title: Both the folate cycle and betaine-homocysteine methyltransferase contribute methyl groups for DNA methylation in mouse blastocysts.
abstract: The embryonic pattern of global DNA methylation is first established in the inner cell mass (ICM) of the mouse blastocyst. The methyl donor S-adenosylmethionine (SAM) is produced in most cells through the folate cycle, but only a few cell types generate SAM from betaine (N,N,N-trimethylglycine) via betaine-homocysteine methyltransferase (BHMT), which is expressed in the mouse ICM. Here, mean ICM cell numbers decreased from 18-19 in controls to 11-13 when the folate cycle was inhibited by the antifolate methotrexate and to 12-14 when BHMT expression was knocked down by antisense morpholinos. Inhibiting both pathways, however, much more severely affected ICM development (7-8 cells). Total SAM levels in mouse blastocysts decreased significantly only when both pathways were inhibited (from 3.1 to 1.6 pmol/100 blastocysts). DNA methylation, detected as 5-methylcytosine (5-MeC) immunofluorescence in isolated ICMs, was minimally affected by inhibition of either pathway alone but decreased by at least 45-55% when both BHMT and the folate cycle were inhibited simultaneously. Effects on cell numbers and 5-MeC levels in the ICM were completely rescued by methionine (immediate SAM precursor) or SAM. Both the folate cycle and betaine/BHMT appear to contribute to a methyl pool required for normal ICM development and establishing initial embryonic DNA methylation.
authors.affiliation: *Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Departments of Obstetrics and Gynecology, and Cellular and Molecular Medicine, University of Ottawa Faculty of Medicine, Ottawa, Ontario, Canada; Department of Obstetrics and Gynecology, and Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; Children's Health Research Institute, London, Ontario, Canada; Developmental Biology and Cancer Program, University College London Institute of Child Health, London, United Kingdom; Research Institute of the McGill University Health Centre, Montréal Children's Hospital, Montréal, Quebec, Canada; and Departments of Human Genetics, Pediatrics, and Pharmacology and Therapeutics, McGill University, Montréal, Quebec, Canada.
authors.forename: Baohua
authors.initials: B
authors.lastname: Zhang
authors.affiliation: *Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Departments of Obstetrics and Gynecology, and Cellular and Molecular Medicine, University of Ottawa Faculty of Medicine, Ottawa, Ontario, Canada; Department of Obstetrics and Gynecology, and Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; Children's Health Research Institute, London, Ontario, Canada; Developmental Biology and Cancer Program, University College London Institute of Child Health, London, United Kingdom; Research Institute of the McGill University Health Centre, Montréal Children's Hospital, Montréal, Quebec, Canada; and Departments of Human Genetics, Pediatrics, and Pharmacology and Therapeutics, McGill University, Montréal, Quebec, Canada.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Denomme
authors.affiliation: *Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Departments of Obstetrics and Gynecology, and Cellular and Molecular Medicine, University of Ottawa Faculty of Medicine, Ottawa, Ontario, Canada; Department of Obstetrics and Gynecology, and Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; Children's Health Research Institute, London, Ontario, Canada; Developmental Biology and Cancer Program, University College London Institute of Child Health, London, United Kingdom; Research Institute of the McGill University Health Centre, Montréal Children's Hospital, Montréal, Quebec, Canada; and Departments of Human Genetics, Pediatrics, and Pharmacology and Therapeutics, McGill University, Montréal, Quebec, Canada.
authors.forename: Carlee R
authors.initials: CR
authors.lastname: White
authors.affiliation: *Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Departments of Obstetrics and Gynecology, and Cellular and Molecular Medicine, University of Ottawa Faculty of Medicine, Ottawa, Ontario, Canada; Department of Obstetrics and Gynecology, and Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; Children's Health Research Institute, London, Ontario, Canada; Developmental Biology and Cancer Program, University College London Institute of Child Health, London, United Kingdom; Research Institute of the McGill University Health Centre, Montréal Children's Hospital, Montréal, Quebec, Canada; and Departments of Human Genetics, Pediatrics, and Pharmacology and Therapeutics, McGill University, Montréal, Quebec, Canada.
authors.forename: Kit-Yi
authors.initials: KY
authors.lastname: Leung
authors.affiliation: *Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Departments of Obstetrics and Gynecology, and Cellular and Molecular Medicine, University of Ottawa Faculty of Medicine, Ottawa, Ontario, Canada; Department of Obstetrics and Gynecology, and Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; Children's Health Research Institute, London, Ontario, Canada; Developmental Biology and Cancer Program, University College London Institute of Child Health, London, United Kingdom; Research Institute of the McGill University Health Centre, Montréal Children's Hospital, Montréal, Quebec, Canada; and Departments of Human Genetics, Pediatrics, and Pharmacology and Therapeutics, McGill University, Montréal, Quebec, Canada.
authors.forename: Martin B
authors.initials: MB
authors.lastname: Lee
authors.affiliation: *Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Departments of Obstetrics and Gynecology, and Cellular and Molecular Medicine, University of Ottawa Faculty of Medicine, Ottawa, Ontario, Canada; Department of Obstetrics and Gynecology, and Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; Children's Health Research Institute, London, Ontario, Canada; Developmental Biology and Cancer Program, University College London Institute of Child Health, London, United Kingdom; Research Institute of the McGill University Health Centre, Montréal Children's Hospital, Montréal, Quebec, Canada; and Departments of Human Genetics, Pediatrics, and Pharmacology and Therapeutics, McGill University, Montréal, Quebec, Canada.
authors.forename: Nicholas D E
authors.initials: ND
authors.lastname: Greene
authors.affiliation: *Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Departments of Obstetrics and Gynecology, and Cellular and Molecular Medicine, University of Ottawa Faculty of Medicine, Ottawa, Ontario, Canada; Department of Obstetrics and Gynecology, and Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; Children's Health Research Institute, London, Ontario, Canada; Developmental Biology and Cancer Program, University College London Institute of Child Health, London, United Kingdom; Research Institute of the McGill University Health Centre, Montréal Children's Hospital, Montréal, Quebec, Canada; and Departments of Human Genetics, Pediatrics, and Pharmacology and Therapeutics, McGill University, Montréal, Quebec, Canada.
authors.forename: Mellissa R W
authors.initials: MR
authors.lastname: Mann
authors.affiliation: *Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Departments of Obstetrics and Gynecology, and Cellular and Molecular Medicine, University of Ottawa Faculty of Medicine, Ottawa, Ontario, Canada; Department of Obstetrics and Gynecology, and Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; Children's Health Research Institute, London, Ontario, Canada; Developmental Biology and Cancer Program, University College London Institute of Child Health, London, United Kingdom; Research Institute of the McGill University Health Centre, Montréal Children's Hospital, Montréal, Quebec, Canada; and Departments of Human Genetics, Pediatrics, and Pharmacology and Therapeutics, McGill University, Montréal, Quebec, Canada.
authors.forename: Jacquetta M
authors.initials: JM
authors.lastname: Trasler
authors.affiliation: *Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Departments of Obstetrics and Gynecology, and Cellular and Molecular Medicine, University of Ottawa Faculty of Medicine, Ottawa, Ontario, Canada; Department of Obstetrics and Gynecology, and Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; Children's Health Research Institute, London, Ontario, Canada; Developmental Biology and Cancer Program, University College London Institute of Child Health, London, United Kingdom; Research Institute of the McGill University Health Centre, Montréal Children's Hospital, Montréal, Quebec, Canada; and Departments of Human Genetics, Pediatrics, and Pharmacology and Therapeutics, McGill University, Montréal, Quebec, Canada jbaltz@ohri.ca.
authors.forename: Jay M
authors.initials: JM
authors.lastname: Baltz
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D050940:Betaine-Homocysteine S-Methyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019070:Cell Lineage; D002478:Cells, Cultured; D019175:DNA Methylation; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique; D005492:Folic Acid / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D012436:S-Adenosylmethionine / Q000378:metabolism*; D055517:snRNP Core Proteins / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D055517:snRNP Core Proteins; D044503:5-Methylcytosine; D012436:S-Adenosylmethionine; D005492:Folic Acid; D050940:Betaine-Homocysteine S-Methyltransferase; D008727:Methotrexate
keywords: S-adenosylmethionine; embryo; epigenetics; inner cell mass; methionine