title: Selective impairment of methylation maintenance is the major cause of DNA methylation reprogramming in the early embryo.
abstract: BACKGROUND
  DNA methylomes are extensively reprogrammed during mouse pre-implantation and early germ cell development. The main feature of this reprogramming is a genome-wide decrease in 5-methylcytosine (5mC). Standard high-resolution single-stranded bisulfite sequencing techniques do not allow discrimination of the underlying passive (replication-dependent) or active enzymatic mechanisms of 5mC loss. We approached this problem by generating high-resolution deep hairpin bisulfite sequencing (DHBS) maps, allowing us to follow the patterns of symmetric DNA methylation at CpGs dyads on both DNA strands over single replications.
  
  
  RESULTS
  We compared DHBS maps of repetitive elements in the developing zygote, the early embryo, and primordial germ cells (PGCs) at defined stages of development. In the zygote, we observed distinct effects in paternal and maternal chromosomes. A significant loss of paternal DNA methylation was linked to replication and to an increase in continuous and dispersed hemimethylated CpG dyad patterns. Overall methylation levels at maternal copies remained largely unchanged, but showed an increased level of dispersed hemi-methylated CpG dyads. After the first cell cycle, the combined DHBS patterns of paternal and maternal chromosomes remained unchanged over the next three cell divisions. By contrast, in PGCs the DNA demethylation process was continuous, as seen by a consistent decrease in fully methylated CpG dyads over consecutive cell divisions.
  
  
  CONCLUSIONS
  The main driver of DNA demethylation in germ cells and in the zygote is partial impairment of maintenance of symmetric DNA methylation at CpG dyads. In the embryo, this passive demethylation is restricted to the first cell division, whereas it continues over several cell divisions in germ cells. The dispersed patterns of CpG dyads in the early-cleavage embryo suggest a continuous partial (and to a low extent active) loss of methylation apparently compensated for by selective de novo methylation. We conclude that a combination of passive and active demethylation events counteracted by de novo methylation are involved in the distinct reprogramming dynamics of DNA methylomes in the zygote, the early embryo, and PGCs.
authors.affiliation: University of Saarland, FR 8.3, Biological Sciences, Genetics/Epigenetics, Campus A2.4, 66123 Saarbrücken, Germany ; Departments of Pediatrics and Genetics, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305 USA.
authors.forename: Julia
authors.initials: J
authors.lastname: Arand
authors.affiliation: University of Saarland, FR 8.3, Biological Sciences, Genetics/Epigenetics, Campus A2.4, 66123 Saarbrücken, Germany ; Departments of Genetics and Obstetrics & Gynaecology, Stanford University School of Medicine, Institute for Stem Cell Biology & Regenerative Medicine, 265 Campus Drive, Stanford, CA 94305 USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Wossidlo
authors.affiliation: University of Saarland, FR 8.3, Biological Sciences, Genetics/Epigenetics, Campus A2.4, 66123 Saarbrücken, Germany.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Lepikhov
authors.affiliation: Epigenetics Programme, The Babraham Institute, Cambridge, CB22 3AT UK.
authors.forename: Julian R
authors.initials: JR
authors.lastname: Peat
authors.affiliation: Epigenetics Programme, The Babraham Institute, Cambridge, CB22 3AT UK ; Wellcome Trust Sanger Institute, Hinxton, CB10 1SA UK.
authors.forename: Wolf
authors.initials: W
authors.lastname: Reik
authors.affiliation: University of Saarland, FR 8.3, Biological Sciences, Genetics/Epigenetics, Campus A2.4, 66123 Saarbrücken, Germany.
authors.forename: Jörn
authors.initials: J
authors.lastname: Walter
keywords: DNA methylation pattern; DNA methylation reprogramming; Deep hairpin bisulfite sequencing; Pre-implantation development