Evidence for stabilizing selection in a eukaryotic enhancer element

Eukaryotic gene expression is mediated by compact cis-regulatory modules, o r enhancers, which are bound by specific sets of transcription factors(1). The combinatorial interaction of these bound transcription factors determin es time- and tissue-specific gene activation or repression. The even-skippe d stripe 2 element controls the expression of the second transverse stripe of a even-skipped messenger RNA in Drosophila melanogaster embryos, and is one of the best characterized eukaryotic enhancers(2-4). Although even-skip ped stripe 2 expression is strongly conserved in Drosophila, the stripe 2 e lement itself has undergone considerable evolutionary change in its binding -site sequences and the spacing between them. We have investigated this app arent contradiction, and here we show that two chimaeric enhancers, constru cted by swapping the 5' and 3' halves of the native stripe 2 elements of tw o species, no longer drive expression of a reporter gene in the wildtype pa ttern. Sequence differences between species have functional consequences, t herefore, but they are masked by other coevolved differences. On the basis of these results, we present a model for the evolution of eukaryotic regula tory sequences.