Figure 3RIF1 association with the Xist promoter assessed by ChIP-qPCR in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female cell lines, in ESCs (A) P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 and 2 are two intergenic regions that serve as negative controls. Peak and cRAD represent two previously identified regions of RIF1 association (positive control). See Appendix Fig. S2C for primer positions within Xist. Mean ±standard deviation from 3 independent experiments (A) and 2 independent experiments (B). p calculated by Student's two-tailed, paired t test comparing RIF1 association in Rif1+/+ cells on Xist P2 and P1 versus 5'. *p ≤ 0.05, ****p ≤ 0.0001 and ns=not significant. B). RIF1 association with the Xist promoter assessed by ChIP-qPCR in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female cell lines, at 2 days of EB differentiation (B). P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 and 2 are two intergenic regions that serve as negative controls. Peak and cRAD represent two previously identified regions of RIF1 association (positive control). See Appendix Fig. S2C for primer positions within Xist. Mean ±standard deviation from 3 independent experiments (A) and 2 independent experiments (B). p calculated by Student's two-tailed, paired t test comparing RIF1 association in Rif1+/+ cells on Xist P2 and P1 versus 5'. *p ≤ 0.05, ****p ≤ 0.0001 and ns=not significant. (C). Rif1 deletion decreases the efficiency of upregulation of a Luciferase reporter under the control of Xist promoter (Xist-2p-luc), at 2 days of EB differentiation. As a control (Luc), empty luciferase reporter vector, was transfected in parallel. The average of three independent experiments is shown. Error bars indicate the standard deviation. p calculated by Student's two-tailed, unpaired t test, for comparison of fold activation of Xist-2p-Luc normalised to empty vector (Luc) in Rif1+/+ versus Rif1-/- cells. **p ≤ 0.01. See Appendix Material and Methods for details about the normalisation. (D). Association of RIF1 with Xist P2 in the Fa2L cells (black) and a wild type female mESC line (grey), also harbouring one castaneus and one 129 X chromosome. Allele-specific ChIP-qPCR primers were used, cast indicates association with the castaneus Xist P2 and 129 indicates association with the 129 Xist P2. Enrichments are presented relative to input DNA. Mean ±standard deviation from 3 independent experiments. p calculated by Student's two-tailed, paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2, ***p ≤ 0.001. Below, schematic of the Xist/Tsix alleles in the Fa2L undifferentiated cells. (E). Association of RIF1 with Xist P2 in the Fa2L cells (black) upon differentiation. The analysis was performed as in (D). Mean ±standard deviation from 3 independent experiments. p calculated by Student's two-tailed, paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2. *p ≤ 0.05. Below, schematic of the Xist/Tsix alleles in the Fa2L differentiated cells. ( (F). Quantification by ChIP-qPCR of RIF1 association with the indicated regions in the Fa2L cell line, following treatment with DMSO only (black) or flavopiridol (grey). Primers as in (E). (G). Same as in (F) but for a wild type female mESC line. All enrichments are presented relative to input DNA. Mean ±standard deviation from three (F) and two (G) independent experiments are presented. Statistical significance was determined using Student's two-tailed, paired t test (*p ≤ 0.05, **p ≤ 0.01 and ns=not significant).