Dataset Information

DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry.

ABSTRACT: Following fertilization in mammals, paternal genomic 5-methyl-2'-deoxycytidine (5?mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2'-deoxycytidine (5?hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5?mC and 5?hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5?mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10?h after fertilization, 5?mC levels had declined by ~40%, consistent with active genomic DNA demethylation. Levels of 5?mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5?mC loss in the first 6?h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5?mC 10-48?h after fertilization, implying that any passive 'demethylation' following DNA replication was balanced by active 5?mC maintenance methylation. However, levels of 5?mC declined during development after 48?h, to 1% (measured as a fraction of G-residues) in blastocysts (~96?h). 5?hmC levels were consistently low (<0.2% of G-residues) throughout development in normal diploid embryos. This work directly quantifies the dynamics of global genomic DNA modification in mouse preimplantation embryos, suggesting that SMM will be applicable to other biomedical situations with limiting sample sizes.

SUBMITTER: Okamoto Y

PROVIDER: S-EPMC4707515 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry.

Okamoto Yoshinori Y Yoshida Naoko N Suzuki Toru T Shimozawa Nobuhiro N Asami Maki M Matsuda Tomonari T Kojima Nakao N Perry Anthony C F AC Takada Tatsuyuki T

Scientific reports 20160111

Following fertilization in mammals, paternal genomic 5-methyl-2'-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2'-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in ...[more]

PMID: 26750605

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Project description:The methylation of cytosine at CG sites in the mammalian genome is dynamically reprogrammed during gametogenesis and preimplantation development. It was previously shown that oocyte-derived DNMT1 (a maintenance methyltransferase) is essential for maintaining and propagating CG methylation at imprinting control regions in preimplantation embryos. In mammalian somatic cells, hemimethylated-CG-binding protein UHRF1 plays a critical role in maintaining CG methylation by recruiting DNMT1 to hemimethylated CG sites. However, the role of UHRF1 in oogenesis and preimplantation development is unknown. In the present study, we show that UHRF1 is mainly, but not exclusively, localized in the cytoplasm of oocytes and preimplantation embryos. However, smaller amounts of UHRF1 existed in the nucleus, consistent with the expected role in DNA methylation. We then generated oocyte-specific Uhrf1 knockout (KO) mice and found that, although oogenesis was itself unaffected, a large proportion of the embryos derived from the KO oocytes died before reaching the blastocyst stage (a maternal effect). Whole genome bisulfite sequencing revealed that blastocysts derived from KO oocytes have a greatly reduced level of CG methylation, suggesting that maternal UHRF1 is essential for maintaining CG methylation, particularly at the imprinting control regions, in preimplantation embryos. Surprisingly, UHRF1 was also found to contribute to de novo CG and non-CG methylation during oocyte growth: in Uhrf1 KO oocytes, transcriptionally-inactive regions gained less methylation, while actively transcribed regions, including the imprinting control regions, were unaffected or only slightly affected. We also found that de novo methylation was defective during the late stage of oocyte growth. To the best of our knowledge, this is the first study to demonstrate the role of UHRF1 in de novo DNA methylation in vivo. Our study reveals multiple functions of UHRF1 during the global epigenetic reprogramming of oocytes and early embryos.

Dataset Information

DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry.

Publications

DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry.

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