Xist ribonucleoproteins promote female sex-biased autoimmunity.

Autoimmune diseases disproportionately affect females more than males. The XX sex chromosome complement is strongly associated with susceptibility to autoimmunity. Xist long non-coding RNA (lncRNA) is expressed only in females to randomly inactivate one of the two X chromosomes to achieve gene dosage compensation. Here, we show that the Xist ribonucleoprotein (RNP) complex comprising numerous autoantigenic components is an important driver of sex-biased autoimmunity. Inducible transgenic expression of a non-silencing form of Xist in male mice introduced Xist RNP complexes and sufficed to produce autoantibodies. Male SJL/J mice expressing transgenic Xist developed more severe multi-organ pathology in a pristane-induced lupus model than wild-type males. Xist expression in males reprogrammed T and B cell populations and chromatin states to more resemble wild-type females. Human patients with autoimmune diseases displayed significant autoantibodies to multiple components of XIST RNP. Thus, a sex-specific lncRNA scaffolds ubiquitous RNP components to drive sex-biased immunity.

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.

XIST loss impairs mammary stem cell differentiation and increases tumorigenicity through Mediator hyperactivation.

X inactivation (XCI) is triggered by upregulation of XIST, which coats the chromosome in cis, promoting formation of a heterochromatic domain (Xi). XIST role beyond initiation of XCI is only beginning to be elucidated. Here, we demonstrate that XIST loss impairs differentiation of human mammary stem cells (MaSCs) and promotes emergence of highly tumorigenic and metastatic carcinomas. On the Xi, XIST deficiency triggers epigenetic changes and reactivation of genes overlapping Polycomb domains, including Mediator subunit MED14. MED14 overdosage results in increased Mediator levels and hyperactivation of the MaSC enhancer landscape and transcriptional program, making differentiation less favorable. We further demonstrate that loss of XIST and Xi transcriptional instability is common among human breast tumors of poor prognosis. We conclude that XIST is a gatekeeper of human mammary epithelium homeostasis, thus unveiling a paradigm in the control of somatic cell identity with potential consequences for our understanding of gender-specific malignancies.

Xist nucleates local protein gradients to propagate silencing across the X chromosome.

The lncRNA Xist forms ∼50 diffraction-limited foci to transcriptionally silence one X chromosome. How this small number of RNA foci and interacting proteins regulate a much larger number of X-linked genes is unknown. We show that Xist foci are locally confined, contain ∼2 RNA molecules, and nucleate supramolecular complexes (SMACs) that include many copies of the critical silencing protein SPEN. Aggregation and exchange of SMAC proteins generate local protein gradients that regulate broad, proximal chromatin regions. Partitioning of numerous SPEN molecules into SMACs is mediated by their intrinsically disordered regions and essential for transcriptional repression. Polycomb deposition via SMACs induces chromatin compaction and the increase in SMACs density around genes, which propagates silencing across the X chromosome. Our findings introduce a mechanism for functional nuclear compartmentalization whereby crowding of transcriptional and architectural regulators enables the silencing of many target genes by few RNA molecules.

The Implication of Early Chromatin Changes in X Chromosome Inactivation.

During 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.

SMCHD1 Merges Chromosome Compartments and Assists Formation of Super-Structures on the Inactive X.

Mammalian chromosomes are partitioned into A/B compartments and topologically associated domains (TADs). The inactive X (Xi) chromosome, however, adopts a distinct conformation without evident compartments or TADs. Here, through exploration of an architectural protein, structural-maintenance-of-chromosomes hinge domain containing 1 (SMCHD1), we probe how the Xi is reconfigured during X chromosome inactivation. A/B compartments are first fused into "S1" and "S2" compartments, coinciding with Xist spreading into gene-rich domains. SMCHD1 then binds S1/S2 compartments and merges them to create a compartment-less architecture. Contrary to current views, TADs remain on the Xi but in an attenuated state. Ablating SMCHD1 results in a persistent S1/S2 organization and strengthening of TADs. Furthermore, loss of SMCHD1 causes regional defects in Xist spreading and erosion of heterochromatic silencing. We present a stepwise model for Xi folding, where SMCHD1 attenuates a hidden layer of Xi architecture to facilitate Xist spreading.

G-quadruplex folding in Xist RNA antagonizes PRC2 activity for stepwise regulation of X chromosome inactivation.

How Polycomb repressive complex 2 (PRC2) is regulated by RNA remains an unsolved problem. Although PRC2 binds G-tracts with the potential to form RNA G-quadruplexes (rG4s), whether rG4s fold extensively in vivo and whether PRC2 binds folded or unfolded rG4 are unknown. Using the X-inactivation model in mouse embryonic stem cells, here we identify multiple folded rG4s in Xist RNA and demonstrate that PRC2 preferentially binds folded rG4s. High-affinity rG4 binding inhibits PRC2's histone methyltransferase activity, and stabilizing rG4 in vivo antagonizes H3 at lysine 27 (H3K27me3) enrichment on the inactive X chromosome. Surprisingly, mutagenizing the rG4 does not affect PRC2 recruitment but promotes its release and catalytic activation on chromatin. H3K27me3 marks are misplaced, however, and gene silencing is compromised. Xist-PRC2 complexes become entrapped in the S1 chromosome compartment, precluding the required translocation into the S2 compartment. Thus, Xist rG4 folding controls PRC2 activity, H3K27me3 enrichment, and the stepwise regulation of chromosome-wide gene silencing.

Imprinted X chromosome inactivation at the gamete-to-embryo transition.

In mammals, dosage compensation involves two parallel processes: (1) X inactivation, which equalizes X chromosome dosage between males and females, and (2) X hyperactivation, which upregulates the active X for X-autosome balance. The field currently favors models whereby dosage compensation initiates "de novo" during mouse development. Here, we develop "So-Smart-seq" to revisit the question and interrogate a comprehensive transcriptome including noncoding genes and repeats in mice. Intriguingly, de novo silencing pertains only to a subset of Xp genes. Evolutionarily older genes and repetitive elements demonstrate constitutive Xp silencing, adopt distinct signatures, and do not require Xist to initiate silencing. We trace Xp silencing backward in developmental time to meiotic sex chromosome inactivation in the male germ line and observe that Xm hyperactivation is timed to Xp silencing on a gene-by-gene basis. Thus, during the gamete-to-embryo transition, older Xp genes are transmitted in a "pre-inactivated" state. These findings have implications for the evolution of imprinting.

Distal and proximal cis-regulatory elements sense X chromosome dosage and developmental state at the Xist locus.

Developmental genes such as Xist, which initiates X chromosome inactivation, are controlled by complex cis-regulatory landscapes, which decode multiple signals to establish specific spatiotemporal expression patterns. Xist integrates information on X chromosome dosage and developmental stage to trigger X inactivation in the epiblast specifically in female embryos. Through a pooled CRISPR screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist at the onset of random X inactivation. Chromatin profiling reveals that X-dosage controls the promoter-proximal region, while differentiation cues activate several distal enhancers. The strongest distal element lies in an enhancer cluster associated with a previously unannotated Xist-enhancing regulatory transcript, which we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. With this study, we start to disentangle how multiple, functionally distinct regulatory elements interact to generate complex expression patterns in mammals.

Noncanonical imprinting sustains embryonic development and restrains placental overgrowth.

Genomic imprinting regulates parental origin-dependent monoallelic gene expression. It is mediated by either germline differential methylation of DNA (canonical imprinting) or oocyte-derived H3K27me3 (noncanonical imprinting) in mice. Depletion of Eed, an essential component of Polycomb repressive complex 2, results in genome-wide loss of H3K27me3 in oocytes, which causes loss of noncanonical imprinting (LOI) in embryos. Although Eed maternal KO (matKO) embryos show partial lethality after implantation, it is unknown whether LOI itself contributes to the developmental phenotypes of these embryos, which makes it unclear whether noncanonical imprinting is developmentally relevant. Here, by combinatorial matKO of Xist, a noncanonical imprinted gene whose LOI causes aberrant transient maternal X-chromosome inactivation (XCI) at preimplantation, we show that prevention of the transient maternal XCI greatly restores the development of Eed matKO embryos. Moreover, we found that the placentae of Eed matKO embryos are remarkably enlarged in a manner independent of Xist LOI. Heterozygous deletion screening of individual autosomal noncanonical imprinted genes suggests that LOI of the Sfmbt2 miRNA cluster chromosome 2 miRNA cluster (C2MC), solute carrier family 38 member 4 (Slc38a4), and Gm32885 contributes to the placental enlargement. Taken together, our study provides evidence that Xist imprinting sustains embryonic development and that autosomal noncanonical imprinting restrains placental overgrowth.

Nascent RNA scaffolds contribute to chromosome territory architecture and counter chromatin compaction.

Nuclear chromosomes transcribe far more RNA than required to encode protein. Here we investigate whether non-coding RNA broadly contributes to cytological-scale chromosome territory architecture. We develop a procedure that depletes soluble proteins, chromatin, and most nuclear RNA from the nucleus but does not delocalize XIST, a known architectural RNA, from an insoluble chromosome "scaffold." RNA-seq analysis reveals that most RNA in the nuclear scaffold is repeat-rich, non-coding, and derived predominantly from introns of nascent transcripts. Insoluble, repeat-rich (C0T-1) RNA co-distributes with known scaffold proteins including scaffold attachment factor A (SAF-A), and distribution of these components inversely correlates with chromatin compaction in normal and experimentally manipulated nuclei. We further show that RNA is required for SAF-A to interact with chromatin and for enrichment of structurally embedded "scaffold attachment regions" prevalent in euchromatin. Collectively, the results indicate that long nascent transcripts contribute a dynamic structural role that promotes the open architecture of active chromosome territories.

Balancing cohesin eviction and retention prevents aberrant chromosomal interactions, Polycomb-mediated repression, and X-inactivation.

Depletion of architectural factors globally alters chromatin structure but only modestly affects gene expression. We revisit the structure-function relationship using the inactive X chromosome (Xi) as a model. We investigate cohesin imbalances by forcing its depletion or retention using degron-tagged RAD21 (cohesin subunit) or WAPL (cohesin release factor). Cohesin loss disrupts the Xi superstructure, unveiling superloops between escapee genes with minimal effect on gene repression. By contrast, forced cohesin retention markedly affects Xi superstructure, compromises spreading of Xist RNA-Polycomb complexes, and attenuates Xi silencing. Effects are greatest at distal chromosomal ends, where looping contacts with the Xist locus are weakened. Surprisingly, cohesin loss creates an Xi superloop, and cohesin retention creates Xi megadomains on the active X chromosome. Across the genome, a proper cohesin balance protects against aberrant inter-chromosomal interactions and tempers Polycomb-mediated repression. We conclude that a balance of cohesin eviction and retention regulates X inactivation and inter-chromosomal interactions across the genome.

A disproportionate impact of G9a methyltransferase deficiency on the X chromosome.

G9a is a histone methyltransferase responsible for the dimethylation of histone H3 at lysine 9 (H3K9me2). G9a plays key roles in transcriptional silencing of developmentally regulated genes, but its role in X-chromosome inactivation (XCI) has been under debate. Here, we uncover a female-specific function of G9a and demonstrate that deleting G9a has a disproportionate impact on the X chromosome relative to the rest of the genome. G9a deficiency causes a failure of XCI and female-specific hypersensitivity to drug inhibition of H3K9me2. We show that G9a interacts with Tsix and Xist RNAs, and that competitive inhibition of the G9a-RNA interaction recapitulates the XCI defect. During XCI, Xist recruits G9a to silence X-linked genes on the future inactive X. In parallel on the future Xa, Tsix recruits G9a to silence Xist in cis Thus, RNA tethers G9a for allele-specific targeting of the H3K9me2 modification and the G9a-RNA interaction is essential for XCI.

Noncoding RNAs and epigenetic mechanisms during X-chromosome inactivation.

In mammals, the process of X-chromosome inactivation ensures equivalent levels of X-linked gene expression between males and females through the silencing of one of the two X chromosomes in female cells. The process is established early in development and is initiated by a unique locus, which produces a long noncoding RNA, Xist. The Xist transcript triggers gene silencing in cis by coating the future inactive X chromosome. It also induces a cascade of chromatin changes, including posttranslational histone modifications and DNA methylation, and leads to the stable repression of all X-linked genes throughout development and adult life. We review here recent progress in our understanding of the molecular mechanisms involved in the initiation of Xist expression, the propagation of the Xist RNA along the chromosome, and the cis-elements and trans-acting factors involved in the maintenance of the repressed state. We also describe the diverse strategies used by nonplacental mammals for X-chromosome dosage compensation and highlight the common features and differences between eutherians and metatherians, in particular regarding the involvement of long noncoding RNAs.

A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary.

cis-Regulatory communication is crucial in mammalian development and is thought to be restricted by the spatial partitioning of the genome in topologically associating domains (TADs). Here, we discovered that the Xist locus is regulated by sequences in the neighboring TAD. In particular, the promoter of the noncoding RNA Linx (LinxP) acts as a long-range silencer and influences the choice of X chromosome to be inactivated. This is independent of Linx transcription and independent of any effect on Tsix, the antisense regulator of Xist that shares the same TAD as Linx. Unlike Tsix, LinxP is well conserved across mammals, suggesting an ancestral mechanism for random monoallelic Xist regulation. When introduced in the same TAD as Xist, LinxP switches from a silencer to an enhancer. Our study uncovers an unsuspected regulatory axis for X chromosome inactivation and a class of cis-regulatory effects that may exploit TAD partitioning to modulate developmental decisions.

Progress toward understanding chromosome silencing by Xist RNA.

The X inactive-specific transcript (Xist) gene is the master regulator of X chromosome inactivation in mammals. Xist produces a long noncoding (lnc)RNA that accumulates over the entire length of the chromosome from which it is transcribed, recruiting factors to modify underlying chromatin and silence X-linked genes in cis Recent years have seen significant progress in identifying important functional elements in Xist RNA, their associated RNA-binding proteins (RBPs), and the downstream pathways for chromatin modification and gene silencing. In this review, we summarize progress in understanding both how these pathways function in Xist-mediated silencing and the complex interplay between them.

Xist Deletional Analysis Reveals an Interdependency between Xist RNA and Polycomb Complexes for Spreading along the Inactive X.

During X-inactivation, Xist RNA spreads along an entire chromosome to establish silencing. However, the mechanism and functional RNA elements involved in spreading remain undefined. By performing a comprehensive endogenous Xist deletion screen, we identify Repeat B as crucial for spreading Xist and maintaining Polycomb repressive complexes 1 and 2 (PRC1/PRC2) along the inactive X (Xi). Unexpectedly, spreading of these three factors is inextricably linked. Deleting Repeat B or its direct binding partner, HNRNPK, compromises recruitment of PRC1 and PRC2. In turn, ablating PRC1 or PRC2 impairs Xist spreading. Therefore, Xist and Polycomb complexes require each other to propagate along the Xi, suggesting a positive feedback mechanism between RNA initiator and protein effectors. Perturbing Xist/Polycomb spreading causes failure of de novo Xi silencing, with partial compensatory downregulation of the active X, and also disrupts topological Xi reconfiguration. Thus, Repeat B is a multifunctional element that integrates interdependent Xist/Polycomb spreading, silencing, and changes in chromosome architecture.

lncRNA-Induced Spread of Polycomb Controlled by Genome Architecture, RNA Abundance, and CpG Island DNA.

Long noncoding RNAs (lncRNAs) cause Polycomb repressive complexes (PRCs) to spread over broad regions of the mammalian genome. We report that in mouse trophoblast stem cells, the Airn and Kcnq1ot1 lncRNAs induce PRC-dependent chromatin modifications over multi-megabase domains. Throughout the Airn-targeted domain, the extent of PRC-dependent modification correlated with intra-nuclear distance to the Airn locus, preexisting genome architecture, and the abundance of Airn itself. Specific CpG islands (CGIs) displayed characteristics indicating that they nucleate the spread of PRCs upon exposure to Airn. Chromatin environments surrounding Xist, Airn, and Kcnq1ot1 suggest common mechanisms of PRC engagement and spreading. Our data indicate that lncRNA potency can be tightly linked to lncRNA abundance and that within lncRNA-targeted domains, PRCs are recruited to CGIs via lncRNA-independent mechanisms. We propose that CGIs that autonomously recruit PRCs interact with lncRNAs and their associated proteins through three-dimensional space to nucleate the spread of PRCs in lncRNA-targeted domains.

X-chromosome inactivation: a historic topic that's still hot.

The last edition of the X-chromosome inactivation (XCI) meeting was held as an EMBO workshop in Berlin on 19-22 June 2023. The conference took place at the Harnack-haus in the Dahlem district, birthplace of the first modern research campus, where notable scientists such as Lise Meitner, Hans Krebs and, briefly, Albert Einstein conducted their research. This special edition, also accessible online, was organized by Rafael Galupa (Centre for Integrative Biology of Toulouse, France), Joost Gribnau (Erasmus MC Rotterdam, The Netherlands), Claire Rougeulle (Université Paris Cité/CNRS, Epigenetics and Cell Fate Center, Paris, France), Edda Schulz (Max Planck Institute for Molecular Genetics, Berlin, Germany) and James Turner (The Francis Crick Institute, London, UK). Originally scheduled for 2021, to commemorate the 60th anniversary of Mary Lyon's hypothesis on X-chromosome inactivation in mammals and the 30th anniversary of XIST/Xist discovery, the meeting had to be postponed because of the COVID-19 pandemic. Seven years after the latest XCI meeting in London, the enthusiasm and expectations of the community were at their highest, bringing together over 160 scientists from around the world to share and discuss their research. Eighty posters and more than 40 talks were presented at this event, in a collegial and collaborative atmosphere. A historical session and several breakout discussions were also organized, as well as the now traditional boat trip, all thanks to great organization. Here, we debrief readers on this fantastic conference.

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.