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Human embryonic stem cells do not change their X-inactivation status during differentiation [RRBS-Seq]

ABSTRACT: Female human ESC-lines can carry active X-chromosomes (Xa) or an XIST-RNA-coated inactive X-chromosome (XiXIST+). Additionally, many ESC lines have abnormal X-chromosomeinactivation (XCI)-states where the Xi no longer expresses XIST-RNA and has transcriptionally active regions (eroded Xi=Xe). The fate of each XCI-state upon differentiation is unclear because individual lines often contain a mixture of XCI-states. Here, we established homogeneous XiXa, XeXa, and XaXa ESC-lines. Employing RNA-FISH, RNA-sequencing and DNA methylation analyses, we found that these lines were unable to initiate XIST-expression and X-chromosome-wide silencing upon differentiation indicating that the ESC XCI-state is maintained in differentiated cells. Consequently, differentiated XeXa and XaXa cells displayed higher levels of X-linked gene-expression than XiXa cells. Although global transcriptional compensation between X-chromosomes and autosomes is not required for female ESC-differentiation, the degree of X-chromosome-silencing influences differentiation efficiencies. Our data suggest that the XiXIST+Xa state is inherent to human ESCs and that all other XCI-states, including XaXa, are abnormal and arise during ESC-derivation or maintenance.

ORGANISM(S): Homo sapiens

PROVIDER: GSE88932 | GEO | 2017/01/26

SECONDARY ACCESSION(S): PRJNA349149

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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Human embryonic stem cells do not change their X-inactivation status during differentiation [RRBS-Seq]

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