Target gene regulation by the Broad-Complex at the onset of Drosophila metamorphosis

The steroid hormone ecdysone controls Drosophila metamorphosis by initiating a cascade of gene expression during which a small group of primary response transcriptional regulators activate a large set of tissue-specific effector genes. The Broad-Complex (BR-C) primary response gene encodes a family of related zinc finger-containing proteins and is genetically defined by several subfunctions required for target gene transcription. This thesis explores the interaction of BR-C proteins with several target genes at two developmental times in order to test the hypothesis that BR-C products directly regulate the transcription of these target genes. The intermolt genes are induced during the mid third instar larval period and depend on the $rbp\sp{+}$ and 2Bc$\sp{+}$ BR-C genetic functions for correctly timed, high level expression. BR-C proteins were shown by mobility shift and DNase I footprinting assays to bind to several functionally important sites in an essential regulatory element of the Sgs-4 intermolt gene, suggesting direct regulation of Sgs-4 by the BR-C. The L71 late genes are induced in salivary glands in response to the premetamorphic ecdysone pulse and are completely dependent on the $rbp\sp{+}$ function of the BR-C. Ectopic expression of BR-C Z1 isoforms using P element transgenic animals restored L71 gene expression in rbp mutants, correlating the Z1 isoforms with the $rbp\sp{+}$ function. L71 gene rescue was restricted to the prepupal salivary gland, and did not occur in BR-C null animals, suggesting the involvement of at least one additional factor in L71 gene regulation. Z1 isoform binding sites were localized to regulatory regions of the L71-6 gene, and alteration of these sites eliminated L71-6 expression in a P element reporter gene construct, demonstrating a direct effect of BR-C Z1 protein on L71-6 gene expression. Thus, the BR-C directly regulates target genes at two developmental times. A model of BR-C protein function at the onset of metamorphosis is proposed