Abstract
DNA methyltransferase 3A (DNMT3A) is a de novo cytosine methyltransferase responsible for establishing proper DNA methylation during mammalian development. Loss-of-function (LOF) mutations to DNMT3A, including the hotspot mutation R882H, frequently occur in developmental growth disorders and hematological diseases, including clonal hematopoiesis and acute myeloid leukemia. Accordingly, identifying mechanisms that activate DNMT3A is of both fundamental and therapeutic interest. Here, we applied a base editor mutational scanning strategy with an improved DNA methylation reporter to systematically identify DNMT3A activating mutations in cells. By integrating an optimized cellular recruitment strategy with paired isogenic cell lines with or without the LOF hotspot R882H mutation, we identify and validate three distinct hyperactivating mutations within or interacting with the regulatory ADD domain of DNMT3A, nominating these regions as potential functional target sites for pharmacological intervention. Notably, these mutations are still activating in the context of a heterozygous R882H mutation. Altogether, we showcase the utility of base editor scanning for discovering functional regions of target proteins.