A unique regulatory phase of DNA methylation in the early mammalian embryo


DNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methylcytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and so far no base-resolution maps exist to support and refine it. Here we generate genome-scale DNA methylation maps in mouse gametes and from the zygote through post-implantation. We find that the oocyte already exhibits global hypomethylation, particularly at specific families of long interspersed element 1 and long terminal repeat retroelements, which are disparately methylated between gametes and have lower methylation values in the zygote than in sperm. Surprisingly, the oocyte contributes a unique set of differentially methylated regions (DMRs)—including many CpG island promoters—that are maintained in the early embryo but are lost upon specification and absent from somatic cells. In contrast, sperm-contributed DMRs are largely intergenic and become hypermethylated after the blastocyst stage. Our data provide a genome-scale, base-resolution timeline of DNA methylation in the pre-specified embryo, when this epigenetic modification is most dynamic, before returning to the canonical somatic pattern.

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Figure 1: Global CpG methylation dynamics across early murine embryogenesis.
Figure 2: Major transitions in DNA methylation levels during early development.
Figure 3: Specific families of LINE and LTR retroelements exhibit the most dramatic methylation changes in the sperm to zygote transition.
Figure 4: Differentially methylated regions represent discrete gamete-specific feature classes.
Figure 5: DMRs resolve after cleavage to univalent hyper- or hypomethylated values in a gamete-of-origin-specific fashion.

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Primary accessions

Gene Expression Omnibus

Data deposits

RRBS data is deposited at the Gene Expression Omnibus under accession number GSE34864.


  1. 1

    Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21 (2002)

    CAS  Article  Google Scholar 

  2. 2

    Jaenisch, R. & Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nature Genet. 33 (Suppl.). 245–254 (2003)

    CAS  Article  Google Scholar 

  3. 3

    Suzuki, M. M. & Bird, A. DNA methylation landscapes: provocative insights from epigenomics. Nature Rev. Genet. 9, 465–476 (2008)

    CAS  Article  Google Scholar 

  4. 4

    Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–770 (2008)

    ADS  CAS  Article  Google Scholar 

  5. 5

    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 

  6. 6

    Weber, M. et al. Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nature Genet. 39, 457–466 (2007)

    CAS  Article  Google Scholar 

  7. 7

    Ji, H. et al. Comprehensive methylome map of lineage commitment from haematopoietic progenitors. Nature 467, 338–342 (2010)

    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

    Gu, T. P. et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature 477, 606–610 (2011)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Inoue, A. & Zhang, Y. Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos. Science 334, 194 (2011)

    ADS  CAS  Article  Google Scholar 

  11. 11

    Reik, W., Dean, W. & Walter, J. Epigenetic reprogramming in mammalian development. Science 293, 1089–1093 (2001)

    CAS  Article  Google Scholar 

  12. 12

    Kafri, T. et al. Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes Dev. 6, 705–714 (1992)

    CAS  Article  Google Scholar 

  13. 13

    Borgel, J. et al. Targets and dynamics of promoter DNA methylation during early mouse development. Nature Genet. 42, 1093–1100 (2010)

    CAS  Article  Google Scholar 

  14. 14

    Meissner, A. Epigenetic modifications in pluripotent and differentiated cells. Nature Biotechnol. 28, 1079–1088 (2010)

    CAS  Article  Google Scholar 

  15. 15

    Razin, A. & Shemer, R. DNA methylation in early development. Hum. Mol. Genet. 4, 1751–1755 (1995)

    CAS  Article  Google Scholar 

  16. 16

    Monk, M., Boubelik, M. & Lehnert, S. Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99, 371–382 (1987)

    CAS  PubMed  Google Scholar 

  17. 17

    Rougier, N. et al. Chromosome methylation patterns during mammalian preimplantation development. Genes Dev. 12, 2108–2113 (1998)

    CAS  Article  Google Scholar 

  18. 18

    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 

  19. 19

    Lane, N. et al. Resistance of IAPs to methylation reprogramming may provide a mechanism for epigenetic inheritance in the mouse. Genesis 35, 88–93 (2003)

    CAS  Article  Google Scholar 

  20. 20

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

    CAS  Article  Google Scholar 

  21. 21

    Santos, F., Hendrich, B., Reik, W. & Dean, W. Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev. Biol. 241, 172–182 (2002)

    CAS  Article  Google Scholar 

  22. 22

    Kim, S. H. et al. Differential DNA methylation reprogramming of various repetitive sequences in mouse preimplantation embryos. Biochem. Biophys. Res. Commun. 324, 58–63 (2004)

    CAS  Article  Google Scholar 

  23. 23

    Bock, C. et al. Quantitative comparison of genome-wide DNA methylation mapping technologies. Nature Biotechnol. 28, 1106–1114 (2010)

    CAS  Article  Google Scholar 

  24. 24

    Harris, R. A. et al. Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications. Nature Biotechnol. 28, 1097–1105 (2010)

    CAS  Article  Google Scholar 

  25. 25

    Smallwood, S. A. et al. Dynamic CpG island methylation landscape in oocytes and preimplantation embryos. Nature Genet. 43, 811–814 (2011)

    CAS  Article  Google Scholar 

  26. 26

    Davis, T. & Vaisvila, R. High sensitivity 5-hydroxymethylcytosine detection in Balb/C brain tissue. J. Vis. Exp. 48, (2011)

  27. 27

    Ficz, G. et al. Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473, 398–402 (2011)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Szulwach, K. E. et al. Integrating 5-hydroxymethylcytosine into the epigenomic landscape of human embryonic stem cells. PLoS Genet. 7, e1002154 (2011)

    CAS  Article  Google Scholar 

  29. 29

    Williams, K. et al. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 473, 343–348 (2011)

    ADS  CAS  Article  Google Scholar 

  30. 30

    Wu, H. et al. Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. Genes Dev. 25, 679–684 (2011)

    CAS  Article  Google Scholar 

  31. 31

    Xu, Y. et al. Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells. Mol. Cell 42, 451–464 (2011)

    CAS  Article  Google Scholar 

  32. 32

    Branco, M. R., Ficz, G. & Reik, W. Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nature Rev. Genet. 13, 7–13 (2011)

    Article  Google Scholar 

  33. 33

    Santos, F., Hendrich, B., Reik, W. & Dean, W. Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev. Biol. 241, 172–182 (2002)

    CAS  Article  Google Scholar 

  34. 34

    Hajkova, P. et al. Genome-wide reprogramming in the mouse germ line entails the base excision repair pathway. Science 329, 78–82 (2010)

    ADS  CAS  Article  Google Scholar 

  35. 35

    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 

  36. 36

    Popp, C. et al. Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency. Nature 463, 1101–1105 (2010)

    ADS  CAS  Article  Google Scholar 

  37. 37

    Mouse Genome Sequencing Consortium Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562 (2002)

    Article  Google Scholar 

  38. 38

    Ichiyanagi, K. et al. Locus- and domain-dependent control of DNA methylation at mouse B1 retrotransposons during male germ cell development. Genome Res. 21, 2058–2066 (2011)

    CAS  Article  Google Scholar 

  39. 39

    Goodier, J. L., Ostertag, E. M., Du, K. & Kazazian, H. H., Jr A novel active L1 retrotransposon subfamily in the mouse. Genome Res. 11, 1677–1685 (2001)

    CAS  Article  Google Scholar 

  40. 40

    Edwards, C. A. & Ferguson-Smith, A. Mechanisms regulating imprinted genes in clusters. Curr. Opin. Cell Biol. 19, 281–289 (2007)

    CAS  Article  Google Scholar 

  41. 41

    Bestor, T. H. The DNA methyltransferases of mammals. Hum. Mol. Genet. 9, 2395–2402 (2000)

    CAS  Article  Google Scholar 

  42. 42

    Hirasawa, R. et al. Maternal and zygotic Dnmt1 are necessary and sufficient for the maintenance of DNA methylation imprints during preimplantation development. Genes Dev. 22, 1607–1616 (2008)

    CAS  Article  Google Scholar 

  43. 43

    Lucifero, D. et al. Coordinate regulation of DNA methyltransferase expression during oogenesis. BMC Dev. Biol. 7, 36 (2007)

    Article  Google Scholar 

  44. 44

    Ramsahoye, B. H. et al. Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc. Natl Acad. Sci. USA 97, 5237–5242 (2000)

    ADS  CAS  Article  Google Scholar 

  45. 45

    Ziller, M. J. et al. Genomic distribution and inter-sample variation of non-CpG methylation across human cell types. PLoS Genet. 7, e1002389 (2011)

    CAS  Article  Google Scholar 

  46. 46

    Haines, T., Rodenhiser, D. & Ainsworth, P. Allele-specific non-CpG methylation of the Nf1 gene during early mouse development. Dev. Biol. 240, 585–598 (2001)

    CAS  Article  Google Scholar 

  47. 47

    Tomizawa, S. et al. Dynamic stage-specific changes in imprinted differentially methylated regions during early mammalian development and prevalence of non-CpG methylation in oocytes. Development 138, 811–820 (2011)

    CAS  Article  Google Scholar 

  48. 48

    Beraldi, R., Pittoggi, C., Sciamanna, I., Mattei, E. & Spadafora, C. Expression of LINE-1 retroposons is essential for murine preimplantation development. Mol. Reprod. Dev. 73, 279–287 (2006)

    CAS  Article  Google Scholar 

  49. 49

    Kigami, D., Minami, N., Takayama, H. & Imai, H. MuERV-L is one of the earliest transcribed genes in mouse one-cell embryos. Biol. Reprod. 68, 651–654 (2003)

    CAS  Article  Google Scholar 

  50. 50

    Okada, Y., Yamagata, K., Hong, K., Wakayama, T. & Zhang, Y. A role for the elongator complex in zygotic paternal genome demethylation. Nature 463, 554–558 (2010)

    ADS  CAS  Article  Google Scholar 

  51. 51

    Nagy, A. Manipulating the Mouse Embryo: a Laboratory Manual 3rd edn (Cold Spring Harbor Laboratory Press, 2003)

    Google Scholar 

  52. 52

    Smith, Z. D., Gu, H., Bock, C., Gnirke, A. & Meissner, A. High-throughput bisulfite sequencing in mammalian genomes. Methods 48, 226–232 (2009)

    CAS  Article  Google Scholar 

  53. 53

    Gu, H. et al. Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling. Nature Protocols 6, 468–481 (2011)

    CAS  Article  Google Scholar 

  54. 54

    Gu, H. et al. Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution. Nature Methods 7, 133–136 (2010)

    CAS  Article  Google Scholar 

  55. 55

    Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300 (1995)

    MathSciNet  MATH  Google Scholar 

  56. 56

    Blake, J. A., Bult, C. J., Kadin, J. A., Richardson, J. E. & Eppig, J. T. The Mouse Genome Database (MGD): premier model organism resource for mammalian genomics and genetics. Nucleic Acids Res. 39, D842–D848 (2011)

    CAS  Article  Google Scholar 

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We would like to thank all members of the Meissner and Regev laboratories. M. Garber, N. Yosef, J. Ye, R. Koche, C. Bock, R. Maehr and D. Egli for technical advice and discussion. We thank all members of the Broad Sequencing Platform, in particular F. Kelly and J. Meldrim, T. Fennel, K. Tibbetts and J. Fostel. We also thank S. Levine, M. Gravina and K. Thai from the MIT BioMicro Center. A.R. is an investigator of the Merkin Foundation for Stem Cell Research at the Broad Institute. This work was supported by the NIH Pioneer Award (5DP1OD003958), the Burroughs Wellcome Career Award at the Scientific Interface and HHMI (to A.R.), the Harvard Stem Cell Institute (to T.S.M.) and the NIH (5RC1AA019317, U01ES017155 and P01GM099117), the Massachusetts Life Science Center and the Pew Charitable Trusts (to A.M.) and a Center for Excellence in Genome Science from the NHGRI (1P50HG006193-01, to A.R. and A.M.).

Author information




Z.D.S. and A.M. conceived the study and Z.D.S., M.M.C. and A.M. facilitated its design. Z.D.S. collected samples and performed methylation profiling, M.M.C. performed all analysis with assistance from T.S.M. and Z.D.S. H.G. and A.G. provided critical technical assistance and expertise. Z.D.S., M.M.C., T.S.M., A.R. and A.M. interpreted the data. Z.D.S., M.M.C. and A.M. wrote the paper with the assistance of the other authors.

Corresponding author

Correspondence to Alexander Meissner.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-11 and full legends for Supplementary Tables 1- 4 and Supplementary Movie 1. (PDF 1636 kb)

Supplementary Table 1

This file contains promoter methylation levels at fertilization and across early embryonic development - see legend in Supplementary Information file. (XLS 3760 kb)

Supplementary Table 2

This file contains Long Interspersed Element (LINE) retrotransposon feature methylation across early embryonic development - see legend in Supplementary Information file. (XLS 28 kb)

Supplementary Table 3

This file contains Long Terminal Repeat (LTR) retrotransposon feature methylation across early embryonic development - see legend in Supplementary Information file. (XLS 42 kb)

Supplementary Table 4

This file contains feature designation and methylation status for identified oocyte-contributed Differentially Methylated Regions (DMRs) across early embryonic development - see legend in Supplementary Information file. (XLS 149 kb)

Supplementary Movie 1

This file contains a movie of a representative polar body biopsy for zygotes and cleavage stage embryos collected in this study - see legend in Supplementary Information file. (MOV 7166 kb)

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Smith, Z., Chan, M., Mikkelsen, T. et al. A unique regulatory phase of DNA methylation in the early mammalian embryo. Nature 484, 339–344 (2012). https://doi.org/10.1038/nature10960

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