UC Riverside

DNA methylation is a fundamental epigenetic mechanism that shapes genome function, chromatin organization, and cellular differentiation. Although the regulation of 5-methylcytosine (5mC) in higher eukaryotes has been extensively studied, key questions remain regarding how distinct DNA methyltransferases recognize DNA substrates under different chromatin contexts. This dissertation investigates the structure and regulation of representative eukaryotic DNA methyltransferases that establish either 5mC or N6-methyladenine (6mA). First, biochemical analysis of the Neurospora crassa cytosine methyltransferase Defective-In-Methylation-2 (DIM2) shows that its activation is tightly controlled by heterochromatin factors. The highly activated DIM2 requires the cooperativity between HP1 and H3K9me3. Three cryo-EM structures reveal how these multivalent inputs enable region-specific DNA methylation. Second, structural characterization of the Tetrahymena thermophila N6-adenine methyltransferase AMT complex captured in an inhibitor-trapped state reveals how a eukaryotic 6mA writer engages a DNA-mimicking substrate. The structure reveals that the AMT1, AMT7, AMTP1, and AMTP2 form a saddle shape in which the complex undergoes an open-to-closed transition during productive DNA engagement. Together, these studies provide structural and mechanistic insights into how eukaryotic DNA methyltransferases orchestrate protein cofactors in different chromatin contexts to enable DNA recognition for distinct methylation pathways. Together, this work expands the structural framework for understanding the regulation of both 5mC and 6mA methyltransferases across eukaryotes.