HR96 and BR-C modulate the phenobarbital induced transcription of cytochrome P450 CYP6D1 in Drosophila S2 cells

Phenobarbital (PB) is a prototypical inducer used for studies of xenobiotic responses in animals. In vertebrates, transcription factors CAR and PXR have been shown to mediate the PB induction of xenobiotic responsive P450 genes. Much less is known in insects about the transcription factors involved in PB induction of P450 genes, although CAR and PXR have a single homolog, HR96 (hormone receptor-like in 96), in Drosophila. We examined the promoter sequence of the PB inducible P450 CYP6D1 using the dual luciferase reporter assay in Drosophila S2 cells, and identified a region (between -330 and -280, relative to the position of transcription start site, +1) responsible for PB induction. Bioinformatics identified putative binding sites for transcription factors BR-C (broad-complex) and DFD (deformed) within this PB responsive promoter region. We used RNAi to examine the effects of silencing HR96, BR-C, or DFD on PB induction through PB responsive promoter region of CYP6D1. Our results showed that silencing of BR-C significantly increased, and silencing of HR96 significantly decreased the PB induced promoter activity, while silencing of DFD was without effect. These studies represent the first direct functional evidence for the involvement of an activator (HR96) and a repressor (BR-C) in PB mediated gene expression of insect xenobiotic responsive P450 genes, and offer new insights about the molecular basis of xenobiotic responses in insects.