SRA-Domain Proteins Required for DRM2-Mediated De Novo DNA Methylation

Lianna M Johnson,Julie A Law,Steven E Jacobsen,Anuj Khattar,Ian R Henderson,Tetsuji Kakutani

doi:10.1371/journal.pgen.1000280

Abstract

De novo DNA methylation and the maintenance of DNA methylation in asymmetrical sequence contexts is catalyzed by homologous proteins in plants (DRM2) and animals (DNMT3a/b). In plants, targeting of DRM2 depends on small interfering RNAs (siRNAs), although the molecular details are still unclear. Here, we show that two SRA-domain proteins (SUVH9 and SUVH2) are also essential for DRM2-mediated de novo and maintenance DNA methylation in Arabidopsis thaliana. At some loci, SUVH9 and SUVH2 act redundantly, while at other loci only SUVH2 is required, and this locus specificity correlates with the differing DNA-binding affinity of the SRA domains within SUVH9 and SUVH2. Specifically, SUVH9 preferentially binds methylated asymmetric sites, while SUVH2 preferentially binds methylated CG sites. The suvh9 and suvh2 mutations do not eliminate siRNAs, suggesting a role for SUVH9 and SUVH2 late in the RNA-directed DNA methylation pathway. With these new results, it is clear that SRA-domain proteins are involved in each of the three pathways leading to DNA methylation in Arabidopsis.

Highlights

Cytosine methylation is found in the genomes of most eukaryotes and plays a critical role in repression of transposable elements as well as in the epigenetic regulation of select genes [1,2,3,4]
While none of the single T-DNA mutants had any obvious phenotype, a suvh9 suvh2 kyp triple mutant displayed morphological differences from wild type. These morphological defects are indistinguishable from those displayed by a DNA methyltransferase triple mutant drm1 drm2 cmt3 (Figure 1A–B; DRM1 and DRM2 are tightly linked genes and in all cases the double mutant is examined though no activity has been ascribed to DRM1) [20,29]
This phenotype, which consists of curling of the leaves and short stature, has recently been shown to be caused by the ectopic expression of an F-box gene, SUPPRESSOR of DRM1 DRM2 CHROMOMETHYLASE 3 (CMT3) (SDC), which is silenced by non-CG DNA methylation occurring at tandem repeats found in its promoter [30]

Summary

Introduction

Cytosine methylation is found in the genomes of most eukaryotes and plays a critical role in repression of transposable elements as well as in the epigenetic regulation of select genes [1,2,3,4]. Maintenance of CG methylation is catalyzed by MET1, a homolog of mammalian DNMT1 [6,7]. Recent studies have shown that an SRA-domain protein is required to efficiently target DNMT1 to the replication fork and to maintain high levels of CG methylation [9,10,11]. A key to this targeting in mammals was shown to involve preferential binding of the UHRF1 SRA domain to hemi-methylated CG sites [10], which are the physiological substrates for DNMT1 that are generated after replication of methylated DNA. A plant homolog of UHRF1, VIM1/ ORTH2, binds methylated CG sites and is required for maintenance of DNA methylation in Arabidopsis [12,13], suggesting that this pathway is widely conserved in eukaryotes

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Conclusion