Fig. 8: Xist levels modulate X-linked dosage in vivo.
From: Escape from X inactivation is directly modulated by Xist noncoding RNA
a, Experimental outline showing male TX B6 mice (XptetY; R26rtTA/WT) were crossed with WT JF1 females. F1 embryos were collected at E2.5 and E3.5 and cultured for 24 h while adding Dox to the culture media. RNA-seq was performed at E3.5 and E4.5. b, RNA-FISH for Xist RNA (green) in E4.5 XX embryos obtained by crossing male TX B6 mice (XptetY; R26rtTA/rtTA) with WT JF1 females. DNA is stained with DAPI (blue). c, Box plots showing mean allelic ratios for −rtTA and +rtTA embryos for the different escapee categories. The constitutive, facultative (includes genes annotated as facultative in Extended Data Fig. 1h and NPC-specific escapees) and E4.5-specific categories contain 10, 97 and 24 escapee genes, respectively. Biological replicates: for E3.5, −rtTA n = 2 embryos, +rtTA n = 4 embryos; for E4.5, −rtTA n = 17 embryos, +rtTA n = 20 embryos. The adjusted P values for the comparison between −rtTA and +rtTA embryos are Padj = 3.9 × 10−2 for the constitutive category, Padj = 6.60 × 10−16 for facultative and Padj = 8.2 × 10−5 for E4.5-specific escapees (two-sided Wilcoxon rank sum test, adjusted using the Benjamini–Hochberg procedure). d, Experimental outline shows TX/Y males carrying the rtTA transactivator (Xptet/Y; R26rtTA/rtTA or Xptet/Y;R26rtTA/WT) were crossed with WT JF1 females. Xist RNA overexpression was induced by adding Dox to the drinking water of pregnant females for 5 days, from E3.5 to E8.5. RNA-seq was performed using RNA extracted from ExE tissue. e, RNA-seq data showing fold changes in Xist expression (normalized CPM) for rtTA− +Dox (control) and rtTA+ +Dox (Dox). Fold changes are calculated to the mean Xist levels of rtTA+ −Dox samples. Each dot represents an individual embryo. Biological replicates: −rtTA, +Dox n = 2 embryos; +rtTA, +Dox n = 6 embryos; +rtTA, −Dox n = 8 embryos. f, Plot showing average escape for each ExE sample, categorized by rtTA genotypes and Dox treatment. Biological replicates: −rtTA, +Dox n = 2 embryos; +rtTA, +Dox n = 6 embryos; +rtTA, −Dox n = 8 embryos. PCA was performed on the whole transcriptome excluding X chromosomes. g, Heat map showing the mean allelic ratios of 74 escapees in E8.5 ExE tissues across rtTA+ −Dox, rtTA+ +Dox and rtTA− +Dox conditions. Escapees are categorized as in Extended Data Fig. 1h, with NPC-specific genes included in the facultative category. Genes are additionally called ‘E8.5-specific’ if they show an allelic ratio >0.1 and <0.8 in more than 50% of rtTA+ −Dox ExE samples and do not escape in NPCs. The constitutive, facultative (includes genes annotated as facultative in Extended Data Fig. 1h and NPC-specific escapees) and E8.5-specific categories contain 10, 48 and 16 escapee genes, respectively. h, Box plots showing mean allelic ratios rtTA+ −Dox, rtTA+ +Dox and rtTA− +Dox ExE samples for the different escapee categories. The adjusted P values for the comparison between rtTA+ −Dox and rtTA+ +Dox ExE samples are Padj = 7.8 × 10−2 for the constitutive category, Padj = 5.6 × 10−12 for facultative and Padj = 8.5 × 10−4 for E8.5-specific escapees (two-sided Wilcoxon rank sum test, adjusted using the Benjamini–Hochberg procedure). All box plots show the median, 25th and 75th percentile and 1.5× the interquartile range. i, Scatterplot showing the same data as in h but comparing allelic ratios of individual escapee genes between rtTA+ −Dox and rtTA+ +Dox. Biological replicates: −rtTA, +Dox n = 2; +rtTA, +Dox n = 6; +rtTA, −Dox n = 8 (h and i).
