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Abstract

To ensure dosage compensation between the sexes, one randomly chosen X chromosome is silenced in each female cell in the process of X-chromosome inactivation (XCI). XCI is initiated during early development through upregulation of the long non-coding RNA Xist, which mediateschromosome-wide gene silencing. Cell differentiation, Xist upregulation and silencing are thought tobe coupled at multiple levels to ensure inactivation of exactly one out of two X chromosomes. Here we perform an integrated analysis of all three processes through allele-specific single-cellRNA-sequencing. Specifically, we assess the onset of random XCI with high temporal resolution indifferentiating mouse embryonic stem cells, and develop dedicated analysis approaches. By exploitingthe inter-cellular heterogeneity of XCI onset, we identify Nanog downregulation as its main trigger and discover additional putative Xist regulators. Moreover, we confirm several predictions of thestochastic model of XCI where monoallelic silencing is thought to be ensured through negativefeedback regulation. Finally, we show that genetic variation modulates the XCI process at multiplelevels, providing a potential explanation for the long-known Xce effect, which leads to preferentialinactivation of a specific X chromosome in inter-strain crosses. We thus draw a detailed picture of thedifferent levels of regulation that govern the initiation of XCI. The experimental and computationalstrategies we have developed here will allow us to profile random XCI in more physiological contexts,including primary human cells in vivo.