Trimethylation of Histone H3 Lysine 4 is an Epigenetic Mark at Regions Escaping Mammalian X Inactivation

It is now estimated that 150-200 genes clustered in several discrete regions escape X inactivation in somatic cells of human females by unknown mechanisms. Here, we show that although the human female inactive X chromosome is largely devoid of histone 3 lysine 4 trimethylation (H3K4me3), regions that are known to escape X inactivation, including the pseudoautosomal regions, are enriched with this modification. Also, H3K4me3, unlike H3K4me2 and H4 and H3 acetylation, is restricted to discrete regions on metaphase chromosomes. In contrast to humans, there are only a few genes that are known to escape X inactivation in the mouse. Therefore, we examined mouse female somatic cells with H3K4me3 to identify candidate regions with genes that escape X inactivation. We found the mouse female inactive X in somatic cells and the male inactive X in meiosis to have seven discrete regions that are enriched with H3K4me3. Furthermore, RNA polymerase II is largely excluded from the XY body at male pachytene except for several discrete regions on the X and Y suggesting the presence of regions that also escape sex chromosome inactivation during male meiosis.     

Histone H4 acetylation analyses in patients with polysomy X: implications for the mechanism of X inactivation

In humans, it is thought that the X-inactivation phenomenon occurs no matter how many X chromosomes are present, and that only one of them remains active. Nevertheless, individuals who have an abnormal number of X chromosomes show a wide spectrum of abnormalities, which increase with the number of X chromosomes present in a given individual. It has been shown that the inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, and that this could be used as an accessible marker for distinguishing between Xi and Xa in spreads of metaphase chromosomes. We studied three X-polysomic patients for the presence of active chromatin by analysis of histone H4 acetylation on unfixed metaphase spreads. Using antisera to H4 acetylated at lysines 16, 8 and 5, respectively, we observed frequencies different from those expected from cells with only one underacetylated X chromosome. In particular, when antiserum to H4 acetylated at lysine 16 was used about 90% of the cells showed acetylation of all X chromosomes. This suggests a possible disturbance in the deacetylation process, probably due to the presence of multiple Xs. Received: 25 April 1997 / Accepted: 15 March 1998     

Differential underacetylation of histones H2A,H3 and H4 on the inactive X chromosome in human female cells

It has previously been shown that the acetylated forms of histone H4 are depleted or absent in both constitutive, centric heterochromatin and in the facultative heterochromatin of the inactive X chromosome (Xi) in female cells. By immunostaining of metaphase chromosomes from human lymphocytes with antibodies to the acetylated isoforms of histones H2A and H3, we now show that these histones too are underacetylated in both Xi and centric heterochromatin. Xi shows two prominent regions of residual H3 acetylation, one encompassing the pseudoautosomal region at the end of the short arm and one at about Xg22. Both these regions have been shown previously to be sites of residual H4 acetylation. H2A acetylation on Xi is higher overall than that of H3 or H4 and is particularly high around the pseudoautosomal region, but not at Xg22. The results suggest that the acetylated isoforms of H3 and H4 have at least some effects on chromosomal structure and function that are not shared by acetylated H2A.     

Ring1b-mediated H2A ubiquitination associates with inactive X chromosomes and is involved in initiation of X inactivation

Histone modifications are thought to serve as epigenetic markers that mediate dynamic changes in chromatin structure and regulation of gene expression. As a model system for understanding epigenetic silencing, X chromosome inactivation has been previously linked to a number of histone modifications including methylation and hypoacetylation. In this study, we provide evidence that supports H2A ubiquitination as a novel epigenetic marker for the inactive X chromosome (Xi) and links H2A ubiquitination to initiation of X inactivation. We found that the H2A-K119 ubiquitin E3 ligase Ring1b, a Polycomb group protein, is enriched on Xi in female trophoblast stem (TS) cells as well as differentiating embryonic stem (ES) cells. Consistent with Ring1b mediating H2A ubiquitination, ubiquitinated H2A (ubH2A) is also enriched on the Xi of both TS and ES cells. We demonstrate that the enrichment of Ring1b and ubH2A on Xi is transient during TS and ES cell differentiation, suggesting that the Ring1b and ubH2A are involved in the initiation of both imprinted and random X inactivation. Furthermore, we showed that the association of Ring1b and ubH2A with Xi is mitotically stable in non-differentiated TS cells.     

Caenorhabditis elegans dosage compensation regulates histone H4 chromatin state on X chromosomes

Dosage compensation equalizes X-linked gene expression between the sexes. This process is achieved in Caenorhabditis elegans by hermaphrodite-specific, dosage compensation complex (DCC)-mediated, 2-fold X chromosome downregulation. How the DCC downregulates gene expression is not known. By analyzing the distribution of histone modifications in nuclei using quantitative fluorescence microscopy, we found that H4K16 acetylation (H4K16ac) is underrepresented and H4K20 monomethylation (H4K20me1) is enriched on hermaphrodite X chromosomes in a DCC-dependent manner. Depletion of H4K16ac also requires the conserved histone deacetylase SIR-2.1, while enrichment of H4K20me1 requires the activities of the histone methyltransferases SET-1 and SET-4. Our data suggest that the mechanism of dosage compensation in C. elegans involves redistribution of chromatin-modifying activities, leading to a depletion of H4K16ac and an enrichment of H4K20me1 on the X chromosomes. These results support conserved roles for histone H4 chromatin modification in worm dosage compensation analogous to those seen in flies, using similar elements and opposing strategies to achieve differential 2-fold changes in X-linked gene expression.     

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.     

Dynamic relocalization of histone MacroH2A1 from centrosomes to inactive X chromosomes during X inactivation

Histone variant macroH2A1 (macroH2A1) contains an NH(2)-terminal domain that is highly similar to core histone H2A and a larger COOH-terminal domain of unknown function. MacroH2A1 is expressed at similar levels in male and female embryonic stem (ES) cells and adult tissues, but a portion of total macroH2A1 protein localizes to the inactive X chromosomes (Xi) of differentiated female cells in concentrations called macrochromatin bodies. Here, we show that centrosomes of undifferentiated male and female ES cells harbor a substantial store of macroH2A1 as a nonchromatin-associated pool. Greater than 95% of centrosomes from undifferentiated ES cells contain macroH2A1. Cell fractionation experiments confirmed that macroH2A1 resides at a pericentrosomal location in close proximity to the known centrosomal proteins gamma-tubulin and Skp1. Retention of macroH2A1 at centrosomes was partially labile in the presence of nocodazole suggesting that intact microtubules are necessary for accumulation of macroH2A1 at centrosomes. Upon differentiation of female ES cells, Xist RNA expression became upregulated and monoallelic as judged by fluorescent in situ hybridization, but early Xist signals lacked associated macroH2A1. Xi acquired macroH2A1 soon thereafter as indicated by the colocalization of Xist RNA and macroH2A1. Accumulation of macroH2A1 on X chromosomes occurred with a corresponding loss of centrosomal macroH2A1. Our results define a sequence for the loading of macroH2A1 on the Xi and place this event in the context of differentiation and Xist expression. Furthermore, these results suggest a role for the centrosome in the X inactivation process.     

Active and repressive chromatin are interspersed without spreading in an imprinted gene cluster in the mammalian genome

The Igf2r imprinted cluster is an epigenetic silencing model in which expression of a ncRNA silences multiple genes in cis. Here, we map a 250 kb region in mouse embryonic fibroblast cells to show that histone modifications associated with expressed and silent genes are mutually exclusive and localized to discrete regions. Expressed genes were modified at promoter regions by H3K4me3 + H3K4me2 + H3K9Ac and on putative regulatory elements flanking active promoters by H3K4me2 + H3K9Ac. Silent genes showed two types of nonoverlapping profile. One type spread over large domains of tissue-specific silent genes and contained H3K27me3 alone. A second type formed localized foci on silent imprinted gene promoters and a nonexpressed pseudogene and contained H3K9me3 + H4K20me3 +/- HP1. Thus, mammalian chromosome arms contain active chromatin interspersed with repressive chromatin resembling the type of heterochromatin previously considered a feature of centromeres, telomeres, and the inactive X chromosome.