Search

Chromosomes are segmented into domains and compartments, but how these structures are spatially related in three dimensions (3D) is unclear. Here, we developed tools that directly extract 3D information from Hi-C experiments and integrate the data across time. With our "4DHiC" method, we use X chromosome inactivation (XCI) as a model to examine the time evolution of 3D chromosome architecture during large-scale changes in gene expression. Our modeling resulted in several insights. Both A/B and S1/S2 compartments divide the X chromosome into hemisphere-like structures suggestive of a spatial phase-separation. During the XCI, the X chromosome transits through A/B, S1/S2, and megadomain structures by undergoing only partial mixing to assume new structures. Interestingly, when an active X chromosome (Xa) is reorganized into an inactive X chromosome (Xi), original underlying compartment structures are not fully eliminated within the Xi superstructure. Our study affirms slow mixing dynamics in the inner chromosome core and faster dynamics near the surface where escapees reside. Once established, the Xa and Xi resemble glassy polymers where mixing no longer occurs. Finally, Xist RNA molecules initially reside within the A compartment but transition to the interface between the A and B hemispheres and then spread between hemispheres via both surface and core to establish the Xi., Competing Interests: Competing interest statement: J.T.L. is a cofounder of Translate Bio and Fulcrum, and is an advisor to Skyhawk Therapeutics.

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., Competing Interests: Declaration of interests J.T.L. is a cofounder of Translate Bio and Fulcrum Therapeutics and is an advisor to Skyhawk Therapeutics. B.N. is an inventor on patent applications related to the dTAG system (WO/2017/024318, WO/2017/024319, WO/2018/148440, and WO/2018/148443)., (Copyright © 2021 Elsevier Inc. All rights reserved.)

How allelic asymmetry is generated remains a major unsolved problem in epigenetics. Here we model the problem using X-chromosome inactivation by developing "BioRBP", an enzymatic RNA-proteomic method that enables probing of low-abundance interactions and an allelic RNA-depletion and -tagging system. We identify messenger RNA-decapping enzyme 1A (DCP1A) as a key regulator of Tsix, a noncoding RNA implicated in allelic choice through X-chromosome pairing. DCP1A controls Tsix half-life and transcription elongation. Depleting DCP1A causes accumulation of X-X pairs and perturbs the transition to monoallelic Tsix expression required for Xist upregulation. While ablating DCP1A causes hyperpairing, forcing Tsix degradation resolves pairing and enables Xist upregulation. We link pairing to allelic partitioning of CCCTC-binding factor (CTCF) and show that tethering DCP1A to one Tsix allele is sufficient to drive monoallelic Xist expression. Thus, DCP1A flips a bistable switch for the mutually exclusive determination of active and inactive Xs.

Long thought to be dispensable after establishing X chromosome inactivation (XCI), Xist RNA is now known to also maintain the inactive X (Xi). To what extent somatic X reactivation causes physiological abnormalities is an active area of inquiry. Here, we use multiple mouse models to investigate in vivo consequences. First, when Xist is deleted systemically in post-XCI embryonic cells using the Meox2-Cre driver, female pups exhibit no morbidity or mortality despite partial X reactivation. Second, when Xist is conditionally deleted in epithelial cells using Keratin14-Cre or in B cells using CD19-Cre, female mice have a normal life span without obvious illness. Third, when Xist is deleted in gut using Villin-Cre, female mice remain healthy despite significant X-autosome dosage imbalance. Finally, when the gut is acutely stressed by azoxymethane/dextran sulfate (AOM/DSS) exposure, both Xist -deleted and wild-type mice develop gastrointestinal tumors. Intriguingly, however, under prolonged stress, mutant mice develop larger tumors and have a higher tumor burden. The effect is female specific. Altogether, these observations reveal a surprising systemic tolerance to Xist loss but importantly reveal that Xist and XCI are protective to females during chronic stress., Competing Interests: Competing interest statement: J.T.L. is a cofounder of Translate Bio and Fulcrum Therapeutics, and an advisor to Skyhawk Therapeutics. To our knowledge, none of the companies is presently working on Xi reactivation.

X-chromosome inactivation triggers fusion of A/B compartments to inactive X (Xi)-specific structures known as S1 and S2 compartments. SMCHD1 then merges S1/S2s to form the Xi super-structure. Here, we ask how S1/S2 compartments form and reveal that Xist RNA drives their formation via recruitment of Polycomb repressive complex 1 (PRC1). Ablating Smchd1 in post-XCI cells unveils S1/S2 structures. Loss of SMCHD1 leads to trapping Xist in the S1 compartment, impairing RNA spreading into S2. On the other hand, depleting Xist, PRC1, or HNRNPK precludes re-emergence of S1/S2 structures, and loss of S1/S2 compartments paradoxically strengthens the partition between Xi megadomains. Finally, Xi-reactivation in post-XCI cells can be enhanced by depleting both SMCHD1 and DNA methylation. We conclude that Xist, PRC1, and SMCHD1 collaborate in an obligatory, sequential manner to partition, fuse, and direct self-association of Xi compartments required for proper spreading of Xist RNA.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The noncoding RNA Xist recruits silencing factors to the inactive X chromosome (Xi) and facilitates re-organization of Xi structure. Here, we examine the mouse epigenomic landscape of Xi and assess how Xist alters chromatin accessibility. Xist deletion triggers a gain of accessibility of select chromatin regions that is regulated by BRG1, an ATPase subunit of the SWI/SNF chromatin-remodeling complex. In vitro, RNA binding inhibits nucleosome-remodeling and ATPase activities of BRG1, while in cell culture Xist directly interacts with BRG1 and expels BRG1 from the Xi. Xist ablation leads to a selective return of BRG1 in cis, starting from pre-existing BRG1 sites that are free of Xist. BRG1 re-association correlates with cohesin binding and restoration of topologically associated domains (TADs) and results in the formation of de novo Xi 'superloops'. Thus, Xist binding inhibits BRG1's nucleosome-remodeling activity and results in expulsion of the SWI/SNF complex from the Xi.

X chromosome inactivation is an epigenetic dosage compensation mechanism in female mammals driven by the long noncoding RNA, Xist. Although recent genomic and proteomic approaches have provided a more global view of Xist's function, how Xist RNA localizes to the inactive X chromosome (Xi) and spreads in cis remains unclear. Here, we report that the CDKN1-interacting zinc finger protein CIZ1 is critical for localization of Xist RNA to the Xi chromosome territory. Stochastic optical reconstruction microscopy (STORM) shows a tight association of CIZ1 with Xist RNA at the single-molecule level. CIZ1 interacts with a specific region within Xist exon 7-namely, the highly repetitive Repeat E motif. Using genetic analysis, we show that loss of CIZ1 or deletion of Repeat E in female cells phenocopies one another in causing Xist RNA to delocalize from the Xi and disperse into the nucleoplasm. Interestingly, this interaction is exquisitely sensitive to CIZ1 levels, as overexpression of CIZ1 likewise results in Xist delocalization. As a consequence, this delocalization is accompanied by a decrease in H3K27me3 on the Xi. Our data reveal that CIZ1 plays a major role in ensuring stable association of Xist RNA within the Xi territory., Competing Interests: The authors declare no conflict of interest.

Extensive 3D folding is required to package a genome into the tiny nuclear space, and this packaging must be compatible with proper gene expression. Thus, in the well-hierarchized nucleus, chromosomes occupy discrete territories and adopt specific 3D organizational structures that facilitate interactions between regulatory elements for gene expression. The mammalian X chromosome exemplifies this structure-function relationship. Recent studies have shown that, upon X-chromosome inactivation, active and inactive X chromosomes localize to different subnuclear positions and adopt distinct chromosomal architectures that reflect their activity states. Here, we review the roles of long non-coding RNAs, chromosomal organizational structures and the subnuclear localization of chromosomes as they relate to X-linked gene expression.

The inactive X chromosome (Xi) serves as a model to understand gene silencing on a global scale. Here, we perform "identification of direct RNA interacting proteins" (iDRiP) to isolate a comprehensive protein interactome for Xist, an RNA required for Xi silencing. We discover multiple classes of interactors-including cohesins, condensins, topoisomerases, RNA helicases, chromatin remodelers, and modifiers-that synergistically repress Xi transcription. Inhibiting two or three interactors destabilizes silencing. Although Xist attracts some interactors, it repels architectural factors. Xist evicts cohesins from the Xi and directs an Xi-specific chromosome conformation. Upon deleting Xist, the Xi acquires the cohesin-binding and chromosomal architecture of the active X. Our study unveils many layers of Xi repression and demonstrates a central role for RNA in the topological organization of mammalian chromosomes., (Copyright © 2015, American Association for the Advancement of Science.)

CTCF is a master regulator that plays important roles in genome architecture and gene expression. How CTCF is recruited in a locus-specific manner is not fully understood. Evidence from epigenetic processes, such as X chromosome inactivation (XCI), indicates that CTCF associates functionally with RNA. Using genome-wide approaches to investigate the relationship between its RNA interactome and epigenomic landscape, here we report that CTCF binds thousands of transcripts in mouse embryonic stem cells, many in close proximity to CTCF's genomic binding sites. CTCF is a specific and high-affinity RNA-binding protein (Kd < 1 nM). During XCI, CTCF differentially binds the active and inactive X chromosomes and interacts directly with Tsix, Xite, and Xist RNAs. Tsix and Xite RNAs target CTCF to the X inactivation center, thereby inducing homologous X chromosome pairing. Our work elucidates one mechanism by which CTCF is recruited in a locus-specific manner and implicates CTCF-RNA interactions in long-range chromosomal interactions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

During meiosis, DNA damage response (DDR) proteins induce transcriptional silencing of unsynapsed chromatin, including the constitutively unsynapsed XY chromosomes in males. DDR proteins are also implicated in double strand break repair during meiotic recombination. Here, we address the function of the breast cancer susceptibility gene Brca1 in meiotic silencing and recombination in mice. Unlike in somatic cells, in which homologous recombination defects of Brca1 mutants are rescued by 53bp1 deletion, the absence of 53BP1 did not rescue the meiotic failure seen in Brca1 mutant males. Further, BRCA1 promotes amplification and spreading of DDR components, including ATR and TOPBP1, along XY chromosome axes and promotes establishment of pericentric heterochromatin on the X chromosome. We propose that BRCA1-dependent establishment of X-pericentric heterochromatin is critical for XY body morphogenesis and subsequent meiotic progression. In contrast, BRCA1 plays a relatively minor role in meiotic recombination, and female Brca1 mutants are fertile. We infer that the major meiotic role of BRCA1 is to promote the dramatic chromatin changes required for formation and function of the XY body., (© 2014 Broering et al.)

The Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation (XCI), the process by which mammals compensate for unequal numbers of sex chromosomes. During XCI, Xist coats the future inactive X chromosome (Xi) and recruits Polycomb repressive complex 2 (PRC2) to the X-inactivation centre (Xic). How Xist spreads silencing on a 150-megabases scale is unclear. Here we generate high-resolution maps of Xist binding on the X chromosome across a developmental time course using CHART-seq. In female cells undergoing XCI de novo, Xist follows a two-step mechanism, initially targeting gene-rich islands before spreading to intervening gene-poor domains. Xist is depleted from genes that escape XCI but may concentrate near escapee boundaries. Xist binding is linearly proportional to PRC2 density and H3 lysine 27 trimethylation (H3K27me3), indicating co-migration of Xist and PRC2. Interestingly, when Xist is acutely stripped off from the Xi in post-XCI cells, Xist recovers quickly within both gene-rich and gene-poor domains on a timescale of hours instead of days, indicating a previously primed Xi chromatin state. We conclude that Xist spreading takes distinct stage-specific forms. During initial establishment, Xist follows a two-step mechanism, but during maintenance, Xist spreads rapidly to both gene-rich and gene-poor regions.

Dosage compensation in mammals occurs at two levels. In addition to balancing X-chromosome dosage between males and females via X inactivation, mammals also balance dosage of Xs and autosomes. It has been proposed that X-autosome equalization occurs by upregulation of Xa (active X). To investigate mechanism, we perform allele-specific ChIP-seq for chromatin epitopes and analyze RNA-seq data. The hypertranscribed Xa demonstrates enrichment of active chromatin marks relative to autosomes. We derive predictive models for relationships among Pol II occupancy, active mark densities and gene expression, and we suggest that Xa upregulation involves increased transcription initiation and elongation. Enrichment of active marks on Xa does not scale proportionally with transcription output, a disparity explained by nonlinear quantitative dependencies among active histone marks, Pol II occupancy and transcription. Notably, the trend of nonlinear upregulation also occurs on autosomes. Thus, Xa upregulation involves combined increases of active histone marks and Pol II occupancy, without invoking X-specific dependencies between chromatin states and transcription.

X-chromosome inactivation is an epigenetic hallmark of mammalian development. Chromosome-wide regulation of the X-chromosome is essential in embryonic and germ cell development. In the male germline, the X-chromosome goes through meiotic sex chromosome inactivation, and the chromosome-wide silencing is maintained from meiosis into spermatids before the transmission to female embryos. In early female mouse embryos, X-inactivation is imprinted to occur on the paternal X-chromosome, representing the epigenetic programs acquired in both parental germlines. Recent advances revealed that the inactive X-chromosome in both females and males can be dissected into two elements: repeat elements versus unique coding genes. The inactive paternal X in female preimplantation embryos is reactivated in the inner cell mass of blastocysts in order to subsequently allow the random form of X-inactivation in the female embryo, by which both Xs have an equal chance of being inactivated. X-chromosome reactivation is regulated by pluripotency factors and also occurs in early female germ cells and in pluripotent stem cells, where X-reactivation is a stringent marker of naive ground state pluripotency. Here we summarize recent progress in the study of X-inactivation and X-reactivation during mammalian reproduction and development as well as in pluripotent stem cells.

The long noncoding Xist RNA inactivates one X chromosome in the female mammal. Current models posit that Xist induces silencing as it spreads along X and recruits Polycomb complexes. However, the mechanisms for Xist loading and spreading are currently unknown. Here, we define the nucleation center for Xist RNA and show that YY1 docks Xist particles onto the X chromosome. YY1 is a "bivalent" protein, capable of binding both RNA and DNA through different sequence motifs. Xist's exclusive attachment to the inactive X is determined by an epigenetically regulated trio of YY1 sites as well as allelic origin. Specific YY1-to-RNA and YY1-to-DNA contacts are required to load Xist particles onto X. YY1 interacts with Xist RNA through Repeat C. We propose that YY1 acts as adaptor between regulatory RNA and chromatin targets., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Acquisition of the pluripotent state coincides with epigenetic reprogramming of the X-chromosome. Female embryonic stem cells are characterized by the presence of two active X-chromosomes, cell differentiation by inactivation of one of the two Xs, and induced pluripotent stem cells by reactivation of the inactivated X-chromosome in the originating somatic cell. The tight linkage between X- and stem cell reprogramming occurs through pluripotency factors acting on noncoding genes of the X-inactivation center. This review article will discuss the latest advances in our understanding at the molecular level. Mouse embryonic stem cells provide a standard for defining the pluripotent ground state, which is characterized by low levels of the noncoding Xist RNA and the absence of heterochromatin marks on the X-chromosome. Human pluripotent stem cells, however, exhibit X-chromosome epigenetic instability that may have implications for their use in regenerative medicine. XIST RNA and heterochromatin marks on the X-chromosome indicate whether human pluripotent stem cells are developmentally 'naïve', with characteristics of the pluripotent ground state. X-chromosome status and determination thereof via noncoding RNA expression thus provide valuable benchmarks of the epigenetic quality of pluripotent stem cells, an important consideration given their enormous potential for stem cell therapy., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A large fraction of the mammalian genome is transcribed into long noncoding RNAs. The RNAs remain largely uncharacterized as the field awaits new technologies to aid functional analysis. Here, we describe a unique use of locked nucleic acids (LNAs) for studying nuclear long noncoding RNA, an RNA subclass that has been less amenable to traditional knockdown techniques. We target LNAs at Xist RNA and show displacement from the X chromosome with fast kinetics. Xist transcript stability is not affected. By targeting different Xist regions, we identify a localization domain and show that polycomb repressive complex 2 (PRC2) is displaced together with Xist. Thus, PRC2 depends on RNA for both initial targeting to and stable association with chromatin. H3K27-trimethyl marks and gene silencing remain stable. Time-course analysis of RNA relocalization suggests that Xist and PRC2 bind to different regions of the X at the same time but do not reach saturating levels immediately. Thus, LNAs provide a tool for studying an emerging class of regulatory RNA and offer a window of opportunity to target epigenetic modifications with possible therapeutic applications.

The X-linked region now known as the "X-inactivation center" (Xic) was once dominated by protein-coding genes but, with the rise of Eutherian mammals some 150-200 million years ago, became infiltrated by genes that produce long noncoding RNA (ncRNA). Some of the noncoding genes have been shown to play crucial roles during X-chromosome inactivation (XCI), including the targeting of chromatin modifiers to the X. The rapid establishment of ncRNA hints at a possible preference for long transcripts in some aspects of epigenetic regulation. This article discusses the role of RNA in XCI and considers the advantages RNA offers in delivering allelic, cis-limited, and locus-specific control. Unlike proteins and small RNAs, long ncRNAs are tethered to the site of transcription and effectively tag the allele of origin. Furthermore, long ncRNAs are drawn from larger sequence space than proteins and can mark a unique region in a complex genome. Thus, like their small RNA cousins, long ncRNAs may emerge as versatile and powerful regulators of the epigenome.

Mammals compensate for unequal X-linked gene dosages between the sexes by inactivating one X chromosome in the female. In marsupials and in the early mouse embryo, X chromosome inactivation (XCI) is imprinted to occur selectively on the paternal X chromosome (X(P)). The mechanisms and events underlying X(P) imprinting remain unclear. Here, we find that the imprinted X(P) can be functionally divided into two domains, one comprising traditional coding genes (genic) and the other comprising intergenic repetitive elements. X(P) repetitive element silencing occurs by the two-cell stage, does not require Xist, and occurs several divisions prior to genic silencing. In contrast, genic silencing initiates at the morula-to-blastocyst stage and absolutely requires Xist. Genes translocate into the presilenced repeat region as they are inactivated, whereas active genes remain outside. Thus, during the gamete-embryo transition, imprinted XCI occurs in two steps, with repeat silencing preceding genic inactivation. Nucleolar association may underlie the epigenetic asymmetry of X(P) and X(M). We hypothesize that transgenerational information (the imprint) is carried by repeats from the paternal germ line or that, alternatively, repetitive elements are silenced at the two-cell stage in a parent-of-origin-specific manner. Our model incorporates aspects of the so-called classical, de novo, and preinactivation hypotheses and suggests that Xist RNA functions relatively late during preimplantation mouse development.

Pluripotency of embryonic stem (ES) cells is controlled by defined transcription factors. During differentiation, mouse ES cells undergo global epigenetic reprogramming, as exemplified by X-chromosome inactivation (XCI) in which one female X chromosome is silenced to achieve gene dosage parity between the sexes. Somatic XCI is regulated by homologous X-chromosome pairing and counting, and by the random choice of future active and inactive X chromosomes. XCI and cell differentiation are tightly coupled, as blocking one process compromises the other and dedifferentiation of somatic cells to induced pluripotent stem cells is accompanied by X chromosome reactivation. Recent evidence suggests coupling of Xist expression to pluripotency factors occurs, but how the two are interconnected remains unknown. Here we show that Oct4 (also known as Pou5f1) lies at the top of the XCI hierarchy, and regulates XCI by triggering X-chromosome pairing and counting. Oct4 directly binds Tsix and Xite, two regulatory noncoding RNA genes of the X-inactivation centre, and also complexes with XCI trans-factors, Ctcf and Yy1 (ref. 17), through protein-protein interactions. Depletion of Oct4 blocks homologous X-chromosome pairing and results in the inactivation of both X chromosomes in female cells. Thus, we have identified the first trans-factor that regulates counting, and ascribed new functions to Oct4 during X-chromosome reprogramming.

To equalize X-chromosome dosages between the sexes, the female mammal inactivates one of her two X chromosomes. X-chromosome inactivation (XCI) is initiated by expression of Xist, a 17-kb noncoding RNA (ncRNA) that accumulates on the X in cis. Because interacting factors have not been isolated, the mechanism by which Xist induces silencing remains unknown. We discovered a 1.6-kilobase ncRNA (RepA) within Xist and identified the Polycomb complex, PRC2, as its direct target. PRC2 is initially recruited to the X by RepA RNA, with Ezh2 serving as the RNA binding subunit. The antisense Tsix RNA inhibits this interaction. RepA depletion abolishes full-length Xist induction and trimethylation on lysine 27 of histone H3 of the X. Likewise, PRC2 deficiency compromises Xist up-regulation. Therefore, RepA, together with PRC2, is required for the initiation and spread of XCI. We conclude that a ncRNA cofactor recruits Polycomb complexes to their target locus.

In mammals, dosage compensation is achieved by X-chromosome inactivation (XCI) in the female. The noncoding Xist gene initiates silencing of the X chromosome, whereas its antisense partner Tsix blocks silencing. The complementarity of Xist and Tsix RNAs has long suggested a role for RNA interference (RNAi). Here, we report that murine Xist and Tsix form duplexes in vivo. During XCI, the duplexes are processed to small RNAs (sRNAs), most likely on the active X (Xa) in a Dicer-dependent manner. Deleting Dicer compromises sRNA production and derepresses Xist. Furthermore, without Dicer, Xist RNA cannot accumulate and histone 3 lysine 27 trimethylation is blocked on the inactive X (Xi). The defects are partially rescued by truncating Tsix. Thus, XCI and RNAi intersect, down-regulating Xist on Xa and spreading silencing on Xi.

Dosage compensation, the mechanism by which organisms equalize the relative gene expression of dimorphic sex chromosomes, requires action of a diverse range of epigenetic mechanisms. The mammalian form, 'named X-chromosome inactivation' (XCI), involves silencing of one X chromosome in the female cell and regulation by genes that make noncoding RNAs (ncRNA). With large-scale genomic and transcriptome studies pointing to a crucial role for noncoding elements in organizing the epigenome, XCI emerges as a major paradigm and a focus of active research worldwide. With more surprising twists, recent advances point to the significance of RNA-directed chromatin change, chromosomal trans-interactions, nuclear organization, and evolutionary change. These findings have impacted our understanding of general gene regulation and are discussed herein.

X-chromosome inactivation (XCI) ensures the equality of X-chromosome dosages in male and female mammals by silencing one X in the female. To achieve the mutually exclusive designation of active X (Xa) and inactive X (Xi), the process necessitates that two Xs communicate in trans through homologous pairing. Pairing depends on a 15-kb region within the genes Tsix and Xite. Here, we dissect molecular requirements and find that pairing can be recapitulated by 1- to 2-kb subfragments of Tsix or Xite with little sequence similarity. However, a common denominator among them is the presence of the protein Ctcf, a chromatin insulator that we find to be essential for pairing. By contrast, the Ctcf-interacting partner, Yy1 (ref. 8), is not required. Pairing also depends on transcription. Transcriptional inhibition prevents new pair formation but does not perturb existing pairs. The kinetics suggest a pairing half-life of <1 h. We propose that pairing requires Ctcf binding and co-transcriptional activity of Tsix and Xite.

In mammalian females, two X chromosomes are epigenetically distinguished as active and inactive chromosomes to balance X-linked gene dosages between males and females. How the Xs are maintained differently in the same nucleus remains unknown. Here, we demonstrate that the inactive X (Xi) is targeted to a distinct nuclear compartment following pairing with its homologous partner. During mid-to-late S phase, 80%-90% of Xi contact the nucleolus and reside within a Snf2h-enriched ring. Autosomes carrying ectopic X-inactivation center sequences are also targeted to the perinucleolar compartment. Deleting Xist results in a loss of nucleolar association and an inability to maintain Xi heterochromatin, leading to Xi reactivation at the single gene level. We propose that the Xi must continuously visit the perinucleolar compartment to maintain its epigenetic state. These data raise a mechanism by which chromatin states can be replicated by spatial and temporal separation in the nucleus.

In mammals, inactivation of one X chromosome in the female equalizes gene dosages between XX females and XY males. Two noncoding loci, Tsix and Xite, together regulate X chromosome fate by controlling homologous chromosome pairing, counting, and mutually exclusive choice. Following choice, the asymmetry of Xite and Tsix expression drives divergent chromosome fates, but how this pattern becomes established is currently unknown. Although no proven trans-acting factors have been identified, a likely candidate is Ctcf, a chromatin insulator with essential function in autosomal imprinting. Here, we search for trans-factors and identify Yy1 as a required cofactor for Ctcf. Paired Ctcf-Yy1 elements are highly clustered within the counting/choice and imprinting domain of Tsix. A deficiency of Yy1 leads to aberrant Tsix and Xist expression, resulting in a deficit of male and female embryos. Yy1 and Ctcf associate through specific protein-protein interactions and together transactivate Tsix. We propose that the Ctcf-Yy1-Tsix complex functions as a key component of the X chromosome binary switch.

X-chromosome inactivation (XCI) evolved in mammals to deal with X-chromosome dosage imbalance between the XX female and the XY male. In eutherian mammals, random XCI of the soma requires a master regulatory locus known as the 'X-inactivation center' (XIC/Xic), wherein lies the noncoding XIST/Xist silencer RNA and its regulatory antisense Tsix gene. By contrast, marsupial XCI is imprinted to occur on the paternal X chromosome. To determine whether marsupials and eutherians share the XIC-driven mechanism, we search for the sequence equivalents in the genome of the South American opossum, Monodelphis domestica. Positional cloning and bioinformatic analysis reveal several interesting findings. First, protein-coding genes that flank the eutherian XIC are well-conserved in M. domestica, as well as in chicken, frog, and pufferfish. However, in M. domestica we fail to identify any recognizable XIST or TSIX equivalents. Moreover, cytogenetic mapping shows a surprising break in synteny with eutherian mammals and other vertebrates. Therefore, during the evolution of the marsupial X chromosome, one or more rearrangements broke up an otherwise evolutionarily conserved block of vertebrate genes. The failure to find XIST/TSIX in M. domestica may suggest that the ancestral XIC is too divergent to allow for detection by current methods. Alternatively, the XIC may have arisen relatively late in mammalian evolution, possibly in eutherians with the emergence of random XCI. The latter argues that marsupial XCI does not require XIST and opens the search for alternative mechanisms of dosage compensation.

In mammals, few DNA replication origins have been identified. Although there appears to be an association between origins and epigenetic regulation, their underlying link to monoallelic gene expression remains unclear. Here, we identify novel origins of DNA replication (ORIs) within the X-inactivation center (Xic). We analyze 86 kb of the Xic using an unbiased approach and find an unexpectedly large number of functional ORIs. Although there has been a tight correlation between ORIs and CpG islands, we find that ORIs are not restricted to CpG islands and there is no dependence on transcriptional activity. Interestingly, these ORIs colocalize to important genetic elements or genes involved in X-chromosome inactivation. One prominent ORI maps to the imprinting center and to a domain within Tsix known to be required for X-chromosome counting and choice. Location and/or activity of ORIs appear to be modulated by removal of specific Xic elements. These data provide a foundation for testing potential relationships between DNA replication and epigenetic regulation in future studies.

In mammals, the X and Y chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during prophase I in the male germline, but their status thereafter is currently unclear. An abundance of X-linked spermatogenesis genes has spawned the view that the X must be active . On the other hand, the idea that the imprinted paternal X of the early embryo may be preinactivated by MSCI suggests that silencing may persist longer . To clarify this issue, we establish a comprehensive X-expression profile during mouse spermatogenesis. Here, we discover that the X and Y occupy a novel compartment in the postmeiotic spermatid and adopt a non-Rabl configuration. We demonstrate that this postmeiotic sex chromatin (PMSC) persists throughout spermiogenesis into mature sperm and exhibits epigenetic similarity to the XY body. In the spermatid, 87% of X-linked genes remain suppressed postmeiotically, while autosomes are largely active. We conclude that chromosome-wide X silencing continues from meiosis to the end of spermiogenesis, and we discuss implications for proposed mechanisms of imprinted X-inactivation.

Mammalian X inactivation turns off one female X chromosome to enact dosage compensation between XX and XY individuals. X inactivation is known to be regulated in cis by Xite, Tsix, and Xist, but in principle the two Xs must also be regulated in trans to ensure mutually exclusive silencing. Here, we demonstrate that interchromosomal pairing mediates this communication. Pairing occurs transiently at the onset of X inactivation and is specific to the X-inactivation center. Deleting Xite and Tsix perturbs pairing and counting/choice, whereas their autosomal insertion induces de novo X-autosome pairing. Ectopic X-autosome interactions inhibit endogenous X-X pairing and block the initiation of X-chromosome inactivation. Thus, Tsix and Xite function both in cis and in trans. We propose that Tsix and Xite regulate counting and mutually exclusive choice through X-X pairing.

Loci associated with noncoding RNAs have important roles in X-chromosome inactivation (XCI), the dosage compensation mechanism by which one of two X chromosomes in female cells becomes transcriptionally silenced. The Xs start out as epigenetically equivalent chromosomes, but XCI requires a cell to treat two identical X chromosomes in completely different ways: One X chromosome must remain transcriptionally active while the other becomes repressed. In the embryo of eutherian mammals, the choice to inactivate the maternal or paternal X chromosome is random. The fact that the Xs always adopt opposite fates hints at the existence of a trans-sensing mechanism to ensure the mutually exclusive silencing of one of the two Xs. This paper highlights recent evidence supporting a model for mutually exclusive choice that involves homologous chromosome pairing and the placement of asymmetric chromatin marks on the two Xs.

In mammals, X-inactivation establishes X-chromosome dosage parity between males and females. How X-chromosome counting regulates this process remains elusive, because neither the hypothesized inactivation "blocking factor" nor the required cis-elements have been defined. Here, a mouse knockout and transgenic analysis identified DNA sequences within the noncoding Tsix and Xite genes as numerators. Homozygous deficiency of Tsix resulted in "chaotic choice" and a variable number of inactive X's, whereas overdosage of Tsix/Xite inhibited X-inactivation. Thus, counting was affected by specific Tsix/Xite mutations, suggesting that counting is genetically separable from but molecularly coupled to choice. The mutations affect XX and XY cells differently, demonstrating that counting and choice are regulated not by one "blocking factor," but by both a "blocking" and a "competence" factor.

In mammals, sex is determined by differential inheritance of a pair of dimorphic chromosomes: the gene-rich X chromosome and the gene-poor Y chromosome. To balance the unequal X-chromosome dosage between the XX female and XY male, mammals have adopted a unique form of dosage compensation in which one of the two X chromosomes is inactivated in the female. This mechanism involves a complex, highly coordinated sequence of events and is a very different strategy from those used by other organisms, such as the fruitfly and the worm. Why did mammals choose an inactivation mechanism when other, perhaps simpler, means could have been used? Recent data offer a compelling link between ontogeny and phylogeny. Here, we propose that X-chromosome inactivation and imprinting might have evolved from an ancient genome-defence mechanism that silences unpaired DNA.

X chromosome inactivation silences one of two X chromosomes in the mammalian female cell and is controlled by a binary switch that involves interactions between Xist and Tsix, a sense-antisense pair of noncoding genes. On the future active X chromosome, Tsix expression suppresses Xist upregulation, while on the future inactive X chromosome, Tsix repression is required for Xist-mediated chromosome silencing. Thus, understanding the binary switch mechanism depends on ascertaining how Tsix expression is regulated. Here we have taken an unbiased approach toward identifying Tsix regulatory elements within the X chromosome inactivation center. First, we defined the major Tsix promoter and found that it cannot fully recapitulate the developmental dynamics of Tsix expression, indicating a requirement for additional regulatory elements. We then delineated two enhancers, one classical enhancer mapping upstream of Tsix and a bipartite enhancer that flanks the major Tsix promoter. These experiments revealed the intergenic transcription element Xite as an enhancer of Tsix and the repeat element DXPas34 as a component of the bipartite enhancer. Each enhancer contains DNase I-hypersensitive sites and appears to confer developmental specificity to Tsix expression. Characterization of these enhancers will facilitate the identification of trans-acting regulatory factors for X chromosome counting and choice.

In mammals, dosage compensation ensures equal X-chromosome expression between males (XY) and females (XX) by transcriptionally silencing one X chromosome in XX embryos. In the prevailing view, the XX zygote inherits two active X chromosomes, one each from the mother and father, and X inactivation does not occur until after implantation. Here, we report evidence to the contrary in mice. We find that one X chromosome is already silent at zygotic gene activation (2-cell stage). This X chromosome is paternal in origin and exhibits a gradient of silencing. Genes close to the X-inactivation centre show the greatest degree of inactivation, whereas more distal genes show variable inactivation and can partially escape silencing. After implantation, imprinted silencing in extraembryonic tissues becomes globalized and more complete on a gene-by-gene basis. These results argue that the XX embryo is in fact dosage compensated at conception along much of the X chromosome. We propose that imprinted X inactivation results from inheritance of a pre-inactivated X chromosome from the paternal germ line.

In classical Mendelian inheritance, each parent donates a set of chromosomes to its offspring so that maternally and paternally encoded information is expressed equally. The phenomena of X-chromosome inactivation (XCI) and autosomal imprinting in mammals violate this dogma of genetic equality. In XCI, one of the two female X chromosomes is silenced to equalize X-linked gene dosage between XX and XY individuals. In genomic imprinting, parental marks determine which of the embryo's two autosomal alleles will be expressed. Although XCI and imprinting appear distinct, molecular evidence now shows that they share a surprising number of features. Among them are cis-acting control centers, long-distance regulation and differential DNA methylation. Perhaps one of the most intriguing similarities between XCI and imprinting has been their association with noncoding and antisense RNAs. Very recent data also suggest the common involvement of histone modifications and chromatin-associated factors such as CTCF. Collectively, the evidence suggests that XCI and genomic imprinting may have a common origin. Here, I hypothesize that the need for X-linked dosage compensation was a major driving force in the evolution of genomic imprinting in mammals. I propose that imprinting was first fixed on the X chromosome for XCI and subsequently acquired by autosomes.

Allelic expression differences contribute to phenotypic variation. In X chromosome inactivation (XCI), unfavorable XCI ratios promote X-linked disease penetrance in females. During XCI, one X is randomly silenced by Xist. X chromosome choice is determined by asymmetric expression of Tsix whose antisense action represses Xist. Here, we discover a cis element in the mouse X-inactivation center that regulates Tsix. Xite harbors intergenic transcription start sites and DNaseI hypersensitive sites with allelic differences. At the onset of XCI, deleting Xite downregulates Tsix in cis and skews XCI ratios, suggesting that Xite promotes Tsix persistence on the active X. Truncating Xite RNA is inconsequential, indicating that Xite action does not require intact transcripts. We propose that allele-specific Xite action promotes Tsix asymmetry and generates X chromosome inequality. Therefore, Xite is a candidate for the Xce, the classical modifier of XCI ratios.

In dosage compensation of female mammals, the accumulation of Xist RNA initiates silencing of one X-chromosome. Xist action is repressed by the antisense gene, Tsix, whose full-length RNA product is complementary to Xist RNA in mice. While previous work showed that Tsix transcription blocks the accumulation of Xist RNA, it is still unclear whether this repression requires the antisense RNA product or whether the antisense transcriptional movement is sufficient. A better understanding of potential mechanisms requires elucidation of Tsix RNA structure and determination of Tsix RNA copy number relative to that of Xist RNA. Previous work indicated that at least some of murine Tsix is spliced and that human TSIX truncates within the 3' end of XIST. Here, further characterization and quantitation of murine Tsix RNA reveal three new findings: first, in undifferentiated embryonic stem cells, Tsix RNA is present at 10-100-fold molar excess over Xist RNA. Second, only 30-60% of Tsix RNA is spliced at known exon-intron junctions. The nearly equal abundance of spliced and unspliced species leaves open possible roles for both isoforms. Finally, Tsix is spliced heterogeneously at the 5' end and most detectable splice variants exhibit only a 1.9 kb region of complementarity between sense and antisense RNAs. Implications for Tsix's possible mechanisms of action are discussed.

X-chromosome inactivation leads to divergent fates for two homologous chromosomes. Whether one X remains active or becomes silenced depends on the activity of Xist, a gene expressed only from the inactive X and whose RNA product 'paints' the X in cis. Recent work argues that Xist RNA itself is the acting agent for initiating the silencing step. Xist RNA contains separable domains for RNA localization and chromosome silencing. While no Xist RNA-interacting factors have been identified, a growing collection of chromatin alterations have been identified on the inactive X, including variant histone H2A composition and histone H3 methylation. Some or all of these changes may be critical for chromosome-wide silencing. As none of the silencing proteins identified so far is unique to X chromosome inactivation, the specificity must partly reside in Xist RNA whose spread along the X orchestrates general silencing factors for this specific task.

In mammals, X-inactivation silences one of two female X chromosomes. Silencing depends on the noncoding gene, Xist (inactive X-specific transcript), and is blocked by the antisense gene, Tsix. Deleting the choice/imprinting center in Tsix affects X-chromosome selection. Here, we identify the insulator and transcription factor, CTCF, as a candidate trans-acting factor for X-chromosome selection. The choice/imprinting center contains tandem CTCF binding sites that function in an enhancer-blocking assay. In vitro binding is reduced by CpG methylation and abolished by including non-CpG methylation. We postulate that Tsix and CTCF together establish a regulatable epigenetic switch for X-inactivation.

In mammals, X-chromosome inactivation (XCI) ensures equal expression of X-linked genes in XX and XY individuals by transcriptionally silencing one X-chromosome in female cells. In this review, we discuss an imprinted form of X-inactivation in which the paternal X (Xp) is preferentially silenced. Believed to be the ancestral mechanism of dosage compensation in mammals, imprinted X-inactivation can still be observed in modern-day marsupials and in the extraembryonic tissues of some eutherians such as the mouse. Recent experiments have addressed the nature of the gametic imprint and focused on the regulatory interaction between the noncoding RNA gene, Xist, and its antisense partner, Tsix. Our review of the literature has inspired an unconventional view of imprinted XCI in mice. First, the evidence strongly argues that imprinted XCI is inabsolute, so that a stochastic number of extraembryonic cells escape imprinting. Second, contrary to conventional thinking, we would like to consider the possibility that the paternal X might actually be transmitted to the zygote as a pre-inactivated chromosome. In this model, the gamete initiates and establishes imprinted XCI, while the zygote maintains the pre-established pattern of gametic inactivation. Finally, we hypothesize that the inabsolute nature of imprinting is caused by imperfect zygotic maintenance. We propose that the mouse represents a transitional stage in the evolution of random XCI from an absolutely imprinted mechanism.

In 1961, Mary Lyon first put forth the hypothesis that one X chromosome is inactivated in each cell of the female mammal. As we enter the new millennium and complete 40 years of study, the field of X-inactivation is rich with ideas and many contrasting viewpoints. This review will focus on the random form of X-inactivation and present the latest views on its mechanism. Much attention has been focused on the genetic parsing of X-chromosome counting, choice, silencing and maintenance. It is now known that counting is functionally distinct from choice and that initiation and establishment of silencing are distinct from maintenance. Since Xist's seminal discovery 10 years ago, significant progress has been made towards understanding its function. Required only for initiation and establishment, Xist must act within a narrow developmental window, but its precise mode of action remains elusive. The ongoing search for Xist RNA-binding factors and effector proteins for silencing has led to members of the macroH2A family of histone variants. Finally, the recent discovery of Tsix implicates regulation of Xist expression by an antisense mechanism. Required for choice but not counting, Tsix blocks Xist RNA accumulation and hence blocks initiation of silencing on the future active X.

We recently characterized a genomic region located upstream of the mouse pre-T cell receptor alpha (pTa) gene, which controls pTa expression in pre-T cells. We now report an unexpected homology between this region and a region in the mouse X chromosome inactivation center between the 3' end of the Xist gene and the start of an antisense transcript Tsix. The homology is extended over 4 kb of genomic sequence split by an expanded repeat region and is observed only in the mouse, not in the rat. Despite high sequence similarity to the pTa transcriptional enhancer, the homologous X chromosome fragment appears to have lost its enhancer activity. These data underscore the complex organization of the mouse genome and, in particular, of the X chromosome inactivation center., (Copyright 2000 Academic Press.)

The onset of X inactivation coincides with accumulation of Xist RNA along the future inactive X chromosome. A recent hypothesis proposed that accumulation is initiated by a promoter switch within Xist. In this hypothesis, an upstream promoter (P(0)) produces an unstable transcript, while the known downstream promoter (P(1)) produces a stable RNA. To test this hypothesis, we examined expression and half-life of Xist RNA produced from an Xist transgene lacking P(0) but retaining P(1). We confirm the previous finding that P(0) is dispensable for Xist expression in undifferentiated cells and that P(1) can be used in both undifferentiated and differentiated cells. Herein, we show that Xist RNA initiated at P(1) is unstable and does not accumulate. Further analysis indicates that the transcriptional boundary at P(0) does not represent the 5' end of a distinct Xist isoform. Instead, P(0) is an artifact of cross-amplification caused by a pseudogene of the highly expressed ribosomal protein S12 gene Rps12. Using strand-specific techniques, we find that transcription upstream of P(1) originates from the DNA strand opposite Xist and represents the 3' end of the antisense Tsix RNA. Thus, these data do not support the existence of a P(0) promoter and suggest that mechanisms other than switching of functionally distinct promoters control the up-regulation of Xist.

Dosage compensation in mammals occurs by X inactivation, a silencing mechanism regulated in cis by the X inactivation center (Xic). In response to developmental cues, the Xic orchestrates events of X inactivation, including chromosome counting and choice, initiation, spread, and establishment of silencing. It remains unclear what elements make up the Xic. We previously showed that the Xic is contained within a 450-kb sequence that includes Xist, an RNA-encoding gene required for X inactivation. To characterize the Xic further, we performed deletional analysis across the 450-kb region by yeast-artificial-chromosome fragmentation and phage P1 cloning. We tested Xic deletions for cis inactivation potential by using a transgene (Tg)-based approach and found that an 80-kb subregion also enacted somatic X inactivation on autosomes. Xist RNA coated the autosome but skipped the Xic Tg, raising the possibility that X chromosome domains escape inactivation by excluding Xist RNA binding. The autosomes became late-replicating and hypoacetylated on histone H4. A deletion of the Xist 5' sequence resulted in the loss of somatic X inactivation without abolishing Xist expression in undifferentiated cells. Thus, Xist expression in undifferentiated cells can be separated genetically from somatic silencing. Analysis of multiple Xic constructs and insertion sites indicated that long-range Xic effects can be generalized to different autosomes, thereby supporting the feasibility of a Tg-based approach for studying X inactivation.

In mammals, the X chromosome is uniquely capable of complete inactivation. Research in the past two years has validated the long-held hypothesis that the 'X-inactivation center' (Xic) controls events of X inactivation and that its resident gene Xist is not only required but is at least partially responsible for the cis-restriction of X inactivation. Progress has also been made in identifying genes within the Xic. Although Xist remains the only known required element, evidence now suggests that a separate element for X counting must exist and that the Xic may be entirely contained within a 450 kb sequence. This small region may be sufficient for both initiation and establishment of X inactivation.

X inactivation results in inactivation of one X chromosome to compensate for gene dosage differences between mammalian females and males. It requires the X-inactivation center (Xic) and Xist in cis. We report that introducing 450 kb of murine Xic/Xist sequences onto autosomes activates female dosage compensation in male ES cells. Xist is induced upon differentiation and can be expressed from both endogenous and ectopic loci, suggesting that elements for counting and choosing Xs are present in the transgene. Differentiating transgenic ES cells undergo excessive cell death. Postnatally, Xist is expressed only from the transgene. Ectopic Xist RNA structurally associates with the autosome and may inactivate a marker gene in cis. These results argue that the Xic is contained within 450 kb and that these sequences are sufficient for chromosome counting, choosing, and initiation of X inactivation.

We describe the construction and characterization of a human X-chromosome-specific yeast artificial chromosome (YAC) library. Starting with 60 micrograms of hybrid cell line genomic DNA, we generated over 150,000 recombinants, over 90% of which range from 150 to 500 kb. From these recombinants, 3300 human-positive YACs (representing coverage of 4.5 X chromosomes) were identified by genomic human DNA hybridization. Mapping of random clones revealed that they are derived from the X chromosome in a regionally unbiased fashion, and screening with single-copy X-chromosome probes has repeatedly produced YACs from the library. By determining the frequency of YAC clones containing both hamster and human repetitive sequences, we estimated that approximately 11% of clones contain discontiguous sequences. Taken together, the low cocloning rate, the unbiased coverage, and a consistent recovery of YACs using specific X-chromosome markers indicate that YAC technology can be used for extensive cloning and mapping purposes. Because a certain amount of genomic rearrangement is present in YAC libraries, chromosome walking must be undertaken with a degree of caution.