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Sex-Specific Silencing of X-Linked Genes by Xist RNA PNAS PLUS

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Sex-Specific Silencing of X-Linked Genes by Xist RNA PNAS PLUS Sex-specific silencing of X-linked genes by Xist RNA PNAS PLUS Srimonta Gayen, Emily Maclary, Michael Hinten, and Sundeep Kalantry1 Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109 Edited by David C. Page, Whitehead Institute, Cambridge, MA, and approved December 11, 2015 (received for review August 11, 2015) X-inactive specific transcript (Xist) long noncoding RNA (lncRNA) We therefore sought to systematically test the impact of Xist is thought to catalyze silencing of X-linked genes in cis during RNA on gene silencing by ectopically inducing Xist from the X-chromosome inactivation, which equalizes X-linked gene dosage endogenous locus, thus ensuring that the cis-regulatory elements between male and female mammals. To test the impact of Xist necessary for robust Xist expression are intact. We previously RNA on X-linked gene silencing, we ectopically induced endoge- generated male and female embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) that harbor an X chromosome with nous Xist by ablating the antisense repressor Tsix in mice. We find Δ a null mutation in the Xist antisense repressor Tsix (X Tsix) (30). that ectopic Xist RNA induction and subsequent X-linked gene Δ Δ A subset of differentiating X TsixY and X TsixX cells display ec- silencing is sex specific in embryos and in differentiating embry- ΔTsix onic stem cells (ESCs) and epiblast stem cells (EpiSCs). A higher topic Xist RNA coating of the X ; thus, male cells harbor a Δ frequency of X TsixY male cells displayed ectopic Xist RNA coating single Xist RNA coat and females possess two Xist coats. These compared with XΔTsixX female cells. This increase reflected the in- populations enabled us to assess the ability of Xist RNA to si- ability of XΔTsixY cells to efficiently silence X-linked genes com- lence X-linked genes in males and in females. ΔTsix Unexpectedly, we observed sex-specific differences in the pared with X X cells, despite equivalent Xist RNA induction Δ frequency of cells that induced Xist from the active X Tsix and and coating. Silencing of genes on both Xs resulted in significantly ΔTsix silenced X-linked genes once Xist was ectopically induced, both reduced proliferation and increased cell death in X X female ΔTsix Δ in vitro and in vivo. We found that a higher percentage of X Y cells relative to X TsixY male cells. Thus, whereas Xist RNA can Δ cells displayed ectopic Xist RNA coating compared with X TsixX inactivate the X chromosome in females it may not do so in males. ΔTsix Δ cells. This increase reflected the inability of X Y cells to effi- We further found comparable silencing in differentiating X TsixY ΔTsix ΔTsix ciently silence X-linked genes upon ectopic Xist induction com- and 39,X (X O) ESCs, excluding the Y chromosome and in- ΔTsix pared with X X cells, despite equivalent levels of Xist expression GENETICS stead implicating the X-chromosome dose as the source of the sex- and RNA coating. We discuss possible underlying reasons for these XΔTsixX specific differences. Because female embryonic epiblast differences, including the requirement of two X chromosomes to cells and EpiSCs harbor an inactivated X chromosome prior to ec- ΔTsix ΔTsix physically interact, epigenetic variation on the X between the topic inactivation of the active X X chromosome, we propose sexes, and differences in developmental timing between male and that the increased expression of one or more X-inactivation escapees female embryos. The comparative analysis compels us to propose activates Xist and, separately, helps trigger X-linked gene silencing. that the higher X-chromosomal dose in females, potentially acting through gene(s) that escape X inactivation, induces Xist and, Xist | Tsix | X inactivation | embryonic stem cells | epiblast stem cells separately, silences X-linked genes once Xist is induced. The in- creased dosage of such a factor(s) in females compared with males inactivation represents a paradigm of epigenetic regulation may explain why females undergo X inactivation and males do not. Xand long noncoding RNA (lncRNA) function. In XX female cells, one of the two X chromosomes undergoes transcriptional Results silencing (1). Moreover, replicated copies of the active and in- ESCs Display a Sex-Specific Difference in the Frequency of Ectopic Xist RNA Coating. To ectopically induce Xist, we differentiated mul- active X chromosomes faithfully maintain their respective tran- Δ – tiple control wild-type (WT) XY and XX and mutant X TsixY and scriptional states through many cell division cycles (2 5). ΔTsix X inactivation requires the X-inactive specific transcript (Xist) X X ESC lines (see SI Appendix, Fig. S1 for map of the ΔTsix (6–8), a lncRNA that is selectively expressed from and physically coats the future inactive X chromosome (9–12). Xist RNA en- Significance ables X-linked gene silencing by recruiting protein complexes to – the inactive X (13 15). Female mouse embryos that inherit a In mammals, the inequality posed by the difference in the paternal Xist mutation die due to defects in imprinted X in- number of X chromosomes between XX females and XY males activation of the paternal X chromosome in extraembryonic is remedied by silencing genes along one of the two X chro- tissues (8, 16, 17). Xist is also required in the epiblast-derived mosomes in females. This process, termed X-chromosome in- embryonic cells, which undergo random X inactivation of either the maternal or the paternal X chromosome. Xist heterozygote activation, is believed to be triggered by X-inactive specific fetal cells exhibit inactivation of only the X chromosome with an transcript (Xist) RNA. Here we find that Xist RNA can silence intact Xist locus, suggesting that Xist is necessary to choose the X X-linked genes efficiently in females but not in males. Thus, chromosome to be inactivated (7, 18, 19). That the Xist-mutant X Xist RNA is insufficient to inactivate the X chromosome. Our chromosome is not selected for inactivation, however, precludes results further suggest that both Xist induction and X-linked assigning to Xist RNA a gene silencing role in the epiblast lineage. gene silencing are orchestrated by the handful of genes that do Ectopic expression studies have, however, demonstrated that not undergo X inactivation in females. The increased dosage of Xist RNA can silence genes, albeit in a context-dependent one or more such factors in females vs. males may explain why manner. Xist transgenes integrated into autosomes can silence females undergo X inactivation and males do not. neighboring autosomal sequences, but the effect is quite variable. Whereas multicopy Xist transgenes or transgenes driven by ar- Author contributions: S.G., E.M., and S.K. designed research; S.G. and E.M. performed tificial promoters often display Xist RNA induction and coating research; M.H. contributed new reagents/analytic tools; S.G., E.M., and S.K. analyzed data; of autosomes in cis accompanied by a degree of silencing of and S.G., E.M., and S.K. wrote the paper. adjacent host sequences (20–28), large single-copy Xist genomic The authors declare no conflict of interest. transgenes do not (19, 28, 29). The sequence composition and This article is a PNAS Direct Submission. the chromatin context at the site of transgene integration as well 1To whom correspondence should be addressed. Email: [email protected]. as the level of Xist expression are confounding variables that may This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. influence the ability of transgenic Xist RNA to silence. 1073/pnas.1515971113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1515971113 PNAS Early Edition | 1of10 Downloaded by guest on October 2, 2021 Δ mutant allele) (30, 31). We assessed ectopic Xist RNA coating Xist RNA coating altogether. Strong Xist RNA-decorated X TsixY every 2 d over a period of 10 d by RNA fluorescence in situ nuclei reached a maximum of between 40% and 58% of all nuclei hybridization (FISH). WT male XY ESC lines did not display at day 6 (d6) of differentiation, before decreasing to ∼30% at d10 Δ Xist RNA-coated nuclei during differentiation. However, mu- (Fig. 1A). Weak Xist RNA-coated X TsixY nuclei peaked at 18– Δ tant male X TsixY lines exhibited three classes of nuclei: some 22% at d4 and decreased to ∼8% at d10 of differentiation. Δ had strong Xist RNA coating, resembling Xist RNA coating in Undifferentiated female WT XX and mutant X TsixX ESCs har- female cells, some had weak Xist RNA coating, and some lacked bor two active X chromosomes, which are randomly inactivated A B Fig. 1. Differential Xist RNA coating in XΔTsixY vs. XΔTsixX differentiating ESCs. (A, Left) RNA FISH detection of Xist (white) and Tsix (green) RNAs followed by Δ Xist DNA FISH (red) in representative XY and X TsixY differentiated ESCs without or with strong or weak Xist RNA coats. Nuclei are stained blue with DAPI. Δ (Right) Quantification of nuclei with strong or weak Xist RNA coats during differentiation of two XY and four X TsixY ESC lines. (B, Left) Xist/Tsix RNA FISH Δ followed by Xist DNA FISH in representative XX and X TsixX differentiated ESCs. (Right) Quantification of nuclei with strong or weak ectopic Xist RNA coats Δ during differentiation in three XX and three X TsixX ESC lines. Only nuclei with a single Xist locus in males or two Xist loci in females detected by DNA FISH were quantified. n = 100 nuclei per cell line per day of differentiation. (Scale bar, 2 μm.) Related data are included in SI Appendix, Fig. S1. 2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1515971113 Gayen et al.
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