Abstract

Autoregulation has been implicated in the expression of many patterning genes in Drosophila, but the molecular details of this process are largely unknown. In the case of the segmentation gene even-skipped (eve), autoregulation is important for the specification of sharp stripes of gene expression at the onset of gastrulation. Here, we use a combination of DNA binding and P-transformation assays to characterize the cis- and trans-acting factors responsible for autoregulation. We show that eve autoregulation is mediated, at least in part, by a 100-bp minimal autoregulatory sequence (MAS) located approximately 5 kb upstream from the eve transcription start site. Multimerization of a 200-bp DNA fragment that encompasses the MAS drives optimal autoregulatory activity, comparable to that obtained with the native distal enhancer element located between -5.9 and -5.2 kb. The MAS contains two eve protein-binding sites, as well as binding sites for two nuclear factors present in early embryos. Directed mutagenesis of these binding sites suggests that both the eve protein and nuclear factors are essential for autoregulation. These results provide evidence that the eve protein acts combinatorially with other transcription factors to enhance its own expression.