Adenine DNA methylation associated with transcriptionally permissive chromatin is widespread across eukaryotes.

DNA methylation is a key regulator of eukaryotic genomes, most commonly through 5-methylcytosine (5mC). In contrast, the existence and function of N(6)-methyladenine (6mA) in eukaryotes have been controversial, with conflicting reports resulting from methodological artifacts. Nevertheless, some unicellular lineages, including ciliates, early-branching fungi and the alga Chlamydomonas, show robust 6mA signals, raising questions about their origin and evolutionary role. Here we apply Oxford Nanopore sequencing to profile 6mA at base-pair resolution across 18 unicellular eukaryotes representing all major supergroups. We find that robust 6mA patterns occur only in species that encode the adenine methyltransferase AMT1. Notably, 6mA consistently accumulates downstream of transcriptional start sites, positioned between H3K4me3-marked nucleosomes, indicating a conserved association with transcriptional activation. Our results support the idea that the last eukaryotic common ancestor had a dual methylation system, with transcription-linked 6mA and repressive 5mC, which has been repeatedly simplified in both multicellular and unicellular lineages through the loss of the AMT1 pathway.