GSE119602
Systematic Analysis of Allelic Silencing Defines the Interplay of Key Pathways in Xist-mediated Chromosome Inactivation
Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome silencing through the stepwise recruitment of factors that modify underlying chromatin structure.  Whilst considerable progress has been made towards defining key silencing factors and the elements to which they bind, their relative contribution to silencing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in ES cell-based models. We show that Spen, recruited through the Xist A-repeat, plays a central role, being critical for silencing of all except a subset of low expressed genes.  Polycomb, recruited through the Xist B/C-repeat, also plays a key role, favouring silencing of genes with pre-existing H3K27me3 chromatin.  LBR and the Rbm15-Mettl3/14 m6A-methyltransferase complex, previously proposed to have a central role, make at most a minor contribution to gene silencing.  We integrate our findings in a comprehensive model for Xist-mediated chromosome silencing. Overall design: We isolated chromatin-associated RNA (ChrRNA) to analyze the allelic ratio, aiming to understand Xist-mediated Silencing. The ChrRNA was isolated from cells with trangenic Xist model (iXist-Chr3) or endogenous Xist model (iXist-ChrX) derived from mouse embryonic stem cells. Xist is composed of several repetitive elements, which act by interacting with different factors. Cell lines with different factor knockout or Xist domain deletions were generated. The allelic ratios were systematically and comprehensively compared.