Letter | Published:

Structural insight into autoinhibition and histone H3-induced activation of DNMT3A

Nature volume 517, pages 640644 (29 January 2015) | Download Citation


DNA methylation is an important epigenetic modification that is essential for various developmental processes through regulating gene expression, genomic imprinting, and epigenetic inheritance1,2,3,4,5. Mammalian genomic DNA methylation is established during embryogenesis by de novo DNA methyltransferases, DNMT3A and DNMT3B6,7,8, and the methylation patterns vary with developmental stages and cell types9,10,11,12. DNA methyltransferase 3-like protein (DNMT3L) is a catalytically inactive paralogue of DNMT3 enzymes, which stimulates the enzymatic activity of Dnmt3a13. Recent studies have established a connection between DNA methylation and histone modifications, and revealed a histone-guided mechanism for the establishment of DNA methylation14. The ATRX–DNMT3–DNMT3L (ADD) domain of Dnmt3a recognizes unmethylated histone H3 (H3K4me0)15,16,17. The histone H3 tail stimulates the enzymatic activity of Dnmt3a in vitro17,18, whereas the molecular mechanism remains elusive. Here we show that DNMT3A exists in an autoinhibitory form and that the histone H3 tail stimulates its activity in a DNMT3L-independent manner. We determine the crystal structures of DNMT3A–DNMT3L (autoinhibitory form) and DNMT3A–DNMT3L-H3 (active form) complexes at 3.82 and 2.90 Å resolution, respectively. Structural and biochemical analyses indicate that the ADD domain of DNMT3A interacts with and inhibits enzymatic activity of the catalytic domain (CD) through blocking its DNA-binding affinity. Histone H3 (but not H3K4me3) disrupts ADD–CD interaction, induces a large movement of the ADD domain, and thus releases the autoinhibition of DNMT3A. The finding adds another layer of regulation of DNA methylation to ensure that the enzyme is mainly activated at proper targeting loci when unmethylated H3K4 is present, and strongly supports a negative correlation between H3K4me3 and DNA methylation across the mammalian genome9,10,19,20. Our study provides a new insight into an unexpected autoinhibition and histone H3-induced activation of the de novo DNA methyltransferase after its initial genomic positioning.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions

Data deposits

The coordinates and structure factors for the ADD–CD–CDNMT3L and ADD–CD–CDNMT3L-H3 structures have been deposited in the Protein Data Bank under accession numbers 4U7P and 4U7T, respectively.


  1. 1.

    & Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nature Genet. 33 (suppl.). 245–254 (2003)

  2. 2.

    & DNA methylation: roles in mammalian development. Nature Rev. Genet. 14, 204–220 (2013)

  3. 3.

    & Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nature Rev. Genet. 11, 204–220 (2010)

  4. 4.

    , & Dynamic regulation of DNA methylation during mammalian development. Epigenomics 1, 81–98 (2009)

  5. 5.

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

  6. 6.

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

  7. 7.

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

  8. 8.

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

  9. 9.

    et al. Dynamic changes in the human methylome during differentiation. Genome Res. 20, 320–331 (2010)

  10. 10.

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

  11. 11.

    & Variable patterns of total DNA and rDNA methylation in animals. Nucleic Acids Res. 8, 1485–1497 (1980)

  12. 12.

    et al. Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells. Nucleic Acids Res. 10, 2709–2721 (1982)

  13. 13.

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

  14. 14.

    & Linking DNA methylation and histone modification: patterns and paradigms. Nature Rev. Genet. 10, 295–304 (2009)

  15. 15.

    et al. DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature 448, 714–717 (2007)

  16. 16.

    et al. Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX–DNMT3–DNMT3L domain. EMBO Rep. 10, 1235–1241 (2009)

  17. 17.

    et al. Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail. Nucleic Acids Res. 38, 4246–4253 (2010)

  18. 18.

    et al. Histone tails regulate DNA methylation by allosterically activating de novo methyltransferase. Cell Res. 21, 1172–1181 (2011)

  19. 19.

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

  20. 20.

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

  21. 21.

    , , & A novel Dnmt3a isoform produced from an alternative promoter localizes to euchromatin and its expression correlates with active de novo methylation. J. Biol. Chem. 277, 38746–38754 (2002)

  22. 22.

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

  23. 23.

    , , & HhaI methyltransferase flips its target base out of the DNA helix. Cell 76, 357–369 (1994)

  24. 24.

    , , & Function and disruption of DNA methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation. Nucleic Acids Res. 40, 569–580 (2012)

  25. 25.

    et al. Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded 15N/19F-labeled unnatural amino acid. Biochem. Biophys. Res. Commun. 402, 461–466 (2010)

  26. 26.

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

  27. 27.

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

  28. 28.

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

  29. 29.

    & MOLREP: an automated program for molecular replacement. J. Appl. Cryst. 30, 1022–1025 (1997)

  30. 30.

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

  31. 31.

    , , & PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993)

  32. 32.

    The PyMOL Molecular Graphics System. (2002)

  33. 33.

    & Biotin-avidin microplate assay for the quantitative analysis of enzymatic methylation of DNA by DNA methyltransferases. Biol. Chem. 381, 269–272 (2000)

  34. 34.

    , & Expression and purification of recombinant histones and nucleosome reconstitution. Methods Mol. Biol. 119, 1–16 (1999)

  35. 35.

    et al. The site-specific installation of methyl-lysine analogs into recombinant histones. Cell 128, 1003–1012 (2007)

  36. 36.

    et al. Site-specific (1)(9)F NMR chemical shift and side chain relaxation analysis of a membrane protein labeled with an unnatural amino acid. Protein Sci. 20, 224–228 (2011)

  37. 37.

    et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007)

  38. 38.

    , & EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999)

  39. 39.

    , , , & A 11.5 Å single particle reconstruction of GroEL using EMAN. JMB (2001)

  40. 40.

    , , , & A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996)

  41. 41.

    & Single particle analysis at high resolution. Methods Enzymol. 482, 211–235 (2010)

  42. 42.

    et al. 4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement. Proc. Natl Acad. Sci. USA 107, 4967–4972 (2010)

  43. 43.

    et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

Download references


We thank staff of beamline BL17U at Shanghai Synchrotron Radiation Facility, China, for their assistance in data collection, and H. Wang for help on electron microscopy analyses. We thank staff of the Biomedical Core Facility, Fudan University, for their help on biochemical analyses, and A. D. Riggs for providing the complementary DNAs of DNMT3A and DNMT3L. This work was supported by grants from the National Basic Research Program of China (2011CB965300, 2009CB918600, 2013CB910401), the National Science & Technology Major Project ‘Key New Drug Creation and Manufacturing Program’ of China (2014ZX09507-002, 2011ZX09506-001), the National Natural Science Foundation of China (31270779, 91419301, 31030019, U1432242, 31270771, 31222016, 31300685, U1332138), the Basic Research Project of Shanghai Science and Technology Commission (12JC1402700, 13JC1406300), the Fok Ying Tung Education Foundation (20090071220012), and the Chinese Academy of Sciences Pilot Strategic Science and Technology Projects B (numbers XDB08030201, XDB08030302). Y.C. is a scholar of the Hundred Talents Program of the Chinese Academy of Sciences.

Author information

Author notes

    • Xue Guo
    •  & Ling Wang

    These authors contributed equally to this work.


  1. Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China

    • Xue Guo
    • , Ling Wang
    • , Jie Li
    • , Jianxiong Xiao
    • , Xiaotong Yin
    • , Shuang He
    •  & Yanhui Xu
  2. State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China

    • Xue Guo
    • , Ling Wang
    •  & Yanhui Xu
  3. National Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

    • Zhanyu Ding
    •  & Yao Cong
  4. High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei 230031, China

    • Pan Shi
    •  & Changlin Tian
  5. National Laboratory for Physical Science at the Microscale, University of Science and Technology of China, Hefei 230026, China

    • Pan Shi
    •  & Changlin Tian
  6. School of Life Sciences, University of Science and Technology of China, Hefei 230026, China

    • Pan Shi
    •  & Changlin Tian
  7. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100101, China

    • Liping Dong
    •  & Guohong Li
  8. University of Chinese Academy of Science, Beijing 100049, China

    • Liping Dong
  9. State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084, China

    • Jiawei Wang


  1. Search for Xue Guo in:

  2. Search for Ling Wang in:

  3. Search for Jie Li in:

  4. Search for Zhanyu Ding in:

  5. Search for Jianxiong Xiao in:

  6. Search for Xiaotong Yin in:

  7. Search for Shuang He in:

  8. Search for Pan Shi in:

  9. Search for Liping Dong in:

  10. Search for Guohong Li in:

  11. Search for Changlin Tian in:

  12. Search for Jiawei Wang in:

  13. Search for Yao Cong in:

  14. Search for Yanhui Xu in:


X.G., L.W., and Y.X. designed the experiments. X.G., L.W., and X.Y. performed protein purification and crystallization of ADD–CD–CDNMT3L-H3 complex; X.G. collected the data and determined the crystal structure. L.W., J.X., and S.H. performed protein purification, crystallization, and data collection of ADD–CD–CDNMT3L. J.L. and J.W. determined the crystal structure. X.G. and L.W. performed enzymatic assays, fluorescence polarization and pull-down assays. L.D. and G.L. prepared nucleosomes for assays. C.T. and P.S. performed and analysed the 19F NMR measurements. Z.D. and Y.C. performed and analysed the electron microscopy measurements. X.G., L.W., and Y.X. analysed the data and wrote the manuscript. Y.X. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Yanhui Xu.

Extended data

Supplementary information


  1. 1.

    Conformational changes of DNMT3A induced by histone H3 tail

    This video illustrates the conformational changes of DNMT3A induced by histone H3 tail. DNMT3A exists in an autoinhibitory form, in which the ADD domain (green) binds to and inhibits the DNA-binding affinity of the CD domain (purple). Histone H3 (yellow) disrupts ADD-CD interaction, induces a large movement of the ADD domain, and releases the autoinhibition of DNMT3A. In the active form of DNMT3A, the ADD domain has no steric hindrance for DNA recognition by the CD domain. As pointed out in the main text, residues R790, R792, D529, and D531 of DNMT3A and H3K4 are shown as sticks.

About this article

Publication history






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.