Genetic interactions between DNA demethylation and methylation in Arabidopsis - PubMed (original) (raw)

Genetic interactions between DNA demethylation and methylation in Arabidopsis

Jon Penterman et al. Plant Physiol. 2007 Dec.

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.

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Figures

Figure 1.

Figure 1.

DML-target loci have features associated with DNA methylation. A, Percentage of DML-target genes and intergenic loci with siRNAs, transposons and repetitive elements, or both. B, Percentage of DML-target loci with 20-, 21-, 22-, 23-, and 24-nucleotide siRNAs.

Figure 2.

Figure 2.

ROS1 removes DNA methylation established and maintained by the PolIV/RDR2/DCL3/AGO4 pathway and DRM2. A to F, Percentage of methylation at At1g26400 (A and D), At1g34245 (B and E), and At5g38550 (C and F) in wild-type, ros1-3, ros1-3; rdr2-1, and ros1-3; drm2-1 plants. The ros1-3; rdr2-1 genotype in A to C was generated by selfing a ros1-3; rdr2-1 F2 line, and the ros1-3; rdr2-1 genotype in D to F was generated by selfing a ros1-3; RDR2/rdr2-1 plant. The ros1-3; drm2-1 F3 plant was generated by selfing a ros1-3; drm2-1 F2 line.

Figure 3.

Figure 3.

ROS1-target loci are hypermethylated in rdr2-1 and drm2-1 mutant plants. A to C, Percentage of methylation at At1g26400 (A), At1g34245 (B), and At5g38550 (C) in wild-type, ros1-3, rdr2-1, drm2-1, and rdr6-11 plants.

Figure 4.

Figure 4.

ROS1 expression is down-regulated in rdr2-1 and drm2-1 mutants. Shown is the expression of ROS1 and ACTIN11 (Actin) in ros1-3, rdr2-1, ros1-3/ROS1; rdr2-1/RDR2, ros1-3/ROS1; rdr2-1, drm2-1, and rdr6-11 plants (lanes 1–6, respectively).

Figure 5.

Figure 5.

RDR2 is required for ROS1 DNA demethylation. A, Inheritance of hypermethylated loci by siblings that differ in the absence or presence of an RDR2 allele. B to D, Percentage of methylation at At1g26400, At1g34245, and At5g38550 in the parental genotypes (rdr2 and ros1-3; RDR2/rdr2-1) and their progeny (ROS1/ros1-3; RDR2/rdr2-1 and ROS1/ros1-3; rdr2-1). Eight to 10 clones from the parents were sequenced, and 17 to 20 clones from the progeny were sequenced.

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