Extended Data Fig. 2: Depletion of PTBP1, MATR3, CELF1 and TDP-43 affects Xist localization during XCI initiation without strongly altering Xist processing.

a, Proportion of Xist-positive cells with co-localizing exclusion of RNA Pol II or enrichment of H3K27me3 or the PRC2 components EZH2, EED, SUZ12 on the Xi, in female ES cells differentiated for 3 days and treated with siGFP or siPTBP1 (n = 50 from one experiment). b, Percentage of Xist-positive cells with H3K27me3 Xi-enrichment in day 3 differentiated female ES cells treated with siGFP, siPTBP1, siMATR3, siTDP-43 or siCELF1 (n = 100, from one experiment). The siPTBP1 sample is independent from that in a. c, Xist splicing events assessed below. d, Histogram showing Xist abundance (exon 1 PCR above) upon siRNA-mediated knockdown of indicated RBPs in female ES cells at differentiation day 3. e, As in d, except for the abundance of spliced Xist exon 1–2 and exon 6–7 amplicons upon knockdown. For d and e, samples were normalized against siGFP and Snrnp27 RNA and assessed in triplicate from three independent experiments. Error bars represent s.e.m; *P < 0.05, two-tailed Student’s t-test. f, Snapshot of expected spliced exon 6 (green) to exon 7 (red) sequence. Correct exon 6–7 ligation occurs after 72 h of siGFP or siPTBP1 treatment in differentiating female ES cells (black sequence) in two independent experiments. g (i), A tet-inducible full-length Xist cDNA transgene was inserted into the X-linked Hprt locus in male ES cells. (ii), Percentage of cells with an Xist cloud after 48 h of siPTBP1, and dox treatment starting at 24 h of siRNA treatment, in cells described in (i) (n = 80, from one experiment). (iii), Representative RNA FISH images of Xist, co-immunostained for PTBP1 and DAPI labelled, in cells described and treated as in (i), (ii). Note Xist dispersal upon PTBP1 knockdown, despite the absence of Xist  splicing.