Systematic Allelic Analysis Defines the Interplay of Key Pathways in X Chromosome Inactivation

Abstract

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 weakly 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/ 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. required for Xist-mediated silencing 17 . Two other factors whose recruitment is linked to the A-repeat are Wtap, a regulatory subunit of the N6-Methyladenosine (m6A) methyltransferase complex 13,14 , and Rbm15, an RBP related to Spen (Patil et al., 2016;.Wtap and Rbm15 have moreover been linked with one another, with the latter directly recruiting the m6A-methyltransferase complex via binding to Xist A-repeat (Patil et al., 2016).Accordingly, several studies have identified sites of m6A deposition in Xist RNA 18-21 . Both Rbm15 and the m6A-methyltransferase (m6A-MTase) complex have been implicated in Xistmediated silencing 13,14,21 .

Dosage Sensing, Threshold Responses, and Epigenetic Memory: A Systems Biology Perspective on Random X‐Chromosome Inactivation

“…Interestingly, deletion of Spen or its binding site in Xist reduce Xist RNA levels even in an inducible system. [ 6 ] Also methylation of N6‐methyladenosine residues (m6A) in the Xist RNA has been suggested to contribute, albeit to a lesser extent, to Xist's silencing ability. [ 6,116 ] Accordingly, Xist upregulation is indeed impaired upon knockdown of components of the m6A methylation machinery.…”

“…[ 6 ] Also methylation of N6‐methyladenosine residues (m6A) in the Xist RNA has been suggested to contribute, albeit to a lesser extent, to Xist's silencing ability. [ 6,116 ] Accordingly, Xist upregulation is indeed impaired upon knockdown of components of the m6A methylation machinery. [ 116 ] Taken together, silencing of X‐linked genes seems to indeed enforce Xist expression, supporting the notion of a silencing‐dependent self‐reinforcing feedback.…”

“…Acting as a scaffold for a multitude of RNA binding proteins and potentially forming a phase‐separated compartment, Xist recruits members of different gene‐silencing pathways, orchestrating chromosome‐wide gene repression. [ 6–10 ] A small subset of genes however can resist silencing and thus escape X inactivation. [ 11,12 ]…”

A single N6-methyladenosine site in lncRNA HOTAIR regulates its function in breast cancer cells

“…Apart from the YTH family of proteins that directly read m6A, a handful of non-canonical indirect m6A readers have been suggested 7 . In the case of Xist, the m6A reader YTHDC1 interacts with Xist to mediate repression; however, it is still unclear mechanistically how m6A and YTHDC1 contribute to repression by Xist 6,8 . In contrast, m6A on chromatin-associated regulatory RNAs leads to their YTHDC1-dependent degradation, preventing transcription of downstream genes 9 .…”

RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

“…Strikingly, regions with high RNP-MaP site density in Xist/XIST corresponded to the A, B, C, D, and E regions ( Fig. 4a), which are each known to contain distinct repetitive sequences important for Xist localization, assembly, and chromatin silencing [28][29][30][31][32][33][34][35] . High RNP-MaP density was observed at each of these repetitive regions despite changes in copy number (human XIST contains two copies of the B region) and changes in size, relative position, and sequence (C, D, and E regions differ extensively between mice and humans) 36 .…”