Fig. 1: Impaired Xist upregulation in Spen^−/− ESCs upon monolayer differentiation.

a Overview of the endogenous doxycycline-inducible Xist hybrid system used in this study. Addition of doxycycline leads to inactivation of the Cast X chromosome, while no addition of the drug leads to inactivation of the 129 X chromosome. b Violin plots depicting the distribution of the allelic ratios ((Xi)/(Xi+Xa)) of X-linked genes in Wt and Spen^−/− ESCs, untreated or treated with doxycycline at days 0, 3, and 7 of differentiation. This figure summarizes data in Supplementary Fig. 1e. The box plots inside the violin plots display the median (central white dot), the interquartile range (box limits) and the whiskers represent 1.5x of the interquartile range. Two-sided Mann–Whitney test corrected with Benjamini–Hochberg for multiple testing (α < 0.05). n = genes with more than 20 reads. Xa = active X chromosome. c Percentage of Rnf12 allelic expression at different time points of monolayer differentiation of two independent Wt and Spen^−/− ESC lines treated with doxycycline, determined by RT-qPCR. Relative Rnf12 allelic (129 and Cast) expression was normalized to Rnf12 total expression and averaged ± standard deviation (SD). Reference gene: Hist2h2aa1. d Relative allele-specific Xist expression of two independent Wt and Spen^−/− ESC lines at different time points of monolayer differentiation treated with doxycycline. Average expression ± SD. Reference gene: Hist2h2aa1. e Same as displayed in (c) without doxycycline. f Same as displayed in (d) without doxycycline. g Xist RNA FISH (red) of Wt and Spen^−/− ESC lines at days 3 and 5 of differentiation. Both Tsix pinpoints (arrowheads) and Xist clouds (asterisks) are visible. DNA is stained with DAPI (blue). Scale bar: 10 µm. h Quantification of (g), displaying the average percentage of nuclei with Xist clouds. Average percentage ± SD, the total number of counted nuclei per clone is indicated. i Relative allele-specific Tsix expression of Wt and Spen^−/− ESCs at different time points of monolayer differentiation, determined by RT-qPCR. Average expression ± SD. Two-sided Mann–Whitney test (α < 0.05). Reference gene: Hist2h2aa1. (c–f, h, i) n = 4 biological replicates including 2 independent clones (squares vs. circles) per condition.