Project description:The Polycomb repressive complexes PRC1 and PRC2 play a key role in chromosome silencing by Xist RNA. Previously we have shown that initation of Polycomb recruitment is mediated by the PCGF3/5-PRC1 complex, which catalyses chromosome-wide H2A ubiquitylation (H2AK119u1), signalling recruitment of other PRC1 complexes, and PRC2. However, the molecular basis for PCGF3/5-PRC1 recruitment by Xist RNA remains unknown. Here we define the Xist RNA Polycomb Interaction Domain (XR-PID), a 600 nt element encompassing the Xist B-repeat element. XR-PID is required for Polycomb recruitment by Xist RNA, Xist-mediated chromosome silencing. We identify the RNA-binding protein hnRNPK as the principal XR-PID binding factor required to recruit PCGF3/5-PRC1. Accordingly, synthetically tethering hnRNPK to Xist RNA lacking the B-repeat is sufficient for Xist-dependent Polycomb recruitment. Our findings resolve the molecular mechanism for Polycomb recruitment by Xist RNA, providing key insights into chromatin modification by non-coding RNA.
Project description:Xist lacking of XR-PID (B-repeat and part of C-repeat) can't recruit the polycomb during X chromsome inactivation. The majoring of Xist-mediated chromosomal silencing wouldn’t be achieved. We found that hnRNPK was the one of main contributors for binding B-repeat of Xist. When we tethered hnRNPK to Xist which lacks of XR-PID region, the silencing and polycomb recruitment are restored.
Project description:The noncoding Xist RNA could mediate chromosome inaccessiblity, especially for the pre-open chromatin regions (enhancer, promoter, CTCF). However, Xist lacking the B-repeats loss the ability of closing the chromatin accessibility. ATAC-seq is consistent with the observation by ATAC-see. XR-PID denotes the Xist RNA polycomb interacting domain, including the entire B-repeats and part of C repeats.
Project description: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.