X chromosomes alternate between two states prior to random X-inactivation

Early in the development of female mammals, one of the two X chromosomes is silenced in half of cells and the other X chromosome is silenced in the remaining half. The basis of this apparent randomness is not understood. We show that before X-inactivation, the two X chromosomes appear to exist in distinct states that correspond to their fates as the active and inactive X chromosomes. Xist and Tsix, noncoding RNAs that control X chromosome fates upon X-inactivation, also determine the states of the X chromosomes prior to X-inactivation. In wild-type ES cells, X chromosomes switch between states; among the progeny of a single cell, a given X chromosome exhibits each state with equal frequency. We propose a model in which the concerted switching of homologous X chromosomes between mutually exclusive future active and future inactive states provides the basis for the apparently random silencing of one X chromosome in female cells.     

A Chromosomal Memory Triggered by Xist Regulates Histone Methylation in X Inactivation

We have elucidated the kinetics of histone methylation during X inactivation using an inducible Xist expression system in mouse embryonic stem (ES) cells. Previous reports showed that the ability of Xist to trigger silencing is restricted to an early window in ES cell differentiation. Here we show that this window is also important for establishing methylation patterns on the potential inactive X chromosome. By immunofluorescence and chromatin immunoprecipitation experiments we show that histone H3 lysine 27 trimethylation (H3K27m3) and H4 lysine 20 monomethylation (H4K20m1) are associated with Xist expression in undifferentiated ES cells and mark the initiation of X inactivation. Both marks depend on Xist RNA localisation but are independent of silencing. Induction of Xist expression after the initiation window leads to a markedly reduced ability to induce H3K27m3, whereas expression before the restrictive time point allows efficient H3K27m3 establishment. Our data show that Xist expression early in ES cell differentiation establishes a chromosomal memory, which is maintained in the absence of silencing. One consequence of this memory is the ability to introduce H3K27m3 efficiently after the restrictive time point on the chromosome that has expressed Xist early. Our results suggest that this silencing-independent chromosomal memory has important implications for the maintenance of X inactivation, where previously self-perpetuating heterochromatin structures were viewed as the principal form of memory.     

Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome

To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.     

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.     

X inactivation and disease

X inactivation is the mechanism by which mammals adjust the X-linked gene dosage between the sexes. The dosage difference between XX females and XY males is functionally equalized by silencing one of the two X chromosomes in female cells. This dosage-compensation mechanism is based on the long functional Xist RNA. Here, we review our understanding of dosage compensation and Xist function in the context of disease.     

A chromosomal memory triggered by Xist regulates histone methylation in X inactivation

We have elucidated the kinetics of histone methylation during X inactivation using an inducible Xist expression system in mouse embryonic stem (ES) cells. Previous reports showed that the ability of Xist to trigger silencing is restricted to an early window in ES cell differentiation. Here we show that this window is also important for establishing methylation patterns on the potential inactive X chromosome. By immunofluorescence and chromatin immunoprecipitation experiments we show that histone H3 lysine 27 trimethylation (H3K27m3) and H4 lysine 20 monomethylation (H4K20m1) are associated with Xist expression in undifferentiated ES cells and mark the initiation of X inactivation. Both marks depend on Xist RNA localisation but are independent of silencing. Induction of Xist expression after the initiation window leads to a markedly reduced ability to induce H3K27m3, whereas expression before the restrictive time point allows efficient H3K27m3 establishment. Our data show that Xist expression early in ES cell differentiation establishes a chromosomal memory, which is maintained in the absence of silencing. One consequence of this memory is the ability to introduce H3K27m3 efficiently after the restrictive time point on the chromosome that has expressed Xist early. Our results suggest that this silencing-independent chromosomal memory has important implications for the maintenance of X inactivation, where previously self-perpetuating heterochromatin structures were viewed as the principal form of memory.     

Genetic activity of X chromosomes in pluripotent female teratocarcinoma cells and their differentiated progeny

We have induced teratocarcinomas from female embryos heterozygous for electrophoretic variants of the X-linked gene coding for phosphoglycerate kinase (PGK). An embryonal carcinoma cell line, P10, has been isolated from such a teratocarcinoma. It has a normal female karyotype and cultures contain both PGK isoenzymic forms. Clonal populations derived from P10 also contain both PGK electrophoretic variants. In addition, both X chromosomes in these cells replicate in synchrony with the autosomes during early S phase of the cell cycle. These data indicate that the undifferentiated P10 embryonal carcinoma cells contain two active X chromosomes. When cultured under the appropriate conditions, the P10 cells differentiate to form a variety of tissue types. At least some of these differentiated cells contain an inactive X chromosome as determined by cytogenetic analysis. Apparently X chromosome inactivation accompanies the differentiation of these female embryonal carcinoma cells.     

X-chromosome inactivation in monkey embryos and pluripotent stem cells

Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.     

An embryonic story: Analysis of the gene regulative network controlling Xist expression in mouse embryonic stem cells

In mice, dosage compensation of X‐linked gene expression is achieved through the inactivation of one of the two X‐chromosomes in XX female cells. The complex epigenetic process leading to X‐inactivation is largely controlled by Xist and Tsix, two non‐coding genes of opposing function. Xist RNA triggers X‐inactivation by coating the inactive X, while Tsix is critical for the designation of the active X‐chromosome through cis‐repression of Xist RNA accumulation. Recently, a plethora of trans‐acting factors and cis‐regulating elements have been suggested to act as key regulators of either Xist, Tsix or both; these include ubiquitous factors such as Yy1 and Ctcf, developmental proteins such as Nanog, Oct4 and Sox2, and X‐linked regulators such as Rnf12. In this paper we summarise recent advances in our knowledge of the regulation of Xist and Tsix in embryonic stem (ES) and differentiating ES cells.     

Evolutionary steps of sex chromosomes are reflected in retrogenes

It has been shown that selective pressure to compensate for the silencing of the sex chromosomes during male meiosis resulted in many X-linked genes being duplicated as functional retrogenes on autosomes. The silencing of male sex chromosomes was probably stratified during evolution, in accordance with their stratified diversification. Here I show that the timing of the retrocopying events is associated with the timing of the X-Y differentiation of the region of the X chromosome housing the parental copy of the gene.     

X Chromosome Inactivation during Drosophila Spermatogenesis

Genes with male- and testis-enriched expression are under-represented on the Drosophila melanogaster X chromosome. There is also an excess of retrotransposed genes, many of which are expressed in testis, that have “escaped” the X chromosome and moved to the autosomes. It has been proposed that inactivation of the X chromosome during spermatogenesis contributes to these patterns: genes with a beneficial function late in spermatogenesis should be selectively favored to be autosomal in order to avoid inactivation. However, conclusive evidence for X inactivation in the male germline has been lacking. To test for such inactivation, we used a transgenic construct in which expression of a lacZ reporter gene was driven by the promoter sequence of the autosomal, testis-specific ocnus gene. Autosomal insertions of this transgene showed the expected pattern of male- and testis-specific expression. X-linked insertions, in contrast, showed only very low levels of reporter gene expression. Thus, we find that X linkage inhibits the activity of a testis-specific promoter. We obtained the same result using a vector in which the transgene was flanked by chromosomal insulator sequences. These results are consistent with global inactivation of the X chromosome in the male germline and support a selective explanation for X chromosome avoidance of genes with beneficial effects late in spermatogenesis.