A distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription.

Rediscovered as a potential eukaryotic epigenetic mark, DNA N-6-adenine methylation (6mA) varies across species in abundance and its relationships with transcription. Here we characterize AMT1-representing a distinct MT-A70 family methyltransferase-in the ciliate Tetrahymena thermophila. AMT1 loss-of-function leads to severe defects in growth and development. Single Molecule, Real-Time (SMRT) sequencing reveals that AMT1 is required for the bulk of 6mA and all symmetric methylation at the ApT dinucleotides. The detection of hemi-methylated ApT sites suggests a semiconservative mechanism for maintaining symmetric methylation. AMT1 affects expression of many genes; in particular, RAB46, encoding a Rab family GTPase involved in contractile vacuole function, is likely a direct target. The distribution of 6mA resembles H3K4 methylation and H2A.Z, two conserved epigenetic marks associated with RNA polymerase II transcription. Furthermore, strong 6mA and nucleosome positioning in wild-type cells is attenuated in Delta AMT1 cells. Our results support that AMT1-catalyzed 6mA is an integral part of the transcriptionassociated epigenetic landscape. AMT1 homologues are generally found in protists and basal fungi featuring ApT hyper-methylation associated with transcription, which are missing in animals, plants, and true fungi. This dichotomy of 6mA functions and the underlying molecular mechanisms may have implications in eukaryotic diversification.