Extended Data Fig. 9: A site-specific recombination-based approach to rescue phenotypes associated with loss of the E-repeat.

a, Flp-In approach taken to constitutively express Flag-tagged MCP fusion proteins in ES cells (Methods). The Flag–MCP–GFP fusion protein was only expressed in wild-type ES cells. All other rescue constructs were expressed in ΔE ES cells. b, Flag–MCP–GFP fusion protein recruitment to Xist^MS2 in wild-type cells at differentiation day 7 shown with representative epifluorescence images. Arrows indicate MS2+Xist¹²⁹ clouds with co-localizing Flag–MCP–GFP enrichment. c, Tsix expression was used as a proxy to confirm presence of two X chromosomes in rescue ES cell lines. d (i), PTBP1-probed immunoblot on lysates from undifferentiated ΔE ES cells expressing full-length MCP–PTBP1 or MCP-PTBP1 mutants. (ii), As in d (i) except for MATR3 immunoblot for various MATR3 rescue lines. (iii), TDP-43-probed immunoblot on lysates from undifferentiated ΔE ES cells expressing MCP–TDP-43. (iv), CELF1-probed immunoblot on lysates from undifferentiated ΔE ES cells expressing MCP–CELF1. e, Histogram of the percentage of Xist^MS2 clouds that also show enrichment of H3K27me3 in wild-type or ΔE cells, or ΔE cells expressing the indicated MCP-fusion protein at differentiation day 7 (n = 80, from one experiment). f, Representative epifluorescence images of RNA FISH against Xist (green) and MS2 (red) in day 7 differentiated ΔE cell lines expressing the indicated variants of MCP fusion proteins. Inset, enlargement of the marked area. Arrowheads indicate wild-type Xist clouds in ΔE cells, derived from the cas allele. g, Immunoprecipitation of PTBP1, MATR3, CELF1, TDP-43 and CIZ1 from ES cell nuclear extracts (RNase treated) and detection of co-precipitated proteins with the same antibodies by immunoblotting (to accompany Fig. 3f). For images in d and g, see Supplementary Fig. 1 for source data.