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Structure of nucleosome-bound DNA methyltransferases DNMT3A and DNMT3B

Nature volume 586, pages 151–155 (2020)Cite this article

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Abstract

CpG methylation by de novo DNA methyltransferases (DNMTs) 3A and 3B is essential for mammalian development and differentiation and is frequently dysregulated in cancer1. These two DNMTs preferentially bind to nucleosomes, yet cannot methylate the DNA wrapped around the nucleosome core2, and they favour the methylation of linker DNA at positioned nucleosomes3,4. Here we present the cryo-electron microscopy structure of a ternary complex of catalytically competent DNMT3A2, the catalytically inactive accessory subunit DNMT3B3 and a nucleosome core particle flanked by linker DNA. The catalytic-like domain of the accessory DNMT3B3 binds to the acidic patch of the nucleosome core, which orients the binding of DNMT3A2 to the linker DNA. The steric constraints of this arrangement suggest that nucleosomal DNA must be moved relative to the nucleosome core for de novo methylation to occur.

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Fig. 1: Cryo-EM structure of the human DNMT3A2–DNMT3B3–NCP complex.
Fig. 2: Details of the acidic patch and DNA–catalytic domain interactions.
Fig. 3: The TRD mediates DNA binding but prevents NCP core binding.
Fig. 4: The DNMT3B3–acidic patch interaction is required for chromatin recruitment.

Data availability

Density maps and structure coordinates have been deposited in the Electron Microscopy Data Bank with accession codes EMD-20281 and EMD-21689 and the Protein Data Bank with accession code 6PA7. The DNA methylation and MNase data have been deposited in the GEO database with accession code GSE152640. No restrictions are placed on data availability. Source data are provided with this paper.

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Acknowledgements

Cryo-EM data was collected at the David Van Andel Advanced Cryo-Electron Microscopy Suite at the Van Andel Institute (VAI). We thank X. Meng for cryo-EM data collection and EM technical support, the HPC team at VAI for computational support, D. Nadziejka for language editing of the manuscript, the VAI genomics core for performing the Illumina Infinium methylation EPIC array assay and J.-H. Min (Washington State University) and S. Baylin (John Hopkins University) for the indicated plasmids. This work was supported by the VAI (H.E.X., K.M., P.A.J.) and US National Cancer Institute (NCI; grants R35CA209859 to G.L., K.M. and P.A.J. and R50CA243878 to M.L.).

Author information

Authors and Affiliations

  1. Center for Cancer and Cell Biology, Program for Structural Biology, Van Andel Institute, Grand Rapids, MI, USA

    Ting-Hai Xu, X. Edward Zhou & Karsten Melcher

  2. Center for Epigenetics, Van Andel Institute, Grand Rapids, MI, USA

    Ting-Hai Xu, Minmin Liu & Peter A. Jones

  3. Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, Los Angeles, CA, USA

    Gangning Liang

  4. David Van Andel Advanced Cryo-Electron Microscopy Suite, Van Andel Institute, Grand Rapids, MI, USA

    Gongpu Zhao

  5. Center for Structure and Function of Drug Targets, CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    H. Eric Xu

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Contributions

H.E.X., K.M. and P.A.J. designed research. T.-H.X. expressed and purified proteins, prepared and screened samples and collected and processed cryo-EM data. T.-H.X. and M.L. performed biochemistry experiments. T.-H.X. and X.E.Z. built the atomic models and performed the refinements. T.-H.X., M.L., X.E.Z., G.L., G.Z., H.E.X., K.M. and P.A.J. analysed data. H.E.X., K.M. and P.A.J. wrote the paper with input from all authors.

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Correspondence to H. Eric Xu, Karsten Melcher or Peter A. Jones.

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Extended data figures and tables

Extended Data Fig. 1 Protein expression and nucleosome reconstitution.

a, Histone octamer expression and purification. Histones H3 and H4 and His6-Sumo-tagged H2A and H3 were coexpressed from a polycistronic expression vector. The octamer was purified by native Ni-affinity chromatography, and then cleavage of the His6-Sumo tags with ULP1 Sumo protease was followed by size exclusion chromatography (SEC). The SDS–PAGE gel shows analysis of histone octamer purification steps. b, SEC profile of histone octamer with indicated size standards. c, Native gel analysis of NCP reconstitution. NCPs reconstituted by double dialysis were separated by native gel electrophoresis (lane 1) together with a size standard (lane 2) and free nucleosomal DNA (lane 3). The gel was stained for both DNA (left) and protein (right). d, SEC profile of cleaved (red) and non-cleaved (blue) DNMT3 complex. e, SDS–PAGE analysis of GFP-tagged DNMT and untagged DNMT purification steps. The GFP tag allowed easy DNMT3B3 visualization in DNMT–NCP complexes. For gel source data, see Supplementary Fig. 3.

Extended Data Fig. 2 DNMT3A2, DNMT3B3 and NCP form a stable complex.

a, DNMT3 and NCP stably interact in an AlphaScreen luminescence proximity assay. Top panel, cartoon of AlphaScreen assay; bottom panel, AlphaScreen interaction data. The nucleosomal DNA of the NCP is biotinylated for immobilization on AlphaScreen streptavidin donor beads, and DNMT3B3 is His8-GFP-tagged for immobilization of Ni-chelating AlphaScreen acceptor beads. Donor beads contained a photosensitizer that, upon activation at 680 nm, converts ambient oxygen to singlet oxygen. If donor and acceptor beads are brought into close proximity by DNMT–NCP interaction, energy is transferred from singlet oxygen to thioxene derivatives in the acceptor beads, resulting in light emission at 520–620 nm. Data are mean ± s.e.m., n = 10. b, Increasing concentrations of GFP-tagged DNMT3A2–DNMT3B3 complex supershift the NCP band. M, DNA size marker. Panels 1–4 show native gel with indicated binding reactions in (1) the ethidium bromide channel to visualize the DNA of the NCP and (2) the fluorescence channel to visualize the GFP-tagged DNMT3A2–DNMT3B3 complex; (3) a merged image of the two channels (yellow bands indicates presence of both DNMT complex and NCP; and (4) gel stained with Coomassie blue. For gel source data, see Supplementary Fig. 4.

Source Data

Extended Data Fig. 3 DNMT3A–DNMT3B3 methylation activity and nucleosome interaction.

a, Free DNA methylation activity of acidic-patch-interacting and control mutant DNMT3B3 proteins. Histone-free (naked) 301-bp nucleosomal DNA was incubated with the indicated DNMT3A2–DNMT3B3 complexes containing wild-type or mutant DNMT3B3. Shown is the percentage methylation. Data are mean with individual values, n = 2. The total number of CpGs in the DNA is 24, of which a subset was preferentially methylated. b, Binding strength and structure resolution of DNMT3A2–DNMT3B3–NCP complexes with varying linker DNA lengths. Left, competition binding curves. AlphaScreen interaction between biotin-tagged NCPs and His6-tagged DNMT3A2–DNMT3B3 in the presence of increasing concentrations of untagged DNMT3A2–DNMT3B3. Data were generated from 3 independent experiments (n = 3) and normalized in each group with highest data point as 100%. Right, IC50 calculated in each experiment and represented in bar graphs. Data are mean ± s.e.m., n = 3, P values determined by non-parametric test with Dunn’s multiple comparisons test. ns, not significant. The table below compares binding strengths (IC50) and cryo-EM resolution of DNMT3A2–DNMT3B3 complexes with NCPs containing different length DNAs. The 200-kV data were collected on a Talos Arctica microscope with a Falcon 3 detector and the 300-kV data on a Titan Krios microscope with a K2 detector.

Source Data

Extended Data Fig. 4 Overall structure of DNMT bound to the nucleosome.

a, Left, local resolution map of DNMT–nucleosome core complex (global resolution of 2.94 Å); middle, cutaway view; right, corrected Fourier shell correlation (FSC) curves of the 3D reconstruction. b, Left, overview of DNMT–nucleosome complex with ADD domains; middle, cartoon representation. Right, overlay of distal ADD EM density with aligned ADD in the auto-inhibitory conformation from PDB 4U7P. The inhibitory ADD loop, residues 526–533, is highlighted in blue. c, Overall map fitting of the nucleosome, DNMT ADD-CD/CLD dimer and DNMT CD/CLD dimer with local resolution colour code. The resolutions were determined using the 0.143 FSC criterion. d, Density map of the CD–DNA interaction region generated in PyMOL and contoured at 8σ (left) and 5.5σ (right). The dashed line indicates the position of the TRD loop, which is only visible at the lower σ level and we have omitted in the final model. e, Density maps of the two SAH ligands generated in PyMOL and contoured at 8σ.

Extended Data Fig. 5 Cryo-EM 3D reconstruction and refinement.

a, Representative micrograph, with one particle highlighted in a green circle of 250 Å diameter. b, Representative 2D class averages. c, Workflow for cryo-electron microscopy data processing by cryoSPARC and RELION3.0. Boxed 3D classes were selected for further processing. The final global nominal resolution for the DNMT–NCP complex was 2.94 Å.

Extended Data Fig. 6 Focused refinement of DNMT and NCP.

About 559,000 good particles were selected from 3D reconstructions for further focused refinement. The final resolution for the DNMT3A2–DNMT3B3 complex including the ADD domains was 3.40 Å, for the DNMT3A2 CD–DNMT3B3 CLD complex was 3.24 Å and for the NCP was 2.78 Å.

Extended Data Fig. 7 Conformation and density asymmetry of linker DNA (CD-bound versus free) and CLD (NCP-bound versus free).

a, b, Structure models overlaid with density maps (grey mesh) generated in PyMOL and contoured at 8σ (a) and 6σ (b). Note that at the 8σ contoured level, complete density is only visible for the CD-bound linker DNA and the histone-octamer-bound CLD. c, While the CD-bound linker DNA adopts a single conformation, the unbound linker DNA adopts four distinguishable conformations. Left, composite model; right, four different conformations shown as cryo-EM densities with their relative frequencies. d, Model of the DNMT3A2–DNMT3B3 complex with extended linker DNA and 3A2 ADD domain in its active conformation. The ADD has been modelled by structural alignment with PDB 4U7T. The structure model is fully compatible with the extended linker DNA. e, Model of the DNMT3A2–DNMT3B3 complex with extended linker DNA and 3A2 ADD domain in the autoinhibitory conformation. The ADD has been modelled by structural alignment with PDB 4U7P. This conformation is not compatible with the extended linker DNA.

Extended Data Fig. 8 MNase footprinting assay.

a, b, NCP301 alone (a) or in complex with DNMT3A2–DNMT3B3 (b) was subjected to MNase footprinting followed by DNA isolation and sequencing. Sequence reads shown by IGV viewer were aligned to the 301-bp reference to reveal the position of DNMT3A2–DNMT3B3 on individual nucleosomal DNA molecules (green, A; red, T; blue, C; brown, G). Magenta oval, position of the histone octamer (Widom 601 147-bp nucleosomal sequence); cyan bars, flexible linker region. Dashed lines demarcate the 10-bp linker regions of NCP167, and red dashed boxes represent the main DNMT protection region (protection of the DNMT–NCP complex relative to the DNMT-free NCP). The percentages of reads covering the DNMT protection regions are shown in red.

Extended Data Fig. 9 Histone H3 K36cMe3 did not improve the stability of ADD domains.

a, The H3K36cMe3 methyl-lysine analogue (MLA) peptide and H2A–H2B–H3K36cMe3 MLA–H4 histone octamer, but not the wild-type (WT) octamer, are recognized by anti-H3K36Me3 antibody. Four titrations (H3K36me3 peptide) or three titrations (H3Kc36me3 octamer) with different amounts of proteins were performed. b, AlphaScreen interaction assay between His-tagged DNMT3A2–DNMT3B3 complexes and biotinylated NCPs. The histone H3 K36cMe3 methyl analogue does not increase DNMT3A2–DNMT3B–NCP binding in the reconstituted system. Data are mean ± s.e.m., n = 3. c, Cryo-EM structure of the DNMT3A2–DNMT3B–H3K36cMe3 MLA NCP. The structure shows no additional density relative to that of the WT NCP complex. The ADD density is still poor and no reliable PWWP density is observed. d, Structural alignment of the DNMT–NCP WT (grey) and DNMT–NCP K36cMe3 (yellow) density maps shows their similar overall architecture. For gel source data, see Supplementary Fig. 5.

Source Data

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table

Supplementary information

Supplementary Information

This file contains: Supplementary Fig. 1: Sequence alignment; Supplementary Figs S2-S5: uncropped gels and size marker indications related to Fig. 4b and Extended Data Figs. 1a, 1c, 1e, 2b, and 9a and Supplementary Table 1: List of primers.

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Video 1

Cryo-EM density map for human DNMT3A (CD) and DNMT3B3 (CLD) with NCP. Cryo-EM map and molecular model fitted in transparent density. The same color code is used as in Fig. 1c.

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Xu, TH., Liu, M., Zhou, X.E. et al. Structure of nucleosome-bound DNA methyltransferases DNMT3A and DNMT3B. Nature 586, 151–155 (2020). https://doi.org/10.1038/s41586-020-2747-1

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