Transcriptome regulation during the X chromosome inactivation process

Abstract

In mammals, female cells achieve dosage compensation between the sexes randomly chosing and transcriptionally silencing one of the two X chromosomes through a process known as X-chromosome inactivation (XCI). This process is initiated during early development through up-regulation of the long non-coding RNA Xist, which mediates chromosome-wide gene silencing of the future inactive chromosome (Xi) in cis. Upon completion of the XCI process Xi will maintain its silenced state in all daughter cells, which results in the genetic mosaicism of female organisms. Cell differentiation, Xist up-regulation and gene silencing are thought to be coupled at multiple levels to ensure inactivation of exactly one out of two X chromosomes.

In this thesis I performed an integrated analysis of all three processes through the analysis of allele-specific single-cell RNA-sequencing data. Specifically, I investigated the endogenous random XCI process in hybrid mouse embryonic stem cells at different time points throughout cellular differentiation developing dedicated analysis approaches that rely on the high number of polymorphisms between the two parental strains.

Putative Xist regulators were identified exploiting the inter-cellular heterogeneity of XCI onset. A large fraction of cells transiently expressed Xist on both X chromosomes which resulted in biallelic gene silencing right before being resolved to a monoallelic state, confirming a prediction of the stochastic model of XCI. The two X chromosomes showed different gene silencing dynamics, and a number of strain-specific escapees (namely, genes that escape transcriptional silencing) were identified and experimentally validated. These results suggest that genetic variation modulates the XCI process at multiple levels, providing a potential explanation for the long-known X-controlling element (Xce) effect, which leads to preferential inactivation of a specific X chromosome in inter-strain crosses.

Overall, this work provides a detailed picture of the different levels of regulation that govern both Xist up-regulation and the initiation of XCI.