DEFORMED PROTEIN-BINDING SITES AND COFACTOR BINDING-SITES ARE REQUIRED FOR THE FUNCTION OF A SMALL SEGMENT-SPECIFIC REGULATORY ELEMENT IN DROSOPHILA EMBRYOS

How each of the homeotic selector proteins can regulate distinct sets of DNA target elements in embryos is not understood. Here we describe a detailed functional dissection of a small element that is specifical ly regulated by the Deformed homeotic protein. This 120 bp element (mo dule E) is part of a larger 2.7 kb autoregulatory enhancer that mainta ins Deformed (Dfd) transcription in the epidermis of the maxillary and mandibular segments of Drosophila embryos. In vitro binding assays sh ow that module E contains only one Dfd protein binding site. Mutations in the Dfd binding site that increase or decrease its in vitro affini ty for Dfd protein generate parallel changes in the regulatory activit y of module E in transgenic embryos, strong evidence that the in vitro -defined binding site is a direct target of Dfd protein in embryos. Ho wever, a monomer or multimer of the Dfd binding region alone is not su fficient to supply Dfd-dependent, segment-specific reporter gene expre ssion. An analysis of a systematic series of clustered point mutations in module E revealed that an additional region containing an imperfec t inverted repeat sequence is also required for the function of this h omeotic protein response element. The Dfd binding site and the putativ e cofactor binding site(s) in the region of the inverted repeat are bo th necessary and in combination sufficient for the function of module E.