The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes


Sperm and eggs carry distinctive epigenetic modifications that are adjusted by reprogramming after fertilization1. The paternal genome in a zygote undergoes active DNA demethylation before the first mitosis2,3. The biological significance and mechanisms of this paternal epigenome remodelling have remained unclear4. Here we report that, within mouse zygotes, oxidation of 5-methylcytosine (5mC) occurs on the paternal genome, changing 5mC into 5-hydroxymethylcytosine (5hmC). Furthermore, we demonstrate that the dioxygenase Tet3 (ref. 5) is enriched specifically in the male pronucleus. In Tet3-deficient zygotes from conditional knockout mice, paternal-genome conversion of 5mC into 5hmC fails to occur and the level of 5mC remains constant. Deficiency of Tet3 also impedes the demethylation process of the paternal Oct4 and Nanog genes and delays the subsequent activation of a paternally derived Oct4 transgene in early embryos. Female mice depleted of Tet3 in the germ line show severely reduced fecundity and their heterozygous mutant offspring lacking maternal Tet3 suffer an increased incidence of developmental failure. Oocytes lacking Tet3 also seem to have a reduced ability to reprogram the injected nuclei from somatic cells. Therefore, Tet3-mediated DNA hydroxylation is involved in epigenetic reprogramming of the zygotic paternal DNA following natural fertilization and may also contribute to somatic cell nuclear reprogramming during animal cloning.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Specific oxidation of methylcytosine and Tet3 distribution in the zygotic male pronucleus.
Figure 2: The role of Tet3 in 5mC oxidation, demethylation of paternal DNA, and activation of the paternal Oct4 allele.
Figure 3: Maternal Tet3 deficiency compromises embryonic development.
Figure 4: Tet3 contributes to reprogram the somatic nucleus transferred into oocytes.


  1. 1

    Surani, M. A., Hayashi, K. & Hajkova, P. Genetic and epigenetic regulators of pluripotency. Cell 128, 747–762 (2007)

    CAS  Article  Google Scholar 

  2. 2

    Mayer, W., Niveleau, A., Walter, J., Fundele, R. & Haaf, T. Demethylation of the zygotic paternal genome. Nature 403, 501–502 (2000)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Oswald, J. et al. Active demethylation of the paternal genome in the mouse zygote. Curr. Biol. 10, 475–478 (2000)

    CAS  Article  Google Scholar 

  4. 4

    Ooi, S. K. & Bestor, T. H. The colorful history of active DNA demethylation. Cell 133, 1145–1148 (2008)

    CAS  Article  Google Scholar 

  5. 5

    Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Kriaucionis, S. & Heintz, N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324, 929–930 (2009)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Iqbal, K., Jin, S. G., Pfeifer, G. P. & Szabo, P. E. Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine. Proc. Natl Acad. Sci. USA 108, 3642–3647 (2011)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Wossidlo, M. et al. 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming. Nature Commun. 2, 241 (2011)

    Article  Google Scholar 

  9. 9

    Wu, S. C. & Zhang, Y. Active DNA demethylation: many roads lead to Rome. Nature Rev. Mol. Cell Biol. 11, 607–620 (2010)

    CAS  Article  Google Scholar 

  10. 10

    Jamil, A. Z., Iqbal, K., Fawad Ur, R. & Mirza, K. A. Effect of phacoemulsification on intraocular pressure. J. Coll. Physicians Surg. Pak. 21, 347–350 (2011)

    PubMed  Google Scholar 

  11. 11

    Wossidlo, M. et al. Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes. EMBO J. 29, 1877–1888 (2010)

    CAS  Article  Google Scholar 

  12. 12

    Jin, S. G., Kadam, S. & Pfeifer, G. P. Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Res. 38, e125 (2010)

    Article  Google Scholar 

  13. 13

    Huang, Y. et al. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS ONE 5, e8888 (2010)

    ADS  Article  Google Scholar 

  14. 14

    Hattori, N. et al. Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells. J. Biol. Chem. 279, 17063–17069 (2004)

    CAS  Article  Google Scholar 

  15. 15

    Imamura, M. et al. Transcriptional repression and DNA hypermethylation of a small set of ES cell marker genes in male germline stem cells. BMC Dev. Biol. 6, 34 (2006)

    Article  Google Scholar 

  16. 16

    Farthing, C. R. et al. Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes. PLoS Genet. 4, e1000116 (2008)

    Article  Google Scholar 

  17. 17

    Iqbal, K. et al. Subcutaneous panniculitis-like T-cell lymphoma in association with sarcoidosis. Clin. Exp. Dermatol. 36, 677–679 (2011)

    CAS  Article  Google Scholar 

  18. 18

    Bui, H. T. et al. The cytoplasm of mouse germinal vesicle stage oocytes can enhance somatic cell nuclear reprogramming. Development 135, 3935–3945 (2008)

    CAS  Article  Google Scholar 

  19. 19

    Yang, H. et al. High-efficiency somatic reprogramming induced by intact MII oocytes. Cell Res. 20, 1034–1042 (2010)

    Article  Google Scholar 

  20. 20

    Egli, D., Rosains, J., Birkhoff, G. & Eggan, K. Developmental reprogramming after chromosome transfer into mitotic mouse zygotes. Nature 447, 679–685 (2007)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Egli, D., Sandler, V. M., Shinohara, M. L., Cantor, H. & Eggan, K. Reprogramming after chromosome transfer into mouse blastomeres. Curr. Biol. 19, 1403–1409 (2009)

    CAS  Article  Google Scholar 

  22. 22

    Seidman, J. G. & Seidman, C. Transcription factor haploinsufficiency: when half a loaf is not enough. J. Clin. Invest. 109, 451–455 (2002)

    CAS  Article  Google Scholar 

  23. 23

    Li, L. et al. The mildiomycin biosynthesis: initial steps for sequential generation of 5-hydroxymethylcytidine 5′-monophosphate and 5-hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119. ChemBioChem 9, 1286–1294 (2008)

    CAS  Article  Google Scholar 

  24. 24

    Erlanger, B. F. & Beiser, S. M. Antibodies specific for ribonucleosides and ribonucleotides and their reaction with DNA. Proc. Natl Acad. Sci. USA 52, 68–74 (1964)

    ADS  CAS  Article  Google Scholar 

  25. 25

    de Vries, W. N. et al. Expression of Cre recombinase in mouse oocytes: a means to study maternal effect genes. Genesis 26, 110–112 (2000)

    CAS  Article  Google Scholar 

  26. 26

    Ohbo, K. et al. Identification and characterization of stem cells in prepubertal spermatogenesis in mice. Dev. Biol. 258, 209–225 (2003)

    CAS  Article  Google Scholar 

  27. 27

    Ge, Y. Z. et al. Chromatin targeting of de novo DNA methyltransferases by the PWWP domain. J. Biol. Chem. 279, 25447–25454 (2004)

    CAS  Article  Google Scholar 

  28. 28

    Liu, P., Jenkins, N. A. & Copeland, N. G. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res. 13, 476–484 (2003)

    CAS  Article  Google Scholar 

  29. 29

    Rodriguez, C. I. et al. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP . Nature Genet. 25, 139–140 (2000)

    CAS  Article  Google Scholar 

  30. 30

    Lomelí, H., Ramos-Mejia, V., Gertsenstein, M., Lobe, C. G. & Nagy, A. Targeted insertion of Cre recombinase into the TNAP gene: excision in primordial germ cells. Genesis 26, 116–117 (2000)

    Article  Google Scholar 

  31. 31

    Lewandoski, M., Wassarman, K. M. & Martin, G. R. Zp3cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line. Curr. Biol. 7, 148–151 (1997)

    CAS  Article  Google Scholar 

  32. 32

    Szabó, P. E., Hubner, K., Scholer, H. & Mann, J. R. Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech. Dev. 115, 157–160 (2002)

    Article  Google Scholar 

  33. 33

    Kimura, Y. & Yanagimachi, R. Intracytoplasmic sperm injection in the mouse. Biol. Reprod. 52, 709–720 (1995)

    CAS  Article  Google Scholar 

  34. 34

    Abràmoff, M. D., Magalhães, P. J. & Ram, S. J. Image processing with ImageJ. Biophotonics Int. 11, 36–42 (2004)

    Google Scholar 

  35. 35

    Hajkova, P. et al. DNA-methylation analysis by the bisulfite-assisted genomic sequencing method. Methods Mol. Biol. 200, 143–154 (2002)

    CAS  PubMed  Google Scholar 

  36. 36

    Weber, M. et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nature Genet. 37, 853–862 (2005)

    CAS  Article  Google Scholar 

  37. 37

    Tsumura, A. et al. Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes Cells 11, 805–814 (2006)

    CAS  Article  Google Scholar 

  38. 38

    Wakayama, T., Perry, A. C., Zuccotti, M., Johnson, K. R. & Yanagimachi, R. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–374 (1998)

    ADS  CAS  Article  Google Scholar 

Download references


We thank C. Walsh and M. Rots for critical reading of the manuscript, J. Walter for discussions, H. Qi for providing cDNA of mouse oocytes, R. Zhang & Q. Cui for Tet3 cDNA, L. Li for help with 5hmCMP synthesis, Shanghai Research Center for Model Organisms for blastocyst injection, and J. Gao for mouse work. This study was supported by grants from the Ministry of Science and Technology China (2007CB947503 to G.-L.X., 2007CB947101 to J.L., and 2009CB941101 to G.-L.X. and J.L.), National Science Foundation of China (30730059 to G.-L.X. and 30871430 to J.L.), the Chinese Academy of Sciences (XDA01010301 to G.-L.X.; XDA01010403 and KSCX2-YW-R-110 to J.L.) and the NIH (GM078458 to Y.G.S.).

Author information




G.-L.X. and J.L. conceived the projects. Y.G.S., H.-P.W. and G.-L.X. contributed to the knockout design. F.G., T.-P.G., H.-P.W., G.-F.X., and W.L. performed the experiments on early embryos. X.H. and Z.D. contributed to the synthesis of the 5hmC hapten. H.Y. and L.S. performed the nuclear transfer and embryo transfer experiments. S.-g.J., K.I., P.E.S., G.P.P. and Z.-G.X. characterized Tet3 expression in PGCs and ovaries. G.-L.X. wrote and G.P.P. revised the manuscript.

Corresponding authors

Correspondence to Jinsong Li or Guo-Liang Xu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-16 with legends, Supplementary Tables 1-6 and additional references. (PDF 10491 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gu, TP., Guo, F., Yang, H. et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature 477, 606–610 (2011).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing