Mechanism Of MBD7on Regulating DNA Demethylation Mediated By ROS1in Arabidopsis

DNA methylation is one of the most important epigenetic marks in animals and plants, which is dynamically regulated through establishing, maintaining and removing. The dynamic regulation of DNA methylation in animals and plants makes their genomes plastic to grow, reproduce and survive from changing environments successfully. Although some active DNA demethylases have been found, such as ROS1, DME, DML2and DML3in Arabidopsis thaliana, the mechanism of how they are specifically recruited to their targets is still not well understood.In this study, we EMS mutagenized the C24transgenic plants that carry pro35S::NPTII and proRD29A::LUC transgenes, and screened for mutants in which the pro35S::NPTII transgene was silenced. In this screen, several new alleles of known anti-silencing factors have been found, such as the DNA demethylase ROS1. We also identified several new genes involved in anti-silencing pathway, such as ROS4/IDM1and ROS5/BDM2. ROS4/IDM1is a histone acetyltransferase that is required for ROS1function in DNA demethylation, and ROS5/IDM2is a small heat shock protein that interacts with ROS4/IDM1and is also involved in DNA demethylation. Here, we discovered a new anti-silencing protein MBD7(METHYL-CpG-BINDING DOMAIN7), a member of methyl-CpG binding domain proteins in Arabidopsis. Through genome-wide bisulfite sequencing, we found that the DNA methylation level at3’-NOS terminal region of pro35S::NPTII was obviously increased, and2664endogenous hypermethylated DMRs (Differentially Methylated Regions) were identified in mbd7mutant, despite the mild difference between C24and mbd7mutant on the whole genome, suggesting that MBD7is involved in DNA demethylation in specific genome loci.We found that MBD7can physically interact with ROS5/IDM2in vitro (using yeast two hybrid and pull-down asssays) and in vivo (using Co-IP). What’s more, several hypermethylated DMRs in ros5-1mutants were also hypermethylated in mbd7, and there were no additive effects observed in ros5-1mbd7double mutants. All these results indicate that MBD7and ROS5/IDM2may work together to regulate DNA demethylation. By analyzing the whole genome bisulfite sequencing data of mbd7, ros1-1, ros4and ros5-1mutants, we identified some overlapped hypermethylated loci in all four mutants. These results suggest that MBD7, ROS1, ROS4/IDM1and ROS5/IDM2function in the same DNA demethylation pathway at specific loci. MBD7is known to bind to methylated CG sites specifically in vitro. Here, we showed that MBD7can specifically bind to mbd7hypermethylated DMRs with high density DNA methylation around.In conclusion, MBD7binds to densely methylated DNA sequences and interacts with ROS5/IDM2, which can facilitate the rescuitment of ROS1for DNA demethylation. Our studies contribute to further understanding of DNA demethylation and increase the knowledge towards MBD proteins in plants.