A mutation in theDrosophila melanogaster evestripe 2 minimal enhancer is buffered by flanking sequences

AbstractEnhancers are DNA sequences composed of transcription factor binding sites that drive complex patterns of gene expression in space and time. Until recently, studying enhancers in their genomic context was technically challenging. Therefore, minimal enhancers, the shortest pieces of DNA that can drive an expression pattern that resembles a gene’s endogenous pattern, are often used to study features of enhancer function. However, evidence suggests that some enhancers require sequences outside the minimal enhancer to maintain function under environmental perturbations. We hypothesized that these additional sequences also prevent misexpression caused by a transcription factor binding site mutation within a minimal enhancer. Using theDrosophila melanogaster even-skippedstripe 2 enhancer as a case study, we tested the effect of a Giant binding site mutation (gt-2) on the expression patterns driven by minimal and extended enhancer reporter constructs. We found that, in contrast to the misexpression caused by the gt-2 binding site mutation in the minimal enhancer, the same gt-2 binding site mutation in the extended enhancer did not have an effect on expression. The buffering of expression levels, but not expression pattern, is partially explained by an additional Giant binding site outside the minimal enhancer. Mutating the gt-2 binding site in the endogenous locus had no significant effect on stripe 2 expression. Our results indicate that rules derived from mutating enhancer reporter constructs may not represent what occurs in the endogenous context.