Ancient transcriptional regulators can easily evolve new pair-wise cooperativity

We investigate how a protein-protein interaction between two ancient transcriptional regulators (a homeodomain and MADS box protein) was gained approximately 200 million years ago in a clade of ascomycete yeasts that includes Saccharomyces cerevisiae. We combine deep mutational scanning of Matα2 with a functional selection for cooperative gene expression and tested millions of possible alternative evolutionary solutions to this interaction interface. Pooled Matα2 mutants were deeply sequenced before and after selection for functional Matα2-Mcm1 interaction. The changes in frequency of each variant allowed us to determine their relative fitness. Targeted short-read sequencing of pooled Matα2 mutants, including from the starting library, following growth in rich media, and following growth in canavanine to select for functional Matα2-Mcm1 interactions.