227RESEARCH ARTICLE

INTRODUCTIONAn unexpected new view of the function of RNA involved in manybiological phenomena has emerged from recent advances intranscriptome analysis revealing that much of the genome inhumans, mice, and other organisms is transcribed into RNAs.Although these RNAs are often subject to splicing andpolyadenylation, like common protein-coding mRNAs, most ofthem do not encode proteins. In addition, these noncoding RNAs areoften transcribed in an antisense orientation relative to known genesor transcription units, suggesting that they play an important role intranscriptional regulation of the sense partner.

The noncoding Xist (X-inactive specific transcripts) gene(Borsani et al., 1991; Brockdorff et al., 1991; Brown et al., 1991) andits antisense Tsix gene (Lee et al., 1999) comprise one of the sense-antisense pairs that has been most extensively studied so far. Thesetwo noncoding genes play a pivotal role in X inactivation inmammals via a mechanism by which the dosage differences of X-linked genes between males and females are equalized byinactivating one of the two X chromosomes in females during earlydevelopment (Lyon, 1961). Although either the paternal or maternalX chromosome is silenced in a basically random fashion (random Xinactivation) in the embryonic tissues, which give rise to the fetus,X inactivation is imprinted in the extraembryonic tissues, fromwhich the placenta and a part of the extraembryonic membranes arederived, in such a way that the paternally derived X is preferentiallyinactivated (imprinted X inactivation) (Takagi and Sasaki, 1975). Atthe onset of X inactivation, Xist expression is induced on one of thetwo X chromosomes, and its 17-kb noncoding transcripts associatein cis along the chromosome and make it transcriptionally inactive

by recruiting protein complexes involved in heterochromatinization.Xist and Tsix are reciprocally imprinted in the extraembryonic tissuesso as to be expressed from the paternal and the maternal Xchromosome, respectively (Kay et al., 1993; Lee, 2000; Sado et al.,2001). Since Xist is essential for X inactivation to occur in cis, anXist-deficient X never undergoes inactivation in either embryonic orextraembryonic tissues (Marahrens et al., 1997; Penny et al., 1996).Paternal transmission of the mutated X, therefore, results in thefailure of imprinted X inactivation and eventually a selective loss offemale embryos soon after implantation owing to the presence oftwo active X chromosomes in the extraembryonic tissues(Marahrens et al., 1997). By contrast, Tsix deficiency, whenmaternally inherited, induces the expression of the normally silentmaternal copy of Xist and subsequent inactivation of the mutatedmaternal X in the extraembryonic tissues (Lee, 2000; Sado et al.,2001). This ends up causing functional nullisomy of the Xchromosome in these particular tissues of both male and femaleembryos, and eventually in death at the early postimplantation stage.It therefore seems likely that at the onset of X inactivation, whilecessation of Tsix expression allows upregulation of Xist and triggersthe inactivation process on the future inactive X, continuedexpression of Tsix is required for the future active X to preclude theactivation of Xist on it. This is consistent with the fact that the Tsix-deficient X is inactivated in all cells of the embryonic tissues ordifferentiating female ES cells heterozygous for the mutation (Leeand Lu, 1999; Sado et al., 2001). Interestingly, mutant maleembryos, even though they inherit the Tsix deficiency from themother, can survive if the wild-type extraembryonic tissues areprovided by an experimental reconstruction using wild-typetetraploid embryos, implying the existence of a Tsix-independentmechanism for Xist silencing in the embryonic tissues (Ohhata etal., 2006). All these facts taken together indicate that theextraembryonic tissues appear to depend more on Tsix to regulateXist than the embryonic tissues do.

Although it is clear that Tsix is a negative regulator of Xist effectiveonly in cis, until recently the molecular mechanism of antisenseregulation by Tsix at the Xist locus remained poorly understood.Recent analyses of the Tsix-deficient X in embryos or ES cells,however, suggested that Tsix silences Xist through modification of thechromatin structure (Navarro et al., 2005; Sado et al., 2005; Sun et al.,

Crucial role of antisense transcription across the Xistpromoter in Tsix-mediated Xist chromatin modificationTatsuya Ohhata1,2,*, Yuko Hoki1,2, Hiroyuki Sasaki2,3 and Takashi Sado1,2,3,†

Expression of Xist, which triggers X inactivation, is negatively regulated in cis by an antisense gene, Tsix, transcribed along theentire Xist gene. We recently demonstrated that Tsix silences Xist through modification of the chromatin structure in the Xistpromoter region. This finding prompted us to investigate the role of antisense transcription across the Xist promoter in Tsix-mediated silencing. Here, we prematurely terminated Tsix transcription before the Xist promoter and addressed its effect on Xistsilencing in mouse embryos. We found that although 93% of the region encoding Tsix was transcribed, truncation of Tsix abolishedthe antisense regulation of Xist. This resulted in a failure to establish the repressive chromatin configuration at the Xist promoteron the mutated X, including DNA methylation and repressive histone modifications, especially in extraembryonic tissues. Theseresults suggest a crucial role for antisense transcription across the Xist promoter in Xist silencing.

KEY WORDS: X inactivation, Antisense regulation, Chromatin modification, Gene targeting, Mouse embryos

Development 135, 227-235 (2008) doi:10.1242/dev.008490

1PRESTO, Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi,Saitama, 332-0012, Japan. 2Division of Human Genetics, National Institute ofGenetics, Research Organization of Information and Systems, 1111 Yata, Mishima,411-8540, Japan. 3Department of Genetics, the Graduate University for AdvancedStudies, 1111, Yata, Mishima, 411-8540, Japan.

*Present address: Research Institute of Molecular Pathology, Dr Bohr-Gasse 7,1030 Vienna, Austria†Author for correspondence (e-mail: tsado@lab.nig.ac.jp)

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2006). Furthermore, our recent study demonstrated that splicingproducts of Tsix RNA are dispensable for Tsix-mediated Xist silencingin mouse embryos (Sado et al., 2006). Therefore, we are particularlyinterested in the biological significance of the antisense transcriptionacross the Xist promoter. To address this issue, in the present study westudied a mutation in mouse causing termination of Tsix transcriptionin exon 4 before the transcription proceeded across the Xist promoterregion. This mutation, which essentially eliminated antisense activityin the upstream region of Xist, caused inappropriate activation of Xistand aberrant chromatin modification in the 5� region of Xist, like theloss of function mutation of Tsix previously reported (Sado et al.,2005). These findings demonstrate that the antisense transcriptionacross the Xist promoter plays a crucial role in Tsix-mediated silencingof the Xist gene.

MATERIALS AND METHODSConstruction of targeting vectorsThe second intron of the human �-globin gene (IVS2) was amplified by PCRusing primers Hu.Gl.IVS2-1(+)35SalI and Hu.Gl.IVS2-1(–)33SalI andcloned into pBluescriptII-SK(–) to produce pIVS2. A fragment containinga polyadenylation sequence that originated from the mouse Pgk1 gene anda triple polyadenylation sequence (a multiple polyadenylation sequence,mpA) amplified from the genome of a Rosa26R mouse using tpA-1(–)41NotI and tpA-2(+)37XhoI as primers was placed 3� to a floxedpuromycin-resistance gene driven by the mouse Pgk1 promoter (PgkPurocassette) to produce pfloxPgkPuro-mpA. The floxPgkPuro-mpA cassettethus created was inserted at the unique PvuII site present in the IVS2fragment in an antisense orientation with respect to IVS2. A 9.0 kb genomicEcoRI-BamHI fragment (104,306-113,284 in GenBank J421479) wascloned into pBluescriptII-SK (–) lacking an XhoI and a SalI site (p�XS-EB9.0). The IVS2 fragment containing the floxPgkPuro-mpA cassette wassubsequently introduced into p�XS-EB9.0 at the unique XhoI site presentin exon 1 of Xist to generate pTsix-mpA. Similarly, the IVS fragmentcontaining only the PgkPuro cassette was inserted at the XhoI site in p�XS-EB9.0 to generate pCont-X.

Generation of mice and ES cells carrying each of the TsixpA andXistIVS allelesEach of the targeting vectors (pTsix-mpA and pCont-X) was electroporatedinto R1 ES cells (Nagy et al., 1993), and selection with puromycin wascarried out as previously described (Sado et al., 2005). The expectedhomologous recombination was found in 1 out of 192 colonies and 4 out of240 colonies when pTsix-mpA or pCont-X was introduced, respectively.Chimeric males were produced and serially crossed with femalesheterozygous for the Tsix-deficient X (XX�Tsix) to transmit the mutatedpaternal X to female offspring as previously described (Sado et al., 2005).The floxed puromycin-resistance gene was subsequently removed bycrossing females heterozygous for either TsixpA2lox or XistIVS2lox with males,which ubiquitously expressed cre recombinase, to derive the TsixpA andXistIVS allele, respectively. Removal of the floxed puromycin cassette in EScells was carried out by transfection of cre-expressing plasmid pBS185.

Expression analysesThe visceral endoderm was separated from the yolk sac mesodermaccording to Dandolo et al. (Dandolo et al., 1993). RNA was prepared fromthe embryo proper, visceral endoderm, placenta and ES cells using RNeasy(Qiagen). Thermal conditions for all the following PCRs will be providedupon request.

For the analysis of antisense transcription in the region distal to the mpAsequence in ES cells, cDNA was synthesized from 5 �g total RNA usingThermoscript (Invitrogen) at 56°C with each of the following three differentprimers: (1) AS90RS, (2) R1910J and (3) R371P1 for Tsix-specific primingin combination with GapdR for Gapd, and subsequently PCR was carriedout on 1/25 of the cDNA thus prepared. Primers Xist1175F and Xist1472Rwere used for cDNA primed by (1) AS90RS, and primers R700P2 and Xist-6(–)20 were used for cDNA primed by (2) R1910J and (3) R371P1. A Gapdsequence was amplified as a control using GapdF/GapdR2.

For the analysis of allelic expression of Xist, cDNA was prepared fromtotal RNA of E13.5 embryo proper and visceral endoderm in a strand-specific manner as described above using primers Xist-7(–)20 and GapdR,and PCR was carried out using primer sets R371P1 and Xist-6(–)20, andGapdF and GapdR2 for Xist and Gapd, respectively. An amplified productof Xist was digested with PvuII and electrophoresed on a 4% NuSieve 3:1agarose gel.

For the analysis of Tsix expression in the placenta, RT-PCR was carriedout on cDNA prepared by random priming as previously described (Sado etal., 2005). A proximal and a distal part of Tsix were amplified using primersets Tsix2F and Tsix2R, and Xist (–540) and Xist-10(–)20, respectively (seebelow).

A northern blot was prepared using 20 �g total RNA from the embryoproper at E13.5 as previously described (Sado et al., 2005) and seriallyprobed with pR97E1 for Xist (Sado et al., 1996) and a cDNA fragment forGapd.

Methylation analysisGenomic DNA isolated from the embryo proper and visceral endoderm atE13.5 was digested with BclI in combination with either methylation-sensitive restriction endonuclease SacII, HhaI, PmaCI or HpaII,electrophoresed on a 0.7% agarose gel, blotted on a nylon membrane andhybridized with either HS0.7 or BE0.6 as a probe (Sado et al., 2005).

Chromatin immunoprecipitationAbout 2�106 undifferentiated ES cells were used as a source of chromatinfor ChIP assays using an antibody against the CTD of RNA polymerase IIlarge subunit phosphorylated at serine 2 (H5) (Abcam) and normal mouseIgG (Sigma) as a mock control. Sonicated soluble chromatin was incubatedwith 3 �g of either H5 or mouse normal IgG overnight at 4°C. Chromatinprecipitated with H5 was purified by incubating for another 3 hours at 4°Cwith anti-mouse IgM antibody raised in goat (Sigma), which had beenpreviously bound to Dynabead-Protein-G (Invitrogen). Washing and elutionof immunoprecipitated chromatin and extraction of DNA were carried outas previously described (Sado et al., 2005).

The visceral endoderm separated from the yolk sac mesoderm wasindividually suspended in 10 mM Tris-HCl, pH 7.4, 3 mM CaCl2, 2 mMMgCl2 including proteinase inhibitor Complete (Roche), to which an equalvolume of 10 mM Tris-HCl, pH 7.4, 3 mM CaCl2, 2 mM MgCl2,1% NP-40,Complete was added. Following crosslinking by formaldehyde, cells werecollected by centrifugation and resuspended in SDS lysis buffer. Subsequentprocedures for the preparation of soluble chromatin were carried out in thesame manner as previously described (Sado et al., 2005). To avoid potentialvariation in the efficiency of immunoprecipitation among different lots ofcommercially available polyclonal antibodies, we used monoclonalantibodies raised against H3K4me2, H3K4me3, H3K9me2 and H3K27me3(gifts from Hiroshi Kimura and Naohito Nozaki), each of which had beenpreviously bound to anti-mouse IgG-Dynabeads 280. Normal mouse IgGwas used as a mock control.

Real-time PCRFor quantitative analysis, real-time PCR was carried out as previouslydescribed (Sado et al., 2006). The primers used were: Tsix2F/Tsix2R (forregion d in Fig. 2A), Xist (–540)F/8692R (for region g in Fig. 2A),8111F/8418R (for region h in Fig. 2A), and Xist1F/Xist101R (for Fig. 6).

PrimersPrimer sequences were as follows: Hu.Gl.IVS2-1(+)35SalI, 5�-GGGTCGACTCAAGGTGAGTCCAGGAGATGTTTCAG-3�; Hu.Gl.I -VS2-1(-)33SalI, 5�-GGGTCGACAGGAGCTGTTGAGATGA AAGG -AGAC-3�; tpA-1(-)41NotI, 5�-TTATATTAAGGCGGCCGCATCAGCT -TGATGGGGATCCAGAC-3�; tpA-2(+)37XhoI, 5�-TTCTAACTC GAG -CAGA AGCTTGCAGATCTGCGACTCT-3�; AS90R2, 5�-TGCGGGAT -TCGCCTTGATTT-3�; Xist1472R, 5�-AGGGCAGGTCACATGACTTC-3�; Xist-10(-)10, 5�-ACAAAATGGCTCCTTGGTTC-3�; 21b80F,5�-CCTGCAAGCGCTACACACTT-3�; F748P1, 5�-CAGGTAGTGCA -ATAACTCACAA-3�; 8111F, 5�-CTGCCACCTGCTGGTTTATT-3�;8418R, 5�-ACATGAAAGAGATCAGACAC-3�; Xist1F, 5�-TGTTT -GCTCGTTTCCCGTGGAT-3�; Xist101R, 5�-CATAAGGCT TGGTG -

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GTAGGG-3�; GapdF, GapdR, GapdR2, R1910J, R371P1, Xist21F,Xist(-540)F, Xist1175F, Xist1554R, Xist-6(-)20, Xist-7(-)20, R700R2 and8692R were previously described (Sado et al., 2005). Tsix2F and Tsix2Rwere described by Sado et al. (Sado et al., 2006).

RESULTSTsix was truncated by introducing a multiplepolyadenylation sequenceWe attempted to address the impact of premature termination of theantisense transcription on the effect of Tsix by placing a multiplepolyadenylation sequence (mpA) in exon 4 of Tsix (Fig. 1A).However, introduction of the mpA cassette and a floxed puromycin-resistance gene would inevitably disrupt the Xist gene. We thereforeembedded these sequences in an intron derived from the human �-globin locus (IVS2) and inserted them in exon 1 of Xist in anappropriate orientation in the targeting vector, as shown in Fig. 1B.We assumed that the inserted fragment would be spliced out upontranscription as an intron from the primary transcript of Xist and thatthe resultant processed transcript would behave as a functional XistRNA. ES cells harboring this allele (TsixpA2lox) were produced by

gene targeting (Fig. 1B,C). In parallel, we also used gene targetingto generate another mutant allele, XistIVS2lox, which harbored thesame intron without the mpA cassette at exactly the same positionas the TsixpA2lox allele (see Fig. S1 in the supplementary material).The selection marker was subsequently removed from both mutatedalleles by transient expression of cre recombinase to generate TsixpA

and XistIVS, which were subjected to further analyses.

Transcription of Tsix was prematurely terminatedin the Xist geneTo test whether Tsix transcription was effectively terminated beforereaching the Xist promoter on the X chromosome carrying the TsixpA

allele (XpA) but not on the X carrying the XistIVS allele (XIVS), strand-specific RT-PCR was carried out in the respective mutant ES cells.As shown in Fig. 2A,B, the expected fragment (PCR product e inFig. 2A) was amplified in all cases in which cDNA was synthesizedwith primer a located proximal to the mpA cassette. By contrast,PCR product f was barely detectable in ES cells carrying TsixpA

when either primer b or c located downstream of the mpA cassettewas used for cDNA synthesis. The antisense transcription was

229RESEARCH ARTICLEAntisense regulation at the Xist locus

Fig. 1. Tsix was truncated in the Xist gene by the introduction of a multiple polyadenylation sequence. (A) The genomic structure of theTsixpA allele is shown below the overall structure of the Xist/Tsix loci. The second intron of the human �-globin gene, which harbors an mpAcassette in an antisense orientation with respect to Xist transcription, was introduced at the XhoI site (107228-107233 in GenBank Acc. No.AJ421479). SD, splicing donor; SA, splicing acceptor. (B) Targeting scheme for generating the TsixpA allele. Positions of the recognition sites of therelevant restriction enzymes and the probes used in C and D are shown. B, BamHI; E, EcoRI; H, HindIII; P, PvuII; X, XhoI. (C) Homologousrecombination in ES cells was confirmed by Southern blotting. Probes and restriction enzymes used are shown below the blot. Appropriaterecombination in the 5� region was confirmed using a 5�probe, Xist5-5, located outside of the fragment used for the short arm of the targetingvector. Since the recombination event in the 3� region could not be properly addressed with a single restriction enzyme, it was confirmed by twosteps. Although use of probe XB verified the recombination event by the appearance of the expected 1.7 kb band upon BamHI digestion, thepresence of a 5.6 kb band detected using probe 3� inner upon EcoRI digestion, which was also seen in parental R1 ES cells, verified that there wasno other rearrangement in the fragment used for the long arm of the targeting vector. (D) Excision of the puromycin-resistance cassette by crerecombinase was confirmed by Southern blotting using tail DNA. PvuII digestion and subsequent hybridization with XB as a probe alloweddifferentiation of the wild-type, TsixpA2lox and TsixpA alleles.

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evident in the regions proximal and distal to the inserted intron in EScells harboring the XistIVS allele (Fig. 2B), demonstrating that thepresence of the intron itself did not affect the transcriptionalelongation of Tsix in the relevant region. These results suggested thatthe antisense transcription beyond the inserted mpA cassette wasdramatically reduced on XpA.

Effective attenuation of Tsix transcription was further examinedby chromatin immunoprecipitation (ChIP) assays using amonoclonal antibody against the C-terminal domain (CTD) of RNApolymerase II (pol II) phosphorylated at Ser2, which has beenwidely used for immunoprecipitating the elongation form of RNApol II (Ahn et al., 2004; Morris et al., 2005). Immunoprecipitatedchromatin prepared from ES cells was subjected to PCR using aprimer set located in the proximal region of exon 2 of Tsix (PCRproduct d) and primer sets located in the promoter region of Xist(PCR products g and h) (Fig. 2A,C). In addition to wild-type XY,XpAY and XIVSY male ES cells, male ES cells carrying Xdc, onwhich Tsix is truncated owing to the insertion of an IRES�geocassette at the distal region of exon 2 (Sado et al., 2005; Sado et al.,

2001), were included in the assay. The elongation form of RNA polII was evenly distributed in exon 2 of Tsix in all types of ES cells,whereas it was significantly reduced in the promoter region of Xistin XpAY to a level comparable with that in XdcY, which essentiallylacks Tsix transcription (Fig. 2C). The distribution of RNA pol IIphosphorylated at Ser2 was higher in the distal region of Tsix (regiong and h) than in the proximal region (region d) in XY and XIVSY EScells, a pattern that is commonly observed on transcriptionally activegenes (Ahn et al., 2004; Morris et al., 2005). It therefore seemsreasonable to conclude that the majority of the antisensetranscription beyond the mpA cassette was significantly diminishedas expected.

The TsixpA allele prematurely terminates antisensetranscription in vivoThe TsixpA2lox and XistIVS2lox alleles were introduced into mice throughchimeric males crossed with females heterozygous for Tsix deficiency,and the puromycin-resistance gene was subsequently removed togenerate the TsixpA and XistIVS allele, respectively, as previously

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Fig. 2. Antisense transcription across the Xist promoter is diminished on XpA. (A) Positions of the primers used for cDNA synthesis (a,b,cindicated as arrows) and the region amplified by PCR (d,e,f,g,h) are shown relative to the introduced intron at the XhoI site. Primer a, AS90R2;primer b, R1910J; primer c, R371P1. PCR products were amplified using the following primer sets: d, Tsix2F/Tsix2R; e, Xist1175F/Xist1472R; f,700P2/Xist-6(–)20; g, Xist (–540)F/8692R; h, 8111F/8418R. (B) The presence or absence of the antisense transcription in the region distal to theintron introduced at the XhoI site was examined by strand-specific RT-PCR in undifferentiated ES cells harboring each of the TsixpA and XistIVS alleles.Product e was amplified on cDNA primed by a. Product f was amplified on cDNA primed by either b or c. cDNA synthesis was carried out in eitherthe presence (+) or absence (–) of reverse transcriptase. (C) The presence or absence of the elongation form of RNA pol II in the region distal to thempA cassette was analyzed by ChIP assays using a monoclonal antibody (H5) against CTD phosphorylated at Ser2 in undifferentiated XY, XdcY, XpAYand XIVSY ES cells. The relative abundance of the chromatin immunoprecipitated by the antibody to the input was determined by real-time PCR.Although the elongation form of pol II was similarly distributed in the vicinity of the major transcription start site of Tsix (product d) among all typesof ES cells, the level of the polymerase in the region downstream of the mpA cassette (product g and h) was significantly diminished in not onlyXdcY but also XpAY ES cells compared with wild-type and XIVSY ES cells. (D) RT-PCR was carried out on cDNA prepared from total RNA isolated fromthe placenta at E12.5. Although the proximal region of Tsix was amplified in all cases, the region distal to the mpA was barely amplified in XpAY andXpAX, indicating that antisense transcription in the Xist promoter region was efficiently attenuated by the mpA cassette in the placenta of thedeveloping embryos, as expected.

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described (Sado et al., 2005). We first examined whether these allelesbehaved in the same manner as in the mutant ES cells. Since Tsix isimprinted in the extraembryonic tissues to be expressed from thematernal allele in both sexes (Lee, 2000; Sado et al., 2001), we usedtotal RNA isolated from embryonic day (E) 12.5 placentas of maleembryos recovered from TsixpA/+ and XistIVS/+ females crossed withwild-type males for RT-PCR. Transcription of Tsix was evident in theproximal region of Tsix on the mutated X carrying either TsixpA orXistIVS (Fig. 2D). Similarly, spliced products of Tsix were alsoproduced from these mutated X chromosomes (data not shown).When PCR was performed using a primer set located in the promoterregion of Xist, by contrast, the expected fragment was readilydetectable in XIVSY as well as wild-type XY placenta, but wassignificantly reduced in XpAY (Fig. 2D). Similarly, antisensetranscription was barely detected in the promoter region of Xist in theplacenta isolated from XpAX females. These results indicate that theantisense transcription runs across the Xist promoter on XIVS as on thewild-type X, but is effectively attenuated on XpA in vivo as expected.

The TsixpA allele abolished the function of theoverlapping Xist geneIf the primary transcript of Xist from XpA was processed as weexpected, the splicing product would be functional and competentto induce X inactivation. To examine whether or not XpA couldundergo inactivation, males hemizygous for TsixpA were crossedwith wild-type females. Recovery of live female pups from thiscross would indicate that Xist on the mutated paternal X isfunctional, because Xist deficiency should cause failure of imprintedX inactivation and result in a selective loss of female embryos atearly postimplantation stages (Marahrens et al., 1997). Of 176offspring obtained from 43 litters, 174 were males and 2 werefemales (Fig. 3A). One of the two females was XO and theremaining one had inherited the mutated X. The strong bias towardmale pups suggested that Xist on the mutated X was dysfunctionaland therefore unable to induce X inactivation. When examined atE7.5, female embryos, although found at a reasonable ratio,exhibited abnormal morphology typical of those suffering from the

failure of imprinted X inactivation (Fig. 3B,C) (Marahrens et al.,1997). In the reciprocal crosses, the mutated X was maternallytransmitted to both male and female pups with the Mendelian ratio(Fig. 3D). Taking advantage of an X-linked GFP transgene (XGFP),the X inactivation pattern was examined in female embryosheterozygous for TsixpA. As shown in Fig. 3E, XpAXGFP femalesrecovered at E8.5 were virtually negative for GFP fluorescence,indicating that XGFP had been uniformly inactivated even in theembryonic lineage, where X inactivation should occur basically atrandom. Taking all these findings together, we concluded that theinsertion of the extra intron unexpectedly made the Xist genedysfunctional and that as a result, XpA was essentially defective inundergoing inactivation. Similarly, paternal transmission of XistIVS

resulted in embryonic lethality (data not shown), demonstrating thatthe function of Xist on XIVS was also impaired.

Loss of Tsix transcription in the Xist promoterregion results in ectopic activation of the XistgeneAlthough Xist on these mutated X chromosomes turned out to bedysfunctional, the TsixpA allele successfully terminated the antisensetranscription as expected. This allowed us to fully address the effectsbrought about by the loss of Tsix transcription in the Xist promoterregion on Xist silencing by using a previously described approach(Sado et al., 2005; Sado et al., 2006).

We first examined whether the Xist locus became ectopicallyactivated by the truncation of Tsix in E13.5 male embryos. Northernblotting of total RNA demonstrated that although wild-type maleembryos did not express Xist at all, a significant amount of Xist RNAwas expressed in XpAY embryos (Fig. 4A). We also carried out strand-specific RT-PCR using RNA isolated from the embryos proper(embryonic lineage) and the visceral endoderm (extraembryoniclineage) in both sexes recovered at E13.5 from TsixpA/+ femalescrossed with males carrying a Mus. m. molossinus (JF1)-derived Xchromosome (XJF1). A PvuII site polymorphism between thelaboratory strains and JF1 allowed us to assess the allelic expressionof Xist in females. As shown in Fig. 4B, Xist on the X chromosome of

231RESEARCH ARTICLEAntisense regulation at the Xist locus

Fig. 3. The Xist gene on XpA isdysfunctional. (A) The number of male andfemale pups born to wild-type females crossedwith males hemizygous for the TsixpA allele. Thesex ratio was extremely biased toward males.One of two females turned out to be XO.(B) The number of male and female embryosrecovered at E7.5 from wild-type femalescrossed with XpAY males. Two of the embryoswere genotyped as XXpAY. (C) Grossmorphology of the typical XXpA embryorecovered at E7.5 is shown together with thatof a male littermate. This phenotype is quitesimilar to that of females that inherit an Xist-deficient X from the father. e, embryonicectoderm; ec ectoplacental cone; rm, Reichert’smembrane. Scale bar: 0.5 mm. (D) Thenumbers of male and female pups born tofemales heterozygous for TsixpA crossed withwild-type males are shown. The TsixpA allele istransmitted to both male and female pups atthe expected ratio. (E) Females heterozygous for TsixpA were crossed with XGFPY males and embryos recovered at E8.5 were examined for GFPexpression. Although GFP fluorescence is uniformly observed in wild-type XXGFP embryos because cells that did not select XGFP as the inactive X aredistributed throughout the body, XpAXGFP embryos are substantially negative for GFP, indicating that XGFP is invariably inactivated. This demonstratesthat the Xist gene on XpA is dysfunctional. ys, yolk sac; am, amnion; al, allantois. Scale bar: 0.8 mm.

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the laboratory strain (Xlab) was predominantly expressed in theembryo proper of wild-type females, probably owing to the Xce effect,which is known to cause some bias in the choice of chromosome forX inactivation, whereas the great majority of Xist detected in thevisceral endoderm was derived from XJF1, in agreement withimprinted paternal X inactivation. In both XpAXJF1 and XIVSXJF1

females, the mutated X should remain active in every cell because ofthe inability of Xist to induce inactivation. Xist on the active X isusually repressed in somatic cells. However, although the Xist locuson the mutated X in XIVSXJF1 females was appropriately repressed, itwas ectopically activated in XpAXJF1 females in both embryonic andextraembryonic tissues (Fig. 4B). This was also the case in XpAY maleembryos (Fig. 4B). By contrast, such ectopic activation of Xist was notobserved on XIVS in either embryonic or extraembryonic lineages ineither sex (Fig. 4B). It should be noted that although Xist on themutated XpA was incompetent for inducing chromosomal inactivation,as mentioned above, the introduced intron was spliced out from theprimary transcript as expected (data not shown). Nonetheless, theseresults suggest that the premature termination of Tsix transcriptioncompromises the Tsix-mediated Xist silencing.

Since the truncation of Tsix might have distinct impact on theembryonic and extraembryonic tissues, we compared the levels ofectopically expressed Xist in the embryo proper and in the visceralendoderm of XpAY male embryos by real-time PCR. Intriguingly,

ectopic expression of Xist was reproducibly higher in the visceralendoderm than in the embryo proper (Fig. 4C), suggesting that theextraembryonic tissues were more susceptible than the embryonictissues to the truncation of Tsix in terms of Xist silencing. However,the ectopic expression in the visceral endoderm was about 30-40%of the level of Xist expression in the visceral endoderm of wild-typefemales. The reason why the Xist locus on XpA was not fullyactivated is currently unknown.

Loss of Tsix transcription across the Xist promoterregion results in reduced CpG methylation in the5� region of XistNext, we studied the effect of the premature termination of Tsix inexon 4 on the chromatin in the Xist promoter region. It is known thatCpG sites in the Xist promoter of the transcriptionally active alleleon the inactive X are largely unmethylated, whereas thetranscriptionally inactive allele on the active X is highly methylated(Norris et al., 1994). Our previous study demonstrated that Tsix isrequired for the establishment of CpG methylation in the Xistpromoter region (Sado et al., 2005). We therefore examined whetherthis Tsix function is impaired by the premature termination. Themethylation profile of the Xist promoter region was examined in theembryo proper and in the visceral endoderm at E13.5 by Southernblotting using four different methylation-sensitive restriction

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Fig. 4. The Xist locus is ectopically activated on the active XpA. (A) Total RNA prepared from E13.5 embryos was analyzed by northernblotting. Ectopic expression of Xist was evident from the single active X in XpAY male embryos. (B) Allelic expression of Xist was analyzed by RT-PCRin the embryo proper and the visceral endoderm. Females heterozygous for either TsixpA or XistIVS were crossed with JF1 males (XJF1Y), and RNAprepared from the embryo proper and visceral endoderm at E13.5 was subjected to RT-PCR on cDNA primed by Xist7(–)20 in a strand-specificmanner. PvuII digestion of the amplified product revealed that in spite of the fact that XpA stayed active in essentially every cell in XpAXJF1 femaleembryos, Xist was expressed from the active XpA as well as the inactivated XJF1 in both the embryo proper and the visceral endoderm (upper panel).Similar activation of the Xist locus on XpA was also seen in both tissues of XpAY males. Such ectopic activation was never observed on XIVS in eithertissue in either sex (lower panel). cDNA synthesis was carried out in the presence (+) or absence (–) of reverse transcriptase. (C) The levels of ectopicexpression of Xist in the embryo proper and the visceral endoderm of XpAY males (XpAY-1 and XpAY-2) were compared with those of Xist in wild-type XX and XY embryos by real-time PCR using cDNA synthesized in B. Xist and Gapd sequences were amplified using primer setR700P2/Xist6(–)20 and GapdF/Gapdr2, respectively, and the expression level was normalized by the value for Gapd. Values are means ± s.d.

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enzymes in combination with methylation-insensitive BclI.Although this region on XIVS, which stays active, was completelymethylated, like that on the wild-type active X, in both the embryoproper and visceral endoderm (see Fig. S2 in the supplementarymaterial), a reduction in methylation was evident on XpA (Fig. 5),which also stays active, especially in the extraembryonic tissues.Intriguingly, the decrease in CpG methylation was much moreprominent in the visceral endoderm than in the embryo proper inboth sexes, which is consistent with the difference in the level of Xistexpressed ectopically in these tissues. These results suggest that theloss of Tsix transcription across the Xist promoter region diminishesthe function of Tsix and causes a reduction in CpG methylation inthe 5� region of Xist, as was seen on Xdc (Sado et al., 2005).

Loss of Tsix transcription across the Xist promoterregion leads to aberrant histone modifications inthe 5� region of XistThe results described so far suggest that the premature terminationof Tsix, which apparently compromised the function of Tsix, hadmore profound effects on Xist silencing in the extraembryoniclineages than in the embryonic lineage. We therefore used chromatinprepared from the visceral endoderm of the male yolk sac to analyzethe histone modifications in the 5� region of Xist using the ChIPassay. We used monoclonal antibodies raised against histone H3 di-and trimethylated on Lys4 (H3K4me2 and H3K4me3, respectively),dimethylated on Lys9 (H3K9me2) and trimethylated on Lys27

(H3K27me3), whose specificities in the ChIP assay have beenextensively studied elsewhere (Kimura et al., personalcommunication). H3K4me2 and H3K4me3 are known to beassociated with transcriptionally active regions, and H3K9me2 andH3K27me3 with transcriptionally inactive regions. Real-time PCRwas performed to amplify the region immediately downstream ofthe transcription start site of Xist (+1 to +101 in GenBank Acc. No.L04961). On the wild-type active X in the visceral endoderm, thisregion was highly enriched in H3K9me2 and H3K27me3 but largelydevoid of H3K4me2 (Fig. 6), consistent with the fact that Xist isrepressed on the active X. The same region on the mutated active Xin XpAY showed an enrichment of H3K4me2 and exclusion ofH3K9me2 and H3K27me3 (Fig. 6). By contrast, this region inXIVSY possessed histone modifications similar to those on the wild-type X (Fig. 6). The distribution of H3K4me3 did not appear to beaffected by the presence or absence of Tsix, and stayed at a low level.These results suggest that it is the truncation of Tsix, not the presenceof the extra intron in exon 1 of Xist, that facilitated the activemodifications instead of the repressive ones in the 5� region of Xist.

DISCUSSIONAntisense transcription across the Xist promoteris crucial for Tsix-mediated Xist silencingIn this study we examined the functional significance of theantisense transcription across the Xist promoter in Tsix-mediated Xistsilencing. Premature termination of Tsix transcription in the Xist

233RESEARCH ARTICLEAntisense regulation at the Xist locus

Fig. 5. Methylation level of CpG sites in the 5� region of Xist is reduced on XpA. (A) The genomic structure of the 5� region of Xist on thewild-type X and XpA is shown along with recognition sites of the methylation-sensitive restriction enzymes and positions of probes used in thisassay. (B-D) The methylation level in the 5� region of Xist was examined by Southern blotting in the embryo proper and the visceral endoderm ofE13.5 embryos recovered from XpAX females crossed with wild-type males. Genomic DNA was digested with one of the four methylation-sensitiverestriction enzymes shown in A in combination with BclI and probed with either HS0.7 or BE0.6. BclI digestion produced an 8.4 kb (blackarrowhead shown on the right of the blot) and a 10.0-kb (white arrowhead shown on the right) band from the wild-type X and XpA, respectively.Although a decrease in CpG methylation was observed on XpA in both tissues of both male and female embryos, it was more prominent in thevisceral endoderm. Since the restriction fragments derived from XpA digested by HhaI and SacII, when probed with HS0.7, migrated together withthose from the wild-type X, the reduction of CpG methylation on XpA was less clear in the embryo proper of XpAX on the blot probed by HS0.7 (B),the same blot was reprobed with BE0.6 to visualize the fragments derived from XpA (indicated by asterisks), which appear only when the relevantrestriction sites are unmethylated (C). Probing with HS0.7 was sufficient to show hypomethylation of the locus in the visceral endoderm (D). Hh,HhaI; S, SacII; Hp, HpaII; and P, PmaCI.

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gene resulted in inappropriate activation of Xist in cis on the mutatedactive X in embryos and MEFs (data not shown), as was the case ona Tsix-deficient X (Sado et al., 2005). This was accompanied byreduced CpG methylation and aberrant histone modifications in the5� region of Xist, especially in the extraembryonic tissues. Bycontrast, neither ectopic activation of Xist nor aberrant chromatinmodifications were observed on the active X bearing XistIVS, whichretains the antisense transcription across the Xist promoter. Thesefindings strongly suggest that it was the transcriptional attenuationof Tsix, not the introduction of the exogenous DNA sequences,that compromised the function of Tsix in establishing thetranscriptionally repressive chromatin configuration in the 5� regionof Xist. However, we cannot completely exclude the possibility thatthe observed phenotype was caused by disruption of not only Tsixfunction but also Xist function.

The transcription of Tsix primarily initiates about 13 kbdownstream of Xist and extends as far as 50 kb along the entire Xistgene beyond the promoter region. Previous studies demonstratedthat the truncation of Tsix in the region downstream of Xist abolishesthe function of Tsix in both embryos and differentiating ES cells(Luikenhuis et al., 2001; Sado et al., 2001). Shibata and Lee furthersuggested that Tsix-mediated Xist silencing requires concurrentantiparallel transcription across the Xist gene (Shibata and Lee,2004). Here we have demonstrated that TsixpA, which still retainedthe transcription of the proximal 93% of the region encoding the Tsixgene, failed to establish Xist silencing, suggesting that Tsix has to betranscribed across the Xist promoter to establish silencing of Xist.Although Air and Kcnq1ot1 noncoding antisense RNAs have alsobeen suggested to be involved in chromatin modification, truncationof these RNAs impairs not only expression of the respective sensepartner but also allelic expression of neighboring imprinted genes inthe respective imprinted domains (Mancini-Dinardo et al., 2006;Sleutels et al., 2002). This implies that the actions of Tsix and thesenoncoding genes are distinct in terms of mediating the chromatinmodifications.

The extraembryonic lineages primarily depend onTsix to prevent Xist activationThe truncation of Tsix had distinct impact on the Xist silencingmechanism in the embryo proper versus the visceral endoderm,which belong to the embryonic and extraembryonic lineages,respectively. Real-time PCR revealed that the level of ectopicexpression of Xist was higher in the visceral endoderm than in theembryo proper. Although a reduction in CpG methylation in the 5�region of Xist was detected in both lineages, it was more prominentin the visceral endoderm than in the embryo proper in both sexes.Similarly, aberrant histone modifications were much more evident

in the visceral endoderm than in MEF (data not shown). It thereforeseemed likely that the truncation of Tsix had more profound effectson the Xist silencing mechanism in the extraembryonic tissues thanin the embryonic tissues. We recently suggested that the embryonictissues but not the extraembryonic tissues possess a mechanism toshut off Xist in a Tsix-independent manner if Xist becomesectopically expressed (Ohhata et al., 2006). Since the majority ofXist RNA transcribed from XpA is subject to the expected splicing ofthe intron introduced in exon 1, this transcript is almost the same asthat transcribed from the wild-type Xist allele, except for the additionof some flanking sequences of the intron. It is possible that thepresence of such transcripts activates the putative Tsix-independentXist silencing mechanism and induces some repressivemodifications in the Xist promoter region in the embryonic tissues.Since this mechanism is missing in the extraembryonic tissues, thevisceral endoderm would exhibit severer effects on Xist silencingthan the embryo proper or MEF as a result of the truncation of Tsix.

The TsixpA allele renders the overlapping Xist genedysfunctionalAlthough the truncation of Tsix inevitably altered the genomicstructure of the overlapping Xist gene, we assumed that the functionof Xist would be relieved if the exogenous sequence containing thempA cassette was removed by splicing. However, the TsixpA alleleaccordingly designed did not act in the expected way. Paternaltransmission of XpA resulted in lethality of female offspring at peri-implantation stages, probably owing to the failure of imprintedpaternal X inactivation in the extraembryonic lineages, suggestingthat the Xist on XpA was dysfunctional. Although the expectedsplicing event was observed in both the mutant male ES cells andthe visceral endoderm isolated from the XpAY embryos (data notshown), it is possible that the excision of this intron was not asefficient as we expected. The splicing products were retained in thenucleus, like those transcribed from the wild-type Xist allele, rulingout the possibility that the exogenous intron derived from theprotein-coding gene enhanced export of the transcripts to thecytoplasm. Another possibility is that the ectopic expression of Xiston XpA was not high enough to induce X inactivation. In fact, theectopic expression of Xist in the visceral endoderm of XpAYembryos was about 30-40% of the level of Xist expression in thevisceral endoderm of wild-type female embryos, and RNAfluorescent in situ hybridization (FISH) failed to detectaccumulation of Xist RNA in the mutant male MEFs (data notshown). Although we cannot rule out the possibility that theantisense transcription across the Xist promoter is not the sole eventresponsible for Tsix-mediated Xist silencing, the incompleteactivation of Xist could be due to the structural alteration of a

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Fig. 6. The Xist locus on XpA fails to establish the repressive histone modification. Chromatin of the visceral endoderm was used for ChIPassays with the antibodies against the respective histone modifications. The immunoprecipitated chromatin was quantified by real-time PCR usingXist1F and Xist101R as primers, which amplify the first 101 bp sequence of Xist exon 1. The value for each modification, which is shown as relativeabundance of the chromatin immunoprecipitated by each antibody compared with the input, was obtained from two or three independentexperiments. Values are means ± s.d.

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putative cis element required for the full activation of Xist. Theinsertion of the intron, in fact, increased the physical distancebetween the Xist promoter and the YY1 binding sites recentlyreported to be necessary for Xist expression (Kim et al., 2006). Thiscould have diminished the appropriate interaction between themrequired to fully activate Xist. It is also formally possible that thepresence of the extra sequence left after the expected splicing eventhas a negative impact on the function of Xist RNA. Further analyseswill shed light on the mechanism by which Xist becomes fullyactivated and mediates the chromosomal silencing upon thecessation of Tsix transcription on the same chromosome.

We are grateful to Hiroshi Kimura and Naohito Nozaki for sharing themonoclonal antibodies against histone modifications. We thank MinakoKanbayashi for genotyping of mice and maintenance of the mouse colonies.This work was supported by grants from PRESTO, Japan Science andTechnology Agency (JST) and from the Ministry of Education, Science, Sports,and Culture of Japan to T.S.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/135/2/227/DC1

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