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• W1557776005 abstract "•Tsix lncRNA does not affect primary choice during X-chromosome inactivation•Tsix prevents ectopic inactivation of the active-X during epiblast differentiation•Selection against cells that ectopically inactivate the XΔTsix biases X-inactivation•EpiSCs temporally capture embryonic epiblasts just after X-inactivation initiates Differentiating pluripotent epiblast cells in eutherians undergo random X-inactivation, which equalizes X-linked gene expression between the sexes by silencing one of the two X-chromosomes in females. Tsix RNA is believed to orchestrate the initiation of X-inactivation, influencing the choice of which X remains active by preventing expression of the antisense Xist RNA, which is required to silence the inactive-X. Here we profile X-chromosome activity in Tsix-mutant (XΔTsix) mouse embryonic epiblasts, epiblast stem cells, and embryonic stem cells. Unexpectedly, we find that Xist is stably repressed on the XΔTsix in both sexes in undifferentiated epiblast cells in vivo and in vitro, resulting in stochastic X-inactivation in females despite Tsix-heterozygosity. Tsix is instead required to silence Xist on the active-X as epiblast cells differentiate in both males and females. Thus, Tsix is not required at the onset of random X-inactivation; instead, it protects the active-X from ectopic silencing once X-inactivation has commenced. Differentiating pluripotent epiblast cells in eutherians undergo random X-inactivation, which equalizes X-linked gene expression between the sexes by silencing one of the two X-chromosomes in females. Tsix RNA is believed to orchestrate the initiation of X-inactivation, influencing the choice of which X remains active by preventing expression of the antisense Xist RNA, which is required to silence the inactive-X. Here we profile X-chromosome activity in Tsix-mutant (XΔTsix) mouse embryonic epiblasts, epiblast stem cells, and embryonic stem cells. Unexpectedly, we find that Xist is stably repressed on the XΔTsix in both sexes in undifferentiated epiblast cells in vivo and in vitro, resulting in stochastic X-inactivation in females despite Tsix-heterozygosity. Tsix is instead required to silence Xist on the active-X as epiblast cells differentiate in both males and females. Thus, Tsix is not required at the onset of random X-inactivation; instead, it protects the active-X from ectopic silencing once X-inactivation has commenced. X-chromosome inactivation results in the mitotically stable transcriptional silencing of genes along one of the two X-chromosomes in female mammals (Lyon, 1961Lyon M.F. Gene action in the X-chromosome of the mouse (Mus musculus L.).Nature. 1961; 190: 372-373Crossref PubMed Scopus (2567) Google Scholar). In the pluripotential mouse epiblast cells, which will form the embryo proper, the selection of which X to inactivate is random. Molecularly, random X-inactivation is posited to be controlled in cis by a pair of oppositely transcribed X-linked long non-coding (lnc) RNAs, Xist and Tsix (Barakat and Gribnau, 2012Barakat T.S. Gribnau J. X chromosome inactivation in the cycle of life.Development. 2012; 139: 2085-2089Crossref PubMed Scopus (39) Google Scholar). Xist RNA is believed to initiate epigenetic silencing of genes in cis by physically coating the X-chromosome from which it is transcribed and recruiting proteins that catalyze heterochromatin formation (Payer and Lee, 2008Payer B. Lee J.T. X chromosome dosage compensation: how mammals keep the balance.Annu. Rev. Genet. 2008; 42: 733-772Crossref PubMed Scopus (365) Google Scholar). Tsix transcription across the Xist promoter, conversely, is proposed to inhibit Xist expression (Lee, 2000Lee J.T. Disruption of imprinted X inactivation by parent-of-origin effects at Tsix.Cell. 2000; 103: 17-27Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Lee and Lu, 1999Lee J.T. Lu N. Targeted mutagenesis of Tsix leads to nonrandom X inactivation.Cell. 1999; 99: 47-57Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, Luikenhuis et al., 2001Luikenhuis S. Wutz A. Jaenisch R. Antisense transcription through the Xist locus mediates Tsix function in embryonic stem cells.Mol. Cell. Biol. 2001; 21: 8512-8520Crossref PubMed Scopus (156) Google Scholar, Navarro et al., 2005Navarro P. Pichard S. Ciaudo C. Avner P. Rougeulle C. Tsix transcription across the Xist gene alters chromatin conformation without affecting Xist transcription: implications for X-chromosome inactivation.Genes Dev. 2005; 19: 1474-1484Crossref PubMed Scopus (148) Google Scholar, Sado et al., 2001Sado T. Wang Z. Sasaki H. Li E. Regulation of imprinted X-chromosome inactivation in mice by Tsix.Development. 2001; 128: 1275-1286PubMed Google Scholar, Sado et al., 2005Sado T. Hoki Y. Sasaki H. Tsix silences Xist through modification of chromatin structure.Dev. Cell. 2005; 9: 159-165Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Because of its ability to repress Xist, the Tsix locus is postulated to be the site where molecular signals converge to help ensure that one X-chromosome remains active in both males and females (Clerc and Avner, 1998Clerc P. Avner P. Role of the region 3′ to Xist exon 6 in the counting process of X-chromosome inactivation.Nat. Genet. 1998; 19: 249-253Crossref PubMed Scopus (144) Google Scholar, Cohen et al., 2007Cohen D.E. Davidow L.S. Erwin J.A. Xu N. Warshawsky D. Lee J.T. The DXPas34 repeat regulates random and imprinted X inactivation.Dev. Cell. 2007; 12: 57-71Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, Debrand et al., 1999Debrand E. Chureau C. Arnaud D. Avner P. Heard E. Functional analysis of the DXPas34 locus, a 3′ regulator of Xist expression.Mol. Cell. Biol. 1999; 19: 8513-8525PubMed Google Scholar, Gontan et al., 2012Gontan C. Achame E.M. Demmers J. Barakat T.S. Rentmeester E. van IJcken W. Grootegoed J.A. Gribnau J. RNF12 initiates X-chromosome inactivation by targeting REX1 for degradation.Nature. 2012; 485: 386-390Crossref PubMed Scopus (145) Google Scholar, Lee, 2005Lee J.T. Regulation of X-chromosome counting by Tsix and Xite sequences.Science. 2005; 309: 768-771Crossref PubMed Scopus (117) Google Scholar, Luikenhuis et al., 2001Luikenhuis S. Wutz A. Jaenisch R. Antisense transcription through the Xist locus mediates Tsix function in embryonic stem cells.Mol. Cell. Biol. 2001; 21: 8512-8520Crossref PubMed Scopus (156) Google Scholar, Morey et al., 2004Morey C. Navarro P. Debrand E. Avner P. Rougeulle C. Clerc P. The region 3′ to Xist mediates X chromosome counting and H3 Lys-4 dimethylation within the Xist gene.EMBO J. 2004; 23: 594-604Crossref PubMed Scopus (65) Google Scholar, Navarro et al., 2010Navarro P. Oldfield A. Legoupi J. Festuccia N. Dubois A. Attia M. Schoorlemmer J. Rougeulle C. Chambers I. Avner P. Molecular coupling of Tsix regulation and pluripotency.Nature. 2010; 468: 457-460Crossref PubMed Scopus (145) Google Scholar, Stavropoulos et al., 2005Stavropoulos N. Rowntree R.K. Lee J.T. Identification of developmentally specific enhancers for Tsix in the regulation of X chromosome inactivation.Mol. Cell. Biol. 2005; 25: 2757-2769Crossref PubMed Scopus (67) Google Scholar, Vigneau et al., 2006Vigneau S. Augui S. Navarro P. Avner P. Clerc P. An essential role for the DXPas34 tandem repeat and Tsix transcription in the counting process of X chromosome inactivation.Proc. Natl. Acad. Sci. USA. 2006; 103: 7390-7395Crossref PubMed Scopus (68) Google Scholar). Investigations of mutations that reduce or abrogate Tsix RNA expression, however, have resulted in disparate outcomes. In differentiating male embryonic stem cells (ESCs), a cell culture model of X-inactivation some Tsix mutations display ectopic Xist induction, consistent with Tsix serving to inhibit Xist and thereby X-inactivation (Clerc and Avner, 1998Clerc P. Avner P. Role of the region 3′ to Xist exon 6 in the counting process of X-chromosome inactivation.Nat. Genet. 1998; 19: 249-253Crossref PubMed Scopus (144) Google Scholar, Debrand et al., 1999Debrand E. Chureau C. Arnaud D. Avner P. Heard E. Functional analysis of the DXPas34 locus, a 3′ regulator of Xist expression.Mol. Cell. Biol. 1999; 19: 8513-8525PubMed Google Scholar, Luikenhuis et al., 2001Luikenhuis S. Wutz A. Jaenisch R. Antisense transcription through the Xist locus mediates Tsix function in embryonic stem cells.Mol. Cell. Biol. 2001; 21: 8512-8520Crossref PubMed Scopus (156) Google Scholar, Morey et al., 2004Morey C. Navarro P. Debrand E. Avner P. Rougeulle C. Clerc P. The region 3′ to Xist mediates X chromosome counting and H3 Lys-4 dimethylation within the Xist gene.EMBO J. 2004; 23: 594-604Crossref PubMed Scopus (65) Google Scholar, Sado et al., 2002Sado T. Li E. Sasaki H. Effect of TSIX disruption on XIST expression in male ES cells.Cytogenet. Genome Res. 2002; 99: 115-118Crossref PubMed Scopus (26) Google Scholar, Vigneau et al., 2006Vigneau S. Augui S. Navarro P. Avner P. Clerc P. An essential role for the DXPas34 tandem repeat and Tsix transcription in the counting process of X chromosome inactivation.Proc. Natl. Acad. Sci. USA. 2006; 103: 7390-7395Crossref PubMed Scopus (68) Google Scholar). Other Tsix mutant male ESCs, though, do not exhibit Xist expression upon differentiation (Cohen et al., 2007Cohen D.E. Davidow L.S. Erwin J.A. Xu N. Warshawsky D. Lee J.T. The DXPas34 repeat regulates random and imprinted X inactivation.Dev. Cell. 2007; 12: 57-71Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, Lee, 2000Lee J.T. Disruption of imprinted X inactivation by parent-of-origin effects at Tsix.Cell. 2000; 103: 17-27Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Lee and Lu, 1999Lee J.T. Lu N. Targeted mutagenesis of Tsix leads to nonrandom X inactivation.Cell. 1999; 99: 47-57Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, Minkovsky et al., 2013Minkovsky A. Barakat T.S. Sellami N. Chin M.H. Gunhanlar N. Gribnau J. Plath K. The pluripotency factor-bound intron 1 of Xist is dispensable for X chromosome inactivation and reactivation in vitro and in vivo.Cell Rep. 2013; 3: 905-918Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The differences observed between the mutant ESC lines may reflect residual Tsix expression due to the incomplete ablation of Tsix or differences in the protocols employed to differentiate ESCs. Whereas ectopic X-inactivation may or may not occur in Tsix mutant males, the choice of which X to inactivate appears absolutely biased in Tsix-heterozygous females (Cohen et al., 2007Cohen D.E. Davidow L.S. Erwin J.A. Xu N. Warshawsky D. Lee J.T. The DXPas34 repeat regulates random and imprinted X inactivation.Dev. Cell. 2007; 12: 57-71Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, Kalantry and Magnuson, 2006Kalantry S. Magnuson T. The Polycomb group protein EED is dispensable for the initiation of random X-chromosome inactivation.PLoS Genet. 2006; 2: e66Crossref PubMed Scopus (94) Google Scholar, Lee, 2000Lee J.T. Disruption of imprinted X inactivation by parent-of-origin effects at Tsix.Cell. 2000; 103: 17-27Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Sado et al., 2001Sado T. Wang Z. Sasaki H. Li E. Regulation of imprinted X-chromosome inactivation in mice by Tsix.Development. 2001; 128: 1275-1286PubMed Google Scholar). In these mice, the Tsix-mutant X-chromosome is inactive in all cells of the differentiating epiblast lineage, which would otherwise undergo random X-inactivation. This bias in choice has been explained by the preferential induction of Xist from the Tsix-mutant X-chromosome prior to or at the onset of X-inactivation in the epiblast lineage. Despite the proposed models of Tsix function, the significance of Tsix RNA remains unclear in both males and females. In the course of a previous study, we noticed that the epiblast in XΔTsixY post-implantation embryos appeared to ectopically express Xist in the absence of Tsix (Maclary et al., 2014Maclary E. Buttigieg E. Hinten M. Gayen S. Harris C. Sarkar M.K. Purushothaman S. Kalantry S. Differentiation-dependent requirement of Tsix long non-coding RNA in imprinted X-chromosome inactivation.Nat. Commun. 2014; 5: 4209Crossref PubMed Scopus (32) Google Scholar). We therefore hypothesized that Tsix-heterozygous females might also aberrantly express Xist during development. Thus, an alternative explanation for the apparent lack of ectopic Xist expression and skewed X-inactivation in Tsix heterozygotes is that a secondary cell-selection effect rapidly removes cells with two inactive-Xs from the population. Because of the tight coupling of X-inactivation with epiblast differentiation (Monk and Harper, 1979Monk M. Harper M.I. Sequential X chromosome inactivation coupled with cellular differentiation in early mouse embryos.Nature. 1979; 281: 311-313Crossref PubMed Scopus (218) Google Scholar), ectopic silencing of the previously active XΔTsix may occur concurrently with or shortly after the initiation of random X-inactivation. Inactivation of both Xs in females would render the cells effectively nullizygous for many X-linked genes, thus compromising proliferation and viability. Later stage epiblast and ESC derivatives would therefore consist only of cells with an active WT X-chromosome. Here, we investigate Tsix function by profiling embryos harboring a Tsix null allele at the onset of random X-inactivation and by deriving Tsix hemizygous male and heterozygous female EpiSC and ESC lines. Random X-inactivation initiates in epiblast cells between embryonic day (E) 4.5–6.5 in mice, just as the pluripotential epiblast cells begin to differentiate (Gardner and Lyon, 1971Gardner R.L. Lyon M.F. X chromosome inactivation studied by injection of a single cell into the mouse blastocyst.Nature. 1971; 231: 385-386Crossref PubMed Scopus (93) Google Scholar, Kalantry and Magnuson, 2006Kalantry S. Magnuson T. The Polycomb group protein EED is dispensable for the initiation of random X-chromosome inactivation.PLoS Genet. 2006; 2: e66Crossref PubMed Scopus (94) Google Scholar, McMahon et al., 1983McMahon A. Fosten M. Monk M. X-chromosome inactivation mosaicism in the three germ layers and the germ line of the mouse embryo.J. Embryol. Exp. Morphol. 1983; 74: 207-220PubMed Google Scholar, Rastan, 1982Rastan S. Timing of X-chromosome inactivation in postimplantation mouse embryos.J. Embryol. Exp. Morphol. 1982; 71: 11-24PubMed Google Scholar). To examine the role of Tsix RNA at the onset of X-inactivation, we generated E5.25 post-implantation stage embryos that inherit either a WT or a Tsix-null maternal X-chromosome from Tsix-heterozygous females. The previously described Tsix mutation, TsixAA2Δ1.7 (herein referred to as XΔTsix) (Sado et al., 2001Sado T. Wang Z. Sasaki H. Li E. Regulation of imprinted X-chromosome inactivation in mice by Tsix.Development. 2001; 128: 1275-1286PubMed Google Scholar), terminates the Tsix transcript in exon 2 and also deletes the critical DXPas34 repeat thought to serve as a platform to drive Tsix expression (Figure 1A) (Cohen et al., 2007Cohen D.E. Davidow L.S. Erwin J.A. Xu N. Warshawsky D. Lee J.T. The DXPas34 repeat regulates random and imprinted X inactivation.Dev. Cell. 2007; 12: 57-71Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, Maclary et al., 2014Maclary E. Buttigieg E. Hinten M. Gayen S. Harris C. Sarkar M.K. Purushothaman S. Kalantry S. Differentiation-dependent requirement of Tsix long non-coding RNA in imprinted X-chromosome inactivation.Nat. Commun. 2014; 5: 4209Crossref PubMed Scopus (32) Google Scholar, Navarro et al., 2010Navarro P. Oldfield A. Legoupi J. Festuccia N. Dubois A. Attia M. Schoorlemmer J. Rougeulle C. Chambers I. Avner P. Molecular coupling of Tsix regulation and pluripotency.Nature. 2010; 468: 457-460Crossref PubMed Scopus (145) Google Scholar, Stavropoulos et al., 2005Stavropoulos N. Rowntree R.K. Lee J.T. Identification of developmentally specific enhancers for Tsix in the regulation of X chromosome inactivation.Mol. Cell. Biol. 2005; 25: 2757-2769Crossref PubMed Scopus (67) Google Scholar, Vigneau et al., 2006Vigneau S. Augui S. Navarro P. Avner P. Clerc P. An essential role for the DXPas34 tandem repeat and Tsix transcription in the counting process of X chromosome inactivation.Proc. Natl. Acad. Sci. USA. 2006; 103: 7390-7395Crossref PubMed Scopus (68) Google Scholar). Since transcription across the Xist promoter region is required for the Tsix RNA to inhibit Xist expression (Navarro et al., 2005Navarro P. Pichard S. Ciaudo C. Avner P. Rougeulle C. Tsix transcription across the Xist gene alters chromatin conformation without affecting Xist transcription: implications for X-chromosome inactivation.Genes Dev. 2005; 19: 1474-1484Crossref PubMed Scopus (148) Google Scholar, Sado et al., 2005Sado T. Hoki Y. Sasaki H. Tsix silences Xist through modification of chromatin structure.Dev. Cell. 2005; 9: 159-165Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), XΔTsix is a bona fide null Tsix mutation (Figure 1B) (Maclary et al., 2014Maclary E. Buttigieg E. Hinten M. Gayen S. Harris C. Sarkar M.K. Purushothaman S. Kalantry S. Differentiation-dependent requirement of Tsix long non-coding RNA in imprinted X-chromosome inactivation.Nat. Commun. 2014; 5: 4209Crossref PubMed Scopus (32) Google Scholar, Sado et al., 2001Sado T. Wang Z. Sasaki H. Li E. Regulation of imprinted X-chromosome inactivation in mice by Tsix.Development. 2001; 128: 1275-1286PubMed Google Scholar). We first tested whether the absence of Tsix RNA led to Xist induction in male epiblasts by RT-PCR. Whereas WT E5.25 XY epiblasts exhibited Tsix but not Xist expression, XΔTsixY epiblasts displayed the opposite pattern (Figure 1B). We next independently assessed Xist induction and X-inactivation in E5.25 XY and XΔTsixY epiblast cells by immunofluorescence (IF) coupled with RNA fluorescence in situ hybridization (RNA FISH). We first marked epiblast cells via IF detection of NANOG, which distinguishes the epiblast from the extra-embryonic cells (Figure 1C). In the same samples, using strand-specific RNA FISH probes, we also assayed expression of Tsix and Xist RNAs. In WT XY epiblasts, Tsix RNA signal but not Xist RNA coating was detectable from the sole X-chromosome (Figure 1C). In contrast, in XΔTsixY mutant embryos, ∼34% of the nuclei displayed Xist RNA coating (Figure 1C). Moreover, Xist coating resulted in the accumulation of histone H3 lysine 27 trimethylation (H3-K27me3), a chromatin mark catalyzed by the Polycomb repressive complex 2 that is associated with the inactive-X heterochromatin (Figure 1D) (Plath et al., 2003Plath K. Fang J. Mlynarczyk-Evans S.K. Cao R. Worringer K.A. Wang H. de la Cruz C.C. Otte A.P. Panning B. Zhang Y. Role of histone H3 lysine 27 methylation in X inactivation.Science. 2003; 300: 131-135Crossref PubMed Scopus (933) Google Scholar, Silva et al., 2003Silva J. Mak W. Zvetkova I. Appanah R. Nesterova T.B. Webster Z. Peters A.H. Jenuwein T. Otte A.P. Brockdorff N. Establishment of histone h3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 polycomb group complexes.Dev. Cell. 2003; 4: 481-495Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar) and accompanied silencing of the X-linked Pgk1 gene (Figure 1E). Thus, Tsix absence leads to Xist RNA induction and coating as well as gene silencing on the single X-chromosome in male epiblast cells. To further explore the requirement of Tsix in the epiblast, we derived WT XY and mutant XΔTsixY epiblast stem cells (EpiSCs; Figures S1A–S1C; Table S1). EpiSCs are thought to represent an early phase of X-inactivation (Bernemann et al., 2011Bernemann C. Greber B. Ko K. Sterneckert J. Han D.W. Araúzo-Bravo M.J. Schöler H.R. Distinct developmental ground states of epiblast stem cell lines determine different pluripotency features.Stem Cells. 2011; 29: 1496-1503Crossref PubMed Scopus (81) Google Scholar, Brons et al., 2007Brons I.G. Smithers L.E. Trotter M.W. Rugg-Gunn P. Sun B. Chuva de Sousa Lopes S.M. Howlett S.K. Clarkson A. Ahrlund-Richter L. Pedersen R.A. Vallier L. Derivation of pluripotent epiblast stem cells from mammalian embryos.Nature. 2007; 448: 191-195Crossref PubMed Scopus (1509) Google Scholar, Han et al., 2011Han D.W. Greber B. Wu G. Tapia N. Araúzo-Bravo M.J. Ko K. Bernemann C. Stehling M. Schöler H.R. Direct reprogramming of fibroblasts into epiblast stem cells.Nat. Cell Biol. 2011; 13: 66-71Crossref PubMed Scopus (98) Google Scholar, Pasque et al., 2011aPasque V. Gillich A. Garrett N. Gurdon J.B. Histone variant macroH2A confers resistance to nuclear reprogramming.EMBO J. 2011; 30: 2373-2387Crossref PubMed Scopus (113) Google Scholar, Pasque et al., 2011bPasque V. Halley-Stott R.P. Gillich A. Garrett N. Gurdon J.B. Epigenetic stability of repressed states involving the histone variant macroH2A revealed by nuclear transfer to Xenopus oocytes.Nucleus. 2011; 2: 533-539Crossref PubMed Scopus (23) Google Scholar, Tesar et al., 2007Tesar P.J. Chenoweth J.G. Brook F.A. Davies T.J. Evans E.P. Mack D.L. Gardner R.L. McKay R.D. New cell lines from mouse epiblast share defining features with human embryonic stem cells.Nature. 2007; 448: 196-199Crossref PubMed Scopus (1639) Google Scholar). If Tsix negatively regulates Xist in undifferentiated epiblast cells, EpiSCs lacking Tsix are expected to display aberrant Xist activation. In assaying Xist expression by RT-PCR, we found that Xist RNA was undetectable in the WT XY EpiSC lines (Figure 2A). In XΔTsixY EpiSC lines, however, Xist RNA was expressed at minimally detectable levels (Figure 2A). This low level of Xist expression may reflect the induction of Xist in the small fraction of differentiated cells that are often found in stem cell cultures. This notion prompted us to test whether Xist would be induced to high levels if we actively differentiated XΔTsixY EpiSCs (Figure S1D). Indeed, Xist expression in XΔTsixY but not XY cells increased markedly upon differentiation (Figure 2A). To examine whether the ectopic Xist expression coincided with coating of the X-chromosome, we performed Xist RNA FISH on undifferentiated and differentiated EpiSCs. As expected, neither undifferentiated nor differentiated XY EpiSC lines exhibited any Xist RNA-coated X-chromosomes (Figure 2B). In all four of the XΔTsixY EpiSC lines, we observed a similar lack of Xist RNA coating in the undifferentiated cells (Figure 2B). However, upon differentiation, a significant percentage of the mutant cells displayed Xist RNA coating (29%–35%; Figures 2B and 2C). As in E5.25 mutant epiblast cells, many Xist RNA-coated XΔTsixY cells still expressed NANOG (38%–42%) (Figure S1E). Xist RNA coating also resulted in the accumulation of histone H3-K27me3 and silencing of Pgk1 on the XΔTsix in a vast majority of the mutant cells (84%–94%) (Figures 2D and 2E). Together, the RT-PCR and RNA FISH data from XΔTsixY EpiSCs prompt the conclusion that Tsix RNA does not participate in repressing Xist in undifferentiated male EpiSCs. Instead, Tsix is required to prevent ectopic Xist induction and X linked gene silencing during the differentiation of male epiblast progenitor cells. That XΔTsixY EpiSCs displayed robust Xist induction only upon differentiation is incongruous with some previous studies with Tsix mutant male ESCs. Tsix deficiency in male ESCs is suggested to either be innocuous in both undifferentiated and differentiated cells (Cohen et al., 2007Cohen D.E. Davidow L.S. Erwin J.A. Xu N. Warshawsky D. Lee J.T. The DXPas34 repeat regulates random and imprinted X inactivation.Dev. Cell. 2007; 12: 57-71Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, Lee, 2000Lee J.T. Disruption of imprinted X inactivation by parent-of-origin effects at Tsix.Cell. 2000; 103: 17-27Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Lee and Lu, 1999Lee J.T. Lu N. Targeted mutagenesis of Tsix leads to nonrandom X inactivation.Cell. 1999; 99: 47-57Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, Minkovsky et al., 2013Minkovsky A. Barakat T.S. Sellami N. Chin M.H. Gunhanlar N. Gribnau J. Plath K. The pluripotency factor-bound intron 1 of Xist is dispensable for X chromosome inactivation and reactivation in vitro and in vivo.Cell Rep. 2013; 3: 905-918Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, Ohhata et al., 2006Ohhata T. Hoki Y. Sasaki H. Sado T. Tsix-deficient X chromosome does not undergo inactivation in the embryonic lineage in males: implications for Tsix-independent silencing of Xist.Cytogenet. Genome Res. 2006; 113: 345-349Crossref PubMed Scopus (27) Google Scholar, Sado et al., 2001Sado T. Wang Z. Sasaki H. Li E. Regulation of imprinted X-chromosome inactivation in mice by Tsix.Development. 2001; 128: 1275-1286PubMed Google Scholar, Sado et al., 2002Sado T. Li E. Sasaki H. Effect of TSIX disruption on XIST expression in male ES cells.Cytogenet. Genome Res. 2002; 99: 115-118Crossref PubMed Scopus (26) Google Scholar) or, conversely, result in ectopic Xist RNA coating of the Tsix-mutant X during differentiation (Debrand et al., 1999Debrand E. Chureau C. Arnaud D. Avner P. Heard E. Functional analysis of the DXPas34 locus, a 3′ regulator of Xist expression.Mol. Cell. Biol. 1999; 19: 8513-8525PubMed Google Scholar, Luikenhuis et al., 2001Luikenhuis S. Wutz A. Jaenisch R. Antisense transcription through the Xist locus mediates Tsix function in embryonic stem cells.Mol. Cell. Biol. 2001; 21: 8512-8520Crossref PubMed Scopus (156) Google Scholar, Morey et al., 2004Morey C. Navarro P. Debrand E. Avner P. Rougeulle C. Clerc P. The region 3′ to Xist mediates X chromosome counting and H3 Lys-4 dimethylation within the Xist gene.EMBO J. 2004; 23: 594-604Crossref PubMed Scopus (65) Google Scholar, Navarro and Avner, 2010Navarro P. Avner P. An embryonic story: analysis of the gene regulative network controlling Xist expression in mouse embryonic stem cells.BioEssays. 2010; 32: 581-588Crossref PubMed Scopus (26) Google Scholar, Vigneau et al., 2006Vigneau S. Augui S. Navarro P. Avner P. Clerc P. An essential role for the DXPas34 tandem repeat and Tsix transcription in the counting process of X chromosome inactivation.Proc. Natl. Acad. Sci. USA. 2006; 103: 7390-7395Crossref PubMed Scopus (68) Google Scholar). We therefore derived XY and XΔTsixY ESC lines (Figure S1) and tested Xist induction in both undifferentiated and differentiated cells by RT-PCR and RNA FISH. As with EpiSCs, we found that Xist remained silenced in undifferentiated XY as well as in XΔTsixY ESCs (Figures 2F and 2G); however, upon differentiation, Xist RNA was induced in XΔTsixY but not XY ESCs (Figures 2F–2H). To distinguish whether Xist induction in XΔTsixY ESCs occurred at the onset of differentiation or later, we transiently differentiated the ESCs into epiblast-like cells (EpiLCs) (Hayashi et al., 2011Hayashi K. Ohta H. Kurimoto K. Aramaki S. Saitou M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.Cell. 2011; 146: 519-532Abstract Full Text Full Text PDF PubMed Scopus (870) Google Scholar). EpiLCs arise early during ESC differentiation and share key features with EpiSCs (Figures S2A–S2C) (Buecker et al., 2014Buecker C. Srinivasan R. Wu Z. Calo E. Acampora D. Faial T. Simeone A. Tan M. Swigut T. Wysocka J. Reorganization of enhancer patterns in transition from naive to primed pluripotency.Cell Stem Cell. 2014; 14: 838-853Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). We found that the mutant EpiLCs displayed low-level Xist expression by RT-PCR, with only a few cells displaying Xist RNA coating (10%) (Figures S2C–S2E). The Xist RNA-coated cells appeared to have differentiated beyond the EpiLC state, as suggested by reduced NANOG expression (Figure S2E). When the EpiLCs were differentiated further, significantly more cells displayed Xist RNA coating (27%–36%) (Figure S2F), consistent with the EpiSC data. We next examined the impact of the XΔTsix mutation in females. The two X-chromosomes in inbred XX epiblast cells are normally equally likely to undergo inactivation; in heterozygous Tsix mutant epiblasts, however, previous work has concluded that only the XΔTsix X-chromosome is chosen for inactivation (Lee, 2000Lee J.T. Disruption of imprinted X inactivation by parent-of-origin effects at Tsix.Cell. 2000; 103: 17-27Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Sado et al., 2001Sado T. Wang Z. Sasaki H. Li E. Regulation of imprinted X-chromosome inactivation in mice by Tsix.Development. 2001; 128: 1275-1286PubMed Google Scholar). This model of biased inactivation in favor of the XΔTsix is borne out by allele-specific Xist RT-PCR analyses of F1 hybrid WT and Tsix-heterozygous E6.5 epiblasts (Figures 3A and 3B ). The X-chromosomes in these embryos are derived from two divergent mouse strains and are polymorphic, thereby allowing allele-specific expression analysis. Both Sanger sequencing (Figure 3A) and Pyrosequencing (Figure 3B), which quantifies allele-specific expression, of the cDNAs revealed that Xist is transcribed from either X in WT XLabXJF1 and XJF1XLab embryos (by convention, the maternal allele precedes the paternal allele), whereas in XΔTsixXJF1 and XJF1XΔTsix epiblasts, Xist is expressed almost exclusively from the XΔTsix. To evaluate the expression of Xist and Tsix in XX, XΔTsixX and XXΔTsix E6.5 epiblasts at the single-cell resolution, we performed strand-specific RNA FISH. As with male embryos, we again confirmed the identity of epiblast cells by first assaying expression of NANOG by IF. We observed Xist RNA coating of both Xs by RNA FISH in a small fraction of XΔTsixX and XXΔTsix mutant (∼2%), but not WT XX E6.5 epiblast cells (Figure 3C). Based on this observation and the hypothesis that cells with ectopic inactivation" @default.
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• W1557776005 date "2015-05-01" @default.
• W1557776005 modified "2023-10-13" @default.
• W1557776005 title "A Primary Role for the Tsix lncRNA in Maintaining Random X-Chromosome Inactivation" @default.
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• W1557776005 doi "https://doi.org/10.1016/j.celrep.2015.04.039" @default.
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