Base Editor Scanning Reveals Activating Mutations of DNMT3A

DNAmethyltransferase 3A (DNMT3A) is a de novo cytosinemethyltransferase responsible for establishing proper DNAmethylation during mammalian development. Loss-of-function (LOF) mutationsto DNMT3A, including the hotspot mutation R882H, frequently occurin developmental growth disorders and hematological diseases, includingclonal hematopoiesis and acute myeloid leukemia. Accordingly, identifyingmechanisms that activate DNMT3A is of both fundamental and therapeuticinterest. Here, we applied a base editor mutational scanning strategywith an improved DNA methylation reporter to systematically identifyDNMT3A activating mutations in cells. By integrating an optimizedcellular recruitment strategy with paired isogenic cell lines withor without the LOF hotspot R882H mutation, we identify and validatethree distinct hyperactivating mutations within or interacting withthe regulatory ADD domain of DNMT3A, nominating these regions as potentialfunctional target sites for pharmacological intervention. Notably,these mutations are still activating in the context of a heterozygousR882H mutation. Altogether, we showcase the utility of base editorscanning for discovering functional regions of target proteins.