Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Active and Passive Demethylation of Male and Female Pronuclear DNA in the Mammalian Zygote

ABSTRACT: The epigenomes of mammalian sperm and oocytes, characterized by gamete-specific 5-methylcytosine (5mC) patterns, are reprogrammed during early embryogenesis to establish full developmental potential. Previous studies have suggested that the paternal genome is actively demethylated in the zygote while the maternal genome undergoes subsequent passive demethylation via DNA replication during cleavage. Active demethylation is known to depend on 5mC oxidation by Tet dioxygenases and excision of oxidized bases by thymine DNA glycosylase (TDG). Here we show that both maternal and paternal genomes undergo widespread active and passive demethylation in zygotes before the first mitotic division. Passive demethylation was blocked by the replication inhibitor aphidicolin, and active demethylation was abrogated by deletion of Tet3 in both pronuclei. At actively demethylated loci, 5mCs were processed to unmodified cytosines. Surprisingly, the demethylation process was unaffected by the deletion of TDG from the zygote, suggesting the existence of other demethylation mechanisms downstream of Tet3-mediated oxidation. The dataset includes RRBS anlysis of 2 MII oocyte samples, 3 WT female pronuclei samples PN3-4 stage, 2 Tet3 KO female pronuclei samples and 2 Aphidicolin treated female pronuclei samples. Also as male counterpart, a Sperm sample, 2 WT male pronuclei samples PN3-4 stage, 2 Tet3 KO male pronuclei samples and 2 Aphidicolin treated male pronuclei samples were included.

ORGANISM(S): Mus musculus

SUBMITTER: Xianlong Li

PROVIDER: E-GEOD-56650 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Fertilization triggers a global erasure of 5-methylcytosine from paternal DNA as part of extensive epigenetic reprogramming during the transition from gametic specialization to totipotency. This active removal has been shown to involve oxidation by TET3, but the targeting of this pathway and the wider context of demethylation remain poorly understood. We optimized a novel technique for whole-genome bisulfite sequencing and applied it to wild-type and TET3-deficient zygotes, using SNPs to access paternal alleles. As the great majority of sperm-contributed methylation lies outside the CpG islands (CGIs) and promoters analysed to date, these genome-wide methylation profiles allow paternal methylation trajectories in the zygote to be comprehensively examined for the first time. This global view revealed that in addition to pervasive loss of methylation from intergenic sequences and most repetitive elements, gene bodies constitute a major target of zygotic demethylation. Methylation loss is associated with zygotic genome activation, and at gene bodies is also linked to increased transcriptional noise in the early embryo. Our data maps the primary contribution of oxidative demethylation to a subset of gene bodies and single-copy intergenic sequences, and implicates the action of redundant pathways at many loci. We further uncover a novel function for TET3 in protection from de novo methylation at CGIs and promoters. This work adds new breadth to our understanding of the molecular events involved in reprogramming gametic identity following fertilization. Three independent collections of zygotes were performed for each genotype (control and TET3 deletion), giving a total of 225 control and 237 TET3 deletion zygotes, of which 120 and 129 were derived from 129S2/SvHsd studs for control and TET3 samples, respectively. Zygotes were pooled and whole-genome bisulfite libraries were prepared using a post-bisulfite adaptor tagging strategy optimized from (Miura et al. Nucleic Acids Research 2012, 40:e136)

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Active and Passive Demethylation of Male and Female Pronuclear DNA in the Mammalian Zygote

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