Changes in locus wide repression underlie the evolution of Drosophila abdominal pigmentation.

Changes in gene regulation represent an important path to generate developmental differences affecting anatomical traits. Interspecific divergence in gene expression often results from changes in transcription-stimulating enhancer elements. While gene repression is crucial for precise spatiotemporal expression patterns, the relative contribution of repressive transcriptional silencers to regulatory evolution remains to be addressed. Here, we show that the Drosophila pigmentation gene ebony has mainly evolved through changes in the spatial domains of silencers patterning its abdominal expression. By precisely editing the endogenous ebony locus of D. melanogaster, we demonstrate the requirement of two redundant abdominal enhancers and three silencers that repress the redundant enhancers in a patterned manner. We observe a role for changes in these silencers in every case of ebony evolution observed to date. Our findings suggest that negative regulation by silencers likely has an under-appreciated role in gene regulatory evolution. Author summary: A central concept in the evo-devo field is that morphological evolution often involves changes in gene regulation. For a variety of reasons, most of the work has focused on the function of enhancers which activate gene expression in discrete patterns. However, this is because enhancers are easier to identify and characterize than other cis-regulatory elements that depend on the activity of multiple elements through long-range interactions. Here, we examined the role of interacting cis-regulatory elements in the regulation and evolution of the pigmentation gene ebony in Drosophila. We showed that in D. melanogaster, ebony abdominal expression is regulated by five cis-regulatory elements. Surprisingly, we found that evolutionary changes to the ebony silencers are sufficient to explain inter specific differences in expression patterns. Our results highlight the importance of silencers in the evolution of ebony regulation and point to a broader possible impact of silencers in the evolution of gene expression that may be prevalent but yet unnoticed. [ABSTRACT FROM AUTHOR]