Crystal structures of REF6 and its complex with DNA reveal diverse recognition mechanisms

Abstract

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.

Removal of H3K27me3 by JMJ Proteins Controls Plant Development and Environmental Responses in Arabidopsis

“…Consistent with this, REF6 functions as a DNA sequence-specific H3K27me3 demethylase. Genomewide REF6 binding studies and crystal structure analysis revealed that the ZnF domains of REF6 recognize the CTCTGYTY DNA motif for H3K27me3 removal (Figures 1B, 2A) (Cui et al, 2016;Li et al, 2016;Tian et al, 2020). The ZnF domains of REF6 complex with NAC004 double-stranded (ds) DNA by forming a half-cross-braced structure (Figure 1B).…”

“…Interactions at the interface between REF6 and dsDNA, such as hydrogen bonds, electrostatic interactions, and hydrophobic interactions, strengthen their binding. dsDNA binding induces profound conformation changes (Tian et al, 2020). Conformational plasticity of DNA allows REF6 to recognize diverse target genes.…”

“…Methylation of CHG within the motif attenuates REF6-binding affinity (Figure 2F), and the minor groove width of each nucleotide in the structure of the complex differs considerably. This difference affects recognition of the CTCTGYTY motifs by REF6 and its binding affinity in CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 (Tian et al, 2020). The possibility that factors other than DNA sequence may contribute to REF6 binding affinity is supported by the fact that REF6 recognizes only 15% of the CTCTGYTY motifs in the Arabidopsis genome.…”

Histone Demethylases as Counterbalance to H3K27me3 Silencing in Plants

“…Genomic DNA motif discovery analyses defined that REF6 binds to a specific CTCTGYTY motif, where Y represents T or C ( Cui et al., 2016 ; Li et al., 2016 , 2018a ). This DNA-protein interaction has been proposed to define a novel half-cross braced ZnFn-type domain ( Tian et al., 2020 ). Interestingly, REF6 binding to DNA is inhibited by DNA methylation which explains why REF6 is depleted from heterochromatin regions in the genome ( Qiu et al., 2019 ; Tian et al., 2020 ).…”

“…This DNA-protein interaction has been proposed to define a novel half-cross braced ZnFn-type domain ( Tian et al., 2020 ). Interestingly, REF6 binding to DNA is inhibited by DNA methylation which explains why REF6 is depleted from heterochromatin regions in the genome ( Qiu et al., 2019 ; Tian et al., 2020 ).…”