Fig. 6: Silencing of most facultative escapees becomes Xist independent after prolonged Xist upregulation.
From: Escape from X inactivation is directly modulated by Xist noncoding RNA
a, Experimental outline showing Xist upregulation was induced by Dox treatment for 7, 14, and 21 days following 7 days of Dox washout. b, RNA-seq data showing fold changes in Xist expression (normalized CPM) compared with untreated control cells after 7, 14 and 21 days of Dox treatment, followed by 7 days of Dox washout. Data relative to the mean of measurements in clone E6 are shown. c, FISH for Xist RNA (green) in NPC clone E6 in the untreated condition (control), after 21 days of Dox treatment (Dox (21 d)) and following 7 days of Dox washout (Dox (21 d)−washout (7 d)). DNA is stained with DAPI (blue). d, Heat map showing allelic ratios of 133 escapees identified in clone E6 upon Dox treatment for 7, 14, and 21 days and following 7 days of Dox washout after these timepoints. Escapees are assigned to three different categories as shown in Extended Data Fig. 1h and described in the Methods. Gene groups are defined as contiguous groups of escapees within 100 kb of each other as in Fig. 1f. e, Box plots quantifying the reversibility of escape per gene (constitutive, 12; facultative, 84 and NPC-specific, 37 genes) for each timepoint by computing the ratio of the allelic ratio after washout divided by the allelic ratio in untreated samples. Differences between Dox (7 d) and Dox (14 d) and between Dox (7 d) and Dox (21 d) are significant for the facultative and NPC categories (Dox (14 d): facultative P = 1.46 × 10−6, NPC-specific P = 0,00608; Dox (21 d): facultative P = 2.08 × 10−12, NPC-specific P = 0,00421). P values are calculated using a paired Wilcoxon’s rank sum test and adjusted using the Benjamini–Hochberg procedure. All box plots show the median, 25th and 75th percentile as well as 1.5× the interquartile range. f, Stack histogrammes showing the classification of genes into irreversible, partially irreversible and fully reversible escapees across the silencing time course. Genes are considered reversible when they reach at least 50% of untreated escape as well as an allelic ratio >0.1 after washout. Similarly, they are considered partially irreversible when they reach 10–50% of untreated escape and an allelic ratio >0.1, and they are considered irreversible otherwise. g, Scatterplot comparing silencing half-lives (Fig. 1) to fold recovery after washout for the gene groups (74 genes in total) indicated in d. Genes in a subset of local gene groups are highlighted to show their coordinated behaviour. Reversible, partially irreversible and irreversible fold recovery thresholds are indicated. R indicates Pearson’s correlation and the P value is given by a correlation test.
