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Molecular Coupling of Tsix Regulation and Pluripotency

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Molecular Coupling of Tsix Regulation and Pluripotency LETTER doi:10.1038/nature09496 Molecular coupling of Tsix regulation and pluripotency Pablo Navarro1,2, Andrew Oldfield1,3, Julie Legoupi1, Nicola Festuccia2, Agne`s Dubois1, Mikael Attia1, Jon Schoorlemmer4, Claire Rougeulle1,3, Ian Chambers2 & Philip Avner1 The reprogramming of X-chromosome inactivation during the Nanog, at DXPas34 (ref. 17) and at Xite18 two enhancers of Tsix19.Oct4 acquisition of pluripotency in vivo and in vitro1 is accompanied by and Sox2, by simultaneously controlling Xist and Tsix, could be acting at the repression of Xist2, the trigger of X-inactivation3, and the upre- the top of the X-inactivation regulatory hierarchy in pluripotent cells. gulation of its antisense counterpart Tsix4. We have shown that key Uncertainties, however, surround this hypothesis because (1) the factors supporting pluripotency—Nanog, Oct4 and Sox2—bind reported binding of Oct4 and Sox2 to DXPas34,thestrongembry- within Xist intron 1 in undifferentiated embryonic stem cells (ESC) onic-stem-specific enhancer of Tsix, is not reproducible (Supplemen- to repress Xist transcription5. However, the relationship between tary Fig. 1), (2) binding levels at Xite are very low (Supplementary Fig. 1) transcription factors of the pluripotency network and Tsix regulation and (3) Xite is a weak enhancer of Tsix18. It appears likely that Oct4 and has remained unclear5,6. Here we show that Tsix upregulation in Sox2 play a minor role, if any, in the establishment of Tsix transcription embryonic stem cells depends on the recruitment of the pluripotent in undifferentiated ESC. In agreement, Tsix remains unaffected after marker Rex1, and of the reprogramming-associated factors Klf4 and 24 h of Oct4 knockdown, whereas Xist upregulation is already estab- c-Myc, by the DXPas34 minisatellite associated with the Tsix pro- lished (Supplementary Fig. 1). Thus additional factors are implicated in moter. Upon deletion of DXPas34, binding of the three factors is controlling Tsix transcription in undifferentiated ESC. Three pluripo- abrogated and the transcriptional machinery is no longer efficiently tency factors, Klf4, c-Myc and Rex1, attracted our attention. recruited to the Tsix promoter. Additional analyses including knock- Because Oct4 and Sox2 directly mediate repression of Xist in ESC, down experiments further demonstrate that Rex1 is critically import- we were interested to extend our analysis to Klf4 and c-Myc, the two ant for efficient transcription elongation of Tsix. Hence, distinct other factors that are commonly used to generate iPS cells13. Rex1 is a embryonic-stem-cell-specific complexes couple X-inactivation repro- marker of pluripotency whose deletion is associated with decreased gramming and pluripotency, with Nanog, Oct4 and Sox2 repressing Tsix expression, as revealed by microarray analysis20. During iPS cell Xist to facilitate the reactivation of the inactive X, and Klf4, c-Myc generation, Tsix re-expression temporally correlates with the induc- and Rex1 activating Tsix to remodel Xist chromatin7–10 and ensure tion of Rex1 (ref. 21). random X-inactivation upon differentiation1. The holistic pattern of We initially determined whether binding activity of Rex1, Klf4 and Xist/Tsix regulation by pluripotent factors that we have identified c-Myc could be detected at the Xist/Tsix region (Fig. 1a). We found suggests a general direct governance of complex epigenetic processes binding to the Tsix 59 region for all three factors, in both female (Fig. 1 by the machinery dedicated to pluripotency. d, g, j) and male (Fig. 1 e, h, k) embryonic stem cells. Only Rex1 displays X-inactivation reprogramming in female mice is a model for the binding at both ends of DXPas34, which suggests that Rex1 is recruited epigenetic processes underlying the acquisition of pluripotency1.The within DXPas34 itself whereas Klf4 and c-Myc are bound between inactivation of the paternal X chromosome that characterizes the earliest DXPas34 and the Tsix promoter. As expected given its chromatin immu- cleavage-stages of development is followed by X-chromosome reactiva- noprecipitation (ChIP) profile, Rex1 binding is lost upon the targeted tion in the pluripotent inner cell mass of the blastocyst11,12. During deletion of DXPas34 in male embryonic stem cells (DPas34 cell line17; differentiation, X-inactivation is established randomly on either the Fig. 1c, f). Strikingly, Klf4 and c-Myc binding is similarly affected in paternal or the maternal X chromosome1. The developmental plasticity DPas34 (Fig. 1i, l), which suggests that DXPas34 influences Klf4 and of X-inactivation during early embryogenesis is paralleled by female c-Myc recruitment. This drastic perturbation in transcription factor induced pluripotent stem (iPS) cells13: the inactive X is reactivated in binding correlates with a 90% reduction in Tsix RNA levels17, mediated iPS cells and random X-inactivation de novo established upon loss of by a strong reduction of RNAPII recruitment at the Tsix promoter pluripotency14. Hence, two distinct processes affect X-inactivation (Supplementary Fig. 2). Thus DXPas34 orchestrates the recruitment of during the generation of pluripotency: the reactivation of the inactive Rex1, Klf4 and c-Myc to activate Tsix transcription in ESC. X per se, probably initiated by the repression of Xist by Nanog, Oct4, and We next silenced Oct4 in ZhbTc4.1 ESC to induce the loss of plur- Sox2 (refs 5,15), and the acquisition by both X chromosomes of an equal ipotency22 and the consequent downregulation of Rex1 and Klf4 competence for future random X-inactivation, a process directly con- (Fig. 2a). Tsix was downregulated (Fig. 2a) by transcriptional mechan- trolled by Tsix. Indeed, invalidation of Tsix in ESC8 and embryos10 leads isms (Fig. 2b, c), and binding of Rex1, Klf4 and c-Myc was reduced to drastic, stable remodelling of Xist chromatin in cis7–10, associated with (Fig. 2d–f). Similar observations were made in terminally differen- the systematic upregulation of Xist from the Tsix-null allele upon dif- tiated, Tsix-silenced9, mouse embryonic fibroblasts (Fig. 2g–i). Rex1, ferentiation. Thus maximal Tsix activity is required in pluripotent cells Klf4 and c-Myc are therefore critically important to couple Tsix regu- to erase inherited Xist chromatin modifications and provide each X lation to pluripotency. We also analysed trophectoderm stem cells, chromosome with equal probabilities of Xist upregulation during which display reduced Tsix transcription9, and found that only Rex1 differentiation16. is absent from the Tsix 59 region (Fig. 2g–i). Although the existence of It has been proposed that the high levels of Tsix transcription char- differentiation-dependent repressors or additional embryonic-stem- acterizing ESC depend on binding of Oct4 and Sox2 (ref. 6), but not specific activators cannot be excluded, this suggests that Rex1 plays 1Unite´ de Ge´ne´tique Mole´culaire Murine, URA 2578, Institut Pasteur, 75724 Paris Cedex 15, France. 2Medical Research Council (MRC) Centre Development in Stem Cell Biology, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JQ, UK. 3UMR 7216 Epige´ne´tique et Destin Cellulaire, CNRS/Universite´ Paris Diderot, Case 7042, 75205 Paris Cedex 13, France. 4ARAID Foundation and Instituto Aragone´s de Ciencias de la Salud, Departamento de Anatomı´a y Embriologı´a, Facultad de Veterinaria, 50013 Zaragoza, Spain. 18 NOVEMBER 2010 | VOL 468 | NATURE | 457 ©2010 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER abcab13 6 c1.0 Relative mRNA levels 10 RNAPII TFIIB 7 –Tc –Tc 0.2 0.2 0.2 –Tc +Tc +Tc deRex1 Rex1 fRex1 Female Male DPas34 +Tc ES cells ES cells ES cells 1 3 0.5 0.1 0.1 0.1 0.5 0 ′ ′ Klf4 Rex1 0 0 c–Myc Tsix5 Tsix3 0 0 0 31303233 34 35 36 37 31303233 34 35 36 37 –3 –2 –1 0 1 2 11 12 31 32 33 34 35 36 37 313032 33 34 35 36 37 313032 33 34 35 36 de f gh0.4 0.2 i0.2 0.2 0.6 0.2 Klf4 Klf4 Klf4 Rex1 Klf4 c-Myc Female Male DPas34 –Tc –Tc –Tc ES cells ES cells ES cells +Tc +Tc +Tc 0.2 0.1 0.1 0.1 0.3 0.1 0 0 0 –3–2–1 0 1 2 11 12 31 32 33 34 35 36 37 313032 33 34 35 36 37 313032 33 34 35 36 jk0.4 0.4 l0.4 0 0 0 c-Myc c-Myc c-Myc 31303233 34 35 36 37 31303233 34 35 36 37 31303233 34 35 36 37 Female Male DPas34 ES cells ES cells ES cells gh0.2 0.6 i0.4 Rex1 Klf4 c-Myc ES ES ES TS TS TS 0.2 0.2 0.2 MEFs MEFs MEFs 0.1 0.3 0.2 0 0 0 –3 –2 –1 0 1 2 11 12 31 32 33 34 35 36 37 313032 33 34 35 36 37 313032 33 34 35 36 Figure 1 | DXPas34 orchestrates Rex1, Klf4 and c-Myc recruitment to the Tsix 59 region in pluripotent ESC. a–c, Schematic representation of the sub- 0 0 0 regions of the Xist/Tsix locus analysed by ChIP in female ESC (a), male ESC 31303233 34 35 36 37 31303233 34 35 36 37 313032 33 34 35 36 37 (b) and male DPas34 ESC (c). Xist exons are in green, the arrows indicate the Figure 2 | Developmentally induced loss of Rex1, Klf4 and c-Myc binding direction of transcription of Xist (top) and Tsix (bottom). DXPas34 is shown in correlates with Tsix repression. a, Relative RNA levels of the indicated genes blue. The location of each primer pair is indicated by a black circle. In b, the blue in undifferentiated (2Tc, black, set to one) and differentiating (96 h Tc circles show the primer pairs flanking DXPas34 that are absent in DPas34 ESC, treatment, 1Tc, red) ZhbTc4.1 cells (n 5 4). b–f, ChIP analysis of RNAPII whereas in c the red triangle shows the location of the loxP site remaining after (b), TFIIB (c), Rex1 (d), Klf4 (e) and c-Myc (f) across the Tsix 59 region in the DXPas34 deletion, and the new primer pair designed at the position shown in same cellular conditions (n 5 2).
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