The study of transcriptional enhancers that generate expression patterns poses unique challenges due to the irregularity of enhancer organization; however, studying the evolution of enhancer activity may further our knowledge of the mechanisms that underlie gene regulation. Recently, our laboratory discovered a novel expression pattern of Neprylisin1 (Nep1) in the visual system of Drosophila santomea that is not present in its closest relative, D. yakuba. By recreating and testing the enhancer sequences of the ancestor of D. yakuba and D. santomea, it was revealed that the strong santomea enhancer activity evolved from a weak latent activity of the gene’s intron. There are four mutations that have occurred within this enhancer between the yakuba/santomea ancestor and modern-day santomea, all of which contribute to enhancer activity. Studying these sites, along with the existing activities of the yakuba/santomea ancestral enhancer, should grant insights into the molecular mechanisms that regulate enhancers.
The novel Nep1 optical lobe enhancer in D. santomea overlaps several existing enhancers, and most likely evolved through the co-option of existing enhancer activity; however, new enhancer activity may also evolve through transposition, promoter switching or de novo. We have set up a new process for discovering such instances of novel activity that uses high-throughput RNA-sequencing (RNA-seq) to screen the entire transcriptomes of the imaginal wing discs from four closely related Drosophila species: D. mauritiana, D. sechellia, D. melanogaster and D. simulans. Genes upregulated in one species but not in its closest relatives are candidates for novel activity. We plan to study compelling examples of novel activity using the same techniques deployed to examine the evolution of the Nep1 optical lobe enhancer.