DNA demethylase augments RNA-directed DNA methylation by enhancing CLSY gene expression in maize and Arabidopsis.

DNA methylation and demethylation activities are coordinated to maintain DNA methylation patterns. However, the interplay between them remains to be explored. In this study, we found that loss of DNA demethylase in maize results in a significant decrease in CHH methylation, a hallmark of RNA-directed DNA methylation (RdDM). Further analyses suggested that this is caused by reduced expression in the DNA demethylase mutant of three CLASSY (CLSY) genes, which encode chromatin remodelers controlling small RNA production. Series of molecular assays confirm that the expression of three maize CLSY genes is sensitive to the DNA methylation levels at their promoter regions, which are regulated by both the DNA demethylase and the RdDM pathway. Moreover, we revealed that Arabidopsis DNA demethylase mutants also show decreased CHH methylation and reduced expression of CLSY1, one of four CLSY genes in Arabidopsis. Similar to the observations in maize, the expression of CLSY1 is associated with DNA methylation levels of its promoter that is targeted by both DNA demethylase and RdDM pathways. Taken together, these results suggest a conserved interplay between DNA demethylation and RdDM pathways, revealing a mechanism to maintain the homeostasis of DNA methylation levels across plants.