A key mechanism determining how a fertilized egg develops into an adult animal is the spatial and temporal regulation of gene expression. This process is probably best understood in Drosophila, where a temporally ordered hierarchy of developmental control genes act to control patterns of gene expression. Our long term goal is understand the biochemical mechanisms regulating transcription of these developmental control genes, particularly the Ultrabithorax (Ubx) gene. Many of the developmental control genes, including many that regulate Ubx, share a homologous DNA binding domain, termed the homeodomain. Previously it has been impossible to rigorously determine the target DNAs bound by homeodomain proteins in embryos. To overcome this difficulty, we have developed an improved in vivo UV cross linking method. This method reveals that the homeodomain protein even skipped (eve) binds throughout the length of its target genes and at only ten fold lower levels to genes it was not initially expected to bind. We now propose, to use this method to probe the range of target genes bound by eve and other homeodomain proteins in vivo and to assess the importance of different mechanisms, including cooperative binding, to achieve occupancy of such a broad range of DNA sites. Since our experiments indicate that eve protein binds to promoter regions it had not previously been considered to regulate, we will use genetic and molecular experiments to examine the function of homeodomain proteins bound to these regions. Biochemical assays and electron microscopy will also be used to relate the in vitro properties of eve and other homeodomain proteins to the broad DNA binding observed in vivo. To better understand detailed biochemical mechanisms by which homeodomain proteins control transcription, we are studying how eve protein represses transcription in vitro. eve protein contains a repression domain, and we now propose to determine how this interacts with the general transcription factors to inhibit their function. These studies should provide a clear molecular description of how homeodomain proteins execute their biological function. The final pattern of Ubx transcription also requires the coordinate action of many other regulators which do not contain a homeodomain. We are studying how three of these factors, zeste, GAGA and NTF-1, regulate specific aspects of Ubx transcription. zeste and NTF-1 appear to be redundant regulators of Ubx, and we propose to use genetic experiments to examine the nature of this redundancy. It has been suggested that zeste mediates the action of distant enhancer elements, and in vivo UV cross linking will be used to determine if this occurs by cooperative binding at a distance. Finally, a possible role for GAGA in mediating repression of Ubx by polycomb will be examined.