Identification of upstream stimulatory factor as an activator of the humandipeptidyl peptidase IV gene in Caco-2 cells

The 5' upstream region (-448/-443) of the human dipeptidyl peptidase IV gen e promoter containing a consensus E-box (CACGTG) was shown to bind upstream stimulatory factor using nuclear extracts from mouse (3T3) fibroblasts and the human intestinal and hepatic epithelial cell lines Caco-2 and HepG2. S upershift analysis with specific antibodies to USF-I and USF-2 indicates th at USF-I is the primary isoform binding to the E-box in nuclear extracts fr om these cells. Using cell culture, transient cotransfection of USF express ion vectors with dipeptidyl peptidase IV promoter constructs revealed that both USF-1 and USF-2 caused an approximately tenfold increase in reporter g ene expression in Caco-2 cells. Mutant forms of USF-1 and -2 lacking the DN A binding or transcriptional activation domains were unable to stimulate re porter gene expression. Mutation of the E-box prevented binding of USF, alt hough stimulation of reporter gene expression by cotransfection with USF wa s reduced by only 50%. By using a series of deletion constructs in cotransf ection experiments, a second possible site of USF interaction with the dipe ptidyl peptidase IV promoter was localized to the -119/-88 region. (C) 2000 Elsevier Science B.V. All rights reserved.