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.     

Methylation of histone H3 lysine 9 occurs during translation

Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9), a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed.     

Methylation changes of lysine 9 of histone H3 during preimplantation mouse development

Immediately after fertilization, a chromatin remodeling process in the oocyte cytoplasm extracts protamine molecules from the sperm-derived DNA and loads histones onto it. We examined how the histone H3-lysine 9 methylation system is established on the remodeled sperm chromatin in mice. We found that the paternal pronucleus was not stained for dimethylated H3-K9 (H3-m2K9) during pronucleus development, while the maternal genome stained intensively. Such H3-m2K9 asymmetry between the parental pronuclei was independent of HP1b localization and, much like DNA methylation, was preserved to the two-cell stage when the nucleus appeared to be compartmentalized for H3-m2K9. A conspicuous increase in H3-m2K9 level was observed at the four-cell stage, and then the level was maintained without a visible change up to the blastocyst stage. The behavior of H3-m2K9 was very similar, but not identical, to that of 5-methylcytosine during preimplantation development, suggesting that there is some connection between methylation of histone and of DNA in early mouse development.     

Deacetylation and methylation at histone H3 lysine 9 (H3K9) coordinate chromosome condensation during cell cycle progression

Interphasic chromatin condenses into the chromosomes in order to facilitate the correct segregation of genetic information. It has been previously reported that the phosphorylation and methylation of the N-terminal tail of histone H3 are responsible for chromosome condensation. In this study, we demonstrate that the deacetylation and methylation of histone H3 lysine 9 (H3K9) are required for proper chromosome condensation. We confirmed that H3K9ac levels were reduced, whereas H3K9me3 levels were increased in mitotic cells, via immunofluorescence and Western blot analysis. Nocodazole treatment induced G2/M arrest but co-treatment with TSA, an HDAC inhibitor, delayed cell cycle progression. However, the HMTase inhibitor, AdoX, had no effect on nocodazole-induced G2/M arrest, thereby indicating that sequential modifications of H3K9 are required for proper chromosome condensation. The expression of SUV39H1 and SETDB1, H3K9me3-responsible HMTases, are specifically increased along with H3K9me3 in nocodazole-arrested buoyant cells, which suggests that the increased expression of those proteins is an important step in chromosome condensation. H3K9me3 was highly concentrated in the vertical chromosomal axis during prophase and prometaphase. Collectively, the results of this study indicate that sequential modifications at H3K9 are associated with correct chromosome condensation, and that H3K9me3 may be relevant to the condensation of chromosome length.     

Methylation at arginine 17 of histone H3 is linked to gene activation

The nuclear hormone receptor co-activator CARM1 has the potential to methylate histone H3 at arginine residues in vitro. The methyltransferase activity of CARM1 is necessary for its co-activator functions in transient transfection assays. However, the role of this methyltransferase in vivo is unclear, given that methylation of arginines is not easily detectable on histones. We have raised an antibody that specifically recognizes methylated arginine 17 (R17) of histone H3, the major site of methylation by CARM1. Using this antibody we show that methylated R17 exists in vivo. Chromatin immunoprecipitation analysis shows that R17 methylation on histone H3 is dramatically upregulated when the estrogen receptor-regulated pS2 gene is activated. Coincident with the appearance of methylated R17, CARM1 is found associated with the histones on the pS2 gene. Together these results demonstrate that CARM1 is recruited to an active promoter and that CARM1-mediated R17 methylation on histone H3 takes place in vivo during this active state.     

Methylation of the Hprt gene on the inactive X occurs after chromosome inactivation

DNA sequences have previously been identified in the first intron of the mouse Hprt gene that are methylated on the inactive but not the active X chromosome. The temporal relationship between methylation of these sequences and X-inactivation was studied in teratocarcinoma cells and postimplantation mouse embryos: the sequences are unmethylated prior to X-inactivation and do not become methylated on the inactive X in most fetal cells until several days postinactivation. Such inactive X-specific methylation occurs in a significantly smaller proportion of the cells in the extra-embryonic tissues, yolk sac mesoderm and endoderm, than in the fetus. These data suggest that the inactive X-specific methylation of sequences such as those in the first intron of the Hprt gene does not play any role in the primary events of X-inactivation, but may function as part of a secondary, tissue-specific mechanism for maintaining the inactive state.     

Distinct methylation patterns in histone H3 at Lys-4 and Lys-9 correlate with up- & down-regulation of genes by ethanol in hepatocytes

Ethanol induced liver injury is associated with a global change in gene expression but its mechanisms are not known. We studied whether alcohol-induced gene expression is associated with post-translational methylations of histone H3. Primary culture of rat hepatocytes was treated with ethanol (50 or 100 mM) for 24 h and the status of methylation of H3 at lys 4 (H3dimeK4) or lys 9 (H3dimeK9) was monitored by Western blotting using antibodies to dimethylated histone H3 at lys 4 or lys 9. The cells exposed to ethanol showed strikingly opposing behaviors in methylation patterns; H3dimeK9 methylation was decreased whereas H3dimeK4 increased. Similar results were obtained in the interphase nuclei. Their binding on the metaphase chromosomes exhibits distinct site specific pattern of accumulation. Next, chromatin immunoprecipitation of the ethanol treated samples with antibodies for methylated lys 4 or lys 9 histone H3 followed by amplification of the immunoprecipitated DNA, was used to determine their association with the promoters of genes up- or downregulated by ethanol. Lys4 methylation was associated with ethanol upregulated genes (Adh, GST-yc2) whereas lys 9 methylation with downregulated genes (Lsdh, cytP4502c11) demonstrating a difference between these two methylations. These results suggest that exposure of hepatocytes to ethanol changes the expression of several susceptible genes which are associated with site specific modification of dimethylated forms of histone H3 amino termini at their regulatory regions.     

Methylation of lysine 4 on histone H3: intricacy of writing and reading a single epigenetic mark

Cells employ elaborate mechanisms to introduce structural and chemical variation into chromatin. Here, we focus on one such element of variation: methylation of lysine 4 in histone H3 (H3K4). We assess a growing body of literature, including treatment of how the mark is established, the patterns of methylation, and the functional consequences of this epigenetic signature. We discuss structural aspects of the H3K4 methyl recognition by the downstream effectors and propose a distinction between sequence-specific recruitment mechanisms and stabilization on chromatin through methyl-lysine recognition. Finally, we hypothesize how the unique properties of the polyvalent chromatin fiber and associated effectors may amplify small differences in methyl-lysine recognition, simultaneously allowing for a dynamic chromatin architecture.     

Reduced levels of histone H3 acetylation on the inactive X chromosome in human females

Novel antibodies were generated that are highly selective for either acetylated or unacetylated iso-forms of histone H3, or the acetylated form of histone H4 in organisms as diverse asTetrahymena and humans. Using these antibodies as pair-wise sets in immunocytological analyses, we demonstrate that the inactive X chromosome is hypoacetylated for both histone H3 and H4 in female mammalian cells, whereas the antibody that recognizes the unacetylated form of histone H3 identifies all chromosomes uniformly. These data verify and extend previous results and suggest that hypoacetylation of core histones may be a general feature of the chromatin along the inactive X chromosome. Edited by: D. Bazett-Jones     

Evidence for the inactivation of an X chromosome early in the development of the human female.

Studies of somatic tissues and cultured cells, including fibroblast clones, from human embryos heterozygous for the electrophoretic variants of glucose-6-phosphate dehydrogenase confirm that one X chromosome is inactivated very early in embryonic development and indicate that X inactivation has occurred in the majority of cells from a variety of tissues at least by 5 weeks from conception.     

The region 3' to Xist mediates X chromosome counting and H3 Lys-4 dimethylation within the Xist gene

A counting process senses the X chromosome/autosome ratio and ensures that X chromosome inactivation (XCI) initiates in the female (XX) but not in the male (XY) mouse embryo. Counting is regulated by the X-inactivation centre, which contains the Xist gene. Deleting 65 kb 3' to Xist in XO embryonic stem (ES) cells affects counting and results in inappropriate XCI upon differentiation. We show here that normal counting can be rescued in these deleted ES cells using cre/loxP re-insertion, and refine the location of elements controlling counting within a 20 kb bipartite domain. Furthermore, we show that the 65 kb deletion also leads to inappropriate XCI in XY differentiated ES cells, which excludes the involvement of sex-specific mechanisms in the initiation of XCI. At the chromatin level, we have found that the Xist gene corresponds to a peak of H3 Lys-4 dimethylation, which is dramatically and specifically affected by the deletion 3' to Xist. Our results raise the possibility that H3 Lys-4 dimethylation within Xist may be functionally implicated in the counting process.     

Methylation of histone H3 mediates the association of the NuA3 histone acetyltransferase with chromatin

The SAS3-dependent NuA3 histone acetyltransferase complex was originally identified on the basis of its ability to acetylate histone H3 in vitro. Whether NuA3 is capable of acetylating histones in vivo, or how the complex is targeted to the nucleosomes that it modifies, was unknown. To address this question, we asked whether NuA3 is associated with chromatin in vivo and how this association is regulated. With a chromatin pulldown assay, we found that NuA3 interacts with the histone H3 amino-terminal tail, and loss of the H3 tail recapitulates phenotypes associated with loss of SAS3. Moreover, mutation of histone H3 lysine 14, the preferred site of acetylation by NuA3 in vitro, phenocopies a unique sas3Delta phenotype, suggesting that modification of this residue is important for NuA3 function. The interaction of NuA3 with chromatin is dependent on the Set1p and Set2p histone methyltransferases, as well as their substrates, histone H3 lysines 4 and 36, respectively. These results confirm that NuA3 is functioning as a histone acetyltransferase in vivo and that histone H3 methylation provides a mark for the recruitment of NuA3 to nucleosomes.     

Human X-autosome translocations: differential inactivation of the X chromosome in a kindred with an X-9 translocation.

A kindred with an X-autosome translocation and differential inactivation of the X chromosome is described. The phenotypically normal mother has a reciprocal translocation [46,X,rcp(X;9) (q11;q32)] while the daughter's karyotype is unbalanced [46,X,--X,+der(9),rcp(X;9) (q11;q32)mat], indicating adjacent-two type of segregation in the mother. In the mother's cells the normal X is late replicating, while in the daughter's cells almost the entire der(9) is late replicating, indicating the presence of autosomal inactivation. The daughter's abnormal phenotype can be explained by her sex chromosomal complement and the absence of effective trisomy 9. At this stage there is no simple explanation to account for all types of inactivation patterns encountered in the 14 balanced and 15 unbalanced cases of X-autosome translocations reported to date. Selection of X inactivation is not an inherent characteristic of the X chromosome per se, and it is not dependent on the direction of chromosomal exchange, as was suggested previously. Correlation of the phenotypic and cytogenetic features of these patients suggests a pattern of X and autosomal inactivation consistent with the least amount of genotypic and phenotypic imbalance in most cases. The data are most consistent with random X inactivation followed by selection of the most viable cell line.