Fig. 4: Ectopic KDM5C expression induces Xist in male ESCs.
From: Activation of Xist by an evolutionarily conserved function of KDM5C demethylase
a Schematic of ectopic expression of HA-tagged transgenes in XY male ESCs. b Quantification of nuclei with Xist RNA coating in transgenic (Tg) XY male ESC lines (n = 3) expressing HA-tagged mouse KDM5C (Tg-mKdm5c; n = 292, 437, and 210); human KDM5C (Tg-hKDM5C; n = 154, 212, and 184); enzymatically inactive KDM5C (Tg-hKDM5C(H514A);45 n = 292, 295, and 316); and, KDM5D (Tg-hKDM5D; n = 262, 222, and 232) proteins. Representative nuclei with IF detection of the HA epitope tag (red) coupled with FISH detection of Xist RNA (green) in the XY male ESCs. Data are presented as mean \(\pm\) SEM. P values, Welch’s two-sided t test. n.s., not significant; *P ≤ 0.05; **P < 0.01; ***P < 0.001. See Supplementary Table 5 for all pairwise statistical comparisons. c Schematic of ectopic Xist RNA induction in day (d) 2 differentiated XΔTsixY male ESCs. See also Supplementary Fig. 2c, d. d Quantification of ectopic Xist RNA expression by RT-qPCR (left) and Xist RNA-coated nuclei by RNA FISH (right) in d2 differentiated XΔTsixY EpiLCs ectopically expressing mouse KDM5C (Tg-mKdm5c;XΔTsixY); human KDM5C (Tg-hKDM5C;XΔTsixY); enzymatically inactive KDM5C (Tg-hKDM5C(H514A);XΔTsixY); KDM5D (Tg-hKDM5D;XΔTsixY); and, XΔTsixY EpiLCs lacking KDM5C (XΔTsix;ΔKdm5cY). For both the Xist RNA FISH and the Xist RT-qPCR data, three independent ESC lines/genotype were differentiated into d2 differentiated EpiLCs/genotype. For Xist RNA coating analysis, n = 100 nuclei per sample. Data are presented as mean \(\pm\) SEM. For RT-qPCR, each sample was analyzed in triplicate. Data are presented as mean \(\pm\) standard deviation between technical replicates. P values, Welch’s two-sided t test on ΔCt values of biological replicates. *P ≤ 0.05; **P < 0.01; ***P < 0.001. See Supplementary Table 6 for all pairwise statistical comparisons. e DNA FISH following RNA FISH demonstrates the presence of one X chromosome in nuclei with ectopic Xist RNA coating in the differentiated EpiLCs. n = 100 nuclei from three independent ESC lines differentiated into EpiLCs/genotype. Data are presented as mean \(\pm\) SEM. See also Supplementary Fig. 3. Source data are provided as a Source Data file.
