Identi!cation of genes required for de novo DNA methylation in Arabidopsis

Cytosine DNA methylation is an ancient form of epigenetic information found in all kingdoms of eukaryotes. Functions for DNA methylation-mediated gene silencing include imprinting and repression of transposable elements. In mammals, methylation occurs almost exclusively in the CG context. Maintenance of the methyl-mark persists through function of the DNA methyltransferase, DNMT1, whereas the initial, or de novo, methylation of unmodified DNA occurs through DNMT3 activity. The importance of DNA methylation for proper development is underscored by embryonic lethality in knockout mice for either type of methyltransferase.1,2 De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late-!owering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM e"ectors AGO6 and NRPE5a for e#cient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1 and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation. Identi!cation of genes required for de novo DNA methylation in Arabidopsis