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

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. The majority of DNA in large eukaryotic genomes (such as the human genome) consists of transposons, sequences that can reproduce at the expense of their host. Plants and animals mark transposon DNA with a chemical modification called DNA methylation. DNA methylation prevents the functional information in transposons from being copied into RNA and utilized—this process is termed “gene silencing.” Using a flowering plant called Arabidopsis, the authors created mutants lacking a particular type of DNA methylation, and found that these plants had defects in leaf shape, plant height, and fertility. This shows that a gene-silencing mechanism used to defend the genome from transposons is also important for normal plant development. When the mutated genes are restored, plant development returns to normal, showing that one type of DNA methylation can be efficiently re-established (other gene-silencing marks can be lost irreversibly). Small RNA molecules are important for targeting DNA methylation to transposons and harmful DNAs. Mutants in genes that are important for making small RNAs have similar developmental defects to those lacking DNA methylation. This implies that normal plant development requires DNA methylation that is targeted by small RNAs.