Transcriptional regulator coding-sequence evolution preceded cis-regulatory changes in the origin of a new transcriptional circuit

Life often diversifies through changes in gene expression patterns. These patterns evolve via changes in transcriptional regulatory circuits that are determined by transcriptional regulatory proteins and the cis-regulatory sequences they bind in the genome. While it has long been known that changes in cis-regulatory sequences can affect the evolution of gene expression patterns and that transcriptional regulatory proteins can themselves evolve, we know little of how these two types of regulatory changes occur together to generate new circuits. I discerned a stepwise order of evolutionary events in which both regulator protein-coding and cis-regulatory changes were necessary to evolve a new transcriptional regulatory circuit (repression of the a-specific genes by Mat⍺2 in yeast). The two changes evolved at separate points in time, millions of years apart. First to evolve were coding-sequence changes in the regulator that formed new protein-protein interaction regions. In one lineage, these new protein-protein interactions became necessary for Mat⍺2’s ancestral gene regulatory function (repression of the haploid-specific genes with Mata1). In another lineage, millions of years after the coding-sequence changes to Mat⍺2, cis-regulatory changes occurred in the a-specific genes, thereby co-opting Mat⍺2 for regulation of this new set of target genes. We propose that this evolutionary trajectory is an example of constructive neutral evolution in that Mat⍺2’s new protein-protein interactions initially had no consequence to the logic of cell-type specific gene regulation, but eventually allowed for the creation of a novel circuit (Chapter 2). In the course of these investigations, I also observed additional coding-sequence changes in the DNA-binding domain of Mat⍺2 (Chapter 3), and evolutionary changes in the identities of some of the yeast cell-type specific genes (Chapter 4). The results presented here add to our understanding of the ways in which transcriptional regulatory circuits diversify