| Evolutionary change in gene expression is thought to underlie the evolution of many developmental and morphological traits, While evolution of the cis-regulatory sequences which regulate transcription of genes is likely to be the basis for much of this change, we know very little about the evolutionary dynamics of these DNA sequences. Because the functional structure of cis-regulatory sequences, or promoters, cannot be inferred directly from the sequence, evolutionary studies must take advantage of those genes in which the regulation of transcription is well understood. In the studies described here, I examine genetic variation and molecular evolution within the cis-regulatory region of the sea urchin gene endo16, the transcriptional regulation of which has been previously elucidated in unparalleled detail.; By characterizing cis-regulatory genetic variation of endo16 within the species Strongylocentrotus purpuratus, as well as among species in the family Strongylocentrotidae, I show that distinct regulatory modules within the promoter exhibit markedly different levels of sequence polymorphism and evolution. Surprisingly, the functional units of cis-regulation, the short sequences targeted by transcription factor proteins, are found to be no more conserved than intervening nonfunctional sequences. Additionally, two previously characterized regulatory modules, composed of 16 transcription factor binding sites, were found to be the product of a single recent insertion. Lastly, using functional assays of protein binding, I show that much of the sequence polymorphism within transcription factor binding sites of S. purpuratus results in detectable variation in the interaction of proteins with those sites, a potential source of evolutionary variation in transcription. These studies reveal rapid evolution of functionally important sites within cis-regulatory sequences, and show that changes within these sites have the potential to underlie evolutionary variation in transcriptional regulation. |
