Temporal regulation of the mid-prepupal gene FTZ-F1: DHR3 early late gene product is one of the plural positive regulators

Background: Various ecdysteroid responsive genes play important roles in insect moulting and metamorphosis. Late FTZ-F1, a member of the nuclear receptor superfamily, is a unique transcription factor which is induced by a pulse exposure of 20-hydroxyecdysone. Elucidation of the regulation mechanism of this gene during prepupal period will help our understanding of metamorphosis at a molecular level. Results: Using transgenic fly lines carrying various transcription regulatory regions of the FTZ-F1 gene fused to the LacZ gene, we investigated cis-regulatory elements in the late FTZ-F1 transcription unit. The region which governs the stage-specific expression during pre-pupal period was narrowed down to 1.2kb, from -0.7 to +0.5 kb relative to the tran-scription start site. Electrophoresis mobility shift assays using staged extracts and various probes within the stage-specific region allowed us to identify binding sites for DHR3, an early late gene product, around 170 and 450 bp downstream of the transcription initiation site. Mutations disrupting these binding sites reduced the reporter gene expression without affecting the stage specificity. Conclusions: Our deletion and mutation studies of the cis-regulatory element of the FTZ-F1 gene suggest that the DHR3 binding sites located in the 5′ non-coding region are involved in the prepupal expression of the gene. These DHR3 binding sites confer high level expression while other elements are also involved in stage-specific expression.