Abstract
The role of the trans-acting factor HNF-1α in activating CYP2E1 gene expression was confirmed by transient co-transfection of an HNF-1α expression plasmid and the CYP2E1 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene. Only HNF-1α, and not HNF-1β, HNF-4, C/EBPα, C/EBPβ, or DBP, was able to activate the CYP2E1 promoter. The extent of activation was proportional to the number of copies of the HNF-1 binding sequence upstream of the promoter. Removal or mutation of the HNF-1 binding sequence led to inactivation of the promoter in response to HNF-1α. Gel-shift Western blot analysis using a synthetic HNF-1 binding sequence derived from CYP2E1 and rat liver nuclear extract revealed that the protein–DNA complex obtained with adult rat liver nuclear extract consisted of both HNF-1α and HNF-1β proteins. The shifted bands produced by nuclear extracts from adult, where the endogenous CYP2E1 gene is active, and fetal rat liver, where the gene is inactive, were found to migrate differently, suggesting that the population of factors, possibly including different ratios of HNF-1α and HNF-1β proteins, may change during development. However, the co-transfection study did not show cooperativity between the two factors. Elements upstream of the HNF-1 binding site were found to affect the activity of the promoter negatively in the transfection assay. DNase I hypersensitive site mapping revealed a hypersensitive site in this inhibiting element in the adult rat liver sample but not in liver from newborn animals.
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