Molecular Mechanism Of DEMR1 In Regulation Of Nuclear And Mitochondrial Genomes DNA Demethylation

As one of the most important epigenetic modifications,the role of DNA methylation and DNA demethylation in participating in abiotic stress response has attracted widespread attention.Compared with DNA methylation,there are few studies on DNA demethylation.In recent years,although there have some studies on the regulatory factors of DNA demethylation in plants,there are few reports on the regulatory factors of abiotic stress response.Therefore,identification of new DNA demethylation regulatory factors involved in abiotic stress response is crucial to reveal the epigenetic regulation mechanism of DNA methylation in plants.In plants,active demethylation plays a more important role than passive demethylation.In order to identify new DNA demethylation regulators,we used methylation sensitive polymerase chain reaction（CHOP-PCR）technology,DT-77（Demethylation Target-77）as a marker locus to conduct genetic screening for mutants with increased DNA methylation levels.We screened multiple Arabidopsis mutants associated with abiotic stress and with elevated DNA methylation levels.Among them,an abscisic acid（ABA）sensitive mutant exhibits a slow growth and early flowering phenotype.We named it demr1（demethylation regulator 1）.Using the demr1 mutant,we mainly explored its regulatory role in active DNA demethylation and the research results are as follows:（1）Screening and identification of DNA demethylation defect mutant demr1The DNA methylation level of demr1 mutant at DT-77 locus was significantly higher than that of wild type when tested by CHOP-PCR.In order to confirm that the increase of DNA methylation levels of demr1 mutant was caused by the loss function of DEMR1 protein,we regenerated complementation lines under the background of demr1 mutant.The results of BSP（bisulfite sequencing PCR）and CHOP-PCR showed that the DNA methylation levels of the complementation lines at multiple detected loci such as DT-77 could recover to the levels of wild type.It indicates that DEMR1 protein is likely to participate in the regulation of genomic DNA methylation levels.（2）DEMR1 promotes ROS1 transcription and nuclear genome DNA demethylation by targeting ROS1 promoterRT-PCR and BSP analysis showed that in the demr1 mutant,the transcript levels of the DNA demethylase gene ROS1 was reduced by 50%,and the DNA methylation levels at the ROS1 promoter was reduced by 60%.It has been demonstrated that DEMR1 could directly bind to ROS1 promoter and promote ROS1 transcription in vivo and in vitro by LUC,MST,EMSA,and Ch IP-q PCR,and the binding was independent of ROS1 and SUVH complex.The binding of DEMR1 to the ROS1 promoter and the promotive effect of DEMR1 on ROS1transcription was not dependent on the DNA methylation levels of the ROS1 promoter.Overexpression of ROS1 gene in the demr1 mutant could inhibit the hyper-methylation levels at multiple loci.The whole genome bisulfite sequencing results of WT and demr1 mutant showed that the levels of DNA methylation in the whole genome of the demr1 mutant was significantly higher than that of the WT,and the demr1 mutant had the highest methylation levels in the transposon element（TE）regions.DEMR1 reduced DNA methylation levels at 172（approximately 40%）highly differentiated methylation regions（hyper-DMRs）in the nuclear genome through ROS1,indicating that DEMR1 is an important factor in the regulation of overall genomic methylation levels.（3）DEMR1 interacts with histone H4 to affect nuclear genome DNA demethylationDEMR1 could interact with histone H4 by LCI,Bi FC,pull-down and MST assays,forming the DEMR1-histone-ROS1 complex and participates in the demethylation process of genomic DNA.The interaction region was the second helical position of H4 which exposed outside the nucleosome.We found that the interaction between DEMR1 and histone H4 might not affect the methylation levels of H4 protein.（4）DEMR1 acts upstream of ROS1 to positively regulate ABA responsePhenotypic analysis revealed that the demr1 mutant and the ros1-4 mutant exhibited similar highly sensitive phenotypes to ABA.To demonstrate the relationship between DEMR1 and ROS1 in regulating ABA signaling pathways,we constructed double mutant demr1 ros1-4.Genetic analysis showed that the phenotypes of the ros1-4 mutant and the demr1 ros1-4 double mutant were more similar,indicating that DEMR1 acts upstream of ROS1 to positively regulate ABA response during seed germination and seedling establishment stages.（5）The loss of DEMR1 function leads to the increase of mitochondrial genomic DNA methylation levels and affects plant developmentGenome-wide bisulfite sequencing revealed that the mitochondrial genome is more highly methylated in the demr1 mutant than in the wild type,DEMR1 could also affect the methylation and expression levels of mitochondrial-related genes in the nuclear genome.We observed that the mitochondrial outer membrane structure of demr1 mutant was damaged by transmission electron microscope,and the biochemical testing showed that the ATP synthase activity in demr1 mutant was decreased.The regulation of DNA methylation levels of mitochondrial genome by DEMR1 might depend on ROS1 family members.In addition,DEMR1 could mitigate the H₂O₂ levels in plants to promote plant growth and development.Compared with WT,flowering time was about 2 days earlier and the number of rosette leaves at flowering was about 3 fewer,the diameter of rosette leaves decreased by about 11%in the demr1 mutant.In summary,our study shows that DEMR1 is a new demethylation regulator of nuclear and mitochondrial genomes DNA,participating in plant development and stress response.