Erickson R H, Lai R S, Lotterman C D, Kim Y S
Gastrointestinal Research Laboratory, Department of Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121, USA.
Gene. 2000 Nov 27;258(1-2):77-84. doi: 10.1016/s0378-1119(00)00422-4.
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.
人二肽基肽酶IV基因启动子的5'上游区域（-448/-443）含有一个共有E盒（CACGTG），研究表明，利用小鼠（3T3）成纤维细胞以及人肠道和肝上皮细胞系Caco-2和HepG2的核提取物，该区域可与上游刺激因子结合。用针对USF-1和USF-2的特异性抗体进行的超迁移分析表明，USF-1是这些细胞核提取物中与E盒结合的主要异构体。通过细胞培养，将USF表达载体与二肽基肽酶IV启动子构建体进行瞬时共转染，结果显示，USF-1和USF-2均可使Caco-2细胞中的报告基因表达增加约10倍。缺乏DNA结合或转录激活结构域的USF-1和-2突变形式无法刺激报告基因表达。E盒的突变阻止了USF的结合，不过与USF共转染对报告基因表达的刺激仅降低了50%。在共转染实验中使用一系列缺失构建体，确定了USF与二肽基肽酶IV启动子相互作用的另一个可能位点位于-119/-88区域。
