, which is conserved among H3.1, H3.2 and H3.3, but not in H3t, is the essential residue for the hNap1-mediated nucleosome assembly. These results suggest that H3t may be incorporated into chromatin by a specific chaperone-mediated pathway.     

Characterization of histone H2A and H2B variants and their post-translational modifications by mass spectrometry

The nucleosome, the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2A and H2B variants derived from Jurkat cells. A combination of mass spectrometric techniques, HPLC separations, and enzymatic digestions using endoproteinase Glu-C, endoproteinase Arg-C, and trypsin were used to identify histone H2A and H2B subtypes and their modifications. We identified nine histone H2A and 11 histone H2B subtypes, among them proteins that only had been postulated at the gene level. The two main H2A variants, H2AO and H2AC, as well as H2AL were either acetylated at Lys-5 or phosphorylated at Ser-1. For the replacement histone H2AZ, acetylation at Lys-4 and Lys-7 was found. The main histone H2B variant, H2BA, was acetylated at Lys-12, -15, and -20. The analysis of core histone subtypes with their modifications provides a first step toward an understanding of the functional significance of the diversity of histone structures.     

Changes in the nuclear deposition of histone H2A variants during pre-implantation development in mice

Histone H2A has several variants, and changes in chromatin composition associated with their replacement might involve chromatin structure remodeling. We examined the dynamics of the canonical histone H2A and its three variants, H2A.X, H2A.Z and macroH2A, in the mouse during oogenesis and pre-implantation development when genome remodeling occurs. Immunocytochemistry with specific antibodies revealed that, although H2A and all variants were deposited in the nuclei of full-grown oocytes, only histone H2A.X was abundant in the pronuclei of one-cell embryos after fertilization, in contrast with the low abundance of histone H2A and the absence of H2A.Z. The decline in H2A and the depletion of H2A.Z and macroH2A after fertilization were confirmed using Flag epitope-tagged H2A, H2A.Z and macroH2A transgenic mouse lines. Microinjection experiments with mRNA encoding the Flag-tagged proteins revealed a similar pattern of nuclear incorporation of the H2A variants. Fusion protein experiments using H2A, H2A.Z and macroH2A fused with the C-terminal 23 amino acids of H2A.X showed that the C-terminal amino acids of H2A.X function specifically to target this variant histone into chromatin in embryos after fertilization and that the absence of H2A.Z and macroH2A from the chromatin is required for normal development. These results suggest that global changes in the composition of histone H2A variants in chromatin play a role in genome remodeling after fertilization.     

Identification of histone 3 variant 2 interacting factors

The epigenome is defined as a type of information that can be transmitted independently of the DNA sequence, at the chromatin level, through post-translational modifications present on histone tails. Recent advances in the identification of histone 3 variants suggest a new model of information transmission through deposition of specific histone variants. To date, several non-centromeric histone 3 variants have been identified in mammals. Despite protein sequence similarity, specific deposition complexes have been characterized for both histone 3.1 (H3.1) and histone 3.3 (H3.3), whereas no deposition complex for histone 3.2 (H3.2) has been identified to date. Here, we identified human H3.2 partners by immunopurification of nuclear H3.2 complexes followed by mass spectrometry analysis. Further biochemical analyses highlighted two major complexes associated with H3.2, one containing chromatin associated factor-1 subunits and the other consisting of a subcomplex of mini chromosome maintenance helicases, together with Asf1. The purified complexes could associate with a DNA template in vitro.