Systematic allelic analysis defines the interplay of key pathways in X chromosome inactivation

Tatyana B Nesterova,Qi Pan,Joseph S Bowness,Mafalda Almeida,Chun-Xiao Song,Benoit Moindrot,Emma J Carter,Guifeng Wei,Tianyi Zhang,Ying Bi,Greta Pintacuda,Ines Alvarez Rodrigo,Bianca Bloechl,Neil Brockdorff,Heather Coker

doi:10.1038/s41467-019-11171-3

Tatyana B Nesterova, Qi Pan + Show 13 more

Open Access

https://doi.org/10.1038/s41467-019-11171-3

Copy DOI

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.

Highlights

X inactive specific transcript (Xist) RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome-wide silencing
To define the contribution of different pathways in Xist-mediated silencing we analysed two complementary models, a previously described XY interspecific M.m. domesticus (129S) × M.m. castaneus (Cast) mouse embryonic stem cells (mESCs) line with a multicopy tetracycline inducible Xist transgene randomly integrated on chromosome 3, referred to as iXist-Chr[3], and a novel 129S × Cast XX ES cell line with a single endogenous Xist allele driven by a tetracycline inducible promoter (Fig. 1a)
In this study we provide insights into the interplay and relative importance of different pathways required for the establishment of chromosome silencing by Xist RNA

Summary

Introduction

Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome-wide silencing. Xist RNA recruits several factors that collectively modify chromatin/chromosome structure to silence transcription[6]. Xist-mediated chromosome silencing occurs in a stepwise manner with specific chromatin/chromosome modifications occurring concurrent with the onset of Xist RNA expression, and others at later timepoints[11]. The RBP Spen has been identified as an important factor for establishment of Xist-mediated silencing[12,13,14,15], functioning by recruiting the NCoR-. 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 Rbm[15], an RBP related to Spen[13,19].

Methods

Results

Conclusion