Identification and characterization of cis-acting elements conferring insulin responsiveness on hamster cholesterol 7α-hydroxylase gene promoter

摘要

pBile acid biosynthesis occurs primarily through a pathway initiated by the 7α-hydroxylation of cholesterol, catalysed by cholesterol 7α-hydroxylase (encoded by iCYP7A1/i). Insulin down-regulates iCYP7A1/i transcription. The aim of our study was to characterize the sequences of hamster iCYP7A1/i promoter, mediating the response to insulin. We therefore performed transient transfection assays with iCYP7A1/i promoter/luciferase chimaeras mutated at putative response elements and studied protein-DNA interactions by means of gel electrophoresis mobility-shift assay. Here we show that two sequences confer insulin responsiveness on hamster iCYP7A1/i promoter: a canonical insulin response sequence TGTTTTG overlapping a binding site for hepatocyte nuclear factor 3 (HNF-3) (at nt -235 to -224) and a binding site for HNF-4 at nt -203 to -191. In particular we show that the hamster iCYP7A1/i insulin response sequence is part of a complex unit involved in specific interactions with multiple transcription factors such as members of the HNF-3 family; this region does not bind very strongly to HNF-3 and as a consequence partly contributes to the transactivation of the gene. Another sequence located at nt -138 to -128 binds to HNF-3 and is involved in the tissue-specific regulation of hamster iCYP7A1/i. The sequence at nt -203 to -191 is not only essential for insulin effect but also has a major role in the liver-specific expression of iCYP7A1/i; it is the target of HNF-4. Therefore the binding sites for liver-enriched factors, present in the hamster iCYP7A1/i proximal promoter in close vicinity and conserved between species, constitute a regulatory unit important for basal hepatic expression and tissue restriction of the action of hormones such as insulin./p