Functional regulatory evolution outside of the minimal stripe 2 enhancer.

Transcriptional enhancers are regions of DNA that drive precise patterns of gene expression. Although many studies have elucidated how individual enhancers can evolve, most of this work has focused on what are called 'minimal' enhancers, the smallest DNA regions that drive expression that approximates an aspect of native gene expression. Here, we explore how the () locus has evolved by testing its activity in the divergent genome. We found, as has been reported previously, that the stripe 2 enhancer () fails to drive appreciable expression in However, we found that a large transgene carrying the entire locus drives normal expression, including in stripe 2. We performed a functional dissection of the region upstream of the region and found multiple Zelda motifs that are required for normal expression. Our results illustrate how sequences outside of minimal enhancer regions can evolve functionally through mechanisms other than changes in transcription factor-binding sites that drive patterning.