: Transcriptional up-regulation of mammalian
CYP1A1 genes by
dioxin is known to require binding of
dioxin to the
Ah receptor (AHR), subsequent interaction 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 (Oncorhyncus 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' flanking region. The transcription start site was determined by primer extension analysis. Five putative AHREs were found between -451 and -1820, with an overlap 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 transiently transfected into mouse
hepatoma Hepa-1c1c7 wild-type (wt) cell cultures and three
benzo[a]pyrene-resistant mutant lines: c2, containing less than 10% levels of functional AHR; c4, defective in ARNT; and c37, deficient in
CYP1A1 metabolism. We compared the trout
CYP1A3 promoter-luc constructs with mouse and human
CYP1A1 promoter-luc constructs. All of our trout
CYP1A3 promoter data are consistent with
dioxin-inducible
luciferase activity being controlled by two or more AHREs via cooperativity with a GC-rich region (-1852)-as has previously been demonstrated for AHREs in mammalian
CYP1A1 promoters. The dependence of trout
CYP1A3 promoter activity on the AHR and on the ARNT, and the enhancement of
CYP1A3 promoter activity in the absence of
CYP1A1 metabolic capacity, are all similar to that with mammalian CYP1A promoters. These findings indicate that the DNA motifs in trout, and the mouse liver
proteins that bind to these motifs, are evolutionarily conserved elements.