A PIWI protein-dependent DNA N6-adenine methylation pathway in <i>Oxytricha</i> protects genomic sequences from deletion

The ciliate <i>Oxytricha</i> undergoes massive genome rearrangements during development to produce a functional nucleus from an encrypted zygotic genome. PIWI-interacting small RNAs protect DNA regions against deletion, but how that protective mark is established has been a mystery. Recently our lab discovered MTA1, a methyltransferase that catalyzes DNA N6-adenine (6mA) methylation. Both MTA1 and the <i>Oxytricha</i> Piwi protein, Otiwi1, are required for development, and Otiwi1 mutation eliminates 6mA signal. To examine the role of 6mA, we analyzed its genome-wide distribution across development in wild-type and MTA1 mutant backcrossed cells. We find specific and abundant enrichment on retained sequences, suggesting a protective role for this epigenetic mark. Furthermore, programmed retention of a DNA region that is normally deleted leads to accumulation of new 6mA marks on the ectopically retained DNA sequence. Together, these results suggest that piRNA-guided 6mA DNA methylation leads to protection of DNA sequences against deletion during nuclear differentiation.