Selection and characterization of sequences with high affinity for the engrailed protein of Drosophila

The engrailed gene helps to direct Drosophila melanogaster development by encoding a homeodomain-containing DNA binding protein. To identify genes whose transcription engrailed regulates, we developed a method to isolate genomic sequences to which engrailed protein binds with high affinity. Fragments of genomic DNA were fractionated on an engrailed protein affinity column, and fragments that were retained in the presence of 0.4-1.0 M KCl were isolated and cloned. The isolated fragments include regions of the engrailed and cubitus interruptus gene promoters, both of which are candidate targets of engrailed, and most fragments contain regions that engrailed protein protects from DNaseI digestion. Chromosomal deletions that remove some of the engrailed binding sites (located either at 64D, 96B or 99D) interact genetically with engrailed. Characterization of a transcript encoded in region 64D revealed its dependence on engrailed protein.