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.     

Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin

The chromodomain (CD) of the Drosophila Polycomb protein exhibits preferential binding affinity for histone H3 when trimethylated at lysine 27. Here we have investigated the five mouse Polycomb homologs known as Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8. Despite a high degree of conservation, the Cbx chromodomains display significant differences in binding preferences. Not all CDs bind preferentially to K27me3; rather, some display affinity towards both histone H3 trimethylated at K9 and H3K27me3, and one CD prefers K9me3. Cbx7, in particular, displays strong affinity for both H3K9me3 and H3K27me3 and is developmentally regulated in its association with chromatin. Cbx7 associates with facultative heterochromatin and, more specifically, is enriched on the inactive X chromosome. Finally, we find that, in vitro, the chromodomain of Cbx7 can bind RNA and that, in vivo, the interaction of Cbx7 with chromatin, and the inactive X chromosome in particular, depends partly on its association with RNA. We propose that the capacity of this mouse Polycomb homolog to associate with the inactive X chromosome, or any other region of chromatin, depends not only on its chromodomain but also on the combination of histone modifications and RNA molecules present at its target sites.     

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.     

G9α‐dependent histone H3K9me3 hypomethylation promotes overexpression of cardiomyogenesis‐related genes in foetal mice

Alcohol consumption during pregnancy can cause foetal alcohol syndrome and congenital heart disease. Nonetheless, the underlying mechanism of alcohol‐induced cardiac dysplasia remains unknown. We previously reported that alcohol exposure during pregnancy can cause abnormal expression of cardiomyogenesis‐related genes, and histone H3K9me3 hypomethylation was observed in alcohol‐treated foetal mouse heart. Hence, an imbalance in histone methylation may be involved in alcohol‐induced cardiac dysplasia. In this study, we investigated the involvement of G9α histone methyltransferase in alcohol‐induced cardiac dysplasia in vivo and in vitro using heart tissues of foetal mice and primary cardiomyocytes of neonatal mice. Western blotting revealed that alcohol caused histone H3K9me3 hypomethylation by altering G9α histone methyltransferase expression in cardiomyocytes. Moreover, overexpression of cardiomyogenesis‐related genes (MEF2C, Cx43, ANP and β‐MHC) was observed in alcohol‐exposed foetal mouse heart. Additionally, we demonstrated that G9α histone methyltransferase directly interacted with histone H3K9me3 and altered its methylation. Notably, alcohol did not down‐regulate H3K9me3 methylation after G9α suppression by short hairpin RNA in primary mouse cardiomyocytes, preventing MEF2C, Cx43, ANP and β‐MHC overexpression. These findings suggest that G9α histone methyltransferase‐mediated imbalance in histone H3K9me3 methylation plays a critical role in alcohol‐induced abnormal expression cardiomyogenesis‐related genes during pregnancy. Therefore, G9α histone methyltransferase may be an intervention target for congenital heart disease.     

Differential histone H3 Lys-9 and Lys-27 methylation profiles on the X chromosome

Histone H3 tail modifications are among the earliest chromatin changes in the X-chromosome inactivation process. In this study we investigated the relative profiles of two important repressive marks on the X chromosome: methylation of H3 lysine 9 (K9) and 27 (K27). We found that both H3K9 dimethylation and K27 trimethylation characterize the inactive X in somatic cells and that their relative kinetics of enrichment on the X chromosome as it undergoes inactivation are similar. However, dynamic changes of H3K9 and H3K27 methylation on the inactivating X chromosome compared to the rest of the genome are distinct, suggesting that these two modifications play complementary and perhaps nonredundant roles in the establishment and/or maintenance of X inactivation. Furthermore, we show that a hotspot of H3K9 dimethylation 5' to Xist also displays high levels of H3 tri-meK27. However, analysis of this region in G9a mutant embryonic stem cells shows that these two methyl marks are dependent on different histone methyltransferases.     

Histone H3 lysine 4 monomethylation (H3K4me1) and H3 lysine 9 monomethylation (H3K9me1): Distribution and their association in regulating gene expression under hyperglycaemic/hyperinsulinemic conditions in 3T3 cells

Hyperglycemia/hyperinsulinemia are leading cause for the induction type 2 diabetes and the role of post-translational histone modifications in dysregulating the expression of genes has emerged as potential important contributor in the progression of disease. The paradoxical nature of histone H3-Lysine 4 and Lysine 9 mono-methylation (H3K4me1 and H3K9me1) in both gene activation and repression motivated us to elucidate the functional relationship of these histone modifications in regulating expression of genes under hyperglycaemic/hyperinsulinemic condition. Chromatin immunoprecipitation–microarray analysis (ChIP-chip) was performed with H3 acetylation, H3K4me1 and H3K9me1 antibody. CLUSTER analysis of ChIP-chip (Chromatin immunoprecipitation–microarray analysis) data showed that mRNA expression and H3 acetylation/H3K4me1 levels on genes were inversely correlated with H3K9me1 levels on the transcribed regions, after 30 min of insulin stimulation under hyperglycaemic condition. Interestingly, we provide first evidence regarding regulation of histone de/acetylases and de/methylases; Myst4, Jmjd2b, Aof1 and Set by H3Ac, H3K4me1 and H3K9me1 under hyperinsulinemic/hyperglycaemic condition. ChIP–qPCR analysis shows association of increased H3Ac/H3K4me1 and decreased levels of H3K9me1 in up regulation of Myst4, Jmjd2, Set and Aof1 genes. We further analyse promoter occupancy of histone modifications by ChIP walking and observed increased occupancy of H3Ac/H3K4me1 on promoter region (−1000 to −1) of active genes and H3K9me1 on inactive genes under hyperglycemic/hyperinsulinemic condition. To best of our knowledge this is the first report that shows regulation of chromatin remodelling genes by alteration in the occupancy of histone H3Ac/H3K4/K9me on both promoter and transcribed regions.     

Hdac3, Setdb1, and Kap1 mark H3K9me3/H3K14ac bivalent regions in young and aged liver

Post‐translational modifications of histone tails play a crucial role in gene regulation. Here, we performed chromatin profiling by quantitative targeted mass spectrometry to assess all possible modifications of the core histones. We identified a bivalent combination, a dually marked H3K9me3/H3K14ac modification in the liver, that is significantly decreased in old hepatocytes. Subsequent sequential ChIP‐Seq identified dually marked single nucleosome regions, with reduced number of sites and decreased signal in old livers, confirming mass spectrometry results. We detected H3K9me3 and H3K14ac bulk ChIP‐Seq signal in reChIP nucleosome regions, suggesting a correlation between H3K9me3/H3K14ac bulk bivalent genomic regions and dually marked single nucleosomes. Histone H3K9 deacetylase Hdac3, as well as H3K9 methyltransferase Setdb1, found in complex Kap1, occupied both bulk and single nucleosome bivalent regions in both young and old livers, correlating to presence of H3K9me3. Expression of genes associated with bivalent regions in young liver, including those regulating cholesterol secretion and triglyceride synthesis, is upregulated in old liver once the bivalency is lost. Hence, H3K9me3/H3K14ac dually marked regions define a poised inactive state that is resolved with loss of one or both of the chromatin marks, which subsequently leads to change in gene expression.     

Repressive but not activating epigenetic modifications are aberrant on the inactive X chromosome in live cloned cattle

X inactivation is the process of a chromosome-wide silencing of the majority of genes on the X chromosome during early mammalian development. This process may be aberrant in cloned animals. Here we show that repressive modifications, such as methylation of DNA, and the presence of methylated histones, H3K9me2 and H3K27me3, exhibit distinct aberrance on the inactive X chromosome in live clones. In contrast, H3K4me3, an active gene marker, is obviously missing from the inactive X chromosome in all cattle studied. This suggests that the disappearance of active histone modifications (H3K4me3) seems to be more important for X inactivation than deposition of marks associated with heterochromatin (DNA methylation, H3K27me3 and H3K9me2). It also implies that even apparently normal clones may have subtle abnormalities in repressive, but not activating epigenetic modifications on the inactive X when they survive to term. We also found that the histone H3 methylations were enriched and co-localized at q21-31 of the active X chromosome, which may be associated with an abundance of LINE1 repeat elements.     

The HP1α–CAF1–SetDB1‐containing complex provides H3K9me1 for Suv39‐mediated K9me3 in pericentric heterochromatin

Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1α (HP1α)–chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non‐nucleosomal histone H3. Therefore, the heterochromatic HP1α–CAF1–SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1α with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.     

H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensable for inducing gene silencing

During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long non‐coding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2‐dependent H3K27me3 and SETD8‐dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the involvement of H4K20me1 in initiating gene silencing. To follow XCI dynamics in living cells, we developed a genetically encoded, H3K27me3‐specific intracellular antibody or H3K27me3‐mintbody. By combining live‐cell imaging of H3K27me3, H4K20me1, the X chromosome and Xist RNA, with ChIP‐seq analysis we uncover concurrent accumulation of both marks during XCI, albeit with distinct genomic distributions. Furthermore, using a Xist B and C repeat mutant, which still shows gene silencing on the X but not H3K27me3 deposition, we also find a complete lack of H4K20me1 enrichment. This demonstrates that H4K20me1 is dispensable for the initiation of gene silencing, although it may have a role in the chromatin compaction that characterises facultative heterochromatin.