title: Identification of genes required for de novo DNA methylation in Arabidopsis.
abstract: 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 flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient 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.
authors.affiliation: Department of Molecular, Cell and Developmental Biology, University of California Davis, USA.
authors.forename: Maxim V C
authors.initials: MV
authors.lastname: Greenberg
authors.forename: Israel
authors.initials: I
authors.lastname: Ausin
authors.forename: Simon W L
authors.initials: SW
authors.lastname: Chan
authors.forename: Shawn J
authors.initials: SJ
authors.lastname: Cokus
authors.forename: Josh T
authors.initials: JT
authors.lastname: Cuperus
authors.forename: Suhua
authors.initials: S
authors.lastname: Feng
authors.forename: Julie A
authors.initials: JA
authors.lastname: Law
authors.forename: Carolyn
authors.initials: C
authors.lastname: Chu
authors.forename: Matteo
authors.initials: M
authors.lastname: Pellegrini
authors.forename: James C
authors.initials: JC
authors.lastname: Carrington
authors.forename: Steven E
authors.initials: SE
authors.lastname: Jacobsen
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D060565:Argonaute Proteins; D019175:DNA Methylation; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D005838:Genotype; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010641:Phenotype; D019076:Transgenes
chemical_list: C521197:AGO6 protein, Arabidopsis; D029681:Arabidopsis Proteins; D060565:Argonaute Proteins; D008780:Methyltransferases; C470732:DRM2 protein, Arabidopsis