Calibrated ChIP-seq for Xist-mediated polycomb recruitment

Calibrated ChIP-seq was employed to quantitatively address the questions of how and where Xist recruits polycomb modifications (H2AK119ub and H3K27me3) to the inactive chromosome. 'Dox' samples were performed after 24 hours of induced Xist expression. Suz12 is the core enzymatic component of PRC2 while Pcgf3/5 are non-canonical PRC1 components which initiate the polycomb cascade in Xist-mediated chromosome inactivation. Xist PID region, encompassing the B-repeat and part of C-repeat, was characterized to bind hnRNPK directly, then recruit the Pcgf3/5. Notably, Suz12 KO does not affect Xist-dependent H2AK119ub deposition too much in a 24hour Xist induction. Overall design: H3K27me3 and H2AK119ub calibrated ChIP-seq for Xist and XistDelPID-induced chromosome inactivation [endogenous Xist model]. H3K27me3 and H2AK119ub calibrated ChIP-seq for WT, Pcgf3/5 acute double knockout, and Suz12KO [transgenic Xist model].