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Proc Natl Acad Sci U S A. 2016 Aug 23;113(34):9545-50. doi: 10.1073/pnas.1610259113. Epub 2016 Aug 2.

Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells.

Author information

1
Massachusetts General Hospital Center for Cancer Research, Charlestown, MA 02129;
2
Massachusetts General Hospital Center for Cancer Research, Charlestown, MA 02129; Institute for Environmental Studies Vrije Universiteit, Amsterdam 1081 HV, The Netherlands;
3
Department of Obstetrics and Gynecology, McGill University and Research Institute of McGill University Health Centre, Montreal, QC, Canada H4A 3J1;
4
Massachusetts General Hospital Center for Cancer Research, Charlestown, MA 02129; Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Cambridge, MA 02138.
5
Massachusetts General Hospital Center for Cancer Research, Charlestown, MA 02129; kisselbacher@mgh.harvard.edu tshioda@mgh.harvard.edu.

Abstract

The genome-wide depletion of 5-methylcytosines (5meCs) caused by passive dilution through DNA synthesis without daughter strand methylation and active enzymatic processes resulting in replacement of 5meCs with unmethylated cytosines is a hallmark of primordial germ cells (PGCs). Although recent studies have shown that in vitro differentiation of pluripotent stem cells (PSCs) to PGC-like cells (PGCLCs) mimics the in vivo differentiation of epiblast cells to PGCs, how DNA methylation status of PGCLCs resembles the dynamics of 5meC erasure in embryonic PGCs remains controversial. Here, by differential detection of genome-wide 5meC and 5-hydroxymethylcytosine (5hmeC) distributions by deep sequencing, we show that PGCLCs derived from mouse PSCs recapitulated the process of genome-wide DNA demethylation in embryonic PGCs, including significant demethylation of imprint control regions (ICRs) associated with increased mRNA expression of the corresponding imprinted genes. Although 5hmeCs were also significantly diminished in PGCLCs, they retained greater amounts of 5hmeCs than intragonadal PGCs. The genomes of both PGCLCs and PGCs selectively retained both 5meCs and 5hmeCs at a small number of repeat sequences such as GSAT_MM, of which the significant retention of bisulfite-resistant cytosines was corroborated by reanalysis of previously published whole-genome bisulfite sequencing data for intragonadal PGCs. PSCs harboring abnormal hypermethylation at ICRs of the Dlk1-Gtl2-Dio3 imprinting cluster diminished these 5meCs upon differentiation to PGCLCs, resulting in transcriptional reactivation of the Gtl2 gene. These observations support the usefulness of PGCLCs in studying the germline epigenetic erasure including imprinted genes, epimutations, and erasure-resistant loci, which may be involved in transgenerational epigenetic inheritance.

KEYWORDS:

PGCLC; epigenetic reprogramming; epimutation; genetic imprinting

PMID:
27486249
PMCID:
PMC5003264
DOI:
10.1073/pnas.1610259113
[Indexed for MEDLINE]
Free PMC Article

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