X-changing information on X inactivation

In female somatic cells of mammalian species one X chromosome is inactivated to ensure dosage equality of X-encoded genes between females and males, during development and adulthood. X chromosome inactivation (XCI) involves various epigenetic mechanisms, including RNA mediated gene silencing in cis, DNA methylation, and changes in chromatin modifications and composition. XCI therefore provides an attractive paradigm to study epigenetic gene regulation in a more general context. The XCI process starts with counting of the number of X chromosomes present in a nucleus, and initiation of XCI follows if this number exceeds one per diploid genome. Recently, X-encoded RNF12 has been identified as a dose-dependent activator of XCI. In addition, other factors, including the pluripotency factors OCT4, SOX2 and Nanog, have been implicated to play a role in suppression of initiation of XCI. In this review, we highlight and explain these new and old findings in the context of a stochastic model for X chromosome counting and XCI initiation.     

The Probability to Initiate X Chromosome Inactivation Is Determined by the X to Autosomal Ratio and X Chromosome Specific Allelic Properties

Background

In female mammalian cells, random X chromosome inactivation (XCI) equalizes the dosage of X-encoded gene products to that in male cells. XCI is a stochastic process, in which each X chromosome has a probability to be inactivated. To obtain more insight in the factors setting up this probability, we studied the role of the X to autosome (X∶A) ratio in initiation of XCI, and have used the experimental data in a computer simulation model to study the cellular population dynamics of XCI.

Methodology/Principal Findings

To obtain more insight in the role of the X∶A ratio in initiation of XCI, we generated triploid mouse ES cells by fusion of haploid round spermatids with diploid female and male ES cells. These fusion experiments resulted in only XXY triploid ES cells. XYY and XXX ES lines were absent, suggesting cell death related either to insufficient X-chromosomal gene dosage (XYY) or to inheritance of an epigenetically modified X chromosome (XXX). Analysis of active (Xa) and inactive (Xi) X chromosomes in the obtained triploid XXY lines indicated that the initiation frequency of XCI is low, resulting in a mixed population of XaXiY and XaXaY cells, in which the XaXiY cells have a small proliferative advantage. This result, and findings on XCI in diploid and tetraploid ES cell lines with different X∶A ratios, provides evidence that the X∶A ratio determines the probability for a given X chromosome to be inactivated. Furthermore, we found that the kinetics of the XCI process can be simulated using a probability for an X chromosome to be inactivated that is proportional to the X∶A ratio. These simulation studies re-emphasize our hypothesis that the probability is a function of the concentration of an X-encoded activator of XCI, and of X chromosome specific allelic properties determining the threshold for this activator.

Conclusions

The present findings reveal that the probability for an X chromosome to be inactivated is proportional to the X∶A ratio. This finding supports the presence of an X-encoded activator of the XCI process.     

Understanding the X chromosome inactivation cycle in mice: A comprehensive view provided by nuclear transfer

During mouse development, imprinted X chromosome inactivation (XCI) is observed in preimplantation embryos and is inherited to the placental lineage, whereas random XCI is initiated in the embryonic proper. Xist RNA, which triggers XCI, is expressed ectopically in cloned embryos produced by somatic cell nuclear transfer (SCNT). To understand these mechanisms, we undertook a large-scale nuclear transfer study using different donor cells throughout the life cycle. The Xist expression patterns in the reconstructed embryos suggested that the nature of imprinted XCI is the maternal Xist-repressing imprint established at the last stage of oogenesis. Contrary to the prevailing model, this maternal imprint is erased in both the embryonic and extraembryonic lineages. The lack of the Xist-repressing imprint in the postimplantation somatic cells clearly explains how the SCNT embryos undergo ectopic Xist expression. Our data provide a comprehensive view of the XCI cycle in mice, which is essential information for future investigations of XCI mechanisms.     

X inactivation counting and choice is a stochastic process: evidence for involvement of an X-linked activator

Female mammalian cells achieve dosage compensation of X-encoded genes by X chromosome inactivation (XCI). This process is thought to involve X chromosome counting and choice. To explore how this process is initiated, we analyzed XCI in tetraploid XXXX, XXXY, and XXYY embryonic stem cells and found that every X chromosome within a single nucleus has an independent probability to initiate XCI. This finding suggests a stochastic mechanism directing XCI counting and choice. The probability is directly proportional to the X chromosome:ploidy ratio, indicating the presence of an X-encoded activator of XCI, that itself is inactivated by the XCI process. Deletion of a region including Xist, Tsix, and Xite still results in XCI on the remaining wild-type X chromosome in female cells. This result supports a stochastic model in which each X chromosome in a nucleus initiates XCI independently and positions an X-encoded trans-acting XCI-activator outside the deleted region.     

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.     

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.     

X chromosome inactivation in the cycle of life

Female mammalian cells silence one of their two X chromosomes, resulting in equal expression levels of X-encoded genes in female XX and male XY cells. In mice, the X chromosomes in female cells go through sequential steps of inactivation and reactivation. Depending on the developmental time window, imprinted or random X chromosome inactivation (XCI) is initiated, and both processes lead to an inactive X chromosome that is clonally inherited. Here, we review new insights into the life cycle of XCI and provide an overview of the mechanisms regulating X inactivation and reactivation.     

Understanding the X chromosome inactivation cycle in mice

During mouse development, imprinted X chromosome inactivation (XCI) is observed in preimplantation embryos and is inherited to the placental lineage, whereas random XCI is initiated in the embryonic proper. Xist RNA, which triggers XCI, is expressed ectopically in cloned embryos produced by somatic cell nuclear transfer (SCNT). To understand these mechanisms, we undertook a large-scale nuclear transfer study using different donor cells throughout the life cycle. The Xist expression patterns in the reconstructed embryos suggested that the nature of imprinted XCI is the maternal Xist-repressing imprint established at the last stage of oogenesis. Contrary to the prevailing model, this maternal imprint is erased in both the embryonic and extraembryonic lineages. The lack of the Xist-repressing imprint in the postimplantation somatic cells clearly explains how the SCNT embryos undergo ectopic Xist expression. Our data provide a comprehensive view of the XCI cycle in mice, which is essential information for future investigations of XCI mechanisms.     

Origin and evolution of X chromosome inactivation

Evolution of the mammalian sex chromosomes heavily impacts on the expression of X-encoded genes, both in marsupials and placental mammals. The loss of genes from the Y chromosome forced a two-fold upregulation of dose sensitive X-linked homologues. As a corollary, female cells would experience a lethal dose of X-linked genes, if this upregulation was not counteracted by evolution of X chromosome inactivation (XCI) that allows for only one active X chromosome per diploid genome. Marsupials rely on imprinted XCI, which inactivates always the paternally inherited X chromosome. In placental mammals, random XCI (rXCI) is the predominant form, inactivating either the maternal or paternal X. In this review, we discuss recent new insights in the regulation of XCI. Based on these findings, we propose an X inactivation center (Xic), composed of a cis-Xic and trans-Xic that encompass all elements and factors acting to control rXCI either in cis or in trans. We also highlight that XCI may have evolved from a very small nucleation site on the X chromosome in the vicinity of the Sox3 gene. Finally, we discuss the possible evolutionary road maps that resulted in imprinted XCI and rXCI as observed in present day mammals.     

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.     

High-resolution chromosomal microarray analysis of early-stage human embryonic stem cells reveals an association between X chromosome instability and skewed X inactivation

X chromosome inactivation (XCI) is a dosage compensation mechanism that silences the majority of genes on one X chromosome in each female cell via a random process. Skewed XCI is relevant to many diseases, but the mechanism leading to it remains unclear. Human embryonic stem cells (hESCs) derived from the inner cell mass (ICM) of blastocyst-stage embryos have provided an excellent model system for understanding XCI initiation and maintenance. Here, we derived hESC lines with random or skewed XCI patterns from poor-quality embryos and investigated the genome-wide copy number variation (CNV) and loss of heterozygosity (LOH) patterns at the early passages of these two groups of hESC lines. It was found that the average size of CNVs on the X chromosomes in the skewed group is twice as much as that in the random group. Moreover, the LOH regions of the skewed group covered the gene locus of either XIST or XACT, which are master long non-coding RNA (lncRNA) effectors of XCI in human pluripotent stem cells. In conclusion, our work has established an experimentally tractable hESC model for study of skewed XCI and revealed an association between X chromosome instability and skewed XCI.     

Higher order chromatin structure at the X-inactivation center via looping DNA

In mammals, the silencing step of the X-chromosome inactivation (XCI) process is initiated by the non-coding Xist RNA. Xist is known to be controlled by the non-coding Xite and Tsix loci, but the mechanisms by which Tsix and Xite regulate Xist are yet to be fully elucidated. Here, we examine the role of higher order chromatin structure across the 100-kb region of the mouse X-inactivation center (Xic) and map domains of specialized chromatin in vivo. By hypersensitive site mapping and chromosome conformation capture (3C), we identify two domains of higher order chromatin structure. Xite makes looping interactions with Tsix, while Xist makes contacts with Jpx/Enox, another non-coding gene not previously implicated in XCI. These regions interact in a developmentally-specific and sex-specific manner that is consistent with a regulatory role in XCI. We propose that dynamic changes in three-dimensional architecture leads to formation of separate chromatin hubs in Tsix and Xist that together regulate the initiation of X-chromosome inactivation.     

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.     

Dosage compensation in the mouse balances up-regulation and silencing of X-linked genes

Dosage compensation in mammals involves silencing of one X chromosome in XX females and requires expression, in cis, of Xist RNA. The X to be inactivated is randomly chosen in cells of the inner cell mass (ICM) at the blastocyst stage of development. Embryonic stem (ES) cells derived from the ICM of female mice have two active X chromosomes, one of which is inactivated as the cells differentiate in culture, providing a powerful model system to study the dynamics of X inactivation. Using microarrays to assay expression of X-linked genes in undifferentiated female and male mouse ES cells, we detect global up-regulation of expression (1.4- to 1.6-fold) from the active X chromosomes, relative to autosomes. We show a similar up-regulation in ICM from male blastocysts grown in culture. In male ES cells, up-regulation reaches 2-fold after 2–3 weeks of differentiation, thereby balancing expression between the single X and the diploid autosomes. We show that silencing of X-linked genes in female ES cells occurs on a gene-by-gene basis throughout differentiation, with some genes inactivating early, others late, and some escaping altogether. Surprisingly, by allele-specific analysis in hybrid ES cells, we also identified a subgroup of genes that are silenced in undifferentiated cells. We propose that X-linked genes are silenced in female ES cells by spreading of Xist RNA through the X chromosome territory as the cells differentiate, with silencing times for individual genes dependent on their proximity to the Xist locus.