Extended Data Fig. 9: Histone CUT&RUN and DNA methylation analysis upon Dox treatment and washout.
From: Escape from X inactivation is directly modulated by Xist noncoding RNA
a, Box plot showing the enrichment and loss of H3K27me3 on the Xi across 10 kb windows with > 10 average read counts spanning the inactive X chromosome (Xi) during Dox treatment (Dox (7 d), Dox (21 d)) and washout (for 7 days after each treatment (Dox (7 d) wo, Dox (21 d) wo)). Shown is the fold change of normalized counts relative to the untreated condition (Control) in two replicates (n = 1,499 10-kb bins). b, same as (a) for H2AK119Ub (n = 1,499 10-kb bins). c, Box plot comparing H3K27me3 coverage (normalized counts) across promoters and gene bodies of silenced X-linked genes after Dox treatment and washout. Levels are shown separately for the Xa (light red) and Xi (dark red) (n = 6514 intergenic, 1540 promoter, 1262 gene body genomic intervals). d, as in (c) for H2AK119Ub. H2AK119Ub levels are shown separately for the Xa (yellow) and Xi (orange) (n = 6514 intergenic, 1540 promoter, 1262 gene body genomic intervals). e, Density plots showing the correlation between H3K27me3 and H2AK119Ub enrichments across 10 kb windows with > 10 average read counts spanning all autosomes during the Dox treatment time course and washout. The axes represent normalized log counts. f, Scatterplots showing the changes in H3K27me3 (bottom) and H2AK119Ub (top) levels (log2 fold change relative to Control) compared to the changes in gene expression (log2 fold change relative to Control) for autosomal genes after 7 days of Dox treatment in NPC clones CL30, CL31 and E6. g, Scatterplot showing the log2 Fold Change in H2AK119Ub levels in 21 days Dox treated versus washout conditions, compared to the untreated condition (Control) for reversibly (green, n = 30 genes) and irreversibly (grey, n = 86 genes) silenced escapees. The diagonal line represents equal changes between treatment and washout. Genes below the diagonal lose the mark upon washout. h, same as (g) but for H3K27me3. reversible genes (green, n = 30 genes) and irreversible genes (grey, n = 86 genes) i, Violin plots showing allele-specific DNA methylation levels across gene bodies for silenced and escaping genes, as derived from the gene expression data in Fig. 1 (escapees = 145 genes; silenced = 226 genes). Shown are the Xa (grey) and Xi (light blue) chromosomes separately. Differences observed for escapees are significant for the Xi for all comparisons to the untreated condition (Control): 8.83e-8 (Dox (7 d), 2.37e-7 (Dox (7 d) wo), 4.22e-28 (Dox (21 d)), 4.17e–27 (Dox (21 d) wo). P-values are calculated using a paired Wilcoxon’s rank sum test. All box plots show median, 25- and 75-quantiles and 1.5x inter-quartile range. c-i, average data across two biological replicates Source numerical data are available in source data.
