Structure, Function, and Evolution of a Signal-Regulated Enhancer.

Abstract

Enhancers are cis-regulatory elements that control the level, timing, and cell-type specificity of gene expression. Despite the central role that enhancers play in transcriptional regulation, the rules that govern their composition and organization are poorly understood. To elucidate the relationship between enhancer structure and function, we performed an in-depth analysis of a signal-regulated enhancer in D. melanogaster, the D-Pax2 sparkling (spa) enhancer. This enhancer drives cone cell-specific expression in the Drosophila eye, where D-Pax2 is required for cone cell fate specification. Although twelve transcription factor binding sites have been previously identified within spa, we have found that those sites are not sufficient for cone cell-specific gene expression. We have identified novel regulatory sequences within spa that are essential for enhancer function in vivo, revealing significant complexity in the regulatory circuitry of this enhancer. We also found that binding site organization is tightly linked to function in spa; rearrangements of the regulatory elements within spa can affect both the levels and patterning of gene expression. These data indicate that organizational rules constrain spa structure. However, evolutionary comparisons show that the sequence and structure of spa are evolving rapidly, suggesting that some aspects of spa structure are flexible. Our results shed light on the highly combinatorial nature of enhancer regulation, as well as the complex structural rules that govern enhancer function in vivo.