Probing regulatory mechanisms of the de novo DNA methyltransferase DNMT3A.

Abstract

DNA methyltransferase 3A (DNMT3A) is one of the two human de novo DNA methyltransferases responsible for establishing proper DNA methylation during development. Beyond this role, DNMT3A is frequently mutated in hematopoietic disorders, including clonal hematopoiesis and acute myeloid leukemia (AML). One hotspot mutation, R882H, likely causes a dominant loss-of-function phenotype, but its mechanism of doing so remains controversial. DNMT3A is typically found as a tetramer and R882 is found at one of its two oligomerization interfaces. However, it is debated in the field whether this mutation perturbs the multimeric state of DNMT3A, and if so whether it breaks up tetramers or promotes oligomerization. We aim to determine how the R882H mutation causes a dominant negative phenotype and to describe new mechanisms of DNMT3A regulation that might allow for reactivation of the mutant protein. Towards this goal, we have performed in vitro biochemical assays that demonstrate that DNMT3AR882H is more likely to form macro-oligomers than WT DNMT3A. We have also shown that this phenomenon holds true in cell lysate through sucrose gradient ultracentrifugation. In addition, we have performed a base editing mutational scan of DNMT3A in cells with WT DNMT3A as well as in cells harboring a heterozygous DNMT3AR882H mutation and have identified mutations to DNMT3A that boost activity with and without the R882H mutation. By further characterizing these mutants and DNMT3AR882H through crosslinking mass spectrometry, biochemical assays, and reporter-based activity assays in cells, we hope to elucidate novel mechanisms of DNMT3A regulation that could be exploited for the treatment of hematopoietic disorders.