De novo DNA methyltransferases DNMT3A and DNMT3B are essential for XIST silencing for erosion of dosage compensation in pluripotent stem cells

Human pluripotent stem cells (hPSCs) have proven to be valuable tools for both drug discovery and the development of cell-based therapies. However, the long non-coding RNA XIST, which is essential for the establishment and maintenance of X chromosome inactivation, is repressed during culture, thereby causing erosion of dosage compensation in female hPSCs. Here, we report that the de novo DNA methyltransferases DNMT3A/3B are necessary for XIST repression in female hPSCs. We found that the deletion of both genes, but not the individual genes, inhibited XIST silencing, maintained the heterochromatin mark of H3K27me3, and did not cause global overdosage in X-linked genes. Meanwhile, DNMT3A/3B deletion after XIST repression failed to restore X chromosome inactivation. Our findings revealed that de novo DNA methyltransferases are primary factors responsible for initiating erosion of dosage compensation in female hPSCs, and XIST silencing is stably maintained in a de novo DNA-methylation-independent manner. [Display omitted] •XIST expression is linked to DNA methylation at the promoter regions in female hPSCs•De novo DNA methyltransferases DNMT3A and DNMT3B are essential for XIST silencing•XIST silencing is maintained in a de novo DNA-methylation-independent manner In this report, Fukuda and colleagues show that the de novo DNA methyltransferases DNMT3A/3B are essential for long non-coding RNA XIST silencing, which is inevitable in conventional culture and causes the erosion of dosage compensation of X chromosome inactivation in female human pluripotent stem cells. Deletion of both enzymes prior to XIST repression can prevent the erosion from happening.