IDENTIFICATION OF A NOVEL CIS-ELEMENT REQUIRED FOR THE CONSTITUTIVE ACTIVITY AND OSMOTIC RESPONSE OF THE RAT ALDOSE REDUCTASE PROMOTER

A new and essential cis-element AEE (aldose reductase enhancer element ), necessary for the constitutive activity and the osmotic stress resp onse of rat aldose reductase transcription in a rat liver cell line, h as been identified. In transient transfection assays, an increase in p romoter activity, up to 3.8-fold, was observed with osmotic stress (60 0 mosm/kg H2O) using a luciferase reporter gene construct containing a ldose reductase promoter sequence from -1,094 base pair (bp) to +23 bp . A deletion between -1,071 and -895 bp reduced the constitutive activ ity and abolished the osmotic response of the promoter. Exonuclease II I mediated in vivo DNA footprinting and dimethyl sulfate in vivo footp rinting revealed DNA protection of a 32-bp region and two guanosines ( G) within this region protected from methylation, respectively. Electr ophoretic gel mobility shift assays using whole liver cell extracts sh owed protein binding, under both normal and stressed conditions. Delet ion of the sequence between the two guanosines protected by in vivo di methyl sulfate DNA footprinting (GAAGAGTG) in a luciferase construct ( -1,094 bp to +23 bp) abolished the constitutive promoter activity. One copy of AEE fused to the thymidine kinase promoter gave a maximum con stitutive activity of 7.7-fold and a maximum osmotic response activity of 6.7-fold.