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

Atsushi Fukuda; Dane Z Hazelbaker; Nami Motosugi; Jin Hao; Francesco Limone; Amanda Beccard; Patrizia Mazzucato; Angelica Messana; Chisa Okada; Irune Guerra San Juan; Menglu Qian; Akihiro Umezawa; Hidenori Akutsu; Lindy E Barrett; Kevin Eggan

doi:10.1016/j.stemcr.2021.07.015

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

Stem Cell Reports. 2021 Sep 14;16(9):2138-2148. doi: 10.1016/j.stemcr.2021.07.015. Epub 2021 Aug 19.

Authors

Affiliations

¹ The Harvard Stem Cell Institute and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan; The Institute of Medical Science, Tokai University, Kanagawa, Japan; Micro/Nano Technology Center, Tokai University, Hiratsuka, Kanagawa, Japan; Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan. Electronic address: fa972942@tsc.u-tokai.ac.jp.
² Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
³ Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
⁴ The Harvard Stem Cell Institute and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁵ Support Center for Medical Research and Education, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
⁶ Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
⁷ The Harvard Stem Cell Institute and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA. Electronic address: eggan@mcb.harvard.edu.

Abstract

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.

Keywords: DNA methylation; DNMT3A/3B; Lnc RNA XIST; dosage compensation; pluripotent stem cells.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Chromatin Assembly and Disassembly
DNA (Cytosine-5-)-Methyltransferases / genetics*
DNA (Cytosine-5-)-Methyltransferases / metabolism
DNA Methylation
DNA Methyltransferase 3A / genetics*
DNA Methyltransferase 3A / metabolism
DNA Methyltransferase 3B
Dosage Compensation, Genetic
Epigenesis, Genetic
Gene Expression Profiling
Gene Expression Regulation*
Gene Silencing*
Genes, X-Linked
Genetic Background
Heterochromatin / genetics
Heterochromatin / metabolism
Humans
Models, Biological
Pluripotent Stem Cells / cytology
Pluripotent Stem Cells / metabolism*
RNA, Long Noncoding / genetics*

Substances

DNMT3A protein, human
Heterochromatin
RNA, Long Noncoding
XIST non-coding RNA
DNA (Cytosine-5-)-Methyltransferases
DNA Methyltransferase 3A

Grants and funding

P01 GM099117/GM/NIGMS NIH HHS/United States