REVIEW

X Chromosome Inactivation: A Silence That Needs to beBroken

Reelina Basu and Li-Feng Zhang*

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore

Received 28 June 2011; Revised 4 August 2011; Accepted 6 August 2011

Summary: Each mammalian female cell transcription-ally inactivates one X chromosome to balance X-linkedgene dosage between males and females. This phe-nomenon, called X chromosome inactivation, is a per-fect epigenetic event, in which two chromosomes withidentical DNA sequences are solely distinguished byepigenetic modifications. In this case, epigeneticmarks, such as histone modifications, histone variants,DNA methylation, and ncRNAs, are all enriched on onechromosome, the inactive X chromosome (Xi), to estab-lish its chromosome-wide gene silencing. At face value,it seems that the gene silencing mechanism of Xi is wellunderstood. However, the ‘‘silence’’ of Xi in somaticcells is so tightly maintained that it remains largelyintact even after almost all known epigenetic modifica-tions are artificially depleted. To understand how thegene silence of Xi is maintained in soma is a major chal-lenge in current research. We summarize the currentknowledge related with this issue and discuss futureresearch directions. genesis 49:821–834, 2011. VVC 2011

Wiley Periodicals, Inc.

Key words: epigenetics; noncoding RNA; chromatin;nuclear organization

INTRODUCTION

In mammals, each female cell independently silencesthe transcription of one X chromosome to balance theX-linked gene dosage between males (XY) and females(XX). This phenomenon, called X chromosome inactiva-tion (XCI), has been studied for half a century sinceMary Lyon first put forth the hypothesis of XCI in 1961(Lyon, 1961). XCI remains an intriguing topic in mod-ern biology. Four aspects of XCI research attract atten-tion of scientists from different research fields. (1) XCIis regarded as a perfect epigenetic event. The two Xs in

each female cell are identical in DNA sequences. How-ever, one is active in transcription, the other is silenced.The regulation of gene expression in this case solelydepends on epigenetic mechanisms. (2) In each undif-ferentiated female embryonic stem (ES) cell, the two Xsare both transcriptionally active. Based on currentknowledge, they are genetically and epigenetically iden-tical at this stage. Upon the onset of XCI, one of the twoXs is randomly silenced in each female cell. How is thisallele-specificity achieved during XCI? This question,also regarded as ‘‘symmetry break’’ or ‘‘counting/choice,’’ is being intensively investigated in the XCI field(Barakat et al., 2010). (3) It is known that, at least in themouse, the regulations of XCI and pluripotency aremechanistically coupled. When ES cells are differenti-ated, pluripotency is lost and XCI occurs. When somaticcells are dedifferentiated to regenerate pluripotency, forexample in iPS cells (induced pluripotent stem cell) andPGCs (primordial germ cell), the inactive X chromosome(Xi) is reactivated. Therefore, XCI serves as a goodresearch subject or experimental readout in studies onmolecular mechanism of pluripotency (Orkin andHochedlinger, 2011). (4) XCI is an interesting topic froman evolutionary point of view (Chaumeil et al., 2011).Dosage compensation of genes located on sex chromo-somes is a common task faced by all heterogameticorganisms. Different strategies of dosage compensationwere established by different organisms. XCI, as oneway of dosage compensation, is specific to mammals.

* Correspondence to: Li-Feng Zhang, School of Biological Sciences,

Nanyang Technological University, 60 Nanyang Drive, Singapore.

E-mail: zhanglf@ntu.edu.sg

Contract grant sponsor: Nanyang Technological University; Contract

grant sponsor: Singapore Stem Cell Consortium.

Published online 26 August 2011 in

Wiley Online Library (wileyonlinelibrary.com).

DOI: 10.1002/dvg.20792

' 2011 Wiley Periodicals, Inc. genesis 49:821–834 (2011)

In fact, XCI mechanism evolves rapidly even within mam-mals. Different mammals utilize different strategies toachieve XCI (Okamoto et al., 2011).

In this review, we focus on the first aspect. For morein-depth information on other XCI related topics, werefer readers to the more specialized literatures cited.

XCI, the Perfect Epigenetic Event

Epigenetics is defined as a heritable regulation ofgene expression unrelated to DNA sequence. Epigeneticmodifications include DNA methylation, histone modifi-cations, histone variants, and higher order of chromatinstructures. Other than DNA and histone modifications,long noncoding RNAs (ncRNA) are also frequentlyobserved in many epigenetic events. These epigeneticmodifications are regarded as important informationwritten on top of the genetic code (the DNA sequence)to annotate the genome and to regulate gene expres-sion. Epigenetic studies help to explain selective, adapt-ive, and inheritable gene expression in different celltypes of a multicellular organism. Therefore, epigeneticsis related to many fundamental questions in biology.

The inactive X chromosome (Xi) has been known asa peculiar structure in cytological stains for a long time.Xi was first observed by Barr and colleagues in 1949(Barr and Bertram, 1949). The structure was identifiedin female cat neurons stained by basic dyes such as cre-syl violet. Nowadays, it is clear that the peculiar charac-ters of Xi in cytological stains are due to its heavy epige-netic coat. Epigenetic marks, such as histone modifica-tions, histone variants, DNA methylation, and ncRNAs,are all enriched on one chromosome, the Xi, to estab-lish its chromosome-wide and allele-specific gene silenc-ing. Many epigenetic modifications on Xi have beenidentified. Most of them can be used as good markers tohighlight the Xi in cytological stains.

At face value, it seems the gene silencing mechanismof Xi is well understood. However, the silence of Xi insomatic cells is so tightly maintained that it remainedlargely intact even after almost all known epigeneticmodifications were artificially depleted (Csankovszkiet al., 1999, 2001; Zhang et al., 2007). Currently, westill do not have any artificial method to efficientlybreak down the silencing of Xi in somatic cells. Theunderstanding of the epigenetic mechanisms involvedin maintaining Xi silencing in soma has important bio-logical significance. It is also directly relevant to the bio-medical aspect of X-linked genetic disorders in female,for example the Rett syndrome (Guy et al., 2007). Here,we will first summarize all the factors (proteins, RNAs,or other molecular identities), which are known to beinvolved in the maintenance of Xi silencing in soma. Af-ter that, we will discuss new perspectives and futureresearch directions.

Xist, the Noncoding RNA

Xist is a critical factor involved in XCI. Xist stands forXi specific transcript, which is a long (18 kb) ncRNA al-lele specifically transcribed from the Xi (Borsani et al.,1991; Brockdorff et al., 1991; Brown et al., 1991). TheXist gene is located on the X chromosome. The onset ofXCI occurs in early embryonic development. In eachfemale cell of the inner cell mass in E4.5 blastocyst,both X chromosomes are transcriptionally active. Atthis time point, both Xs have Xist gene expressed at alow level. The Xist RNA transcripts associate in cis withthe DNA region from which they are transcribed. InRNA FISH (fluorescence in situ hybridization), Xist

RNAs can be visualized as two pinpoint RNA signals(Lee et al., 1999). At early postimplantation stage(�E5.5–6.5), each cell independently and randomlyselects one X as the future Xi. On the chosen Xi, Xistexpression is allele-specifically upregulated. Upregu-lated Xist RNA transcripts spread and coat the X chro-mosome territory (Clemson et al., 1996). At this timepoint, Xist RNAs can be visualized as a cloud signal coat-ing the X chromosome territory in RNA FISH. Coatingof Xist RNA on the Xi further recruits other gene silenc-ing factors to establish the allele-specific and chromo-some-wide gene silencing. On the chosen Xa (active X),Xist expression is allele-specifically repressed. Theonset of XCI can be recapitulated in vitro by differentia-tion of ES cells.

The critical role of Xist in establishing gene silencingof Xi during the initiation stages of XCI was demon-strated by gene knockout studies (Marahrens et al.,1997; Penny et al., 1996). Loss of Xist caused XCI fail-ure and cell death. However, the role of Xist in main-taining gene silencing of Xi in somatic cell was shownto be minimal if not dispensable. When the Xist genewas conditionally removed from the Xi in somatic cells,gene silencing of Xi remained largely intact (Costanziet al., 2000; Csankovszki et al., 2001). Using an Xi-linked GFP reporter, it was shown that only 0.05% ofthe cells reactivated the GFP gene after Xist deletion insomatic cells. The reactivation rate was merely raised to0.8% when the mutant cells were further treated with5-azadC to induce limited DNA demethylation and TSA(Trichostatin A) to inhibit histone deacetylase. The func-tional role of Xist in establishing and maintaining genesilencing of Xi was further studied by inducible expres-sion of Xist. It was found that gene silencing dependedon Xist expression during an early time window,roughly the first 72 h of in vitro differentiation of EScells (Wutz and Jaenisch, 2000). After this time window,the gene silencing status becomes irreversible and inde-pendent of Xist expression.

How does Xist RNA establish gene silencing of Xi dur-ing the early developmental time window? This criticalquestion is yet to be answered. One observation closely

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related to this issue is a so-called ‘‘chromosomal mem-ory’’ established by Xist RNA during the early develop-mental time window. Chromosomal memory can beexemplified by Xist-induced H3K27me3 enrichment onthe Xi. H3K27me3 (trimethylation of histone H3 lysine27) is one layer of epigenetic coating of the Xi. Enrich-ment of H3K27me3 on Xi depends on Xist RNA. WhenXist RNA is deleted from the Xi, the enrichment ofH3K27me3 is also depleted (Plath et al., 2004; Zhanget al., 2007). Using the inducible expression system,Xist expression was turned on, off, then back on againduring ES cell differentiation. As expected, H3K27me3enrichment on Xi was depleted after Xist expressionwas turned off. Interestingly, it was found that earlyexpression of Xist enabled the RNA to reestablishH3K27me3 enrichment on the Xi at a later time point(Kohlmaier et al., 2004). This property was termed as‘‘chromosomal memory.’’ Chromosomal memory isestablished by Xist during the early developmental timewindow. The molecular nature of chromosomal mem-ory is unknown. Unfortunately, chromosomal memorywas found to be independent of gene silencing. Using atruncated Xist allele, which is unable to induce genesilencing, to repeat the experiment, it was found thatthe mutant Xist RNA successfully established chromo-somal memory (Kohlmaier et al., 2004). Using a differ-ent experimental system, a recent study reported thatH3K27me3 enrichment could be established by autoso-mal transgenic Xist expression in somatic cells (Jeonand Lee, 2011).

Besides Xist, there are other ncRNAs involved in XCI.Tsix, as its name suggests, is the antisense transcript ofXist (Lee, 2000; Lee et al., 1999). The expression ofTsix from one X allele counteracts Xist expression fromthe same allele. Therefore, Tsix helps to establish allele-specific expression of Xist (choice), and Xist helps toestablish chromosome-wide gene inactivation(silencing). Other than Xist and Tsix, there are otherncRNAs identified from the nearby DNA region, forexample Jpx (Tian et al., 2010) and Xite (Ogawa andLee, 2003). Jpx and Xite involve in XCI by affecting Xist

and Tsix expression. Tsix and Xite RNAs are notexpressed in somatic cells. Therefore they are notinvolved in maintaining Xi gene silencing in soma. Jpxis expressed in both male and female somatic cells. It isunclear whether Jpx is involved in maintaining Xi genesilencing in soma.

DNA Methylation

The cytosine residues of the CpG island of a gene’spromoter region are often methylated when the gene issilenced. DNA methylation is a common mechanismwidely involved in regulation of many genes: for exam-ple, the silencing of retroviral genome during mouseembryogenesis (Jahner et al., 1982) and the allele-spe-

cific expression of genetically imprinted genes. Geneticimprinting occurs on a small group of genes (about 100genes), which are allele-specifically expressed from ei-ther the paternal or the maternal allele. Obviously,genetic imprinting is an epigenetic event. Parentallyinherited epigenetic marks (genetic imprints) areresponsible for the allele-specific expression pattern ofimprinted genes. The critical role of DNA methylationin genetic imprinting has been clearly demonstrated (Liet al., 1993).

Interestingly, an imprinted form of X inactivationexists, in which the paternal X is non-randomly inacti-vated (Sharman, 1971). Compared to random XCI,imprinted XCI is born with an obvious genetic disad-vantage. Because the paternal X is always silenced inimprinted XCI, a maternally inherited defective X alleleinevitably causes trouble. Imprinted XCI is believed tobe the evolutionarily older form of XCI. In corrobora-tion with this notion, imprinted XCI occurs exclusivelyin all tissues of marsupials (ancient mammals, such askangaroos). In the mouse, imprinted XCI can bedetected in early embryogenesis starting from the 4- to8-cell stage embryo (Huynh and Lee, 2003; Mak et al.,2004; Okamoto et al., 2004). In blastocyst (E3.5–4.5),imprinted XCI is only maintained in the trophectodermand primitive endoderm, which further carry imprintedXCI into the future extraembryonic tissues (Takagi andSasaki, 1975). In the inner cell mass, imprinted XCI iserased and random XCI is set to occur around E5.5–6.5(Mak et al., 2004) in all cells of the embryonic tissues.Unlike the situation in mouse, recent data suggest thatthe Xist gene expression is not subject to imprinting inrabbit and human embryos (Okamoto et al., 2011).

Interestingly, the DNA methylation pattern of Xist

gene promoter correlates with its gene activity (Arielet al., 1995; Norris et al., 1994; Zuccotti and Monk,1995), which suggests that DNA methylation is involvedin XCI. To test this idea, a series of functional analysiswere done using gene knockout mutants of Dnmt1

(maintenance DNA methylase) and Dnmt 3a/3b (denovo DNA methylases). In these mutants, DNA hypome-thylation status was confirmed both at the genome leveland locally on the Xist gene. Mutant embryos or mutantES cell lines were examined in various aspects for XCIdefects, such as imprinted XCI status, random XCI sta-tus, allele-specific expression pattern of Xist gene, andallele-specific silencing of other X-linked genes.

Mutants of Dnmt gene knockouts did show mis-regu-lated Xist expression (up-regulation of Xist from the sin-gle X in male and from both Xs in female). However,this only occurred in a low percentage of cells in mu-tant embryos (Beard et al., 1995; Panning and Jaenisch,1996; Sado et al., 2004). Consistent results wereobtained from mutant ES cell lines. Undifferentiated mu-tant ES cell lines did not upregulate Xist expression(Beard et al., 1995; Panning and Jaenisch, 1996; Sado

823X CHROMOSOME INACTIVATION IN SOMATIC CELLS

et al., 2004). Xist misregulation only occurred in mu-tant ES cells after prolonged in vitro differentiation (Pan-ning and Jaenisch, 1996; Sado et al., 2004). Theseresults suggest that DNA methylation may only berequired in repressing Xist expression in vitro in a latedifferentiation ES population. In addition, Xist upregula-tion in late-differentiation ES cells did not induce genesilencing (Sado et al., 2004), as Xist-mediated genesilencing requires a critical time window during earlydifferentiation. Thus, current data revealed a dispensa-ble role of DNA methylation in regulating the allele-spe-cific expression pattern of Xist.

Dnmt gene knockouts also showed limited effect onthe gene silencing status of other X-linked genes. Mu-tant embryos were able to survive to E9.5 stage, whichsuggests that both imprinted XCI and random XCI werelargely intact in the mutant embryos. In addition, ifDNA demethylation activated the inactive X chromo-some in mutant embryos, mutant females with imbal-anced X-linked gene dosage are expected to die earlierthan mutant males. However, this was not observed(Beard et al., 1995). In Dnmt1 (maintenance DNAmethylase) knockout mutants, an X-linked lacZ trans-gene was tightly silenced in extraembryonic tissuesupon paternal transmission (Sado et al., 2000), whichshowed that imprinted XCI was intact in Dnmt1mutantembryos. The same transgene only showed leakyexpression in embryonic tissues. This result showedthat Dnmt1 was dispensable for both forms of XCI. Onthe other hand, the dispensable role of de novo DNAmethylases in XCI was also confirmed by double geneknockout of the two de novo DNA methylases (Dnmt3a/3b), (Sado et al., 2004). Taken together, a series ofgene knockout studies on Dnmt enzymes revealed thedispensable role of DNA methylation in XCI.

Besides DNA methylation, methylated DNA bindingprotein Mbd2 also showed limited effect in repressingXist expression in somatic cells (Barr et al., 2007).Mbd2 was shown to physically interact with Xist gene.In male mutant fibroblasts of Mbd2 gene knockout, alow level of Xist expression was detected. Other meth-ylated DNA binding proteins, Mbd1, MeCP2, and Kaiso,had no effect on Xist expression.

Heterochromatic Histone Modifications(H3K27me3 and H2AK119ub)

Polycomb group (PcG) proteins were identified ingenetic screening of Drosophila melanogaster as fac-tors involved in heritable silencing of homeotic genes(Schuettengruber et al., 2007). These proteins are con-served in evolution and play extensive biological roles.Interestingly, PcG proteins are also involved in X chro-mosome inactivation and genetic imprinting.

Mammalian PcG proteins form two multiprotein com-plexes, polycomb repressive Complexes 1 and 2 (PRC1

and PRC2). PRC1 ubiquitylates histone H2A at lysine119 (Wang et al., 2004). PRC1 proteins andH2AK119ub are enriched on the Xi (de Napoles et al.,2004; Fang et al., 2004; Plath et al., 2004). PRC2 cata-lyzes tri-methylation of histone H3 lysine 27 (Cao et al.,2002; Czermin et al., 2002; Kuzmichev et al., 2002; Mul-ler et al., 2002). PRC2 proteins (namely Ezh2, Eed andSuz12) and H3K27me3 also showed enriched localiza-tion on the inactive X chromosome (de la Cruz et al.,2005; Erhardt et al., 2003; Mak et al., 2002; Plath et al.,2003; Silva et al., 2003). Despite a well-established func-tional role in heritable gene silencing in many biologicalevents, the functional roles of PcG proteins and theirassociated histone modifications in XCI remain elusive.

It is clear that PcG proteins and their associated his-tone modifications are dispensable for maintaining Xisilencing in soma. In Drosophila, a DNA regulatory ele-ment called PcG response element (PRE) is responsiblefor recruiting PcG proteins. In XCI, recruiting PcG pro-teins and the Xi enrichment of PcG-associated histonemodifications depend on Xist RNA (Plath et al., 2004;Schoeftner et al., 2006; Zhang et al., 2007). BecauseXist is dispensable in somatic cells, so are the PcG pro-teins and their associated histone modifications.

Current data also ruled out a critical role of PcG pro-teins in establishing random XCI. First, in Ring1B (aPRC 1 protein) and Eed (a PRC2 protein) deficient EScells, enrichment of H2AK119ub and H3K27me3 onthe Xi were depleted, but random XCI was undisrupted(Kalantry and Magnuson, 2006; Leeb and Wutz, 2007).Second, a truncated Xist RNA, which lost its ability tointroduce gene silencing, was able to recruitH3K27me3 (Kohlmaier et al., 2004).

The role of PcG proteins in imprinted XCI remainslargely unknown. Attempts to address this question areprecluded by maternally expressed proteins in preim-plantation embryos. Currently, evidence is available tosupport a functional role of Eed in maintaining thesilencing status of the paternal X in differentiated TScells, which represents trophectoderm cell lineage(Kalantry et al., 2006; Wang et al., 2001). Meanwhile, itwas found that PRC2 proteins were not enriched on theimprinted paternal Xi in XEN cells, which representsextraembryonic endoderm cell lineage (Kunath et al.,2005).

PcG proteins and trithorax group proteins (trxG)were originally identified in Drosophila melanogaster

as repressors and activators of Hox genes. While manyPcG proteins showed Xi-enrichment, a trxG proteinAsh2I (absent, small or homeotic discs 2) showed sur-prising Xi-enrichment (Pullirsch et al., 2010). However,it is believed that, as a trxG protein, Ash2I only plays aregulatory role unrelated to gene activation. Xi-enrich-ment of Ash2I is Xist-dependent and independent ofgene silencing. The enrichment is also regulated bychromosomal memory, such that Xist expression in

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early ES cell differentiation enables Xist to efficientlyrecruit Ash2I at a later time point of differentiation.

Polycomblike protein 2 (Pcl2), one of the three Pclproteins in the mouse, was recently identified as a pro-tein enriched on Xi (Casanova et al., 2011). Pcl2 inter-acts with PRC2. After shRNA knockdown of Pcl2, theXist RNA foci were unaffected, however the Ezh2enrichment on Xi was diminished. Therefore, Pcl2 facili-tates the loading of PRC2 complex onto the Xi.

Euchromatic Histone Modifications (H3K4me3and H4Ac)

The PcG protein associated histone modifications(H3K27me3 and H2AK119ub) are usually associatedwith gene silencing, therefore, they are considered asheterochromatin marks. In contrast, histone modifica-tions such as H3K4me3 and H4Ac (histone 4 acetyla-tion) are generally associated with active transcription,therefore, they are considered as euchromatin marks.The chromatin of Xi is defined by an enrichment of het-erochromatin marks (H3K27me3 and H2AK119ub) anda dearth of euchromatin marks (H3K4me3 and H4Ac).Enrichment of H3K27me3 and H2AK119ub on the Xi isinduced by Xist RNA. It is currently unclear what mech-anism is involved in generating the dearth of euchroma-tin marks on the Xi. Upon Xist deletion from Xi in so-matic cells, enrichment of H3K27me3 and H2AK119ubwere depleted, however the dearth of euchromatinmarks on Xi remained unchanged (Zhang et al., 2007).When Xist expression was induced from the Xa in so-matic cells by DNA demethylation, the Xist cloud colo-calized with the X chromosome territory. However, itdid not result in gene silencing and dearth of euchroma-tin marks on the chromosome (Thorogood and Brown,2010). When a mutant form of Xist, which lacked genesilencing capacity, was induced in ES cells, it reas-sembled heterochromatic modifications on the chromo-some territory (Pullirsch et al., 2010). Such a chromo-some territory, with all the heterochromatic modifica-tions resembling Xi but without gene silencing, wastermed as Xiag (Xi with active genes). Hypoacetylationof histone 4 was also established to a certain degreealong Xiag. However, on comparing the immunostainpattern of H4Ac on the metaphase chromosomespreads of Xiag and Xi, numerous H4Ac ‘‘bright bands’’could be recognized along the overall hypoacetylatedXiag. It was believed that such acetylated chromatinregions corresponded to active gene activity. In sum-mary, the current results show that the dearth ofeuchromatin marks along the Xi is independent of Xistfunction. It will be interesting to test if the dearth ofeuchromatin marks on Xi is responsible for the tightgene silencing maintained on Xi after Xist deletion.

Another observation related to euchromatic modifica-tion along the Xi is that the gene silencing along the Xi

is not complete. Profiling of the human inactive X(Carrel and Willard, 2005) showed that 75% of geneswere inactivated, 15% escape inactivation (escapegenes) and 10% escape inactivation in some females(heterogeneous genes). Compared to the human, thegene silencing along the mouse Xi is more complete.Among the murine X-linked genes, which escape inacti-vation, are Smcx, Sts, Utx (Agulnik et al., 1994; Green-field et al., 1998; Salido et al., 1996). The fact thathumans have a much higher number of active genes onthe inactive X may explain the phenotype differencebetween the Turners syndrome patients and the XOmice. The DNA loci of escapee genes carry euchromaticmodifications. One possible mechanism to escape het-erochromatization on the inactive X is the presence ofboundary elements as DNA insulators for shieldinggenes from the position effect of their neighboringregions (Filippova et al., 2005).

macroH2A, the Histone Variant

macroH2A (mH2A) is a histone2A variant with 370amino acid residues, three times the size of H2A (130a.a.), (Costanzi and Pehrson, 1998). mH2A has threevariants (mH2A1.1, mH2A1.2 and mH2A2) encoded bytwo genes (Chadwick and Willard, 2001; Costanzi andPehrson, 1998). The N-terminal 120 a.a. of mH2A showshomology with H2A. The rest of the peptide forms a do-main called the ‘‘macro’’ domain, which showed ADP-ribose binding ability (Karras et al., 2005) and is homol-ogous with other ADP-ribose binding proteins such aspoly-ADP-ribose polymerases (PARPs).

mH2A showed enriched localization on Xi in somaticcells (Chadwick and Willard, 2001; Costanzi and Pehr-son, 1998), preimplantation embryos (Costanzi et al.,2000) and primordial germ cells (Nesterova et al.,2002). Using mH2A-GFP fusion proteins, it was shownthat the H2A-homologous domain of mH2A alone wasable to cause enrichment on the Xi (Chadwick et al.,2001). The mH2A-dense focus in cell nucleus wasnamed as MCB (macrochromatin bodies). Interestingly,before the onset of XCI, MCB was observed in bothmale and female ES cells as a structure not associatedwith the X chromosome (Mermoud et al., 1999), butassociated with the centrosome (Rasmussen et al.,2000). Time course study further showed that enrich-ment of mH2A on the Xi was a late event (Mermoudet al., 1999). This result argued against a functional roleof mH2A in the initiation of random XCI. When the Xist

gene is conditionally deleted in somatic cells, mH2Aenrichment on the Xi was also depleted (Csankovszkiet al., 1999). Therefore, mH2A is dispensable in main-taining XCI in somatic cells. In corroboration with thisnotion, the gene knockout of mH2A1 showed nodefects of X chromosome inactivation (Changolkaret al., 2007).

825X CHROMOSOME INACTIVATION IN SOMATIC CELLS

Other Factors

PARP-1. Poly(ADP-ribose)polymerase 1 (Parp-1) is anuclear enzyme, which transfers and polymerizes ADP-ribose units from donor nicotinamide adenine dinucleo-tide (NAD1) molecules to target proteins. PARP pro-teins are involved in modulating chromatin structuresand regulating gene expression. For example, H1 andH2B are poly(ADP-ribosyl)ated in response to DNAdamage.

Female-specific embryonic lethality at E9.5 wasobserved in Parp-1

1/2Parp-2

2/2 mutant mice due to aspecific instability of the X chromosome (Menissier de

Murcia et al., 2003). Parp-1 showed enriched localiza-tion within the nucleolus in wild-type cells (Mederet al., 2005) and was identified as one of the proteinscoimmunoprecipitated with macroH2A1.2 (mH2A1.2)(Nusinow et al., 2007). Interestingly, when mH2A1.2was tagged with GFP, Parp-1 showed a stronger interac-

tion with the tagged mH2A1.2 in the coimmunoprecipi-tation assay and an enriched localization on the Xi to-gether with the tagged mH2A1 (Nusinow et al., 2007).The same study also showed that mH2A inhibited theenzyme activity of Parp-1. When somatic cells weretreated with shRNA against Parp-1, together with 5-

azadC and TSA, about 7% of cells showed reactivationof a Xi-linked GFP reporter (Nusinow et al., 2007).

Cullin3/Spop E3 ubiquitin ligase. Identified asproteins interacting with Bmi1 (a PRC1 protein), theCullin3/Spop Ubiquitin E3 ligase was shown to have alink with XCI (Hernandez-Munoz et al., 2005), Cullin3/Spop ubiquitinate Bmi1, and macroH2A1. When cellswere treated with 5-azadC, TSA, and shRNA againstCullin3 or Spop, about 3–6% of cells showed reactiva-tion of a Xi-linked GFP reporter (Hernandez-Munozet al., 2005).

SmcHD1. Multiple copy transgenes often displaymosaic or variegated expression patterns in individualcells (Martin and Whitelaw, 1996). Clearly, epigeneticmodifications play a role in this phenomenon. A mecha-nistic link between XCI and variegated transgeneexpression was speculated (Blewitt et al., 2005).SmcHD1 (structural maintenance of chromosomeshinge domain containing 1) was identified through arandom mutagenesis genetic screening of mice insearch for defects of a variegated expression pattern ofa GFP transgene (Blewitt et al., 2005). In femaleSmcHD1 mutant embryos, leakiness of both imprintedand random XCI was observed using an X-linked GFP re-porter (Blewitt et al., 2008). It was also reported thatreactivation of a small sampling of X-linked genes wasdetected using quantitative RT-PCR. The DNA hyper-methylation status of the promoter region of genes sub-jected to X chromosome inactivation was disrupted inmutants. Immunofluorescence showed that theSmcHD1 protein was enriched on the Xi. However, in

SmcHD1 knockout cells, other epigenetic characters ofXi, such as the coating of Xist cloud and enrichment ofH3K27me3, were not affected. The biochemical func-tion of SmcHD1 is currently unclear.

ATRX. The alpha thalassemia/mental retardation X-linked (ATRX) protein belongs to the SWI/SNF2 ATP-de-pendent helicase family, which is involved in a varietyof cellular activities through its chromatin remodelingfunction (Gibbons, 2006). Male embryos carrying anATRX-null allele failed to form a normal placenta (Gar-rick et al., 2006). Surprisingly, individual femaleembryos carrying a maternally inherited ATRX-null al-lele showed ATRX expression in extraembryonic tis-sues. This result showed that the paternally inheritedwild type ATRX allele escaped the imprinted XCI in cer-tain animals. Leakiness of the imprinted form of XCIwas observed in previous studies, for example in thecase of maternally inherited Tsix (antisense transcript ofXist) mutant allele (Lee, 2000). Therefore, the resultdoes not directly support a role of ATRX protein in XCI.However, ATRX protein showed enriched localizationon the Xi in immunostain (Baumann and De La Fuente,2009). The Xi enrichment was more easily recognizablein metaphase chromosome spreads than in interphasenuclei, as the protein also showed enrichment on cen-tromeric heterochromatin.

RNF12. Ring finger protein 12 (RNF12) was identi-fied initially as an XCI activator (Jonkers et al., 2009).RNF12 gene is X-linked. The expression level of RNF12helps cells to sense X chromosome copy numbers percell. Over-expression of RNF12 in male cells resulted inectopic XCI in male cells. RNF12 is known as an E3ubiquitin ligase. It was shown that RNF12 directlyaffected Xist expression (Barakat et al., 2011). Interest-ingly, heterozygous RNF12 gene knockout ES cellsshowed a biased XCI toward the mutant X allele.Because RNF12 gene knockout showed no deleteriouseffect in males (Shin et al., 2010), this result suggestedRNF12 was required in maintaining Xist expression. Ifan RNF121/2 cell chooses to inactivate the wild typeX allele, the single functional RNF12 allele is silenced.As a consequence, Xist expression cannot be main-tained properly on the wild type X allele. In corrobora-tion with this notion, maternally inherited RNF12 wasrequired for imprinted Xist expression from the pater-nal X allele (Shin et al., 2010). However, RNF12 expres-sion decreases and eventually disappears during ES celldifferentiation (Jonkers et al., 2009). Therefore, the roleof RNF12 in maintaining Xist expression is only essen-tial during initiation of XCI. RNF12 is not required forXist expression in somatic cells and is unrelated tomaintenance of Xi gene silencing.

Terra. Terra (telomeric repeat-containing RNA) isRNA transcribed from the telomeric DNA region. Detec-tion of Terra has been reported in various organisms,such as birds (Solovei et al., 1994), trypanosomes

826 BASU AND ZHANG

(Rudenko and Van der Ploeg, 1989), and mammals(Azzalin et al., 2007; Schoeftner and Blasco, 2008). InRNA FISH, Terra RNA could be visualized as pinpointfoci overlapping with the telomere DNA. Terra RNAfoci number per cell varies per cell. Not every telomereis associated with Terra RNA. Among all the Terra RNAfoci detected in each cell, bright and major Terra RNAsignals could be clearly distinguished. Interestingly,such bright Terra signals consistently showed up incells, and these signals were associated with sex chro-mosomes (Zhang et al., 2009). The expression patternof Terra RNA was developmentally regulated. In undif-ferentiated ES cells, bright Terra foci were associatedwith all sex chromosomes (the two Xs in female, and X/Y in males). In somatic cells, bright Terra RNA signalswere allele-specifically associated with the Xi in femaleand the Y chromosome in male. The allele-specificexpression pattern of Terra suggests the RNA may beinvolved in maintaining Xi silencing in somatic cells. Todate, Terra’s functional role has been studied in differ-ent aspects of telomere biology (Luke and Lingner,2009). Its role in XCI remains unclear.

Higher Order of Chromatin Structures

Besides histone and DNA modifications, higher orderof chromatin structure is also regarded as epigeneticmodification. Currently, a growing body of evidencesuggests that nuclear organization or nuclear matrixmay play an important role in XCI. Noticeably, tworecently identified proteins Saf-A and SATB1 are bothnuclear matrix proteins. In addition, studies on LINE-1elements (long interspersed nuclear element) suggestthat they participate in chromosome territory organiza-tion of Xi.

SATB1. SATB1 (special AT-rich binding protein) wasidentified as a factor involved in Xist-mediated genesilencing through a sophisticated genetic screen. Xist-mediated gene silencing requires strict developmentalcontext (Wutz and Jaenisch, 2000). Using an inducibleXist cDNA transgene, it was found that, other than EScells, hematopoietic precursor cells also provided a per-missive environment for Xist-mediated gene silencing(Savarese et al., 2006). The inducible Xist system wasthen combined with a thymic lymphoma model (Agreloet al., 2009). The inducible Xist cDNA transgene on thesingle male X chromosome was able to repress tumordevelopment upon doxycycline induction. Dox-resist-ant lymphoma cells evolved after the tumor cells werecultured in vitro for 1 month. Global gene expressionprofiling identified that SATB1 was down-regulated indox-resistant tumor cells. The role of SATB1 in Xist-mediated gene silencing was confirmed by induction ofSATB1 expression in fibroblast cells. Induced expres-sion of Xist in fibroblasts cannot initiate gene silencing.However, SATB1 expression alone is sufficient to create

a permissive environment for Xist-mediated genesilencing.

Interestingly, it was observed that SATB1 and Xist

influenced each other’s pattern of localization. In nor-mal thymocyte nuclei, SATB1 forms a cage-like structurecircumscribing heterochromatin (Cai et al., 2003). Inmore than 50% of the female thymocytes, Xist did notcoat the Xi, but localized along the SATB1 cage. InSATB1-deficient mice, more female thymocytes showeda Xist cloud coating Xi. Upon Xist induction on bothfemale Xs in thymocytes, SATB1 delocalized from a cagestructure and showed a two-focus pattern overlappingwith two Xist clouds (Agrelo et al., 2009).

These results demonstrated a functional role ofSATB1 in Xist-mediated gene silencing in lymphomacells and fibroblast. However, the role of SATB1 in em-bryonic stem cells is less clear. SATB1 does not colocal-ize with Xist RNA in ES cells. Single gene disruption ofeither SATB1 or SATB2 was compatible with female de-velopment (Alvarez et al., 2000; Britanova et al., 2006;Dobreva et al., 2006). Data of double gene disruption ofSATB1 and SATB2 are currently not available. In addi-tion, SATB1 is not involved in maintenance of Xi genesilencing in somatic cells, as SATB1 is not expressed inall somatic cell types (Agrelo et al., 2009).

Saf-A. Saf-A, also known as hnRNP U or SP120, is anuclear matrix protein, which was found to be concen-trated on the inactive X chromosome (Helbig and Fack-elmayer, 2003). FRAP (fluorescence recovery after pho-tobleaching) experiments showed that the enrichmentof Saf-A on Xi was stable with low protein mobility(Fackelmayer, 2005). Saf-A enrichment on Xi was Xist-dependent and independent of gene silencing (Pullirschet al., 2010). Interestingly, Saf-A affected Xist RNA coat-ing on Xi. It was found Xist cloud formation was dis-rupted after RNAi knockdown of Saf-A in somatic cells(Hasegawa et al., 2010). Using RNAi to knock down Saf-A in embryonic stem cells, it was also found that Saf-Awas required in establishing XCI (Hasegawa et al.,2010). Homologous Saf-A gene disruption in mouseembryos was post-implantation lethal at around E6.5(Roshon and Ruley, 2005). This result argued against arole of Saf-A in imprinted XCI. It is currently unknownwhether mutant female embryos suffered a more severedefect than males due to failure of random XCI.

It is interesting to note that Saf-A and SATB1 both arenuclear matrix proteins (Dickinson et al., 1992; Romiget al., 1992) and both affect Xist cloud formation. Nu-clear matrix is defined as the insoluble filamentary rem-nant of cell nuclei after cells are treated with DNase Iand high-salt extraction of proteins (Pederson, 2000). Itis long-known that Xist RNA remained with the nuclearmatrix fraction after DNA extraction (Clemson et al.,1996). These results suggest the involvement of nuclearmatrix in Xist cloud formation and XCI. The detailedmechanism awaits future research.

827X CHROMOSOME INACTIVATION IN SOMATIC CELLS

LINE-1. LINE-1 is a major type of repetitive DNA ele-ments in mammalian genome. Full-length LINE-1 ele-ments, when being transcribed, can function as autono-mous retrotransposon. Most of the LINE-1 elements inthe genome, however, are inactive LINE-1 carrying atruncated 50 end. It has been proposed that LINE-1 ele-ments work as ‘‘way stations’’ facilitating the spreading ofXistmediated gene silencing along the host chromosome(Lyon, 1998). Consistent with this hypothesis, studies onthe localization of genes within the Xi chromosome terri-tory showed that a gene silencing compartment wasformed by intergenic DNA sequences, and X-linked geneswere moved into this compartment when being silenced(Chaumeil et al., 2006). The distribution profiles of LINE-1 were shown to be consistent with the distribution ofXist-mediated gene silencing (Bailey et al., 2000; Carrelet al., 2006; Chow et al., 2010; Tang et al., 2010). InRNA FISH, large LINE-1 RNA foci (LINE-1 RNA cloud)were detected in differentiating ES cells (Chow et al.,2010). In Day 4 in vitro differentiation cell population,some cells showed two LINE-1 clouds adjacent to Xa andXi. On Day 8, some cells showed one LINE-1 cloud spe-cifically overlapping with Xi chromosome territory. LINE-1 clouds were not detected in the fully differentiatedMEF cells (mouse embryonic fibroblast). It is important

to extend this finding to human cells, as LINE-1 elementsshow particularly interesting behavior in humans(Ostertag and Kazazian, 2001). First, LINE-1 distributionis significantly more enriched on human X chromosomethan human autosomes. Second, 11 of 14 recent (79%)L1 insertions in the human genome are insertions intothe X chromosome. It is also important to extend thefindings in mouse ES cells to early mouse embryos, as invitro differentiation of ES cell is a highly artificial process.Observations made in the late in vitro differentiationpopulation might be artificial. Furthermore, it is neces-sary to confirm whether LINE-1 transposon activities areinvolved in XCI, as the LINE-1 activities detected in differ-entiating ES cells were confirmed as full-length LINE-1RNAs, which could be functionally active. Nonetheless,mounting evidence suggests LINE-1 elements areinvolved in the chromosome territory organization of Xi.

YY1. YY1 (yin-yang 1) was first reported as a DNA-binding protein, which represses the adeno-associatedvirus P5 promoter (Shi et al., 1991). YY1 is alsoinvolved in transcription regulation of other genes,including the LINE-1 elements (Becker et al., 1993; Kur-ose et al., 1995; Minakami et al., 1992). Immunostain ofYY1 did not show an enrichment of YY1 on the Xi(unpublished data). Interestingly, a recent study showedthat YY1 was involved in Xist RNA coating of the Xichromosome territory (Jeon and Lee, 2011). shRNAknockdown of YY-1 in female somatic cells disruptedXist cloud formation. Therefore, emerging evidencesuggests the involvement of LINE-1 elements and nu-clear architecture in XCI.

Perspectives and Future Research Directions

All protein and RNA factors discussed in this revieware summarized in Figure 1. Detailed information abouteach factor including its full-name, function and key ref-erence papers are listed in Table 1. Here, we discusspossible future research directions.

Most of the epigenetic modifications on Xi depend onXist expression. However the dearth of euchromatinmarks on Xi is independent of Xist expression. It will beinteresting to find out whether the lack of euchromaticmarks is responsible for maintaining gene silencing alongXi in somatic cells after Xist deletion. It is currentlyunclear whether there is a molecular mechanism, whichactively depletes euchromatin marks from the Xi afterthe onset of XCI. It is possible that lack of euchromatinmarks is merely a consequence of transcription silencing.The transcription machinery is known to be able to de-posit euchromatin marks along its DNA template region(MacDonald and Howe, 2009). Therefore, the euchroma-tin marks could be passively depleted from the Xi aftergene silencing. In this regard, it will be interesting tocheck whether the sporadic gene reactivation along Xiafter Xist deletion in somatic cells could introduce

FIG. 1. Factors involved in the maintenance of Xi silencing in so-matic cells. Protein and RNA factors are divided into two groups:Xi-markers and nonmarkers. The Xi-markers include factors specif-ically enriched on, depleted from or associated with the Xi chromo-some territory. The Xi-markers and nonmarkers are further groupedby their relationship with Xist RNA.

828 BASU AND ZHANG

euchromatic marks into corresponding DNA regions,and whether such euchromatic ‘‘lesions’’ could graduallyspread into neighboring regions after a number of celldivisions.

A growing list of recent discoveries shows that nu-clear matrix, nuclear organization or higher order ofchromatin structures may play important roles in XCI.Clearly, future research is required in this direction. It

should be noted that nuclear organization can only bestable for one cell cycle. Upon cell division, correct nu-clear organization has to be re-established in eachdaughter cell. If Xi is specifically organized into a cer-tain nuclear compartment but not Xa, there must besome kind of epigenetic modifications on the Xi, whichcan be recognized by cells in rebuilding the Xi nuclearcompartment after each cell cycle. Such an epigenetic

Table 1Factors Involved in Xi Silencing in Somatic Cell

Name Full name Function/identity Involvement in XCI Reference

Ash2l absent, small or homeoticdiscs 2

belongs to trithoraxgroup protein

enrichment on Xi Pullirsch et al., 2010

ATRX alpha thalassemia/mentalretardation X-linked

belongs to SWI/SNF2ATP-dependenthelicase family

enrichment on Xi Baumann et al., 2009

Cullin3/Spop N/A E3 ubiquitin ligase ubiquitinate macroH2A1 Hernandez-Munozet al., 2005

Dnmt1 DNA methylatranferase 1 maintenance DNAmethylase

regulate Xist and other X-linked gene expression

Beard et al., 1995;Panning andJaenisch, 1996;Sado et al., 2000

Dnmt3a/3b DNA methylatranferase3a/3b

de novo DNAmethylases

regulate Xist and other X-linked gene expression

Sado et al., 2004

H2AK119ub histone2A lysine119ubiquitination

histone modification enrichment on Xi Plath et al., 2004; Fanget al., 2004; deNapoles et al., 2004

H3K4Me3 histone3 lysine4trimethylation

histone modification depletion from Xi Zhang et al., 2007

H3K27Me3 histone3 lysine27trimethylation

histone modification enrichment on Xi Silva et al., 2003; Plathet al., 2003; Erhardtet al., 2003; Maket al., 2002; de laCruz et al., 2005

H4Ac histone4 acetylation histone modification depletion from Xi Pullirsch et al., 2010Line-1 long interspersed nuclear

elementretrotransposon facilitate spreading of Xist

along X chromosome;involved in Xi chromosometerritory organization

Chow et al., 2010

macroH2A macro histone2A histone variant enrichment on Xi Costanzi and Pehrson,1998

Mbd2 methylated DNA bindingprotein 2

methylated DNAbinding protein

regulate Xist expression Barr et al., 2007

PARP-1 Poly(ADP-ribose)polymerase 1

Poly(ADP-ribose)polymerase

interact with macroH2A1.2 Nusinow et al., 2007

Pcl2 Polycomblike Protein 2 Facilitate loading ofPRC2 proteins on Xi

Enrichment on Xi Casanova et al., 2011

Saf-A scaffold attachmentfactor A

nuclear matrix protein enrichment on Xi; influenceXist localization

Helbig andFackelmayer, 2003;Pullirsch et al., 2010;Hasegawa et al.,2010

SATB1 special AT-rich bindingprotein

nuclear matrix protein influence Xist localization Agrelo et al., 2009

SmcHD1 structural maintenance ofchromosomes hingedomain containing 1

structural maintenanceof chromosomeshinge domaincontaining protein

enrichment on Xi Blewitt et al., 2008

Terra telomeric repeat-containing RNA

ncRNA large and bright Terra RNAfoci attach to Xichromosome territory

Zhang et al., 2009

Xist Xi specific transcript ncRNA play critical role duringinitiation of XCI

Costanzi et al., 2000;Csankovszki et al.,2001

YY1 Yin Yang-1 transcription factor Required for loading Xist RNAon Xi

Yesu Jeon and Lee,2011

829X CHROMOSOME INACTIVATION IN SOMATIC CELLS

modification may still be missing in our current list. Itshould also be noted that there might not be a nuclearcompartment specially built just for Xi. Xi may belongto a nuclear environment that is generally shared by fac-ultative heterochromatin. The perinucleolar region maybe such a nuclear domain. Xi is frequently localizedwithin the perinucleolar region and evidence suggeststhat Xi is targeted to perinucleolar region during the Sphase (Zhang et al., 2007).

Based on our current knowledge, marsupials undertakeimprinted XCI in all tissues, but they do not have Xist

gene. Eutherians (placental mammals) have Xist gene andundertake random XCI in all somatic tissues. It is reasona-ble to argue that Xist was evolved to achieve the ‘‘symme-try break’’ required in random XCI. If so, the Xist-depend-ent XCI may be built up upon a core gene silencing mech-anism established in the ancient imprinted XCI ofmarsupials. Therefore, studies on imprinted XCI in marsu-pials may eventually provide an answer for the genesilencing mechanism in random XCI.

Because of the lack of knowledge on gene silencingmechanism of XCI, we still do not have an artificialmethod to efficiently breakdown gene silencing on Xi.Xi can only be reactivated in vivo in three known bio-logical events. First, the imprinted XCI in early mouseembryos is reactivated during epiblast lineage specifi-cation in the inner cell mass. Second, Xi is reactivatedin PGCs, right after PGC specification. Third, Xi reacti-vation occurs in generation of iPS cells. Studies on Xireactivation will no doubt help us understand thegene silencing mechanism of XCI. A recent study onepiblast specification has shown that gene reactiva-tion occurred on Xi when its chromosome territorywas still covered by the Xist cloud (Williams et al.,2011). This result is consistent with the existence of aXist-independent gene silencing mechanism. Such agene silencing mechanism is established by Xist dur-ing the initiation stage of XCI. However, in somaticcells, the gene silencing mechanism can efficientlymaintain gene silencing without depending on Xist

expression. During epiblast specification, the silencedgenes on Xi can also be reactivated regardless of Xistexpression.

CONCLUSION

The inactive X chromosome carries multiple layers ofepigenetic modifications to achieve its chromosome-wide gene silencing. Many of the Xi-associated epigeneticmodifications and their associated proteins have beenidentified. However, the Xi silencing remained largelyintact even after almost all known factors were artificiallydepleted from the Xi. Current data showed that Xi silenc-ing in soma was tightly controlled by a multilayer mecha-nism. The difficulty in breaking down Xi silencing couldbe a result of mechanistic redundancy. On the other

hand, there might be unknown epigenetic factors/mech-anisms responsible for maintaining Xi silencing. Tounderstand how Xi silencing is epigenetically maintainedin soma is a challenging question bearing important bio-logical and biomedical significance.

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