Plants maintain cytosine methylation at CG and non-CG residues to control gene expression and genome stability. In a screen for Arabidopsis mutants that alter methylation and silencing of a densely methylated endogenous reporter gene, we recovered 11 loss-of-function alleles in the CMT3 chromomethylase gene. The cmt3 mutants displayed enhanced expression and reduced methylation of the reporter, particularly at non-CG cytosines. CNG methylation was also reduced at repetitive centromeric sequences. Thus, CMT3 is a key determinant for non-CG methylation. The lack of CMT homologs in animal genomes could account for the observation that in contrast to plants, animals maintain primarily CG methylation.

Cytosine methylation is critical for correct development and genome stability in mammals and plants. In order to elucidate the factors that control genomic DNA methylation patterning, a genetic screen for mutations that disrupt methylation-correlated silencing of the endogenous gene PAI2 was conducted in Arabidopsis. This screen yielded seven loss-of-function alleles in a SET domain protein with histone H3 Lys9 methyltransferase activity, SUVH4. The mutations conferred reduced cytosine methylation on PAI2, especially in non-CG sequence contexts, but did not affect methylation on another PAI locus carrying two genes arranged as an inverted repeat. Moreover, an unmethylated PAI2 gene could be methylated de novo in the suvh4 mutant background. These results suggest that SUVH4 is involved in maintenance but not establishment of methylation at particular genomic regions. In contrast, a heterochromatin protein 1 homolog, LHP1, had no effect on PAI methylation.

Transcribed inverted repeats are potent triggers for RNA interference and RNA-directed DNA methylation in plants through the production of double-stranded RNA (dsRNA). For example, a transcribed inverted repeat of endogenous genes in Arabidopsis thaliana, PAI1-PAI4, guides methylation of itself as well as two unlinked duplicated PAI genes, PAI2 and PAI3. In previous work, we found that mutations in the SUVH4/KYP histone H3 lysine 9 (H3 K9) methyltransferase cause a loss of DNA methylation on PAI2 and PAI3, but not on the inverted repeat. Here we use chromatin immunoprecipitation analysis to show that the transcribed inverted repeat carries H3 K9 methylation, which is maintained even in an suvh4 mutant. PAI1-PAI4 H3 K9 methylation and DNA methylation are also maintained in an suvh6 mutant, which is defective for a gene closely related to SUVH4. However, both epigenetic modifications are reduced at this locus in an suvh4 suvh6 double mutant. In contrast, SUVH6 does not play a significant role in maintenance of H3 K9 or DNA methylation on PAI2, transposon sequences, or centromere repeat sequences. Thus, SUVH6 is preferentially active at a dsRNA source locus versus targets for RNA-directed chromatin modifications.In eukaryotic genomes, condensed transcriptionally silent heterochromatin domains serve key roles in gene regulation and genome stability. In mammals and plants, heterochromatin is associated with cytosine methylation, as well as with histone tail modifications including H3 methylated at lysine 9 (H3 mK9). In many cases, these heterochromatin-associated modifications are directed to target sequences by a doublestranded RNA (dsRNA)-derived signal (26). For example, in plants, sources of dsRNA that can be converted into small RNAs by dicer RNase action, such as RNA viruses, products of RNA-dependent RNA polymerase synthesis, or transcripts derived from inverted repeat templates, direct DNA methylation and H3 mK9 of identical genomic sequences. However, the factors that connect RNA signals with DNA methylation and H3 mK9 remain to be elucidated.Plant RNA-directed DNA methylation typically affects cytosines in all sequence contexts (26). Genetic studies in Arabidopsis thaliana indicate that methylation in the symmetric context 5Ј CG 3Ј is maintained by the MET1 cytosine methyltransferase (MTase) (16, 37). Maintenance of methylation in non-CG contexts is more complex, with contributions from both the DRM1/DRM2 and CMT3 cytosine MTases. DRM1 and DRM2 control the initiation of new methylation imprints (7) but have different roles in the maintenance of non-CG methylation at different genomic regions (6). For example, although DRM1 and DRM2 are required to maintain non-CG methylation at direct repeat sequences that lie 3Ј to the MEA gene, they do not obviously contribute to maintenance of non-CG methylation at the Ta3 retrotransposon or at centromere-associated repeats. Instead, CMT3 is the major enzyme involved in maintenance of non-CG methylation at these targets (2,6,20), as well as at other transposon sequences ...