Initiation Of X-chromosome Inactivation Research Articles

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.

IndiSPENsable for X Chromosome Inactivation and Gene Silencing.

For about 30 years, SPEN has been the subject of research in many different fields due to its variety of functions and its conservation throughout a wide spectrum of species, like worms, arthropods, and vertebrates. To date, 216 orthologues have been documented. SPEN had been studied for its role in gene regulation in the context of cell signaling, including the NOTCH or nuclear hormone receptor signaling pathways. More recently, SPEN has been identified as a major regulator of initiation of chromosome-wide gene silencing during X chromosome inactivation (XCI) in mammals, where its function remains to be fully understood. Dependent on the biological context, SPEN functions via mechanisms which include different domains. While some domains of SPEN are highly conserved in sequence and secondary structure, species-to-species differences exist that might lead to mechanistic differences. Initiation of XCI appears to be different between humans and mice, which raises additional questions about the extent of generalization of SPEN's function in XCI. In this review, we dissect the mechanism of SPEN in XCI. We discuss its subregions and domains, focusing on its role as a major regulator. We further highlight species-related research, specifically of mouse and human SPEN, with the aim to reveal and clarify potential species-to-species differences in SPEN's function.

RNA polymerase II depletion from the inactive X chromosome territory is not mediated by physical compartmentalization.

Subnuclear compartmentalization has been proposed to play an important role in gene regulation by segregating active and inactive parts of the genome in distinct physical and biochemical environments. During X chromosome inactivation (XCI), the noncoding Xist RNA coats the X chromosome, triggers gene silencing and forms a dense body of heterochromatin from which the transcription machinery appears to be excluded. Phase separation has been proposed to be involved in XCI, and might explain the exclusion of the transcription machinery by preventing its diffusion into the Xist-coated territory. Here, using quantitative fluorescence microscopy and single-particle tracking, we show that RNA polymerase II (RNAPII) freely accesses the Xist territory during the initiation of XCI. Instead, the apparent depletion of RNAPII is due to the loss of its chromatin stably bound fraction. These findings indicate that initial exclusion of RNAPII from the inactive X reflects the absence of actively transcribing RNAPII, rather than a consequence of putative physical compartmentalization of the inactive X heterochromatin domain.

Xist exerts gene-specific silencing during XCI maintenance and impacts lineage-specific cell differentiation and proliferation during hematopoiesis

X chromosome inactivation (XCI) is a dosage compensation phenomenon that occurs in females. Initiation of XCI depends on Xist RNA, which triggers silencing of one of the two X chromosomes, except for XCI escape genes that continue to be biallelically expressed. In the soma XCI is stably maintained with continuous Xist expression. How Xist impacts XCI maintenance remains an open question. Here we conditionally delete Xist in hematopoietic system of mice and report differentiation and cell cycle defects in female hematopoietic stem and progenitor cells (HSPCs). By utilizing female HSPCs and mouse embryonic fibroblasts, we find that X-linked genes show variable tolerance to Xist loss. Specifically, XCI escape genes exhibit preferential transcriptional upregulation, which associates with low H3K27me3 occupancy and high chromatin accessibility that accommodates preexisting binding of transcription factors such as Yin Yang 1 (YY1) at the basal state. We conclude that Xist is necessary for gene-specific silencing during XCI maintenance and impacts lineage-specific cell differentiation and proliferation during hematopoiesis.

Gene regulation in time and space during X-chromosome inactivation.

X-chromosome inactivation (XCI) is the epigenetic mechanism that ensures X-linked dosage compensation between cells of females (XX karyotype) and males (XY). XCI is essential for female embryos to survive through development and requires the accurate spatiotemporal regulation of many different factors to achieve remarkable chromosome-wide gene silencing. As a result of XCI, the active and inactive X chromosomes are functionally and structurally different, with the inactive X chromosome undergoing a major conformational reorganization within the nucleus. In this Review, we discuss the multiple layers of genetic and epigenetic regulation that underlie initiation of XCI during development and then maintain it throughout life, in light of the most recent findings in this rapidly advancing field. We discuss exciting new insights into the regulation of X inactive-specific transcript (XIST), the trigger and master regulator of XCI, and into the mechanisms and dynamics that underlie the silencing of nearly all X-linked genes. Finally, given the increasing interest in understanding the impact of chromosome organization on gene regulation, we provide an overview of the factors that are thought to reshape the 3D structure of the inactive X chromosome and of the relevance of such structural changes for XCI establishment and maintenance.

The neo-X does not form a Barr body but shows a slightly condensed structure in the Okinawa spiny rat (Tokudaia muenninki).

X chromosome inactivation (XCI) is an essential mechanism for gene dosage compensation between male and female cells in mammals. The Okinawa spiny rat (Tokudaia muenninki) is a native rodent in Japan with XX/XY sex chromosomes, like most mammals; however, the X chromosome has acquired a neo-X region (Xp) by fusion with an autosome. We previously reported that dosage compensation has not yet evolved in the neo-X region; however, X-inactive-specific transcript (Xist) RNA (long non-coding RNA required for the initiation of XCI) is partially localized in the region. Here, we show that the neo-X region represents an early chromosomal state in the acquisition of XCI by analyses of heterochromatin and Barr body formation. We found no evidence for heterochromatin formation in the neo-X region by RBA (R-banding by acridine orange) assays and immunostaining of H3K27me3. Double-immunostaining of H3K27me3 and HP1, a component of the Barr body, revealed that the entire ancestral-X chromosome region (Xq) showed a bipartite folded structure. By contrast, HP1 was not localized to the neo-X region. However, BAC FISH revealed that the signals of genes on the neo-X region of the inactive X chromosome were concentrated in a narrow region. These findings indicated that although the neo-X region of the inactive X chromosome does not form a complete Barr body structure (e.g., it lacks HP1), it forms a slightly condensed structure. These findings combined with the previously reported partial binding of Xist RNA suggest that the neo-X region exhibits incomplete inactivation. This may represent an early chromosomal state in the acquisition of the XCI mechanism.

SPEN is required for Xist upregulation during initiation of X chromosome inactivation

At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen−/− ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in initiation of the XCI process.

The Molecular and Nuclear Dynamics of X-Chromosome Inactivation.

In female eutherian mammals, dosage compensation of X-linked gene expression is achieved during development through transcriptional silencing of one of the two X chromosomes. Following X chromosome inactivation (XCI), the inactive X chromosome remains faithfully silenced throughout somatic cell divisions. XCI is dependent on Xist, a long noncoding RNA that coats and silences the X chromosome from which it is transcribed. Xist coating triggers a cascade of chromosome-wide changes occurring at the levels of transcription, chromatin composition, chromosome structure, and spatial organization within the nucleus. XCI has emerged as a paradigm for the study of such crucial nuclear processes and the dissection of their functional interplay. In the past decade, the advent of tools to characterize and perturb these processes have provided an unprecedented understanding into their roles during XCI. The mechanisms orchestrating the initiation of XCI as well as its maintenance are thus being unraveled, although many questions still remain. Here, we introduce key aspects of the XCI process and review the recent discoveries about its molecular basis.

Integrated analysis of Xist upregulation and X-chromosome inactivation with single-cell and single-allele resolution

To ensure dosage compensation between the sexes, one randomly chosen X chromosome is silenced in each female cell in the process of X-chromosome inactivation (XCI). XCI is initiated during early development through upregulation of the long non-coding RNA Xist, which mediates chromosome-wide gene silencing. Cell differentiation, Xist upregulation and gene silencing are thought to be coupled at multiple levels to ensure inactivation of exactly one out of two X chromosomes. Here we perform an integrated analysis of all three processes through allele-specific single-cell RNA-sequencing. Specifically, we assess the onset of random XCI in differentiating mouse embryonic stem cells, and develop dedicated analysis approaches. By exploiting the inter-cellular heterogeneity of XCI onset, we identify putative Xist regulators. Moreover, we show that transient Xist upregulation from both X chromosomes results in biallelic gene silencing right before transitioning to the monoallelic state, confirming a prediction of the stochastic model of XCI. Finally, we show that genetic variation modulates the XCI process at multiple levels, providing a potential explanation for the long-known X-controlling element (Xce) effect, which leads to preferential inactivation of a specific X chromosome in inter-strain crosses. We thus draw a detailed picture of the different levels of regulation that govern the initiation of XCI. The experimental and computational strategies we have developed here will allow us to profile random XCI in more physiological contexts, including primary human cells in vivo.

Loss of <i>XIST</i> Impairs Human Mammary Stem Cell Differentiation and Increases Tumorigenicity Through Enhancer and Mediator Complex Hyperactivation

X-chromosome inactivation (XCI) is triggered by up-regulation of XIST, which coats the chromosome in cis and promotes recruitment of chromatin modifiers that transform the X into a silent heterochromatic domain. Whether XIST plays a role beyond initiation of XCI is unclear. Here, we demonstrate that XIST loss impairs differentiation of human mammary stem cells (MaSC) and, upon oncogenic transformation, promotes emergence of highly metastatic carcinomas. On the Xi, XIST-deficient MaSC display epigenetic erosion and reactivation of genes overlapping Polycomb domains. Among reactivated loci we identify MED14 , a critical backbone of Mediator, and show that MED14 overdosage is sufficient to explain how defective XCI maintenance can stabilize MaSC enhancer landscape and transcriptional program, making differentiation less favorable. We conclude that XIST is a gatekeeper of mammary epithelium homeostasis, thus unveiling a new paradigm in the control of somatic cell identity with potential consequences in our understanding of gender-specific malignancies.

Initiation of X Chromosome Inactivation during Bovine Embryo Development.

X-chromosome inactivation (XCI) is a developmental process that aims to equalize the dosage of X-linked gene products between XY males and XX females in eutherian mammals. In female mouse embryos, paternal XCI is initiated at the 4-cell stage; however, the X chromosome is reactivated in the inner cell mass cells of blastocysts, and random XCI is subsequently initiated in epiblast cells. However, recent findings show that the patterns of XCI are not conserved among mammals. In this study, we used quantitative RT-PCR and RNA in situ hybridization combined with immunofluorescence to investigate the pattern of XCI during bovine embryo development. Expression of XIST (X-inactive specific transcript) RNA was significantly upregulated at the morula stage. For the first time, we demonstrate that XIST accumulation in bovine embryos starts in nuclei of female morulae, but its colocalization with histone H3 lysine 27 trimethylation was first detected in day 7 blastocysts. Both in the inner cell mass and in putative epiblast precursors, we observed a proportion of cells with XIST RNA and H3K27me3 colocalization. Surprisingly, the onset of XCI did not lead to a global downregulation of X-linked genes, even in day 9 blastocysts. Together, our findings confirm that diverse patterns of XCI initiation exist among developing mammalian embryos.

SPEN integrates transcriptional and epigenetic control of X-inactivation

SummaryXist represents a paradigm for long non-coding RNA function in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Multiple Xist-RNA binding proteins have recently been identified, including SPEN1–3, the loss of which has been associated with deficient XCI at multiple loci2–6. Here we demonstrate that SPEN is a key orchestrator of XCI in vivo and unravel its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and embryonic stem cells. SPEN is dispensable for maintenance of XCI in neural progenitors, although it significantly dampens expression of genes that escape XCI. We show that SPEN is immediately recruited to the X-chromosome upon Xist up-regulation, and is targeted to enhancers and promoters of active genes. SPEN rapidly disengages from chromatin upon gene silencing, implying a need for active transcription to tether it to chromatin. We define SPEN’s SPOC domain as a major effector of SPEN’s gene silencing function, and show that tethering SPOC to Xist RNA is sufficient to mediate gene silencing. We identify SPOC’s protein partners which include NCOR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for initiation of XCI, bridging Xist RNA with the transcription machinery as well as nucleosome remodelers and histone deacetylases, at active enhancers and promoters.

The B-side of Xist.

Female mammals express the long noncoding X inactivation-specific transcript ( Xist) RNA to initiate X chromosome inactivation (XCI) that eventually results in the formation of the Barr body. Xist encompasses half a dozen repeated sequence stretches containing motifs for RNA-binding proteins that recruit effector complexes with functions for silencing genes and establishing a repressive chromatin configuration. Functional characterization of these effector proteins unveils the cooperation of a number of pathways to repress genes on the inactive X chromosome. Mechanistic insights can be extended to other noncoding RNAs with similar structure and open avenues for the design of new therapies to switch off gene expression. Here we review recent advances in the understanding of Xist and on this basis try to synthesize a model for the initiation of XCI.

The role of Xist-mediated Polycomb recruitment in the initiation of X-chromosome inactivation.

Xist RNA has been established as the master regulator of X‐chromosome inactivation (XCI) in female eutherian mammals, but its mechanism of action remains unclear. By creating novel Xist‐inducible mutants at the endogenous locus in male mouse embryonic stem (ES) cells, we dissect the role of the conserved A‐B‐C‐F repeats in the initiation of XCI. We find that transcriptional silencing can be largely uncoupled from Polycomb repressive complex 1 and complex 2 (PRC1/2) recruitment, which requires B and C repeats. Xist ΔB+C RNA specifically loses interaction with PCGF3/5 subunits of PRC1, while binding of other Xist partners is largely unaffected. However, a slight relaxation of transcriptional silencing in Xist ΔB+C indicates a role for PRC1/2 proteins in early stabilization of gene repression. Distinct modules within the Xist RNA are therefore involved in the convergence of independent chromatin modification and gene repression pathways. In this context, Polycomb recruitment seems to be of moderate relevance in the initiation of silencing.

Varying levels of X chromosome coalescence in female somatic cells alters the balance of X-linked dosage compensation and is implicated in female-dominant systemic lupus erythematosus

The three-dimensional organization of the genome in mammalian interphase nuclei is intrinsically linked to the regulation of gene expression. Whole chromosome territories and their encoded gene loci occupy preferential positions within the nucleus that changes according to the expression profile of a given cell lineage or stage. To further illuminate the relationship between chromosome organization, epigenetic environment, and gene expression, here we examine the functional organization of chromosome X and corresponding X-linked genes in a variety of healthy human and disease state X diploid (XX) cells. We observe high frequencies of homologous chromosome X colocalization (or coalescence), typically associated with initiation of X-chromosome inactivation, occurring in XX cells outside of early embryogenesis. Moreover, during chromosome X coalescence significant changes in Xist, H3K27me3, and X-linked gene expression occur, suggesting the potential exchange of gene regulatory information between the active and inactive X chromosomes. We also observe significant differences in chromosome X coalescence in disease-implicated lymphocytes isolated from systemic lupus erythematosus (SLE) patients compared to healthy controls. These results demonstrate that X chromosomes can functionally interact outside of embryogenesis when X inactivation is initiated and suggest a potential gene regulatory mechanism aberration underlying the increased frequency of autoimmunity in XX individuals.

A novel approach to differentiate rat embryonic stem cells in vitro reveals a role for RNF12 in activation of X chromosome inactivation

X chromosome inactivation (XCI) is a mammalian specific, developmentally regulated process relying on several mechanisms including antisense transcription, non-coding RNA-mediated silencing, and recruitment of chromatin remodeling complexes. In vitro modeling of XCI, through differentiation of embryonic stem cells (ESCs), provides a powerful tool to study the dynamics of XCI, overcoming the need for embryos, and facilitating genetic modification of key regulatory players. However, to date, robust initiation of XCI in vitro has been mostly limited to mouse pluripotent stem cells. Here, we adapted existing protocols to establish a novel monolayer differentiation protocol for rat ESCs to study XCI. We show that differentiating rat ESCs properly downregulate pluripotency factor genes, and present female specific Xist RNA accumulation and silencing of X-linked genes. We also demonstrate that RNF12 seems to be an important player in regulation of initiation of XCI in rat, acting as an Xist activator. Our work provides the basis to investigate the mechanisms directing the XCI process in a model organism different from the mouse.

DNA methylation and functional characterization of the XIST gene during in vitro early embryo development in cattle

ABSTRACTXIST, in association with the shorter ncRNA RepA, are essential for the initiation of X chromosome inactivation (XCI) in mice. The molecular mechanisms controlling XIST and RepA expression are well characterized in that specie. However, little is known in livestock. We aimed to characterize the DNA methylation status along the 5’ portion of XIST and to characterize its transcriptional profile during early development in cattle. Three genomic regions of XIST named here as promoter, RepA and DMR1 had their DNA methylation status characterized in gametes and embryos. Expression profile of XIST was evaluated, including sense and antisense transcription. Oocytes showed higher levels of methylation than spermatozoa that was demethylated. DMR1 was hypermethylated throughout oogenesis. At the 8–16-cell embryo stage DMR1 was completed demethylated. Interestingly, RepA gain methylation during oocyte maturation and was demethylated at the blastocyst stage, later than DMR1. These results suggest that DMR1 and RepA are transient differentially methylated regions in cattle. XIST RNA was detected in matured oocytes and in single cells from the 2-cell to the morula stage, confirming the presence of maternal and embryonic transcripts. Sense and antisense transcripts were detected along the XIST in blastocyst. In silico analysis identified 63 novel transcript candidates at bovine XIST locus from both the plus and minus strands. Taking together these results improve our understanding of the molecular mechanisms involved in XCI initiation in cattle. This information may be useful for the improvement of assisted reproductive technologies in livestock considering that in vitro conditions may impair epigenetic reprogramming.

The Implication of Early Chromatin Changes in X Chromosome Inactivation

SummaryDuring development, the precise relationships between transcription and chromatin modifications often remain unclear. We use the X chromosome inactivation (XCI) paradigm to explore the implication of chromatin changes in gene silencing. Using female mouse embryonic stem cells, we initiate XCI by inducing Xist and then monitor the temporal changes in transcription and chromatin by allele-specific profiling. This reveals histone deacetylation and H2AK119 ubiquitination as the earliest chromatin alterations during XCI. We show that HDAC3 is pre-bound on the X chromosome and that, upon Xist coating, its activity is required for efficient gene silencing. We also reveal that first PRC1-associated H2AK119Ub and then PRC2-associated H3K27me3 accumulate initially at large intergenic domains that can then spread into genes only in the context of histone deacetylation and gene silencing. Our results reveal the hierarchy of chromatin events during the initiation of XCI and identify key roles for chromatin in the early steps of transcriptional silencing.

Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males

SummaryA hallmark of naive pluripotency is the presence of two active X chromosomes in females. It is not clear whether prevention of X chromosome inactivation (XCI) is mediated by gene networks that preserve the naive state. Here, we show that robust naive pluripotent stem cell (nPSC) self-renewal represses expression of Xist, the master regulator of XCI. We found that nPSCs accumulate Xist on the male X chromosome and on both female X chromosomes as they become NANOG negative at the onset of differentiation. This is accompanied by the appearance of a repressive chromatin signature and partial X-linked gene silencing, suggesting a transient and rapid XCI-like state in male nPSCs. In the embryo, Xist is transiently expressed in males and in females from both X chromosomes at the onset of naive epiblast differentiation. In conclusion, we propose that XCI initiation is gender independent and triggered by destabilization of naive identity, suggesting that gender-specific mechanisms follow, rather than precede, XCI initiation.

Genetic Intersection of Tsix and Hedgehog Signaling during the Initiation of X-Chromosome Inactivation

X-chromosome inactivation (XCI) silences one X chromosome in the female mammal and is essential to peri-implantation development. XCI is thought to be cell autonomous, with all factors required being produced within each cell. Nevertheless, external cues may exist. Here, we search for such developmental signals by combining bioinformatic, biochemical, and genetic approaches. Using exvivo and invivo models, we identify the Hedgehog (HH) paracrine system as a candidate signaling cascade. HH signaling keeps XCI in check in pluripotent cells and is transduced by GLI transcription factors to binding sites in Tsix, the antisense repressor of XCI. GLI potentiates Tsix expression and impedes XCI. Invivo, mutating Indian Hedgehog results in a sex ratio bias against females, and the female lethality is rescued by a second-site mutation in Tsix. These data demonstrate a genetic and functional intersection between HH and XCI and support a role for intercellular signaling during XCI.