Trout CYP1A3 gene: Recognition of fish DNA motifs by mouse regulatory proteins

Transcriptional up-regulation of mammalian CYP1A1 genes by dioxin is known to require binding of dioxin to the Ah receptor (AHR), subsequent interacti on of this ligand-receptor complex with the AHR nuclear translocator (ARNT) , and binding of this heterodimer to aromatic hydrocarbon response elements (AHREs) located in the 5' flanking sequences. From the rainbow trout (Onco rhyncus mykiss), we have isolated and sequenced the CYP1A3 gene-spanning 4. 0 kb and containing seven exons and six introns-and 1897 bp of the 5' flank ing region. The transcription start site was determined by primer extension analysis. Five putative AHREs were found between -451 and -1820, with an o verlap of AHRE3 and AHRE4 sharing 1 bp. The 5' flanking region of the trout CYP1A3 gene was fused to the firefly luciferase (luc) reporter gene and tr ansiently transfected into mouse hepatoma Hepa-1c1c7 wild-type (wt) cell cu ltures and three benzo[a]pyrene-resistant mutant lines: c2, containing less than 10% levels of functional AHR; c4, defective in ARNT; and c37, deficie nt in CYP1A1 metabolism. We compared the trout CYP1A3 promoter-luc construc ts with mouse and human CYP1A1 promoter-luc constructs. Ah of our trout CYP 1A3 promoter data are consistent with dioxin-inducible luciferase activity being controlled by two or more AHREs via cooperativity with a GC-rich regi on (-1852)-as has previously been demonstrated for AHREs in mammalian CYP1A 1 promoters. The dependence of trout CYP1A3 promoter activity on the AHR an d on the ARNT, and the enhancement of CYP1A3 promoter activity in the absen ce of CYP1A1 metabolic capacity, are all similar to that with mammalian CYP 1A promoters. These findings indicate that the DNA motifs in trout, and the mouse liver proteins that bind to these motifs, are evolutionarily conserv ed elements.