Crystal Structures of REF6 and Its Complex with DNA Reveal Diverse Recognition Mechanisms

Abstract

RELATIVE OF EARLY FLOWERING 6 (REF6) is a DNA-sequence-specific H3K27me3/2 demethylase containing four zinc finger (ZnF) domains, targeting several thousand genes in Arabidopsis thaliana. The ZnF domains are essential for binding target genes, but the structural basis and mechanisms remain unclear. Here, we determined crystal structures of the ZnF domains and its dsDNA complex. It is composed of a unique REF6-family-specific half-cross-braced ZnF (RCZ) domain and two C2H2-type ZnFs. DNA binding induces a profound conformational change of REF6 at the hinge region. Each REF6 recognizes six bases and DNA methylation reduces the binding affinity. Both the acidic region and basic region are crucial for the self-association of REF6. The binding ability and specificity are enhanced by combining base and shape readout, and cooperativity. The resulting conformational plasticity enables REF6 to function as a global transcriptional regulator that directly binds many diverse genes, revealing the structural basis of recognizing epigenetic modification.