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

Changes in both the coding sequence of transcriptional regulators and in the cis-regulatory sequences recognized by these regulators have been implicated in the evolution of transcriptional circuits. However, little is known about how they evolved in concert. We describe an evolutionary pathway in fungi where a new transcriptional circuit (a-specific gene repression by the homeodomain protein Matα2) evolved by coding changes in this ancient regulator, followed millions of years later by cis-regulatory sequence changes in the genes of its future regulon. By analyzing a group of species that has acquired the coding changes but not the cis-regulatory sites, we show that the coding changes became necessary for the regulator's deeply conserved function, thereby poising the regulator to jump-start formation of the new circuit.