Xist Function Research Articles

Unraveling the role of Xist in X chromosome inactivation: insights from rabbit model and deletion analysis of exons and repeat A

X chromosome inactivation (XCI) is a process that equalizes the expression of X-linked genes between males and females. It relies on Xist, continuously expressed in somatic cells during XCI maintenance. However, how Xist impacts XCI maintenance and its functional motifs remain unclear. In this study, we conducted a comprehensive analysis of Xist, using rabbits as an ideal non-primate model. Homozygous knockout of exon 1, exon 6, and repeat A in female rabbits resulted in embryonic lethality. However, X∆ReAX females, with intact X chromosome expressing Xist, showed no abnormalities. Interestingly, there were no significant differences between females with homozygous knockout of exons 2–5 and wild-type rabbits, suggesting that exons 2, 3, 4, and 5 are less important for XCI. These findings provide evolutionary insights into Xist function.

XIST directly regulates X-linked and autosomal genes in naive human pluripotent cells

X chromosome inactivation (XCI) serves as a paradigm for RNA-mediated regulation of gene expression, wherein the long non-coding RNA XIST spreads across the X chromosome in cis to mediate gene silencing chromosome-wide. In female naive human pluripotent stem cells (hPSCs), XIST is in a dispersed configuration, and XCI does not occur, raising questions about XIST's function. We found that XIST spreads across the X chromosome and induces dampening of X-linked gene expression in naive hPSCs. Surprisingly, XIST also targets specific autosomal regions, where it induces repressive chromatin changes and gene expression dampening. Thereby, XIST equalizes X-linked gene dosage between male and female cells while inducing differences in autosomes. The dispersed Xist configuration and autosomal localization also occur transiently during XCI initiation in mouse PSCs. Together, our study identifies XIST as the regulator of X chromosome dampening, uncovers an evolutionarily conserved trans-acting role of XIST/Xist, and reveals a correlation between XIST/Xist dispersal and autosomal targeting.

28. Trisomy 21 silencing in hematopoietic and neural cells: New insights and prospects

We previously showed that a single gene, XIST, inserted into one chromosome 21 in trisomic iPS cells can silence the whole chromosome. This was shown in pluripotent stem cells, the optimal developmental context for XIST function, hence it was important to extend studies to differentiated cell systems. Two recent studies investigate trisomy silencing during in vitro hematopoiesis or in neural differentiated cells, both of which illustrate the power of inducible trisomy 21 silencing as an experimental approach to understand DS cell pathologies.

Stochastic gene expression and chromosome interactions in protecting the human active X from silencing by XIST

ABSTRACT Mammals use X chromosome inactivation to compensate for the sex difference in numbers of X chromosomes. A relatively unexplored question is how the active X is protected from inactivation by its own XIST gene, the long non-coding RNA, which initiates silence of the inactive X. Previous studies of autosomal duplications show that human chromosome 19 plays a critical role in protecting the active X. I proposed that it genetically interacts with the X chromosome to repress XIST function on the future active X. Here, I show that the type of chromosome 19 duplication influences the outcome of the interaction: the presence of three chromosome 19s is tolerated whereas duplications affecting only one chromosome 19 are not. The different outcomes have mechanistic implications for how chromosome 19 interacts with the future active X, pointing to a role for stochastic gene expression and possibly physical interaction.

Cdk8 is required for establishment of H3K27me3 and gene repression by Xist and mouse development.

ABSTRACTWe previously identified the cyclin dependent kinase Cdk8 as a putative silencing factor for Xist. To investigate its role in X inactivation, we engineered a Cdk8 mutation in mouse embryonic stem cells (ESCs) carrying an inducible system for studying Xist function. We found that Xist repressed X-linked genes at half of the expression level in Cdk8 mutant cells, whereas they were almost completely silenced in the controls. Lack of Cdk8 impaired Ezh2 recruitment and the establishment of histone H3 lysine 27 tri-methylation but not PRC1 recruitment by Xist. Transgenic expression of wild-type but not catalytically inactive Cdk8 restored efficient gene repression and PRC2 recruitment. Mutation of the paralogous kinase Cdk19 did not affect Xist function, and combined mutations of Cdk8 and Cdk19 resembled the Cdk8 mutation. In mice, a Cdk8 mutation caused post-implantation lethality. We observed that homozygous Cdk8 mutant female embryos showed a greater developmental delay than males on day 10.5. Together with the inefficient repression of X-linked genes in differentiating Cdk8 mutant female ESCs, these data show a requirement for Cdk8 in the initiation of X inactivation.

Mechanistic insights in X-chromosome inactivation.

X-chromosome inactivation (XCI) is a critical epigenetic mechanism for balancing gene dosage between XY males and XX females in eutherian mammals. A long non-coding RNA (lncRNA), XIST, and its associated proteins orchestrate this multi-step process, resulting in the inheritable silencing of one of the two X-chromosomes in females. The XIST RNA is large and complex, exemplifying the unique challenges associated with the structural and functional analysis of lncRNAs. Recent technological advances in the analysis of macromolecular structure and interactions have enabled us to systematically dissect the XIST ribonucleoprotein complex, which is larger than the ribosome, and its place of action, the inactive X-chromosome. These studies shed light on key mechanisms of XCI, such as XIST coating of the X-chromosome, recruitment of DNA, RNA and histone modification enzymes, and compaction and compartmentalization of the inactive X. Here, we summarize recent studies on XCI, highlight the critical contributions of new technologies and propose a unifying model for XIST function in XCI where modular domains serve as the structural and functional units in both lncRNA-protein complexes and DNA-protein complexes in chromatin.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'.

Repeat E anchors Xist RNA to the inactive X chromosomal compartment through CDKN1A-interacting protein (CIZ1)

X chromosome inactivation is an epigenetic dosage compensation mechanism in female mammals driven by the long noncoding RNA, Xist. Although recent genomic and proteomic approaches have provided a more global view of Xist's function, how Xist RNA localizes to the inactive X chromosome (Xi) and spreads in cis remains unclear. Here, we report that the CDKN1-interacting zinc finger protein CIZ1 is critical for localization of Xist RNA to the Xi chromosome territory. Stochastic optical reconstruction microscopy (STORM) shows a tight association of CIZ1 with Xist RNA at the single-molecule level. CIZ1 interacts with a specific region within Xist exon 7-namely, the highly repetitive Repeat E motif. Using genetic analysis, we show that loss of CIZ1 or deletion of Repeat E in female cells phenocopies one another in causing Xist RNA to delocalize from the Xi and disperse into the nucleoplasm. Interestingly, this interaction is exquisitely sensitive to CIZ1 levels, as overexpression of CIZ1 likewise results in Xist delocalization. As a consequence, this delocalization is accompanied by a decrease in H3K27me3 on the Xi. Our data reveal that CIZ1 plays a major role in ensuring stable association of Xist RNA within the Xi territory.

Probing Xist RNA Structure in Cells Using Targeted Structure-Seq.

The long non-coding RNA (lncRNA) Xist is a master regulator of X-chromosome inactivation in mammalian cells. Models for how Xist and other lncRNAs function depend on thermodynamically stable secondary and higher-order structures that RNAs can form in the context of a cell. Probing accessible RNA bases can provide data to build models of RNA conformation that provide insight into RNA function, molecular evolution, and modularity. To study the structure of Xist in cells, we built upon recent advances in RNA secondary structure mapping and modeling to develop Targeted Structure-Seq, which combines chemical probing of RNA structure in cells with target-specific massively parallel sequencing. By enriching for signals from the RNA of interest, Targeted Structure-Seq achieves high coverage of the target RNA with relatively few sequencing reads, thus providing a targeted and scalable approach to analyze RNA conformation in cells. We use this approach to probe the full-length Xist lncRNA to develop new models for functional elements within Xist, including the repeat A element in the 5’-end of Xist. This analysis also identified new structural elements in Xist that are evolutionarily conserved, including a new element proximal to the C repeats that is important for Xist function.

Xist localization and function: new insights from multiple levels.

In female m ammals, one of the two X chromosomes in each cell is transcriptionally silenced in order to achieve dosage compensation between the genders in a process called X chromosome inactivation. The master regulator of this process is the long non-coding RNA Xist. During X-inactivation, Xist accumulates in cis on the future inactive X chromosome, triggering a cascade of events that provoke the stable silencing of the entire chromosome, with relatively few genes remaining active. How Xist spreads, what are its binding sites, how it recruits silencing factors and how it induces a specific topological and nuclear organization of the chromatin all remain largely unanswered questions. Recent studies have improved our understanding of Xist localization and the proteins with which it interacts, allowing a reappraisal of ideas about Xist function. We discuss recent advances in our knowledge of Xist-mediated silencing, focusing on Xist spreading, the nuclear organization of the inactive X chromosome, recruitment of the polycomb complex and the role of the nuclear matrix in the process of X chromosome inactivation.

Identification of Spen as a Crucial Factor for Xist Function through Forward Genetic Screening in Haploid Embryonic Stem Cells.

SummaryIn mammals, the noncoding Xist RNA triggers transcriptional silencing of one of the two X chromosomes in female cells. Here, we report a genetic screen for silencing factors in X chromosome inactivation using haploid mouse embryonic stem cells (ESCs) that carry an engineered selectable reporter system. This system was able to identify several candidate factors that are genetically required for chromosomal repression by Xist. Among the list of candidates, we identify the RNA-binding protein Spen, the homolog of split ends. Independent validation through gene deletion in ESCs confirms that Spen is required for gene repression by Xist. However, Spen is not required for Xist RNA localization and the recruitment of chromatin modifications, including Polycomb protein Ezh2. The identification of Spen opens avenues for further investigation into the gene-silencing pathway of Xist and shows the usefulness of haploid ESCs for genetic screening of epigenetic pathways.

X Chromosome Inactivation and Xist Evolution in a Rodent Lacking LINE-1 Activity

Dosage compensation in eutherian mammals occurs by inactivation of one X chromosome in females. Silencing of that X chromosome is initiated by Xist, a large non-coding RNA, whose coating of the chromosome extends in cis from the X inactivation center. LINE-1 (L1) retrotransposons have been implicated as possible players for propagation of the Xist signal, but it has remained unclear whether they are essential components. We previously identified a group of South American rodents in which L1 retrotransposition ceased over 8 million years ago and have now determined that at least one species of these rodents, Oryzomys palustris, still retains X inactivation. We have also isolated and analyzed the majority of the Xist RNA from O. palustris and a sister species retaining L1 activity, Sigmodon hispidus, to determine if evolution in these sequences has left signatures that might suggest a critical role for L1 elements in Xist function. Comparison of rates of Xist evolution in the two species fails to support L1 involvement, although other explanations are possible. Similarly, comparison of known repeats and potential RNA secondary structures reveals no major differences with the exception of a new repeat in O. palustris that has potential to form new secondary structures.

Meiotic sex chromosome inactivation in male mice with targeted disruptions of Xist.

X chromosome inactivation occurs twice during the life cycle of placental mammals. In normal females, one X chromosome in each cell is inactivated early in embryogenesis, while in the male, the X chromosome is inactivated together with the Y chromosome in spermatogenic cells shortly before or during early meiotic prophase. Inactivation of one X chromosome in somatic cells of females serves to equalise X-linked gene dosage between males and females, but the role of male meiotic sex chromosome inactivation (MSCI) is unknown. The inactive X-chromosome of somatic cells and male meiotic cells share similar properties such as late replication and enrichment for histone macroH2A1.2, suggesting a common mechanism of inactivation. This possibility is supported by the fact that Xist RNA that mediates somatic X-inactivation is expressed in the testis of male mice and humans. In the present study we show that both Xist RNA and Tsix RNA, an antisense RNA that controls Xist function in the soma, are expressed in the testis in a germ-cell-dependent manner. However, our finding that MSCI and sex-body formation are unaltered in mice with targeted mutations of Xist that prevent somatic X inactivation suggests that somatic X-inactivation and MSCI occur by fundamentally different mechanisms.