Structural Insights into DNMT3A-3L-Mediated de novo DNA Methylation on Chromatin

DNA methylation is one of the critical epigenetic mechanisms, yet its establishment and regulation during mammalian embryonic development remain poorly understood. DNA methyltransferases DNMT3A and DNMT3B require catalytically inactive variants such as DNMT3L or DNMT3B3 to function effectively during gametogenesis and early embryogenesis. Abnormal DNA methylation can lead to genomic instability and oncogenesis, underscoring the importance of deciphering its molecular mechanisms, especially during embryonic development, where DNMT3L is primarily expressed. Here, we present a high-resolution cryo-EM structure of the nucleosome-bound full-length DNMT3A2-3L and oligomeric structures of the DNMT3A2-3L complex in its nucleosome-free state. Notably, in the nucleosome-bound complex, we observed a 180° rotational difference in the C-terminal “Switching Helix” of DNMT3L compared to its counterpart in DNMT3B3 within the DNMT3A2-3B3-NCP complex, eliminating the direct interactions with the nucleosome acidic patch. Instead, we identify a previously unrecognized mechanism in which the DNMT3L ADD domain significantly contributes to nucleosome engagement, while the DNMT3A PWWP domain inhibits nucleosome binding, suggesting an additional layer of regulatory complexity. Furthermore, our first-ever discovery of the novel oligomeric arrangement of DNMT3A2-3L in its nucleosome-free state highlights its dynamic assembly and indicates potential allosteric regulation. Based on the structures and supporting biophysical data, we propose that the DNMT3A-3L complex senses histone tail modifications and dynamically searches for and engages with linker DNA by selecting or reorganizing the complex to a more stable and active heterotetrameric state, shedding light on its role in co-regulating DNA methylation on chromatin. To assess the role of DNMT3L C-terminal “Switching Helix” and ADD domain in restoring DNA methylation in cells, we analyzed DNA methylation in DKO8 cells expressing either wild-type or mutant DNMT3L 56 days post-infection using the Infinium MethylationEPIC BeadChip array.