Niu, J.; Yu, H.; Liu, Y.; Nie, L.; Nan, B.; Zhang, W.; Song, N.; Gao, S.

DNA N6-methyladenine (6mA) has recently been rediscovered as an epigenetic mark in eukaryotes. Several demethylases responsible for 6mA removal have been identified in multicellular eukaryotes. However, despite the much higher abundance of 6mA in unicellular species, no such enzymes have been reported and the biological significance of 6mA removal in these organisms remain unclear. Here, we identify and functionally characterize DMT3 as a dedicated 6mA demethylase in unicellular eukaryotes. DMT3 targets both fully and hemi-methylated ApT dinucleotides on DNA. It is predominantly localized at transcription start sites (TSS), where it antagonizes the activity of the 6mA methyltransferase AMT1. Deletion or catalytic inactivation of DMT3 leads to genome-wide ectopic 6mA deposition specifically at TSS regions, driven by AMT1, and is accompanied by increased chromatin accessibility and elevated enrichment of transcription-associated histone marks. These epigenetic alterations disrupt gene expression, reduce osmotic stress resistance, and impair the initiation of sexual reproduction. Together, our findings uncover a critical 6mA demethylase in unicellular eukaryotes, and highlight the essential role of active 6mA removal in maintaining transcriptional and developmental integrity.