Identification of a novel cis-acting negative regulatory element affecting expression of the CYP1A1 gene in rat...

Polycyclic aromatic hydrocarbons such as 3-methylcholanthrene are toxic to rat epidermal cells in low passages (3 to 6), but cultures of high passage (>/=15) are resistant. Since such compounds can be metabolically activated by cytochrome P4501A1, we have examined the regulation of this gene in low and high passage cells. Consistent with this difference, little or no 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible P4501A1 mRNA or enzyme activity was observed in high passage as compared to low passage cultures. Similarly, transfection of a luciferase reporter construct containing -1317 to +256 base pairs of the 5'-flanking region of the murine CYP1A1 gene was TCDD-inducible in low but not high passage cells. Ligand binding and transfection experiments demonstrated the presence of functional Ah receptor complexes in both high and low passage cells. Deletion analysis identified a 26-base pair negative regulatory DNA (NeRD) element contained within the upstream regulatory region of the CYP1A1 gene responsible for this effect. Nuclear extracts from both low and high passage cells contain a protein which specifically binds to NeRD-containing DNA. Thus, the loss of polycyclic aromatic hydrocarbon sensitivity in high passage rat epidermal cells appears to be due to decreased expression of CYP1A1, and this effect may be mediated by an altered NeRD binding factor(s) present in these cells.