DNA methylation is a critical epigenetic modification controlling transcriptional regulation in mammals. New 5-methylcytosine (5mC) patterns are created by DNMT3A and the closely related DNMT3B, but the mechanisms that control their activity and genomic site selection is not understood. To understand how DNA methylation patterns are created, the enzymatic properties of DNMT3A were determined and it was examined how they are regulated by homo- and hetero-protein complexes, environmental conditions (pH), DNA topology, and noncoding RNA.

First, DNMT3A was probed to determine its ability to methylate multiple sites before dissociating from the DNA. Using chase assays and mathematical modeling, DNMT3A was shown to methylate human promoters processively. Processive methylation was also enhanced by the catalytically inactive homolog DNMT3L and modulated by DNA topology. Processive de novo DNA methylation would provide a mechanism for transcriptional regulation and silencing of newly integrated viral DNA.

The ability of DNMT3A to be processive is due to oligomerization at both the dimer and termer interfaces; shown by testing single amino acid mutations, including those implicated in the development of acute myeloid leukemia (AML), at the homotetramer, homodimer, and DNMT3A:DNMT3L heterotetramer interfaces. A model for the DNMT3A homotetramer was developed via computational interface scanning and tested using light scattering and electrophoretic mobility shift assays. Distinct oligomeric states were functionally characterized using fluorescence anisotropy and steady state kinetics. Replacement of residues that result in DNMT3A dimers, including those identified in AML patients, show minor changes in methylation activity but lose the capacity for processive catalysis on multisite DNA substrates, unlike the processive wild type enzyme. The dimer interface of DNMT3A is also dynamic and changes with protein concentration, DNA, tetramer interface occupation, DNMT3L, pH, and somatic mutations. Our results are consistent with the bimodal distribution of DNA methylation in vivo and provide a possible mechanism that accounts for the changes in methylation patterns that are observed in AML patients with DNMT3A mutations that may contribute to oncogenesis and its progression.

Lastly, two modes of RNA regulation is observed in vitro, including RNA binding allosterically and causing no change in catalysis and another RNA molecule that binds tightly to the catalytic domain in a structurally dependent fashion and causing potent inhibition.