Maintenance Of X-chromosome Inactivation Research Articles

Altered X-chromosome inactivation predisposes to autoimmunity.

In mammals, males and females show marked differences in immune responses. Males are globally more sensitive to infectious diseases, while females are more susceptible to systemic autoimmunity. X-chromosome inactivation (XCI), the epigenetic mechanism ensuring the silencing of one X in females, may participate in these sex biases. We perturbed the expression of the trigger of XCI, the noncoding RNA Xist, in female mice. This resulted in reactivation of genes on the inactive X, including members of the Toll-like receptor 7 (TLR7) signaling pathway, in monocyte/macrophages and dendritic and B cells. Consequently, female mice spontaneously developed inflammatory signs typical of lupus, including anti-nucleic acid autoantibodies, increased frequencies of age-associated and germinal center B cells, and expansion of monocyte/macrophages and dendritic cells. Mechanistically, TLR7 signaling is dysregulated in macrophages, leading to sustained expression of target genes upon stimulation. These findings provide a direct link between maintenance of XCI and female-biased autoimmune manifestations and highlight altered XCI as a cause of autoimmunity.

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.

Impaired dynamic X-chromosome inactivation maintenance in T cells is a feature of spontaneous murine SLE that is exacerbated in female-biased models

ObjectiveSystemic lupus erythematosus (SLE) is a highly female-biased systemic autoimmune disease, but the molecular basis for this female bias remains incompletely elucidated. B and T lymphocytes from patients with SLE and female-biased mouse models of SLE exhibit features of epigenetic dysregulation on the X chromosome which may contribute to this strong female bias. We therefore examined the fidelity of dynamic X-chromosome inactivation maintenance (dXCIm) in the pathogenesis of two murine models of spontaneous lupus—NZM2328 and MRL/lpr—with disparate levels of female-bias to determine whether impaired dXCIm contributes to the female bias of disease. MethodsCD23+ B cells and CD3+ T cells were purified from age-matched C57BL/6 (B6), MRL/lpr, and NZM2328 male and female mice, activated in vitro, and processed for Xist RNA fluorescence in situ hybridization, H3K27me3 immunofluorescence imaging, qPCR, and RNA sequencing analyses. ResultsThe dynamic relocalization of Xist RNA and the canonical heterochromatin mark, H3K27me3, to the inactive X chromosome was preserved in CD23+ B cells, but impaired in activated CD3+ T cells from the MRL/lpr model (p < 0.01 vs. B6), and even more impaired in the heavily female-biased NZM2328 model (p < 0.001 vs. B6; p < 0.05 vs. MRL/lpr). RNAseq of activated T cells from NZM2328 mice revealed the female-biased upregulation of 32 X-linked genes distributed broadly across the X chromosome, many of which have roles in immune function. Many genes encoding Xist RNA-interacting proteins were also differentially expressed and predominantly downregulated, which may account for the observed mislocalization of Xist RNA to the inactive X chromosome. ConclusionsAlthough evident in T cells from both the MRL/lpr and NZM2328 models of spontaneous SLE, impaired dXCIm is more severe in the heavily female-biased NZM2328 model. The aberrant X-linked gene dosage in female NZM2328 mice may contribute towards the development of female-biased immune responses in SLE-prone hosts. These findings provide important insights into the epigenetic mechanisms contributing to female-biased autoimmunity.

Unusual X chromosome inactivation maintenance in female alveolar type 2 cells is correlated with increased numbers of X-linked escape genes and sex-biased gene expression.

Sex differences exist for many lung pathologies, including COVID-19 and pulmonary fibrosis, but the mechanistic basis for this remains unclear. Alveolar type 2 cells (AT2s), which play a key role in alveolar lung regeneration, express the X-linked Ace2 gene that has roles in lung repair and SARS-CoV-2 pathogenesis, suggesting that X chromosome inactivation (XCI) in AT2s might impact sex-biased lung pathology. Here we investigate XCI maintenance and sex-specific gene expression profiles using male and female AT2s. Remarkably, the inactive X chromosome (Xi) lacks robust canonical Xist RNA "clouds" and less enrichment of heterochromatic modifications in human and mouse AT2s. We demonstrate that about 68% of expressed X-linked genes in mouse AT2s, including Ace2, escape XCI. There are genome-wide expression differences between male and female AT2s, likely influencing both lung physiology and pathophysiologic responses. These studies support a renewed focus on AT2s as a potential contributor to sex-biased differences in lung disease.

Polycomb repressive complexes 1 and 2 are each essential for maintenance of X inactivation in extra-embryonic lineages.

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.

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.

HiMoRNA: A Comprehensive Database of Human lncRNAs Involved in Genome-Wide Epigenetic Regulation.

Long non-coding RNAs (lncRNAs) play an important role in genome regulation. Specifically, many lncRNAs interact with chromatin, recruit epigenetic complexes and in this way affect large-scale gene expression programs. However, the experimental data about lncRNA-chromatin interactions is still limited. The majority of experimental protocols do not provide any insight into the mechanics of lncRNA-based genome-wide epigenetic regulation. Here we present the HiMoRNA (Histone-Modifying RNA) database, a resource containing correlated lncRNA–epigenetic changes in specific genomic locations genome-wide. HiMoRNA integrates a large amount of multi-omics data to characterize the effects of lncRNA on epigenetic modifications and gene expression. The current release of HiMoRNA includes more than five million associations in humans for ten histone modifications in multiple genomic loci and 4145 lncRNAs. HiMoRNA provides a user-friendly interface to facilitate browsing, searching and retrieving of lncRNAs associated with epigenetic profiles of various chromatin loci. Analysis of the HiMoRNA data suggests that several lncRNA including JPX might be involved not only in regulation of XIST locus but also in direct establishment or maintenance of X-chromosome inactivation. We believe that HiMoRNA is a convenient and valuable resource that can provide valuable biological insights and greatly facilitate functional annotation of lncRNAs.

The dynamic epigenetic regulation of the inactive X chromosome in healthy human B cells is dysregulated in lupus patients

Systemic lupus erythematous (SLE) is a female-predominant disease characterized by autoimmune B cells and pathogenic autoantibody production. Individuals with two or more X chromosomes are at increased risk for SLE, suggesting that X-linked genes contribute to the observed sex bias of this disease. To normalize X-linked gene expression between sexes, one X in female cells is randomly selected for transcriptional silencing through X-chromosome inactivation (XCI), resulting in allele-specific enrichment of epigenetic modifications, including histone methylation and the long noncoding RNA XIST/Xist on the inactive X (Xi). As we have previously shown that epigenetic regulation of the Xi in female lymphocytes from mice is unexpectedly dynamic, we used RNA fluorescence in situ hybridization and immunofluorescence to profile epigenetic features of the Xi at the single-cell level in human B cell subsets from pediatric and adult SLE patients and healthy controls. Our data reveal that abnormal XCI maintenance in B cells is a feature of SLE. Using single-cell and bulk-cell RNA sequencing datasets, we found that X-linked immunity genes escape XCI in specific healthy human B cell subsets and that human SLE B cells exhibit aberrant expression of X-linked genes and XIST RNA interactome genes. Our data reveal that mislocalized XIST RNA, coupled with a dramatic reduction in heterochromatic modifications at the Xi in SLE, predispose for aberrant X-linked gene expression from the Xi, thus defining a genetic and epigenetic pathway that affects X-linked gene expression in human SLE B cells and likely contributes to the female bias in SLE.

Loss of &lt;i&gt;XIST&lt;/i&gt; 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.

A small-molecule screen reveals novel modulators of MeCP2 and X-chromosome inactivation maintenance

BackgroundRett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MeCP2) gene. While MeCP2 mutations are lethal in most males, females survive birth but show severe neurological defects. Because X-chromosome inactivation (XCI) is a random process, approximately 50% of the cells silence the wild-type (WT) copy of the MeCP2 gene. Thus, reactivating the silent WT copy of MeCP2 could provide therapeutic intervention for RTT.MethodsToward this goal, we screened ~ 28,000 small-molecule compounds from several libraries using a MeCP2-luciferase reporter cell line and cortical neurons from a MeCP2-EGFP mouse model. We used gain/increase of luminescence or fluorescence as a readout of MeCP2 reactivation and tested the efficacy of these drugs under different drug regimens, conditions, and cellular contexts.ResultsWe identified inhibitors of the JAK/STAT pathway as XCI-reactivating agents, both by in vitro and ex vivo assays. In particular, we show that AG-490, a Janus Kinase 2 (JAK2) kinase inhibitor, and Jaki, a pan JAK/STAT inhibitor, are capable of reactivating MeCP2 from the inactive X chromosome, in different cellular contexts.ConclusionsOur results suggest that inhibition of the JAK/STAT pathway is a new potential pathway to reinstate MeCP2 gene expression as an efficient RTT treatment.

Investigating nuclear organization of the inactive X in female lymphocytes

Abstract Autoimmune diseases are devastating, incurable disorders that affect over 20 million people in the US, and many of these disorders have a striking female bias. The genetic basis for this sex-bias arises from the X chromosome. Female mammals, with two X chromosomes, utilize the epigenetic process of X Chromosome Inactivation (XCI), which transcriptionally silences one X. Expression of the long noncoding RNA Xist from the inactive X (Xi) reorganizes the Xi into a compact, bipartite structure, and targets the Xi to the nuclear periphery in somatic cells. The paradigm of XCI maintenance in somatic cells is stable association of Xist RNA and heterochromatic marks on the Xi, which function to maintain the Xi structure. Recently, we found a dynamic mechanism of XCI maintenance in female lymphocytes. In naïve female B cells, Xist RNA is not localized to the Xi, but after stimulation Xist RNA and heterochromatin marks return to the Xi. How this dynamic XCI process impacts Xi structure in lymphocytes is unknown, including which factors regulate Xi nuclear organization in this system. Using a novel allele-specific imaging system, we will present new findings on the compaction and organization of the Xi in lymphocytes. Additionally, we are investigating the regulatory roles of chromatin organization and nuclear structure proteins for lymphocyte-specific XCI maintenance, and how these factors regulate X-linked gene expression. Together, these results underscore the importance of Xi structure for the fidelity of XCI in female lymphocytes.

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.

XIST Recruits BRCA1 to Ubiquitinate macroH2A for X Chromosome Inactivation in Proliferating Breast Cells

X chromosome inactivation (XCI) in females needs to be maintained in newly synthesized chromosome during each cell proliferation cycle, and improper XCI may reactivate X chromosome oncogenes which links to carcinogenesis. However, mechanisms maintaining XCI during cell proliferation remain unclear. During XCI, PRC1 canonical or PRC1-like proteins are assumed to ubiquitinate H2A, which forms a reversibly silenced chromatin, while during the maintenance stage of XCI, macroH2As instead of H2As are enriched in the inactivated X chromosome to form a stable silencing status. Recently studies suggested that Breast cancer type 1 (BRCA1), a tumor suppressor commonly mutated in breast cancer cells, can catalyze macroH2A ubiquitination, which prompted us to hypothesize that BRCA1 instead of PRC1 mediates macroH2A ubiquitination and maintains XCI during cell proliferation. Based on RNA immunoprecipitation and confocal microscopic imaging, we found that BRCA1 directly interacts with XIST, a key LncRNA mediating XCI, which colocalizes with mH2A in inactivated X chromosome. Loss of either XIST or BRCA1 decreased the ubiquitination level of macroH2A, suppressing DNA methylation of X chromosome genes, which leads to failure of heterochromatin formation in proliferating breast cancer and mammary gland epithelial cells. Furthermore, this ubiquitination by BRCA1 was not replaceable by PRC1. Using domain-specific deletion, the interaction between XIST and BRCA1 was narrowed to exon 2-7. Our data showed that BRCA1 interacts with XIST to ubiquitinate the macroH2A on X chromosome, which is required for XCI maintenance in mammary gland epithelial cells during cell proliferation. These data provide a novel mechanism explaining the frequent BRCA1 mutations occurred during breast carcinogenesis.

When the balance is broken: X-linked gene dosage from two X chromosomes and female-biased autoimmunity.

Women and men exhibit differences in innate and adaptive immunity, and women are more susceptible to numerous autoimmune disorders. Two or more X chromosomes increases the risk for some autoimmune diseases, and increased expression of some X-linked immune genes is frequently observed in female lymphocytes from autoimmune patients. Evidence from mouse models of autoimmunity also supports the idea that increased expression of X-linked genes is a feature of female-biased autoimmunity. Recent studies have begun to elucidate the correlation between abnormal X-chromosome inactivation (XCI), an essential mechanism female somatic cells use to equalize X-linked gene dosage between the sexes, and autoimmunity in lymphocytes. In this review, we highlight research describing overexpression of X-linked immunity-related genes and female-biased autoimmunity in both humans and mouse models, and make connections with our recent work elucidating lymphocyte-specific mechanisms of XCI maintenance that become altered in lupus patients.

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome.

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a XistΔ:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

X-chromosome inactivation maintenance is perturbed in T cells from lupus mouse models and human lupus patients

Abstract Sex differences exist with immune responses, yet females are more susceptible to autoimmune and inflammatory disorders. One important biological factor underlying this sex bias is the X-chromosome, which is enriched for immunity-related genes. Female mammals use X-Chromosome Inactivation (XCI) to equalize gene expression between the sexes, which generates a transcriptionally silent inactive X-chromosome enriched with heterochromatic modifications and the long noncoding RNA Xist. We have discovered that mature naïve T cells are missing Xist RNA and heterochromatin marks on the inactive X (Xi), and these marks return to most cells with in vitro stimulation. During T cell development in the thymus, Xist RNA is not localized to the Xi and heterochromatin marks are progressively lost. Xist RNA returns to the Xi in mature T cell subsets between days 2 and 3 of in vitro activation. During lupus disease progression in the female-biased mouse model NZB/W F1, Xist RNA signals aberrantly appear in naïve T cells and Xist RNA becomes mis-localized from the Xi in activated T cells. These abnormal XIST RNA localization patterns are also observed in activated T cells from pediatric lupus patients in disease remission. Using next-generation sequencing, we found significant overexpression of X-linked genes across the X in female SLE T cells compared to healthy females. We propose that the dynamic mechanism of XCI maintenance female lymphocytes predisposes the Xi to become partially reactivated and to overexpress immunity-related genes. Our work provides the first mechanistic evidence for the female-specific enhanced immune responses and increased susceptibility for autoimmunity.

Altered X-chromosome inactivation in T cells may promote sex-biased autoimmune diseases.

Systemic lupus erythematosus (SLE) is an autoimmune disorder that predominantly affects women and is driven by autoreactive T cell-mediated inflammation. It is known that individuals with multiple X-chromosomes are at increased risk for developing SLE; however, the mechanisms underlying this genetic basis are unclear. Here, we use single cell imaging to determine the epigenetic features of the inactive X (Xi) in developing thymocytes, mature T cell subsets, and T cells from SLE patients and mice. We show that Xist RNA and heterochromatin modifications transiently reappear at the Xi and are missing in mature single positive T cells. Activation of mature T cells restores Xist RNA and heterochromatin marks simultaneously back to the Xi. Notably, X-chromosome inactivation (XCI) maintenance is altered in T cells of SLE patients and late-stage-disease NZB/W F1 female mice, and we show that X-linked genes are abnormally upregulated in SLE patient T cells. SLE T cells also have altered expression of XIST RNA interactome genes, accounting for perturbations of Xi epigenetic features. Thus, abnormal XCI maintenance is a feature of SLE disease, and we propose that Xist RNA localization at the Xi could be an important factor for maintaining dosage compensation of X-linked genes in T cells.

X-Chromosome Inactivation: A Crossroads Between Chromosome Architecture and Gene Regulation.

In somatic nuclei of female therian mammals, the two X chromosomes display very different chromatin states: One X is typically euchromatic and transcriptionally active, and the other is mostly silent and forms a cytologically detectable heterochromatic structure termed the Barr body. These differences, which arise during female development as a result of X-chromosome inactivation (XCI), have been the focus of research for many decades. Initial approaches to define the structure of the inactive X chromosome (Xi) and its relationship to gene expression mainly involved microscopy-based approaches. More recently, with the advent of genomic techniques such as chromosome conformation capture, molecular details of the structure and expression of the Xi have been revealed. Here, we review our current knowledge of the 3D organization of the mammalian X-chromosome chromatin and discuss its relationship with gene activity in light of the initiation, spreading, and maintenance of XCI, as well as escape from gene silencing.

Loss of Xist RNA from the inactive X during B cell development is restored in a dynamic YY1-dependent two-step process in activated B cells.

X-chromosome inactivation (XCI) in female lymphocytes is uniquely regulated, as the inactive X (Xi) chromosome lacks localized Xist RNA and heterochromatin modifications. Epigenetic profiling reveals that Xist RNA is lost from the Xi at the pro-B cell stage and that additional heterochromatic modifications are gradually lost during B cell development. Activation of mature B cells restores Xist RNA and heterochromatin to the Xi in a dynamic two-step process that differs in timing and pattern, depending on the method of B cell stimulation. Finally, we find that DNA binding domain of YY1 is necessary for XCI in activated B cells, as ex-vivo YY1 deletion results in loss of Xi heterochromatin marks and up-regulation of X-linked genes. Ectopic expression of the YY1 zinc finger domain is sufficient to restore Xist RNA localization during B cell activation. Together, our results indicate that Xist RNA localization is critical for maintaining XCI in female lymphocytes, and that chromatin changes on the Xi during B cell development and the dynamic nature of YY1-dependent XCI maintenance in mature B cells predisposes X-linked immunity genes to reactivation.

Unusual maintenance of X chromosome inactivation predisposes female lymphocytes for increased expression from the inactive X

Females have a greater immunological advantage than men, yet they are more prone to autoimmune disorders. The basis for this sex bias lies in the X chromosome, which contains many immunity-related genes. Female mammals use X chromosome inactivation (XCI) to generate a transcriptionally silent inactive X chromosome (Xi) enriched with heterochromatic modifications and XIST/Xist RNA, which equalizes gene expression between the sexes. Here, we examine the maintenance of XCI in lymphocytes from females in mice and humans. Strikingly, we find that mature naïve T and B cells have dispersed patterns of XIST/Xist RNA, and they lack the typical heterochromatic modifications of the Xi. In vitro activation of lymphocytes triggers the return of XIST/Xist RNA transcripts and some chromatin marks (H3K27me3, ubiquitin-H2A) to the Xi. Single-cell RNA FISH analysis of female T cells revealed that the X-linked immunity genes CD40LG and CXCR3 are biallelically expressed in some cells. Using knockout and knockdown approaches, we find that Xist RNA-binding proteins, YY1 and hnRNPU, are critical for recruitment of XIST/Xist RNA back to the Xi. Furthermore, we examined B cells from patients with systemic lupus erythematosus, an autoimmune disorder with a strong female bias, and observed different XIST RNA localization patterns, evidence of biallelic expression of immunity-related genes, and increased transcription of these genes. We propose that the Xi in female lymphocytes is predisposed to become partially reactivated and to overexpress immunity-related genes, providing the first mechanistic evidence to our knowledge for the enhanced immunity of females and their increased susceptibility for autoimmunity.