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DNA methylation is essential for gene regulation, transposon silencing and imprinting. Although the generation of specific DNA methylation patterns is critical for these processes, how methylation is regulated at individual loci remains unclear. Here we show that a family of four putative chromatin remodeling factors, CLASSY (CLSY) 1-4, are required for both locus-specific and global regulation of DNA methylation in Arabidopsis thaliana. Mechanistically, these factors act in connection with RNA polymerase-IV (Pol-IV) to control the production of 24-nucleotide small interfering RNAs (24nt-siRNAs), which guide DNA methylation. Individually, the CLSYs regulate Pol-IV-chromatin association and 24nt-siRNA production at thousands of distinct loci, and together, they regulate essentially all 24nt-siRNAs. Depending on the CLSYs involved, this regulation relies on different repressive chromatin modifications to facilitate locus-specific control of DNA methylation. Given the conservation between methylation systems in plants and mammals, analogous pathways may operate in a broad range of organisms.

DNA methylation is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation are known, relatively little is understood about how DNA methylation influences gene expression. Here we identified a METHYL-CpG-BINDING DOMAIN 7 (MBD7) complex in Arabidopsis thaliana that suppresses the transcriptional silencing of two LUCIFERASE (LUC) reporters via a mechanism that is largely downstream of DNA methylation. Although mutations in components of the MBD7 complex resulted in modest increases in DNA methylation concomitant with decreased LUC expression, we found that these hyper-methylation and gene expression phenotypes can be genetically uncoupled. This finding, along with genome-wide profiling experiments showing minimal changes in DNA methylation upon disruption of the MBD7 complex, places the MBD7 complex amongst a small number of factors acting downstream of DNA methylation. This complex, however, is unique as it functions to suppress, rather than enforce, DNA methylation-mediated gene silencing. DOI: http://dx.doi.org/10.7554/eLife.19893.001