Project Summary: Phenotypic diversity and adaptation can result from changes in protein function or changes in gene expression. The importance of the former has always been recognized, but a growing body of work over the last two decades has established the equal or greater importance of the latter as well. Variation in cis- regulatory sequences—such as promoters and enhancers—has often been found to underlie differences in gene expression and cause evolutionary divergence across a wide range of traits, from metabolism to morphology. Despite many case studies implicating cis-regulatory changes in phenotypic divergence, the specific mutations and mechanisms responsible for changes in cis-regulatory activity often remain unknown. In addition, experimental investigations of how cis-regulatory changes coevolve with their cognate proteins and cause either evolutionary divergence or maintain functional conservation remain sparse. This knowledge gap has largely been caused by a combination of technical limitations and the circumstantial division of most empirical evolutionary research into programs that focus on either the study of gene expression or protein function, though the value of a combined approach is well-recognized. These limitations can now be overcome in some systems because of advances in technology and the capacity to use integrative approaches that characterize variation in gene expression and protein function. Consequently, key questions such as how changes in cis-regulatory sequences change gene expression, and how the effects of such mutations are shaped by genetic interactions and coevolution with proteins are now amenable to direct experimental investigation. Here, I propose to address these questions by studying gene expression divergence of TDH3 in Saccharomyces yeast, an ideal system in which the Wittkopp Laboratory’s expertise in gene expression can be combined with my background in protein evolution to gain novel, more complete insight into the process of phenotypic divergence. Specifically, I will use variation in TDH3 promoter function between two closely related species to investigate 1) how cis-regulatory mutations cause gene expression divergence, 2) how mutations in protein coding sequences coevolved with these changes in the cis-regulatory sequence and with paralogous genes to impact metabolic function, and 3) how epistasis causes cis-regulatory mutations to have different effects in different genetic contexts, in turn affecting the role of coevolution and mutational contingency in gene expression divergence. The proposed research will provide a rare nucleotide-level case study of how mutations and their context-dependent interactions cause functional divergence of a cis-regulatory sequence and contribute mechanistic insight into how evolution of regulatory sequences and their cognate proteins affect phenotypic evolution. The results of this study will also advance our basic understanding of regulatory mutations and epi...