Multimodal bHLH-PAS DNA binding controls specificity and drives obesity.

The basic-helix-loop-helix Per-Arnt-Sim (PAS) homology domain (bHLH-PAS) transcription factor (TF) family comprises critical sensors or actuators of physiological (hypoxia, tryptophan metabolites, neuronal activity, and appetite) and environmental (diet-derived metabolites and pollutants) stimuli regulating genes involved in signal adaptation and homeostasis. Despite the importance of this TF family, the mechanisms underlying specificity of DNA binding and target gene regulation by the bHLH-PAS subfamily remain unresolved. We systematically analysed cognate DNA binding hierarchies of prototypical bHLH-PAS family members (ARNT, ARNT2, HIF1α, HIF2α, AhR, NPAS4, SIM1), revealing large DNA binding footprints (12-15 bp) and unique mechanisms of DNA binding specificity involving preferential DNA sequences flanking the core motif. Flank-encoded DNA binding specificity discerns otherwise identical core sequence binding by SIM1 and the HIFs, mediated through N-terminal HIFα-DNA interactions. We also reveal an intimate relationship between DNA shape and core and flank TF binding that allows motif sequence flexibility and underpins multimodal mechanisms for achieving TF binding specificity. Furthermore, novel downstream SIM1 PAS-loop/DNA interactions are associated with AT-rich sequences contributing to DNA binding and transcriptional activity; these interactions are critical for TF biological function underpinning a monogenic cause of human hyperphagic obesity in a recapitulated SIM1.R171H knock-in mouse model.