Extended Data Fig. 2. GATA1 is a potent Xist activator.
(a-c) Individual overexpression of screen hits with CRISPRa in E14-STNΔTsixP mESCs using a single guide RNA per gene that had performed well in the screen. (a) The cells were treated with doxycycline 24 h before differentiation by LIF withdrawal for 2 days. (b) Expression levels of the targeted genes were measured by qRT-PCR. (c) Xist expression measured by Flow-FISH. Dashed lines mark the 99th percentile of undifferentiated NTC-transduced E14-STNΔTsixP cells (Xist- population). The percentage of Xist+ cells is indicated. (d) Xist expression was measured via Flow-FISH in female TX1072 cell line and in male E14-STNΔTsixP cells transduced with multiguide expression vectors of three sgRNAs against the Gata1 promoter region or with NTCs. TX1072 cells were cultured in naive conditions (2i/LIF) and E14-STNΔTsixP in conventional ESC medium (LIF). The cells were differentiated (2i/LIF or LIF withdrawal) for 2 days. E14-STNΔTsixP were treated with doxycycline 24 h before and during differentiation. Dashed lines mark the 99th percentile of the TX1072 undifferentiated (2i/LIF) sample and the percentage of Xist+ cells in each sample is indicated. (e) Heatmap showing expression levels assessed by RNA-seq (mean of 3 biological replicates) of the most enriched genes in the screen (Fig. 1d) in XX and XO TX1072 mESCs differentiated by 2i/LIF withdrawal. (f-h) Gata1 knock-down by CRISPRi in female mESCs. (f) Schematic representation of an ABA-inducible CRISPRi system in female TX-SP107 mESCs. Gata1 knock-down efficiency (g) and effect on Xist (h) quantified by qRT-PCR after 2 days of differentiation. SgRNAs targeting the Xist TSS and NTCs were included as controls. Horizontal dashes indicate the mean of 3 biological replicates (dots); asterisks indicate p < 0.05 for two-sided paired Student’s T-test. (i) Expression of screen hits during preimplantation development42,43. Xist could not be quantified (grey) because the employed protocol was not strand-specific, such that Xist could not be distinguished from its antisense transcript Tsix. In (e) and (i) Xist and known Xist regulators are coloured in yellow. Source numerical data and exact p-values are available in source data.