Targets and dynamics of promoter DNA methylation during early mouse development

Journal name:
Nature Genetics
Volume:
42,
Pages:
1093–1100
Year published:
DOI:
doi:10.1038/ng.708
Received
Accepted
Published online

Abstract

DNA methylation is extensively reprogrammed during the early phases of mammalian development, yet genomic targets of this process are largely unknown. We optimized methylated DNA immunoprecipitation for low numbers of cells and profiled DNA methylation during early development of the mouse embryonic lineage in vivo. We observed a major epigenetic switch during implantation at the transition from the blastocyst to the postimplantation epiblast. During this period, DNA methylation is primarily targeted to repress the germline expression program. DNA methylation in the epiblast is also targeted to promoters of lineage-specific genes such as hematopoietic genes, which are subsequently demethylated during terminal differentiation. De novo methylation during early embryogenesis is catalyzed by Dnmt3b, and absence of DNA methylation leads to ectopic gene activation in the embryo. Finally, we identify nonimprinted genes that inherit promoter DNA methylation from parental gametes, suggesting that escape of post-fertilization DNA methylation reprogramming is prevalent in the mouse genome.

At a glance

Figures

  1. Profiling of DNA methylation during early mouse embryogenesis.
    Figure 1: Profiling of DNA methylation during early mouse embryogenesis.

    (a) Comparison of MeDIP-WGA on 150 ng of DNA and of pooled unamplified MeDIPs on 2 μg of the same DNA (Supplementary Fig. 1). The scatter plot compares average log2 ratios in −400 bp to +400 bp relative to all TSS. (b) We hybridized MeDIP samples from E3.5 blastocysts, E6.5 epiblasts (EPB) and total E9.5 embryos on NimbleGen HD2 arrays covering 11 kb of all mouse promoters. (c) The top graph shows the fraction of tiles with a methylated region as a function of the distance to the TSS (black arrow). For comparison, the average CpG count per kilobase along the tiles is shown (red dotted line, right axis). The bottom graph shows the fraction of tiles with a de novo methylation peak as a function of the distance to the TSS. (d) MeDIP profiles at the imprinted gene Plagl1 confirm the presence of a germline methylation mark47. The graphs show smoothed MeDIP over input ratios of individual oligonucleotides. Here and in all figures, the MeDIP profiles we obtained with unamplified pooled MeDIPs at E9.5 are also shown for validation. The gene is shown below the graphs as a gray box, and the transcription start site is shown as a gray arrow. Red bars represent the position of the CpGs. (e) The Heatmap shows the dynamics of DNA methylation at 691 genes with a methylated promoter in E9.5 embryos. Group I genes are de novo methylated in early embryos, whereas group II genes are already hypermethylated in preimplantation blastocysts.

  2. De novo CpG island methylation in epiblast cells.
    Figure 2: De novo CpG island methylation in epiblast cells.

    (a) De novo methylation of the pluripotency gene Tcl1 in epiblast (EPB) cells. Other examples of de novo methylated pluripotency genes are given in Supplementary Figure 4. The graphs show smoothed MeDIP over input ratios of individual oligonucleotides. Red bars represent the position of CpGs. (b) Examples of germline-specific genes de novo methylated during implantation in epiblast cells. More examples are given in Supplementary Figure 5. (c) Validation of promoter DNA methylation by COBRA. All five tested germline-specific genes are de novo methylated at E6.5 in the EPB and the extraembryonic ectoderm (EE). The promoter of Oct4, which has been shown to be methylated in extraembryonic lineages48 and partially de novo methylated in E9.5 embryos38, is used as a control. Here and in all figures, the number of TaqαI sites in the amplified fragment is indicated in parenthesis, and asterisks mark restriction fragments representing end products of the digestion. (d) Bisulfite sequencing in the promoters of Dazl and Sycp1 confirms de novo methylation during implantation in epiblast cells. Other validations by bisulfite sequencing are shown in Supplementary Figure 7. Circles represent CpG dinucleotides either unmethylated (open) or methylated (closed).

  3. Promoter DNA methylation at hematopoietic genes is erased during hematopoietic differentiation.
    Figure 3: Promoter DNA methylation at hematopoietic genes is erased during hematopoietic differentiation.

    (a) The hematopoietic genes Pou2af1, Tlr6 and Cytip gain promoter DNA methylation in EPB cells. The graphs show smoothed MeDIP over input ratios of individual oligonucleotides. Red bars represent the position of CpGs. (b) Validation by COBRA confirms that all three tested hematopoietic genes gain promoter DNA methylation during implantation and are hypermethylated at E6.5 in the EPB, the EE and in E9.5 embryos. All genes also show substantial promoter DNA methylation in hematopoietic stem cells (HSCs) isolated from E10.5 embryos. Subsequently in adults (Ad), Cytip and Tlr6 promoter methylation is lost in bone marrow HSCs, B cells and T cells but is maintained in other tissues such as liver. For the B-cell–specific gene Pou2af1, promoter methylation is specifically erased during differentiation of adult HSCs into B cells.

  4. Inheritance of promoter DNA methylation from oocytes at nonimprinted genes.
    Figure 4: Inheritance of promoter DNA methylation from oocytes at nonimprinted genes.

    (a) Examples of hematopoietic genes (Fyb) and germline-specific genes (Piwil1 and Csnka2ip) with high levels of promoter DNA methylation throughout early development. More examples are given in Supplementary Figure 10. The graphs show smoothed MeDIP over input ratios of individual oligonucleotides. Red bars represent the position of CpGs. (b) Validation of promoter DNA methylation by COBRA. All tested genes show hypermethylation in EPB and EE in E6.5 embryos. E3.5 blastocysts and E2.5 morulas show a consistent pattern of mixed methylated and unmethylated alleles. (c) Bisulfite sequencing in the promoter of Piwil1 in gametes and early embryos. (d) Bisulfite sequencing in the Piwil1 promoter in BL6 × JF1 E3.5 blastocysts shows that only maternal alleles carry DNA methylation. Mat, maternal alleles; pat, paternal alleles. (e) Bisulfite sequencing in the promoter of Tssk2 in gametes and early embryos. (f) Bisulfite sequencing in the Tssk2 promoter in BL6 × JF1 E3.5 blastocysts shows that only maternal alleles carry DNA methylation. Circles represent CpG dinucleotides either unmethylated (open) or methylated (closed).

  5. Promoter DNA methylation mediated by Dnmt3b maintains gene repression in vivo.
    Figure 5: Promoter DNA methylation mediated by Dnmt3b maintains gene repression in vivo.

    (a) DNA methylation in the promoter of the indicated germline-specific genes was analyzed by COBRA in wildtype (WT) and mutant E9.5 embryos heterozygous or homozygous for Dnmt3 deletions. Most tested genes showed severe reduction of promoter DNA methylation in Dnmt3b−/− embryos but were unaffected in Dnmt3a−/− embryos. Additional validations by bisulfite sequencing are shown in Supplementary Figure 12. (b) Promoter DNA methylation by COBRA in wildtype and Dnmt3 mutant E9.5 embryos at pluripotency genes (left), hematopoietic genes (middle) and eye genes (right). (c) Absence of promoter de novo methylation is associated with gene reactivation. Expression of indicated genes was measured by real-time qPCR in wildtype (WT), Dnmt3a−/− and Dnmt3b−/− E9.5 embryos. Values are arbitrary units after normalization to three housekeeping genes (Gapdh, Rpl13A and Actb). Error bars represent standard deviations from two or three independent experiments.

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References

  1. Reik, W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447, 425432 (2007).
  2. Lei, H. et al. De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development 122, 31953205 (1996).
  3. Okano, M., Bell, D.W., Haber, D.A. & Li, E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247257 (1999).
  4. Mayer, W., Niveleau, A., Walter, J., Fundele, R. & Haaf, T. Demethylation of the zygotic paternal genome. Nature 403, 501502 (2000).
  5. Oswald, J. et al. Active demethylation of the paternal genome in the mouse zygote. Curr. Biol. 10, 475478 (2000).
  6. Rougier, N. et al. Chromosome methylation patterns during mammalian preimplantation development. Genes Dev. 12, 21082113 (1998).
  7. Dean, W. et al. Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos. Proc. Natl. Acad. Sci. USA 98, 1373413738 (2001).
  8. Illingworth, R. et al. A novel CpG island set identifies tissue-specific methylation at developmental gene loci. PLoS Biol. 6, e22 (2008).
  9. Weber, M. et al. Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat. Genet. 39, 457466 (2007).
  10. Shen, L. et al. Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters. PLoS Genet. 3, 20232036 (2007).
  11. Farthing, C.R. et al. Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes. PLoS Genet. 4, e1000116 (2008).
  12. Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766770 (2008).
  13. Mohn, F. et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 30, 755766 (2008).
  14. Bhutani, N. et al. Reprogramming towards pluripotency requires AID-dependent DNA demethylation. Nature 463, 10421047 (2009).
  15. Mikkelsen, T.S. et al. Dissecting direct reprogramming through integrative genomic analysis. Nature 454, 4955 (2008).
  16. Oda, M. et al. DNA methylation regulates long-range gene silencing of an X-linked homeobox gene cluster in a lineage-specific manner. Genes Dev. 20, 33823394 (2006).
  17. Brunner, A.L. et al. Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver. Genome Res. 19, 10441056 (2009).
  18. Jirtle, R.L. & Skinner, M.K. Environmental epigenomics and disease susceptibility. Nat. Rev. Genet. 8, 253262 (2007).
  19. Lane, N. et al. Resistance of IAPs to methylation reprogramming may provide a mechanism for epigenetic inheritance in the mouse. Genesis 35, 8893 (2003).
  20. Hammoud, S.S. et al. Distinctive chromatin in human sperm packages genes for embryo development. Nature 460, 473478 (2009).
  21. Puschendorf, M. et al. PRC1 and Suv39h specify parental asymmetry at constitutive heterochromatin in early mouse embryos. Nat. Genet. 40, 411420 (2008).
  22. Lister, R. et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315322 (2009).
  23. Popp, C. et al. Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency. Nature 463, 11011105 (2010).
  24. Weber, M. et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat. Genet. 37, 853862 (2005).
  25. Ball, M.P. et al. Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells. Nat. Biotechnol. 27, 361368 (2009).
  26. Laurent, L. et al. Dynamic changes in the human methylome during differentiation. Genome Res. 20, 320331 (2010).
  27. Ng, R.K. et al. Epigenetic restriction of embryonic cell lineage fate by methylation of Elf5. Nat. Cell Biol. 10, 12801290 (2008).
  28. Kim, M.S. et al. DNA demethylation in hormone-induced transcriptional derepression. Nature 461, 10071012 (2009).
  29. Métivier, R. et al. Cyclical DNA methylation of a transcriptionally active promoter. Nature 452, 4550 (2008).
  30. Kato, Y. et al. Role of the Dnmt3 family in de novo methylation of imprinted and repetitive sequences during male germ cell development in the mouse. Hum. Mol. Genet. 16, 22722280 (2007).
  31. Watanabe, D., Suetake, I., Tada, T. & Tajima, S. Stage- and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis. Mech. Dev. 118, 187190 (2002).
  32. Nichols, J., Silva, J., Roode, M. & Smith, A. Suppression of Erk signaling promotes ground state pluripotency in the mouse embryo. Development 136, 32153222 (2009).
  33. Brons, I.G. et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191195 (2007).
  34. Tesar, P.J. et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196199 (2007).
  35. Bao, S. et al. Epigenetic reversion of post-implantation epiblast to pluripotent embryonic stem cells. Nature 461, 12921295 (2009).
  36. Dahl, J.A., Reiner, A.H., Klungland, A., Wakayama, T. & Collas, P. Histone H3 lysine 27 methylation asymmetry on developmentally-regulated promoters distinguish the first two lineages in mouse preimplantation embryos. PLoS ONE 5, e9150 (2010).
  37. Hayashi, K., Lopes, S.M., Tang, F. & Surani, M.A. Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell Stem Cell 3, 391401 (2008).
  38. Li, J.Y. et al. Synergistic function of DNA methyltransferases Dnmt3a and Dnmt3b in the methylation of Oct4 and Nanog. Mol. Cell. Biol. 27, 87488759 (2007).
  39. Maatouk, D.M. et al. DNA methylation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell lineages. Development 133, 34113418 (2006).
  40. Straussman, R. et al. Developmental programming of CpG island methylation profiles in the human genome. Nat. Struct. Mol. Biol. 16, 564571 (2009).
  41. Simpson, A.J., Caballero, O.L., Jungbluth, A., Chen, Y.T. & Old, L.J. Cancer/testis antigens, gametogenesis and cancer. Nat. Rev. Cancer 5, 615625 (2005).
  42. Waterland, R.A. et al. Epigenomic profiling indicates a role for DNA methylation in early postnatal liver development. Hum. Mol. Genet. 18, 30263038 (2009).
  43. Boyer, L.A. et al. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441, 349353 (2006).
  44. Lee, T.I. et al. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125, 301313 (2006).
  45. 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, 16071616 (2008).
  46. Skinner, M.K. & Guerrero-Bosagna, C. Environmental signals and transgenerational epigenetics. Epigenomics 1, 111117 (2009).
  47. Smith, R.J. et al. The mouse Zac1 locus: basis for imprinting and comparison with human ZAC. Gene 292, 101112 (2002).
  48. Hattori, N. et al. Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells. J. Biol. Chem. 279, 1706317069 (2004).
  49. Huang da, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 4457 (2009).
  50. Smyth, G.K. & Speed, T. Normalization of cDNA microarray data. Methods 31, 265273 (2003).

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Author information

Affiliations

  1. Institute of Molecular Genetics, Centre National de la Recherche Scientifique (CNRS) UMR 5535, Université Montpellier 2, Université Montpellier 1, Montpellier, France.

    • Julie Borgel,
    • Sylvain Guibert,
    • Thierry Forné &
    • Michael Weber
  2. Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan.

    • Yufeng Li,
    • Hatsune Chiba &
    • Hiroyuki Sasaki
  3. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

    • Dirk Schübeler

Contributions

J.B. performed all experiments and data analysis and contributed to the writing of the manuscript. S.G. developed R scripts and participated in data analysis. Y.L., H.C. and H.S. prepared samples from Dnmt mutant embryos. D.S. and T.F. participated in the study design and writing of the manuscript. M.W. designed and supervised the study, participated in data analysis and wrote the manuscript.

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The authors declare no competing financial interests.

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Supplementary information

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  1. Supplementary Text and Figures (4M)

    Supplementary Figures 1–13 and Supplementary Table 2

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  1. Supplementary Table 1 (72K)

    Genes with methylated promoters identified in early mouse embryos.

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