Hormonal regulation of the E75 gene in Drosophila: identifying functional regulatory elements through computational and biological analysis.

Drosophila development is regulated by two hormones, 20-hydroxyecdysone (ecdysone) and juvenile hormone. We previously found that expression of the E75 gene is induced by both hormones in cultured S2 cells. E75 occupies over 100 kb of genomic DNA; it has four alternative promoters producing isoforms E75A, E75B, E75C, and E75D. To identify hormone response elements in the 60-kb noncoding area upstream of the E75A transcription start site, we developed a novel approach combining in vitro, in vivo, and in silico techniques. Using chromatin immunoprecipitation coupled with quantitative real-time PCR, we identified five putative enhancers marked with H3K4 monomethylation and depletion of H3. Four of these are ecdysone-regulated enhancers, which possess hormone-responsive chromatin and contain sequences sufficient to confer ecdysone inducibility to a reporter gene. Using EvoPrinterHD- and Multiple Expectation Maximization for Motif Elicitation-based computational analysis, we first created a database of short sequences that are highly conserved among 12 Drosophila species. Within this database, we then identified a set of putative ecdysone response elements (EcREs). Seven of these elements represent in vivo binding sites for the ecdysone receptor and are necessary for hormone-mediated activation of gene expression in cultured cells. We found that each EcRE exhibits different binding and activation properties, and at least some of them function cooperatively.We propose that the presence of multiple EcREs with distinct features provides flexibility to the rapid and powerful response of E75A to ecdysone during Drosophila development.