Evolution of a Eukaryotic Transcription Factor’s co-TF Dependence Involves Multiple Intrinsically Disordered Regions Affecting Activation and Autoinhibition

Combinatorial control by multiple transcription factors (TFs) allows for increased specificity and information integration. Despite its prevalence in eukaryotes and crucial roles in gene regulation, the reason why eukaryotic TFs often depend on one another, and whether and how such codependency evolves, are not well understood. We exploit natural variation in co-TF dependence in the phosphate starvation response between related yeasts to address this question. In Saccharomyces cerevisiae , a bHLH TF, Pho4, relies on the homeodomain co-TF Pho2 to regulate ∼28 genes. By contrast, Pho4 in a related yeast pathogen, Candida glabrata , exhibits significantly reduced Pho2 dependence and an expanded target set of ∼70 genes. Biochemical analyses showed C. glabrata Pho4 (CgPho4) binds to the same consensus motif with a 3-4 fold higher affinity than ScPho4. A machine-learning-based prediction and yeast one-hybrid assay identified two Intrinsically Disordered Regions (IDRs) in CgPho4 that can boost the activity of the main activation domain but showed little to no activity on their own. We also found evidence for autoinhibition as a mechanism for the co-TF dependence. We identified an IDR in ScPho4 next to its DNA binding domain that functions as a double-edged sword: it both allows for enhanced TF activity with Pho2, but also constrains the activity when the co-TF is absent. Together, our results provide a detailed molecular picture of how co-TF dependence is mediated and how evolution in functional domains, including in the IDR of the TF, can lead to changes in codependency and result in significant rewiring of the regulatory targets. ### Competing Interest Statement The authors have declared no competing interest.