ROS3 is an RNA-binding protein required for DNA demethylation in Arabidopsis

Abstract

DNA methylation is an important epigenetic mark for transcriptional gene silencing (TGS) in diverse organisms [1][2][3][4][5][6] . Recent studies suggest that the methylation status of a number of genes is dynamically regulated by methylation and demethylation [7][8][9][10] . In Arabidopsis, active DNA demethylation is mediated by the ROS1 (repressor of silencing 1) subfamily of 5-methylcytosine DNA glycosylases through a base excision repair pathway 8,[10][11][12][13] . These demethylases play critical roles in erasing DNA methylation and preventing TGS of target genes 7,8,10 . However, it is not known how the demethylases are targeted to specific sequences. We report here the identification of ROS3, an essential regulator of DNA demethylation that contains an RNA recognition motif. Analysis of ros3 mutant and ros1ros3 double mutant suggests that ROS3 acts in the same genetic pathway as ROS1 to prevent DNA hypermethylation and TGS. Gel mobility shift assays and analysis of ROS3 immunoprecipitate from plant extracts showed that ROS3 binds to small RNAs in vitro and in vivo. Immunostaining shows that ROS3 and ROS1 proteins colocalize in discrete foci dispersed throughout the nucleus. These results demonstrate a critical role for ROS3 in preventing DNA hypermethylation and suggest that DNA demethylation by ROS1 may be guided by RNAs bound to ROS3.We developed a sensitive assay system in Arabidopsis to genetically dissect active DNA demethylation 10,14 . The system consists of the RD29A-LUC transgene (firefly luciferase reporter driven by the stress-responsive RD29A promoter) and the non-allelic endogenous RD29A gene. The RD29A promoter is subjected to continuous siRNA-directed DNA methylation such that active DNA demethylation is required to keep the RD29A and RD29A-LUC genes transcriptionally active. In ros1 mutants, the RD29A promoter for both the transgene and endogenous gene becomes hypermethylated and both genes are silenced 10 . In addition, the 35S-NPTII transgene linked to RD29A-LUC is also silenced such that ros1 mutant plants are sensitive to kanamycin. We isolated the ros3 mutant from a T-DNA mutagenized population 15 Fig. 1a and Supplementary Fig. 1a and 1b) as well as sensitivity to kanamycin (Fig. 1b). Genetic analysis indicated that the ros3 mutation is recessive and affects a nuclear gene (data not shown).Northern blot ( Fig. 1c) and nuclear run-on ( Fig. 1d) Fig. 2a and 2b). Treatment with the cytosine methylation inhibitor 5-aza-2'-deoxycytidine increased RD29A-LUC expression in the ros3 mutant to the wild type level ( Supplementary Fig. 3). These results suggest that DNA hypermethylation is responsible for the TGS in ros3 mutant plants.The nrpd1a-1 mutation in the largest subunit of RNA polymerase IVa blocks the accumulation of 24-nt siRNAs corresponding to the RD29A promoter (data not shown). Analysis of nrpd1aros3 double mutant showed that the nrpd1a mutation causes a significant increase in RD29A-LUC expression ( Fig. 2c and 2d) and substantial decrease in CpG, CpNpG and CpNpN methyl...

Repeat elements and the Arabidopsis DNA methylation landscape

“…In contrast, the three other DNA demethylase genes are ubiquitously expressed and their protein products seem to be targeted to a limited number of loci (Penterman et al, 2007b;Lister et al, 2008). Although the targeting mechanism is unknown, genetic evidence suggest that small RNAs are involved (Mosher et al, 2008;Zheng et al, 2008;Hsieh et al, 2009).…”

“…These observations imply that at some loci, DNA methylation patterns result from the opposing action of RdDM and DNA demethylation. The identification of the small RNA-binding protein ROS3 as a component of the DNA demethylation machinery (Zheng et al, 2008) should help to define the number of loci targeted by the two pathways, as well as the factors involved in the balance between methylation and demethylation at each locus.…”

“…In contrast, the three other DNA demethylase genes are ubiquitously expressed and their protein products seem to be targeted to a limited number of loci (Penterman et al, 2007b;Lister et al, 2008). Although the targeting mechanism is unknown, genetic evidence suggest that small RNAs are involved (Mosher et al, 2008;Zheng et al, 2008;Hsieh et al, 2009).Interplay between DNA methylation and demethylation mechanismsAs expected from their distinct roles, the RdDM and maintenance DNA methylation pathways are, to a large extent, genetically independent. Thus, CG methylation is nearly unchanged in RdDM mutants, and significant amounts of CHH methylation and 24-nt siRNAs persist in ddm1 and met1 mutant plants Mathieu et al, 2007;Lister et al, 2008;Blevins et al, 2009;Slotkin et al, 2009;Teixeira et al, 2009).…”

Finding the fifth base: Genome-wide sequencing of cytosine methylation

“…Post-replicative maintenance of DNA methylation at CG sites is catalyzed by the DNA methyltransferase DNMT1 in mammals and its ortholog METHYL-TRANSFERASE 1 (MET1) in plants, while the plant-specific DNA methyltransferase CHROMOMETHYLASE 3 is required for maintenance of DNA methylation at CHG sites in plants (Finnegan and Dennis 1993;Bartee et al 2001;Bird 2002;Jackson et al 2002;Kankel et al 2003;Saze et al 2003;Goll and Bestor 2005). Furthermore, plants possess the demethylase enzymes ROS1, DME, DML2, and DML3 that remove methylcytosines by a baseexcision-mediated repair process (Gong et al 2002;Penterman et al 2007), and a recent study indicates that small RNA molecules can target the ROS1 demethylase to specific genomic target regions via the RNA-binding protein AT5G58130 (also known as ROS3) (Zheng et al 2008). Cokus et al (2008) generated a map of cytosine methylation at single-base resolution of the aerial tissues of A. thaliana, implementing a unique approach dubbed ''BS-seq'' to generate complex libraries of short BS-converted fragments of genomic DNA amenable to sequencing on the Illumina Genome Analyzer.…”

Mechanisms and evolution of genomic imprinting in plants

“…siRNAs in plants target de novo methylation at CHG (H is A, C or T) and CHH sites by the RNA interference machinery, involving the de novo methyltransferase DRM2 (Henderson and Jacobsen, 2007). However, also DNA demethylation might be targeted by siRNAs, based on the recent discovery of the siRNA binding protein ROS3, which acts in the DNA demethylation pathway involving the DME homolog ROS1 (Zheng et al, 2008). These findings raise the interesting possibility that maternal-specific siRNAs guide genome-wide hypomethylation in the endosperm.…”