Histone H4


Replacement variant genes for different histones have been described for most higher eukaryotes. A novel histone H4 encoding gene, H4r, has been isolated which displays all the properties of a histone replacement variant gene: it contains introns, generates polyadenylated mRNA, represents the predominant H4 transcript in non-dividing tissues and is present in the genome as a single copy. The encoded polypeptide is identical to the Drosophila cell-cycle regulated histone H4. The fact that it is a single copy gene makes it prone to genetic analysis and it might prove a useful tool for studying nucleosome structure and function (Akhmanova, 1996).

A cDNA encoding a rat transcription factor IID (TFIID) subunit (p80), with histone H4 homology, was isolated and sequenced. The deduced amino acid sequence predicts a 678-aa protein with 97% identity to the human and 42% to the Drosophila melanogaster homologs. Homologies between three species indicate the presence of three distinct regions (Kida, 1995).

Histone 4 in yeast

The hydrophilic amino-terminal sequences of histones H3 and H4 extend from the highly structured nucleosome core. Histone amino termini are important for nucleosome assembly in vivo and in vitro. Deletion, in yeast, of both tails, a lethal event, alters micrococcal nuclease-generated nucleosomal ladders, plasmid superhelicity in whole cells, and nucleosome assembly in cell extracts. The H3 and H4 amino-terminal tails have redundant functions in this regard because the presence of either tail allows assembly and cellular viability. Moreover, the tails need not be attached to their native carboxy-terminal core. Their exchange re-establishes both cellular viability and nucleosome assembly. In contrast, the regulation of GAL1 and the silent mating loci by the H3 and H4 tails is highly disrupted by exchange of the histone amino termini (Ling, 1996).

In S. cerevisiae, a temperature-sensitive lethal histone H4 mutant has been shown to be defective in mitotic chromosome transmission. The mutant requires two amino acid substitutions in histone H4: a lethal Thr-to-Ile change at position 82, which lies within one of the DNA-binding surfaces of the protein, and a substitution of Ala to Val at position 89 that is an intragenic suppressor. Genetic and biochemical evidence shows that the mutant histone H4 is temperature sensitive for function but not for synthesis, deposition, or stability. The chromatin structure of 2 micrometer circle minichromosomes is temperature sensitive in vivo, consistent with a defect in H4-DNA interactions. The mutant also has defects in transcription, displaying weak Spt- phenotypes. At the restrictive temperature, mutant cells arrest in the cell cycle at nuclear division, with a large bud, a single nucleus with 2C DNA content, and a short bipolar spindle. At semipermissive temperatures, the frequency of chromosome loss is elevated 60-fold in the mutant, while DNA recombination frequencies are unaffected. High-copy CSE4, encoding an H3 variant related to the mammalian CENP-A kinetochore antigen, suppresses the temperature sensitivity of the mutant without suppressing the Spt- transcription defect. These genetic, biochemical, and phenotypic results indicate that this novel histone H4 mutant defines one or more chromatin-dependent steps in chromosome segregation (Smith, 1996).

Repression of yeast mating type "a" cell-specific genes by the global repressor Ssn6/Tup1 (see Drosophila Groucho) has been linked to a specific organization of chromatin. Tup1 directly interacts with the amino-terminal tails of histones H3 and H4, providing a molecular basis for this connection. This interaction appears to be required for Tup1 function because amino-terminal mutations in H3 and H4 that weaken interactions with Tup1 cause derepression of both "a" cell-specific and DNA damage-inducible genes. Moreover, the Tup1 histone-binding domain coincides with the Tup1 repression domain. Tup1/histone interactions are negatively influenced by high levels of histone acetylation, suggesting a mechanism whereby the organization of chromatin may be modulated in response to changing environmental signals (Edmondson, 1996).

The Saccharomyces cerevisiae alpha2 repressor controls two classes of cell-type-specific genes in yeast through association with different partners. alpha2-Mcm1 complexes repress a cell-specific gene expression in haploid alpha cells and diploid a/alpha cells, while a1-alpha2 complexes repress haploid-specific genes in diploid cells. In both cases, repression is mediated through Ssn6-Tup1 corepressor complexes that are recruited via direct interactions with alpha2. Nucleosomes are positioned adjacent to the alpha2-Mcm1 operator under conditions of repression and Tup1 interacts directly with histones H3 and H4. An examination was carried out of the role of chromatin in a1-alpha2 repression to determine if chromatin is a general feature of repression by Ssn6-Tup1. Mutations in the amino terminus of histone H4 cause a 4- to 11-fold derepression of a reporter gene under a1-alpha2 control, while truncation of the H3 amino terminus has a more modest (3-fold or less) effect. Strikingly, combination of the H3 truncation with an H4 mutation causes a 40-fold decrease in repression, clearly indicating a central role for these histones in a1-alpha2-mediated repression. However, in contrast to the ordered positioning of nucleosomes adjacent to the alpha2-Mcm1 operator, nucleosomes are not positioned adjacent to the a1-alpha2 operator in diploid cells. These data indicate that chromatin is important to Ssn6-Tup1-mediated repression but that the degrees of chromatin organization directed by these proteins differ at different promoters (Huang, 1997).

RNA polymerase I (Pol I) transcription in the yeast Saccharomyces cerevisiae, which is responsible for ribosomal RNA synthesis, is greatly stimulated in vivo and in vitro by the multiprotein complex, upstream activation factor (UAF). UAF binds tightly to the upstream element of the rDNA promoter: once bound (in vitro), UAF does not readily exchange onto a competing template. UAF binding to the template is necessary for subsequent binding of TATA box-binding protein and core factor into a stable preinitiation complex. Of the polypeptides previously identified in purified UAF, three are encoded by genes required for Pol I transcription in vivo: RRN5, RRN9, and RRN10. Two others, p30 and p18, have remained uncharacterized. However, the N-terminal amino acid sequence, its mobility in gel electrophoresis, and the immunoreactivity of p18 all indicate that p18 is the same as histone H3. Histone H4 was found in UAF, and myc-tagged histone H4 can be used to affinity-purify UAF. Histones H2A and H2B are not detectable in UAF. These results suggest that histones H3 and H4 probably account for the strong binding of UAF to DNA and may offer a means by which general nuclear regulatory signals could be transmitted to Pol I (Keener, 1997).

Transcription of many yeast genes requires the SWI/SNF regulatory complex (See Brahma and ISWI). Prior studies have shown that reduced transcription of the HO gene in swi and snf mutants is partially relieved by mutations in the SIN1 and SIN2 genes. SIN2 is identical to HHT1, one of the two genes coding for histone H3; mutations in either gene can result in a Sin- phenotype. These mutations are partially dominant to wild type and cause amino acid substitutions in three conserved positions in the structured domain of histone H3. Partially dominant sin mutations have been identified that affect two conserved positions in the histone-fold domain of histone H4. Three sin mutations affect surface residues proposed to interact with DNA and may reduce affinity of DNA for the histone octamer. Two sin mutations affect residues at or near interfaces between (H2A-H2B) dimer and (H3-H4)*2 tetramer subunits of the histone octamer and may affect nucleosome stability or conformation. The ability of mutations affecting the structure of the histone octamer to relieve the need for SWI and SNF products supports the proposal that the SWI/SNF complex stimulates transcription by altering chromatin structure and can account for the apparent conservation of SWI and SNF proteins in eukaryotes other than yeast (Kruger, 1995).

Yeast histone H4 has been mutagenized at several amino acid positions that participate in the globular core of the nucleosome. The native protein contains residues at these positions, which are either invariant or highly conserved over all known H4 sequences, whether from yeast, Tetrahymena or higher eukaryotes. Nonetheless, the protein is tolerant of non-conservative mutations. At the level of cell function the mutant proteins cause no significant change in the length of the cell cycle of mating efficiency. At the level of chromatin structure no effect is observed on the internucleosomal spacing of chromatin or the pattern of hydroxyl radical cleavage of nucleosomal DNA (Agarwal, 1996).

Within the core histone octamer each histone H4 interacts with each H2A-H2B dimer subunit through two binding surfaces. Tyrosines play a central role in these interactions with H4 tyrosines 72 and 88 contacting one H2A-H2B dimer subunit, and tyrosine 98 contacting the other. To investigate the roles of these interactions in vivo, site-directed amino acid substitutions were made at each of these tyrosine residues of the yeast S. cerevisiae. Elimination of either set of interactions is lethal, suggesting that binding of the tetramer to both dimers is essential. Temperature-sensitive mutants were obtained through single amino acid substitutions at each of the tyrosines. The mutants show both strong positive and negative effects on transcription. Positive effects include Spt- and Sin-phenotypes resulting from mutations at each of the three tyrosines. Spt-phenotypes are known to be due to alterations of the relative ratio of the H3-H4 and H2A-H2B histone gene pairs and can have strong effects on a variety of cell functions, including mitotic chromosome transmission. Sin-phenotypes render regulated target genes SWI-SNF independent. One allele has a strong negative effect on the expression of genes essential for the G1 cell cycle transition. At restrictive temperature, mutant cells fail to express the cyclins CLN1 and CLN2 and also fail to express SWI4 and SWI6 genes. Mutants also have reduced levels of CLN3 mRNA. These results demonstrate the critical role of histone dimer-tetramer interactions in vivo, and define their essential role in the expression of genes regulating G1 cell cycle progression. The finding that histone dimer-tetramer subunit interactions also affect the G1-S transition, through expression of the G1 cyclins, suggests that the regulatory components of the cell division cycle may have evolved to depend on multiple aspects of chromatin structure (Santisteban, 1997).

A search for proteins that interact genetically with histone H3 and H4 amino termini uncovers novel regulators of the Swe1 kinase in S. cerevisiae. Three new gene products have been found that regulate the S. cerevisiae Swe1 kinase. The Swe1 protein kinase phosphorylates tyrosine residue 19 of Cdc28 and inhibits its activity. One histone synthetic-lethal gene, HSL1, encodes a putative protein kinase that has high sequence and functional homology to fission yeast cdr1/nim1, an inhibitory kinase of wee1. Another gene, HSL7, is a novel negative regulator of Swe1 function. Sequences similar to Hsl7 exist in both C. elegans and humans. In addition, a dosage-dependent suppressor (OSS1) of hsl1 and hsl7 has been isolated. OSS1 is important for the transcriptional repression of SWE1 and CLN2 in G2. Mutations in HSL1 and HSL7 therefore cause hyperactivity of the Swe1 kinase, which in turn decreases mitotic Cdc28 kinase activity. Moreover, HSL5 is identical to CDC28, further suggesting that it is the decreased Cdc28 kinase activity in these hsl mutants that causes lethality in the histone mutant background. Yeast cell cycle regulators function in a pathway upstream of both histones H3 and H4, thereby modulating histone function in the cell cycle (Ma, 1996).

Histone H4 can be acetylated at N-terminal lysines K5, K8, K12, and K16, but newly synthesized H4 is diacetylated at K5/K12 in diverse organisms. This pattern is widely thought to be important for histone deposition onto replicating DNA. To investigate the importance of K5/K12 these lysines have been mutagenized in yeast and nucleosome assembly has been assayed. Assaying was done in the absence of the histone H3 N terminus, which has functions redundant with those of H4 in histone deposition. Nucleosome assembly was assayed by three methods. Because nucleosome depletion may be lethal, cell viability was assayed. Nucleosome assembly was also assayed in vivo and in vitro by examining plasmid superhelicity density in whole cells and supercoiling in yeast cell extracts. All three approaches demonstrate that mutagenizing K5 and K12 together does not prevent cell growth and histone deposition in vivo or in vitro. Therefore, K5/K12 cannot be required for nucleosome assembly in yeast. It is only when the first three sites of acetylation-K5, K8, and K12-are mutagenized simultaneously that lethality occurs and assembly is most strongly decreased both in vivo and in vitro. These data argue for the redundancy of sites K5, K8, and K12 in the deposition of yeast histone H4 (Ma, 1998).

Heterochromatin in metazoans induces transcriptional silencing, as exemplified by position effect variegation in Drosophila and X-chromosome inactivation in mammals. Heterochromatic DNA is packaged in nucleosomes that are distinct in their acetylation pattern from those present in euchromatin, although the role these differences play in the structure of heterochromatin or in the effects of heterochromatin on transcriptional activity is unclear. As observed in the facultative heterochromatin of the inactive X chromosome in female mammalian cells, histones H3 and H4 in chromatin spanning the transcriptionally silenced mating-type cassettes of the yeast Saccharomyces cerevisiae are hypoacetylated relative to histones H3 and H4 of transcriptionally active regions of the genome. By immunoprecipitation of chromatin fragments with antibodies specific for H4 acetylated at particular lysine residues, only three of the four lysine residues in the amino-terminal domain of histone H4 spanning the silent cassettes are found to be hypoacetylated. Lysine 12 shows significant acetylation levels. This is identical to the pattern of histone H4 acetylation observed in centric heterochromatin of D. melanogaster. These two observations provide additional evidence that the silent cassettes are encompassed in the yeast equivalent of metazoan heterochromatin. Further, mutational analysis of the amino-terminal domain of histone H4 in S. cerevisiae demonstrate that this observed pattern of histone H4 acetylation is required for transcriptional silencing. This result, in conjunction with prior mutational analyses of yeast histones H3 and H4, indicates that the particular pattern of nucleosome acetylation found in heterochromatin is required for its effects on transcription and is not simply a side effect of heterochromatin formation (Braunstein, 1996).

The target of rapamycin (TOR) protein is a conserved regulator of ribosome biogenesis, an important process for cell growth and proliferation. However, how TOR is involved remains poorly understood. Rapamycin and nutrient starvation, conditions inhibiting TOR, are found to lead to significant nucleolar size reduction in both yeast and mammalian cells. In yeast, this morphological change is accompanied by release of RNA polymerase I (Pol I) from the nucleolus and inhibition of ribosomal DNA (rDNA) transcription. Evidence is presented that TOR regulates association of Rpd3-Sin3 histone deacetylase (HDAC) with rDNA chromatin, leading to site-specific deacetylation of histone H4. Moreover, histone H4 hypoacetylation mutations cause nucleolar size reduction and Pol I delocalization, while rpd3Delta and histone H4 hyperacetylation mutations block the nucleolar changes as a result of TOR inhibition. Taken together, these results suggest a chromatin-mediated mechanism by which TOR modulates nucleolar structure, RNA Pol I localization and rRNA gene expression in response to nutrient availability (Tsang, 2003).

Saccharomyces cerevisiae has a global pattern of histone acetylation in which histone H3 and H4 acetylation levels are lower at protein-coding sequences than at promoter regions. The loss of Eaf3, a subunit of the NuA4 histone acetylase and Rpd3 histone deacetylase complexes, greatly alters the genomic profile of histone acetylation, with the effects on H4 appearing to be more pronounced than those on H3. Specifically, the loss of Eaf3 causes increases in H3 and H4 acetylation at coding sequences and decreases at promoters, such that histone acetylation levels become evenly distributed across the genome. Eaf3 does not affect the overall level of H4 acetylation, the recruitment of the NuA4 catalytic subunit Esa1 to target promoters, or the level of transcription of the genes analyzed for histone acetylation. Whole-genome transcriptional profiling indicates that Eaf3 plays a positive, but quantitatively modest, role in the transcription of a small subset of genes, whereas it has a negative effect on very few genes. It is suggested that Eaf3 regulates the genomic profile of histone H3 and H4 acetylation in a manner that does not involve targeted recruitment and is independent of transcriptional activity (Reid, 2004).

The budding yeast histone H3 variant, Cse4, replaces conventional histone H3 in centromeric chromatin and, together with centromere-specific DNA-binding factors, directs assembly of the kinetochore, a multiprotein complex mediating chromosome segregation. Scm3, a nonhistone protein that colocalizes with Cse4 is required for its centromeric association. Bacterially expressed Scm3 binds directly to and reconstitutes a stoichiometric complex with Cse4 and histone H4 but not with conventional histone H3 and H4. A conserved acidic domain of Scm3 is responsible for directing the Cse4-specific interaction. Strikingly, binding of Scm3 can replace histones H2A-H2B from preassembled Cse4-containing histone octamers. This incompatibility between Scm3 and histones H2A-H2B is correlated with diminished in vivo occupancy of histone H2B, H2A, and H2AZ at centromeres. These findings indicate that nonhistone Scm3 serves to assemble and maintain Cse4-H4 at centromeres and may replace histone H2A-H2B dimers in a centromere-specific nucleosome core (Mizuguchi, 2007).

In the eukaryotic genome, transcriptionally silent chromatin tends to propagate along a chromosome and encroach upon adjacent active chromatin. The silencing machinery can be stopped by chromatin boundary elements. A screen was performed in Saccharomyces cerevisiae for proteins that may contribute to the establishment of a chromatin boundary. It was found that disruption of histone deacetylase Rpd3p results in defective boundary activity, leading to a Sir-dependent local propagation of transcriptional repression. In rpd3δ cells, the amount of Sir2p that was normally found in the nucleolus decreased and the amount of Sir2p found at telomeres and at HM and its adjacent loci increased, leading to an extension of silent chromatin in those areas. In addition, Rpd3p interacts directly with chromatin at boundary regions to deacetylate histone H4 at lysine 5 and at lysine 12. Either the mutation of histone H4 at lysine 5 or a decrease in the histone acetyltransferase (HAT) activity of Esa1p abrogated the silencing phenotype associated with rpd3 mutation, suggesting a novel role for the H4 amino terminus in Rpd3p-mediated heterochromatin boundary regulation. Together, these data provide insight into the molecular mechanisms for the anti-silencing functions of Rpd3p during the formation of heterochromatin boundaries (Zhou, 2009).

The chromatin landscape of Drosophila: comparisons between species, sexes, and chromosomes

The chromatin landscape is key for gene regulation, but little is known about how it differs between sexes or between species. This paper examines the sex-specific chromatin landscape of Drosophila miranda, a species with young sex chromosomes, and compares it with Drosophila melanogaster. Six histone modifications were examined in male and female larvae of D. miranda (H3K4me1, H3K4me3, H3K36me3, H4K16ac, H3K27me3, and H3K9me2), and seven biologically meaningful chromatin states were defined that show different enrichments for transcribed and silent genes, repetitive elements, housekeeping, and tissue-specific genes. The genome-wide distribution of both active and repressive chromatin states differs between males and females. In males, active chromatin is enriched on the X, relative to females, due to dosage compensation of the hemizygous X. Furthermore, a smaller fraction of the euchromatic portion of the genome is in a repressive chromatin state in males relative to females. However, sex-specific chromatin states appear not to explain sex-biased expression of genes. Overall, conservation of chromatin states between male and female D. miranda is comparable to conservation between D. miranda and D. melanogaster, which diverged >30 MY ago. Active chromatin states are more highly conserved across species, while heterochromatin shows very low levels of conservation. Divergence in chromatin profiles contributes to expression divergence between species, with approximately 26% of genes in different chromatin states in the two species showing species-specific or species-biased expression, an enrichment of approximately threefold over null expectation. These data suggest that heteromorphic sex chromosomes in males (that is, a hypertranscribed X and an inactivated Y) may contribute to global redistribution of active and repressive chromatin marks between chromosomes and sexes (Brown, 2014).

Structure of a mammalian CENP-A-histone H4 heterodimer in complex with chaperone HJURP

In higher eukaryotes, the centromere is epigenetically specified by the histone H3 variant Centromere Protein-A (CENP-A). Deposition of CENP-A to the centromere requires histone chaperone HJURP (Holliday junction recognition protein). The crystal structure of an HJURP-CENP-A-histone H4 complex shows that HJURP binds a CENP-A-H4 heterodimer. The C-terminal β-sheet domain of HJURP caps the DNA-binding region of the histone heterodimer, preventing it from spontaneous association with DNA. This analysis also revealed a novel site in CENP-A that distinguishes it from histone H3 in its ability to bind HJURP. These findings provide key information for specific recognition of CENP-A and mechanistic insights into the process of centromeric chromatin assembly (Hu, 2011).

HJURP has been identified is distantly related to the yeast centromeric protein Scm3. HJURP interacts directly with CENP-A and histone H4, localizes CENP-A to the centromere in a cell cycle-dependent manner, and enables the deposition of newly synthesized CENP-A into the centromeric nucleosome. An approximately 80-amino-acid CENP-A-binding domain (CBD) at the N terminus of HJURP is necessary and sufficient for binding CENP-A, and a region encompassing loop-1 and helix α2 of the histone fold domain of CENP-A, known as the CENP-A targeting domain (CATD), is required for interaction with HJURP. This study provide the structural basis for the recognition of CENP-A by HJURP, as well as mechanistic insights into the histone chaperone activity of HJURP (Hu, 2011).

It is striking that the Ser 68 site outside of the CENP-A CATD plays a critical role for HJURP recognition, as previous studies have shown that the H3CATD chimera protein recapitulated essential functions of CENP-A, and the current in vitro results also confirmed direct binding of H3CATD to HJURP. Surprisingly, analyses of the HJURP-CENP-A-H4 and H3-H4 structures revealed no hindrance of HJURP binding in the histone H3 region corresponding to CATD. One scenario, which has been called a 'yin-yang' model, is that CATD provides major binding affinities for HJURP, and the Ser 68 site serves as a principal determinant of CENP-A specificity. In this model, the histone H3 region corresponding to the CATD of CENP-A can interact with HJURP, perhaps suboptimally, but Gln 68 pushes away HJURP; the latter force wins and there is no binding. In H3CATD, the artificially introduced CATD can overcome the energy barrier caused by the unfavorable contact of Gln 68, resulting in the binding of HJURP. Less intuitive in this mode is how the CATD overpowers Gln 68 in H3CATD, as the S68Q mutant of CENP-A with a native CATD loses the ability to bind HJURP. It should be pointed out that structural change may play an important role in the resolution of this puzzle, as it is evident from the deuterium exchange experiments that a non-CATD region in helix α1 of H3CATD has a different conformation from the corresponding region in histone H3 or CENP-A. It is also evident from structural comparisons that the α1-L1 region is the most variable region among the structures of histone H3-H4 and CENP-A-H4 complexes. Thus, it is possible that introduction of a CATD into H3 resulted in an environment that remedied the adverse effect of Gln 68. A cocrystal structure of HJURP CBD in complex with the H3CATD-H4 complex should clarify the role of Gln 68 in H3CATD, and, together with in vivo experiments, will provide further tests of the 'yin-yang' model of centromere targeting proposed in this study (Hu, 2011).

The discovery that HJURP binds a heterodimeric form of the CENP-A-H4 complex also has profound implications in understanding the molecular mechanism of assembly of CENP-A-containing nucleosomes. The precise model of CENP-A-containing nucleosomes is still a matter of debate. A closely relevant point here is whether a centromeric nucleosome contains a CENP-A-H4 heterodimer or heterotetramer. The structural result cannot distinguish whether a CENP-A nucleosome is octameric or a hemisome. However, it does point out that, if CENP-A nucleosomes were octameric, additional processes or regulations would be required to ensure that the CENP-A-containing nucleosomes are predominantly homotypic, as heterotypic nucleosomes with both CENP-A and histone H3 are a minority species (Hu, 2011).

In summary, this structural and biochemical analyses of the HJURP CBD-CENP-A-histone H4 complex have provided novel insights into the specificity of CENP-A recognition by HJURP, and advanced understanding of the histone chaperone activities of HJURP in preventing the formation of a CEN-A-histone H4 tetramer and modulating the DNA-binding activity of the CENP-A-histone H4 complex. The results of this study should facilitate in-depth analyses of the molecular mechanism of centromeric chromatin assembly (Hu, 2011).

Transcriptional regulation of Histone H4

The CCAAT displacement protein (CDP-cut/CUTL1/cux) performs a key proliferation-related function as the DNA binding subunit of the cell cycle controlled HiNF-D complex. HiNF-D interacts with all five classes (H1, H2A, H2B, H3, and H4) of the cell-cycle dependent histone genes, which are transcriptionally and coordinately activated at the G(1)/S phase transition independent of E2F. The tumor suppressor pRB/p105 is an intrinsic component of the HiNF-D complex. However, the molecular interactions that enable CDP and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters must be further defined. Using transient transfections, it has been shown that CDP represses the H4 gene promoter and that pRB functions with CDP as a co-repressor. Direct physical interaction between CDP and pRB was observed in glutathione-S-transferase (GST) pull-down assays. Furthermore, interactions between these proteins were established by yeast and mammalian two-hybrid experiments and co-immunoprecipitation assays. Confocal microscopy shows that subsets of each protein are co-localized in situ. Using a series of pRB mutants, it has been found that the CDP/pRB interaction, similar to the E2F/pRB interaction, utilizes the A/B large pocket (LP) of pRB. Thus, several converging lines of evidence indicate that complexes between CDP and pRB repress cell cycle regulated histone gene promoters (Gupta, 2003).

Histone H4 and early development

In the mouse embryo, transcriptional activation begins during S/G2 phase of the first cell cycle when paternal and maternal chromatin are still in separate nuclear entities within the same cytoplasm. At this time, the male pronucleus exhibits greater transcriptional activity than the female pronucleus. Since acetylation of histones in the nucleosome octamer exerts a regulatory influence on gene expression, changes in histone acetylation were investigated during the remodeling of paternal and maternal chromatin from sperm entry through to minor genome activation and mitosis. Neither mature sperm nor metaphase II maternal chromatin stain for hyperacetylated histone H4. Immediately following fertilization, hyperacetylated H4 is associated with paternal but not maternal chromatin, while maternal chromatin becomes hyperacetylated in parthenogenetically activated oocytes. In zygotes, differential levels and patterns of hyperacetylated H4 between male and female pronuclei persist throughout most of G1 with histone deacetylases and acetyltransferases already active at this time. When transcriptional differences are observed in S/G2, male and female pronuclei have equivalent levels of H4 hyperacetylation: DNA replication is not required to attain this equivalence. In contrast to the lack of H4 hyperacetylation on gametic chromatin, chromosomes at the first mitosis showed distinct banding patterns of H4 hyperacetylation. These results suggest (1) that sperm chromatin initially out-competes maternal chromatin for the pool of hyperacetylated H4 in the oocyte; (2) that hyperacetylated H4 participates in the process of histone-protamine exchange in the zygote, and (3) that differences in H4 acetylation in male and female pronuclei during G1 are translated across DNA replication to transcriptional differences in S/G2. Prior to fertilization, neither paternal nor maternal chromatin show memory of H4 hyperacetylation patterns, but by the end of the first cell cycle, before major zygotic genome activation at the 2-cell stage, chromosomes already show hyperacetylated H4 banding patterns. It is suggested that core histone acetylation is an important regulatory parameter for transcription in a chromatin context. The greater transcriptional activity of the male as compared to the female pronucleus might be related to differences in the progression of histone H4 acetylation patterns. In this context histone acetylation might be a mechanism for maintaining memory of cellular transcriptional patterns during mitosis (Adenot, 1997).

The transition from a late 1-cell mouse embryo to a 4-cell embryo, the period when zygotic gene expression begins, is accompanied by an increasing ability to repress the activities of promoters and replication origins. Since this repression can be relieved by either butyrate or enhancers, it appears to be mediated through chromatin structure. Oocytes, which can repress promoter activity, synthesize a full complement of histones, and histone synthesis up to the early 2-cell stage originate from mRNA inherited from the oocyte. However, while histones H3 and H4 continue to be synthesized in early 1-cell embryos, synthesis of histones H2A, H2B and H1 (proteins required for chromatin condensation) is delayed until the late 1-cell stage, reaching amaximum rate in early 2-cell embryos. Histone H4 in both 1-cell and 2-cell embryos is predominantly diacetylated (a modification that facilitates transcription). Deacetylation towards the unacetylated and monoacetylated H4 population in fibroblasts begin at the late 2-cell to 4-cell stage. Arresting development at the beginning of S-phase in 1-cell embryos prevents both the appearance of chromatin-mediated repression of transcription in paternal pronuclei and synthesis of new histones. These changes correlate with the establishment of chromatin-mediated repression during formation of a 2-cell embryo, and the increase in repression from the 2-cell to 4-cell stage as linker histone H1 accumulates and core histones are deacetylated (Wiekowski, 1997).

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).

H4K20 methylation is a broad chromatin modification that has been linked with diverse epigenetic functions. Several enzymes target H4K20 methylation, consistent with distinct mono-, di-, and trimethylation states controlling different biological outputs. To analyze the roles of H4K20 methylation states, conditional null alleles were generated for the two Suv4-20h histone methyltransferase (HMTase) genes in the mouse. Suv4-20h-double-null (dn) mice are perinatally lethal and have lost nearly all H4K20me3 and H4K20me2 states. The genome-wide transition to an H4K20me1 state results in increased sensitivity to damaging stress, since Suv4-20h-dn chromatin is less efficient for DNA double-strand break (DSB) repair and prone to chromosomal aberrations. Notably, Suv4-20h-dn B cells are defective in immunoglobulin class-switch recombination, and Suv4-20h-dn deficiency impairs the stem cell pool of lymphoid progenitors. Thus, conversion to an H4K20me1 state results in compromised chromatin that is insufficient to protect genome integrity and to process a DNA-rearranging differentiation program in the mouse (Schotta, 2008).

Multiple spatially distinct types of facultative heterochromatin on the human inactive X chromosome

Heterochromatin is defined classically by condensation throughout the cell cycle, replication in late S phase and gene inactivity. Facultative heterochromatin is of particular interest, because its formation is developmentally regulated as a result of cellular differentiation. The most extensive example of facultative heterochromatin is the mammalian inactive X chromosome (Xi). A variety of histone variants and covalent histone modifications have been implicated in defining the organization of the Xi heterochromatic state, and the features of Xi heterochromatin have been widely interpreted as reflecting a redundant system of gene silencing. The human Xi is packaged into at least two nonoverlapping heterochromatin types, each characterized by specific Xi features: one defined by the presence of Xi-specific transcript RNA, the histone variant macroH2A, and histone H3 trimethylated at lysine 27 and the other defined by H3 trimethylated at lysine 9, heterochromatin protein 1, and histone H4 trimethylated at lysine 20. Furthermore, regions of the Xi packaged in different heterochromatin types are characterized by different patterns of replication in late S phase. The arrangement of facultative heterochromatin into spatially and temporally distinct domains has implications for both the establishment and maintenance of the Xi and adds a previously unsuspected degree of epigenetic complexity (Chadwick, 2004).

Histone H4 Acetylation and chromatin assembly

Continued: see Histone 4 - Evolutionary Homologs part 2/3 ! part 3/3

Histone H4: Biological Overview | Regulation | Developmental Biology | References

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