DNA demethylation by DNA glycosylases in Arabidopsis

Plants defend their genomes by using DNA methylation to repress parasitic transposable elements. DNA methylation is very stable and reports of active demethylation have been limited. In the flowering plant Arabidopsis thaliana, DNA glycosylases of the DEMETER (DME) family remove methylcytosines from DNA. Demethylation by DME is necessary for genomic imprinting, and demethylation by a DME-LIKE (DML) protein, REPRESSOR OF SILENCING 1 (ROS 1), prevents gene silencing in a transgenic background. Prior to this work, the function of DML DNA glycosylases in wild type plants was not known.;Here I show that DML genes are expressed in every major plant organ. Reporter gene analysis shows that ROS1 and DML2 are expressed in a tissue-specific and age-dependent manner. Evidence for subnuclear ROS1 localization was also uncovered. Under certain conditions, the DML genes are required for normal plant growth and development, and ROS1 is required for normal resistance to a bacterial pathogen.;Using genome-tiling microarrays, ROS1, DML2, and DML3 were found to actively demethylate approximately 179 loci. More than 80% of these loci were at or near genes, which as a set were representative of the Arabidopsis genome. Mutations in DML genes lead to DNA hypermethylation and, in some cases, transcriptional repression of genes. Genic demethylation by DML enzymes primarily occurs at the 5' and 3' ends, a pattern opposite to the overall distribution of WT DNA methylation. These results imply that DML demethylation defines the genomic methylation profile of Arabidopsis genes.;RNA interference (RNAi) pathways establish and maintain DNA methylation. Genetic experiments confirmed that ROS1 removes methylation established by an RNAi pathway involving RNA DEPENDENT RNA POLYMERASE 2 (RDR2). Genes demethylated by DML enzymes were associated with transposons and enriched for small RNAs, which the RDR2-dependent RNAi pathway likely generates. Interestingly, the RDR2-dependent RNAi pathway was found to be required for ROS1 expression and hence demethylation by ROS1.;My results show that DML demethylation protects genes from potentially deleterious methylation established by plant's genome defense pathway. I propose demethylation by DML enzymes enables Arabidopsis to support robust pathways for genome defense.