Integrated analysis of Xist upregulation and X-chromosome inactivation with single-cell and single-allele resolution

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 mediates chromosome-wide gene silencing. Cell differentiation, Xist upregulation and gene silencing are thought to be 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-cell RNA-sequencing. Specifically, we assess the onset of random XCI in differentiating mouse embryonic stem cells, and develop dedicated analysis approaches. By exploiting the inter-cellular heterogeneity of XCI onset, we identify putative Xist regulators. Moreover, we show that transient Xist upregulation from both X chromosomes results in biallelic gene silencing right before transitioning to the monoallelic state, confirming a prediction of the stochastic model of XCI. Finally, we show 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. We thus draw a detailed picture of the different levels of regulation that govern the initiation of XCI. The experimental and computational strategies we have developed here will allow us to profile random XCI in more physiological contexts, including primary human cells in vivo.

Single-cell analysis reveals X upregulation is not global in pre-gastrulation embryos

“…In view of large number of autosomal genes compare to the small set of X-linked genes (14442-15497 autosomal genes, 798-733 X-linked genes), we calculated the X:A ratio for different lineages of pre-gastrulation embryo through bootstrapping procedure adapted from previous studies ( Pacini et al., 2021 ). For each cell, we divided expression of X-linked genes with same number of randomly selected autosomal genes and this step was repeated 1000 times and median of this 1000 values were considered for X/A expression ratio.…”

GATA transcription factors drive initial Xist upregulation after fertilization through direct activation of a distal enhancer element

“…Differentiating TX1072 XO mESCs (clone H7/A3) were profiled in three biological replicates by bulk RNA-seq as described previously for TX1072 XX mESCs (40). RNA-seq libraries were generated using the Tru-Seq Stranded Total RNA library preparation kit (Illumina) with 1 μg starting material for rRNA-depletion and amplified with 15 Cycles of PCR.…”

“…RNA-seq data during the differentiation of female TX1072 mESCs (XX) was acquired from GSE151009 (40). (Single-cell)-RNA-seq data during mouse embryonic development (Deng et al 2014; Zhang et al 2018) was similarly acquired from GEO (GSE45719, GSE76505).…”

“…The scRNA-seq data from mouse embryos analyzed in this study (41,56), are available on Github (https://github.com/rargelaguet/scnmt_gastrulation) and on GEO (GSE121708, GSE45719). Bulk RNA-seq for TX1072 XX (40) and ChIP-seq for FLAG-tagged GATA6 (48) are available via GEO (GSE151009, GSE69323).…”

The Molecular and Nuclear Dynamics of X-Chromosome Inactivation

“…Although several factors involved in Xist regulation are yet to be characterized (Pacini et al 2021), the regulatory network unraveled so far already provides a molecular basis for the temporal link observed between pluripotency exit and Xist expression (Fig. 3).…”