Abstract

Gene regulatory evolution provides a significant source of material for phenotypical change. However, there is only a limited understanding of what paths are possible for regulatory evolution, as most evidence is limited to either standing variation or biased perturbations of transcriptional enhancers. Using a synthetic mutation library for a developmental enhancer in Drosophila melanogaster, and an automated robotics pipeline, we show that most nucleotide mutations in a minimal enhancer cause changes in gene expression. These changes include transcription levels, probability, timing, and spatial patterns. We demonstrate that this pipeline can be used to identify novel transcription factor binding sites. Based on these sites, we present evidence for transcriptional cooperativity that makes the enhancer sensitive to nucleotide polymorphisms. We find that sets of mutations often simultaneously change levels and locations of expression. Together, our results suggest that the parameters of gene expression are not independent variables but rather convolved within an enhancer, and this codependency can constrain the evolvability of developmental enhancers.