Histone modifications associated with both A and B chromosomes of maize

We report the distribution of several histone modifications along the arms and in centromeric regions of somatic chromosomes of maize, including the supernumerary B chromosome. Acetylated H3 and H4 as well as H3K4me2, modifications associated with euchromatin, were enriched in the distal parts of the A chromosomes, but were progressively depleted toward the centromeres of the A chromosomes and were depleted in the heterochromatic portions of the B chromosome. Classical histone modifications associated with heterochromatin, including H3K9me2, H3K27me1 and H3K27me2, were distributed throughout both A and B chromosomes. However, H3K27me2 showed a reduced level on the B chromosome compared with the A chromosomes and was not associated with some classes of constitutive heterochromatin. We monitored the presence of each histone modification in the centromeric regions using a YFP-tagged centromere-specific histone, CENH3. We observed the presence of H3K9me2 and absence of H3K4me2 in the centromeric regions of both A and B chromosomes of maize, which is in contrast to the presence of H3K4me2 and absence of H3K9me2 in animal centromeres. These results show a diversity of epigenetic modifications associated with centromeric chromatin in different eukaryotes.     

The chromosomal distribution of histone methylation marks in gymnosperms differs from that of angiosperms

The chromosomal distribution of seven histone methylation marks (H3K4me2, H3K9me1,2,3 and H3K27me1,2,3) was analysed in the gymnosperm species Pinus sylvestris and Picea abies. Similarly to the situation in other investigated eukaryotes, dimethylation of lysine 4 of histone H3 is restricted to euchromatin in gymnosperms. Surprisingly, also H3K9me1-a mark classified as heterochromatin-specific in angiosperms-labels the euchromatin in P. sylvestris and P. abies. The other investigated methylation marks are either equally distributed along the chromosomes, as H3K9me2 and H3K27me1 (in both species) and H3K9me3 (in P. abies), or enriched at specific types of heterochromatin, as H3K9me3 (in P. sylvestris) and H3K27me2 and H3K27me3 in both species. Although the methylation marks themselves are apparently conserved, their functional specificity within the frame of the 'epigenetic code' might have diverged during evolution.     

Broad chromosomal domains of histone modification patterns in C. elegans

Chromatin immunoprecipitation identifies specific interactions between genomic DNA and proteins, advancing our understanding of gene-level and chromosome-level regulation. Based on chromatin immunoprecipitation experiments using validated antibodies, we define the genome-wide distributions of 19 histone modifications, one histone variant, and eight chromatin-associated proteins in Caenorhabditis elegans embryos and L3 larvae. Cluster analysis identified five groups of chromatin marks with shared features: Two groups correlate with gene repression, two with gene activation, and one with the X chromosome. The X chromosome displays numerous unique properties, including enrichment of monomethylated H4K20 and H3K27, which correlate with the different repressive mechanisms that operate in somatic tissues and germ cells, respectively. The data also revealed striking differences in chromatin composition between the autosomes and between chromosome arms and centers. Chromosomes I and III are globally enriched for marks of active genes, consistent with containing more highly expressed genes, compared to chromosomes II, IV, and especially V. Consistent with the absence of cytological heterochromatin and the holocentric nature of C. elegans chromosomes, markers of heterochromatin such as H3K9 methylation are not concentrated at a single region on each chromosome. Instead, H3K9 methylation is enriched on chromosome arms, coincident with zones of elevated meiotic recombination. Active genes in chromosome arms and centers have very similar histone mark distributions, suggesting that active domains in the arms are interspersed with heterochromatin-like structure. These data, which confirm and extend previous studies, allow for in-depth analysis of the organization and deployment of the C. elegans genome during development.     

Jmjd2b antagonizes H3K9 trimethylation at pericentric heterochromatin in mammalian cells

Histone lysine trimethyl states represent some of the most robust epigenetic modifications in eukaryotic chromatin. Using a candidate approach, we identified the subgroup of murine Jmjd2 proteins to antagonize H3K9me3 at pericentric heterochromatin. H3K27me3 and H4K20me3 marks are not impaired in inducible Jmjd2b-GFP cell lines, but Jmjd2b also reduces H3K36 methylation. Since recombinant Jmjd2b appears as a very poor enzyme, we applied metabolic labeling with heavy methyl groups to demonstrate Jmjd2b-mediated removal of chromosomal H3K9me3 as an active process that occurs well before replication of chromatin. These data reveal that certain members of the jmjC class of hydroxylases can work in a pathway that actively antagonizes a histone lysine trimethyl state.     

Histone modification and the control of heterochromatic gene silencing in Drosophila

Covalent modifications of histones index structurally and functionally distinct chromatin domains in eukaryotic nuclei. Drosophila with its polytene chromosomes and developed genetics allows detailed cytological as well as functional analysis of epigenetic histone modifications involved in the control of gene expression pattern during development. All H3K9 mono- and dimethylation together with all H3K27 methylation states and H4K20 trimethylation are predominant marks of pericentric heterochromatin. In euchromatin, bands and interbands are differentially indexed. H3K4 and H3K36 methylation together with H3S10 phosphorylation are predominant marks of interband regions whereas in bands different H3K27 and H4K20 methylation states are combined with acetylation of H3K9 and H3K14. Genetic dissection of heterochromatic gene silencing in position-effect variegation (PEV) by Su(var) and E(var) mutations allowed identification and functional analysis of key factors controlling the formation of heterochromatin. SU(VAR)3-9 association with heterochromatic sequences followed by H3K9 methylation initiates the establishment of repressive SU(VAR)3-9/HP1/SU(VAR)3-7 protein complexes. Differential enzymatic activities of novel point mutants demonstrate that the silencing potential of SU(VAR)3-9 is mainly determined by the kinetic properties of the HMTase reaction. In Su(var)3-9^ptn a significantly enhanced enzymatic activity results in H3K9 hypermethylation, enhanced gene silencing and extensive chromatin compaction. Mutations in factors controlling active histone modification marks revealed the dynamic balance between euchromatin and heterochromatin. Further analysis and definition of Su(var) and E(var) genes in Drosophila will increase our understanding of the molecular hierarchy of processes controlling higher-order structures in chromatin.     

Disruption of genomic neighbourhood at the imprinted IGF2-H19 locus in Beckwith-Wiedemann syndrome and Silver-Russell syndrome

Hyper- and hypomethylation at the IGF2-H19 imprinting control region (ICR) result in reciprocal changes in IGF2-H19 expression and the two contrasting growth disorders, Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). DNA methylation of the ICR controls the reciprocal imprinting of IGF2 and H19 by preventing the binding of the insulator protein, CTCF. We here show that local changes in histone modifications and CTCF--cohesin binding at the ICR in BWS and SRS together with DNA methylation correlate with the higher order chromatin structure at the locus. In lymphoblastoid cells from control individuals, we found the repressive histone H3K9me3 and H4K20me3 marks associated with the methylated paternal ICR allele and the bivalent H3K4me2/H3K27me3 mark together with H3K9ac and CTCF--cohesin associated with the non-methylated maternal allele. In patient-derived cell lines, the mat/pat asymmetric distribution of these epigenetic marks was lost with H3K9me3 and H4K20me3 becoming biallelic in the BWS and H3K4me2, H3K27me3 and H3K9ac together with CTCF-cohesin becoming biallelic in the SRS. We further show that in BWS and SRS cells, there is opposing chromatin looping conformation mediated by CTCF--cohesin binding sites surrounding the locus. In normal cells, lack of CTCF--cohesin binding at the paternal ICR is associated with monoallelic interaction between two CTCF sites flanking the locus. CTCF--cohesin binding at the maternal ICR blocks this interaction by associating with the CTCF site downstream of the enhancers. The two alternative chromatin conformations are differently favoured in BWS and SRS likely predisposing the locus to the activation of IGF2 or H19, respectively.     

Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk

Cross-talk between DNA methylation and histone modifications drives the establishment of composite epigenetic signatures and is traditionally studied using correlative rather than direct approaches. Here, we present sequential ChIP-bisulfite-sequencing (ChIP-BS-seq) as an approach to quantitatively assess DNA methylation patterns associated with chromatin modifications or chromatin-associated factors directly. A chromatin-immunoprecipitation (ChIP)-capturing step is used to obtain a restricted representation of the genome occupied by the epigenetic feature of interest, for which a single-base resolution DNA methylation map is then generated. When applied to H3 lysine 27 trimethylation (H3K27me3), we found that H3K27me3 and DNA methylation are compatible throughout most of the genome, except for CpG islands, where these two marks are mutually exclusive. Further ChIP-BS-seq-based analysis in Dnmt triple-knockout (TKO) embryonic stem cells revealed that total loss of CpG methylation is associated with alteration of H3K27me3 levels throughout the genome: H3K27me3 in localized peaks is decreased while broad local enrichments (BLOCs) of H3K27me3 are formed. At an even broader scale, these BLOCs correspond to regions of high DNA methylation in wild-type ES cells, suggesting that DNA methylation prevents H3K27me3 deposition locally and at a megabase scale. Our strategy provides a unique way of investigating global interdependencies between DNA methylation and other chromatin features.     

Chromosome-wide regulation of euchromatin-specific 5mC to 5hmC conversion in mouse ES cells and female human somatic cells

DNA cytosine methylation (5mC) is indispensable for a number of cellular processes, including retrotransposon silencing, genomic imprinting, and X chromosome inactivation in mammalian development. Recent studies have focused on 5-hydroxymethylcytosine (5hmC), a new epigenetic mark or intermediate in the DNA demethylation pathway. However, 5hmC itself has no role in pluripotency maintenance in mouse embryonic stem cells (ESCs) lacking Dnmt1, 3a, and 3b. Here, we demonstrated that 5hmC accumulated on euchromatic chromosomal bands that were marked with di- and tri-methylated histone H3 at lysine 4 (H3K4me2/3) in mouse ESCs. By contrast, heterochromatin enriched with H3K9me3, including mouse chromosomal G-bands, pericentric repeats, human satellite 2 and 3, and inactive X chromosomes, was not enriched with 5hmC. Therefore, enzymes that hydroxylate the methyl group of 5mC belonging to the Tet family might be excluded from inactive chromatin, which may restrict 5mC to 5hmC conversion in euchromatin to prevent nonselective de novo DNA methylation.     

The Polycomb Group Protein SUZ12 regulates histone H3 lysine 9 methylation and HP1α distribution

Regulation of histone methylation is critical for proper gene expression and chromosome function. Suppressor of Zeste 12 (SUZ12) is a requisite member of the EED/EZH2 histone methyltransferase complexes, and is required for full activity of these complexes in vitro. In mammals and flies, SUZ12/Su(z)12 is necessary for trimethylation of histone H3 on lysine 27 (H3K27me3) on facultative heterochromatin. However, Su(z)12 is unique among Polycomb Group Proteins in that Su(z)12 mutant flies exhibit gross defects in position effect variegation, suggesting a role for Su(z)12 in constitutive heterochromatin formation. We investigated the role of Suz12 in constitutive heterochromatin and discovered that Suz12 is required for histone H3 lysine 9 tri-methylation (H3K9me3) in differentiated but not undifferentiated mouse embryonic stem cells. Knockdown of SUZ12 in human cells caused a reduction in H3K27me3 and H3K9me3, and altered the distribution of HP1α. In contrast, EZH2 knockdown caused loss of H3K27me3 but not H3K9me3, indicating that SUZ12 regulates H3-K9 methylation in an EZH2-independent fashion. This work uncovers a role for SUZ12 in H3-K9 methylation.     

Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila

Histone lysine methylation is an epigenetic mark to index chromosomal subdomains. In Drosophila, H3-K9 di- and trimethylation is mainly controlled by the heterochromatic SU(VAR)3-9 HMTase, a major regulator of position-effect variegation (PEV). In contrast, H3-K27 methylation states are independently mediated by the Pc-group enzyme E(Z). Isolation of 19 point mutants demonstrates that the silencing potential of Su(var)3-9 increases with its associated HMTase activity. A hyperactive Su(var)3-9 mutant, pitkin(D), displays extensive H3-K9 di- and trimethylation within but also outside pericentric heterochromatin. Notably, mutations in a novel Su(var) gene, Su(var)3-1, severely restrict Su(var)3-9-mediated gene silencing. Su(var)3-1 was identified as "antimorphic" mutants of the euchromatic H3-S10 kinase JIL-1. JIL-1(Su(var)3-1) mutants maintain kinase activity and do not detectably impair repressive histone lysine methylation marks. However, analyses with seven different PEV rearrangements demonstrate a general role of JIL-1(Su(var)3-1) in controlling heterochromatin compaction and expansion. Our data provide evidence for a dynamic balance between heterochromatin and euchromatin, and define two distinct mechanisms for Su(var) gene function. Whereas the majority of Su(var)s encode inherent components of heterochromatin that can establish repressive chromatin structures [intrinsic Su(var)s], Su(var)3-1 reflects gain-of-function mutants of a euchromatic component that antagonize the expansion of heterochromatic subdomains [acquired Su(var)s].     

Analysis of heterochromatic epigenetic markers in the holocentric chromosomes of the aphid <Emphasis Type="Italic">Acyrthosiphon pisum</Emphasis>

Monomethylated-K9 H3 histones (Me9H3) and heterochromatin protein 1 (HP1) are reported as heterochromatin markers in several eukaryotes possessing monocentric chromosomes. In order to confirm that these epigenetic markers are evolutionarily conserved, we sequenced the HP1 cDNA and verified the distribution of Me9H3 histones and HP1 in the holocentric chromosomes of the aphid Acyrthosiphon pisum. Sequencing indicates that A. pisum HP1 cDNA (called ApHP1) is 1623 bp long, including a 170 bp long 5′UTR and a 688 bp long 3′UTR. The ApHP1 protein consists of 254 amino acidic residues, has a predicted molecular mass of 28 kDa and a net negative charge. At the structural level, it shows an N terminal chromo domain and a chromo shadow domain at the C terminus linked by a short hinge region. At the cytogenetic level, ApHP1 is located exclusively in the heterochromatic regions of the chromosomes. The same heterochromatic regions were labelled after immuno-staining with antibodies against Me9H3 histones, confirming that Hp1 and Me9H3 co-localize at heterochromatic chromosomal areas. Surprisingly, aphid heterochromatin lacks DNA methylation and methylated cytosine residues were mainly spread at euchromatic regions. Finally, the absence of DNA methylation is observed also in aphid rDNA genes that have been repeatedly described as mosaic of methylated and unmethylated units in vertebrates.     

Lamin B1 depletion in senescent cells triggers large-scale changes in gene expression and the chromatin landscape

Senescence is a stable proliferation arrest, associated with an altered secretory pathway, thought to promote tumor suppression and tissue aging. While chromatin regulation and lamin B1 down-regulation have been implicated as senescence effectors, functional interactions between them are poorly understood. We compared genome-wide Lys4 trimethylation on histone H3 (H3K4me3) and H3K27me3 distributions between proliferating and senescent human cells and found dramatic differences in senescence, including large-scale domains of H3K4me3- and H3K27me3-enriched “mesas” and H3K27me3-depleted “canyons.” Mesas form at lamin B1-associated domains (LADs) in replicative senescence and oncogene-induced senescence and overlap DNA hypomethylation regions in cancer, suggesting that pre-malignant senescent chromatin changes foreshadow epigenetic cancer changes. Hutchinson-Gilford progeria syndrome fibroblasts (mutant lamin A) also show evidence of H3K4me3 mesas, suggesting a link between premature chromatin changes and accelerated cell senescence. Canyons mostly form between LADs and are enriched in genes and enhancers. H3K27me3 loss is correlated with up-regulation of key senescence genes, indicating a link between global chromatin changes and local gene expression regulation. Lamin B1 reduction in proliferating cells triggers senescence and formation of mesas and canyons. Our data illustrate profound chromatin reorganization during senescence and suggest that lamin B1 down-regulation in senescence is a key trigger of global and local chromatin changes that impact gene expression, aging, and cancer.