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 hyper-methylation 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 1000 genomic regions. SIZ1 physically interacts with ROS1 and mediates the SUMOylation of ROS1. The SUMOylation of ROS1 is reduced in siz1 mutant plants. Compared with that in wild-type plants, the protein level of ROS1 is significantly decreased, whereas there is an increased level of ROS1 transcripts in siz1 mutant plants. Our results suggest that SIZ1-mediated SUMOylation of ROS1 promotes its stability and positively regulates active DNA demethylation.

沉默1（ROS1）介导的活性DNA去甲基化的5-甲基胞嘧啶DNA糖基化酶/裂解酶阻遏物对于在拟南芥中塑造基因组DNA甲基化景观至关重要。ROS1的稳定性是否以及如何通过翻译后修饰来调控尚不清楚。使用基于甲基化敏感PCR（CHOP–PCR）的拟南芥DNA高甲基化突变体的正向遗传筛选，我们确定了SUMO E3连接酶SIZ1是活性DNA去甲基化的关键调节剂。SIZ1的功能障碍导致在大约1000个基因组区域发生高甲基化。SIZ1与ROS1发生物理相互作用，并介导ROS1的SUMOylation。siz1中ROS1的SUMOylation减少突变植物。与野生型植物相比，siz1突变体植物中的ROS1蛋白水平显着降低，而ROS1转录物水平却升高。我们的结果表明，ROS1的SIZ1介导的SUMOylation可以提高其稳定性并积极调节活性DNA的去甲基化。