title: Haploinsufficiency of the paternal-effect gene Dnmt3L results in transient DNA hypomethylation in progenitor cells of the male germline.
abstract: OBJECTIVE
  How does haploinsufficiency of the paternal-effect gene Dnmt3L affect DNA methylation establishment and stability in the male germline?
  
  
  CONCLUSIONS
  Reduced expression of DNMT3L in male germ cells, associated with haploinsufficiency of the paternal-effect gene Dnmt3L, results in abnormal hypomethylation of prenatal germline progenitor cells.
  
  
  BACKGROUND
  The DNA methyltransferase regulator Dnmt3-Like (Dnmt3L) is a paternal-effect gene required for DNA methylation acquisition in male germline stem cells and their precursors. In males, DNMT3L deficiency causes meiotic abnormalities and infertility. While Dnmt3L heterozygous males are fertile, they have abnormalities in X chromosome compaction and postmeiotic gene expression and sire offspring with sex chromosome aneuploidy. It has been proposed that the paternal effects of Dnmt3L haploinsufficiency are due to epigenetic defects in early male germ cells. DNA methylation is an essential epigenetic modification essential for normal germ cell development. Since patterns of DNA methylation across the genome are initially acquired in prenatal male germ cells, perturbations in methylation could contribute to the epigenetic basis of the paternal effects in Dnmt3L(+/-) males.
  
  
  METHODS
  This is a cross-sectional study of DNA methylation in Dnmt3L(+/+) versus Dnmt3L(+/-) male germ cells collected from mice at 16.5 days post-coitum (dpc), Day 6 and Day 70 (n = 3 per genotype, each n represents a pool of 2-20 animals). Additionally, DNA methylation was compared in enriched populations of spermatogonial stem cells (SSC)/progenitor cells from Dnmt3L(+/+) and Dnmt3L(+/-) males following ≈ 2 months in culture.
  
  
  METHODS
  DNA methylation at intergenic loci along chromosomes 9 and X was examined by quantitative analysis of DNA methylation by real-time polymerase chain reaction at the time of initial acquisition of epigenetic patterns in the prenatal male germline (16.5 dpc) and compared with patterns in early post-natal spermatogonia (Day 6) and in spermatozoa in mice. DNA methylation status at CpG-rich sites across the genome was assessed in spermatogonial precursors from Day 4 male mice using restriction landmark genomic scanning.
  
  
  RESULTS
  At 16.5 dpc, 42% of intergenic loci examined along chromosome 9 and 10% of those along chromosome X were hypomethylated in Dnmt3L heterozygotes. By Day 6 and in spermatozoa, germ cell DNA methylation was similar in heterozygous and wild-type mice. DNA methylation stability of acquired patterns in wild-type and Dnmt3L(+/-) SSC/progenitor cell culture was analyzed at numerous loci across the genome in cells cultured in vitro and collected at passages 6-28. While the methylation of most loci was stable in culture over time, differences at ≈ 1% of sites were found between Dnmt3L(+/-) and Dnmt3L(+/+) cultures.
  
  
  CONCLUSIONS
  Evaluation of DNA methylation in SSCs can only be performed after a period of culture limiting the investigation to changes observed during culture when compared with DNA methylation differences between genotypes that could be present at the beginning of culture establishment.
  
  
  CONCLUSIONS
  The DNA methylation defects described here in prenatal male germline progenitor cells and SSC culture are the earliest epigenetic perturbations yet identified for a mammalian paternal-effect gene and may influence downstream epigenetic events in germ cells at later stages of development. Together, the results provide evidence of a 'window' of susceptibility in prenatal male germ cell precursors for the induction of epimutations due to genetic perturbations and, potentially, in utero environmental exposures.
authors.affiliation: Department of Human Genetics,  McGill University, Montréal, Quebec,  Canada.
authors.forename: K M
authors.initials: KM
authors.lastname: Niles
authors.forename: J R
authors.initials: JR
authors.lastname: Yeh
authors.forename: D
authors.initials: D
authors.lastname: Chan
authors.forename: M
authors.initials: M
authors.lastname: Landry
authors.forename: M C
authors.initials: MC
authors.lastname: Nagano
authors.forename: J M
authors.initials: JM
authors.lastname: Trasler
mesh_terms: D000782:Aneuploidy; D000818:Animals; D003430:Cross-Sectional Studies; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005838:Genotype; D057895:Haploinsufficiency; D006579:Heterozygote; D008297:Male; D051379:Mice; D012729:Sex Chromosome Aberrations; D013093:Spermatogonia / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*
chemical_list: C412484:DNMT3L protein, human; D004248:DNA (Cytosine-5-)-Methyltransferases