Naturally Occurring Deletions of Hunchback Binding Sites in the <i>Even-Skipped</i> Stripe 3+7 Enhancer

<div><p>Changes in regulatory DNA contribute to phenotypic differences within and between taxa. Comparative studies show that many transcription factor binding sites (TFBS) are conserved between species whereas functional studies reveal that some mutations segregating within species alter TFBS function. Consistently, in this analysis of 13 regulatory elements in <i>Drosophila melanogaster</i> populations, single base and insertion/deletion polymorphism are rare in characterized regulatory elements. Experimentally defined TFBS are nearly devoid of segregating mutations and, as has been shown before, are quite conserved. For instance 8 of 11 Hunchback binding sites in the stripe 3+7 enhancer of <i>even-skipped</i> are conserved between <i>D. melanogaster</i> and <i>Drosophila virilis</i>. Oddly, we found a 72 bp deletion that removes one of these binding sites (Hb8), segregating within <i>D. melanogaster</i>. Furthermore, a 45 bp deletion polymorphism in the spacer between the stripe 3+7 and stripe 2 enhancers, removes another predicted Hunchback site. These two deletions are separated by ∼250 bp, sit on distinct haplotypes, and segregate at appreciable frequency. The <i>Hb8Δ</i> is at 5 to 35% frequency in the new world, but also shows cosmopolitan distribution. There is depletion of sequence variation on the <i>Hb8Δ</i>-carrying haplotype. Quantitative genetic tests indicate that <i>Hb8Δ</i> affects developmental time, but not viability of offspring. The Eve expression pattern differs between inbred lines, but the stripe 3 and 7 boundaries seem unaffected by <i>Hb8Δ</i>. The data reveal segregating variation in regulatory elements, which may reflect evolutionary turnover of characterized TFBS due to drift or co-evolution.</p>