UC Santa Barbara

DNA Methylation of cytosine to 5-methylcytosine (5mC) is a crucial epigenetic modification that plays a vital role in numerous biological processes, including cellular differentiation, genomic imprinting, and X-chromosome inactivation. De novo DNA methylation by DNMT3A plays a fundamental role in driving cell fate specification using allosteric regulation with nucleosomes and partner proteins. Mutations in DNMT3A are commonly found in patients with Acute Myeloid Leukemia (22%) where disruption of regulatory mechanisms leads to aberrant DNA methylation patterns. This has created a large interest in understanding the complex regulatory mechanisms that control DNMT3A activity and recruitment. Our lab has discovered a novel small molecule phthalazinone derivative allosteric inhibitor of DNMT3A with similar potency to current FDA approved drugs. By generating a library of analogs, we characterized the important pharmacophores and kinetic mechanism of the parent compound using radiochemical activity assays. We also investigated recruitment mechanisms of DNMT3A on nucleosomes using binding assays, radiochemical activity assays and nanopore 5mC sequencing. We found that DNMT3A has multi-valent interactions with nucleosomes utilizing that facilitate DNA methylation of the linker up to 24 bp away from the core. Additionally, our results indicate that DNMT3A1 binding is restricted to a single nucleosome, suggesting that its activity is not allosterically regulated by neighboring nucleosomes. Together these findings provide critical insights into the mechanisms of DNMT3A recruitment to nucleosomes and highlight the therapeutic potential of a novel DNMT3A allosteric inhibitor.