Letter | Published:

Structural basis for DNMT3A-mediated de novo DNA methylation

Nature volume 554, pages 387391 (15 February 2018) | Download Citation

Abstract

DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) at cytosines is essential for genome regulation and development1,2. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-ångström crystal structure of the DNMT3A–DNMT3L–DNA complex in which two DNMT3A monomers simultaneously attack two cytosine–phosphate–guanine (CpG) dinucleotides, with the target sites separated by 14 base pairs within the same DNA duplex. The DNMT3A–DNA interaction involves a target recognition domain, a catalytic loop, and DNMT3A homodimeric interface. Arg836 of the target recognition domain makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Haematological cancer-associated somatic mutations of the substrate-binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of haematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its aetiological link to human disease.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Gene Expression Omnibus

References

  1. 1.

    , , & DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–257 (1999)

  2. 2.

    , & Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat. Genet. 19, 219–220 (1998)

  3. 3.

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

  4. 4.

    & Eukaryotic cytosine methyltransferases. Annu. Rev. Biochem. 74, 481–514 (2005)

  5. 5.

    , , , & Dnmt3L and the establishment of maternal genomic imprints. Science 294, 2536–2539 (2001)

  6. 6.

    , & The DNA methyltransferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a. Proc. Natl Acad. Sci. USA 99, 16916–16921 (2002)

  7. 7.

    , , & Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice. Development 129, 1983–1993 (2002)

  8. 8.

    DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005)

  9. 9.

    , & DNMT3A in haematological malignancies. Nat. Rev. Cancer 15, 152–165 (2015)

  10. 10.

    et al. DNMT3A mutations in acute myeloid leukemia. N. Engl. J. Med. 363, 2424–2433 (2010)

  11. 11.

    et al. Structural insight into autoinhibition and histone H3-induced activation of DNMT3A. Nature 517, 640–644 (2015)

  12. 12.

    , , , & Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. Nature 449, 248–251 (2007)

  13. 13.

    et al. Formation of nucleoprotein filaments by mammalian DNA methyltransferase Dnmt3a in complex with regulator Dnmt3L. Nucleic Acids Res. 36, 6656–6663 (2008)

  14. 14.

    & Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpA sites. J. Mol. Biol. 309, 1201–1208 (2001)

  15. 15.

    et al. Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. J. Mol. Biol. 357, 928–941 (2006)

  16. 16.

    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)

  17. 17.

    et al. Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain. Nat. Neurosci. 17, 215–222 (2014)

  18. 18.

    et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322 (2009)

  19. 19.

    , & The PWWP domain of Dnmt3a and Dnmt3b is required for directing DNA methylation to the major satellite repeats at pericentric heterochromatin. Mol. Cell. Biol. 24, 9048–9058 (2004)

  20. 20.

    et al. COSMIC: exploring the world’s knowledge of somatic mutations in human cancer. Nucleic Acids Res. 43, D805–D811 (2015)

  21. 21.

    et al. Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability. Nat. Genet. 46, 385–388 (2014)

  22. 22.

    , & Mutations in DNA methyltransferase (DNMT3A) observed in acute myeloid leukemia patients disrupt processive methylation. J. Biol. Chem. 287, 30941–30951 (2012)

  23. 23.

    et al. A DNMT3A mutation common in AML exhibits dominant-negative effects in murine ES cells. Blood 122, 4086–4089 (2013)

  24. 24.

    et al. Epigenetic perturbations by Arg882-mutated DNMT3A potentiate aberrant stem cell gene-expression program and acute leukemia development. Cancer Cell 30, 92–107 (2016)

  25. 25.

    et al. The R882H DNMT3A mutation associated with AML dominantly inhibits wild-type DNMT3A by blocking its ability to form active tetramers. Cancer Cell 25, 442–454 (2014)

  26. 26.

    et al. (R)-2-hydroxyglutarate is sufficient to promote leukemogenesis and its effects are reversible. Science 339, 1621–1625 (2013)

  27. 27.

    , , & Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331, 1036–1040 (2011)

  28. 28.

    et al. Zebularine: a novel DNA methylation inhibitor that forms a covalent complex with DNA methyltransferases. J. Mol. Biol. 321, 591–599 (2002)

  29. 29.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  30. 30.

    Xds. Acta Crystallogr. D 66, 125–132 (2010)

  31. 31.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

  32. 32.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

  33. 33.

    . et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

  34. 34.

    et al. A resource for cell line authentication, annotation and quality control. Nature 520, 307–311 (2015)

  35. 35.

    et al. Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nat. Methods 3, 287–293 (2006)

  36. 36.

    et al. Tris(1,3-dichloro-2-propyl)phosphate induces genome-wide hypomethylation within early zebrafish embryos. Environ. Sci. Technol. 50, 10255–10263 (2016)

  37. 37.

    et al. Comprehensive assessment of oxidatively induced modifications of DNA in a rat model of human Wilson’s disease. Mol. Cell. Proteomics 15, 810–817 (2016)

  38. 38.

    & Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27, 1571–1572 (2011)

  39. 39.

    et al. Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays. Bioinformatics 30, 1363–1369 (2014)

Download references

Acknowledgements

We thank X. Cheng for comments on the manuscript, M. Okano, J. Wang, and J.-A. Losman for providing reagents used in the study, and staff members at the Advanced Light Source, Lawrence Berkeley National Laboratory, and at the Advanced Photo Source, Argonne National Laboratory, for access to X-ray beamlines. We are also grateful for the support of University of North Carolina facilities including Genomics Core, which are partly supported by UNC Cancer Center Core Support Grant P30-CA016086. This work was supported by Kimmel Scholar Awards (to J.S. and G.G.W.), the March of Dimes Foundation (1-FY15-345 to J.S.), the DoD Peer-reviewed Cancer Research Program (W81XWH-14-1-0232 to G.G.W.), Gabrielle’s Angel Foundation for Cancer Research (to G.G.W.), Gilead Sciences Research Scholars Program in haematology/oncology (to G.G.W.), University Cancer Research Fund of the N.C. state (to G.G.W.), and the National Institutes of Health (1R35GM119721 to J.S.; R35GM124736 to S.B.R.; 5R21ES025392 to Y.W.; and 1R01CA215284, 1R01CA218600, and 1R01CA211336 to G.G.W.). G.G.W. is a Research Scholar of American Cancer Society and a Junior Faculty Scholar of American Society of Haematology. R.L. was supported by a Lymphoma Research Foundation postdoctoral fellowship.

Author information

Author notes

    • Zhi-Min Zhang

    Present address: School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China.

    • Zhi-Min Zhang
    •  & Rui Lu

    These authors contributed equally to this work.

    • Gang Greg Wang
    •  & Jikui Song

    These authors jointly supervised this work.

Affiliations

  1. Department of Biochemistry, University of California, Riverside, California 92521, USA

    • Zhi-Min Zhang
    •  & Jikui Song
  2. The Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599, USA

    • Rui Lu
    • , Dongliang Chen
    •  & Gang Greg Wang
  3. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599, USA

    • Rui Lu
    • , Dongliang Chen
    • , Scott B Rothbart
    •  & Gang Greg Wang
  4. Environmental Toxicology Graduate Program, University of California, Riverside, California 92521, USA

    • Pengcheng Wang
    • , Yang Yu
    • , Linfeng Gao
    • , Shuo Liu
    • , Yinsheng Wang
    •  & Jikui Song
  5. Department of Chemistry, University of California, Riverside, California 92521, USA

    • Debin Ji
    •  & Yinsheng Wang
  6. Center for Epigenetics, Van Andel Research Institute, Grand Rapids, Michigan 49503, USA

    • Scott B Rothbart

Authors

  1. Search for Zhi-Min Zhang in:

  2. Search for Rui Lu in:

  3. Search for Pengcheng Wang in:

  4. Search for Yang Yu in:

  5. Search for Dongliang Chen in:

  6. Search for Linfeng Gao in:

  7. Search for Shuo Liu in:

  8. Search for Debin Ji in:

  9. Search for Scott B Rothbart in:

  10. Search for Yinsheng Wang in:

  11. Search for Gang Greg Wang in:

  12. Search for Jikui Song in:

Contributions

Z.-M.Z., R.L., P.W., Y.Y., D.C., L.G., S.L., D.J., and J.S. performed experiments. S.B.R. provided technical support. Y.W., G.G.W., and J.S. conceived and organized the study. Z.-M.Z., R.L., G.G.W., and J.S. prepared the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Gang Greg Wang or Jikui Song.

Reviewer Information Nature thanks A. Jeltsch, R. Xu and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

  2. 2.

    Supplementary Figure 1

    This file contains the uncropped western blots from the main and supplementary figures.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature25477

Further reading

Comments

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.