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.

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 .

The X inactive-specific transcript (Xist) gene is the master regulator of X chromosome inactivation in mammals. Xist produces a long noncoding (lnc)RNA that accumulates over the entire length of the chromosome from which it is transcribed, recruiting factors to modify underlying chromatin and silence X-linked genes in cis. Recent years have seen significant progress in identifying important functional elements in Xist RNA, their associated RNA-binding proteins (RBPs), and the downstream pathways for chromatin modification and gene silencing. In this review, we summarize progress in understanding both how these pathways function in Xist-mediated silencing and the complex interplay between them.

RNA N 6 -methyladenosine (m 6 A) modification plays important roles in multiple aspects of RNA regulation. m 6 A is installed co-transcriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here we investigate the presence of m 6 A in nascent RNA of mouse embryonic stem cells. We find that around 10% of m 6 A peaks are located in alternative introns/exons, often close to 5' splice sites. m 6 A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Acute depletion of METTL3 disrupts inclusion of alternative introns/exons in the nascent transcriptome, particularly at 5' splice sites that are proximal to m 6 A peaks. For terminal or variable-length exons, m 6 A peaks are generally located on or immediately downstream of a 5' splice site that is suppressed in the presence of m 6 A, and upstream of a 5' splice site that is promoted in the presence of m 6 A. Genes with the most immediate effects on splicing include several components of the m 6 A pathway, suggesting an autoregulatory function. Collectively, our findings demonstrate crosstalk between the m 6 A machinery and the regulation of RNA splicing.

No abstract