Relative of Early Flowing 6 (REF6) is a DNA-sequence-specific H3K27me3/2 demethylase that contains four zinc finger (ZnF) domains and targets several thousand genes in Arabidopsis thaliana. The ZnF domains are essential for binding target genes, but the structural basis remains unclear. Here, we determined crystal structures of the ZnF domains and REF6-DNA complex, revealing a unique REF6-family-specific half-cross-braced ZnF (RCZ) domain and two C2H2-type ZnFs. DNA-binding induces a profound conformational change in the hinge region of REF6. Each REF6 recognizes six bases and DNA methylation reduces the binding affinity. Both the acidic region and basic region are important for the self-association of REF6. The REF6 DNA-binding affinity is determined by the sequence-dependent conformations of DNA and also the cooperativity in different target motifs. The conformational plasticity enables REF6 to function as a global transcriptional regulator that directly binds to many diverse genes, revealing the structural basis for the epigenetic modification recognition.

Early Flowing 6 的亲属 (REF6) 是一种 DNA 序列特异性 H3K27me3/2 去甲基酶，包含四个锌指 (ZnF) 结构域，靶向拟南芥中​​的数千个基因。ZnF 结构域对于结合靶基因至关重要，但其结构基础仍不清楚。在这里，我们确定了 ZnF 结构域和 REF6-DNA 复合物的晶体结构，揭示了独特的 REF6 家族特异性半交叉支撑 ZnF (RCZ) 结构域和两个 C2H2 型 ZnF。DNA 结合诱导 REF6 铰链区发生深刻的构象变化。每个 REF6 识别 6 个碱基，DNA 甲基化会降低结合亲和力。酸性区域和碱性区域对于 REF6 的自缔合都很重要。REF6 DNA 结合亲和力由 DNA 的序列依赖性构象以及不同目标基序的协同性决定。构象可塑性使 REF6 能够作为全局转录调节因子，直接与许多不同的基因结合，揭示表观遗传修饰识别的结构基础。