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Dissecting the onset of random X-chromosome inactivation at high temporal and allelic 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 a model for cellular decision making and epigenetic gene regulation. The random cell-autonomous nature of XCI however has so far prevented its chromosome-wide investigation in its endogenous context without genetic perturbation. Here we use allele-specific single-cell RNA-sequencing to assess the onset of random XCI with high temporal resolution in differentiating mouse embryonic stem cells, and develop dedicated analysis approaches. We find that XCI is initiated on both chromosomes in a subset of cells, but reverted to the final mono-allelic state once gene silencing is established. By exploiting the heterogeneity of XCI onset, we identify Nanog as its main trigger and discover additional genes that might control upregulation of Xist, the master regulator of XCI. Finally, we show that genetic variation between different mouse strains modulates the XCI process at multiple levels, and validate these findings through an orthogonal experimental approach. We thus draw a detailed picture of how XCI is initiated and the experimental and computational strategies we have developed here will now allow us to profile random XCI in more physiological contexts, including primary human cells in vivo.

ORGANISM(S): Mus musculus

PROVIDER: GSE151009 | GEO | 2020/05/22

REPOSITORIES: GEO

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Project description:In naïve human pluripotent stem cells (hPSCs) that represent the pre-implantation embryonic states, epigenetic regulators for cell identity remain poorly defined. One of the major remaining questions in naïve stem cell biology is how female cells mediate and regulate X chromosome inactivation (XCI). Recent studies incorporating single-cell RNA sequencing (scRNA-seq) have demonstrated that transcript-based technologies are insufficient to unequivocally resolve XCI regulation in a human system. Instead, 3D genomics shows immense promise for studying XCI by interrogating changes to the X chromosomes’ 3D states. Here, we sought to characterize the 3D state of the X chromosome in naïve and primed hPSCs. Using chromatin tracing, we analyzed the folding conformations of whole X chromosomes with megabase genomic resolution in multiple naïve and primed hPSC lines. Our data found that X chromosomes in female naive hPSCs exhibit a range of spatial folding conformations similar to the active X chromosome (Xa) and the inactive X chromosome (Xi) in somatic cells; these data suggest that naïve hPSCs have initiated XCI. As XCI process ultimately generates detectable differences in volume between the Xa and the Xi, we measured the radius of gyration of individual X chromosome copies across various hPSC cell lines and culture conditions. In naïve hPSCs, our results show that the X chromosomes with Xi-like conformations are not more compact than those with Xa-like conformation. This finding is directly counter to the Xi compaction typically observed in post-XCI female somatic cells. These contradictory 3D signatures of XCI suggest that female naïve hPSCs have initiated the XCI process, but are poised before XCI-driven silencing in a metastable state. As observed in H7 naïve hPSCs, this metastable state can be abolished through classically noted means, such as the loss of Xp chromatin, leading to the deleted p X chromosome (dXp). The Xp loss observed here seems to drive XIST expression and accumulation around the dXp chromosome. Overall, our findings provide insight into the previously undefined X chromosome status in naïve hPSCs in single chromosome resolution, and are critical in understanding the unique epigenetic regulation in early embryonic cells.

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Dissecting the onset of random X-chromosome inactivation at high temporal and allelic resolution

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