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

Abstract

Summary 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.

De-erosion of X chromosome dosage compensation by the editing of XIST regulatory regions restores the differentiation potential in hPSCs

“…XIST is expressed from one of two X chromosomes to archive X chromosome inactivation (XCI), and the expression is maintained in female somatic cells ( Augui et al., 2011 ; Penny et al., 1996 ). Previous studies have shown that XIST repression in female hPSCs causes the overexpression of X-linked genes and results in a poor differentiation ability ( Anguera et al., 2012 ; Fukuda et al., 2021 ; Mekhoubad et al., 2012 ; Patel et al., 2017 ). Therefore, XIST dysregulation may be one of the largest and most underestimated sources of variability hampering reproducibility and disease modeling in female hPSC research.…”

Preventing erosion of X-chromosome inactivation in human embryonic stem cells

“…X-inactivation is an experimentally tractable system to dissect epigenetic transcriptional regulation in PSCs. Much prior work has demonstrated that prolonged culture of female hPSCs results in loss of XIST RNA coating and erosion of X-inactivation 29 , 31 , 35 , 36 , 38 , 39 , 42 , 43 , 45 , 57 , 59 . Consistent with previous findings, our data rule out atmospheric O 2 concentration as a cause of XIST RNA loss in hESCs 35 , 43 , 45 , 68 – 70 .…”

“…The loss of XIST RNA expression in the hESCs is irreversible, in agreement with prior findings 35 , 38 , 39 . Of note, a recent report suggests that DNA methylation may contribute to the irreversibility of XIST RNA loss in cultured female hESCs that have undergone X-inactivation erosion 59 .…”

“…This loss of XIST RNA coating and re-expression of silenced X-linked genes has been termed X-inactivation erosion 31 , 34 , 35 , 38 , 42 , 43 , 58 . Loss of XIST RNA coating and the leaky expression of inactive X-linked genes also characterize cultured human induced pluripotent stem cells (hiPSCs) and are thought to be irreversible 35 , 36 , 42 , 45 , 57 , 59 . Increased X-linked gene expression due to X-inactivation erosion or failure can be deleterious to development and differentiation 23 , 45 , 60 .…”

Long noncoding RNA XIST: Mechanisms for X chromosome inactivation, roles in sex-biased diseases, and therapeutic opportunities

“…In human pluripotent stem cells, XIST expression is silenced by the de novo DNA methyltransferases DNMT3A and DNMT3B. 40

Figure 1 The long noncoding Xist RNA and its roles in X chromosome inactivation. (A) Genomic arrangement of XIST / Xist and its regulators in X inactivation center (XIC) in human and mouse.

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