RNAi, DRD1, and Histone Methylation Actively Target Developmentally Important Non-CG DNA Methylation in Arabidopsis

Simon W.-L Chan,Steven E Jacobsen,Xiaoyu Zhang,Jason S.-C Chien,Govind Shah,Ian R Henderson,John Doebley

doi:10.1371/journal.pgen.0020083

Simon W.-L Chan, Steven E Jacobsen + Show 5 more

Open Access

https://doi.org/10.1371/journal.pgen.0020083

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Abstract

Cytosine DNA methylation protects eukaryotic genomes by silencing transposons and harmful DNAs, but also regulates gene expression during normal development. Loss of CG methylation in the Arabidopsis thaliana met1 and ddm1 mutants causes varied and stochastic developmental defects that are often inherited independently of the original met1 or ddm1 mutation. Loss of non-CG methylation in plants with combined mutations in the DRM and CMT3 genes also causes a suite of developmental defects. We show here that the pleiotropic developmental defects of drm1 drm2 cmt3 triple mutant plants are fully recessive, and unlike phenotypes caused by met1 and ddm1, are not inherited independently of the drm and cmt3 mutations. Developmental phenotypes are also reversed when drm1 drm2 cmt3 plants are transformed with DRM2 or CMT3, implying that non-CG DNA methylation is efficiently re-established by sequence-specific signals. We provide evidence that these signals include RNA silencing though the 24-nucleotide short interfering RNA (siRNA) pathway as well as histone H3K9 methylation, both of which converge on the putative chromatin-remodeling protein DRD1. These signals act in at least three partially intersecting pathways that control the locus-specific patterning of non-CG methylation by the DRM2 and CMT3 methyltransferases. Our results suggest that non-CG DNA methylation that is inherited via a network of persistent targeting signals has been co-opted to regulate developmentally important genes.

Highlights

The met1 and ddm1 mutations that affect maintenance of CG DNA methylation cause severe and variable developmental defects, suggesting that DNA methylation can affect many developmental genes [1,2,3,4]
We further show that DRD1 works along with the 24-nucleotide short interfering RNA (siRNA) pathway in the establishment of DNA methylation, and works through both the DRM2 and CMT3 methyltransferases in the maintenance of DNA methylation
Unlike met1 phenotypes, all of these defects were entirely recessive when drm1–1 drm2–1 cmt3–7 was crossed to wild type Landsberg erecta (Ler) (Figure 1A)

Summary

Introduction

The met and ddm mutations that affect maintenance of CG DNA methylation cause severe and variable developmental defects, suggesting that DNA methylation can affect many developmental genes [1,2,3,4]. Our finding is consistent with the fact that multiple mutant alleles of drd were isolated from a screen for plants that could not maintain non-CG DNA methylation and transcriptional gene silencing targeted by an inverted repeat of the soybean a9 promoter [21], yet neither DRM2 nor CMT3 were identified by this screen This suggests that like AtSN1, the target of a9 siRNAs has non-CG DNA methylation that is controlled by DRD1, which acts through the redundant action of the DRM2 and CMT3 methyltransferases. The fact that normal development is largely restored when DRM2 or CMT3 are reintroduced suggests that these enzymes are actively targeted by signals that persist in the drm drm cmt mutant To test this model further, cmt was combined with null mutants in the RNA polymerase IV subunit– encoding gene NRPD2a and in DRD1, both of which are required for RNAi-directed DNA methylation [21,26,27,28]. Both RNAi pathways and the silencing-associated histone modification H3K9me can target non-CG DNA methylation to developmentally important genes

A Model for the Targeting of Locus Specific Non-CG DNA Methylation

Materials and Methods