Fig. 2: Firre RNA acts in trans to maintain H3K27me3 on the Xi.
From: Trans- and cis-acting effects of Firre on epigenetic features of the inactive X chromosome
a–c A total of >300 Patski nuclei were scored per cell type over 3 independent experiments; 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 Xist RNA-FISH (green) and Hoechst 33342 staining (blue). The bar plot shows no significant differences among cell lines. b Examples of nuclei after H3K27me3 immunostaining (red) and Hoechst 33342 staining (blue). The bar plot shows significantly fewer nuclei with a H3K27me3 cluster in ΔFirreXa versus WT (p value = 4.63688e-94), but no change in ΔFirreXi nor InvFirreXi. c Examples of nuclei after macroH2A.1 or H2AK119ubi (red) immunostaining and Hoechst 33342 staining (blue). The bar plots show no significant differences between cell types. d Profiles of H3K27me3 ChIP-seq reads along the Xi in WT (blue), ΔFirreXa (red), and log2 ratio ΔFirreXa/WT (green). Box plots (log2) of H3K27me3 ChIP-seq reads in 100 bp Xi bins show a significantly lower median in ΔFirreXa (red) versus WT (blue) (Wilcoxon test: p value = 2.2e-16). The boxes demarcate the interquartile range (IQR) with median; whiskers ±1.5 times the IQR; outliers plotted as points. e Density histograms of the distribution of allelic proportions (Xa/(Xa+Xi)) of H3K27me3 peaks show a shift for the X chromosomes due to lower H3K27me3 on the Xi in ΔFirreXa (red) compared to WT (blue) (Wilcoxon test: −log10P = inf). f Heatmaps of H3K27me3 ChIP-seq reads 3 kb around transcription start sites (TSS) of genes on the Xi in ΔFirreXa versus WT. g. Metagene plots of average H3K27me3 occupancy at X-linked genes ((TSS to termination site (TES), not at scale)) in ΔFirreXa (Xi red Xa pink) versus WT (Xi blue, Xa purple). h Density histograms of the distribution of allelic proportions (Xa/(Xa+Xi)) of SUZ12 peaks show a shift for the X chromosomes due to lower SUZ12 on the Xi in ΔFirreXa (red) versus WT (blue) (Wilcoxon test: −log10P = 20.98). i Genome tracks demonstrating interactions between hnRNPK, EZH2, and SUZ12 with Firre RNA based on RIP-seq data in trophoblast and embryo stem cells39,40.
