Extended Data Fig. 7: The XistΔE-coated X chromosome displays decreased DAPI staining and less compact H3K27me3 accumulation at differentiation day 7.
From: A protein assembly mediates Xist localization and gene silencing
a, Epifluorescence images of cells immunostained for H3K27me3 and probed for MS2. b, Quantification of XistMS2 RNA FISH clouds with a co-localizing accumulation of H3K27me3 at day 3 or 7 of differentiation in wild-type or ΔE cells (n = 60/coverslip, 3 coverslips over 2 experiments); *P = 0.05, two-tailed Student’s t-test. c (i), Top left, 3D-SIM section of wild-type and ΔE cells at differentiation day 7 stained for H3K27me3 and DAPI and probed for MS2. Inset, DAPI staining of marked region. Right, magnification of inset area with (top) or without DAPI (bottom). Bottom left, Z-stack projection of inset without DAPI. (ii), 3D Amira reconstruction of images in (i). d, Graph showing the number of pixels with indicated DAPI fluorescence intensity from XistMS2-expressing X chromosome in wild-type and ΔE cells, masked by H3K27me3 enrichment (n = 10, from one experiment). e, Epifluorescence images of wild-type and ΔE cells probed for MS2. Arrowheads point to the Xist cloud and highlight the DAPI-bright staining for the X-territory. f (i), Epifluorescence images of wild-type cells stained for EZH2 and Xist, with (left) and without (right) EZH2 Xi-enrichment at differentiation day 7. (ii), Histogram of the percentage of Xist clouds with co-localized EZH2 enrichment (n = 60 per coverslip, 3 coverslips from 2 experiments), *P < 0.05, **P < 0.005, ***P < 0.0005, two-tailed Student’s t-test. g, 3D-SIM sections through day 3.5 differentiated wild-type or ΔE ES cells (EpiLC differentiation), immunostained for RNA Pol II and probed for Xist, showing exclusion of RNA Pol II from the X-territory. Inset, signals derived from marked area. Small images: top left, same as inset without DAPI; bottom left, same as inset with only DAPI; top right, Z-stack projection of the cell; bottom right: Z-stack projection of the Xist-coated X chromosome. Scale bar, 5 μm; inset, 1 μm. h, 3D Amira reconstruction of cells in Fig. 2e. Inset, enlargement of the XistMS2-expressing X. Right, same as left without DAPI. i, Quantification of RNA Pol II exclusion from XistMS2-coated territory (n = 50 per coverslip, 2 coverslips from 1 experiment), *P = 0.05, two-tailed Student’s t-test.
