regulation of x-chromosome inactivation by the x …zider.free.fr/papers/paper (14).pdfabstract |...

Sex chromosome dimorphism leads to a genetic imbal-ance between the homogametic and heterogametic sexes, which mammals compensate for by inactivating one of the two X chromosomes during female development. Although this chromosome-wide silencing process was originally described more than 50 years ago (TIMELINE), the underlying molecular mechanisms remain poorly understood. One of the most intriguing aspects of X-chromosome inactivation (XCI) is that two homol-ogous X chromosomes are differently treated within the same nucleus. How the inactive state is set up and faithfully transmitted through cell division remains a central question for which answers are only now begin-ning to emerge. This Review will focus on the recent progress that has been made in our understanding of the initiation of XCI, as well as the reversibility of the inactive state during specific stages of development and in the context of reprogramming experiments.

In mice, which have been the favoured model for XCI studies, there are two waves of XCI, the first being imprinted (paternal XCI) and the second random. Imprinted inactivation of the paternal X chromosome (Xp) is initiated shortly after fertilization. This silent state is maintained in extra-embryonic tissues but lost in the inner cell mass (ICM), which gives rise to the embryo proper. Shortly after this, random inactivation of either the maternal X chromosome (Xm) or the Xp is initiated in the cells of the ICM. In vitro differentia-tion of mouse embryonic stem cells (ESCs), which are

derived from the ICM and have two active X chromo-somes, is accompanied by random XCI and has been extensively used to dissect the early events underly-ing this process. As such, the regulation of random XCI is more thoroughly understood and is the main focus of the Review, although imprinted XCI is also discussed.

A growing number of new molecular players have been implicated in XCI over recent years. How they function together to control XCI and how this fits in with — or challenges — the original views of the pro-cess remains largely unclear. The aim of this Review is to examine the role of these recently identified molecu-lar players in the context of the initial historical notions underlying the process of XCI; that is, the concepts of counting, choice and sensing/competence (BOX 1). We will focus primarily on the X-inactivation centre (Xic) and the key non-coding X-inactivation specific transcript (Xist) it produces, which represents the trig-ger for chromosome-wide silencing. We first explain briefly how the Xic was functionally and physically identified. We then describe how Xist underlies some, but not all, of the functions attributed to the Xic and review our current knowledge on the increasingly complex regulatory network controlling Xist expres-sion. Finally, we discuss random and imprinted XCI in the context of mouse development and the recent insights that XCI has brought into reprogramming processes.

Mammalian Developmental Epigenetics Group, Unit of Genetics and Developmental Biology, Institut Curie, CNRS UMR3215, INSERM U934, Paris F‑75248, France.*These authors contributed equally to this work.Correspondence to E.H. e‑mail: [email protected]:10.1038/nrg2987

Homogametic and heterogametic sexesIn species with sexual dimorphism, the sex that can produce two different types of gametes (X and Y or Z and W) is called heterogametic, whereas the sex that can produce only one type of gamete (X or Z) is called homogametic.

ImprintedEpigenetic marking of a locus on the basis of its parental origin, which can result in differential expression of the paternal and maternal alleles in specific tissues or developmental stages.

Regulation of X‑chromosome inactivation by the X‑inactivation centreSandrine Augui*, Elphège P. Nora* and Edith Heard

Abstract | X-chromosome inactivation (XCI) ensures dosage compensation in mammals and is a paradigm for allele-specific gene expression on a chromosome-wide scale. Important insights have been made into the developmental dynamics of this process. Recent studies have identified several cis- and trans-acting factors that regulate the initiation of XCI via the X-inactivation centre. Such studies have shed light on the relationship between XCI and pluripotency. They have also revealed the existence of dosage-dependent activators that trigger XCI when more than one X chromosome is present, as well as possible mechanisms underlying the monoallelic regulation of this process. The recent discovery of the plasticity of the inactive state during early development, or during cloning, and induced pluripotency have also contributed to the X chromosome becoming a gold standard in reprogramming studies.

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Polycomb group proteins(PcG proteins). A class of proteins — originally described in Drosophila melanogaster — that form large complexes and maintain the stable and heritable repression of several genes throughout development.

Trithorax group proteins(TrxG proteins). A class of proteins — originally described in Drosophila melanogaster — that form large complexes and maintain the stable and heritable expression of several genes throughout development.

The X‑inactivation centreEarly studies of XCI patterns in mouse embryos or embryonic cells that carried translocated or trun-cated X chromosomes revealed the existence of a single X-linked locus, the Xic, that needs to be physi-cally linked to a chromosome to trigger its inactivation1 (FIG. 1). Random XCI is only triggered in cells with at least two Xic-bearing chromosomes2, suggesting that the two copies of the Xic are able to potentiate each other in trans, a phenomenon that has been referred to as com-petence, or sensing3–5 (BOX 1). In XX cells, either one of the two X chromosomes will be inactivated, a process known as choice (BOX 1). The autosomal ploidy of a cell (the number of sets of autosomes that is contains) also seems to affect the number of X chromosomes that will be inactivated, a phenomenon known as counting (BOX 1). The precise mechanisms underlying these pro-cesses are only now being unravelled and recent data sug-gest that they are highly interconnected, both genetically and molecularly.

Xist RNA triggers cis‑inactivationThe Xic harbours the Xist gene6–8 (FIG. 1B), which pro-duces a non-coding RNA (ncRNA) that is retained in the nucleus and that, in its spliced form, can coat the chromosome from which it is expressed9. It is devoid of any significant ORF and is only expressed from the inactive X chromosome (Xi) in somatic cells. During both female mouse development and in vitro differen-tiation of female mouse ESCs, Xist is monoallelically upregulated. This upregulation is tightly correlated with the onset of XCI and precedes the initiation of silenc-ing (FIG. 2). Deletions of Xist have demonstrated that it is necessary in cis to induce chromosome-wide silenc-ing10,11. Furthermore, inducible expression of Xist cDNA transgenes on autosomes demonstrated that Xist RNA is sufficient to trigger cis-inactivation of the chromosome from which it is expressed during an early developmental time window12.

How exactly Xist RNA induces gene silencing still remains a mystery, but the highly conserved A-repeat region of Xist is crucial for its silencing function, whereas

other parts of the RNA ensure its cis-coating capacity13,14. Expression of an Xist cDNA lacking the A-repeat region in differentiating mouse ESCs has revealed that the tran-script can induce several chromatin modifications on the chromosome that it associates with, independently of transcriptional repression. These modifications include recruitment of Polycomb group proteins (PcG proteins), the histone variant macroH2A, the Trithorax group protein (TrxG protein) ASH2-like (ASH2L) and heterogeneous nuclear ribonucleoprotein U (hnRNPU; also known as SAFA)12,14–19. Wild-type Xist RNA has also been shown to induce the spatial reorganization of the X chromo-some, creating a repressed nuclear compartment that is depleted of the transcription machinery and into which genes are recruited when they are silenced20–22.

Based on the above evidence, Xist activation clearly triggers the establishment of chromosome-wide silenc-ing. Therefore, much of the research into the mecha-nisms of XCI initiation has focused on regulation of this particular gene and the ncRNA it produces. However, an important observation from studies of Xist knockouts is that heterozygous Xist mutants are still able to initiate XCI from the wild-type X chromosome10,11. Thus, Xist sequences alone cannot account for the competence function of the Xic, which means other elements must be responsible for female-specific (XX) Xist activation and XCI initiation. As discussed below, it is now clear that Xist’s unique expression pattern is controlled by a complex interplay of long-range cis-acting elements and trans-acting factors.

Xist regulation during random XCIHow is female-specific, monoallelic Xist upregulation achieved and why does it only occur within a precise time window during development and differentiation? In the following sections we describe what is known about the different levels of control acting on Xist during random XCI (see FIG. 3A for a summary). Xist is expressed at very low levels in undifferentiated male and female ESCs, but becomes upregulated on one X chromosome upon differentiation of female cells. Although it is now clear that Xist is controlled mainly at the transcriptional

Timeline | Landmarks in our understanding of the initiation of random XCI

1949 1960 1961 1963 1967 1983 1991 1996 1999 2000

The Barr body is proposed to be an inactive X chromosome (Xi)141

Discovery of a dense structure in female somatic nuclei called the Barr body140

Based on phenotypic variegation in the coat colours of heterozygous female mice, Lyon proposed that one of the two X chromosomes is stably inactivated in female cells142

Identification of an X-controlling element (Xce), which induces a skew in choice of the Xi40

Discovery of the Xist/XIST gene as a candidate for the Xic6–8

Demonstration that large single-copy Xist transgenes are insufficient for full Xic functions during random XCI34

(1963–1964) Lyon, Russell and Grumbach propose that inactivation spreads from a unique locus (the X-inactivation centre, (Xic)) located on the X chromosome129–131,143

(1983–1985) Definition of the Xic and its functions1,2

(1996–1997) Demonstration that Xist is essential for initiation of XCI in mice10,11 and that multicopy Xist transgenes can induce XCI to some extent125–127,144

Identification of the Xist antisense unit, Tsix56,145,146

Demonstration that Xist RNA is sufficient to initiate cis-inactivation12

(2000–2010) Discovery of numerous Xist molecular regulators (see main text)

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Pluripotency factorsA class of proteins that maintain pluripotency — the capacity to give rise to a wide range of, but not all, cell lineages — of stem cells.

level23, post-transcriptional maturation events may also participate. For example, recent studies have shown that deletion of the A-repeat region of Xist prevents accu-mulation of the spliced form of Xist RNA during dif-ferentiation24 and somehow disrupts the gene’s correct upregulation during development25.

Repression of Xist in undifferentiated ESCsWhat accounts for the low expression level of Xist in undifferentiated ESCs? Several circumstantial lines of evidence have pointed to pluripotency factors as negative regulators of the XCI process (FIG. 3Ba). In mouse ESCs, inducible knockout of Nanog or Oct4 (also known as Pou5f1) leads to ectopic Xist upregulation and chromo-some coating in a fraction of differentiating male ESCs26. Another study reported that knockdown of Oct4 leads to Xist RNA accumulation on both X chromosomes in a fraction of differentiating female ESCs27. The binding of OCT4, NANOG, SOX2, transcription factor 3 (TCF3; also known as TCFE2A) and the PR domain contain-ing protein PRDM14 within the first intron of Xist26,28,29 had led to the proposal that such factors might repress Xist expression, via this region, in undifferentiated ESCs. However, deletion of this intronic region of Xist was recently shown to have no impact on Xist repres-sion in undifferentiated ESCs30, although the chromo-some with the deleted allele is mildly favoured for XCI upon differentiation. Furthermore, it has recently been shown, using reporter assays, that a construct contain-ing just the Xist core promoter can be activated during female mouse ESC differentiation23, and that OCT4, NANOG and SOX2 do not bind the Xist promoter26,28,31.Therefore, these pluripotency factors probably control Xist activity indirectly via intermediate regulators. As we

discuss later, both upregulation of RING finger protein 12 (RNF12; also known as RLIM) and Xic–Xic homolo-gous pairing events during differentiation may represent such intermediates27,32.

Female‑specific activation of XistWhat is the mechanism underlying the specific upregu-lation of Xist in cells with more than one X chromo-some? Several lines of evidence point to the existence of long-range regulatory elements that are required for Xist’s XX-specific upregulation. First, female cells carrying a 58 kb deletion — including Xist — on one X chromosome can still initiate XCI on the wild-type X chromosome33, implying that they can still sense their XX status and are still competent for XCI. Second, large 460 kb single-copy Xist transgenes in male ESCs are unable to trigger Xist during differentiation, either from the transgene or from the endogenous Xic34. This implies that critical Xic sequences that are needed to render cells competent for XCI must be missing from these large DNA fragments (BOX 2). Thus, the sequences underlying XX-specific Xist activation must lie some distance from the gene itself.

In a quest to identify these sequences, investigation of the genomic neighbourhood of Xist led to the identifica-tion of at least three X-linked loci that are possibly impli-cated in the activation of Xist during random XCI in female mouse ESCs. One is the X-pairing region (Xpr), which lies 200–300 kb 5′ to Xist. Xpr is able to medi-ate homologous trans-interactions between the two Xic loci (known as ‘pairing’) in female mouse ESCs before Xist activation. This ability, which is also shown by Xpr single-copy transgenes, was proposed to participate in female-specific Xist expression, as Xpr–Xpr interactions do not normally occur in male cells5. A recent report describing the unusual genomic instability of the Xpr region when present as a transgene in male cells35 could be indicative of recombination pathways being involved in Xpr pairing. However, the mechanisms underlying Xpr pairing in female cells and the impact of this on Xist transactivation remain to be elucidated.

A second locus that has clearly been shown to have a role in XX-specific Xist activation is Rnf12, which lies approximately 500 kb 5′ to Xist (FIG. 3Bb). This gene produces a trans-acting factor, RNF12, which has a ubiquitin ligase activity. Overexpression of RNF12 can induce Xist RNA coating of the single X chromo-some in differentiating male mouse ESCs and of both X chromosomes in differentiating female mouse ESCs32. Based on such observations, it has been proposed that RNF12 can activate Xist when present above a certain threshold. In mouse ESCs, this threshold is proposed to be reached only when two X chromosomes are active. How RNF12 activates Xist remains to be determined, but one possibility is that its ubiquitin ligase activity acts to degrade a repressor of Xist. Recently, RNF12 has been shown to be capable of activating the core promoter of Xist30. Importantly, heterozygous deletion of Rnf12 delays, but does not prevent, XCI in female mouse ESCs32,36, implying that additional Xist activation mechanisms, present in XX but not XY cells, must exist.

Box 1 | Key concepts in X‑chromosome inactivation

Before the discovery of the many molecular actors in X-chromosome inactivation (XCI), some key concepts relating to the steps necessary for inactivation to occur were proposed. Although theoretical, these notions became, to an extent, dogmatic over the years. However, these concepts are now being revised in the face of new molecular insights.

CountingThis refers to the process by which a cell determines its X/autosome (X/A) ratio in order to maintain only a single active X chromosome per diploid autosome set. It was first proposed by Lyon and Grumbach based on humans with abnormal numbers of X chromosomes118,119. A normal XY male, or an XO female, shows no inactivation of the unique X chromosome, whereas XXX and even XXXX individuals display one active X chromosome and inactivation of all supernumerary X chromosomes120–124.

ChoiceRefers to how one of the two X chromosomes is selected for inactivation. During random XCI, the probability that the paternal or the maternal X chromosome will be chosen for XCI is equal, unless mutations or polymorphisms are present within the X-inactivation centre (Xic)41. The selection of one X chromosome for inactivation must somehow preclude the initiation of XCI of the other X chromosome and is thus a part of the trans-function of the Xic.

Sensing/competenceThis describes a permissive state for XCI that occurs only when there is more than one X chromosome present in a cell. It must be noted that sensing/competence is implicit in the original concept of counting (as defined above) and involves both XX-recognition, as well as assessment of the X/A ratio. However, investigation of phenotypes of different Xic mutants has led to a distinction being made between the two concepts4,5,47.

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Figure 1 | The X‑inactivation centre. A | The X-inactivation centre (Xic) has been defined as the minimum region both necessary and sufficient to trigger X-chromosome inactivation (XCI)2. The existence of a unique locus controlling the initiation of XCI was first proposed in 1964, based on studies of individuals or cell lines with balanced X–autosome translocations. Aa | In normal female cells, there is random XCI such that there is an equal probability of either X chromosome undergoing inactivation. Ab | In studies of the reciprocal translocation T(X;16)16H (also known as T16H, Searle’s translocation) only one of the two translocation products was found to be inactivated, suggesting the existence of an X-linked region (the Xic) that is required in cis for XCI to occur129–131. Note that 16X is not found to be inactivated, which is due to secondary counter selection. Ac | Surprisingly, when the same translocation is unbalanced there is no inactivation process at all, suggesting that in the absence of two Xics, a cell does not detect the presence of two X chromosomes132. Ad | Subsequent studies involving female embryonic cells where one of the two X chromosomes was truncated2,132 confirmed this hypothesis. It was revealed that neither the truncated X chromosome (HD2 truncation) nor the intact X chromosome showed any sign of XCI based on cytological staining. This indicates that at least two Xics are required for a cell to initiate XCI. Ae | By contrast, for a truncation that does not remove Xic, random XCI still takes place. B | In addition to providing a functional definition of the Xic, these chromosomal rearrangements define the physical boundaries of the locus. In mice (shown), the minimum candidate region for the Xic has been defined, based on studies in developing mouse embryos or differentiating embryo-derived (EK) cells133. The Xic lies between the T16H breakpoint134,135 and the HD3 breakpoint1,2, a region spanning 8 cM (10–20 Mb). Here, only the elements around Xist are shown. Some of these elements, such as the Xist antisense gene (Tsix) or RING finger protein 12 (Rnf12; also known as Rlim) gene are now known to be involved in Xist regulation. Xist and its antisense Tsix, as well as regulators of Tsix — Xite (X-inactivation intergenic transcription element) and DXPas34 — are shown at higher resolution under the Xic map. In humans (not shown), the XIC has been proposed to map between the T(X:14) and rea(X) breakpoints, a region spanning 700 kb136,137. However, the human XIC has been defined through the analysis of X-inactivation status in somatic cells of patients with X-chromosomal deletions or translocations, rather than in embryonic cells where XCI is actually initiated. Thus, it cannot be excluded that some of these rearrangements could have arisen after initiation of XCI. Cdx4, caudal X-linked gene 4; Chic1, cysteine rich hydrophobic 1; Cnbp2, cellular nucleic acid binding protein 2; Ftx, five prime to Xist; Jpx, also known as Enox (expressed neighbour of Xist); Nap1l2, nucleosome assembly protein 1-like 2; Tsx, testis specific X-linked; Xpct, X-linked PEST-containing transporter.

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Figure 2 | The cycle of XCI in female mouse embryos and ESCs. a | In mice, X-chromosome inactivation (XCI) begins at the four-cell stage — see the middle of the top part of this panel. Inactivation is initially imprinted, with preferential inactivation of the paternal X chromosome (Xp). Studies of parthenogenetic and gynogenetic embryos suggest that this imprint is maternal. This is because, in the presence of two maternal X chromosomes (Xm), there is no XCI until blastocyst formation, implying that the Xm cannot be inactivated prior to this, despite the presence of two X chromosomes89,138. Furthermore, experiments performed with non-growing oocytes showed that an X chromosome from an immature oocyte (prior to establishment of imprinting) behaves like an Xp during embryogenesis and can undergo early X-inactivation104. Once established, the Xp remains inactive in extra-embryonic tissues (trophectoderm and placenta) but is reactivated in the inner cell mass (ICM) of the blastocyst in pre-epiblast cells, which gives rise to the embryo. A second wave of inactivation then occurs in the ICM and randomly affects either the Xp or the Xm. The inactive state is

then stably maintained and transmitted through cell divisions in the soma but the inactive X chromosome (Xi) is reactivated during the formation of the female germ line. Imprinted and random inactivation are both Xist-dependent and both seem to involve RING finger protein 12 (RNF12; also known as RLIM). A maternal pool of RNF12 may be required for initiation of imprinted Xp inactivation and two copies of Rnf12 may be required to activate Xist in female embryos during random XCI (see main text for discussion). b | RNA fluorescent in situ hybridization (FISH) in mouse embryonic stem cells (ESCs). In undifferentiated cells, the two X chromosomes are active, as shown here by biallelic expression of α-thalassaemia/mental retardation syndrome X-linked gene (AtrX). In these cells, Xist is expressed at a low level and is hardly detectable. During differentiation, one of the two Xist alleles is upregulated. Xist RNA coats the X chromosome from which it is produced and triggers X-inactivation, which leads to the monoallelic expression of X-linked genes such as AtrX in differentiated cells. Tsix, Xist antisense gene.

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Figure 3 | Summary of Xist regulation at the onset of XCI. Numerous factors are implicated in Xist regulation. A | Network of genetic interactions. Note that here arrows do not necessarily imply direct regulation. Repression of Xist by SOX2, although not formally assessed, is to be expected, given that it shares the vast majority of its targets with OCT4 and NANOG. B | Possible molecular mechanisms involved in regulating Xist. Ba | Binding sites of pluripotency-associated transcription factors within elements of the network. It is still unclear whether binding to these sites actually mediates control of Xist and Tsix (Xist antisense gene) expression — see main text for details. Bb | The activation of Xist also requires X-linked activators such as RING finger protein 12 (RNF12; also known as RLIM) in a dose-dependent manner. The upregulation of RNF12 during differentiation is thought to activate Xist, thereby triggering cis-inactivation and ultimately lowering RNF12 levels. This feedback loop ensures that one X chromosome remains active. Bc | Different modes of cis-regulation operate on each chromosome. On the future active X chromosome, Tsix expression is stimulated by the X-inactivation intergenic transcription element (Xite) and DXPas34. On the future inactive X chromosome, the A-repeat region is required for the accumulation of the spliced form of Xist, which mediates silencing. However, an effect of this region on the Xist promoter in XX-differentiating cells still cannot be excluded (dashed arrow). Bd | The regulatory activity of the A-repeat region has been proposed to involve the expression of a short RNA, RepA. RepA has been proposed to bind Polycomb repressive complex 2 (PRC2) and recruit it to the Xist promoter, somehow resulting in the activation of Xist. This binding of RepA to PRC2 would be antagonized by Tsix on the future active X chromosome. CTCF, CCCTC-binding factor; Jpx, also known as Enox (expressed neighbour of Xist); OCT4, also known as POU5F1; PcG, Polycomb group; PRDM14, PR domain zinc finger protein 14; REX1, reduced expression protein 1 (also known as ZFP42); YY1, transcriptional repressor protein Yin and Yang 1.

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Intriguingly, contrasting outcomes have been reported in two different studies concerning the effects of homozygous deletion of Rnf12 in ESCs. In one case, complete abrogation of XCI is reported30, whereas in the other only a slightly reduced efficiency of XCI is observed36. Whatever the cause of these differences, RNF12 is clearly a key activator of Xist. It also has an essential role during imprinted XCI, as will be discussed later. However, additional XCI activation mechanisms that can act in a partially redundant fashion during random XCI must exist and remain to be identified.

Another element that has recently been implicated in Xist activation in XX cells is the Jpx locus (also known as Enox (expressed neighbour of Xist)) which is situated immediately 5′ to Xist37. This locus lies within a 120 kb region that is hyperacetylated in undifferentiated female (but not male) mouse ESCs38. Like many other loci within the Xic, Jpx produces an ncRNA and has been proposed to enable female-specific Xist activation, as its heterozygous deletion impedes XCI initiation. However, unlike Rnf12 transgenes, Jpx transgenes that are unlinked to Xist do not activate endogenous Xist expression in male ESCs32. How exactly Jpx, or its surrounding chro-matin environment, influences Xist activation in female cells remains to be determined. A recent study reported that another long ncRNA Ftx (five prime to Xist) can control the expression levels of Xist, Tsix and Jpx in male cells. However, the role of Ftx in XCI remains to be investigated in female cells39.

In conclusion, it is becoming increasingly evident that the competence of XX cells for Xist upregulation is not mediated by one, but by several loci that act at the DNA level and/or at the level of the proteins or RNAs that they produce, with partially redundant activity. It should be emphasized that, to date, no Xic sequence introduced as a single extra copy in a male ESC has yet been reported to induce XCI in a fashion that is reminiscent of normal

XX ESCs (BOX 2). Identifying the remaining unknown Xic elements and factors, and understanding how their interplay controls Xist expression, is an exciting challenge for the future.

Monoallelic regulation of Xist during random XCIDuring random XCI in mice, expression of Xist is restricted to a single allele. This is the result of both counting and choice (BOX 1) and is influenced by reg-ulatory elements within the Xic. Some of these ele-ments are intimately linked to the regulation of XCI by pluripotency factors and the XX competence-regulation mechanisms described above.

The Xce locus. In female inbred mice, the two X chro-mosomes have an equal chance of being chosen for XCI. However, XCI choice can be biased by alleles at the X-linked X-controlling element (Xce), which maps within Xic and causes non-random XCI in heterozy-gotes40–42. At least three natural alleles of Xce have been identified, based on skewed XCI patterns in Xce het-erozygotes. Although Xce has been genetically mapped to the region 3′ to Xist43, its exact nature, location and mechanism of action are still not known. Furthermore, given the complexity of the Xist regulatory landscape, Xce alleles may correspond not just to one, but to several polymorphic controlling elements within this subregion of the Xic44–46.

Tsix-mediated repression of Xist. Analysis of targeted deletions and studies with transgenes have revealed that the region lying immediately 3′ to Xist is essential for correct monoallelic regulation of this gene. Heterozygous deletion of a 65 kb region 3′ to Xist in female mouse ESCs results in nonrandom Xist upregulation and inactivation of the mutated X chromosome47. Subsequent sequence replacement48,49 and reinsertion50,51 strategies have shown that the major Xist repressor in this region is its antisense transcription unit, Tsix (FIG. 1). Indeed, disruption of Tsix transcription by a 3.7 kb deletion encompassing its promoter recapitulates the skewing observed with the 65 kb deletion, although additional elements regulating Xist may lie within this larger region48,50,51. Although deletion of the Tsix promoter and enhancers leads to skewed XCI of the mutated copy of the X chromosome, this does not enhance the overall expression level of Xist50. Thus, antisense transcription appears to control the binary decision of whether to upregulate Xist dur-ing differentiation. In fact, the ratio of sense/antisense transcription across Xist seems to be crucial in deter-mining which allele will be upregulated. When antisense transcription is artificially driven across Xist, this pre-vents its upregulation in cis52,53. Conversely, enforced Xist transcription is sufficient to induce preferential inactiva-tion of the mutated chromosome, without altering Tsix transcript levels54,55 (FIG. 3Bc). However, Xist repression in undifferentiated cells does not rely on Tsix alone. This is because Tsix deletion does not lead to high-frequency ectopic Xist activation before differentiation; rather, it does so only after differentiation is induced, when pluripotency-factor downregulation begins47,48,52,56–58.

Box 2 | Transgenesis studies of the X‑inactivation centre

Numerous experiments involving transgenesis have attempted to identify the minimum region necessary to recapitulate the functions of the X-inactivation centre (Xic). Single-copy Xist transgenes of up to 460 kb are unable to trigger Xist upregulation during differentiation of male embryonic stem cells (ESCs), either from the transgene or from the endogenous Xic34. This shows that crucial Xic sequences required to render cells competent for X-chromosome inactivation (XCI) are missing from these large DNA fragments. Importantly, such single-copy transgenes can trigger imprinted XCI when paternally inherited (see the main text), implying that Xic sequence requirements are different between the two forms of XCI34,96,125. Surprisingly, the lack of random XCI functions for single-copy transgenes can be bypassed at least partially using multicopy arrays, which can initiate inactivation in cis in male and female cells126,127. However, their capacity to trigger Xist expression from the endogenous X chromosome is limited, and inactivation of the endogenous X chromosome or the transgene is neither random nor exclusive in such lines32,34,128. Importantly, female mouse ESCs were also reported to be unable to trigger Xist expression from these single-copy transgenes, even though they are competent to trigger random XCI of their endogenous X chromosomes34,125. Thus, not only do such ectopic single-copy Xic fragments lack sequences to efficiently trigger the endogenous Xist allele(s) in trans, they also cannot respond to trans -activating competence signals, such as RING finger protein 12 (RNF12; also known as RLIM)32, originating from the endogenous X chromosomes in XX cells. Most of these missing elements still remain to be identified.

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DicerAn RNase III family endonuclease that processes dsRNA and precursor microRNAs into small interfering RNAs and microRNAs, respectively.

CCCTC‑binding factor(CTCF). A highly conserved DNA-binding protein with 11 zinc fingers that, in mammalian genomes, binds to regulatory elements such as insulators.

How does Tsix prevent Xist upregulation? Several reports have now clearly established that antisense transcription across the Xist promoter is accompanied by modifications of its chromatin structure23,57,59–63, although the precise function of these chromatin changes is unclear. Whether it is the act of transcription or the Tsix RNA itself that participates in these chroma-tin changes is also still an open question. The possible existence of Xist–Tsix RNA duplexes and small RNAs (~25–42 nucleotides long) that match the Xist promoter and the A-repeat region has been reported in differen-tiating mouse ESCs64, suggesting the potential involve-ment of an RNAi-like mechanism. However, although a Dicer mutation was reported to increase Xist levels in this study, more recent analyses have shown that this is likely to be due to demethylation of the Xist promoter65. This demethylation is due to the downregulation of microRNAs that regulate the DNA methyltransferase 3A (DNMT3A) when the microRNA machinery is impaired65,66.

The repressive action of Tsix has also been proposed to rely on competition for Xist-activating factors. In par-ticular, the Xist A-repeat region, which is essential for Xist RNA accumulation — as well as for Xist-mediated gene silencing in cis at the level of the Xist RNA — has also been reported to produce a 1.6 kb non-coding tran-script, RepA, independently of the main Xist promoter67. It has been proposed that this RepA transcript enhances Xist expression via recruitment of the Polycomb repres-sive complex 2 (PRC2) and that Tsix RNA prevents Xist activation by somehow competing with RepA RNA for PRC2 recruitment23,67 (FIG. 3Bd). However, the exact role that PRC2 serves in the activation of Xist is still unclear. Indeed, it had been suggested that PRC2 also participates in Xist repression in male cells, in synergy with Tsix62. Additionally, PRC2 does not seem to be required for Xist upregulation, as a lack of EED protein — a key com-ponent of PRC2 — does not prevent XCI initiation in female embryos, nor in male Tsix mutants62,68. Thus, the functional relevance of the connections among RepA, PRC2 and the initiation of XCI remains unclear.

Although the exact molecular mechanisms underly-ing the role of Tsix in regulating Xist are still not fully understood, the genetic evidence suggests that antisense transcription has a key role in the cis-regulation of Xist. Importantly, however, Tsix heterozygous mutants do not simply induce Xist expression more frequently from the mutated chromosome. Somehow, inactivation of the Xist wild-type X chromosome also seems to be prevented, or bypassed, owing to accelerated XCI on the deleted X chromosome, which results in rapidly reduced RNF12 levels33. This finding highlights the fact that there must be some mechanism to coordinate XCI between the two chromosomes. Indeed, this coordination seems to be lost in Tsix homozygous mutants, as an increased number of cells activate Xist from both X chromo-somes4. The general picture that is emerging from such studies is that Tsix plays a central part in the accurate monoallelic expression of Xist during random XCI ini-tiation. This, of course, begs the question of how Tsix itself is regulated.

Regulation of Tsix expression. Perhaps surprisingly, the Tsix core promoter is dispensable for basal transcrip-tion of this RNA and its deletion does not skew choice69. Conversely, several critical regulatory elements of Tsix have been defined. For example, the DXPas34 minisatel-lite, which was initially identified based on differential methylation patterns of the active X chromosome versus the Xi44,70, acts as an enhancer of the Tsix promoter in reporter assays71. Furthermore, DXPas34 removal results in loss of Tsix transcription and nonrandom Xist upregu-lation in mouse ESCs and mice56,69,72. This suggests that loss of this regulatory element is responsible for the phe-notypes observed with the larger deletions of the Tsix promoter region that encompass the DXPas34 minisat-ellite47,48. Interestingly, transcription can be initiated at DXPas34 (REF. 69) and the 5′ ends of some Tsix isoforms map to the minisatellite73. In addition, several of the transcription factors that have been shown to regulate Tsix expression, such as CCCTC-binding factor (CTCF) and its PcG co-factor YY1, as well as the stem-cell factor reduced expression protein 1 (REX1; also known as ZPF42), bind to DXPas34 (REFS 31,74).

Another enhancer of Tsix, Xite (X-inactivation inter-genic transcription element), lies 10–15 kb upstream of the Tsix start site. Deletion of Xite results in mildly skewed XCI and accelerated downregulation of Tsix upon differentiation75. Several pluripotency factors have been found to target Xite and their binding sites are nec-essary for Xite reporter constructs to be transactivated27. These pluripotency factors include SOX2 (REFS 27,28), OCT4 (REFS 27,28) and NANOG28.

In summary, Tsix is a regulator of Xist and is itself regulated by several long-range cis-acting elements, as well as by pluripotency factors (FIG. 3A). Although muta-tions in Tsix or Xite on one X chromosome can clearly skew the choice of X chromosome to be inactivated, the question remains as to how asymmetry in Xist expression patterns is established when two genetically identical Tsix alleles are present.

Ensuring monoallelic expression of Xist. After Xist has been upregulated and XCI triggered on one X chromo-some in female cells, repression of the second allele of Xist (on the active X chromosome) is maintained by DNA methylation of its promoter. This is supported by the fact that impairment of both Dnmt3a and Dnmt3b leads to ectopic Xist activation at late developmental stages in both males in females76. However, the mecha-nisms ensuring that only one Xist allele is expressed at the outset of XCI are still not fully understood. One pathway that has been proposed to explain the asym-metric expression of Xist is a negative feedback loop that involves Rnf12 (and other possible X-linked Xist activators)32 and is triggered by Xist expression itself30 (for review, see REF. 77). Upon initiation of XCI on one X chromosome, rapid Xist RNA-mediated silencing of Rnf12 would result in downregulation of the protein, thereby diminishing the activating effect of RNF12 on Xist transcription (FIGS 3Bb,4a). This feedback model is based on the hypothesis that Rnf12 (and other poten-tial X-linked XCI-promoting factors) must be rapidly

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downregulated by Xist RNA to avoid activation of the second Xist allele (FIG. 4a).

Transient homologous Xic pairing has also been pro-posed to play a part in the coordination and asymmetric treatment of the two Xics during initiation of XCI (FIG. 4b). Indeed, initial Xic–Xic pairing events mediated by Xpr are followed by pairing at Tsix/Xite region in differenti-ating XX ESCs at the moment of Xist upregulation5,78,79. Although pairing at Tsix/Xite is clearly not necessary for Xist activation78,79, several observations indicate these events may be linked to the monoallelic regulation of Xist. First, Tsix/Xite trans-interactions occur at the time of Xist activation78,79. Second, Tsix deletions, which have been shown to skew choice, also abrogate pairing at the Tsix/Xite region78,79. Third, single-copy transgenes of the region containing Xist, Tsix and Xite that are unable to activate Xist either in cis or in trans are also unable to associate with the endogenous Xic78. Last, ectopically provided Tsix and Xite multicopy arrays can mediate

efficient pairing with the endogenous Xic regions and inhibit XCI in females4,79,80. Insights into the events immediately downstream of pairing have recently been obtained using live cell imaging of tagged Tsix loci81. Tsix expression was found to become transiently monoallelic after separation of the loci, thus providing a window of opportunity for Xist upregulation in cis to the silent Tsix allele. Depletion of CTCF and OCT4 by knockdown, as well as transcriptional inhibition, has been shown to disrupt Tsix/Xite pairing and perturb Xist expres-sion. However, it is unclear whether the Xist deregula-tion observed is directly due to the disruption of these chromosomal interactions or due to other effects27,80. The mechanisms that drive pairing, and its exact role (or roles) in XCI, remain to be precisely elucidated.

Another mechanism that has been proposed to account for monoallelic Xist upregulation is that Xist activation would occur stochastically at a low frequency and that this would be followed by a counter-selection of

Figure 4 | Models for monoallelic regulation of Xist. Monoallelic Xist expression may be achieved through several (not mutually exclusive) mechanisms. a | The feedback model proposes that each X chromosome produces an X-chromosome inactivation (XCI) promoting factor (or factors), which will activate Xist in a dose-dependent fashion. Initiation of XCI will lead to the downregulation of such a factor on one allele, bringing its concentration below the threshold required to activate the second Xist allele33,77. It has been proposed that RING finger protein 12 (RNF12; also known as RLIM) is one such factor32. To serve as a robust feedback mechanism, the downregulation of the XCI-promoting factors would need to occur very rapidly, before Xist activation on the second allele. b | The X-inactivation centre (Xic) pairing model proposes that physical interactions between the two X-pairing regions (Xpr) may render the two X chromosomes competent for Xist expression. Subsequent trans-interactions between homologous Tsix (Xist antisense gene) and Xite (X-inactivation intergenic transcription element) regions could enable monoallelic Xist upregulation5,78,79 by promoting a symmetry-breaking event between the two alleles139. This model was recently supported by live-cell imaging of Tsix-pairing events followed by Xist/Tsix RNA fluorescent in situ hybridization (FISH)81. c | The stochastic and secondary selection-based model proposes that each X chromosome has a low and intrinsic probability of activating Xist. Because only cells with one active X chromosome per diploid set of autosomes can survive, this leads to counter-selection against cells with two active or two inactive X chromosomes. Counter-selection may either involve cell death or the ability to shift to an XCI pattern that is compatible with cell proliferation33. Such a model requires that cells remain competent for Xist activation for numerous cell cycles85, a property that has not yet been examined in vivo (in peri-implantation mouse embryos). d | Pre-emptive states corresponding to different propensities for Xist activation have been proposed to exist prior to XCI. In this model each chromosome can alternate between these states, which have been proposed to involve alternative structural configurations at the level of sister chromatid cohesion between the X chromosomes82.

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EutheriansMammals in which the development of progeny takes place in the mother’s body thanks to the placenta, a fetal membrane that facilitates nutrient and waste exchange between the fetus and the mother.

Meiotic sex chromosome inactivation(MSCI). Silencing and hetero-chromatinization of sex chromosomes in the male germ line during meiosis.

AndrogeneticAndrogenetic embryos are produced by the fusion of two haploid paternal genomes.

ParthenogenoteA uniparental embryo produced by the development of an unfertilized egg.

GynogenoteAn embryo produced by the fusion of two haploid maternal genomes.

cells that have not established XCI correctly during dif-ferentiation. This means that they have either inactivated both X chromosomes or nei ther X chromosome, both of which would be deleterious situations with aberrant X chromosome dosage (FIG. 4c). However, although some degree of selection following inaccurate XCI may occur during in vitro differentiation of mouse ESCs4,33, it rarely seems to occur in vivo in mice (C. Corbel, I. Okamoto and E.H., unpublished observations).

A final proposed model is that, before random XCI, the two Xic loci are differently poised for Xist activa-tion (FIG. 4d). Indeed, in undifferentiated mouse ESCs, the two X chromosomes exist in alternative — and alternating — structural states at the level of their sister chromatid cohesion82. These states appear to be anticor-related between the two X chromosomes in the same cell. Furthermore, chromosome-wide patterns of asyn-chronous sister chromatid cohesion states are altered by mutations within Xist or Tsix. However, the basis for these coordinated, alternating states and their possible role in random monoallelic choice during XCI remain to be determined.

In conclusion, multiple models have been proposed for the asymmetric expression and monoallelic regula-tion of Xist. These are not mutually exclusive; in fact, they may be exploited at different levels and to varying extents, in order to accomplish appropriate Xist and XCI patterns during development. Indeed, in differ-ent species, some of these routes to achieve monoal-lelic XCI may be exploited more than others, as will be discussed later.

Impact of the X/autosome ratio on XCIExperiments involving triploid and tetraploid embryos demonstrated that the number of inactive X chromo-somes seems to depend on autosomal ploidy, with the majority of cells retaining one active X chromosome per diploid set of autosomes83,84 (see ‘Counting’ in BOX 1). More recently, kinetic measurements revealed that, for the same number of X chromosomes, Xist upregulation happens more rapidly in differentiating mouse ESCs with a high X/autosome (X/A) ratio than in cells with a low X/A ratio85. This suggests that autosomal factors directly regulate the probability of Xist activation; secondary selection is, however, clearly measurable at later stages in these cell populations. What could the nature of these autosomal counting factors be? Given that most pluripo-tency factors are autosomal, it is tempting to speculate that they might be part of this X/A counting mechanism. The exact nature of counting still remains mysterious and it should be noted that all of the Xic mutations so far proposed to affect counting have only been investigated in diploid cells. Therefore, they have not been tested for their sensitivity to autosomal dosage, which is how defects in X/A counting should be assessed.

The regulation of imprinted XCIAlthough random XCI is believed to be the norm in eutherians, in mice XCI is initially subject to imprinting during pre-implantation development, with exclusive inactivation of the Xp (FIG. 2). At the time of zygotic

genome activation (ZGA), both X chromosomes are active but the Xp rapidly initiates XCI following imprinted Xist expression from the 2–4 cell stage onwards86–88. XCI seems to be complete by the blastocyst stage. Imprinted inactivation of the Xp is maintained in the extra-embryonic tissues but is reversed in the ICM, where random XCI subsequently takes place86–87.

How is this imprinted form of XCI controlled? A robust maternal imprint that prevents inactivation of the Xm exists in mice, given that XCI does not occur in Xm disomies, leading to early lethality owing to defects in extra-embryonic development89–91. Xist is clearly essen-tial for imprinted XCI, as a deletion of the paternal Xist allele also leads to early lethality8. However, it has been proposed that the Xp may also be predisposed to silenc-ing due to its heterochromatinization in the XY body during meiotic sex chromosome inactivation (MSCI) in the male germ line92,93. In support of this, a recent study suggested that some genes on the Xp may be silenced independently of Xist during cleavage stages94. However, a subsequent study came to the conclusion that Xist is in fact required for initiation of XCI of X-linked genes95, although silencing of repetitive elements on the Xp may persist independently of Xist from the male germ line into the zygote on the Xp. The demonstration that Xist is suf-ficient for the initiation of imprinted XCI in mice came from a study96 showing that autosomal Xist transgenes can initiate imprinted cis-inactivation independently of MSCI when they are paternally transmitted.

How then is Xist regulated during early mouse embryogenesis? Contrary to the situation for random XCI, Xist expression is strictly dependent on parental origin immediately after fertilization. Whereas Xist is exclusively transcribed from the paternal allele, the maternal allele of Xist is never expressed during early pre-implantation development97–100. Importantly, pater-nal Xist expression occurs regardless of X chromosome number, unlike the situation during random XCI. For example, in androgenetic XX embryos, Xist RNA coating of both X chromosomes is seen, although this is resolved to monoallelic Xist expression by the blastocyst stage101. Furthermore, XO androgenotes also initiate Xist RNA coating, but this later disappears. Conversely, parthe-nogenotes (XmXm) show a complete absence of Xist expression up to the morula stage, after which some Xist upregulation is observed102,103. Taken together, these data suggest that only the paternal — but not the mater-nal — Xist allele can respond to the transcription factor environment present in cleavage-stage mouse embryos. Recently, the maternal pool of RNF12 was shown to be essential for paternal Xist expression, as imprinted XCI is not initiated in Rnf12+/– female embryos derived from Rnf12-deficient oocytes36.

What prevents Xist expression from the Xm in cleavage- stage mouse embryos? Evidence that a repressive imprint is deposited during egg maturation came from the obser-vation that an X chromosome derived from non-grow-ing, rather than fully grown, oocytes can be inactivated in gynogenotes104. However, the nature of this imprint is still unknown. Unlike many autosomal imprinted loci, Xist imprinting does not seem to rely on differential

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gametic DNA methylation105. Given the transient nature of this Xist imprint (it is reversed by the morula or early blastocyst stage), it may rely on the different chroma-tin states of the two parental genomes that have been reported to exist during early embryogenesis106, or else on a maternally bound repressor that becomes gradu-ally diluted. Whatever its nature, the imprint must lie either within Xist or close to it, because paternally or maternally derived 210 kb, single-copy, Xist-containing autosomal transgenes show correctly imprinted Xist expression patterns. Tsix does not appear to represent the maternal repressive Xist imprint that is present only in cleavage-stage embryos, as it is not expressed until the morula stage, well after paternal Xist expression and XCI have initiated49,56 (FIG. 2). However, Tsix does seem to be required later to maintain the silent state of the maternal Xist allele in extra-embryonic male and female tissues107.

In conclusion, although the imprinted form of XCI shares certain conserved features with the random form in the epiblast (such as the implication of Xist and its activation by RNF12), the two processes appear to differ substantially in the mechanism by which Xist is monoal-lelically controlled. The initiation of imprinted XCI in pre-implantation embryos bypasses the requirement for sensing, counting and choice by ensuring parent-of- origin-specific Xist expression through imprinting. By the morula or early blastocyst stage, however, this imprint appears to be lost or ignored and the sensing, counting and choice mechanisms that characterize random XCI seem to be enabled (FIG. 2a). Importantly, a recent study has shown that in rabbit and human embryos, con-trary to the situation in mice, Xist is not imprinted108. Furthermore, this study showed that, unlike mice, both Xist alleles can be activated, often simultaneously, during early embryogenesis in these species. This suggests that very different strategies of Xist regulation are deployed in other mammals, with a lack of imprinting and with a choice of X chromosome to inactivate occurring down-stream of Xist upregulation108. This highlights the evolu-tionary diversity of X-inactivation initiation mechanisms in mammals and suggests that imprinted XCI via an Xist imprint may have evolved specifically in mice.

Reprogramming of the inactive X chromosomeDuring mouse pre-implantation embryogenesis, the inactive Xp is specifically reactivated in the ICM at the blastocyst stage86,87. Another round of Xi reactiva-tion also occurs following random XCI in the female germ line just before meiosis109,110 (FIG. 2a). Reactivation of the Xi can also be induced when somatic cells are fused to ESCs, as well as during cloning, somatic cell nuclear transfer (SCNT) and induced pluripotency. An important question is whether reprogramming is simply a mirror image of the steps involved in the initiation of XCI, or do other pathways come into play?

One key difference in these two reprogramming events is that the epigenetic status of the Xi is prob-ably less firmly ‘locked-in’, in the ICM of the blasto-cyst, compared to primordial germ cells of the embryo. However, in both cases, loss of Xist expression seems to

be a very early event. How is Xist repression achieved? In the blastocyst, Tsix is expressed from the Xp in ICM cells. However, it is not required to reprogramme the Xist locus as deletion of Tsix does not interfere with Xist downregulation in the ICM58. Conversely, Xist repres-sion from the Xp coincides with the increase in NANOG expression86 and Nanog mutants do not show loss of PRC2 recruitment, a marker of the inactive state, on the Xp111. This argues for a role for NANOG in Xp reactiva-tion in the ICM. Similarly, a role for pluripotency fac-tors during X chromosome reactivation in the germ line has also been proposed, based on the early expression of OCT4 in primordial germ cells109,110,112, although this remains to be demonstrated formally. Thus, loss of Xist expression seems to be an early event in both waves of reprogramming that occur in vivo. However, the man-ner in which the reversal of repressive chromatin marks, nuclear reorganization of the Xi and gene reactivation are achieved remains to be defined. Whether similar mech-anisms are exploited in both of these reprogramming waves also remains to be seen.

The Xi can also be reactivated when somatic cells are treated with a cocktail of factors, including some pluripo-tency factors, to generate induced pluripotent stem cells113. However, the degree of reprogramming is often incomplete, as many genes still remain silent despite partial loss of Xist RNA coating and PcG-associated histone H3 lysine 27 trimethylation (H3K27me3)114. In cloning experiments, it has been shown that the inac-tive state of the previously inactive X chromosome is preserved to some extent in pre-implantation stages and extra-embryonic tissues, suggesting that reprogram-ming of Xist and reactivation of X-linked genes is not fully accomplished during SCNT115,116. The fact that at subsequent stages, in post-implantation SCNT embryos, XCI is found to be random presumably indicates that, in the ICM, the inactive state of the previously inactive somatic X chromosome is completely reset115. This is as expected, given the capacity of the ICM to reactivate the Xp during normal mouse development. Interestingly, in pre-implantation SCNT embryos, the silent Xist allele on the previously active somatic X chromosome is often aberrantly upregulated116. This aberrant Xist upregula-tion on the previously active X chromosome, as well as Xist expression on the previously inactive X chro-mosome, presumably results in aberrant XCI of both X chromosomes and has a deleterious effect on early development. A demonstration of this came from a study of Xist deletion, which resulted in dramatically increased cloning efficiencies117. Finally, it should be noted that substantial species differences may exist in the status of the X chromosome in the ICM and possibly in ESCs and in induced pluripotent stem cells. A recent study revealed that, in rabbits, XCI initiates in the ICM rather than X-reactivation occurring in the ICM as hap-pens in mice108. The situation is even more surprising in humans, where XIST RNA is clearly expressed in the ICM but the X chromosome appears to remain globally active108. Thus, the regulation of XIST by pluripotency factors and reprogramming events in blastocysts may be strikingly different between mammals.

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1. Rastan, S. Non‑random X‑chromosome inactivation in mouse X‑autosome translocation embryos—location of the inactivation centre. J. Embryol. Exp. Morphol. 78, 1–22 (1983).Based on the study of X/A translocations, this paper demonstrates that the initiation of cis-inactivation depends on the presence of a unique region of the X chromosome, the Xic, the distal boundary of which maps to the Searle’s translocation breakpoint.

2. Rastan, S. & Robertson, E. J. X‑chromosome deletions in embryo‑derived (EK) cell lines associated with lack of X‑chromosome inactivation. J. Embryol. Exp. Morphol. 90, 379–388 (1985).Based on the analysis of female cells with truncated X chromosomes, this paper demonstrates that at least two copies of the Xic are required for XCI to occur and maps the proximal boundary of the Xic to the HD3 breakpoint.

3. Gartler, S. M. & Riggs, A. D. Mammalian X‑chromosome inactivation. Annu. Rev. Genet. 17, 155–190 (1983).

4. Lee, J. T. Regulation of X‑chromosome counting by Tsix and Xite sequences. Science 309, 768–771 (2005).

5. Augui, S. et al. Sensing X chromosome pairs before X inactivation via a novel X‑pairing region of the Xic. Science 318, 1632–1636 (2007).

6. Brockdorff, N. et al. Conservation of position and exclusive expression of mouse Xist from the inactive X chromosome. Nature 351, 329–331 (1991).

7. Brown, C. J. et al. A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature 349, 38–44 (1991).This paper identifies the human XIST gene, based on its unique expression pattern and location within the candidate XIC region.

8. Borsani, G. et al. Characterization of a murine gene expressed from the inactive X chromosome. Nature 351, 325–329 (1991).

9. Clemson, C. M., McNeil, J. A., Willard, H. F. & Lawrence, J. B. XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell Biol. 132, 259–275 (1996).

10. Penny, G. D., Kay, G. F., Sheardown, S. A., Rastan, S. & Brockdorff, N. Requirement for Xist in X chromosome inactivation. Nature 379, 131–137 (1996).This study demonstrates that Xist expression is required in cis for XCI.

11. Marahrens, Y., Panning, B., Dausman, J., Strauss, W. & Jaenisch, R. Xist‑deficient mice are defective in dosage compensation but not spermatogenesis. Genes Dev. 11, 156–166 (1997).

12. Wutz, A. & Jaenisch, R. A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. Mol. Cell 5, 695–705 (2000).This study demonstrated that Xist RNA expression is sufficient to trigger cis-inactivation, and also defined the developmental time window during which it can trigger silencing.

13. Beletskii, A., Hong, Y. K., Pehrson, J., Egholm, M. & Strauss, W. M. PNA interference mapping demonstrates functional domains in the noncoding RNA Xist. Proc. Natl Acad. Sci. USA 98, 9215–9220 (2001).

14. Wutz, A., Rasmussen, T. P. & Jaenisch, R. Chromosomal silencing and localization are mediated by different domains of Xist RNA. Nature Genet. 30, 167–174 (2002).

15. Rasmussen, T. P., Wutz, A. P., Pehrson, J. R. & Jaenisch, R. R. Expression of Xist RNA is sufficient to initiate macrochromatin body formation. Chromosoma 110, 411–420 (2001).

16. Plath, K. et al. Role of histone H3 lysine 27 methylation in X inactivation. Science 300, 131–135 (2003).

17. Kohlmaier, A. et al. A chromosomal memory triggered by Xist regulates histone methylation in X inactivation. PLoS Biol. 2, e171 (2004).

18. Pullirsch, D. et al. The Trithorax group protein Ash2l and Saf‑A are recruited to the inactive X chromosome at the onset of stable X inactivation. Development 137, 935–943 (2010).

19. Hasegawa, Y. et al. The matrix protein hnRNP U is required for chromosomal localization of Xist RNA. Dev. Cell 19, 469–476 (2010).

20. Chaumeil, J., Le Baccon, P., Wutz, A. & Heard, E. A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced. Genes Dev. 20, 2223–2237 (2006).

21. Clemson, C. M., Hall, L. L., Byron, M., McNeil, J. & Lawrence, J. B. The X chromosome is organized into a gene‑rich outer rim and an internal core containing silenced nongenic sequences. Proc. Natl Acad. Sci. USA 103, 7688–7693 (2006).

22. Chow, J. C. et al. LINE‑1 activity in facultative heterochromatin formation during X chromosome inactivation. Cell 141, 956–969 (2010).

23. Sun, B. K., Deaton, A. M. & Lee, J. T. A transient heterochromatic state in Xist preempts X inactivation choice without RNA stabilization. Mol. Cell 21, 617–628 (2006).

24. Royce‑Tolland, M. E. et al. The A‑repeat links ASF/SF2‑dependent Xist RNA processing with random choice during X inactivation. Nature Struct. Mol. Biol. 17, 948–954 (2010).

25. Hoki, Y. et al. A proximal conserved repeat in the Xist gene is essential as a genomic element for X‑inactivation in mouse. Development 136, 139–146 (2009).

26. Navarro, P. et al. Molecular coupling of Xist regulation and pluripotency. Science 321, 1693–1695 (2008).This study demonstrates that the core pluripotency transcription factors OCT4 and NANOG participate in Xist repression in ESCs.

27. Donohoe, M. E., Silva, S. S., Pinter, S. F., Xu, N. & Lee, J. T. The pluripotency factor Oct4 interacts with Ctcf and also controls X‑chromosome pairing and counting. Nature 460, 128–132 (2009).

28. Marson, A. et al. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell 134, 521–533 (2008).

29. Ma, Z., Swigut, T., Valouev, A., Rada‑Iglesias, A. & Wysocka, J. Sequence‑specific regulator Prdm14 safeguards mouse ESCs from entering extraembryonic endoderm fates. Nature Struct. Mol. Biol. 18, 120–127 (2011).

PerspectivesOur understanding of the molecular events underlying the initiation of XCI has increased substantially over the past decade. It is now apparent that Xist and its regulators are embedded in a network of stem-cell-specific factors, the interplay of which will require genetic and biochem-ical studies in order to be elucidated. The ESC model system for random XCI should allow this goal to be achieved in the near future. However, a full understand-ing of the regulation of imprinted XCI remains a chal-lenge, as no in vitro model exists for the initiation of this process. Biochemical approaches in ESCs will hopefully shed some light on the molecular partners and targets of RNF12 and its role as an XX dosage-sensitive Xist acti-vator. However, it is clear that other dosage-dependent activators exist and will hopefully be identified using genetic screens and transgenesis techniques.

Despite the recent exciting advances in our under-standing of Xist regulation, there are still many gaps in our knowledge of the repertoire of cis- and trans-acting elements involved in regulating the onset of XCI. Much is now known about Xist and its antisense transcript Tsix; however, the full extent of the Xic still remains to be defined. Indeed, the regulatory landscape of Xist that is emerging is highly complex and careful genetic dissection of long-range regulatory elements will be required. Furthermore, the discovery that trans- acting factors such as RNF12 are encoded at locations so closely linked to Xist begs the question of whether

all X-linked loci that are involved in XCI process will be close to, or part of, the Xic.

The monoallelic regulation of random XCI also remains somewhat mysterious. Although Tsix clearly has an important role in mice, the manner in which monoal-lelic Xist expression is achieved in other mammals, such as humans, in which no antisense TSIX transcription across the XIST promoter exists, is not clear. Indeed, the regulation of XCI initiation in eutherians other than mice has now clearly been shown to be different108, and even more so in marsupials, in which no Xist gene has been identified so far and only an imprinted form of XCI takes place. XCI thus provides a beautiful example of the evolutionary tinkering that can be used to achieve the same outcome (dosage compensation) with a diversity of approaches.

Finally, genetic dissection of the molecular pathways underlying reactivation of the Xi in the female ICM and germ line during normal development should provide important insights into the molecular basis of induced pluripotency. Whether repression of Xist is indeed the key initiating event and how the reversal of the inactive state of the X chromosome is achieved are still largely unanswered questions in mice. Furthermore, the situ-ation in other mammals may again be very different108. The integration of molecular genetics, epigenomics and single cell technologies, including live-cell imaging, pro-vides us with the exciting prospect of finally deciphering the regulatory mechanisms that control XCI.

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30. Barakat, T. S. et al. RNF12 activates Xist and is essential for X chromosome inactivation. PLoS Genet. 7, e1002001 (2011).

31. Navarro, P. et al. Molecular coupling of Tsix regulation and pluripotency. Nature 468, 457–460 (2010).

32. Jonkers, I. et al. RNF12 is an X‑encoded dose‑dependent activator of X chromosome inactivation. Cell 139, 999–1011 (2009).This paper identifies RNF12, a ubiquitin ligase encoded by an X-linked gene, as a dose-dependent Xist activator and also provides molecular evidence for the existence of a negative feedback loop involving X inactivation of Rnf12 following Xist expression.

33. Monkhorst, K., Jonkers, I., Rentmeester, E., Grosveld, F. & Gribnau, J. X inactivation counting and choice is a stochastic process: evidence for involvement of an X‑linked activator. Cell 132, 410–421 (2008).This study demonstrates that heterozygous deletion of a region spanning the Xist/Tsix/Xite locus in XX ESCs does not prevent XCI from the wild-type chromosome upon differentiation, thus demonstrating that other X-linked loci must be responsible for the capacity of XX cells to trigger XCI.

34. Heard, E., Mongelard, F., Arnaud, D. & Avner, P. Xist yeast artificial chromosome transgenes function as X‑inactivation centers only in multicopy arrays and not as single copies. Mol. Cell. Biol. 19, 3156–3166 (1999).This work demonstrates that large, single-copy Xist transgenes covering up to 460 kb of neighbouring sequence are unable to trigger Xist expression — either in cis from the transgene or in trans from the endogenous X chromosome — thus highlighting the existence of critical long-range cis- and trans- acting elements in the regulation of Xist.

35. Sun, B. K., Fukue, Y., Nolen, L., Sadreyev, R. I. & Lee, J. T. Characterization of Xpr (Xpct) reveals instability but no effects on X‑chromosome pairing or Xist expression. Transcription 1, 1–11 (2010).

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