title: Widespread recovery of methylation at gametic imprints in hypomethylated mouse stem cells following rescue with DNMT3A2.
abstract: Imprinted loci are paradigms of epigenetic regulation and are associated with a number of genetic disorders in human. A key characteristic of imprints is the presence of a gametic differentially methylated region (gDMR). Previous studies have indicated that DNA methylation lost from gDMRs could not be restored by DNMT1, or the de novo enzymes DNMT3A or 3B in stem cells, indicating that imprinted regions must instead undergo passage through the germline for reprogramming. However, previous studies were non-quantitative, were unclear on the requirement for DNMT3A/B and showed some inconsistencies. In addition, new putative gDMR has recently been described, along with an improved delineation of the existing gDMR locations. We therefore aimed to re-examine the dependence of methylation at gDMRs on the activities of the methyltransferases in mouse embryonic stem cells (ESCs).
  
  
  
  We examined the most complete current set of imprinted gDMRs that could be assessed using quantitative pyrosequencing assays in two types of ESCs: those lacking DNMT1 (1KO) and cells lacking a combination of DNMT3A and DNMT3B (3abKO). We further verified results using clonal analysis and combined bisulfite and restriction analysis. Our results showed that loss of methylation was approximately equivalent in both cell types. 1KO cells rescued with a cDNA-expressing DNMT1 could not restore methylation at the imprinted gDMRs, confirming some previous observations. However, nearly all gDMRs were remethylated in 3abKO cells rescued with a DNMT3A2 expression construct (3abKO + 3a2). Transcriptional activity at theH19/Igf2locus also tracked with the methylation pattern, confirming functional reprogramming in the latter.
  
  
  
  These results suggested (1) a vital role for DNMT3A/B in methylation maintenance at imprints, (2) that loss of DNMT1 and DNMT3A/B had equivalent effects, (3) that rescue with DNMT3A2 can restore imprints in these cells. This may provide a useful system in which to explore factors influencing imprint reprogramming.
authors.affiliation: Genomic Medicine Research Group, Biomedical Sciences Research Institute, Centre for Molecular Biosciences, University of Ulster, Coleraine, BT52 1SA UK ; Terry Fox Laboratory, BC Cancer Agency, 675 W 10th Ave, Vancouver, BC V5Z 1G1 Canada.
authors.forename: Avinash
authors.initials: A
authors.lastname: Thakur
authors.affiliation: Genomic Medicine Research Group, Biomedical Sciences Research Institute, Centre for Molecular Biosciences, University of Ulster, Coleraine, BT52 1SA UK.
authors.forename: Sarah-Jayne
authors.initials: SJ
authors.lastname: Mackin
authors.affiliation: Genomic Medicine Research Group, Biomedical Sciences Research Institute, Centre for Molecular Biosciences, University of Ulster, Coleraine, BT52 1SA UK.
authors.forename: Rachelle E
authors.initials: RE
authors.lastname: Irwin
authors.affiliation: Genomic Medicine Research Group, Biomedical Sciences Research Institute, Centre for Molecular Biosciences, University of Ulster, Coleraine, BT52 1SA UK ; Centre for Experimental Medicine, The Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7AE UK.
authors.forename: Karla M
authors.initials: KM
authors.lastname: O'Neill
authors.affiliation: Genomic Medicine Research Group, Biomedical Sciences Research Institute, Centre for Molecular Biosciences, University of Ulster, Coleraine, BT52 1SA UK.
authors.forename: Gareth
authors.initials: G
authors.lastname: Pollin
authors.affiliation: Genomic Medicine Research Group, Biomedical Sciences Research Institute, Centre for Molecular Biosciences, University of Ulster, Coleraine, BT52 1SA UK.
authors.forename: Colum
authors.identifier: 0000-0001-9921-7506
authors.initials: C
authors.lastname: Walsh
mesh_terms: D000818:Animals; D000076123:DNA (Cytosine-5-)-Methyltransferase 1 / Q000172:deficiency / Q000235:genetics; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D018392:Genomic Imprinting / Q000235:genetics*; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism; D000097584:DNA Methyltransferase 3B
chemical_list: D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D012313:RNA; D062085:RNA, Long Noncoding; D000097584:DNA Methyltransferase 3B; C000717967:DNMT3A protein, human; C000715535:Dnmt3a protein, mouse; C094335:H19 long non-coding RNA; C000619381:Dnmt1 protein, mouse
keywords: DNA methylation; ESC; Imprinting; Reprogramming