SIZ1-mediated SUMOylation of ROS1 Enhances Its Stability and Positively Regulates Active DNA Demethylation in Arabidopsis

The 5-methylcytosine DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1)-mediated active DNA demethylation is critical for shaping the genomic DNA methylation landscape in Arabidopsis. Whether and how the stability of ROS1 may be regulated by post-translational modifications is unknown. Using a methylation-sensitive PCR (CHOP-PCR)-based forward genetic screen for Arabidopsis DNA hypermethylation mutants, we identified the SUMO E3 ligase SIZ1 as a critical regulator of active DNA demethylation. Dysfunction of SIZ1 leads to hyper-methylation at approximately one thousand genomic regions. SIZ1 physically interacts with ROS1 and mediates the SUMOylation of ROS1. The SUMOylation of ROS1 is reduced in siz1 mutant plants. Compared to that in wild type plants, the protein level of ROS1 is significantly decreased, even though there is an increased level of ROS1 transcripts in siz1 mutant plants. Our results suggest that SIZ1 positively regulates active DNA demethylation by promoting the stability of ROS1 protein through SUMOylation. MethylC-Seq: siz1-2 mutant plant examined