Fig. 1: Increased levels of Xist RNA silences Xi escapees in NPCs.
From: Escape from X inactivation is directly modulated by Xist noncoding RNA
a, Scatterplot showing a correlation between average escape and Xist expression using RNA-seq data from 21 NPC clones (129/Sv × Cast/Eij genetic background). Mean escape is calculated as the average allelic ratio (Xi/(Xi+Xa)) across 379 informative genes. Normalized Xist expression is calculated as library-size scaled counts per million (CPM), divided by the value for the lowest clone. R specifies Pearson’s correlation coefficient, and the P value is given by a correlation test. The error band depicts 95% confidence intervals. b, The experimental outline showing that TX ES cells (Cast/Eij × C57BL/6 genetic background) carrying a tetracycline-responsive promoter (ptet) upstream of the Xist gene on the B6 X chromosome were differentiated into NPCs without Dox. Single clones carrying the inactivated B6 allele were picked and expanded, and Xist RNA levels were increased by adding Dox to the culture media. c, FISH for Xist RNA (green) in NPC clone E6 in the untreated condition (control) and after 3 days of Dox treatment (Dox (3 d)). DNA is stained with DAPI (blue). d, RNA-seq data showing the fold change in Xist expression (normalized CPM) compared with untreated cells across the time course of Dox treatment. Data relative to the mean of measurements in clone E6 are shown. Individual biological replicates are shown for each timepoint (control, Dox (3 d) and Dox (7 d): n = 3, Dox (14 d) and Dox (21 d): n = 2 biological replicates. e, Schematic of the mouse X chromosome and heat map showing X-linked transcript allelic ratios in untreated clone E6 and after 3, 7, 14 and 21 days of Dox treatment. Allelic ratio indicates the fraction of reads from the Xi compared with reads from the Xi and Xa (ratio, 1: Xi monoallelic expression; ratio, 0: Xa monoallelic expression; ratio, 0.5: biallelic expression; ratio, >0.1: escape). Gene groups are defined as contiguous groups of escapees within 100 kb of each other (Methods). f, Heat map showing the allelic ratio of 133 escapees identified in clone E6 and shown in e. Escapees are assigned to three different categories as shown in Extended Data Fig. 1h and described in the Methods. The escape category for each gene is indicated below the heat map together with the zoom-in of the gene groups shown in e. g, Box plot showing the changes in allelic ratios for the different escape categories across the time course of Dox treatment. Data of clone E6 are shown. All the differences between control and Dox-treated measurements for escapee sets are significant at Padj < 0.01 (Wilcoxon rank sum test, Benjamini–Hochberg adjusted). Box plots show the median, 25th and 75th percentiles as well as 1.5× the interquartile range. h, Schematic of the exponential decay models used to study gene silencing kinetics. Data can be described by a full silencing model (blue, allelic ratio approaches 0) or a residual escape model (green, allelic ratio approaches value >0.1. The steepness of the curve corresponds to the gene’s silencing half-life). i, Beeswarm plot showing the distributions of silencing half-life fit to all escapees using the offset model and stratified by escape category. P values are calculated using Wilcoxon’s rank sum test (not adjusted for multiple testing). The large dots and error bars depict the median and 25th and 75th percentiles. j, Silencing half-lives per gene group as shown in f. Large dots show the mean and whiskers show the standard deviation across genes in the group. k, Beeswarm plot showing the distributions of residual escape parameters fit to all escapees using the offset model and stratified by escape category. P values are computed using Wilcoxon’s rank sum test (not adjusted for multiple testing). The large dots and error bars depict the median and 25th and 75th percentiles. Averaged data from individual biological replicates per timepoint (control, Dox (3 d) and Dox (7 d): n = 3, Dox (14 d) and Dox (21 d): n = 2 biological replicates) (e–g and i–k). Data from 133 genes (constitutive n = 12; facultative n = 84; NPC-specific n = 37 genes) (g, h, j and k).
