DNA methylation occurs in CG and non-CG sequence contexts. Non-CG methylation is abundant in plants, and is mediated by CHROMOMETHYLASE (CMT) and DOMAINS REARRANGED METHYLTRANSFERASE (DRM) proteins; however its roles remain poorly understood. Here we characterize the roles of non-CG methylation in Arabidopsis thaliana. We show that a poorly characterized methyltransferase, CMT2, is a functional methyltransferase in vitro and in vivo. CMT2 preferentially binds histone H3 lysine 9 (H3K9) dimethylation and methylates non-CG cytosines that are regulated by H3K9 methylation. We revealed the contributions and redundancies between each non-CG methyltransferase in DNA methylation patterning and in regulating transcription. We also demonstrate extensive dependencies of small RNA accumulation and H3K9 methylation patterning on non-CG methylation, suggesting self-reinforcing mechanisms between these epigenetic factors. The results suggest that non-CG methylation patterns are critical in shaping the histone modification and small non-coding RNA landscapes.

Methylation of DNA and of histone 3 at Lys 9 (H3K9) are highly correlated with gene silencing in eukaryotes from fungi to humans. Both of these epigenetic marks need to be established at specific regions of the genome and then maintained at these sites through cell division. Protein structural domains that specifically recognize methylated DNA and methylated histones are key for targeting enzymes that catalyse these marks to appropriate genome sites. Genetic, genomic, structural and biochemical data reveal connections between these two epigenetic marks, and these domains mediate much of the crosstalk.

SUMMARY DNA methylation and histone modification exert epigenetic control over gene expression. CHG methylation by CHROMOMETHYLASE3 (CMT3) depends on histone H3K9 dimethylation (H3K9me2), but the mechanism underlying this relationship is poorly understood. Here, we report multiple lines of evidence that CMT3 interacts with H3K9me2-containing nucleosomes. CMT3 genome locations nearly perfectly correlated with H3K9me2 and CMT3 stably associated with H3K9me2-containing nucleosomes. Crystal structures of maize CMT3 homologue, ZMET2, in complex with H3K9me2 peptides, showed that ZMET2 binds H3K9me2 via both BAH- and chromo-domains. The structures reveal an aromatic cage within both BAH- and chromo-domains as interaction interfaces that capture H3K9me2. Mutations that abolish either interaction disrupt CMT3 binding to nucleosomes, and show a complete loss of CMT3 activity in vivo. Our study establishes dual recognition of H3K9me2 marks by BAH- and chromo-domains, and reveals a novel mechanism of interplay between DNA methylation and histone modification.

DNA methylation is an epigenetic modification that plays critical roles in gene silencing, development, and genome integrity. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and targeted by 24 nt small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM)1. This pathway requires two plant-specific RNA polymerases: Pol-IV, which functions to initiate siRNA biogenesis and Pol-V, which functions to generate scaffold transcripts that recruit downstream RdDM factors1,2. To understand the mechanisms controlling Pol-IV targeting we investigated the function of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1)3,4, a Pol-IV interacting protein3. Here we show that SHH1 acts upstream in the RdDM pathway to enable siRNA production from a large subset of the most active RdDM targets and that SHH1 is required for Pol-IV occupancy at these same loci. We also show that the SHH1 SAWADEE domain is a novel chromatin binding module that adopts a unique tandem Tudor-like fold and functions as a dual lysine reader, probing for both unmethylated K4 and methylated K9 modifications on the histone 3 (H3) tail. Finally, we show that key residues within both lysine binding pockets of SHH1 are required in vivo to maintain siRNA and DNA methylation levels as well as Pol-IV occupancy at RdDM targets, demonstrating a central role for methylated H3K9 binding in SHH1 function and providing the first insights into the mechanism of Pol-IV targeting. Given the parallels between methylation systems in plants and mammals1,5, a further understanding of this early targeting step may aid in our ability to control the expression of endogenous and newly introduced genes, which has broad implications for agriculture and gene therapy.

RNA-directed DNA methylation (RdDM) in Arabidopsis thaliana depends on the upstream synthesis of 24-nucleotide small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV)1,2 and downstream synthesis of non-coding transcripts by Pol V. Pol V transcripts are thought to interact with siRNAs which then recruit DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to methylate DNA3-7. The SU(VAR)3-9 homologs SUVH2 and SUVH9 act in this downstream step but the mechanism of their action is unknown8,9. Here we show that genome-wide Pol V association with chromatin redundantly requires, SUVH2 and SUVH9. Although SUVH2 and SUVH9 resemble histone methyltransferases a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-adenosyl methionine (SAM) binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins8. SUVH2 and SUVH9 both contain SET- and RING-ASSOCIATED (SRA) domains capable of binding methylated DNA8, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of DNA METHYLTRANSFERASE 1 (MET1) causes loss of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. Furthermore, tethering SUVH2 with a zinc finger to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. These results suggest that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of plant genomes for epigenetic silencing.

Small noncoding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the socalled piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that transgenerationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the histone 3 Lys9 trimethylation (H3K9me3) mark on genomic piRNA cluster sequences. The heterochromatin protein 1 (HP1) homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that transgenerationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels: by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors.

SUMMARY DNA methylation is a conserved epigenetic gene regulation mechanism. DOMAINS REARRANGED METHYLTRANSFERASE (DRM) is a key de novo methyltransferase in plants, but how DRM acts mechanistically is poorly understood. Here, we report the crystal structure of the methyltransferase domain of tobacco DRM (NtDRM) and reveal a molecular basis for its rearranged structure. NtDRM forms a functional homo-dimer critical for catalytic activity. We also show that Arabidopsis DRM2 exists in complex with the siRNA effector ARGONAUTE4 (AGO4) and preferentially methylates one DNA strand, likely the strand acting as the template for RNA polymerase V mediated non-coding RNA transcripts. This strand-biased DNA methylation is also positively correlated with strand-biased siRNA accumulation. These data suggest a model in which DRM2 is guided to target loci by AGO4-siRNA and involves base-pairing of associated siRNAs with nascent RNA transcripts.

Some plants acquire competence to flower in spring after experiencing a seasonal temperature drop-winter cold, in a process termed vernalization. In Arabidopsis thaliana, prolonged exposure to cold induces epigenetic silencing of the potent floral repressor locus FLOWERING LOCUS C (FLC) by Polycomb group (PcG) proteins, and this silencing is stably maintained in subsequent growth and development upon return to warm temperatures. Here we show that a cis-regulatory DNA element in the nucleation region for PcG silencing at FLC and two homologous trans-acting epigenome readers, VAL1 and VAL2, control vernalization-mediated FLC silencing. The sequence-specific readers recognize both the cis element (termed the cold memory element) and a repressive mark, trimethylation of histone H3 at lysine 27 (H3K27me3), and directly associate with LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), leading to establishment of the H3K27me3 peak in the nucleation region at FLC during vernalization. Thus, our work describes a mechanism for PcG-mediated silencing by a DNA sequence-specific epigenome reader.