Regulation of X-chromosome inactivation by the X-inactivation centre

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(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.
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Regulation of X‑chromosome inactivation by the X‑inactivation centre Sandrine 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.

Homogametic and heterogametic sexes In 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.

Imprinted Epigenetic 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.

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] doi:10.1038/nrg2987

Sex chromosome dimorphism leads to a genetic imbalance 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 homologous 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 beginning 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 differentiation of mouse embryonic stem cells (ESCs), which are

derived from the ICM and have two active X chromosomes, is accompanied by random XCI and has been extensively used to dissect the early events underlying 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 molecular 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 trigger 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 expression. Finally, we discuss random and imprinted XCI in the context of mouse development and the recent insights that XCI has brought into reprogramming processes.

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

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

1949 1960 1961 1963 1967 1983 1991 1996 1999 2000

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

(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

The X‑inactivation centre Early studies of XCI patterns in mouse embryos or embryonic cells that carried translocated or truncated X  chromosomes revealed the existence of a single X‑linked locus, the Xic, that needs to be physically 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 competence, or sensing 3–5 (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 suggest that they are highly interconnected, both genetically and molecularly.

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.

Xist RNA triggers cis-inactivation The Xic harbours the Xist gene6–8 (FIG. 1B), which produces 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 differentiation of female mouse ESCs, Xist is monoallelically upregulated. This upregulation is tightly correlated with the onset of XCI and precedes the initiation of silencing (FIG. 2). Deletions of Xist have demonstrated that it is necessary in cis to induce chromosome-wide silencing 10,11. 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 window 12. 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

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

Demonstration that Xist RNA is sufficient to initiate cis-inactivation12

other parts of the RNA ensure its cis-coating capacity 13,14. Expression of an Xist cDNA lacking the A‑repeat region in differentiating mouse ESCs has revealed that the transcript 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 chromosome, 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 silencing. Therefore, much of the research into the mechanisms 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 XCI How 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

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REVIEWS 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.

Counting This 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. Choice Refers 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/competence This 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.

level23, post-transcriptional maturation events may also participate. For example, recent studies have shown that deletion of the A‑repeat region of Xist prevents accumulation of the spliced form of Xist RNA during differentiation24 and somehow disrupts the gene’s correct upregulation during development25.

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

Repression of Xist in undifferentiated ESCs What 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 chromosome 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 containing 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 repression in undifferentiated ESCs30, although the chromosome with the deleted allele is mildly favoured for XCI upon differentiation. Furthermore, it has recently been shown, using reporter assays, that a construct containing just the Xist core promoter can be activated during female mouse ESC differentiation23, and that OCT4, NANOG and SOX2 do not bind the Xist promoter 26,28,31. 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 homologous pairing events during differentiation may represent such intermediates27,32.

Female-specific activation of Xist What is the mechanism underlying the specific upregulation of Xist in cells with more than one X chromosome? 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 wildtype 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 identification of at least three X‑linked loci that are possibly implicated 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 mediate 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 chromosome 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.

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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 unique0CVWTG4GXKGYU^)GPGVKEU 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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