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In mammals, dosage compensation of sex chromosomal genes between females (XX) and males (XY) is achieved through X-chromosome inactivation (XCI). The X-linked X-inactive-specific transcript (Xist) long noncoding RNA is indispensable for XCI and initiates the process early during development by spreading in cis across the X chromosome from which it is transcribed. During XCI, Xist RNA triggers gene silencing, recruits a plethora of chromatin modifying factors, and drives a major structural reorganization of the X chromosome. Here, we review our knowledge of the multitude of epigenetic events orchestrated by Xist RNA to allow female mammals to survive through embryonic development by establishing and maintaining proper dosage compensation. In particular, we focus on recent studies characterizing the interaction partners of Xist RNA, and we discuss how they have affected the field by addressing long-standing controversies or by giving rise to new research perspectives that are currently being explored. This review is dedicated to the memory of Denise Barlow, pioneer of genomic imprinting and functional long noncoding RNAs (lncRNAs), whose work has revolutionized the epigenetics field and continues to inspire generations of scientists.

Women develop stronger immune responses than men, with positive effects on the resistance to viral or bacterial infections but magnifying also the susceptibility to autoimmune diseases like systemic lupus erythematosus (SLE). In SLE, the dosage of the endosomal Toll-like receptor 7 (TLR7) is crucial. Murine models have shown that TLR7 overexpression suffices to induce spontaneous lupus-like disease. Conversely, suppressing TLR7 in lupus-prone mice abolishes SLE development. TLR7 is encoded by a gene on the X chromosome gene, denoted TLR7 in humans and Tlr7 in the mouse, and expressed in plasmacytoid dendritic cells (pDC), monocytes/macrophages, and B cells. The receptor recognizes single-stranded RNA, and its engagement promotes B cell maturation and the production of pro-inflammatory cytokines and antibodies. In female mammals, each cell randomly inactivates one of its two X chromosomes to equalize gene dosage with XY males. However, 15 to 23% of X-linked human genes escape X chromosome inactivation so that both alleles can be expressed simultaneously. It has been hypothesized that biallelic expression of X-linked genes could occur in female immune cells, hence fostering harmful autoreactive and inflammatory responses. We review here the current knowledge of the role of TLR7 in SLE, and recent evidence demonstrating that TLR7 escapes from X chromosome inactivation in pDCs, monocytes, and B lymphocytes from women and Klinefelter syndrome men. Female B cells where TLR7 is thus biallelically expressed display higher TLR7-driven functional responses, connecting the presence of two X chromosomes with the enhanced immunity of women and their increased susceptibility to TLR7-dependent autoimmune syndromes.

Xist represents a paradigm for the function of long non-coding RNA in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Several proteins that bind to Xist RNA have recently been identified, including the transcriptional repressor SPEN 1 – 3 , the loss of which has been associated with deficient XCI at multiple loci 2 – 6 . Here we show in mice that SPEN is a key orchestrator of XCI in vivo and we elucidate its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and in embryonic stem cells. SPEN is dispensable for maintenance of XCI in neural progenitors, although it significantly decreases the expression of genes that escape XCI. We show that SPEN is immediately recruited to the X chromosome upon the upregulation of Xist , and is targeted to enhancers and promoters of active genes. SPEN rapidly disengages from chromatin upon gene silencing, suggesting that active transcription is required to tether SPEN to chromatin. We define the SPOC domain as a major effector of the gene-silencing function of SPEN, and show that tethering SPOC to Xist RNA is sufficient to mediate gene silencing. We identify the protein partners of SPOC, including NCoR/SMRT, the m 6 A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in the regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for the initiation of XCI, bridging Xist RNA with the transcription machinery—as well as with nucleosome remodellers and histone deacetylases—at active enhancers and promoters. The transcriptional repressor SPEN bridges the non-coding RNA Xist to transcription machinery, histone deacetylases and chromatin remodelling factors to initiate X-chromosome inactivation.

Recruitment of the Polycomb repressive complexes PRC1 and PRC2 by Xist RNA is an important paradigm for chromatin regulation by long noncoding RNAs. Here, we show that the noncanonical Polycomb group RING finger 3/5 (PCGF3/5)–PRC1 complex initiates recruitment of both PRC1 and PRC2 in response to Xist RNA expression. PCGF3/5–PRC1–mediated ubiquitylation of histone H2A signals recruitment of other noncanonical PRC1 complexes and of PRC2, the latter leading to deposition of histone H3 lysine 27 methylation chromosome-wide. Pcgf3/5 gene knockout results in female-specific embryo lethality and abrogates Xist-mediated gene repression, highlighting a key role for Polycomb in Xist-dependent chromosome silencing. Our findings overturn existing models for Polycomb recruitment by Xist RNA and establish precedence for H2AK119μ1 in initiating Polycomb domain formation in a physiological context.

In female mammals, one of the two X chromosomes becomes inactivated during development by X-chromosome inactivation (XCI). Although Polycomb repressive complex (PRC) 1 and PRC2 have both been implicated in gene silencing, their exact roles in XCI during in vivo development have remained elusive. To this end, we have studied mouse embryos lacking either PRC1 or PRC2. Here we demonstrate that the loss of either PRC has a substantial impact on maintenance of gene silencing on the inactive X chromosome (Xi) in extra-embryonic tissues, with overlapping yet different genes affected, indicating potentially independent roles of the two complexes. Importantly, a lack of PRC1 does not affect PRC2/H3K27me3 accumulation and a lack of PRC2 does not impact PRC1/H2AK119ub1 accumulation on the Xi. Thus PRC1 and PRC2 contribute independently to the maintenance of XCI in early post-implantation extra-embryonic lineages, revealing that both Polycomb complexes can be directly involved and differently deployed in XCI. Polycomb repressive complexes 1 and 2 both regulate maintenance of X inactivation in extra-embryonic lineages of post-implantation embryos by affecting overlapping yet different genes, thus implying potentially independent roles for the two complexes.

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. X-chromosome inactivation (XCI) ensures dosage compensation between the sexes. Here the authors perform allele-specific single-cell RNA sequencing in differentiating mouse embryonic stem cells to provide a detailed profile of the onset of XCI.

Single-cell RNA sequencing of embryos can resolve the transcriptional landscape of development at unprecedented resolution. To date, single-cell RNA-sequencing studies of mammalian embryos have focused exclusively on eutherian species. Analysis of mammalian outgroups has the potential to identify deeply conserved lineage specification and pluripotency factors, and can extend our understanding of X dosage compensation. Metatherian (marsupial) mammals diverged from eutherians around 160 million years ago. They exhibit distinctive developmental features, including late implantation 1 and imprinted X chromosome inactivation 2 , which is associated with expression of the XIST -like noncoding RNA RSX 3 . Here we perform a single-cell RNA-sequencing analysis of embryogenesis and X chromosome inactivation in a marsupial, the grey short-tailed opossum ( Monodelphis domestica ). We resolve the developmental trajectory and transcriptional signatures of the epiblast, primitive endoderm and trophectoderm, and identify deeply conserved lineage-specific markers that pre-date the eutherian–marsupial divergence. RSX coating and inactivation of the X chromosome occurs early and rapidly. This observation supports the hypothesis that—in organisms with early X chromosome inactivation—imprinted X chromosome inactivation prevents biallelic X silencing. We identify XSR , an RSX  antisense transcript expressed from the active X chromosome, as a candidate for the regulator of imprinted X chromosome inactivation. Our datasets provide insights into the evolution of mammalian embryogenesis and X dosage compensation. Single-cell RNA-sequencing analysis of embryogenesis and X chromosome inactivation in the opossum ( Monodelphis domestica ) resolves the developmental trajectory of a marsupial, and sheds light on the evolution of embryogenesis in mammals.

X-chromosome inactivation (XCI) silences one X in female cells to balance sex-differences in X-dosage. A subset of X-linked genes escape XCI, but the extent to which this phenomenon occurs and how it varies across tissues and in a population is as yet unclear. To characterize incidence and variability of escape across individuals and tissues, we conducted a transcriptomic study of escape in adipose, skin, lymphoblastoid cell lines and immune cells in 248 healthy individuals exhibiting skewed XCI. We quantify XCI escape from a linear model of genes’ allelic fold-change and XIST -based degree of XCI skewing. We identify 62 genes, including 19 lncRNAs, with previously unknown patterns of escape. We find a range of tissue-specificity, with 11% of genes escaping XCI constitutively across tissues and 23% demonstrating tissue-restricted escape, including cell type-specific escape across immune cells of the same individual. We also detect substantial inter-individual variability in escape. Monozygotic twins share more similar escape than dizygotic twins, indicating that genetic factors may underlie inter-individual differences in escape. However, discordant escape also occurs within monozygotic co-twins, suggesting environmental factors also influence escape. Altogether, these data indicate that XCI escape is an under-appreciated source of transcriptional differences, and an intricate phenotype impacting variable trait expressivity in females.

During embryonic development, one of the two X chromosomes of a mammalian female cell is randomly inactivated by the X chromosome inactivation mechanism, which is mainly dependent on the regulation of the non-coding RNA X-inactive specific transcript at the X chromosome inactivation center. There are three proteins that are essential for X-inactive specific transcript to function properly: scaffold attachment factor-A, lamin B receptor, and SMRT- and HDAC-associated repressor protein. In addition, the absence of X-inactive specific transcript expression promotes tumor development. During the process of chromosome inactivation, some tumor suppressor genes escape inactivation of the X chromosome and thereby continue to play a role in tumor suppression. A well-functioning tumor suppressor gene on the idle X chromosome in women is one of the reasons they have a lower propensity to develop cancer than men, women thereby benefit from this enhanced tumor suppression. This review will explore the mechanism of X chromosome inactivation, discuss the relationship between X chromosome inactivation and tumorigenesis, and consider the consequent sex differences in cancer.

A male sex bias has emerged in the COVID-19 pandemic, fitting to the sex-biased pattern in other viral infections. Males are 2.84 times more often admitted to the ICU and mortality is 1.39 times higher as a result of COVID-19. Various factors play a role in this, and novel studies suggest that the gene-dose of Toll-Like Receptor (TLR) 7 could contribute to the sex-skewed severity. TLR7 is one of the crucial pattern recognition receptors for SARS-CoV-2 ssRNA and the gene-dose effect is caused by X chromosome inactivation (XCI) escape. Female immune cells with TLR7 XCI escape have biallelic TLR7 expression and produce more type 1 interferon (IFN) upon TLR7 stimulation. In COVID-19, TLR7 in plasmacytoid dendritic cells is one of the pattern recognition receptors responsible for IFN production and a delayed IFN response has been associated with immunopathogenesis and mortality. Here, we provide a hypothesis that females may be protected to some extend against severe COVID-19, due to the biallelic TLR7 expression, allowing them to mount a stronger and more protective IFN response early after infection. Studies exploring COVID-19 treatment via the TLR7-mediated IFN pathway should consider this sex difference. Various factors such as age, sex hormones and escape modulation remain to be investigated concerning the TLR7 gene-dose effect.