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Mechanisms of DNMT3A–3L-mediated de novo DNA methylation on chromatin

Nature Structural & Molecular Biology (2025)Cite this article

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Abstract

De novo DNA methylation is mediated by DNA methyltransferases DNMT3A and DNMT3B, in cooperation with the catalytically inactive paralogs DNMT3L and DNMT3B3. DNMT3L is predominantly expressed in embryonic stem cells to establish methylation patterns and is silenced upon differentiation, with DNMT3B3 substituting in somatic cells. Here we present high-resolution cryo-electron microscopy structures of nucleosome-bound, full-length DNMT3A2–3L and its oligomeric assemblies in the nucleosome-free state. We identified the critical role of DNMT3L as a histone modification sensor, guiding chromatin engagement through a mechanism distinct from DNMT3B3. The structures show a 180° rotated ‘switching helix’ in DNMT3L that prevents direct interaction with the nucleosome acidic patch. Instead, nucleosome binding is mediated by the DNMT3L ADD domain, while the DNMT3A PWWP domain exhibits reduced engagement in the absence of H3K36 methylation. The oligomeric arrangement of DNMT3A2–3L in nucleosome-free states highlights its dynamic assembly and potential allosteric regulation. We further capture dynamic structural movements of DNMT3A2–3L on nucleosomes. These findings uncover a previously unknown mechanism by which DNMT3A–3L mediates de novo DNA methylation on chromatin through complex assembly, histone tail sensing, dynamic DNA search and regulated nucleosome engagement, providing insights into epigenetic regulation.

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Fig. 1: Cryo-EM structure of DNMT3A2–3L in a nucleosome-bound state.
Fig. 2: Cryo-EM structures of DNMT3A2–3L in nucleosome-free state.
Fig. 3: Interactions between DNMT3A2–3L and nucleosomes.
Fig. 4: ADD and PWWP domains are important for nucleosome recruitment and regulation.
Fig. 5: In-cell DNA methylation of DNMT3A2–3L complexes.
Fig. 6: DNMT3A2–3L senses histone tail and co-regulates DNA methylation.
Fig. 7: Dynamic analysis and model for DNMT3A2–3L-mediated de novo DNA methylation on chromatin.

Data availability

The 3D cryo-EM maps have been deposited in the Electron Microscopy Database under accession numbers EMD-48322 (DNMT3A2–3L–NCP consensus map, 3.10 Å), EMD-48492 (DNMT3A2–3L, 3.60 Å), EMD-48493 (NCP, 3.08 Å), EMD-48495 (DNMT3A2–3L–NCP intermediate state map, 3.29 Å), EMD-48494 (DNMT3A2–3L–NCP ‘up’ state map, 3.16 Å), EMD-48498 (combined DNMT3A2–3L–NCP map, 3.60 ~ 3.08 Å), EMD-48496 (DNMT3A2–3L dodecamer, 3.66 Å) and EMD-48497 (DNMT3A2 octamer, 3.62 Å). The structure coordinates have been deposited in the Protein Data Bank under accession numbers PDB 9MPP (DNMT3A2–3L–NCP), PDB 9MP0 (DNMT3A2–3L dodecamer) and PDB 9MPO (DNMT3A2 octamer). The DNA methylation data have been deposited in the GEO database with accession code GSE291793. All other data needed to evaluate the conclusions in the paper are presented in the main text or supplementary materials. No restrictions are placed on data availability. Source data are provided with this paper.

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Acknowledgements

We thank G. Zhao and X. Meng from the Van Andel Institute (VAI) for support with data collection at the David Van Andel Advanced Cryo-Electron Microscopy Suite (RRID: SCR_023210), and the VAI Genomics Core (RRID: SCR_022913) for sequencing and Illumina Infinium MethylationEPIC array support. We are grateful to W. Choi, C. Li and Y. Cheng from the University of California San Francisco for providing the CryoROLE algorithms and assisting in the use of the algorithms; J. C. Eissenberg and E. Di Cera from Saint Louis University and B. M. Dickson from VAI for advice. This work was supported by the Doisy Fund of the Edward A. Doisy Department of Biochemistry and Molecular Biology at Saint Louis University School of Medicine (T.-H.X.), Saint Louis University School of Medicine President’s Research Fund (T.-H.X.) and the National Institutes of Health (NIH) R35CA209859 (P.A.J.), NIH R35GM147261 (E.J.W.) and NIH R50CA243878 (M.L.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, USA

    Yan Yan, Gan-Qiang Lai & Ting-Hai Xu

  2. Department of Structural Biology, Van Andel Institute, Grand Rapids, MI, USA

    X. Edward Zhou & Evan J. Worden

  3. Department of Epigenetics, Van Andel Institute, Grand Rapids, MI, USA

    Stacey L. Thomas, Minmin Liu & Peter A. Jones

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  1. Yan Yan
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  2. X. Edward Zhou
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  3. Stacey L. Thomas
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  7. Peter A. Jones
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  8. Ting-Hai Xu
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Contributions

T.-H.X. and P.A.J. conceived the project. Y.Y. and T.-H.X. prepared protein samples, performed electron microscopy experiments and data analysis. Y.Y. and T.-H.X. performed and interpreted all experiments. Y.Y., T.-H.X. and X.E.Z. built the atomic models and refined the atomic models. M.L. performed DKO8 cell transfection and MethylationEPIC array and data analysis. S.L.T. assisted in experiments. G.-Q.L. assisted in DNA cloning. E.J.W. supervised X.E.Z.; P.A.J. supervised S.L.T. and M.L.; T.-H.X. supervised Y.Y. and G.-Q.L. Y.Y. and T.-H.X. wrote the paper with support from all authors, and the manuscript was reviewed and approved by all authors.

Corresponding authors

Correspondence to Peter A. Jones or Ting-Hai Xu.

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P.A.J. is a paid consultant for Zymo Research. The other authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Hanna Yuan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Conformational states of the DNMT3A2-3L complexes in solution.

a, Size-exclusion chromatography analysis (top) and SDS-PAGE analysis (middle) of the DNMT3A2-3L complex. Bottom: quantification of relative band intensities from SDS-PAGE. b, AlphaScreen interaction assay between His-tagged DNMT3A2-3L and biotinylated NCPs. Data were presented as mean ± s.e.m. of n = 6 biological repeats. c, Native gel analysis of the cross-linked DNMT3A2-3L-NCP complex. For gel source data, see source data file. d, Mass photometry analysis of DNMT3A2-3L, mCherry-labeled DNMT3A2, and sfGFP-labeled DNMT3L. Molecular weights (kDa) are indicated for each peak. Bottom: schematic models for possible subunit arrangements. Dashed outlines indicate subunits not observed in the cryo-EM structures.

Source data

Extended Data Fig. 2 Cryo-EM data collection and structure determination of DNMT3A2-3L-NCP complex from dataset1.

a, Representative micrograph of DNMT3A2-3L-NCP complex with one particle highlighted in a green circle of 250 Å diameter. b, Representative 2D class-averages. c, Workflow diagram of cryo-EM data processing and 3D reconstructions in CryoSPARC (4.6.0) and particle subtraction in RELION 5. Boxed 3D classes were selected for further processing. The final global nominal resolution for the DNMT3A2-3L-NCP complex was 3.14 Å, for the NCP was 3.08 Å, and for the DNMT3A2-3L was 3.70 Å. Color-coded local resolution maps for consensus refinement (d), focused nucleosome refinement(e), and focused DNMT refinement(f). The final global nominal resolution was set at 3.14 Å for DNMT3A2-3L-NCP complex, 3.08 Å for NCP, and 3.70 Å for DNMT3A2-3L at 0.143 FSC. Half-map corrected Fourier Shell Correlation (cFSC) plots and angular distribution plots obtained from CryoSPARC (color indicative of the number of particles, increasing from blue to red, in a defined orientation) were shown at the bottom.

Extended Data Fig. 3 Structure determination of DNMT3A2-3L-NCP in the combined dataset.

a, Cryo-EM data processing workflow by CryoSPARC (4.6.0) and RELION 5 of the combined dataset for the DNMT3A2-3L-NCP complex. Half-map cFSC plots were shown on the bottom for the three distinct conformations: a DNA-engaged state (grey, left), an intermediate state (yellow, middle), and an “up” state (cyan, right). The final global nominal resolution was set at 3.10 Å for the engaged state, 3.29 Å for the intermediate state, and 3.16 Å for the “up” state at 0.143 FSC. Particles from each conformation were further processed with focused refinements of DNMT and NCP separately. Boxed 3D classes were selected for further processing. The final global nominal resolution was set at 3.15 Å, 3.28 Å, and 3.15 Å at 0.143 FSC for NCPs from three conformations, and at 3.60 Å, 5.68 Å, and 4.22 Å at 0.143 FSC for DNMT3A2-3L from three conformations. b, Color-coded local resolution maps in two different orientations, related by a 180˚ rotation around a vertical axis for DNMT3A2-3L complexes in three distinct conformations. Half-map cFSC plots and angular distribution plots obtained from CryoSPARC were shown at the bottom for each conformation.

Extended Data Fig. 4 Cryo-EM data collection and structure determination of DNMT3A2-3L oligomer complex.

a, The UV (280 nm) absorption profile of cross-linked DNMT3A2-3L-NCP sample collected from bottom to top (left). Native gel analysis of cross-linked DNMT3A2-3L oligomer complex (right). For gel source data, see source data file. b, Representative micrograph of DNMT3A2-3L complex with one particle highlighted in green circle of 300 Å diameter. c, Representative 2D class-averages. d, Workflow of cryo-EM data processing and 3D reconstructions in CryoSPARC (4.6.0). Boxed 3D classes were selected for further processing. The final global nominal resolution for the DNMT3A2-3L oligomer complexes was 3.66 Å for the dodecamer and 3.62 Å for the octamer. e, Color-coded local resolution maps for DNMT3A2-3L dodecamer in two different orientations and overall atomic model fitting into the cryo-EM map density. Inlet, overlay of distal EM density with DNMT3L (pink) at lower contour, which was omitted from the final model, indicating the dodecamer. Half-map cFSC plots and angular distribution plots obtained from CryoSPARC were shown at the bottom. f, Color-coded local resolution maps for DNMT3A2-3L octamer in two different orientations and overall atomic model fitting into the cryo-EM map density. Inlet, overlay of distal EM density with DNMT3L (pink) at lower contour, which was omitted from the final model, indicating further oligomerization. Half-map corrected cFSC plots and angular distribution plots obtained from CryoSPARC were shown on the right.

Source data

Extended Data Fig. 5 Structure analysis of DNMT3A2-3L bound to nucleosomes.

a,b, Overall cryo-EM map fittings of NCP (a) and DNMT3A2-3L (b). Inlet, unsharpened density map fitting the distal DNMT3A2 ADD domain. c, Density map fittings of the distal DNMT3L CLD domain. DNMT3L C-ter “Switching Helix” was indicated by the arrow. d, Top: active (PDB ID 4U7T) and autoinhibitory (PDB ID 4U7P) forms of DNMT3A ADD-CD dimers. Bottom: Structure comparison of current nucleosome-bound DNMT3A2-3L with autoinhibitory form of DNMT3A ADD-CD dimer (PDB ID 4U7P). Only DNMT3A2 ADD-CD dimers were shown. e, Structure comparison of proximal DNMT3L with active form (left) and autoinhibitory form (right) of DNMT3A ADD-CD domains (PDB ID 4U7T and 4U7P). f, Interfaces of the DNMT3A (CD)-3L(CLD) in crystal structure (PDB ID 2QRV). DNMT3A CD can form a V-shaped, polar, homodimeric interface (3A-3A RD interface) while with DNMT3L CLD, DNMT3A CD can form a flat, non-polar, heterodimeric interface (3A-3L FF interface) due to lack of the TRD loops in DNMT3L. g, Density map of the CD-DNA interaction region generated in ChimeraX with contour level at 0.1 (left) and 0.005 (right). The expected flipped C base position in the active site was highlighted in red. h, Density maps of the two SAH ligands generated in ChimeraX and level at 0.05.

Extended Data Fig. 6 Detailed structural insights into the interactions of DNMT3A2-3L with nucleosomes.

a, Cryo-EM densities of the nucleosome acidic patch region for the DNMT3A2-3L-NCP complex (left), and the DNMT3A2-3B3-NCP complex (right, EMD-20281). b, Two detailed views of superimposed maps of cryo-EM densities for the CLDs of DNMT3L and DNMT3B3 in the acidic patch region. The CLD regions were shown as cartoon models within the transparent density maps. c, Sequence alignments of the DNMT3 C-terminal region. Similar residues were shaded grey, while identical residues were shaded black. The TRD loop was missing in accessory proteins DNMT3L and DNMT3B3. The acidic patch-interacting arginine finger residues in DNMT3B3 (R740 and R743) were labeled with red stars, and the corresponding residues in DNMT3L (Q348 and Q351) were labeled with blue arrows. The alignment was annotated with secondary structure arrangements for DNMT3A1 (top) and DNMT3L (bottom).

Extended Data Fig. 7 The interactions between DNMT3 complexes and nucleosomes.

a, AlphaScreen photo accounts for the interaction of His-tagged DNMT3A2, DNMT3A2-3B3, DNMT3A2-3L, DNMT3B3, and DNMT3L titrated against modified nucleosomes. Data were presented as mean ± s.e.m. of n = 4-13 biological repeats specified in each experiment. b, The full panel of DNMT3A2 nucleosomes binding strength (EC50) in AlphaScreen assay.

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Extended Data Fig. 8 In-vitro DNMT3 DNA activities and kinetics.

a, In-vitro DNA methylation activities of DNMT3A2 (left), DNMT3A2-3L (middle), DNMT3A2-3B3 (right) in the absence (black) and presence of histone H3 peptide (1-44) unmodified (blue) or carrying K4me3 (red) or K36me3 (green). b, The full panel of DNMT3A2 DNA methylation activities with/without accessory proteins in the absence and presence of histone H3 peptide (1-44). c, Kinetics of DNMT3A2 with/without accessory proteins (DNMT3L or DNMT3B3) in the absence and presence of histone H3 peptide (1-44). d, In-vitro DNA methylation activity of wild-type or mutant DNMT3A2-3L complexes. Shown was the percentage methylation. e, Kinetics of DNMT3A2-3L and DNMT3A2-3B3 in the absence or presence of different H3 peptides. Data were presented as mean ± s.e.m. of n = 8 (a) and 3 (c, d, e) biological repeats. P-values, unpaired two-tailed Student’s t-test.

Source data

Extended Data Fig. 9 CryoROLE and RELION5 multi-body analysis of the nucleosome-bound DNMT3A2-3L complex.

a, Overlays of 3D reconstructions calculated from particles selected along different directions (α, β, and γ) in the conformational landscape. The number of particles used was shown on the left. Relative motion trajectories were shown on the right. The reconstructions were calculated from locations at every 10° from +20° to -20° in α and γ direction, from +20° to -30° in β direction. All reconstructions were calculated from particles within a 5 Å radius around selected coordinates and low-pass filtered by 15 Å. The detailed movements were recorded in Supplementary Video 1. b, Multi-body refinement in RELION5 showed similar motion between the nucleosomes and DNMT3A2-3L, especially the distal part of DNMT3A2-3L. The detailed movements were recorded in Supplementary Video 2.

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Supplementary Video 1

Relative motion between DNMT3A2-3L and nucleosomes revealed by CryoROLE.

Supplementary Video 2

Relative motion between DNMT3A2-3L and nucleosomes revealed by RELION5 Multi-body refinement.

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Source Data Fig. 3

Numerical source data.

Source Data Fig. 4

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Source Data Fig. 5

Uncropped western blots. The boxed areas are the parts shown in the main figures.

Source Data Fig. 6

Numerical source data.

Source Data Fig. 6

Uncropped western blots. The boxed areas are the parts shown in the main figures.

Source Data Extended Data Fig./Table 1

Numerical source data.

Source Data Extended Data Fig. 1

Uncropped gel images. The boxed areas are the parts shown in the main figures.

Source Data Extended Data Fig. 4

Uncropped gel images. The boxed areas are the parts shown in the main figures.

Source Data Extended Data Fig. 7

Numerical source data.

Source Data Extended Data Fig. 8

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Yan, Y., Zhou, X.E., Thomas, S.L. et al. Mechanisms of DNMT3A–3L-mediated de novo DNA methylation on chromatin. Nat Struct Mol Biol (2025). https://doi.org/10.1038/s41594-025-01704-4

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