BioProject

DNA methyltransferases DNMT3A- and DNMT3B-mediated de novo DNA methylation critically regulates epigenomic and transcriptomic patterning during development. More...

DNA methyltransferases DNMT3A- and DNMT3B-mediated de novo DNA methylation critically regulates epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote macro-oligomer formation, leading to aberrant DNA methylation that in turn contributes to pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the hotspot mutation-induced functional mis-regulation of DNMT3A remains unclear. Here, we report the crystal structure of DNMT3A methyltransferase (MTase) domain, revealing a molecular basis for its DNMT3B-distinct oligomerization behavior. Introducing DNMT3B-converting mutations to DNMT3A R882 mutants also led to structure determination of R882H- and R882C-mutated DNMT3A, which show enhanced intermolecular contacts than wild-type DNMT3A. Consistently, our in vitro and genomic DNA methylation analyses reveal that the DNMT3B-converting mutations eliminate the gain-of-function effect of the DNMT3A R882 mutations in cells. Together, this study provides mechanistic insights into DNMT3A R882 mutation-triggered aberrant oligomerization and DNA hypomethylation in AML, with important implications in cancer therapy. Overall design: TF-1 cell line was stably expressed with Myc-tagged DNMT3A (isoform 1), either wild-type (WT) or AML-associated hotspot mutant (R882C or R882H) or the one carrying an additional macro-oligomerization-decreasing mutation, namely, R676K (WT-K), R882C/R676K (R882C/K), R882C/M674T/R676K (CTK), R882H/R676K (R882H/K) or R882H/M674T/R676K (R882H/TK). 100,000 of TF-1 cells were subjected to CUT&Tag using anti-Myc tag antibody (CST #2276s). 5,000 of NIH-3T3 cells with stable expression of Myc-tagged WT DNMT3A1 were added to all TF-1 cell groups as spike-in normalization control. Less...