DNA methylation prevents REF6 binding in Arabidopsis

RELATIVE OF EARLY FLOWERING 6 (REF6/JMJ12), a Jumonji C (JmjC)-domain-containing histone demethylase, directly recognizes the CTCTGYTY motif by its zinc-finger domains to demethylate H3K27me3 at specific loci in Arabidopsis genome. Here we show that the recognition of CTCTGYTY motif by REF6 is prevented by DNA methylation. REF6 prefers to bind DNA hypo-methylated regions in vivo. DNA cytosine-methylation decreases REF6 binding affinity in vitro, and the crystal structure of ZnF-clusters showed that REF6 prefers unmethylated DNA sequences. Furthermore, REF6 displayed ectopic binding on multiple new target loci in drm1 drm2 cmt2 cmt3 (ddcc) quadruple mutants, where non-CG methylation was largely diminished. Collectively, this study reveals a novel targeting crosstalk between an H3K27me3 demethylase and DNA methylation. All mutant lines used in this study were in the Columbia ecotype background. Whole seedlings of 10 days after germination were harvested for ChIP-seq. ChIP-Seq experiments with anti-REF6 antibody were performed with three samples: Col (WT), ddcc (drm1-2; drm2-2; cmt2-7; cmt3-11 quadruple mutant), ref6-5 (mutant,Negative control). ChIP-Bisulfite-Seq experiments with anti-REF6 antibody were performed with three samples: Col (WT), ddcc (drm1-2; drm2-2; cmt2-7; cmt3-11 quadruple mutant), ref6-5 (mutant,Negative control).