4sU-seq analysis of Xist-mediated chromosomal silencing. Mus musculus

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. Overall design: Stable cell lines were derived by transfecting the FLXist, XistDXEv or some truncated Xist into P4D7, a Mus domesticus (129S1) x Mus castaneus F1 hybrid mouse embryonic stem cell (mESC) line. Use of a hybrid genetic background facilitated analysis of Xist-mediated silencing. Xist expression was induced upon the Dox treatment. Due to high amount of SNPs located in intronic region, we isolated the nascent transcripts labelled by 4sU, to quantify the silencing efficiency among different Xist constructs.