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• W2801467871 abstract "•Ftx is required for Xist accumulation and XCI during mouse ESC differentiation•Ftx acts in cis to promote Xist transcriptional activation•Ftx transcription, but not Ftx noncoding transcripts, is required for Xist regulation Accumulation of the Xist long noncoding RNA (lncRNA) on one X chromosome is the trigger for X chromosome inactivation (XCI) in female mammals. Xist expression, which needs to be tightly controlled, involves a cis-acting region, the X-inactivation center (Xic), containing many lncRNA genes that evolved concomitantly to Xist from protein-coding ancestors through pseudogeneization and loss of coding potential. Here, we uncover an essential role for the Xic-linked noncoding gene Ftx in the regulation of Xist expression. We show that Ftx is required in cis to promote Xist transcriptional activation and establishment of XCI. Importantly, we demonstrate that this function depends on Ftx transcription and not on the RNA products. Our findings illustrate the multiplicity of layers operating in the establishment of XCI and highlight the diversity in the modus operandi of the noncoding players. Accumulation of the Xist long noncoding RNA (lncRNA) on one X chromosome is the trigger for X chromosome inactivation (XCI) in female mammals. Xist expression, which needs to be tightly controlled, involves a cis-acting region, the X-inactivation center (Xic), containing many lncRNA genes that evolved concomitantly to Xist from protein-coding ancestors through pseudogeneization and loss of coding potential. Here, we uncover an essential role for the Xic-linked noncoding gene Ftx in the regulation of Xist expression. We show that Ftx is required in cis to promote Xist transcriptional activation and establishment of XCI. Importantly, we demonstrate that this function depends on Ftx transcription and not on the RNA products. Our findings illustrate the multiplicity of layers operating in the establishment of XCI and highlight the diversity in the modus operandi of the noncoding players. X chromosome inactivation (XCI) is a chromosome-wide regulatory process that ensures dosage compensation for X-linked gene expression between female (XX) and male (XY) mammals. This is achieved through the transcriptional inactivation of a single X chromosome in females. XCI is a paradigm for developmentally regulated processes involving long noncoding RNAs (lncRNAs). Xist was one of the first long noncoding transcripts to be discovered in the early 90s (Borsani et al., 1991Borsani G. Tonlorenzi R. Simmler M.-C. Dandolo L. Arnaud D. Capra V. Grompe M. Pizzuti A. Muzny D. Lawrence C. et al.Characterization of a murine gene expressed from the inactive X chromosome.Nature. 1991; 351: 325-329Crossref PubMed Scopus (460) Google Scholar, Brockdorff et al., 1991Brockdorff N. Ashworth A. Kay G.F. Cooper P. Smith S. McCabe V.M. Norris D.P. Penny G.D. Patel D. Rastan S. Conservation of position and exclusive expression of mouse Xist from the inactive X chromosome.Nature. 1991; 351: 329-331Crossref PubMed Scopus (520) Google Scholar), and while its crucial function in XCI has been rapidly demonstrated, it took much longer to start uncovering its mode of action as an RNA molecule. Xist is expressed from the future inactive X chromosome (Xi), where it accumulates in cis, forming a shield-like structure that protects the chromosome from being actively transcribed. Xist interacts with a plethora of protein partners that convey its silencing ability by modifying the architecture of the chromosome and its positioning within the nucleus (da Rocha and Heard, 2017da Rocha S.T. Heard E. Novel players in X inactivation: insights into Xist-mediated gene silencing and chromosome conformation.Nat. Struct. Mol. Biol. 2017; 24: 197-204Crossref PubMed Scopus (96) Google Scholar, Mira-Bontenbal and Gribnau, 2016Mira-Bontenbal H. Gribnau J. New Xist-interacting proteins in X-chromosome inactivation.Curr. Biol. 2016; 26: 1383Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Female-specific, monoallelic Xist upregulation is controlled by a balanced combination of repressors and activators, including trans-acting factors and cis-acting sequences. The latter constitute the X inactivation center (Xic), a region surrounding and encompassing the Xist gene and identified as being both necessary and sufficient to trigger XCI (Augui et al., 2011Augui S. Nora E.P. Heard E. Regulation of X-chromosome inactivation by the X-inactivation centre.Nat. Rev. Genet. 2011; 12: 429-442Crossref PubMed Scopus (268) Google Scholar). A notable feature of the Xic is that it is especially enriched in loci producing lncRNAs including, in addition to Xist, its antisense Tsix and the neighboring genes Linx, Jpx, and Ftx (Chureau et al., 2002Chureau C. Prissette M. Bourdet A. Barbe V. Cattolico L. Jones L. Eggen A. Avner P. Duret L. Comparative sequence analysis of the X-inactivation center region in mouse, human, and bovine.Genome Res. 2002; 12: 894-908Crossref PubMed Google Scholar, Lee et al., 1999Lee J.T. Davidow L.S. Warshawsky D. Tsix, a gene antisense to Xist at the X-inactivation centre.Nat. Genet. 1999; 21: 400-404Crossref PubMed Scopus (651) Google Scholar, Nora et al., 2012Nora E.P. Lajoie B.R. Schulz E.G. Giorgetti L. Okamoto I. Servant N. Piolot T. van Berkum N.L. Meisig J. Sedat J. et al.Spatial partitioning of the regulatory landscape of the X-inactivation centre.Nature. 2012; 485: 381-385Crossref PubMed Scopus (1828) Google Scholar). Most of these noncoding loci, including Xist, evolved from protein-coding genes by pseudogenization, in which reorganization of the loci and insertion of various repeat elements led to the loss of the original coding potential and to the emergence of novel regulatory roles converging toward the control of XCI (Chureau et al., 2002Chureau C. Prissette M. Bourdet A. Barbe V. Cattolico L. Jones L. Eggen A. Avner P. Duret L. Comparative sequence analysis of the X-inactivation center region in mouse, human, and bovine.Genome Res. 2002; 12: 894-908Crossref PubMed Google Scholar, Furlan and Rougeulle, 2016Furlan G. Rougeulle C. Function and evolution of the long noncoding RNA circuitry orchestrating X-chromosome inactivation in mammals.Wiley Interdiscip. Rev. RNA. 2016; 7: 702-722Crossref PubMed Scopus (33) Google Scholar). LncRNA loci are in general rapidly evolving, and their plasticity is thought to contribute to the diversification of their functions and/or mechanisms of action (Furlan and Rougeulle, 2016Furlan G. Rougeulle C. Function and evolution of the long noncoding RNA circuitry orchestrating X-chromosome inactivation in mammals.Wiley Interdiscip. Rev. RNA. 2016; 7: 702-722Crossref PubMed Scopus (33) Google Scholar). How noncoding elements of the Xic regulate Xist expression and XCI is, however, still incompletely understood. In particular, apart from the case of Xist itself, it remains mostly unclear whether lncRNA loci exert their function through lncRNA products, the act of transcription, or associated regulatory DNA elements. Functions of noncoding elements from within the Xic have been mostly studied in the mouse. In this model, the Xic is partitioned, both linearly and spatially, into two topologically associating domains (TADs), whose border maps in between the Xist and Tsix promoters. These two adjacent chromosomal neighborhoods segregate XCI activators (Xist TAD) from XCI repressors (Tsix TAD) (Nora et al., 2012Nora E.P. Lajoie B.R. Schulz E.G. Giorgetti L. Okamoto I. Servant N. Piolot T. van Berkum N.L. Meisig J. Sedat J. et al.Spatial partitioning of the regulatory landscape of the X-inactivation centre.Nature. 2012; 485: 381-385Crossref PubMed Scopus (1828) Google Scholar). At the onset of differentiation, Tsix is required to prevent Xist upregulation in cis, from the future active X (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 (396) 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 (159) Google Scholar, Morey et al., 2001Morey C. Arnaud D. Avner P. Clerc P. Tsix-mediated repression of Xist accumulation is not sufficient for normal random X inactivation.Hum. Mol. Genet. 2001; 10: 1403-1411Crossref PubMed Scopus (64) 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: 1275-1286Crossref PubMed Google Scholar). There are several hypotheses regarding the mechanism of Tsix action, but the prevalent view is that Tsix expression ultimately results in the accumulation of repressive histone modifications at the Xist promoter (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 (150) Google Scholar, Navarro et al., 2006Navarro P. Page D.R. Avner P. Rougeulle C. Tsix-mediated epigenetic switch of a CTCF-flanked region of the Xist promoter determines the Xist transcription program.Genes Dev. 2006; 20: 2787-2792Crossref PubMed Scopus (109) 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 (188) Google Scholar). Whether Tsix RNAs or Tsix transcription mediates the deposition of such chromatin marks remains to be clarified. The noncoding locus Linx has also been proposed to play a negative role in the regulation of XCI, possibly acting through Tsix (Nora et al., 2012Nora E.P. Lajoie B.R. Schulz E.G. Giorgetti L. Okamoto I. Servant N. Piolot T. van Berkum N.L. Meisig J. Sedat J. et al.Spatial partitioning of the regulatory landscape of the X-inactivation centre.Nature. 2012; 485: 381-385Crossref PubMed Scopus (1828) Google Scholar). Within the Xist TAD, the noncoding gene Jpx is upregulated concomitantly with Xist induction (Johnston et al., 2002Johnston C.M. Newall A.E. Brockdorff N. Nesterova T.B. Enox, a novel gene that maps 10 kb upstream of Xist and partially escapes X inactivation.Genomics. 2002; 80: 236-244Crossref PubMed Scopus (49) Google Scholar, Tian et al., 2010Tian D. Sun S. Lee J.T. The long noncoding RNA, Jpx, is a molecular switch for X chromosome inactivation.Cell. 2010; 143: 390-403Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). Jpx is thought to act through its RNA, which was suggested to activate Xist in a dose-dependent manner by evicting the CTCF repressor from the Xist promoter (Sun et al., 2013Sun S. Del Rosario B.C. Szanto A. Ogawa Y. Jeon Y. Lee J.T. Jpx RNA activates Xist by evicting CTCF.Cell. 2013; 153: 1537-1551Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Here, we investigate the function of Ftx, a noncoding locus lying upstream of Jpx within the Xist TAD. Ftx produces various nuclear lncRNA isoforms generated through complex combinations of alternative promoter usage, termination sites, and splicing events. Ftx also contains a microRNA (miR) cluster of unknown function within one intron (Chureau et al., 2011Chureau C. Chantalat S. Romito A. Galvani A. Duret L. Avner P. Rougeulle C. Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region.Hum. Mol. Genet. 2011; 20: 705-718Crossref PubMed Scopus (172) Google Scholar). We have previously shown that the deletion of Ftx results in decreased Xist expression in male cells (Chureau et al., 2011Chureau C. Chantalat S. Romito A. Galvani A. Duret L. Avner P. Rougeulle C. Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region.Hum. Mol. Genet. 2011; 20: 705-718Crossref PubMed Scopus (172) Google Scholar). We now reveal that Ftx is required for robust Xist upregulation and proper XCI progression during the differentiation of female embryonic stem cells (ESCs). We moreover provide evidence that Ftx function is independent of its lncRNA products or of Ftx-embedded miRs, and that Ftx transcription is required, in cis, for Xist transcriptional activation at the onset of differentiation. By identifying Ftx as a regulator of Xist, these results highlight the diversity of mechanisms employed by noncoding players of the Xic to regulate the establishment of XCI. In order to evaluate the contribution of Ftx to XCI dynamics in females, we generated mutant mouse ESC (mESC) lines (LF2 cell line) in which we deleted a 9-kb region including the three putative promoters of Ftx using the CRISPR/Cas9 technology (Figure 1A). Female mESCs recapitulate random XCI upon differentiation, and are thus a model of choice to explore XCI dynamics and identify XCI regulators. We isolated four independent clones, two heterozygous (Ftx+/− a and b) and two homozygous (Ftx−/− A and B), for the deletion. We confirmed the presence of two X chromosomes by DNA-FISH (fluorescence in situ hybridization) (Figure S1A) and verified Ftx targeting by PCR and qPCR using primer pairs within and outside the deleted region (Figures S1B and S1C). Sequencing of PCR products allowed us to precisely characterize the limits of the deletion in each clone and indicated that the corresponding region on the non-targeted Ftx allele is globally intact in both heterozygous clones (Figure S1D). Quantification and visualization of Ftx expression by qRT-PCR and RNA-FISH, respectively, showed that the Ftx promoter deletion completely abrogated Ftx transcription from the targeted chromosome (Figures S1E and S1F). While assessing Ftx expression patterns at the single-cell level by RNA-FISH, we observed that a high proportion of Ftx-positive cells (60%) displayed multiple discrete foci in the nuclear space (Figure S1F), suggesting that the Ftx RNA can be found beyond its transcription site. This pattern seems to be specific, since it was not observed in Ftx−/− cells. Whether these foci co-localize with particular chromatin structures or are located inside specific subnuclear domains is currently unknown. All female cell lines, irrespective of their genotype, appeared morphologically undistinguishable at the ESC state (Figure S1G). However, upon differentiation (from day 3 onward), both Ftx+/− and Ftx−/− mutant lines started to display significant levels of cell death accompanied by a lack of isolated, sharp-edged cells that characterize differentiated populations (Figure 1B). Flow cytometry assessment of the number of dead and living cells in each cell line revealed a high percentage of cell death upon differentiation of Ftx mutant lines (up to 30% of dead cells) compared to wild-type (Figure 1B). Both wild-type and mutant female cells, however, showed a similar downregulation of the pluripotency markers Nanog, Klf4, and Rex1 during the course of differentiation, excluding the possibility of a marked differentiation delay of the surviving Ftx mutant cells (Figure S1H). The lack of such cell death during differentiation of male Ftx−/Y mESCs (Chureau et al., 2011Chureau C. Chantalat S. Romito A. Galvani A. Duret L. Avner P. Rougeulle C. Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region.Hum. Mol. Genet. 2011; 20: 705-718Crossref PubMed Scopus (172) Google Scholar) further indicated that Ftx is not required, per se, for pluripotency exit (Figure 1B). The female-restricted nature of the defect observed during differentiation suggested a link to XCI. To test this hypothesis, we monitored by qRT-PCR and RNA-FISH the level of Xist expression and the percentage of cells showing an Xist accumulation in differentiating Ftx mutant female cell lines compared to wild-type cells (Figures 1C and 1D). In Ftx+/+ cells, Xist was efficiently upregulated (∼10 times at day 4 compared to day 0) and accumulated in ∼32% of nuclei at day 4 of differentiation. In contrast, 15%–18% of Ftx+/− and only 2%–4% of Ftx−/− cells displayed an Xist cloud at this time point. This phenotype persisted later on during differentiation, suggesting that Xist accumulation is blocked rather than just delayed in the mutant lines (day 6, Figure 1D; day 9, data not shown). Although Xist can still be upregulated in a small proportion of Ftx null cells, its accumulation pattern, as detected by RNA-FISH, drastically differed from that of wild-type cells. Among the Ftx−/− cells that we scored as Xist positive, a vast proportion (up to 70%) displayed abnormal Xist domains (Figure 2A). We carefully assessed the size of the Xist RNA territory and its sphericity and concluded that Ftx−/− cells harbored larger and less compact Xist clouds compared to wild-type Xist domains (Figure 2A). We next determined the ability of these abnormal Xist clouds to trigger XCI. PRC2-mediated deposition of H3K27me3 is a hallmark of XCI and one of the earliest events triggered by Xist, contributing to the extensive remodeling of the chromatin structure of the Xi (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 (529) Google Scholar). Indeed, the vast majority of Ftx+/+ cells (∼85%) displayed H3K27me3 accumulation that co-localized with the Xist RNA signal as detected by immunostaining followed by RNA-FISH. In striking contrast, in most Ftx−/− cells, the nuclear territory occupied by Xist was devoid of H3K27me3 enrichment (Figure 2B), suggesting that, in these cells, the abnormal Xist cloud fails to recruit chromatin-modifying activities and is, therefore, not entirely functional. This conclusion was further supported by RNA-FISH analyses investigating the transcriptional status of a selection of X-linked genes, located at various positions along the X chromosome (Abcd1, centromeric; Taf1, central; Huwe1, telomeric) in differentiating mESCs (Figure 2C). In Ftx−/− cells, active transcription of all three genes was detected from the Xist-coated chromosome in a higher percentage of cells compared to wild-type, indicating that the X chromosome is not efficiently silenced in these cells (Figures 2C, S2A, and S2B). These results indicate that Ftx is required for proper Xist expression dynamics and for efficient XCI progression during female mESC differentiation. To gain further insight into the modalities of the Ftx-mediated Xist regulation, we analyzed the expression patterns of Xist relative to Ftx by RNA-FISH during mESC differentiation. In Ftx+/+ cells, Ftx transcription was detected at the Xist-coated chromosome in ∼84% of Xist-positive cells, with ∼70% and ∼14% of the cells showing biallelic or monoallelic Ftx transcription, respectively (Figure 3A). This confirms previous observations that Ftx escapes XCI (Chureau et al., 2011Chureau C. Chantalat S. Romito A. Galvani A. Duret L. Avner P. Rougeulle C. Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region.Hum. Mol. Genet. 2011; 20: 705-718Crossref PubMed Scopus (172) Google Scholar), and may suggest that Ftx is required in cis for Xist accumulation. In differentiating Ftx+/− cells, Ftx transcription still occurred in the majority of cells (∼80%) where it was essentially monoallelic, in agreement with the heterozygous nature of the deletion. We thus used the Ftx pinpoint as a marker of non-targeted alleles. Since in the majority of Xist-positive cells (∼80%) Xist coating preferentially occurred on the Ftx-transcribing, non-targeted X chromosome, we concluded that Ftx likely regulates Xist in cis. Of note, in the small number of cells (5%–9%) showing an Xist accumulation away from the Ftx-transcribing X, the cloud appeared abnormal, suggesting that, when XCI is initiated from the mutated chromosome, the lack of Ftx prevents a normal progression of Xist accumulation (Figure S3A). To confirm the cis-effect, we reproduced the Ftx deletion in hybrid female mESCs (PGK12.1 cell line) carrying X chromosomes from different genetic backgrounds (X129/XPgk) to benefit from SNPs allowing for allelic discrimination. We isolated three independent clones—one homozygous and two heterozygous—for the deletion. We verified the presence of two Xs of each genetic background in these clones prior to and during differentiation using DNA-FISH and allelic genomic qPCR (Figures S3B and S3C) and checked the targeting specificity by PCR and sequencing as previously described for the LF2 clones (Figures S3D and S3E). Allele-specific qPCR analysis showed that either the X129 or the XPgk had been targeted in each heterozygous clone (Figure S3F). Small deletions or point mutations, which do not affect Ftx expression, also occurred on opposite Ftx alleles in each clone (Figures S3E and S3G). As previously observed in the LF2 cell line, Ftx+/− and Ftx−/− PGK12.1 mESCs displayed high mortality rates upon differentiation compared to wild-type cells (Figure S3H) but normal dynamics of pluripotency marker repression (Figure S3I). In addition, Xist expression and RNA accumulation appeared significantly reduced both in Ftx+/− and, more severely, in Ftx−/− cells, further supporting a role for Ftx in Xist upregulation (Figures S3J and S3K). Importantly, in Ftx+/− cells, allelic qRT-PCR analysis showed that Xist expression is unambiguously skewed (70%/30%) toward the non-targeted X chromosome (X129 in ΔFtxPgk cells and XPgk in ΔFtx129 cells), indicating that Ftx mostly regulates Xist in cis (Figure 3B). In support of this conclusion, the residual Xist expression found in Ftx−/− cells originated from either chromosome, thereby reproducing the 50%/50% allelic ratio observed in wild-type cells (Figure 3B). In principle, Ftx could either regulate Xist at the transcriptional level so that, upon Ftx mutation, fewer Xist RNA molecules are produced, or affect Xist accumulation post-transcriptionally by interfering with Xist RNA splicing, stabilization, tethering to the Xi, or spreading. To discriminate between these two possibilities, we compared the profiles of Xist ongoing transcription and of mature Xist RNA accumulation in wild-type and Ftx−/− cells by RNA-FISH, using two sets of strand-specific oligonucleotide probes distinguishing unspliced from spliced transcripts. “Xist IN” probe set specifically detects Xist introns, allowing us to monitor Xist ongoing transcription, while “Xist EX” labels Xist exons and thus mainly reveals the accumulation of Xist spliced forms (Figure 3C). As expected from previous RNA-FISH analyses, with the Xist EX probe set, we observed a strong reduction (∼20% compared to ∼40%) of the percentage of Xist-positive cells in Ftx−/− clones compared to wild-type after 4 days of differentiation (Figure 3C). Likewise, the percentage of cells with Xist IN signals dropped from ∼37% in wild-type cells to 15%–20% in Ftx mutant cells, suggesting that Xist transcription is impaired when Ftx is absent. Thus, Ftx most probably impacts Xist accumulation by regulating Xist transcription. To test whether the regulation of Xist by Ftx is mediated by Ftx lncRNAs, we used a knockdown (KD) approach with LNA-Gapmers, which are known to efficiently target nuclear RNAs. Another advantage of this approach, in contrast to inserting a transcription stop signal, is to dissect out effect of mature RNAs from that of transcription, without the slightest modification at the genomic level. We designed LNA-Gapmers (locked nucleic acid, oligonucleotides antisense to target RNAs) targeting Ftx exon 8, which is conserved across evolution and present in all known Ftx isoforms (Chureau et al., 2011Chureau C. Chantalat S. Romito A. Galvani A. Duret L. Avner P. Rougeulle C. Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region.Hum. Mol. Genet. 2011; 20: 705-718Crossref PubMed Scopus (172) Google Scholar) (Figure 4A). We first carried out Ftx KD in mouse embryonic fibroblasts (MEFs), a context in which XCI has already been established and in which Xist accumulation is, therefore, found in the vast majority of cells. Female MEFs transfected either with non-targeting (nt) control LNA-Gapmer or with Ftx-specific LNA-Gapmers appeared morphologically indistinguishable, despite Ftx spliced transcripts being significantly reduced in cells treated with Ftx LNA-Gapmers specifically (Figures S4A and S4B). Both Xist RNA level, as measured by qRT-PCR, and the percentage of cells displaying an Xist domain in RNA-FISH remained unchanged upon Ftx KD (Figures S4C and S4D). This suggests that Xist accumulation on the Xi is properly maintained in the absence of Ftx transcripts. To investigate the contribution of Ftx transcripts to XCI establishment, we performed a similar KD experiment in differentiating mESCs. Cells were transfected either with the nt LNA-Gapmer or with Ftx-specific LNA-Gapmers just prior to differentiation and analyzed 3.5 days later, a differentiation time point that corresponds to the peak of Ftx expression and to the initiation of XCI. Importantly, Ftx KD appeared efficient from day 0 onward, targeting mostly mature Ftx transcripts, but not unspliced Ftx transcripts as assessed by trans-exonic and intronic qRT-PCR assays (Figures 4B, S4E, and S4F). Unlike what we observed in Ftx deleted cells, mESCs that have been knocked down for Ftx did not show any increased cell death or morphological changes upon differentiation (Figure S4G). Pluripotency factor repression, as assessed by qRT-PCR, occurred similarly upon Ftx KD and in control conditions, indicating a normal progression of differentiation in cells with reduced levels of Ftx lncRNAs (Figure S4H). Consistent with these observations, Xist upregulation measured by qRT-PCR and the number of cells showing Xist accumulation in RNA-FISH did not change significantly upon differentiation of Ftx KD cells compared to control cells (Figures 4B and 4C). Collectively, these results indicate that Ftx mature transcripts are dispensable for proper Xist accumulation, during both the initiation and the maintenance of XCI. We then directly addressed the involvement of the Ftx-embedded Mir421, Mir374b, and Mir374c, the transcription of which is under the control of the Ftx promoter region (Chureau et al., 2011Chureau C. Chantalat S. Romito A. Galvani A. Duret L. Avner P. Rougeulle C. Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region.Hum. Mol. Genet. 2011; 20: 705-718Crossref PubMed Scopus (172) Google Scholar). To do this, we used a CRISPR-Cas9 approach to delete ∼300 bp encompassing the miR cluster in LF2 female ESCs (Figures 4A and S4I). As previously described for Ftx deletion, we thoroughly characterized two independent miR KO clones: miR−/−A and miR−/−B (Figures S4I–S4K). As expected, transcription at the miR cluster appeared abrogated in miR−/− cells (Figure S4L). We then analyzed the effect of the miR deletion on Ftx and Xist expressions using qRT-PCR and RNA-FISH. Ftx and Xist appeared normally upregulated upon differentiation of miR−/− cells, in which Xist accumulates on the Xi as efficiently as in wild-type cells. This clearly indicates that Ftx-embedded miRs do not participate in Xist regulation (Figures 4D and 4E). To determine which player, other than the Ftx ncRNAs, could be responsible for impaired Xist regulation in differentiating Ftx−/− mESCs, we exploited publicly available chromatin immunoprecipitation sequencing (ChIP-seq) datasets of male mESCs to scan the region corresponding to the deletion for potential regulatory elements. This analysis revealed a significant peak of enrichment of CTCF downstream of Ftx promoter 1 that is associated with consensus CTCF-binding sites (Figure 5A). The CTCF zinc-finger protein is a potent transcription factor and a major mediator of chromatin looping that has been shown to bind to the Xist locus (Makhlouf et al., 2014Makhlouf M. Ouimette J.F. Oldfield A. Navarro P. Neuillet D. Rougeulle C. A prominent and conserved role for YY1 in Xist transcriptional activation.Nat. Commun. 2014; 5: 4878Crossref PubMed Scopus (76) Google Scholar, Navarro et al., 2006Navarro P. Page D.R. Avner P. Rougeulle C. Tsix-mediated epigenetic switch of a CTCF-flanked region of the Xist promoter determines the Xist transcription program.Genes Dev. 2006; 20: 2787-2792Crossref PubMed Scopus (109) Google Scholar). By performing Capture-C enriching for genome-wide interactions with a ∼1 kb fragment at the transcription start site of Xist in LF2 or PGK12.1 female ESCs, we detected interactions between the Xist promoter and the CTCF-binding site downstream of Ftx promoter 1, indicating that stable 3D looping brings together Ftx and Xist promoters in undifferentiated female ESCs (Figure 5B). To test whether this interaction could be involved in the regulation of Xist, we designed a CRISPR/Cas9 approach to specifically delete the CTCF enrichment peak without affecting Ftx promoter activity (Figure S5A). The deletion was first achieved in LF2 mESCs, in which two homozygous clones (FtxΔCTCF/ΔCTCF) were extensively characterized at the genomic level and selected for further analysis (Figures S5A–S5C). As expected, the frequency of interactions with the Xist promoter appeared strongly reduced in both FtxΔCTCF/ΔCTCF clones compared to wild-type mESCs (Figure 5B); CTCF binding, monitored by ChIP-qPCR, was significantly lower at positions surrounding the deletion (Ftx in1 down and U" @default.
• W2801467871 created "2018-05-17" @default.
• W2801467871 creator A5018287271 @default.
• W2801467871 creator A5035314440 @default.
• W2801467871 creator A5045322704 @default.
• W2801467871 creator A5048740083 @default.
• W2801467871 creator A5050888688 @default.
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• W2801467871 date "2018-05-01" @default.
• W2801467871 modified "2023-10-17" @default.
• W2801467871 title "The Ftx Noncoding Locus Controls X Chromosome Inactivation Independently of Its RNA Products" @default.
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• W2801467871 doi "https://doi.org/10.1016/j.molcel.2018.03.024" @default.
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