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. 2020 Nov 27;11:6053. doi: 10.1038/s41467-020-19879-3

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a–c A total of >300 nuclei per cell type over 3 independent experiments were scored for the location of the Xi marked by a H3K27me3 cluster or an Xist cloud relative to the nuclear periphery or the nucleolus; significance was determined by one-sided Fisher exact test; bar plots are presented as mean values ± SEM; scale bars represent 10 µm. a Examples of nuclei after H3K27me3 immunostaining (red) to locate the Xi in WT, ΔFirre^Xi, and InvFirre^Xi, or Xist RNA-FISH (red) to locate the Xi in ΔFirre^Xa since there is no H3K27me3 cluster in these nuclei. Nuclei were also immunostained for NPM1 (green) to locate the nucleolus. b Bar plots show a significant decrease in periphery- and nucleolus association of the Xi in ΔFirre^Xa compared to WT (p value = 3.95632e-40 and 3.75497e-14, respectively), but no significant changes in ΔFirre^Xi and InvFirre^Xi (p value = 0.6968). Ectopic expression of a mouse transgene in ΔFirre^{Xa+mtransgene} partly rescues the Xi location to 41% at the periphery and 34% at the nucleolus (p value = 6.62701e-10 and 0.000265481, respectively). c Bar plots show a significant decrease in periphery- and nucleolus association of the Xi after Firre KD in primary MEFs derived from an F1 embryo (BL6 × spretus) (p value = 1.13757e-09 and 0.0000634923, respectively). d Density histograms of the distribution of allelic proportions (Xa/(Xa + Xi)) of CTCF peaks show a shift in the distribution for the X chromosomes due to a decrease in CTCF on the Xi in ΔFirre^Xa (red) compared to WT (blue). In ΔFirre^{Xa+mtransgene} (brown) this distribution becomes binomial due to partial restoration of CTCF on the Xi. e Plots of Xi-associated (common +Xi-specific) CTCF peak density (counts binned within 100 kb windows) along the Xi for WT (blue) and ΔFirre^Xa (red). To account for differences in the number of SNP-covered peaks due to sequencing depth, the binned counts are scaled by a factor obtained from the between-sample ratios of autosomal diploid SNP-covered peaks.