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-wide silencing. Whilst recent studies have defined candidate silencing factors, their relative contribution to repressing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in mouse embryonic stem cell-based models. Using a machine learning approach we identify distance to the Xist locus and prior gene expression levels as key determinants of silencing efficiency. We go on to 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 make only minor contributions to gene silencing. Together our results provide a comprehensive model for Xist-mediated chromosome silencing.

How to silence an X chromosome

“…To interrogate SPEN's role in XCI, Dossin et al first used a biological system known as an auxin-inducible degron to rapidly degrade SPEN in mouse embryonic stem cells. Consistent with a 2019 report 11 , the authors observed that Xist is almost completely unable to silence genes along the X chromosome in the absence of SPEN. In an important first, the authors demonstrated that SPEN is required for successful XCI in vivo in mice.…”

“…Moreover, the authors observed that the SPOC domain interacts with parts of the machinery used for transcription and splicing (the process by which newly made RNA transcripts are turned into messenger RNA), including RNA polymerase II, the enzyme that catalyses transcription. Dossin and colleagues identified interactions with components of the N 6 -methyladenosine (m 6 A) methyltransferase complex, several of which have been linked to XCI 6,11,15 . Accordingly, SPEN and its array of associated proteins might function like a molecular multi-tool to silence genes in various genomic contexts.…”

Progress toward understanding chromosome silencing by Xist RNA

“…(A) Binding profiles of key RBPs discussed in this article. iCLIP/eCLIP data obtained from various sources (Chen et al 2016;Lu et al 2016;Cirillo et al 2017;Nesterova et al 2019) is shown against a map of the Xist gene with tandem repeat elements A-F indicated below. A-repeat and B/C-repeat regions are highlighted with pale-blue shading.…”

“…inactivation. Specifically, deletion of the SPEN RNAbinding domains and/or the entire protein leads to a more dramatic loss of silencing than HDAC3 loss of function (Nesterova et al 2019;Żylicz et al 2019;Dossin et al 2020). Interestingly, deletion of the C-terminal SPOC domain alone results in an intermediate silencing deficit (Dossin et al 2020).…”

“…Further studies are required to determine whether these additional SPEN-linked functions contribute to Xist-mediated silencing. Besides its interactions with chromatin modifying factors, SPEN loss of function has been reported to reduce Xist RNA levels and to disrupt local accumulation over the Xi domain, as has deletion of the Xist A-repeat region (Nesterova et al 2019), and this also could be a contributory factor in abrogated silencing following SPEN loss of function.…”