Abstract
DNA methyltransferase DNMT3A-mediated DNA methylation is important for genomic imprinting and transcriptional regulation. However, how the regulatory domains of DNMT3A cooperate with its methyltransferase domain and histone marks to orchestrate genomic methylation remains unclear. Here we report the cryo-EM structure of DNMT3A2 with regulatory factor DNMT3L, revealing an intricate domain interaction underlying multilayered autoinhibition. The PWWP domain interacts with the ADD and methyltransferase domains to block the target recognition domain and the H3K36me2-binding pocket, thereby coupling the H3K36me2 binding with DNMT3A activation, adding a layer of allosteric regulation distinct from the previously characterized ADD-H3K4me0 regulation. Molecular dynamics simulations of the DNMT3A-DNMT3L complex further reveals that relief of DNMT3A autoinhibition involves disengagement of the CpG-recognition loop of the target recognition domain from autoinhibitory interaction, leading to enhanced accessibility of the target recognition domain loop for DNA binding and DNMT3A activation. Importantly, our combined structural, biochemical and genomic methylation analysis demonstrates that disrupting the PWWP-ADD interaction by disease-associated DNMT3A mutations leads to impaired DNMT3A autoinhibition and substrate specificity, providing a potential explanation to aberrant DNA methylation in disease.
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Data availability
The atomic model for the DNMT3A2-DNMT3L complex has been deposited in the Protein Data Bank under accession code 9PRW. The cryo-EM density map has been deposited in EMDB under the accession number of EMD-71814. The PDB accession codes 2PVC2PV03LLR, 4QBQ, 4U7P, 5CIU, 5YX2 and 8EIH were used in this study. The Cut&Tag and DNA methylation profiling data deposited in NCBI Gene Expression Omnibus under accession number GSE24701913 were used in this study. Source Data are provided in the Source Data file. Source data are provided with this paper.
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Acknowledgements
This work was supported by NIH grants (R35GM119721 to J.S., R35GM138181 and R01CA266978 to C.L., and R01GM109045 to C.A.C.), NSF grant MCB-2437134 to C.A.C. and the Academic Senate Award of UC Riverside to J.S. E.V. and N.K. were supported by GAANN fellowship of Department of Education (P200A210136). N.K. was also supported by the administrative supplement of NIH R35GM119721. A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. Collection of the Cryo-EM data was assisted by Dr. Rose Marie Haynes. We thank Dr. Lingchao Zhu in ACIF NMR facility of UC Riverside for assistance in NMR data collection and UC Riverside High Performance Computer Cluster for providing computation resources.
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J.L., E.V., J.C., K.H.G., N.K. and Z.S. performed the experiments, C.L., C.C. and J.S. supervised the study. J.L. and J.S. wrote the manuscript and all authors approved the manuscript.
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Lu, J., Vig, E., Chen, J. et al. Structural insight into hierarchical DNMT3A autoinhibition and its dysregulation in disease. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69563-1
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DOI: https://doi.org/10.1038/s41467-026-69563-1
