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.

Figure 1. Inheritance of drm1 drm2 cmt3 Developmental Phenotypes

(A) Developmental phenotypes of drm1–1 drm2–1 cmt3–7 are fully recessive when crossed to wild-type Ler. (B) The developmental phenotypes of the original drm1–1 drm2–1 cmt3–7 plants are inherited with the drm2–1 cmt3–7 genotype in the F2 of a backcross. Phenotypes were scored blindly before the plants were genotyped. The p-value was calculated from a chi square test based on the null hypothesis that the developmental phenotypes were segregating independently of the drm2–1 and cmt3–7 genotypes, such that one would only expect 1/16th of the twisted leaf dwarf plants to be drm2–1 cmt3–7 double mutants. The two plants scored as having a mutant phenotype that were not homozygous for drm2–1 cmt3–7 were drm2–1 cmt3–7/CMT3.

Figure 2. drm1 drm2 cmt3 Phenotypes Are Efficiently Restored to Wild Type by Transformed DRM2 or CMT3

(A) Normal rosette leaf shape and stature are restored in drm1–1 drm2–1 cmt3–7 transformed with either DRM2 or CMT3. (B) The sterility of drm1–1 drm2–1 cmt3–7 plants is partially restored in the T2 generation of DRM2 or CMT3 transformants. The length of ten mature siliques on the primary stem was measured; analysis was started at the third silique of the stem. Between eight and 36 individual plants were measured. Error bars show standard error of the mean.

Figure 3. DRD1 Is Required for De Novo DNA Methylation of Tandem Repeats

(A) DRD1 is required for de novo silencing of transformed FWA. Flowering time in untransformed and transformed T1 plants is shown—overexpression of FWA causes late flowering. Col, Columbia ecotype; WT, wild type. (B) DRD1 is required for de novo DNA methylation of transformed FWA. DNA methylation of the FWA transgene was measured by bisulfite genomic sequencing in T1 plants. Graph represents the percentage methylation in different sequence contexts. (C) Several RNAi mutants are competent for de novo silencing of transformed FWA. Flowering time for each transformed mutant is shown adjacent to its corresponding wild-type ecotype.

Figure 4. Role of DRD1 in Maintaining Non-CG DNA Methylation at Endogenous Loci

(A) The drd1–6 mutant cannot maintain non-CG DNA methylation at the endogenous direct repeats FWA and MEA-ISR. (B) drd1–6 loses all non-CG methylation at the SINE transposon AtSN1, and thus phenocopies drm1 drm2 cmt3. DNA methylation was measured by bisulfite genomic sequencing. (C) Southern blot analysis of DNA methylation at the pericentromeric retrotransposon Ta3. HpaII digestion at CCGG is blocked by CG or CNG DNA methylation, whereas MspI digestion at CCGG is blocked by CNG methylation. WT, wild type.

Figure 5. Developmentally Important Gene Regulation by DRM2 and CMT3 Requires RNAi, DRD1, and KRYPTONITE

Rosette leaf shape defects in a variety of multiple mutant combinations are shown. Col (WT), wild-type Columbia ecotype.

Figure 6. A Model for the Inheritance of Non-CG DNA Methylation in Arabidopsis thaliana

Background colors represent different pathways for targeting of non-CG DNA methylation. DRM2 is guided by an RNAi pathway initiated by DNA-dependent RNA polymerase IV. CMT3 is guided by histone H3 lysine 9 dimethylation (H3K9me2) that depends on KRYPTONITE/KYP. At some loci, histone H3 methylation is targeted by RNAi (represented by dotted arrow). DRD1 controls both DRM2 and CMT3, but there is also a DRD1-independent pathway that directs CMT3.