Abstract
Human pluripotent stem cells (hPSCs) have proven to be valuable tools for both drug discovery and the development of cell based therapies. However, silencing of XIST expression followed by inappropriate reactivation and biallelic expression of X chromosome linked genes regularly occurs in female hPSCs, placing them in a state observed in human tumors. This gradual erosion of dosage compensation also occludes cellular phenotypes while modeling X-linked diseases and interferes with directed differentiation, greatly limiting the utility of female hPSCs. Here, we investigate the mechanisms regulating erosion of dosage compensation and whether it can be reversed or prevented. We found that changes to the X chromosome during erosion of dosage compensation both repress XIST through DNA methylation and prevent its RNA from silencing the chromosome, even if XIST expression is induced using CRISPR/Cas9 activation. Encouragingly, we found that elimination of the DNMT3A and genes, but the similarity of global transcriptional status of DNMT3A/3B deleted cell to non41 eroded cell differs in cell lines. While DNMT3A/3B deletion after erosion of dosage compensation failed to restore it. Thus, our study revealed that de novo DNA methyltransferases are primary factors responsible for the initiation of erosion of dosage compensation in female hPSCs and once DNA methylation is imposed on XIST promoterregions, the status is stably maintained to repress XIST by de novo DNA methylation independent manner.
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