Isolation and Characterization of a Dioxin-InducibleCYP1A1Promoter/Enhancer Region from Zebrafish (Danio rerio)

Abstract

To isolate the CYP1A1 promoter/enhancer from zebrafish, a PAC genomic library was screened with sequence derived from the 5'UTR of the zfCYP1A1 cDNA. Sequence was identified that contained CAAT and TATA boxes, had a large intron within the 5'UTR, and showed 100% sequence identity to zfCYP1A1 cDNAs in the 5'UTR and initial 300 bp of the open reading frame. Oligonucleotides complementary to the 5'UTR were used to detect zfCYP1A1 mRNA in zebrafish liver cells (ZFL) exposed to TCDD, thus identifying the gene as a TCDD-inducible CYP1A1. Sequence analysis revealed that the 5' flanking region contained eight putative xenobiotic response elements (XRE) arranged in two distinct clusters. One cluster was localized between –580 and –187 and contained three XREs and two XREs bound zfAHR2 · rtARNTb complexes in vitro. However, this region was incapable of conferring TCDD-responsiveness to an SV40 promoter. In contrast, the region between –2608 and –2100 contained five XREs and was capable of driving TCDD-inducible expression when placed in either a forward or reverse orientation upstream or downstream of an SV40 promoter. Thus, the zfCYP1A1 gene has similar structural features to mammalian CYP1A1s and will be ideal for the analysis of AHR-mediated gene regulation in an aquatic organism.