Histone acetylation and X inactivation

In mammals, the levels of X-linked gene products in males and females are equalised by the silencing, early in development, of most of the genes on one of the two female X chromosomes. Once established, the silent state is stable from one cell generation to the next. In eutherian mammals, the inactive X chromosome (Xi) differs from its active homologue (Xa) in a number of ways, including increased methylation of selected CpGs, replication late in S-phase, expression of the Xistgene with binding of Xist RNA and underacetylation of core histones. The latter is a common property of genetically inactive chromatin but, in the case of Xi, it is not clear whether it is an integral part of the silencing process or simply a consequence of some other property of Xi, such as late replication. The present review describes two approaches that address this problem. The first shows that Xi in marsupial mammals also contains underacetylated H4, even though its properties differ widely from those of the eutherian Xi. The continued presence of histone underacetylation on Xi in these evolutionarily distant mammals argues for its fundamental importance. The second approach uses mouse embryonic stem cells and places H4 deacetylation in a sequence of events leading to complete X inactivation. The results argue that histone underacetylation plays a role in the stabilisation of the inactive state, rather than in its initiation. Dev. Genet. 22:65–73, 1998. © 1998 Wiley-Liss, Inc.     

Histone H3 lysine 4 dimethylation is enriched on the inactive sex chromosomes in male meiosis but absent on the inactive X in female somatic cells

Inactivation of the X chromosome occurs in female somatic cells and in male meiosis. In both cases, the inactive X chromosome undergoes changes in histone modifications including deacetylation of core histone proteins and enrichment with histone H3 lysine 9 (H3-K9) dimethylation. In this study we show that while the inactive X in female somatic cells is largely devoid of H3-K4 dimethylation, the inactive X in male meiosis is enriched with this modification. However, the inactive X chromosome in female somatic cells and the inactive X and Y in male meiosis are devoid of H3-K4 trimethylation. Further, trimethylation of H3-K4 is present at discrete regions along most of the autosomes, while H3-K4 dimethylation shows a more homogenous staining. Also, the Y chromosome is largely devoid of H3-K4 di- and trimethylation in somatic cells of both humans and mice, however, the Y chromosome is enriched with H3-K4 di- but not trimethylation throughout spermatogenesis. Our results provide insights into the differences between female somatic cells and male germ cells in inactivating the X chromosome, and suggest that trimethylation, and not dimethylation, of H3-K4 is a more robust indicator of the active regions of the genome.     

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.     

Micromanipulating dosage compensation: understanding X-chromosome inactivation through nuclear transplantation

Nuclear transfer (NT) studies have provided insight into the functional importance of epigenetic alteration of the X chromosomes during X-inactivation. Uniparental embryos created by NT have been informative as to the time and location at which the imprint controlling extraembryonic X-inactivation is established. Experiments with female somatic cells, have demonstrated that the inactive X chromosome (Xi) is reactivated after NT, leading to random X-inactivation in the embryonic lineages of cloned embryos. However, in the extraembryonic lineages of clones, epigenetic information from the donor cell nucleus persists, leading to preferential inactivation of the donor cell's inactive X in the placenta of cloned animals. These results suggest epigenetic information established during embryonic X-inactivation is functionally equivalent to the gametic imprint.     

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 loss of histone H3 isoforms mono-, di- and tri-methylated at lysine 4 during X-inactivation in female embryonic stem cells

Silencing of genes on one of the two female X chromosomes early in development helps balance expression of X-linked genes between XX females and XY males and involves chromosome-wide changes in histone variants and modifications. Mouse female embryonic stem (ES) cells have two active Xs, one of which is silenced on differentiation, and provide a powerful model for studying the dynamics of X inactivation. Here, we use immunofluorescence microscopy of metaphase chromosomes to study changes in H3 mono-, di- or tri-methylated at lysine 4 (H3K4mel, -2 or -3) on the inactivating X (Xi) in female ES cells. H3K4me3 is absent from Xi in approximately 25% of chromosome spreads by day 2 of differentiation and in 40-50% of spreads by days 4-6, making it one of the earliest detectable changes on Xi. In contrast, loss of H3K4me2 occurs 1-2 days later, when histone acetylation also diminishes. Remarkably, H3K4mel is depleted on both (active) X chromosomes in undifferentiated female ES cells, and on the single X in males, and remains depleted on Xi. Consistent with this, chromatin immunoprecipitation reveals differentiation-related reductions in H3K4me2 and H3K4me3 at the promoter regions of genes undergoing X-inactivation in female ES cells, but no comparable change in H3K4me1.     

Histone macroH2A1.2 is concentrated in the XY-body by the early pachytene stage of spermatogenesis

The pairing of sex chromosomes during meiosis in male mammals is associated with ongoing heterochromatinization and X inactivation. This process occurs in a specific area of the nucleus that can be discerned morphologically: the sex vesicle or XY-body. In contrast to X inactivation in the somatic cells of female mammals the reasons for X inactivation in the male germline remain obscure. We have recently demonstrated that the inactive X chromosome in somatic cells of female mammals is marked by a high concentration of histone macroH2A. Here we investigate X inactivation in the meiotic cells of the male germline. We demonstrate here that macroH2A1.2 is present in the nuclei of germ cells starting first with localization that is largely, if not exclusively, to the developing XY-body in early pachytene spermatocytes. Our results suggest that inactivation of sex chromosomes in the male germ cell includes a major alteration of the nucleosomal structure.     

X-inactivation is stably maintained in mouse embryos deficient for histone methyl transferase G9a

One of the two X chromosomes becomes inactivated during early development of female mammals. Recent studies demonstrate that the inactive X chromosome is rich in histone H3 methylated at Lys-9 and Lys-27, suggesting an important role for these modifications in X-inactivation. It has been shown that in the mouse Eed is required for maintenance of X-inactivation in the extraembryonic lineages. Interestingly, Eed associates with Ezh2 to form a complex possessing histone methyltransferase activity predominantly for H3 Lys-27. We previously showed that G9a is one of the histone methyltransferases specific for H3 Lys-9 and is essential for embryonic development. Here we examined X-inactivation in mouse embryos deficient for G9a. Expression of Xist, which is crucial for the initiation of X-inactivation, was properly regulated and the inactivated X chromosome was stably maintained even in the absence of G9a. These results demonstrate that G9a is not essential for X-inactivation.     

Ubiquitinated proteins including uH2A on the human and mouse inactive X chromosome: enrichment in gene rich bands

The inactive X chromosome (Xi) forms a heterochromatic structure in the nucleus that is known to have several modifications to specific histones involving acetylation or methylation. Using three different antibodies in four different cell lines, we demonstrate that the Xi in human and mouse cells is highly enriched in ubiquitinated protein(s), much of which is polyubiquitinated. This ubiquitination appears specific for the Xi as it was not observed for centromeres or other regions of heterochromatin. Results using an antibody specific to ubiquitinated H2A provide a clear link between H2A ubiquitination and gene repression, as visualized across an entire inactive chromosome. Interestingly, the ubiquitination of the chromosome persists into mitosis and can be seen in a reproducible banded pattern. This pattern matches that of Xist RNA which forms bands as it detaches from the mitotic X chromosome. Both ubiquitination and Xist RNA appear enriched in gene dense regions and depleted in gene poor bands, but do not correlate with L1 LINE elements which have been suggested as key to X-inactivation. These results provide evidence that ubiquitination along with Xist RNA plays an important role in the formation of facultative heterochromatin during X-inactivation.     

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     

Analysis of loss of inactive X chromosomes in interphase cells.

We have developed a method that allows, for the first time, a specific analysis of the inactive X chromosome (Xi) in interphase cells. By combining immunolabeling of acetylated histone H4 with specific antisera and FISH with an X-chromosome centromere-specific DNA probe, micronucleated whole Xis in human female cells may be identified by their lack of histone H4 acetylation. As one example of the potential applications of this methodology in genetic studies in humans, an artifact-free X-chromosome aneuploidy detection in lymphocytes of women of different ages has been performed. Our results indicate that not only the Xi but also the active X chromosome is preferentially lost during aging, indicating that the high frequency of sex-chromosome aneuploidy in human females cannot be explained solely by a lack of negative selection of Xi aneuploid cells. Further applications of the proposed methodology in genetic studies are discussed.