Identification of upstream stimulatory factor as an activator of the human dipeptidyl peptidase IV gene in Caco-2 cells.
The 5' upstream region (-448/-443) of the human dipeptidyl peptidase IV gene 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. Supershift analysis with specific antibodies to USF-1 and USF-2 indicates that USF-1 is the primary isoform binding to the E-box in nuclear extracts from these cells. Using cell culture, transient cotransfection of USF expression vectors with dipeptidyl peptidase IV promoter constructs revealed that both USF-1 and USF-2 caused an approximately tenfold increase in reporter gene expression in Caco-2 cells. Mutant forms of USF-1 and -2 lacking the DNA binding or transcriptional activation domains were unable to stimulate reporter gene expression. Mutation of the E-box prevented binding of USF, although stimulation of reporter gene expression by cotransfection with USF was reduced by only 50%. By using a series of deletion constructs in cotransfection experiments, a second possible site of USF interaction with the dipeptidyl peptidase IV promoter was localized to the -119/-88 region.
Erickson
R H
RH
Gastrointestinal Research Laboratory, Department of Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121, USA. neko@itsa.ucsf.edu
Lai
R S
RS
Lotterman
C D
CD
Kim
Y S
YS
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018938:Caco-2 Cells; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I / Q000378:metabolism; D018819:Dipeptidyl Peptidase 4 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D051720:Upstream Stimulatory Factors
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; D004247:DNA; D003850:Deoxyribonuclease I; D018819:Dipeptidyl Peptidase 4