Activation of BMWCP10 promoter and regulation by BR-C Z2 in wing disc of Bombyx mori.

The cuticle protein gene BMWCP10 is transcriptionally upregulated by ecdysone during development. In the present study, using a transient reporter assay, the activity of various genomic segments at the 5'-flanking region of the BMWCP10 gene in driving gene expression and their involvement in ecdysone-mediated activation were assessed in the Bombyx wing disc. The promoter activity of BMWCP10 was responsive to 20-hydroxyecdysone (20E) in a dose-dependent manner, and the highest luciferase activity was observed in the presence of 2 microg/ml 20E. Furthermore, the upstream BMWCP10 promoter was activated by 20E in a stage-specific manner, and the 2.9-kb promoter contained essential elements for the temporal regulation of BMWCP10 in the Bombyx wing disc. Deletion studies revealed that the -598/-387 bp region was required for high-level transcription. In this region, a BR-C Z2 binding element was identified by electrophoretic mobility shift assay (EMSA). Site-directed mutagenesis of this element in the context of the 598-bp promoter fragment significantly decreased the reporter activity in response to ecdysone treatment. The results confirmed the role of BmBR-C Z2 in the transcription regulation of BMWCP10 and suggested the contribution of BmBR-C Z2 to BMWCP10 induction by 20E.