Abstract
DNA methylation is a conserved epigenetic mark that is critical for many biological processes in plants and mammals. In Arabidopsis, the antagonistic activities of RNA-directed DNA methylation (RdDM) and ROS1-dependent active DNA demethylation are key for the dynamic regulation of locus-specific DNA methylation. However, the molecular factors that coordinate RdDM and active demethylation are largely unknown. Here we report that CLSY4 and its three paralogous SWI2/SNF2-type chromatin-remodeling proteins function in both RdDM and DNA demethylation in Arabidopsis. We initially identified CLSY4 in a genetic screen for DNA demethylation factors and subsequently demonstrated that it also is important in RdDM. Comprehensive genetic analyses using single and high order mutants of CLSY family proteins revealed their roles as double agents in the balance between methylation and demethylation reactions. The four CLSY proteins collectively are necessary for the canonical RdDM pathway; at the same time, each CLSY likely mediates DNA demethylation at specific loci where DNA methylation depends on RdDM. These results indicate that the four chromatin-remodeling proteins have dual functions in regulating genomic DNA methylation, and thus provide new insights into the dynamic regulation of DNA methylation in a model multicellular eukaryotic organism.
Highlights
DNA methylation is an important epigenetic mark that confers transcriptional regulation of genes and transposable elements[1,2,3,4]
In searching for molecular factors involved in active DNA demethylation, we conducted a genetic screen in a T-DNA mutant collection (Supplementary Table S1) using Chop-PCR to assess the DNA methylation status of two genomic loci, Pm36 and Pm27 (Supplementary Fig. S1a)
To confirm the DNA hypermethylation phenotype, we performed bisulfite sequencing at the two loci and found that DNA methylation levels in all cytosine contexts (CG, CHG, and CHH, where H represents A, T, and C) were increased in both clsy4/chr[40] alleles as is the case in the ros[1] mutant (Fig. 1c, d)
Summary
DNA methylation is an important epigenetic mark that confers transcriptional regulation of genes and transposable elements[1,2,3,4]. De novo DNA methylation can be established by the RNA-directed DNA methylation (RdDM) pathway, in which complementary pairing between 24-nt siRNAs and nascent scaffold RNAs, together with protein–protein interactions, recruits the DNA methyltransferase DRM2 for DNA methylation[4,7,8,9,10]. Production of most 24-nt siRNAs depends on the plant-specific RNA polymerase. Pol IV is recruited to RdDM targets in part by a SAWADEE domain-containing protein, SHH1/DTF1, which binds via its Tudor domain to histone H3 with methylated H3K918,19. Both Pol IV and DTF1/SHH1 associate with CLSY118,19, which is a chromatin-remodeling protein involved in Pol IV-dependent siRNA production[20]
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