Dual control of ROS1‐mediated active DNA demethylation by DNA damage‐binding protein 2 (DDB2)

Summary By controlling gene expression, DNA methylation contributes to key regulatory processes during plant development. Genomic methylation patterns are dynamic and must be properly maintained and/or re‐established upon DNA replication and active removal, and therefore require sophisticated control mechanisms. Here we identify direct interplay between the DNA repair factor DNA damage‐binding protein 2 ( DDB 2) and the ROS 1‐mediated active DNA demethylation pathway in Arabidopsis thaliana . We show that DDB 2 forms a complex with ROS 1 and AGO 4 and that they act at the ROS 1 locus to modulate levels of DNA methylation and therefore ROS 1 expression. We found that DDB 2 represses enzymatic activity of ROS 1. DNA demethylation intermediates generated by ROS 1 are processed by the DNA 3′‐phosphatase ZDP and the apurinic/apyrimidinic endonuclease APE 1L, and we also show that DDB 2 interacts with both enzymes and stimulates their activities. Taken together, our results indicate that DDB 2 acts as a critical regulator of ROS 1‐mediated active DNA demethylation.