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

The ciliate Oxytricha 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 Oxytricha 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. HighlightsO_LIDNA N6-methyladenine accumulates on retained DNA regions during Oxytricha development. C_LIO_LImta1 mutant backcrosses have disrupted DNA methylation and a developmental delay. C_LIO_LIDNA N6-adenine methylation during genome rearrangement requires the presence of Otiwi1. C_LIO_LIProgrammed retention of a germline-limited region leads to developmental methylation. C_LI