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Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting

Nature volume 429, pages 900–903 (2004)Cite this article

Abstract

Imprinted genes are epigenetically marked during gametogenesis so that they are exclusively expressed from either the paternal or the maternal allele in offspring1. Imprinting prevents parthenogenesis in mammals and is often disrupted in congenital malformation syndromes, tumours and cloned animals1. Although de novo DNA methyltransferases of the Dnmt3 family are implicated in maternal imprinting2, the lethality of Dnmt3a and Dnmt3b knockout mice3 has precluded further studies. We here report the disruption of Dnmt3a and Dnmt3b in germ cells, with their preservation in somatic cells, by conditional knockout technology4. Offspring from Dnmt3a conditional mutant females die in utero and lack methylation and allele-specific expression at all maternally imprinted loci examined. Dnmt3a conditional mutant males show impaired spermatogenesis and lack methylation at two of three paternally imprinted loci examined in spermatogonia. By contrast, Dnmt3b conditional mutants and their offspring show no apparent phenotype. The phenotype of Dnmt3a conditional mutants is indistinguishable from that of Dnmt3L knockout mice2,5, except for the discrepancy in methylation at one locus. These results indicate that both Dnmt3a and Dnmt3L are required for methylation of most imprinted loci in germ cells, but also suggest the involvement of other factors.

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Figure 1: Conditional knockout of the Dnmt3a locus in the germline by TNAP–Cre.
Figure 2: Developmental defects and loss of maternal imprints in embryos derived from Dnmt3a conditional mutant mothers.
Figure 3: Impaired spermatogenesis and failure of establishment of paternal methylation imprints in Dnmt3a conditional mutant males.
Figure 4: Efficient deletion of Dnmt3b by TNAP–Cre in developing germ cells.

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Acknowledgements

We thank A. Nagy and H. Lomeli (Samuel Lunenfeld Research Institute) for providing the TNAP–Cre mice; K. Shiota and S. Tanaka (The University of Tokyo) for help in laser-microdissection microscopy; K. Kumaki and Y. Kato for help and advice on bisulphite sequencing; and C. Suda, M. Kanbayashi, M. Serizawa and H. Furuumi for technical assistance and mouse maintenance. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to H.S., and grants from the National Institutes of Health to E.L.

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Author notes

  1. En Li

    Present address: Novartis Institute for Biomedical Research, Cambridge, Massachusetts, 02139, USA

Authors and Affiliations

  1. Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information and Systems, Mishima, 411-8540, Japan

    Masahiro Kaneda, Kenichiro Hata, Takashi Sado, Naomi Tsujimoto & Hiroyuki Sasaki

  2. Department of Genetics, School of Life Science, The Graduate University for Advanced Studies (Sokendai), Mishima, 411-8540, Japan

    Masahiro Kaneda, Naomi Tsujimoto & Hiroyuki Sasaki

  3. Laboratory for Mammalian Epigenetic Studies, Center for Developmental Biology, RIKEN, Kobe, 650-0047, Japan

    Masaki Okano

  4. Cardiovascular Research Center, Massachusetts General Hospital, Department of Medicine, Harvard Medical School, Charlestown, Massachusetts, 02129, USA

    Masaki Okano, Naomi Tsujimoto & En Li

  5. Department of Biosystems Science, School of Advanced Sciences, The Graduate University for Advanced Studies (Sokendai), Hayama, 240-0193, Japan

    Takashi Sado

  6. PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, 332-0012, Japan

    Takashi Sado

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Correspondence to En Li or Hiroyuki Sasaki.

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Supplementary information

Supplementary Figure

Includes bisulfite sequencing profiles of IAP LTR sequences of oocytes and spermatogonia lacking Dnmt3a. (PDF 38 kb)

Supplementary Figure Legend (DOC 20 kb)

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Kaneda, M., Okano, M., Hata, K. et al. Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature 429, 900–903 (2004). https://doi.org/10.1038/nature02633

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