Genomic regulation and molecular pathways of X chromosome inactivation

Thesis

X chromosome inactivation (XCI) is a process by which one X chromosome in female mammals is silenced to equalise the dosage of X-linked gene expression between XX females and XY males. XCI is initiated by a long non-coding RNA (lncRNA), Xist, expressed from the future inactive X chromosome (Xi) during embryonic development. Xist spreads in cis to coat the chromosome and recruits molecular pathways which modify the underlying chromatin from an active to a repressive state, leading to complete transcriptional silencing of almost all genes on Xi. XCI is an important paradigm for lncRNA-directed gene repression and its study can inform our understanding of mechanisms of chromatin regulation more widely.

This thesis presents an experimental characterisation of iXist-ChrX, a cellular model that recapitulates the establishment of XCI during early mouse development. I perform quantitative, high-resolution and allele-specific genomic analyses of Xist-mediated changes to chromatin over time courses of Xist induction, providing novel insights into the cis-regulatory features that influence variable silencing dynamics on a gene-by-gene basis. A key finding from these analyses is that slow-silencing genes and genes which escape complete inactivation are marked by binding motifs for the transcription factor YY1. I also document a pilot scRNA-seq experiment to address questions of cellular heterogeneity in Xist-mediated gene silencing and lay the foundations for future investigations at single-cell resolution.

In further experiments, I use CRISPR-Cas9 genome editing to interrogate two key molecular pathways acting downstream of Xist. By disrupting SPEN and Polycomb pathways individually and in combination in iXist-ChrX cells, I dissect the relative contributions of each pathway and demonstrate that they act in parallel, through distinct mechanisms of chromatin modification, and additively to silence X-linked genes. These experiments also reveal that both SPEN and PCGF3/5-PRC1 have secondary roles in ensuring correct localisation of Xist RNA over Xi, and highlight an interplay with cellular differentiation important for the complete establishment of silencing during the later stages of XCI.

Authors: Bowness, JS

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: polycomb; Xist; genomics; gene silencing; X chromosome inactivation; gene regulation; chromatin; long non-coding RNA
• Subjects: Non-coding RNA; Transcription factors; X chromosome; Gene silencing; Molecular biology