Genetic Interactions between DNA Demethylation and Methylation in Arabidopsis

Abstract

DNA demethylation in Arabidopsis (Arabidopsis thaliana) is mediated by DNA glycosylases of the DEMETER family. Three DEMETER-LIKE (DML) proteins, REPRESSOR OF SILENCING1 (ROS1), DML2, and DML3, function to protect genes from potentially deleterious methylation. In Arabidopsis, much of the DNA methylation is directed by RNA interference (RNAi) pathways and used to defend the genome from transposable elements and their remnants, repetitive sequences. Here, we investigated the relationship between DML demethylation and RNAi-mediated DNA methylation. We found that genic regions demethylated by DML enzymes are enriched for small interfering RNAs and generally contain sequence repeats, transposons, or both. The most common class of small interfering RNAs was 24 nucleotides long, suggesting a role for an RNAi pathway that depends on RNA-DEPENDENT RNA POLYMERASE2 (RDR2). We show that ROS1 removes methylation that has multiple, independent origins, including de novo methylation directed by RDR2-dependent and -independent RNAi pathways. Interestingly, in rdr2 and drm2 mutant plants, we found that genes demethylated by ROS1 accumulate CG methylation, and we propose that this hypermethylation is due to the ROS1 down-regulation that occurs in these mutant backgrounds. Our observations support the hypothesis that DNA demethylation by DML enzymes is one mechanism by which Arabidopsis genes are protected from genome defense pathways.

An atypical component of RNA-directed DNA methylation machinery has both DNA methylation-dependent and -independent roles in locus-specific transcriptional gene silencing

“…We tested and found that the dtf1-1 mutation did not affect Pol V-dependent transcripts, while loss of the largest subunit of Pol V, NRPE1, blocked accumulation of the Pol V-dependent transcripts, including AtSN1 B and IGN5 B ( Figure 3A). Previous studies showed that mutations in canonical RdDM components suppress ROS1 expression [21,48,49]. We found that the dtf1-1 mutation also suppressed the expression of ROS1 ( Figure 3A).…”

Distinct size distribution of endogenous siRNAs in maize: Evidence from deep sequencing in the mop1-1 mutant

“…This is surprising because heterochromatic siRNAs are substantially diminished in rdr2, which one might have suspected would lead to stochastic activation of transposons and the generation of epialleles, such as reported for the ddm1 in Arabidopsis that dramatically reduces DNA methylation within repeats (27). However, it has been suggested that the rdr2 mutation causes a decrease in production of the demethylase ROS1, leading to hypermethylation at CG sites and additional protection against the activation of transposons (28). A more complete understanding of transposon reactivation in rdr2 and mop1 mutants will await larger-scale studies of DNA methylation in these mutants.…”

Repeat elements and the Arabidopsis DNA methylation landscape

“…Thus, the local DNA hypermethylation observed in ros1dml2dml3 is frequently associated with overaccumulation of matching siRNAs (Lister et al, 2008). Moreover, many second-site suppressors of ros1-induced hypermethylation and silencing are components of the RdDM machinery (Zheng et al, 2007;Penterman et al, 2007a;He et al, 2009a, b). These observations imply that at some loci, DNA methylation patterns result from the opposing action of RdDM and DNA demethylation.…”