Jpx RNA Activates by Evicting CTCF

Sha Sun,1,2,3 Brian C. Del Rosario,1,2,3 Attila Szanto,1,2,3 Yuya Ogawa,1,2,3,4 Yesu Jeon,1,2,3 and Jeannie T. Lee1,2,3,* 1Howard Hughes Medical Institute, Boston, MA 02114, USA 2Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA 3Department of Genetics, Harvard Medical School, Boston, MA 02114, USA 4Present address: Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2013.05.028

SUMMARY (Scute, SisA, Runt, Upd) activates the master regulator Sxl, and autosomal factors, such as Deadpan, together with nuclear In mammals, dosage compensation between XX volume, oppose the X-linked signals (Barbash and Cline, 1995; and XY individuals occurs through X Cline and Meyer, 1996; Salz and Erickson, 2010). inactivation (XCI). The noncoding Xist RNA is ex- In mammals, the X:A ratio currently exists only as a genetic pressed and initiates XCI only when more than one concept. Evidence derives from studies of aneuploids and poly- is present. Current models invoke a ploids indicating that XXX females establish two inactive X’s (Xi) dependency on the X-to-autosome ratio (X:A), but when diploid but inactivate only one Xi when tetraploid; further- more, XXX triploid females establish one or two Xi, consistent molecular factors remain poorly defined. Here, we with their intermediate X:A ratio (Brown and Chandra, 1973; Gar- demonstrate that molecular titration between an tler et al., 2006). Two conceptual frameworks have been instruc- X-encoded RNA and an autosomally encoded pro- tive. Early thinking favored the ‘‘Blocking Factor Hypothesis,’’ in tein dictates Xist induction. In pre-XCI cells, CTCF which a single complex of autosomal factors binds to and blocks protein represses Xist transcription. At the onset of one Xic and protects that X chromosome from silencing (Ohno, XCI, Jpx RNA is upregulated, binds CTCF, and extri- 1969; Lyon, 1971; Kay et al., 1994; Starmer and Magnuson, cates CTCF from one Xist allele. We demonstrate that 2009). Remaining Xs are unprotected and become silenced by CTCF is an RNA-binding protein and is titrated away default. The alternative ‘‘Two Factors Model’’ postulates the ex- from the Xist promoter by Jpx RNA. Thus, Jpx acti- istence of not only a blocking factor to protect one X but also a vates Xist by evicting CTCF. The functional antago- ‘‘competence factor’’ to trigger silencing on additional Xs (Gartler nism via molecular titration reveals a role for long and Riggs, 1983; Lee and Lu, 1999; Lee, 2005). Thus, regardless of details, all current models imply a molecular titration of noncoding RNA in epigenetic regulation. X-linked and autosomal factors. Consistent with their separate evolution, fly and worm regula- INTRODUCTION tors are not apparently utilized in the mouse. Unlike the inverte- brate systems, mammalian dosage compensation is allelically Dosage compensation balances X chromosome content controlled and involves regulatory switches defined by long non- between females and males in organisms with the XY mecha- coding RNA (lncRNA) within the master ‘‘X-inactivation center’’ nism of sex determination. In fruitflies, the single X chromosome (Xic). Xist RNA emanates from the Xic, coats the future Xi in in males is transcriptionally upregulated 2-fold; in roundworms, cis, and deposits silencing complexes along the chromosome expression from two X is halved in hermaphro- (Brockdorff et al., 1992; Brown et al., 1992). Xist is expressed dites; and in mammals, one of two X’s is transcriptionally only when X:A R1.0, and the number of Xist RNA foci follows silenced in females during X chromosome inactivation (XCI) the ‘‘n-1’’ rule in diploid cells (n = X chromosome number). (Cline and Meyer, 1996; Lucchesi et al., 2005; Payer and Lee, Recent work shows that Xist is both negatively and positively 2008; Wutz, 2011). Although dosage compensation is achieved regulated. It is repressed by the antisense Tsix RNA and the non- differently in various organisms, all three mechanisms depend coding Xite locus (Lee and Lu, 1999; Sado et al., 2001; Ogawa on the ‘‘X-to-autosome ratio’’ (X:A), which triggers the epigenetic and Lee, 2003) but activated by the long noncoding Jpx RNA process only when X:A = 0.5 in fruitflies or X:A R 1.0 in round- (Tian et al., 2010) and the E3 ubiquitin ligase, RNF12 (Jonkers worms and in mammals. Failure to achieve dosage compensa- et al., 2009)(Figure 1A). tion results in early embryonic death. In roundworms, X-linked The identities of X-encoded ‘‘numerators’’ and autosomally factors (SEX-1, CEH-39) and autosomal signals (SEA-1, SEA-2) encoded ‘‘denominators’’ of the X:A ratio have been elusive. In antagonize each other at the master regulatory gene, xol-1 principle, X-linked and autosomal regulators must converge (Meyer, 2010). In fruitflies, accumulation of X signal elements at the Xic—potentially at the Tsix, Xite,orXist locus. Indeed,

Cell 153, 1537–1551, June 20, 2013 ª2013 Elsevier Inc. 1537 A Negative B regulation Cnbp2 Ftx Jpx Xist Xite Tsx Xite Positive Xpct regulation Tsix Tsix Jpx/Enox 10 Kb RepA Probe 2 Probe 1 (BAC5) Tg(Jpx) Xist α Rex1 Rnf12 Tg(EF1 :Jpx)

XCI C 5 Tg(Jpx) 2 Tg(Jpx) E 4 Cells with Cells with Jpx Cells with 2 Xist clouds n Jpx 1.5 1 Xist cloud 3 Xist 1 Xist cloud n Xist Ctrl 7.5% 0% 53 1 2 Ctrl 0% >200 E2 8.6% 1.2% 81 1.8% 168 0.5 Bs2 E1 12.3% 1.4% 212 1 B2 3.5% 115 Ef1 6.8% 5.0% 161 0 0 Ef2 6.3% 7.0% 158 Level Log2(Tg/Ctrl) E2 E1 -1 Bs2 B2 -0.5

D Xist Xic

2 μ Ctrl Tg(Jpx) Bs2 Tg(Jpx) B2 Tg(EF1α:Jpx) Ef1

Ctrl Tg(Jpx) E2 Tg(Jpx) E1 Tg(EF1α:Jpx) Ef2

F Xist Xic

2 μ Tg(Jpx) JTB1 Tg(EF1a:Jpx) JTF4 Tg(Jpx) ETB1 Tg(EF1a:Jpx) ETF2

H G 6 1.5 JTB1 ETB1 JTF4 5 ETF2 5 Cells with Cells with Cells with 4 1.0 1 Xist cloud n 1 Xist cloud 2 Xist clouds n 4 1.0 3 3 TsixTST/Y 5.0% 139 TsixTST/+ 44.2% 4.6% 129 2 0.5 JTB1 7.7% 104 ETB1 47.7% 12.2% 172 2 0.5 JTF4 15.4% 123 ETF2 47.9% 12.9% 194 1 1

Level Log2(Tg/Ctrl) 0 0.0 0 0.0 Jpx Xist Jpx Xist

(legend on next page)

1538 Cell 153, 1537–1551, June 20, 2013 ª2013 Elsevier Inc. integrating extra Xic copies into either a male or female genome lates XCI during cell differentiation. Quantitative RT-PCR mimics the presence of supernumerary Xs and triggers ectopic confirmed overexpression of Jpx RNA between 0.5- to 2-fold XCI (reviewed in Starmer and Magnuson, 2009; Lee, 2011). in multiple independent clones on day 4 of differentiation (d4; One study implicated the Tsix/Xite loci as binding sites for de- Figure 1C), and RNA-DNA fluorescence in situ hybridization nominators without identifying specific autosomal factors (Lee, (FISH) showed ectopic Xist clouds in differentiating embryonic 2005). Another study showed that XCI is sensitive to dosage of stem (ES) cells at d4 (Figures 1D and 1E). Consistent with autosomal OCT4 protein (Donohoe et al., 2009). The X-encoded elevated steady-state levels of Xist shown by qRT-PCR (Fig- E3 ubiquitin ligase, RNF12, was also proposed as a candidate ure 1C), 1%–4% of transgenic male cells possessed an Xist numerator, as excess RNF12 triggers ectopic Xist expression cloud, and 1%–2% of diploid female cells exhibited a second (Jonkers et al., 2009) and RNF12-mediated ubiquitylation of Xist cloud. (Note: tetraploid cells were excluded by performing REX1 occurs at the initiation of XCI (Gontan et al., 2012). DNA FISH using probe 2; Figure 1D.) Ectopic Xist clouds were RNF12’s candidacy as numerator may be complicated by its neither observed in control male nor female cells carrying vector catalytic nature, however. The necessity of precise X:A titration sequences (ctrl). Thus, whereas reducing Jpx expression by half renders catalytic factors conceptually problematic because cat- blocks Xist activation (Tian et al., 2010), 2-fold overexpression of alytic factors with rapid enzymatic rates are unlikely to be limited Jpx caused ectopic Xist expression. by 2-fold molar differences. Indeed, deleting a single allele of The magnitude of overexpression was, however, small. Noting Rnf12—a state that mimics the XY state—delayed but did not a general correlation between Jpx levels and degree of Xist up- abrogate Xist upregulation in mice (Jonkers et al., 2009; Shin regulation (Figures 1C–1E), we asked if increasing Jpx overex- et al., 2010; Barakat et al., 2011). pression caused greater Xist induction. By retrofitting the strong In molecular titration models, numerators are more easily en- Elongation Factor 1a (EF1a) promoter into the Jpx transgene visioned as stoichiometric than as catalytic factors. Candidate (Tg(EF1a:Jpx); Figure 1B), we further enhanced Jpx activity numerators must in theory satisfy several experimental criteria. and observed greater Xist induction. RNA-DNA FISH indicated First, it must be X-linked and escape XCI in order to provide that up to 7% of cells displayed two Xist clouds (Figures 1D numerical information. Second, it should elicit discrete XCI phe- and 1E). Correlation of Jpx and Xist overexpression was also notypes in response to changes in gene copy number. It must observed in later stages of differentiation (Figure S1A available also be haploinsufficient, with the +/ state phenocopying the online; data not shown). Xist expression therefore shows a male state; overexpression should simulate supernumerary X’s dose-dependent response to Jpx expression. and trigger ectopic XCI. Finally, the numerator must act at the Xic. Intriguingly, the noncoding Jpx/Enox gene resides 10 kb Deleting Tsix Augments Jpx’s Effect upstream of Xist (Chureau et al., 2002; Johnston et al., 2002; The transgene experiments showed that, although the number of Chow et al., 2003), escapes XCI, and is required for Xist activa- Xist clouds (10%–14%) was consistently elevated with Jpx over- tion (Tian et al., 2010). Deleting a single Jpx allele in female cells expression, the percentage of ectopic Xist clouds never ex- is sufficient to prevent XCI (Tian et al., 2010). This haploinsuffi- ceeded 7%. We recalled that Tsix and Jpx dually regulate Xist ciency and its escape from XCI raises the intriguing possibility expression (Tian et al., 2010), with Xist being difficult to induce that Jpx RNA may be a numerator. Below, we subject Jpx to when the Tsix allele in cis is intact (Luikenhuis et al., 2001; Stav- the test and define interactions that implicate Jpx in molecular ropoulos et al., 2001). During female differentiation, Tsix’s titration of an autosomal factor. repression on the future Xi enables Xist upregulation, whereas its persistent expression on the future Xa blocks Xist induction. RESULTS Likewise, during male differentiation, persistent Tsix on the single X chromosome prevents Xist expression. Therefore, could an Jpx Induces Xist in a Dose-Dependent Manner intact Tsix allele blunt the effect of Jpx overexpression in the To determine if altering Jpx levels results in quantifiable changes Jpx transgenic male and female cells? of Xist expression, we introduced a 90 kb Jpx transgene (Tg(Jpx); To address this, we introduced Tg(EF1a:Jpx) into ES cells Figure 1B) into wild-type male and female mouse embryonic carrying a truncated Tsix allele (TsixTST)(Ogawa et al., 2008) stem (ES) cells, a well-established ex vivo model that recapitu- and observed that disabling Tsix increased susceptibility to

Figure 1. Jpx Overexpression Results in Ectopic Xist Upregulation (A) Positive-negative regulation of Xist. Arrows, activation; blunt arrows, repression. (B) Map of Xic, FISH probes, and Jpx transgenes. Tg(Jpx). (C) Quantitation of Jpx and Xist expression on d4 in Tg(Jpx) ES cells by qRT-PCR. Representative independent clones (male Bs2, B2; female E2, E1) are shown. Expression levels normalized to ctrl (control clone). Averages ± SE. See also Figure S1. (D) Xist expression examined by RNA-DNA FISH in d4 ES cells. FITC (green, probe 1), Xist RNA cloud. Cy3 (red), BAC5 probe 2 marks Xic. Arrowheads, Tg insertion site. (E) Quantitation of Xist RNA-DNA FISH results in d4 ES cells. Representative clones shown. n, sample size. See also Figure S1. (F) Disabling Tsix augments Xist’s response to Jpx overexpression. RNA-DNA FISH of Tg(EF1a:Jpx) cells in a TsixTST/Y or TsixTST/+ cells on d4 of differentiation. One representative clone shown for each. FITC (green, probe 1), Xist RNA cloud. Cy3 (red), BAC5 probe 2 marks Xic. Arrowheads, Tg insertion site. (G) Quantitation of cells (in F) with the Xist pattern indicated. (H) qRT-PCR of Xist and Jpx RNA levels on d4, normalized to RNA levels in parental TsixTST/Y or TsixTST/+ cells. Averages ± SE. See also Figure S1.

Cell 153, 1537–1551, June 20, 2013 ª2013 Elsevier Inc. 1539 A B

P1 Probe P1 RS14c P2 1 kb Xist_5’ Xist_5’mut P2 P2 mut RS14c Probe Xist RS14c CTCF --+ --+ --+ --+ --+ CTCF --+ --+ GFP 12 3 CTCF binding sites 4 --+--+--+--+--+ GFP - - + --+

1. Xist_5’ ...CATGCTGCCACCTGCTGGTTTATT... 1. Xist_5’ mut ...CATGCTGCTACTTGCTAGTTTATT... 2. P1 ...CCGCCTTCAGCGCCGCGGATCAGTT... * 3. P2 ...ACCTGCCCTCTAGTGGTTTCTTTC... 3. P2 mut ...ACCTGTCCTATAGTTGTTTCTTTC... 4. RS14c ...TGTAGGCCAGCAGAGGGTGTCGGA...

Unbound Unbound probe probe

C P=0.17 0.8 CTCF IgG

0.7 d0 0.6 WT WT d3 0.5 d6

% Input 0.4

0.3 P=0.12 P=0.38 P=0.28 P=0.33 0.2 P=0.48 0.1

0 Rnf12 Xist_5' P1 P2 RS14c H19 Rnf12 Xist_5' P1 P2 RS14c H19

P=0.32 0.6

0.5 WT WT 0.4 P=0.37

% Input 0.3 P=0.40 P=0.008** 0.2 P=0.17

0.1 P=0.25

0 Rnf12 Xist_5' P1 P2 RS14c H19 Rnf12 Xist_5' P1 P2 RS14c H19

D E CTCF P=0.30 TsixTST/+ 0.6 Jpx+/-

0.3 P=0.02* P=0.30 0.5 d0 0.7 d3 P=0.28 P=0.17 0.4 d6 0.6 0.2 CTCF

% Input 0.3 0.5 d0 P=0.18 P=0.14 d3 0.2

% Input 0.4 0.1 d6 0.1 P=0.40 0.3 0 0.2 Rnf12 Xist_5' P1 P2 RS14c H19 0 0.1 cas mus IgG (future Xa) (future Xi) % Input 0 Rnf12 Xist_5' P1 P2 RS14c H19

(legend on next page)

1540 Cell 153, 1537–1551, June 20, 2013 ª2013 Elsevier Inc. Jpx overexpression (Figures 1F and 1G). In male cells, the num- site at RS14c,aTsix-Xist boundary element at Xist’s 30 end ber of ectopic Xist clouds increased from 1%–4% in a wild-type (Spencer et al., 2011). Mutating three nucleotides in the background (Figure 1E) to 7.7% in the TsixTST/Y background consensus motif abolished the shift (Figures 2A and 2B). We (JTB1). The percentage further increased to 15.4% when Jpx conclude that CTCF directly binds Xist_50 and P2 in vitro. was overexpressed from the EF1a promoter in the TsixTST/Y To test binding in vivo, we carried out quantitative chromatin background (JTF4) (Figure 1G; c2 one-tailed, p = 0.01). In female immunoprecipitation (ChIP) for CTCF in male and female ES cells, Tsix deficiency increased ectopic Xist clouds from 1% in cells. In undifferentiated (d0) male and female cells, CTCF occu- a wild-type background (Figure 1E) to 12.2% in the TsixTST/+ pied Xist_50 and P2 (Figure 2C), consistent with EMSA results. background (ETB1). The percentage further increased to Although CTCF could not bind P1 in the gel shift analysis, occu- 12.9% when Jpx was overexpressed from the EF1a promoter pancy was observed by ChIP, suggesting that CTCF occupancy (ETF2) (Figure 1G; c2 one-tailed, p < 0.05). (The effect in females at P1 could be mediated by other factors in vivo. One possibility was less than in males, likely because Tsix remained intact on is that chromatin looping from P2 brought CTCF in proximity to one female X; nonetheless, persistent Tsix allele on the future P1. During differentiation, CTCF enrichment at all three sites re- Xa would eventually be downregulated during differentiation, mained unchanged in male cells. However, in female cells, bind- providing an opportunity for Jpx overexpression to act on the ing to P2 was significantly reduced (p = 0.008) by approximately newly susceptible Xa, resulting in cells with two Xist clouds.) half between d0 and d6 (Figure 2C), the time frame of Xist upre- qRT-PCR showed a further 2-fold increase in Xist levels gulation. Enrichment at P1 was similarly decreased (but with when Jpx is overexpressed from the EF1a promoter after trun- borderline significance; p = 0.17), consistent with the idea of cating Tsix in female cells; in male cells, an even larger increase CTCF looping in from P2. Binding to positive control regions, in Xist was observed (Figure 1H). RS14c and H19, and a negative control, Rnf12, was unaltered We conclude that the dose-dependent response of Xist to Jpx during this time frame. These results demonstrate that CTCF levels is augmented by deleting Tsix, consistent with the notion binding to P2 is dynamically regulated during XCI. that Xist is coregulated by a Jpx-based positive pathway and a Given 2-fold changes in P2 binding, we suspected allelic dif- Tsix-based negative pathway (Lee and Lu, 1999; Sado et al., ferences. To test the possibility of differential binding to Xa and 2001; Tian et al., 2010). The presence of intact Tsix may explain Xi, we performed allele-specific ChIP quantitative PCR in a why Jpx transgenic overexpression in previous studies did not genetically marked female ES line, TsixTST/+. In this cell line, a result in ectopic Xist induction (Augui et al., 2011; Barakat Tsix truncation on the Mus musculus (mus) X chromosome en- et al., 2011). sures that this chromosome will be Xi and the Mus castaneus (cas) X chromosome will be Xa (Ogawa et al., 2008); a strain-spe- CTCF Binds the Xist Promoter and Correlates with Xist cific polymorphism located 200 bp downstream of P2 (Keane Repression et al., 2011) enabled allelic discrimination by PCR. Indeed, Xist is known to be regulated at the transcriptional level (Sun CTCF occupancy was significantly reduced at P2 of the future et al., 2006). Within a 3 kb promoter region of Xist, three binding Xi (Figure 2D), linking the loss of CTCF binding with Xist activa- sites for the zinc finger protein, CTCF (Bell et al., 1999; Ohlsson tion. In the Jpx+/ haploinsufficient ES cell line (Tian et al., et al., 2001), have been proposed, including one upstream of 2010), the inability to upregulate Xist during differentiation corre- mouse Xist (Xist_50; Navarro et al., 2006), one at human XIST’s lated with persistent binding of CTCF to P2 (Figure 2E). Together, first promoter (P1; Pugacheva et al., 2005), and one at promoter these data show that CTCF binding to Xist P2 is allelically regu- P2 in mice (Sheardown et al., 1997; Navarro et al., 2006; Essien lated and is anticorrelated with Xist expression. et al., 2009)(Figure 2A). One study reported a positive correlation between Xist activation and affinity of P1 for CTCF (Pugacheva CTCF Is a Blocking Factor for Xist Upregulation et al., 2005). Another study proposed CTCF as chromatin insu- Anticorrelation of CTCF binding and Xist upregulation suggests lator for P1 (Navarro et al., 2006). To pinpoint CTCF’s role, we that CTCF serves as an Xist repressor. To test this, we overex- asked if CTCF directly binds the three murine sites by performing pressed CTCF in male and female ES cells by creating stable electrophoretic mobility shift analysis (EMSA). Whereas a GFP transgenic lines carrying a doxycycline(dox)-inducible bidirec- control did not shift any probe, purified CTCF strongly shifted tional TRE promoter that, when induced, simultaneously ex- Xist_50 and P2 (Figure 2B). By contrast, CTCF did not shift P1, presses FLAG-tagged CTCF and a GFP marker (Figure 3A). At a site lacking the CTCF consensus motif (Essien et al., 2009). least two independent transgenic male and female clones were The shift was as robust as that observed for the strong binding analyzed, and representative results are shown. Induction was

Figure 2. CTCF Binding to the Xist Promoter Is Anticorrelated with Xist Expression (A) Xist P1 and P2 promoters, CTCF sites, and EMSA probes. Red, CTCF motif. Green bases, nonconforming CTCF motif. Blue, mutated bases in EMSA probes. (B) DNA EMSA using indicated probes with recombinant CTCF or GFP protein. *, DNA-protein shift. (C) CTCF ChIP quantitative PCR analysis on d0, d3, and d6 ES cells as indicated. Rnf12, negative control; RS14c and H19, positive controls. At least three independent experiments were performed for each time point. Averages ± SE. (D) Allele-specific ChIP for CTCF at Xist P2 site in TsixTST/+ female ES cells. cas, M. castaneus. mus, M. musculus. Two independent experiments in triplicates were performed for each time point. Averages ± SE. (E) CTCF ChIP on d0, d3, and d6 female Jpx+/ ES cells. Three independent experiments were performed for each time point. Averages ± SE. P, one-way t test comparing d0 versus d3 and d6 for each site.

Cell 153, 1537–1551, June 20, 2013 ª2013 Elsevier Inc. 1541 ABGFP TRE CTCF-3xFLAG d1 ♀ Phase GFP -FLAG -CTCF DAPI ♂ #11 ♀ #2 #2 API Dox Dox -+ Dox- FLAG FLAG

CTCF CTCF

Dox+ OCT4 actin

♂ #11 ♀ #2 C 100 μ

Dox-

Dox+

♀ D Ctrl #5 ♀ #2 ♀ #1

Dox-

100 μ

Dox+

Figure 3. CTCF Overexpression Results in Female-Specific EB Outgrowth Defect (A) Dox-inducible bidirectional expression of CTCF-3xFLAG and GFP in rtTA-expressing MEF. Normal cell morphology shown by phase-contrast before (Dox) and after (Dox+) induction of GFP and CTCF. FLAG and CTCF immunofluorescence shown. (B) Western blot of representative transgenic clones shows dox-induction of CTCF-3xFLAG in d0 ES cells (left panel) and d1 female ES cells (right panel). (C) CTCF overexpression results in female-specific defect in outgrowth up to d13. Bright field images show EB outgrowth. GFP indicates transgene (CTCF) overexpression. Note GFP-positive cells in male, but not female, outgrowth in Dox+ condition. After induction, GFP signals are confined to the EB center. (D) Female control, ctrl#5, carries a silenced transgene and grows normally, whereas overexpressers, clones #2 and #1, fail to outgrow. d5 images shown. tightly regulated by dox and not obviously toxic (Figures 3A and pression in female clones (e.g., #1, #2) caused severe outgrowth 3B). In male clones (e.g., #11), CTCF overexpression revealed defects after differentiation (Figures 3C and 3D). GFP+ (CTCF+) no phenotype between d0 to d11, as embryoid bodies (EB) cells were confined to the EB center, whereas outgrown cells outgrowth was robust (Figure 3C). By contrast, CTCF overex- were GFP (CTCF), implying viability only when the transgene

1542 Cell 153, 1537–1551, June 20, 2013 ª2013 Elsevier Inc. A Dox- Dox+ Xist Cells with Xic ♀ Xist cloud n ♀ #2 Dox- 1.5 Dox+ Dox- 43% 159 #2 Dox+ 8% 131 1.0 χ2 P<10-4

Dox- 37% 270 #1 0.5 Dox+ 7% 235 χ2 P<10-4 Relative RNA levels Relative RNA 0 Oct4 Bmp4 Xist 5 μ

B d0 d2 d4 d6 d11 d0Dox+ d2 d4 d6 d11 Dox-

100 μ

d0 d2 d4 d6 d11 Dox+ d0Dox+ d2 d4 d6 d11

d0 d2 d4 d6 d11 d0 d2 d4 d6 d11 Dox+ Dox+

Figure 4. CTCF Overexpression Inhibits Xist Induction in a Temporally Sensitive Manner (A) CTCF overexpression blocks Xist upregulation. Left panel: RNA-DNA FISH on d6 female transgenic cells ± Dox. Green, Xist RNA. Red, Xic locus. Middle panel: Quantitation of Xist clouds ± Dox on d6 for two clones. Only full Xist clouds are scored (not pinpoint signals). Right panel: qRT-PCR on d4 cells for Oct4, Bmp4, and Xist RNA. Averages ± SE. (B) Sensitive time window between d2–d4 of EB differentiation. Dox is applied on the days indicated (red bar) during time course analysis. Phase contrast and corresponding GFP images are shown for d11.

was silenced. Defects were strictly correlated with CTCF overex- We next performed Xist RNA-DNA FISH and found that CTCF- pression, as transgenic female clones without overexpression overexpression significantly affected Xist upregulation. Whereas (e.g., #5) showed no growth anomalies (Figure 3D). We conclude 37%–43% of uninduced cells displayed full Xist clouds by d6, that CTCF overexpression is toxic specifically to female cells only 7%–8% of CTCF-overexpressing cells showed robust Xist during differentiation, consistent with an effect on XCI. clouds (Figure 4A). Quantitative RT-PCR confirmed a failure to

Cell 153, 1537–1551, June 20, 2013 ª2013 Elsevier Inc. 1543 fully upregulate Xist, despite progression through cell differenti- erentially interacted with Jpx RNA among total cellular RNA ation as indicated by normal levels of Oct4 and Bmp4 messenger extracted from female MEF (Figure 6B). Conversely, Jpx RNA RNA (mRNA) (Figure 4A). Collectively, these findings demon- preferentially bound CTCF over control FLAG-GFP. Increasing strate that CTCF overexpression blunts Xist upregulation and CTCF levels resulted in greater pull-down of Jpx RNA, demon- argue that CTCF is a blocking factor for Xist. The proposed strating that the CTCF-Jpx interaction is dose dependent. Jpx repressive effect of CTCF is consistent with the observation RNA has a number of alternative splice forms (Chureau et al., that knocking down CTCF results in Xist upregulation (Donohoe 2002; Johnston et al., 2002; Chow et al., 2003), but genetic anal- et al., 2007). ysis indicates that only the region spanning exons 1–3 (E1–E3) is We then asked if CTCF’s effects were restricted to the devel- required for function (Lee et al., 1999). In an electrophoretic opmental window of Xist upregulation. To perform a time course mobility shift assay (EMSA) using purified recombinant protein, analysis, we applied dox at various time intervals and observed the 383 nt E1–E3 isoform of Jpx was robustly shifted by CTCF, large differences in relation to specific periods of induction (Fig- but not GFP (Figure 6C). Increasing CTCF resulted in greater ure 4B). As expected, dox induction between d0–d11 caused binding to Jpx but not an Xist RNA control of the same size, scant EB outgrowths, whereas untreated EB exhibited robust indicating a specific, direct, and dose-dependent interaction outgrowth. When dox was applied only between d0–d2, between CTCF and Jpx. The strong shift required full-length outgrowth was slow to appear but recovered during the dox- E1–E3, as CTCF only weakly shifted E1–E2 and E2–E3 isoforms free interval. When treatment was extended to d0–d4 or applied (Figure 6D). We conclude that CTCF directly binds Jpx RNA. between d2–d11, outgrowth was severely restricted and never recovered. On the other hand, dox treatment after d4 had no Jpx Activates the Xist Promoter by Titrating Away CTCF effect. Thus, female ES cells are sensitive to CTCF overexpres- Binding sion only between d0 and d4, most acutely between d2–d4, The physical interaction (Figures 6A–6D) and opposing effects the time frame corresponding to the initiation of XCI. (Figure 5) of Jpx and CTCF raised the possibility that Jpx could titrate CTCF from Xist P2 and, in so doing, activate Xist. Interest- Jpx RNA Titrates CTCF Binding ingly, EMSA revealed that Jpx RNA and P2 DNA compete for The fact that CTCF persisted at Xist P2 when Jpx is deficient (Fig- binding to CTCF. In an RNA EMSA, the Jpx-CTCF shift was ure 2E) hinted that CTCF and Jpx may be antagonistically linked. diminished by adding cold P2 DNA (Figure 6E). This effect was We asked if overexpressing Jpx could relieve the effect of CTCF attenuated by mutating the P2 DNA competitor. In the reciprocal overexpression by introducing the EF1a:Jpx transgene into DNA EMSA, the P2-CTCF shift was progressively titrated away CTCF-overexpressing female cells (Tg(EF1a:Jpx)/Tg(TRE:Ctcf)). by increasing amounts of Jpx RNA (Figure 6F, left panel), with Indeed, the outgrowth defect was reversed (Figure 5A, note intact E1–E3 being required for maximal effect (Figure 6F, right GFP+ outgrowth), and normal numbers of full Xist clouds were panel). Thus, Jpx RNA and P2 DNA compete for CTCF binding. restored, from 3% to 36% (Figure 5B). qRT-PCR and western To determine whether the competition results in titration of analysis confirmed Jpx and CTCF overexpression (Figures 5C CTCF-P2 binding in vivo, we performed CTCF ChIP at the Xist and 5D). An antagonistic relationship between Jpx and CTCF promoter and asked how CTCF binding is affected by Jpx or was also evident from 2-fold decreases in Xist levels when CTCF levels (Figure 7A). When CTCF alone was overexpressed, CTCF alone was overexpressed (Figure 5C); when Jpx alone P2 binding was significantly boosted (red) and Xist upregulation was overexpressed, Xist levels increased 2-fold; and, finally, was compromised (Figures 4A and 5B). When Jpx alone was when CTCF and Jpx were both overexpressed, Xist was restored overexpressed, CTCF binding at P2 was reduced approximately to wild-type levels. Therefore, Xist defects due to CTCF overex- by half (green), consistent with titration at one Xist allele and pression could be overcome by expressing more Jpx. These ectopic Xist upregulation (Figure 1). When Jpx and CTCF were data indicate that Xist is controlled by a balance of Jpx and overexpressed together (purple), P2 binding was restored. CTCF and that Jpx antagonizes CTCF by titrating CTCF’s Therefore, Jpx indeed titrates CTCF binding at the Xist P2 repressive effect on Xist. promoter. To investigate how the CTCF-Jpx titration affects Xist pro- Jpx RNA Binds CTCF moter activity, we carried out luciferase (Luc) reporter assays To understand the mechanism of titration, we asked if Jpx RNA after modulating CTCF and Jpx levels (Figure 7B). Luciferase and CTCF interact in vivo by performing UV crosslinked RNA constructs fused to P1 and/or P2 were introduced into female immunoprecipitation (UV-RIP) with anti-FLAG antibodies in d12 ES cells and tested during differentiation when Jpx levels nor- female ES cells (Figure 6A). Jpx RNA showed significant coim- mally rise. When introduced into Jpx+/ females, luciferase munoprecipitation with FLAG-CTCF, whereas cyclophilin and expression was reduced to half that of wild-type (WT) females Xist RNA did not. When RIP was performed without UV crosslink- (Figure 7B, left panel, black versus red), consistent with ing, Jpx enrichment was not observed, demonstrating that strin- Jpx+/ cells being male-like and incapable of supporting Xist gent conditions enabled RNA pull-down only when Jpx and upregulation. This defect was overcome by transgenically ex- CTCF were crosslinked. Given that UV crosslinks RNA to protein pressing Jpx RNA (Figure 7B, middle panel). This rescue only when in direct contact, these data suggest that Jpx directly depended on an intact P2 promoter, as mutating P2 abolished interacts with CTCF in vivo. the rescue (P2mut; red versus purple). Finally, Xist promoter We then investigated Jpx-CTCF interactions in vitro. RNA pull- activity was repressed by CTCF overexpression (Figure 7B, down assays using purified FLAG-CTCF showed that CTCF pref- right panel, black versus green), and the repression could be

1544 Cell 153, 1537–1551, June 20, 2013 ª2013 Elsevier Inc. C R-C fJx it n tfRAo 8 omlzdt gTECc)Dox Tg(TRE:Ctcf) to blotting. normalized western d8, by on shown RNA induction Ctcf CTCF-FLAG and (D) Xist, Jpx, of qRT-PCR (C) A vrxrsigJxRArsusotrwhdfcscue yCC vrxrsin hs otatadcrepnigGPsgassono 8of d8 on shown signals GFP corresponding red, and RNA; Xist contrast Green, Phase d8. overexpression. on FISH CTCF RNA-DNA by (B) caused defects CTCF outgrowth Titrating rescues differentiation. by RNA Defect Jpx Outgrowth the Overexpressing Neutralizes (A) RNA Jpx 5. Figure C B A Dox+ Dox- Dox+ Dox- Xist Relative RNA levels Xic 5 10 20 30 40 50 μ 0 Tg(TRE:Ctcf) #E5 J p x Tg(TRE:Ctcf) #E5 100 0.5 1.0 1.5 2.0 2.5 0 μ * Ctcf Xic ou.FS onsaesoni h ih panel. right the in shown are counts FISH locus. Tg(TRE:Ctcf)/Tg(EF1a:Jpx) #E8 * Xist Tg(TRE:Ctcf)/Tg(EF1a:Jpx) Tg(TRE:Ctcf)/Tg(EF1a:Jpx) Tg(TRE:Ctcf) Tg(TRE:Ctcf) Dox+ Dox- stt .) Averages 1.0). to (set Cell Tg(TRE:Ctcf)/Tg(EF1a:Jpx) #E8 Tg(TRE:Ctcf)/Tg(EF1α:Jpx) Tg(TRE:Ctcf)/Tg(EF1α:Jpx) Tg(TRE:Ctcf) Tg(TRE:Ctcf) 153 Dox+ Dox- d8 5715,Jn 0 2013 20, June 1537–1551, , D ± Eo rpiae.* .5(n-a test). t (one-way 0.05 < *p triplicates. of SE LaminB CTCF FLAG Dox Dox + + Tg(TRE:Ctcf) - - ++ Xist cloud Cells with 36% 43% 3% 36% –– Tg(TRE:Ctcf)/Tg(EF1a:Jpx) ª 03Esve Inc. Elsevier 2013 P<10 P<10 χ2 -4 -4 106 100 149 70 n 1545 A B 45 40 6 Jpx UV- 35 Cyclophilin D 5 UV+ 30 Jpx isoforms 4 25 20 12 3 4 5 pA pA

3 % Input 15 (E4-E5 not required) % Input 2 10 E1-E3 1 5 E1-E2 E2-E3 0 0 Jpx Jpx Cyclo- 100 200 500 1000 100 200 500 1000 Xist Jpx Probe E1-E3 E1-E2 E2-E3 E1-E2 E4-E5 philin FLAG-GFP (ng) FLAG-CTCF (ng) bead FLAG CTCF - 14- 14 - 14 pmole

C * --124 -- pmole Probe CTCF Jpx Xist GFP -- -- 124 pmole CTCF - 0.5 1 2- 0.5 1 2 pmole Unbound * RNA probe *

Unbound Unbound Unbound RNA probe RNA probe RNA probe

EF Jpx Xist

Jpx E1-E3 E1-E2; E2-E3 P2 DNA -- -- P2 mut RNA --+ ++++++++ Jpx RNA CTCF - 124 22pmole CTCF --+++++++++ - ++++++++ CTCF - +++++++++ * * *

Unbound RNA probe Unbound Unbound DNA probe DNA probe

Figure 6. Jpx RNA Binds to CTCF and Extricates CTCF from the Xist Promoter (A) UV-RIP using anti-FLAG antibodies, followed by qRT-PCR in d12 female ES cells. qRT-PCR was performed on Jpx exons 1–2 (E1–E2), exons 4–5 (E4–E5), Cyclophilin control, and Xist. UV-negative controls were processed in parallel. Averages ± SE. (B) In vitro RNA pull-down using total RNA extracted from wild-type female MEFs. Purified FLAG-GFP or -CTCF was incubated with RNA. Pull-downs quantified by qRT-PCR. Averages ± SE. (C) RNA EMSA. *, RNA-protein shift. Left panel: Jpx RNA probe (383 nt) with increasing amounts of purified CTCF or GFP. Right panel: CTCF with Jpx versus 383 nt Xist control probe. (D) RNA EMSA using Jpx isoforms. E1–E3, 383 nt (full length). Truncated E1–E2, 220 nt, or E2–E3, 183 nt. E4–E5 are alternative 30 ends, which are not required for Xist activation (Lee et al., 1999). (E) Titration EMSA shows that Jpx RNA and P2 DNA compete for binding to CTCF. A 0.5 pmol of Jpx probe, with 2 pmol cold P2 or P2 mut 80 bp DNA competitor. *, RNA-protein shift. Compare lanes marked by green/red arrows. (F) Reciprocal titration: P2 DNA and Jpx RNA compete for binding to CTCF. CTCF and P2 DNA probe were mixed with cold RNA competitor at 0.1, 0.2, 0.4, and 0.8 mg. *, DNA-protein shift. Left panel: With cold Jpx RNA or Xist RNA competitor. Right panel: With full-length Jpx versus truncated Jpx RNA competitor. Compare lanes marked by red arrows. overcome by mutating P2 (green versus purple). Although the P1 and P2 together enabled full regulation. We conclude that effects were statistically significant with P2 alone (1 kb), the competitive interactions between CTCF, Jpx RNA, and the Xist combination of P1+P2 (2 kb) was more robust, suggesting that promoter regulate Xist transcriptional induction.

1546 Cell 153, 1537–1551, June 20, 2013 ª2013 Elsevier Inc. DISCUSSION such as those resulting from highly cooperative interactions, would be a necessary property of the Jpx-CTCF-P2 interactions. We have shown that Xist transcription is controlled by a dynamic Nonlinear dynamics enable small changes in factor concentra- balance of CTCF and Jpx RNA. Our data argue that CTCF is a tion to elicit large biological effects (e.g., all-or-none binding blocking factor for the Xist promoter, that promoter blockage is and transcription induction) in a way that is not possible with relieved by Jpx-mediated eviction of CTCF, and that promoter linear dynamics (Figure 7F). This concept is exemplified by the P2 is the primary site of action. Jpx meets several conceptual classic biphasic switch of lambda phage. The all-or-none effects criteria for a numerator of the X:A ratio. Jpx resides at the Xic of cro activation is based on cooperative binding of multiple l and escapes XCI (Chureau et al., 2002; Johnston et al., 2002; repressors to three operators within the cro promoter (Ptashne, Chow et al., 2003; Tian et al., 2010). Jpx RNA is also diffusible 2011). At the 50 end of Xist, multiple sites for CTCF and other fac- and trans-acting (Tian et al., 2010), two properties that facilitate tors (e.g., YY1) occur, though the potential for coooperativity has titration. Moreover, Jpx RNA acts directly at the Xic, specifically not been tested. at the Xist promoter, where it titrates away CTCF to induce Xist Long noted for an abundance of lncRNA, the Xic is now known transcription. to regulate XCI through a series of RNA-protein interactions (Lee, The titration model centered on Jpx-CTCF-P2 interactions is 2011). In addition to Xist-Polycomb (Zhao et al., 2008) and Xist- supported by experimental observations (summarized in Fig- YY1 interactions (Jeon and Lee, 2011) for the initiation of ure 7C). In wild-type cells, Jpx is expressed at 2-fold greater silencing, here we have uncovered Jpx-CTCF interactions for levels in female cells than in male cells (Figure S1B). Deleting a molecular titration. Like YY1, CTCF is a ‘‘bivalent’’ protein, single allele of Jpx in females abolishes Xist upregulation and capable of binding both RNA and DNA. Interestingly, CTCF was simulates the XY state (Tian et al., 2010). Conversely, overex- previously found to be in a larger complex containing SRA (Yao pressing Jpx induces ectopic Xist expression, mimicking the et al., 2010). Perhaps, bivalency of other chromatin factors will presence of additional X chromosomes. Modulating levels of also emerge in time as key determinants of epigenetic regulation. the autosomal factor, CTCF, likewise affects Xist induction. Overexpressing CTCF blocks Xist activation, but this blockage EXPERIMENTAL PROCEDURES is overcome by increasing Jpx RNA levels. Conversely, ectopic Xist activation caused by Jpx overexpression is blunted by over- Transgenic ES Cell Lines expressing CTCF. Furthermore, Xist promoter repression by Tg(Jpx) and Tg(EF1a:Jpx) containing full-length Jpx and Neo marker were made by ET-cloning (Yang and Seed, 2003) from BAC 399K20 and pEF1/ CTCF cannot be relieved in Jpx+/ cells, but the defect is V5-His vector (Invitrogen). The Neo vector was used as control. For bidirec- rescued by exogenous Jpx. tional inducible CTCF and EGFP expression, a Hygro cassette flanked by Some curious aspects of our findings are the locus-specific SV40 promoter and polyA was inserted into pTRE-Tight-Bi (Clontech) using and allelic nature of the Jpx-CTCF-P2 interactions. Because PciI; EGFP was inserted using BglII; 3xFLAG cassette was inserted into CTCF is ubiquitous, specificity for P2 binding must involve addi- NotI-SalI sites; and CTCF complementary DNA (cDNA) was cloned in-frame tional factors and proper context. Similarly, Jpx’s exclusivity for using MluI and NotI. Wild-type J1 male and 16.7 female ES cells (Lee and Lu, 1999) and TsixTST/Y and TsixTST/+ ES cells (Ogawa and Lee, 2003) have CTCF bound to P2 must be influenced by proper context. been described previously. Ainv15 male ES cells carrying the tet-activator Furthermore, the monoallelic nature of the titration under physi- rtTA were a gift from G. Daley (Kyba et al., 2002). F1-2.1 female ES cells car- ological conditions is likely linked to the X:A ratio. We suggest rying rtTA were a gift from R. Jaenisch (Luikenhuis et al., 2001). Transgenic ES that Jpx is a component of the competence factor proposed in cells were generated by electroporation (Bio-Rad Genepulser) with stable the Two Factors Model, which postulates that—in addition to clones picked after 8–11 days under G418 (400 mg/ml), hygromycin B an autosomal blocking factor—an X-linked competence factor (200 mg/ml), or puromycin (1 mg/ml) selection. Autosomal integration was m arises only when X:A R1.0 and is essential for inducing Xist confirmed by DNA FISH. Doxycycline induction was performed at 1 g/ml. (Lee and Lu, 1999; Lee, 2005). Our data suggest that CTCF is RNA/DNA FISH and Immunostaining a denominator and subunit of the blocking factor. In support of Simultaneous RNA-DNA FISH has been described previously (Lee and Lu, this, overexpression blocks Xist P2 activity, knockdown results 1999). Immuno-FISH was performed as described previously (Zhang et al., in increased Xist expression (Donohoe et al., 2007), and binding 2007). Cells were blocked with PBS, 0.2% Tween20, and 1% BSA. Anti- to P2 is anticorrelated with Xist expression. In our model, the CTCF (Cell Signaling D31H2) and anti-FLAG M2 (Sigma F1804) were diluted Jpx:CTCF ratio induces Xist transcription only when a critical 1:1,000 and incubated at room temperature for 1–2 hr. threshold is exceeded (Figures 7E and 7F). When expressed from a single X (XY state), Jpx concentrations fail to reach qRT-PCR Total RNA was extracted using TRIzol (Invitrogen), treated with TURBO DNase threshold. When expressed from two X’s (XX state), Jpx concen- (Ambion), and reverse transcribed using SuperScript III (Invitrogen) and trations exceed threshold and result in extrication of CTCF from random primers (Promega). Quantitative PCR was performed using iQ SYBR one Xist allele. Overexpression of Jpx (e.g., in XXX or Tg(Jpx) Green Supermix on a CFX96 (Bio-Rad). PCR primers are as follows. cells) would result in crossing of a second threshold, leading to Jpx: e1-F and e1-R (Tian et al., 2010); mJpx76+(ex1), 50-TTAGCCA 0 eviction of CTCF from both Xist promoters and to biallelic Xist GGCAGCTAGAGGA-3 , and mJpx225-(ex2), AGCCGTATTCCTCCATGGTT. activation. Xist: NS33 and NS66 (Stavropoulos et al., 2001). Oct4: BD151 GGACAT GAAAGCCCTGCAGAAGG and BD152 CGAAGCGACAGATGGTGGTCTGGC. The all-or-none dynamics of this type of stoichiometric inter- Bmp4: BD172 CTGGACACCTCATCACACGACTACTGG and BD173 GGG action would require a robust ‘‘biphasic switch,’’ as proposed CCCAATCTCCACTCCCTTGAGG. Ctcf: BD79 ATCGTAgctagcATGGAAGGT for other bimodal gene expression responses (Cline and Meyer, GAGGCGGTTGAAGCC and BD6 tgacatcctggaccacctcaccc. Gapdh: Gapdh 1996; Ptashne, 2011; Gutierrez et al., 2012). Nonlinear dynamics, F and R (Jeon and Lee, 2011).

Cell 153, 1537–1551, June 20, 2013 ª2013 Elsevier Inc. 1547 P1 P2 Xist A B 1 Kb CTCF Lu c 2 Kb Tg(TRE:Ctcf), Dox- Lu c Tg(TRE:Ctcf), Dox+ WT CTCF- Jpx+/- Tg(TRE:Ctcf)/Tg(EF1α:Jpx), Dox- CTCF-; P2mut 3.0 * WT; JpxRNA Jpx+/- Tg(TRE:Ctcf)/Tg(EF1α:Jpx), Dox+ CTCF+ Jpx+/-; JpxRNA Jpx+/-; JpxRNA CTCF+; P2mut 2.5 * 1.4 ** 2.5 2.0 ** ** * 1.2 ** 1.5 2.0 * 1.0 IP 1.5 ** * *** 1.5 * 0.8 1.0 * ** 1.0 0.6 1.0 0.4 0.5 0.5 0.5 0.2 Relative Luc Activity 0 0 0 0 Xist_5' P1 P2 RS14c 1 Kb 2 Kb 2 KbWT P2mut 1 Kb 2 Kb C Criteria for numerator (X:A ratio) Evidence supporting Jpx RNA

Escape from XCI Jpx fully escapes XCI [Chureau et al., 2002; Johnston et al., 2002; Chow et al., 2003; Tian et al., 2010] Deletion of one allele abolishes Jpx+/- cells cannot induce Xist; Jpx+/- = Jpx+/Y XCI (male-like state) [Tian et al., 2010]. Tg(Jpx) female cells have 2 Xist clouds [Fig. 1]. Overexpression induces ectopic Tg(Jpx) male cells show 1 Xist cloud [Fig. 1]. XCI (simulates supernumerary X’s) Deleting Tsix (repressor) augments Jpx’s activating effect [Fig. 1].

Diffusibility (trans-action) Autosomal Tg(Jpx) rescues Jpx+/- defect [Tian et al., 2010].

CTCF binds and blocks Xist promoter --> Xist ‘blocking factor’ [ChIP, EMSA - Fig. 2, 4A]. Titration of denominator Overexpressing CTCF blocks Xist activation, but blockage is overcome by increasing Jpx levels. Overexpressing Jpx causes ectopic Xist activation, but ovexpressing CTCF blunts effect. Jpx binds and molecularly titrates CTCF [RIP, competitive EMSA - Fig.6]. Jpx relieves CTCF binding to Xist promoter in vivo [ChIP - Fig. 7A]. Jpx relieves CTCF repression of Xist transcription in vivo [Luc assay, RNA FISH - Fig. 7B,5]. In Jpx+/- cells, CTCF-P2 binding cannot be relieved, but is overcome by Jpx overexpression [Fig. 2E].

Jpx:CTCF interaction occurs at Xist promoter P2 [Fig. 2,4,6,7]. Action at Xic P2 mutation abolishes titration [Fig. 2,6,7].

Jpx RNA induced 10-fold during cell differentiation [Tian et al., 2010]. Temporal specificity Cells sensitive to CTCF overexpression from d2-d4 [Fig. 3,4]. Sensitivity overcome by Jpx overexpression [Fig. 5].

D E F CTCF X:A ratio Xist non-linear Jpx Jpx XCI model X Xist threshold XiXi ES differentiation, biallelic OFF induction of Jpx A Xa CTCF binding to P2 Tsix RNA CTCF

Jpx mono RNA Xist XaXi mono titration allelic Xist allelic Jpx RNA

Jpx RNA Xist expression titrates CTCF X model) OFF (linear Xi biallelic A 012 3 4 Xist Xist Jpx RNA level RNA CTCF 2-fold increase 2 Xi X:A ratio Xa crosses 1 Xi threshold Tsix RNA threshold

(legend on next page)

1548 Cell 153, 1537–1551, June 20, 2013 ª2013 Elsevier Inc. FLAG-CTCF and FLAG-GFP Purification tured at 95C for 2 min and incubated at 50C for 5 min, 37C for 15 min, and Ctcf or GFP cDNA (pFA6a-GFP(S65T)-kanMX6) with C-terminal 6xHis tag was RT for 15 min, cooled, and maintained in folding buffer (50 mM NaCl, 2 mM cloned into pFLAG-2 (Sigma-Aldrich) using EcoRI and XhoI. FLAG-CTCF- MgCl2) on ice prior to the binding reaction. Recombinant proteins were incu- 6xHis and FLAG-GFP-6xHis proteins were purified from Rosetta-Gami B cells bated at room temperature with 0.5 pmol RNA probes for 30 min in 10 ml bind- (Novagen). Briefly, cells were induced with 0.2 mM IPTG at 18C (CTCF) or ing buffer containing 1 mg poly dI-dC, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM IPTG (GFP) at 30 C and lysed at 4 C with 50 mM sodium phosphate 5 mM MgCl2, 0.1 mM ZnSO4, 1 mM DTT, 10% glycerol, 0.1% Tween-20, (pH 8.0), 300 mM NaCl, 20 mM imidazole, 0.5% Sarkosyl, and protease inhib- 0.1 mM polyamine, 8 U Rnase Inhibitor (Roche), and 1 mg Yeast transfer itors. Triton X-100 was added to 2% final [v/v]; debris was removed by centri- RNA (tRNA). Samples were resolved at 4C by TBE- 5% or 6% PAGE and fugation; and proteins were eluted from Ni-NTA resin (QIAGEN) with 50 mM imaged on BioMax film (Kodak). In DNA-RNA titration EMSA, protein was incu- sodium phosphate (pH 8.0), 300 mM NaCl, and 250 mM imidazole. Eluates bated with DNA competitors prior to adding RNA probes. For RNA-DNA titra- were dialyzed against 50 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2, 150 mM tion experiments, RNA competitors were mixed with protein and DNA probes

NaCl, 0.1 mM ZnSO4, 1 mM DTT, 0.1% Tween-20, and 10% glycerol. in RNA EMSA binding buffer, including RNase inhibitor and yeast tRNA.

DNA and RNA EMSA Chromatin Immunoprecipitation For DNA EMASA, these double-stranded 80-mer DNA oligonucleotides were ChIP was performed as previously described (Blais et al., 2005), with modifica- used: tions. Approximately 1.5 million ES cells were crosslinked in 1% formaldehyde Xist_50: TTTGGCTTCAGATGCCTTGGAAAGATTCATGCTGCCACCTGCTG at RT for 10 min and quenched with glycine (125 mM final concentration) for GTTTATTCAGTCTGTGTGCATCATTTATTTACTTGT; Xist_50mut: 5 min at RT. Chromatin was sonicated using Bioruptor XL (Diagenode), treated TTTGGCTTCAGATGCCTTGGAAAGATTCATGCTGCTACTTGCTAGTTTAT with RNase A (0.2 mg/ml) at 37C for 30 min, and 3 mg of anti-CTCF (Cell TCAGTCTGTGTGCATCATTTATTTACTTGT; P1: Signaling 2899) or IgG (Cell Signaling 2729) were incubated with chromatin TTGTGGCCACTCCTCTTCTGGTCTCTCCGCCTTCAGCGCCGCGGATCA at 4C overnight. Protein-antibody complexes were recovered with Dynal- GTTAAAGGCGTGCAACGGCTTGCTCCAGCCAT; P2: beads Protein G (Invitrogen), DNA was extracted with phenol/chloroform, GAATGTGTCTAAGATGGCGGAAGTCATGTGACCTGCCCTCTAGTGGTTT and quantitative PCR was performed using these primers: CTTTCAGTGATTTTTTTTTTGGCGGGCTTTA; P2 mut: Rnf12_F, CCCCAGGTGAAAGTACTGAGG; Rnf12_R, CTCTCCAGCTC GAATGTGTCTAAGATGGCGGAAGTCATGTGACCTGTCCTATAGTTGTTTC TATTTTCATCG; Xist_50F, CCCTACCTGAACCACCTCAATAGT; Xist_50R, TTTCAGTGATTTTTTTTTTGGCGGGCTTTA; and AGTTCCCTTTAGGCGTCCCAT (Navarro et al., 2006); P1_F, ACGCGTCATGT RS14C: CTATGCATTAGGTTTGGGTGTTATACCCGTGTAGGCCAGCAGA CACTGAGC; P1_R, AAAACGTCAAAAATCTCGTGG; P2_F, CTCGACAGCC GGGTGTCGGATCCCAAGGAAACCAAGTTACAGACGCC. CAATCTTTGTT; P2_R, ACCAACACTTCCACTTAGCC (Jeon and Lee, 2011); To anneal forward and reverse strands, oligos were heated to 95C and RS14c_F, GGCTACACACAAGATGGCGTCTGTAACTTG; RS14c_R, TAGG slow-cooled to room temperature in 10 mM Tris-HCl (pH 8.3), 1.5 mM TTTGGGTGTTATACCCGTGTAGGC (Spencer et al., 2011); H19_F, GTCACT

MgCl2, and 50 mM KCl. Double-stranded DNA probes were end-labeled CAGGCATAGCATTC; and H19_R, GTCTGCCGAGCAATATGTAG. with ATP[g-32P] and unincorporated ATP removed with Microspin G-50 Allele-specific quantitative PCR at P2 was performed using primers 231_F, columns (GE Healthcare). A 2 pmol of recombinant proteins were incubated TGTGCATTATTGCTTGGTGGCCAG and 371cas_R, TGCGCTCTCCCGA at room temperature (RT) with 1 pmol of probes in 1 mg poly dI-dC, 50 mM CACTTC or 129_R, CATGCGCTCTCCCGACCTG.

Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 0.1 mM ZnSO4, 1 mM DTT, 10% glycerol, and 0.1% Tween-20. Samples were resolved at RT by TBE- RNA Immunoprecipitation 6% or 7% PAGE gel. Crosslink RNA immunoprecipitation (RIP) was performed as previously RNA EMSA was performed as described previously (Cifuentes-Rojas et al., described (Jeon and Lee, 2011)on107 Tg(TRE:Ctcf) ES cells differentiated 2011). RNA probe was in vitro transcribed with T7 RNA polymerase (Ambion) in the presence of 1 mg/ml doxycycline for 12 days. Cells were UV crosslinked off PCR-amplified cDNA templates created using at 254 nm (200 J/cm2) in PBS, collected by scraping, and lysed in PBS supple- T7_Jpx_ex1F, TAATACGACTCACTATAGACGGCACCACCAGGCTTCT; mented with 0.5% NP-40, 0.5% sodium-deoxycholate, RNase inhibitor, and Jpx_ex3R, GAGTTTATTTGGGCTTACAGTTC (Johnston et al., 2002); proteinase inhibitor cocktail at 4C for 30 min. After 15 min at 37C with Jpx_ex2R, AGCCGTATTCCTCCATGGTT; T7_Jpx_ex2F, TAATACGACTCAC 30 U of TURBO DNase, cell debris was removed, and supernatant was used TATAGAACCATGGAGGAATACGGCT; T7_Xist_F, TAATACGACTCACTATA for immunoprecipitation using Flag-agarose beads overnight at 4C. Beads GCTACTTACACCTTGGCCTC; and Xist_R, AAGTAATTTGAGTATCAT were washed three times with PBS containing 1% NP-40, 0.5% sodium-deox- CTGCC. ycholate, and 300 mM NaCl and were resuspended in TURBO DNase buffer In-vitro-transcribed RNA was DNased (TURBO), TRIzol-purified, phospha- with 10 U DNase, RNase inhibitor, and proteinase inhibitor cocktail for tased (Alkaline Phosphatase, New England Biolabs), end-labeled with ATP 30 min at 37C. Beads were further washed twice in the abovementioned [g-32P], and purified through Microspin G-50 columns. RNA probe was dena- wash buffer containing 10 mM EDTA. RNA was eluted by incubating in

Figure 7. Jpx RNA and CTCF Titrate Each Other at the Xist Promoter In Vivo (A) In vivo titration shown by CTCF ChIP analysis on transgenic female ES cells at d0. Enrichment values were normalized against H19 control and compared to Tg(TRE:Ctcf) Dox (set to 1.0) for each site using a one-way t test (*p < 0.05). At least three independent experiments were performed for each cell line. Averages ± SE. (B) Titration and modulation of Xist P1+P2 transcription shown by luciferase assays in d4 female ES cells of indicated genotypes. Map of Xist’s 50 end is shown with luciferase constructs. Left panel: Jpx overexpression achieved by Tg(EF1a:Jpx) in Jpx+/ mutants rescues Xist promoter activity. Middle panel: Rescue is abolished by mutating P2. Right panel: Transcription repression caused by overexpressing CTCF is relieved by mutating P2. CTCF+, CTCF overexpression induced by Dox via Tg(TRE:Ctcf). At least two independent transfections and R4 independent luciferase measurements were performed for each experiment. Averages ± SE. *p < 0.05. **p < 0.01. (One-tail t tests in the pairwise comparison indicated.) (C) Summary of data supporting the Jpx-CTCF titration model. (D) Model: Jpx RNA activates Xist by extricating CTCF from the Xist promoter. (E) X:A titration: BF, blocking factor. CF, competence factor. The precise composition of BF and its Xic binding site is currently unknown. CTCF may be a component of BF. Jpx forms CF only when present in 2-fold excess. (F) Nonlinear dynamics would be a necessary property of the titration model, most probably effected by highly cooperative interactions. A sigmoidal binding curve would enable small changes in factor concentration to elicit large biological effects (e.g., all or none binding), in a way not possible with linear dynamics (broken gray line).

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The product of the mouse Xist gene is of Jpx and a housekeeping mRNA, cyclophilin A, was normalized to input. a 15 kb inactive X-specific transcript containing no conserved ORF and Primers are as follows: mJpx76+(ex1), mJpx225-(ex2), and mJpx339+(ex4), located in the nucleus. Cell 71, 515–526. GGGCTCAACAAAAGAGCAAA; mJpx607-(ex5), TGGAGTTATGGCCTCG Brown, S.W., and Chandra, H.S. (1973). Inactivation system of the mammalian AAGT; CycophilinA_F, CGATGACGAGCCCTTGG; CycophilinA_R, TCTG X chromosome. Proc. Natl. Acad. Sci. USA 70, 195–199. CTGTCTTTGGAACTTTGTC; CycophilinA_probe, FAM-CGCGTCTCCTTT Brown, C.J., Hendrich, B.D., Rupert, J.L., Lafrenie` re, R.G., Xing, Y., Lawrence, GAGCTGTTTGCA-TAMRA. J., and Willard, H.F. (1992). The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within Luciferase Assay the nucleus. Cell 71, 527–542. 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Luciferase activities were of a Ctcf cofactor, Yy1, for the X chromosome binary switch. Mol. Cell 25, analyzed 36 hr posttransfection. Firefly and Renilla luminescence was 43–56. measured on a TopCount NXT Luminescence Counter. Firefly/Renilla lumines- cence ratio was normalized against the promoterless control. Donohoe, M.E., Silva, S.S., Pinter, S.F., Xu, N., and Lee, J.T. (2009). The plu- ripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting. Nature 460, 128–132. SUPPLEMENTAL INFORMATION Essien, K., Vigneau, S., Apreleva, S., Singh, L.N., Bartolomei, M.S., and Han- nenhalli, S. (2009). CTCF binding site classes exhibit distinct evolutionary, Supplemental Information includes one figure and can be found with this genomic, epigenomic and transcriptomic features. Genome Biol. 10, R131. article online at http://dx.doi.org/10.1016/j.cell.2013.05.028. Gartler, S.M., and Riggs, A.D. (1983). Mammalian X-chromosome inactivation. Annu. Rev. 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