Genetic evidence for Dnmt3a-dependent imprinting during oocyte growth obtained by conditional knockout with Zp3-Cre and complete exclusion of Dnmt3b by chimera formation

Authors

  • Masahiro Kaneda,

    Corresponding author
    1. Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information and Systems (ROIS), 1111 Yata, Mishima 411-8540, Japan
    2. Reproductive Biology and Technology Research Team, National Institute of Livestock and Grassland Science (NILGS), National Agriculture and Food Research Organization (NARO), 2 Ikenodai, Tsukuba 305-0901, Japan
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  • Ryutaro Hirasawa,

    1. Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information and Systems (ROIS), 1111 Yata, Mishima 411-8540, Japan
    2. Department of Genetics, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), 1111 Yata, Mishima 411-8540, Japan
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    • a

      Present address: Institute of Molecular Genetics, CNRS UMR-5535, 34293 Montpellier, France.

  • Hatsune Chiba,

    1. Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information and Systems (ROIS), 1111 Yata, Mishima 411-8540, Japan
    2. Department of Genetics, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), 1111 Yata, Mishima 411-8540, Japan
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  • Masaki Okano,

    1. Laboratory for Mammalian Epigenetic Studies, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
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  • En Li,

    1. Epigenetics Program, Novartis Institute for Biomedical Research, 250 Massachusetts Ave., Cambridge, MA 02139, USA
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  • Hiroyuki Sasaki

    1. Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information and Systems (ROIS), 1111 Yata, Mishima 411-8540, Japan
    2. Department of Genetics, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), 1111 Yata, Mishima 411-8540, Japan
    3. Division of Epigenomics, Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
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  • Communicated by: Shunsuke Ishii

* Correspondence:mkaneda@affrc.go.jp

Abstract

In the male and female germ-lines of mice, both of the two de novo DNA methyltransferases Dnmt3a and Dnmt3b are expressed. By the conditional knockout experiments using the Tnap-Cre gene, we previously showed that deletion of Dnmt3a in primordial germ cells disrupts paternal and maternal imprinting, however, Dnmt3b mutants did not show any defect. Here, we have knocked out Dnmt3a after birth in growing oocytes by using the Zp3-Cre gene and obtained genetic evidence that de novo methylation by Dnmt3a during the oocyte growth stage is indispensable for maternal imprinting. We also carried out DNA methylation analysis in the mutant oocytes and embryos and found that hypomethylation of imprinted genes in Dnmt3a-deficient oocytes was directly inherited to the embryos, but repetitive elements were re-methylated during development. Furthermore, we show that Dnmt3b-deficient cells can contribute to the male and female germ-lines in chimeric mice and can produce normal progeny, establishing that Dnmt3b is dispensable for mouse gametogenesis and imprinting. Finally, Dnmt3-related protein Dnmt3L is not only essential for methylation of imprinted genes but also enhances de novo methylation of repetitive elements in growing oocytes.

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