Protein-DNA interactions in the promoter region of the gene encoding diapause hormone and pheromone biosynthesis activating neuropeptide of the cotton bollworm, Helicoverpa armigera.
Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are two crucial neuropeptides which regulate insect development and sex pheromone biosynthesis respectively. These peptides are encoded by a single gene, termed DH-PBAN gene. In this study, we characterized the promoter of the DH-PBAN gene in Helicoverpa armigera (Har). Transient transfection assays using a series of stepwise deletion fragments linked to the luciferase reporter gene indicate that the promoter contains multiple regulator domains that can activate and repress reporter gene expression. The fragment spanning -467 to -371 bp of the DH-PBAN promoter is an activator domain of transcription, whereas the region from -965 to -534 bp represses the promoter activity in the insect cell line BmN. Electrophoretic mobility shift assays demonstrate that at least two nuclear protein factors from the nuclear protein extracts of H. armigera suboesophageal ganglion, Har-DHMBP-1 and-2 (DH-modulator-binding protein) can specifically bind to the activating region. Furthermore, we characterized in detail that the nuclear protein factor Har-DHMBP-3 can specifically bind to a classical E-box, CAGCTG localized at positions -360 to -355 bp, a potential site for interaction with basic helix-loop-helix transcription factors. Mutation of this E-box results in a significant reduction of the promoter activity, suggesting it can modulate the previously identified activator domain. Taken together, multipartite cis-elements and transcription factors in the DH-PBAN promoter are involved in regulation of the gene expression.