ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing.

Constitutive heterochromatin in Arabidopsis thaliana is marked by repressive chromatin modifications, including DNA methylation, histone H3 dimethylation at Lys9 (H3K9me2) and monomethylation at Lys27 (H3K27me1). The enzymes catalyzing DNA methylation and H3K9me2 have been identified; alterations in these proteins lead to reactivation of silenced heterochromatic elements. The enzymes responsible for heterochromatic H3K27me1, in contrast, remain unknown. Here we show that the divergent SET-domain proteins ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6 have H3K27 monomethyltransferase activity, and atxr5 atxr6 double mutants have reduced H3K27me1 in vivo and show partial heterochromatin decondensation. Mutations in atxr5 and atxr6 also lead to transcriptional activation of repressed heterochromatic elements. Notably, H3K9me2 and DNA methylation are unaffected in double mutants. These results indicate that ATXR5 and ATXR6 form a new class of H3K27 methyltransferases and that H3K27me1 represents a previously uncharacterized pathway required for transcriptional repression in Arabidopsis

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

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

DNA methylation pathways and their crosstalk with histone methylation

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

SRA-domain proteins required for DRM2-mediated de novo DNA methylation.

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

DDR complex facilitates global association of RNA polymerase V to promoters and evolutionarily young transposons.

The plant-specific DNA-dependent RNA polymerase V (Pol V) evolved from Pol II to function in an RNA-directed DNA methylation pathway. Here, we have identified targets of Pol V in Arabidopsis thaliana on a genome-wide scale using ChIP-seq of NRPE1, the largest catalytic subunit of Pol V. We found that Pol V is enriched at promoters and evolutionarily recent transposons. This localization pattern is highly correlated with Pol V-dependent DNA methylation and small RNA accumulation. We also show that genome-wide chromatin association of Pol V is dependent on all members of a putative chromatin-remodeling complex termed DDR. Our study presents a genome-wide view of Pol V occupancy and sheds light on the mechanistic basis of Pol V localization. Furthermore, these findings suggest a role for Pol V and RNA-directed DNA methylation in genome surveillance and in responding to genome evolution

A dual flip-out mechanism for 5mC recognition by the Arabidopsis SUVH5 SRA domain and its impact on DNA methylation and H3K9 dimethylation in vivo

Cytosine DNA methylation is evolutionarily ancient, and in eukaryotes this epigenetic modification is associated with gene silencing. Proteins with SRA (SET- or RING-associated) methyl-binding domains are required for the establishment and/or maintenance of DNA methylation in both plants and mammals. The 5-methyl-cytosine (5mC)-binding specificity of several SRA domains have been characterized, and each one has a preference for DNA methylation in different sequence contexts. Here we demonstrate through mobility shift assays and calorimetric measurements that the SU(VAR)3-9 HOMOLOG 5 (SUVH5) SRA domain differs from other SRA domains in that it can bind methylated DNA in all contexts to similar extents. Crystal structures of the SUVH5 SRA domain bound to 5mC-containing DNA in either the fully or hemimethylated CG context or the methylated CHH context revealed a dual flip-out mechanism where both the 5mC and a base (5mC, C, or G, respectively) from the partner strand are simultaneously extruded from the DNA duplex and positioned within binding pockets of individual SRA domains. Our structure-based in vivo studies suggest that a functional SUVH5 SRA domain is required for both DNA methylation and accumulation of the H3K9 dimethyl modification in vivo, suggesting a role for the SRA domain in recruitment of SUVH5 to genomic loci