a previous report, Lee and colleagues showed that Oct4 and Sox2 bind to DXPas34 and Xite

Transcription

1 SUPPLEMENTARY INFORMATION doi:1.138/nature9496 a 1 Relative RNA levels D12 female ES cells 24h Control sirna 24h sirna Oct4 5 b,4 Oct4 Xist (wt) Xist (mut) Tsix 3' (wt) Tsix 5' (wt) Beads Oct4,2 c,2 Xist Exon1 Xist Intron1 Tsix site D Xite Beads Sox2,1 Xist Exon1 Xist Intron1 Tsix site D Xite Supplementary Figure 1: Oct4/Sox2 are not the main regulators of Tsix in ES cells. a, In a previous report, Lee and colleagues showed that Oct4 and Sox2 bind to DXPas34 and Xite to control Tsix transcription in ES cells 1. We have shown that Oct4 and Sox2 bind at Xist 1

2 RESEARCH SUPPLEMENTARY INFORMATION intron 1 to silence Xist 2. Given that Tsix negatively influences Xist expression, the finding by Lee et al. suggests that the upregulation of Xist we had observed upon depletion of Oct4 could be dependent on the regulation of Tsix by Oct4. To test this, we knocked-down Oct4 mrna levels in female D12 cells 3. In these cells, one X-chromosome carries a 65 kb-long deletion that encompasses the Tsix promoter, DXPas34 and Xite. We reasoned that, if the effect of Oct4 knock-down on Xist were dependent on a downregulation of Tsix, then Xist should not be upregulated from the mutant X-chromosome. After 24h post nucleofection, Oct4 mrna levels are greatly reduced and Xist upregulation established from both the wt and mutant X- chromosomes (previously described allelic real-time PCR was used for this assay 3 ). This demonstrates that Tsix is not required for Xist upregulation upon Oct4 knock-down. Importantly, we could not detect, after 24h of interference, any significant modification of Tsix expression, which in this cell line is produced by the wt allele only. Thus, we conclude that Oct4 mediates Xist silencing independently of Tsix. Further, the absence of modification of Tsix expression by Oct4 knock-down indicates that Tsix is not a primary target of Oct4. b, c, Since Tsix expression does not show any major variation upon Oct4 knock-down, and we were not able in our previous report 2 to detect Oct4 binding across the Tsix 5 region, we wished to try to reproduce the ChIP experiments published by Lee and colleagues. To this end we used the same primer pairs amplifying DXPas34 and Xite that had been shown by Lee and colleagues to be positive in their Oct4 and Sox2 ChIP experiments. Oct4 (b) and Sox2 (c) showed very low levels of binding to Xite but not to DXPas34 (Tsix site D). Since binding to DXPas34 has similarly not been reported in published ChIP-Seq experiments 4, we conclude that binding at DXPas34, if any, is unlikely to be of biological significance. Low levels of binding are detected at Xite, which is not, however, a strong enhancer of Tsix. If our results argue against a major role of Oct4 and Sox2 on the regulation of Tsix in undifferentiated ES cells, the crucial function that Lee and colleagues attributed to them during the early stages of differentiation 1, in particular concerning X-chromosome pairing, is not addressed by our work. 2

3 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 2: Binding of TFIIB and RNAPII at Xist and Tsix promoters in wild-type and Pas34 ES cells. ChIP analysis of TFIIB and RNAPII binding to the Xist, Tsix and Arpo P promoters in wild-type and Pas34 ES cells, demonstrating that in the absence of DXPas34 the Tsix promoter is repressed (note that the Xist promoter remains repressed in Pas34 ES cells). 3

4 RESEARCH SUPPLEMENTARY INFORMATION Navarro et al. Supplementary Figure 3 a 1,5 Relative mrna levels Control shrna shrna1 Rex1 shrna2 Rex1 b 1 Relative mrna levels Control shrna shrna1#a shrna1#b shrna1#c shrna1#d shrna2#a shrna2#b 1,75,5,5,25 Supplementary Material - Navarro et al. Molecular coupling of Tsix regulation and pluripotency. Rex1 Klf4 c-m yc Tsix5' Tsix3' Rex1 Tsix we used the same primer pairs amplifying DXPas34 and Xite that had been shown by Lee and, c TFIIB d e f H3K4Me3 H3K9Ac H3K36Me3 colleagues Control to be shrna positive in their Control Oct4 shrna and Sox2 ChIP experiments. Control shrnaoct4 (b) and Control Sox2 shrna (c) shrna Rex1 shrna Rex1 shrna Rex1 shrna Rex1 showed very low levels of binding to Xite but not to DXPas34 (Tsix site D). Since binding to,6,3 6 DXPas34 has similarly not been reported in published ChIP-Seq experiments 4, we conclude that binding at DXPas34, if any, is unlikely to be of biological significance. Low levels of 3 binding are detected at Xite, which is not, however, a strong enhancer of Tsix. If our results argue against a major role of Oct4 and Sox2 on the regulation of Tsix in undifferentiated ES cells, the crucial function 37 that 3Lee 31 32and colleagues attributed to 33them 34 35during the early 9 1stages 11 of g differentiation 1, H3K4Me3 in particular concerning H3K9Ac X-chromosome RNAPII pairing, is H3K36Me3 not addressed by our P=,6378 P=,6988 P=,6 P=,6 work. n=8 n=8 n=12 n= Supplementary Figure 2: Binding of TFIIB and RNAPII at Xist and Tsix promoters in wild-type and Pas34 ES cells. ChIP analysis of TFIIB and RNAPII binding to the Xist, Tsix and Arpo P promoters in wild-type and Pas34 ES cells, demonstrating that in the absence of DXPas34 the Tsix promoter is repressed (note that the Xist promoter remains repressed in Pas34 ES cells). Supplementary Figure 3: Additional analyses of Rex1 knock-down cells. a, Relative gene expression 24 hours after Rex1 knock-down using the independent shrna1rex1 and shrna2rex1 constructs (n=2). b, Relative Rex1 and Tsix expression levels in 4 independent clones stably carrying a vector expressing shrna1rex1 and 2 independent clones stably expressing shrna2rex1, compared to a clone stably expressing a control shrna against Gfp (set here arbitrarily to one). Clone #1a was retained for further analysis. c, d, e, Analysis

5 SUPPLEMENTARY INFORMATION RESEARCH of TFIIB (c, n=2), H3K4Me3 (d, n=2), and H3K9Ac (e, n=2) across the Tsix 5 region, in control and Rex1-interfered cells (Clone #1a). f, ChIP analysis of H3K36Me3 across Xist intron 1, showing reduced levels of enrichment in Rex1 knock-down cells (Clone #1a). The position which shows very low levels of enrichment in both control and knock-down cells corresponds to the region of Nanog, Oct4 and Sox2 binding, where a DnaseI hypersensitive site has also been shown to be located. g, Boxplots of ChIP data corresponding to the factor indicated (in this case, n refers to the number of positions analysed by ChIP and used to generate the boxplots). P-values were obtained with a paired t-test in which the average %IP obtained at each location was compared between control and Rex1 knock-down cells. 5

6 RESEARCH SUPPLEMENTARY INFORMATION control shrna c-myc shrna Supplementary Figure 4: c-myc also controls Tsix transcription elongation. ChIP-seq results of RNAPII binding in control and c-myc knock-down ES cells 5 were uploaded to the UCSC browser ( ) as custom tracks. The top panel shows RNAPII distribution across the Tsix locus. Note that ChIP-Seq identifies the two regions of accumulation (corresponding to the 5 and 3 ends) that we have identified here and elsewhere. The bottom panel shows the distribution of RNAPII in c-myc knock-down cells: the density of signal across Tsix, in particular at the 3 end, is reduced as compared to the control. In contrast, the Tsix promoter remains competent to recruit the RNAPII at high levels. These results are highly similar to that we report here upon Rex1 knock-down (Figure 3, and Supplementary Fig. 3). Note that the orientations of Xist/Tsix are the opposite to that of our figures. 6

7 SUPPLEMENTARY INFORMATION RESEARCH a 1 Yy1 ES cells b,4 Ctcf ES cells,5,2 c ,6 Sp1 ES cells d Rex Klf4,3 Yy1 Sp1 Ctcf c-myc e ,2 Relative Binding,8,4 Xite Tsix 5' region Negative control Xite Tsix 5' region Negative control Xite Tsix 5' region Negative control Rex1 Klf4 c-myc Supplementary Figure 5: Additional binding activities at DXPas34 suggest a protein interaction network. a, b, Validation of previously reported binding of Yy1 (a) and Ctcf (b) to DXPas34. Yy1 has been shown to interact with Rex1 6, Ctcf 7, c-myc 8 and Sp1 9. Sp1 also interacts with Klf4 1 and c-myc 11. The binding of Sp1 to the Tsix 5 region that we report here (c) supports a protein interaction network in which Yy1 and Sp1 play a fundamental structural role (d). e, Klf4 binds at Xite (red bar), although at levels lower than to the Tsix 5 region. Data corresponding to this panel was generated in parallel to that of Fig. 3b. 7

8 RESEARCH SUPPLEMENTARY INFORMATION,3 Nanog CK35 DPas34, ,8 Oct4 CK35 DPas34, ,6 Sox2 CK35 DPas34, Supplementary Figure 6: Binding of Nanog, Oct4 and Sox2 to Xist intron 1 in wild-type and Pas34 ES cells. ChIP analysis of Nanog (a), Oct4 (b), and Sox2 (c) binding across Xist intron 1 in CK35 and Pas34 cells. 8

9 SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 7: Abundant binding across the imprinted locus on chromosome 12qF1. ChIP-seq 4 identified binding sites for Nanog, Oct4, Sox2, Klf4 and Esrrb were uploaded to the UCSC browser ( ) as custom tracks. Known imprinted genes were similarly uploaded. Binding profiles were correlated to enrichment for H3K4Me2 and H3K9Me3 that mark imprinting centres 12. Nanog, Oct4 and Esrrb bind around the upstream region of the non-coding Meg3 gene, where the imprinting centre of the cluster is located. 9

10 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 8: Primer and antibody information. 1

11 SUPPLEMENTARY INFORMATION RESEARCH Supplementary references. 1 Donohoe, M. E. et al., The pluripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting. Nature 46 (7251), 128 (29). 2 Navarro, P. et al., Molecular coupling of Xist regulation and pluripotency. Science 321 (5896), 1693 (28). 3 Clerc, P. and Avner, P., Role of the region 3' to Xist exon 6 in the counting process of X-chromosome inactivation. Nat Genet 19 (3), 249 (1998). 4 Chen, X. et al., Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133 (6), 116 (28). 5 Rahl, P. B. et al., c-myc regulates transcriptional pause release. Cell 141 (3), 432 (21). 6 Wang, J. et al., A protein interaction network for pluripotency of embryonic stem cells. Nature 444 (7117), 364 (26). 7 Donohoe, M. E. et al., Identification of a Ctcf cofactor, Yy1, for the X chromosome binary switch. Mol Cell 25 (1), 43 (27). 8 Stojanova, A. et al., Repression of the human immunodeficiency virus type-1 long terminal repeat by the c-myc oncoprotein. J Cell Biochem 92 (2), 4 (24). 9 Lee, J. S., Galvin, K. M., and Shi, Y., Evidence for physical interaction between the zinc-finger transcription factors YY1 and Sp1. Proc Natl Acad Sci U S A 9 (13), 6145 (1993). 11

12 RESEARCH SUPPLEMENTARY INFORMATION 1 Zhang, W. et al., The gut-enriched Kruppel-like factor suppresses the activity of the CYP1A1 promoter in an Sp1-dependent fashion. J Biol Chem 273 (28), (1998). 11 Gartel, A. L. et al., Myc represses the p21(waf1/cip1) promoter and interacts with Sp1/Sp3. Proc Natl Acad Sci U S A 98 (8), 451 (21). 12 Mikkelsen, T. S. et al., Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448 (7153), 553 (27). 12