Cloning and analysis of the CYP1A promoter from the atlantic killifish (Fundulus heteroclitus)

Abstract

Enzymes in the cytochrome P450 gene family 1 (CYP1) catalyze the metabolic activation of numerous hydrocarbon carcinogens and various natural compounds. CYP1 family members have been identified in several vertebrates, including fish, amphibians, birds, and mammals, and are inducible by aromatic hydrocarbons acting through the aryl hydrocarbon receptor (AHR). Together with its heterodimeric partner ARNT, the ligand-bound AHR binds conserved xenobiotic response elements (XREs) near the promoter of CYP1A and other genes. However, some populations of the Atlantic killifish Fundulus heteroclitus inhabiting highly contaminated sites are refractory to CYP1A induction by aromatic hydrocarbons. To better understand the mechanisms underlying this phenomenon, we are characterizing the AHR-CYP1A signaling pathway in this species. We report here the characterization of a genomic clone containing the 5(') end of the wild-type F. heteroclitus CYP1A gene. The 5(') coding sequence matches that of the F. heteroclitus CYP1A cDNA reported earlier [Comp. Biochem. Physiol. 121C (1998) 231]. Consistent with its inducibility by AHR agonists, the CYP1A gene contains three consensus XREs (5(')CACGC3(')) within 1.6 kb of the putative transcriptional start site. When oligonucleotides containing each of these sites were analyzed in an electrophoretic mobility shift assay, one of these showed a strong, TCDD-inducible mobility shift in the presence of in vitro expressed mouse AHR protein. These sequence data and initial functional characterization provide a valuable tool for the study of genetic variations in CYP1A expression and activity in sensitive and resistant populations. These studies may ultimately shed light on the importance of P4501A activity in xenobiotic toxicity.