title: Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells.
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.
authors.affiliation: Massachusetts General Hospital Center for Cancer Research, Charlestown, MA 02129;
authors.forename: Norikatsu
authors.initials: N
authors.lastname: Miyoshi
authors.affiliation: Massachusetts General Hospital Center for Cancer Research, Charlestown, MA 02129; Institute for Environmental Studies Vrije Universiteit, Amsterdam 1081 HV, The Netherlands;
authors.forename: Jente M
authors.initials: JM
authors.lastname: Stel
authors.affiliation: Massachusetts General Hospital Center for Cancer Research, Charlestown, MA 02129;
authors.forename: Keiko
authors.initials: K
authors.lastname: Shioda
authors.affiliation: Massachusetts General Hospital Center for Cancer Research, Charlestown, MA 02129;
authors.forename: Na
authors.initials: N
authors.lastname: Qu
authors.affiliation: Massachusetts General Hospital Center for Cancer Research, Charlestown, MA 02129;
authors.forename: Junko
authors.initials: J
authors.lastname: Odajima
authors.affiliation: Massachusetts General Hospital Center for Cancer Research, Charlestown, MA 02129;
authors.forename: Shino
authors.initials: S
authors.lastname: Mitsunaga
authors.affiliation: Department of Obstetrics and Gynecology, McGill University and Research Institute of McGill University Health Centre, Montreal, QC, Canada H4A 3J1;
authors.forename: Xiangfan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Obstetrics and Gynecology, McGill University and Research Institute of McGill University Health Centre, Montreal, QC, Canada H4A 3J1;
authors.forename: Makoto
authors.initials: M
authors.lastname: Nagano
authors.affiliation: Massachusetts General Hospital Center for Cancer Research, Charlestown, MA 02129; Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Cambridge, MA 02138.
authors.forename: Konrad
authors.initials: K
authors.lastname: Hochedlinger
authors.affiliation: Massachusetts General Hospital Center for Cancer Research, Charlestown, MA 02129; kisselbacher@mgh.harvard.edu tshioda@mgh.harvard.edu.
authors.forename: Kurt J
authors.initials: KJ
authors.lastname: Isselbacher
authors.affiliation: Massachusetts General Hospital Center for Cancer Research, Charlestown, MA 02129; kisselbacher@mgh.harvard.edu tshioda@mgh.harvard.edu.
authors.forename: Toshi
authors.initials: T
authors.lastname: Shioda
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002135:Calcium-Binding Proteins; D000073399:DNA Demethylation; D019175:DNA Methylation; D004622:Embryo, Mammalian; D044127:Epigenesis, Genetic; D005260:Female; D016678:Genome; D018392:Genomic Imprinting; D005854:Germ Cells / Q000166:cytology / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D007453:Iodide Peroxidase / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D009154:Mutation; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; C512925:Dlk1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; C422882:MEG3 non-coding RNA, mouse; D062085:RNA, Long Noncoding; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; C117274:iodothyronine deiodinase type III; D007453:Iodide Peroxidase
keywords: PGCLC; epigenetic reprogramming; epimutation; genetic imprinting