GSE185769
Calibrated native ChIP-seq for H2AK119ub1 and H3K27me3 in FKBP12-PCGF3/5 mESCs
X chromosome inactivation (XCI) is mediated by the non-coding RNA Xist which directs chromatin modification and gene silencing in cis. The RNA binding protein SPEN and associated corepressors have a central role in Xist-mediated gene silencing. Other silencing factors, notably the Polycomb system, have been reported to function downstream of SPEN. Making use of a SPEN separation-of-function mutation we show that SPEN and Polycomb pathways in fact function in parallel to establish gene silencing. Additionally, we find that differentiation-dependent recruitment of the chromosomal protein SmcHD1 is required for silencing many X-linked genes. Overall design: This series contains datasets of calibrated native ChIP-seq for the Polycomb modifications H2AK119ub1 and H3K27me3. We performed two replicate experiments for untreated, 24h Dox, and 36h dTAG-13 + 24h Dox FKBP12-PCGF3/5 mESCs. mESCs were mixed with 20% Drosophila spike-in (Sg4 cells), native chromatin was extracted, and ChIP was performed with H2AK119ub1 and H3K27me3 antibodies. Calibration of ChIP-seq datasets shows that 36h PCGF3/5 degradation results in a ~30% reduction of genome-wide H2AK119ub1 with little effect of genome-wide H3K27me3. Allelic analysis of ChrX shows widespread deposition of both Polycomb modifications over Xi after 24h of Xist induction. This is abolished by degradation of PCGF3/5 (12h dTAG-13 treatment prior to Xist induction), demonstrating that PCGF3/5-PRC1 complexes are required for all Xist-mediated Polycomb deposition over Xi.