DNA features beyond the transcription factor binding site specify target recognition by plant MYC2-related bHLH proteins

Transcription factors (TFs) regulate gene expression by binding to cis-regulatory sequences in the promoters of target genes. Recent research is helping to decipher in part the cis-regulatory code in eukaryotes, including plants, but it is not yet fully understood how paralogous TFs select their targets. Here we addressed this question by studying several proteins of the basic helix-loop-helix (bHLH) family of plant TFs, all of which recognize the same DNA motif. We focused on the MYC-related group of bHLHs, that redundantly regulate the jasmonate (JA) signaling pathway, and we observed a high correspondence between DNA-binding profiles in vitro and MYC function in vivo. We demonstrated that A/T-rich modules flanking the MYC-binding motif, conserved from bryophytes to higher plants, are essential for TF recognition. We observed particular DNA-shape features associated with A/T modules, indicating that the DNA shape may contribute to MYC DNA binding. We extended this analysis to 20 additional bHLHs and observed correspondence between in vitro binding and protein function, but it could not be attributed to A/T modules as in MYCs. We conclude that different bHLHs may have their own codes for DNA binding and specific selection of targets that, at least in the case of MYCs, depend on the TF-DNA interplay.