Cis-regulatory evolution integrated the Bric-à-brac transcription factors into a novel fruit fly gene regulatory network.

Gene expression evolution through gene regulatory network (GRN) changes has gained appreciation as a driver of morphological evolution. However, understanding how GRNs evolve is hampered by finding relevant cis-regulatory element (CRE) mutations, and interpreting the protein-DNA interactions they alter. We investigated evolutionary changes in the duplicated Bric-à-brac (Bab) transcription factors and a key Bab target gene in a GRN underlying the novel dimorphic pigmentation of D. melanogaster and its relatives. It has remained uncertain how Bab was integrated within the pigmentation GRN. Here, we show that the ancestral transcription factor activity of Bab gained a role in sculpting sex-specific pigmentation through the evolution of binding sites in a CRE of the pigment-promoting yellow gene. This work demonstrates how a new trait can evolve by incorporating existing transcription factors into a GRN through CRE evolution, an evolutionary path likely to predominate newly evolved functions of transcription factors.