Protein-coding changes preceded cis-regulatory gains in newly evolved transcription circuit

While changes in both the coding-sequence of transcriptional regulators and in the cis-regulatory sequences recognized by them have been implicated in the evolution of transcriptional circuits, little is known of how they evolve in concert. We describe an evolutionary pathway in fungi where a new transcriptional circuit (a-specific gene repression by Matα2) evolved by coding changes in an ancient master regulator followed millions of years later by cis-regulatory sequence changes in the genes of its future regulon. We discerned this order of events by analyzing a group of species in which the coding changes in the regulator are present, but the cis-regulatory changes in the target genes have not yet occurred. In this group we show that the coding changes became necessary for the regulator’s deeply conserved function and were therefore preserved. We propose that the changes first arose without altering the overall function of the regulator (although changing the details of its mechanism) and were later co-opted to “jump start” the formation of the new circuit. Gene expression was measured in wildtype a/α and α cell of the yeast W. anomalus, and compared to a/α and α cells with deletions of single genes encoding mating type transcriptional regulators.