AUF1/HnRNP D RNA binding protein functions in telomere maintenance

In the current issue of Molecular Cell, Yu et al. (2012) establish H3K56 monomethylation (H3K56me1) as a new mammalian chromatin mark, imposed by the G9a methyltransferase and recognized by the replication clamp PCNA.     

Predominant expression of H3K9 methyltransferases in prehypertrophic and hypertrophic chondrocytes during mouse growth plate cartilage development

Histone lysine methylation (HKM) is an epigenetic change that establishes cell-specific gene expression and determines cell fates. In this study, we investigated the expression patterns of histone H3 lysine 9 methyltransferases (H3K9MTases) G9a (euchromatic histone lysine N-methyltransferase 2, Ehmt2), GLP (euchromatic histone lysine N-methyltransferase 1, Ehmt1), SETDB1 (SET domain, bifurcated 1), PRDM2 (PR domain containing 2), SUV39H1 (suppressor of variegation 3–9 homolog 1), and SUV39H2, as well as the distribution of 3 types of HKM at histone H3 lysine 9: mono- (H3K9me1), di- (H3K9me2), or tri-methylation (H3K9me3), during mouse growth plate development. In the forelimb cartilage primordial at embryonic day 12.5 (E12.5), none of the H3K9MTases were detected and H3K9me1, H3K9me2, and H3K9me3 were scarcely detected. At E14.5, the H3K9MTases were expressed at low levels in proliferating chondrocytes and at high levels in prehypertrophic and hypertrophic chondrocytes. Among the H3K9 methylations, H3K9me1 and H3K9me3 were markedly noted in these chondrocytes. At E16.5, G9, GLP, SETDB1, PRDM2, SUV39H1, and SUV39H2, as well as H3K9me1, H3K9me2, and H3K9me3, were detected in prehypertrophic and hypertrophic chondrocytes in the growth plate. Western blotting and real-time quantitative polymerase chain reaction analysis revealed the distributions of G9 and GLP proteins and the expression of all the H3K9MTase mRNAs in prehypertrophic and hypertrophic chondrocytes. These data suggest that H3K9 methyltransferases are predominantly expressed in prehypertrophic and hypertrophic chondrocytes, and that they could be involved in the regulation of gene expression and progression of chondrocyte differentiation by affecting the methylation state of histone H3 lysine 9 in the mouse growth plate.     

Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3

Epigenetic indexing of chromatin domains by histone lysine methylation requires the balanced coordination of methyltransferase and demethylase activities. Here, we show that SU(VAR)3-3, the Drosophila homolog of the human LSD1 amine oxidase, demethylates H3K4me2 and H3K4me1 and facilitates subsequent H3K9 methylation by SU(VAR)3-9. Su(var)3-3 mutations suppress heterochromatic gene silencing, display elevated levels of H3K4me2, and prevent extension of H3K9me2 at pericentric heterochromatin. SU(VAR)3-3 colocalizes with H3K4me2 in interband regions and is abundant during embryogenesis and in syncytial blastoderm, where it appears concentrated at prospective heterochromatin during cycle 14. In embryos of Su(var)3-3/+ females, H3K4me2 accumulates in primordial germ cells, and the deregulated expansion of H3K4me2 antagonizes heterochromatic H3K9me2 in blastoderm cells. Our data indicate an early developmental function for the SU(VAR)3-3 demethylase in controlling euchromatic and heterochromatic domains and reveal a hierarchy in which SU(VAR)3-3-mediated removal of activating histone marks is a prerequisite for subsequent heterochromatin formation by H3K9 methylation.     

Phosphorylation of H3S10 blocks the access of H3K9 by specific antibodies and histone methyltransferase. Implication in regulating chromatin dynamics and epigenetic inheritance during mitosis

Post-translational modifications of histones play a critical role in regulating genome structures and integrity. We have focused on the regulatory relationship between covalent modifications of histone H3 lysine 9 (H3K9) and H3S10 during the cell cycle. Immunofluorescence microscopy revealed that H3S10 phosphorylation in HeLa, A549, and HCT116 cells was high during prophase, prometaphase, and metaphase, whereas H3K9 monomethylation (H3K9me1) and dimethylation (H3K9me2), but not H3K9 trimethylation (H3K9me3), were significantly suppressed. When H3S10 phosphorylation started to diminish during anaphase, H3K9me1 and H3K9me2 signals reemerged. Western blot analyses confirmed that mitotic histones, extracted in an SDS-containing buffer, had little H3K9me1 and H3K9me2 signals but abundant H3K9me3 signals. However, when mitotic histones were extracted in the same buffer without SDS, the difference in H3K9me1 and H3K9me2 signals between interphase and mitotic cells disappeared. Removal of H3S10 phosphorylation by pretreatment with lambda-phosphatase unmasked mitotic H3K9me1 and H3K9me2 signals detected by both fluorescence microscopy and Western blotting. Further, H3S10 phosphorylation completely blocked methylation of H3K9 but not demethylation of the same residue in vitro. Given that several conserved motifs consisting of a Lys residue immediately followed by a Ser residue are present in histone tails, our studies reveal a potential new mechanism by which phosphorylation not only regulates selective access of methylated lysines by cellular factors but also serves to preserve methylation patterns and epigenetic programs during cell division.     

Abnormal histone H3K9 dimethylation but normal dimethyltransferase EHMT2 expression in cloned sheep embryos

The objective was to investigate the relationship between histone H3 lysine 9 (H3K9) dimethylation (me2) and the histone methyltransferase EHMT2 (also known as G9A) in ovine embryos cloned by somatic cell nuclear transfer (SCNT). Levels of H3K9me2 or EHMT2 were detected (with immunostaining) and compared between SCNT and IVF-derived preimplantation embryos. In one-cell embryos, SCNT zygotes had significantly higher levels of H3K9me2 and EHMT2 than IVF zygotes. In cloned embryos, H3K9me2 remained hypermethylated relative to IVF embryos at two-cell and late developmental stages (morula and blastocyst), with no difference (P > 0.05) between IVF and SCNT embryos in EHMT2 levels from two-cell to blastocyst stages. The EHMT2-specific inhibitor, BIX01294, reduced global H3K9me2 levels in cultured ovine cells or SCNT embryos, but it was not appropriate for somatic cell nuclear transfer because of its high cellular toxicity. We inferred that abnormal H3K9me2 hypermethylation in SCNT embryos may not completely arise from EHMT2 expression error.     

Differential Localization and Independent Acquisition of the H3K9me2 and H3K9me3 Chromatin Modifications in the Caenorhabditis elegans Adult Germ Line

Histone methylation is a prominent feature of eukaryotic chromatin that modulates multiple aspects of chromosome function. Methyl modification can occur on several different amino acid residues and in distinct mono-, di-, and tri-methyl states. However, the interplay among these distinct modification states is not well understood. Here we investigate the relationships between dimethyl and trimethyl modifications on lysine 9 of histone H3 (H3K9me2 and H3K9me3) in the adult Caenorhabditis elegans germ line. Simultaneous immunofluorescence reveals very different temporal/spatial localization patterns for H3K9me2 and H3K9me3. While H3K9me2 is enriched on unpaired sex chromosomes and undergoes dynamic changes as germ cells progress through meiotic prophase, we demonstrate here that H3K9me3 is not enriched on unpaired sex chromosomes and localizes to all chromosomes in all germ cells in adult hermaphrodites and until the primary spermatocyte stage in males. Moreover, high-copy transgene arrays carrying somatic-cell specific promoters are highly enriched for H3K9me3 (but not H3K9me2) and correlate with DAPI-faint chromatin domains. We further demonstrate that the H3K9me2 and H3K9me3 marks are acquired independently. MET-2, a member of the SETDB histone methyltransferase (HMTase) family, is required for all detectable germline H3K9me2 but is dispensable for H3K9me3 in adult germ cells. Conversely, we show that the HMTase MES-2, an E(z) homolog responsible for H3K27 methylation in adult germ cells, is required for much of the germline H3K9me3 but is dispensable for H3K9me2. Phenotypic analysis of met-2 mutants indicates that MET-2 is nonessential for fertility but inhibits ectopic germ cell proliferation and contributes to the fidelity of chromosome inheritance. Our demonstration of the differential localization and independent acquisition of H3K9me2 and H3K9me3 implies that the trimethyl modification of H3K9 is not built upon the dimethyl modification in this context. Further, these and other data support a model in which these two modifications function independently in adult C. elegans germ cells.     

Variation in Xi chromatin organization and correlation of the H3K27me3 chromatin territories to transcribed sequences by microarray analysis

The heterochromatin of the inactive X chromosome (Xi) is organized into nonoverlapping bands of trimethylated lysine-9 of histone H3 (H3K9me3) and trimethylated lysine-27 of histone H3 (H3K27me3). H3K27me3 chromatin of the Xi is further characterized by ubiquitylated H2A and H4 monomethylated at lysine-20. A detailed examination of the metaphase H3K9me3 pattern revealed that banding along the chromosome arms is not a consistent feature of the Xi in all cell lines, but instead is generally restricted to the centromere and telomeres. However, H3K9me3 does form a reproducible band centered at Xq13 of the active X. In contrast, H3K27me3 banding is a feature of all Xi, but the precise combination and frequency of bands is not consistent. One notable exception is a common band at Xq22–23 that spans 12–15 Mb. The detailed examination of the chromatin territory by microarray analysis refined the H3K27me3 band as well as revealed numerous less extensive clusters of H3K27me3 signals. Furthermore, the microarray analysis indicates that H3K27me3 bands are directly correlated with gene density. The reexamination of the chromosome wide banding indicates that other major H3K27me3 bands closely align with regions of highest gene density.     

HP1a Recruitment to Promoters Is Independent of H3K9 Methylation in Drosophila melanogaster

Heterochromatin protein 1 (HP1) proteins, recognized readers of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me), are important regulators of heterochromatin-mediated gene silencing and chromosome structure. In Drosophila melanogaster three histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9: Su(var)3-9, Setdb1, and G9a. To probe the dependence of HP1a binding on H3K9me, its dependence on these three HKMTs, and the division of labor between the HKMTs, we have examined correlations between HP1a binding and H3K9me patterns in wild type and null mutants of these HKMTs. We show here that Su(var)3-9 controls H3K9me-dependent binding of HP1a in pericentromeric regions, while Setdb1 controls it in cytological region 2L:31 and (together with POF) in chromosome 4. HP1a binds to the promoters and within bodies of active genes in these three regions. More importantly, however, HP1a binding at promoters of active genes is independent of H3K9me and POF. Rather, it is associated with heterochromatin protein 2 (HP2) and open chromatin. Our results support a hypothesis in which HP1a nucleates with high affinity independently of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites.     

The SUVR4 histone lysine methyltransferase binds ubiquitin and converts H3K9me1 to H3K9me3 on transposon chromatin in Arabidopsis

Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs) on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases) can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3) is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation-dependent and -independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state

Histone posttranslational modifications (PTMs) and sequence variants regulate genome function. Although accumulating evidence links particular PTM patterns with specific genomic loci, our knowledge concerning where and when these PTMs are imposed remains limited. Here, we find that lysine methylation is absent prior to histone incorporation into chromatin, except at H3K9. Nonnucleosomal H3.1 and H3.3 show distinct enrichments in H3K9me, such that H3.1 contains more K9me1 than H3.3. In addition, H3.3 presents other modifications, including K9/K14 diacetylated and K9me2. Importantly, H3K9me3 was undetectable in both nonnucleosomal variants. Notably, initial modifications on H3 variants can potentiate the action of enzymes as exemplified with Suv39HMTase to produce H3K9me3 as found in pericentric heterochromatin. Although the set of initial modifications present on H3.1 is permissive for further modifications, in H3.3 a subset cannot be K9me3. Thus, initial modifications impact final PTMs within chromatin.     

Histone H3 methylation patterns in Brassica nigra, Brassica juncea, and Brassica carinata species

Core histones are subjected to various post-translational modifications, and one of them, most intensively studied in plants, is the methylation of histone H3. In the majority of analyzed plant species, dimethylation of H3 at lysine 9 (H3K9me2) is detected in heterochromatin domains, whereas methylation of H3 at lysine 4 (H3K4me2) is detected in euchromatin domains. The distribution of H3K9me2 in the interphase nucleus seems to be correlated with genome size, chromatin organization, but also with tissue specificity. In this paper, we present the analysis of the pattern and level of histone H3 methylation for two allotetraploid and one diploid Brassica species. We have found that the pattern of H3K9me2 in interphase nuclei from root meristematic tissue is comparable within the analyzed species and includes both heterochromatin and euchromatin, but the level of modification differs not only among species but even among nuclei in the same phase of the cell cycle within one species. Moreover, the differences in the level of H3K9me2 are not directly coupled with DNA content in the nuclei and are probably tissue specific.