Trout CYP1A3 Gene: Recognition of Fish DNA Motifs by Mouse Regulatory Proteins

Abstract:

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.