Differential Protein Binding and Transcriptional Activities of HNF-4 Elements in Three Closely RelatedCYP2CGenes

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Abstract
A functional binding site for a liver-enriched transcription factor, hepatocyte nuclear factor-4 (HNF-4), has previously been identified around −100 in the CYP2C2 promoter and proposed to be a common regulatory motif for the hepatic expression of many CYP2 genes. The transcriptional activity of the proximal promoters of three closely related cytochrome P450 genes (CYP2C1, CYP2C2, and CYP2C3) have been compared in HepG2 cells and correlated with the relative binding affinities of the HNF-4 motifs in the genes for proteins in liver, kidney, and HepG2 extracts that react with antisera to HNF-4. Gel-shift assays suggested that these highly similar motifs bound HNF-4 with significantly different affinities. The relative binding affinities to the protein in liver extracts were estimated by competitive gel-shift binding, and the binding affinity of CYP2C2 was two-fold and eight-fold greater than that of CYP2C1 and CYP2C3, respectively. These affinity differences correlated well with the transcriptional activities of either the minimal hepatic promoters (117 to 135 bp of 5′-flanking region) of the genes in HepG2 cells or minimal CYP2C2 promoters containing the HNF-4 motif from each gene. Identification of the CYP2C3 site was somewhat indirect; the HNF-4 element in the CYP2C3 promoter could be converted to a motif with binding affinity and hepatic cell-specific transcriptional activity similar to that of CYP2C2 by a single nucleotide substitution. The activities of the minimal promoters did not correlate well with hepatic expression of each gene in vivo, as estimated previously by the concentration of mRNA in livers of untreated animals. Transcriptional activities with longer promoter constructions (about −250) more closely approximately the relative activities of the genes in vivo. Therefore, although the HNF-4 element is a principal determinant for liver-specific expression of the CYP2C genes, other cis-acting elements contribute to the level of transcription for each individual gene.