Xist-mediated chromatin changes that establish silencing of an entire X chromosome in mammals

X chromosome inactivation (XCI) ensures an equal gene dosage between the sexes in placental mammals. Xist, a modular multi-domain X-encoded long non-coding RNA coats the X chromosome in cis during XCI. Xist recruits chromatin remodelers and repressor complexes ensuring silencing of the inactive X (Xi). Here, we review the recent work focused on the role of Xist functional repeats and interacting RNA-binding factors in the establishment of the silent state. Xist orchestrates recruitment of remodelers and repressors that first facilitate removal of the active chromatin landscape and subsequently direct the transition into a repressive heterochromatic environment. Some of these factors affect silencing on a chromosome-wide scale, while others display gene-specific silencing defects. The temporal order of recruitment shows each silencing step is party dependent on one another. After the Xi is established, many of the factors are dispensable, and a different repertoire of proteins ensure the silenced Xi is maintained and propagated.     

Synergism of Xist RNA, DNA methylation, and histone hypoacetylation in maintaining X chromosome inactivation

Xist RNA expression, methylation of CpG islands, and hypoacetylation of histone H4 are distinguishing features of inactive X chromatin. Here, we show that these silencing mechanisms act synergistically to maintain the inactive state. Xist RNA has been shown to be essential for initiation of X inactivation, but not required for maintenance. We have developed a system in which the reactivation frequency of individual X-linked genes can be assessed quantitatively. Using a conditional mutant Xist allele, we provide direct evidence for that loss of Xist RNA destabilizes the inactive state in somatic cells, leading to an increased reactivation frequency of an X-linked GFP transgene and of the endogenous hypoxanthine phosphoribosyl transferase (Hprt) gene in mouse embryonic fibroblasts. Demethylation of DNA, using 5-azadC or by introducing a mutation in Dnmt1, and inhibition of histone hypoacetylation using trichostatin A further increases reactivation in Xist mutant fibroblasts, indicating a synergistic interaction of X chromosome silencing mechanisms.     

Random X Inactivation and Extensive Mosaicism in Human Placenta Revealed by Analysis of Allele-Specific Gene Expression along the X Chromosome

Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 22 X-linked genes, including XIST, using 27 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this analysis indicated heterogeneous maintenance of gene silencing along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals.     

Analysis of expressed SNPs identifies variable extents of expression from the human inactive X chromosome

Background

X-chromosome inactivation (XCI) results in the silencing of most genes on one X chromosome, yielding mono-allelic expression in individual cells. However, random XCI results in expression of both alleles in most females. Allelic imbalances have been used genome-wide to detect mono-allelically expressed genes. Analysis of X-linked allelic imbalance in females with skewed XCI offers the opportunity to identify genes that escape XCI with bi-allelic expression in contrast to those with mono-allelic expression and which are therefore subject to XCI.

Results

We determine XCI status for 409 genes, all of which have at least five informative females in our dataset. The majority of genes are subject to XCI and genes that escape from XCI show a continuum of expression from the inactive X. Inactive X expression corresponds to differences in the level of histone modification detected by allelic imbalance after chromatin immunoprecipitation. Differences in XCI between populations and between cell lines derived from different tissues are observed.

Conclusions

We demonstrate that allelic imbalance can be used to determine an inactivation status for X-linked genes, even without completely non-random XCI. There is a range of expression from the inactive X. Genes escaping XCI, including those that do so in only a subset of females, cluster together, demonstrating that XCI and location on the X chromosome are related. In addition to revealing mechanisms involved in cis-gene regulation, determining which genes escape XCI can expand our understanding of the contributions of X-linked genes to sexual dimorphism.     

Early loss of Xist RNA expression and inactive X chromosome associated chromatin modification in developing primordial germ cells

BACKGROUND: The inactive X chromosome characteristic of female somatic lineages is reactivated during development of the female germ cell lineage. In mouse, analysis of protein products of X-linked genes and/or transgenes located on the X chromosome has indicated that reactivation occurs after primordial germ cells reach the genital ridges. PRINCIPAL FINDINGS/METHODOLOGY: We present evidence that the epigenetic reprogramming of the inactive X-chromosome is initiated earlier than was previously thought, around the time that primordial germ cells (PGCs) migrate through the hindgut. Specifically, we find that Xist RNA expression, the primary signal for establishment of chromosome silencing, is extinguished in migrating PGCs. This is accompanied by displacement of Polycomb-group repressor proteins Eed and Suz(12), and loss of the inactive X associated histone modification, methylation of histone H3 lysine 27. CONCLUSIONS/SIGNIFICANCE: We conclude that X reactivation in primordial germ cells occurs progressively, initiated by extinction of Xist RNA around the time that germ cells migrate through the hindgut to the genital ridges. The events that we observe are reminiscent of X reactivation of the paternal X chromosome in inner cell mass cells of mouse pre-implantation embryos and suggest a unified model in which execution of the pluripotency program represses Xist RNA thereby triggering progressive reversal of epigenetic silencing of the X chromosome.