Kardassis D, Tzameli I, Hadzopoulou-Cladaras M, Talianidis I, Zannis V
Department of Medicine, University of Crete, Greece.
Arterioscler Thromb Vasc Biol. 1997 Jan;17(1):222-32. doi: 10.1161/01.atv.17.1.222.
Transient transfection assays have shown that the distal apoC-III promoter segments that contain the regulatory elements F to J enhance the strength of the tandemly linked proximal apoA-I promoter 5- to 13-fold in hepatic (HepG2) cells. Activation in intestinal (CaCo-2) cells to levels comparable to those obtained in HepG2 cells requires a larger apoA-I promoter sequence that extends to nucleotide -1500 as well as the presence of hepatic nuclear factor-4 (HNF-4). The distal apoC-III regulatory elements can also enhance 4- to 8-fold the strength of the heterologous apoB promoter in HepG2 and CaCo-2 cells. Finally, these elements in the presence of HNF-4 enhance 14.5- to 18.5-fold the strength of the minimal adenovirus major late promoter linked to two copies of the hormone response element (HRE) AID of apoA-I in both HepG2 and CaCo-2 cells. In vitro mutagenesis of the promoter/enhancer cluster established that the enhancer activity is lost by a mutation in the HRE present in the 3' end of the regulatory element I (-736 to -714) and is reduced significantly by point mutations or deletions in one or more of the regulatory elements F to J of the apoC-III enhancer. The enhancer activity also requires the HREs of the proximal apoA-I promoter. The apoC-III enhancer can also restore the activity of the proximal apoA-I and apoB promoters that have been inactivated by mutations in CCAAT/enhancers binding protein binding sites, indicating that C/EBP may not participate in the synergistic activation of the promoter/enhancer cluster. The findings suggest that the regulatory elements F to J of the apoC-III promoter act as a general modular enhancer that can potentiate the strength of proximal promoters that contain HREs. Such potentiation in the HepG2 cells can be accounted for by synergistic interactions between HNF-4 or other nuclear hormone receptors bound to the proximal and distal HREs and SP1 or other factors bound to the apoC-III enhancer. Additional factors may be required for optimal activity in CaCo-2 cells as well as for the function of this region as an intestinal enhancer.
瞬时转染分析表明，包含调控元件F至J的载脂蛋白C-III启动子远端片段可使串联连接的近端载脂蛋白A-I启动子在肝（HepG2）细胞中的强度增强5至13倍。在肠（CaCo-2）细胞中激活至与HepG2细胞中相当的水平，需要一个延伸至核苷酸-1500的更大的载脂蛋白A-I启动子序列以及肝细胞核因子-4（HNF-4）的存在。载脂蛋白C-III远端调控元件还可使异源载脂蛋白B启动子在HepG2和CaCo-2细胞中的强度增强4至8倍。最后，在HNF-4存在的情况下，这些元件可使与两个载脂蛋白A-I激素反应元件（HRE）AID拷贝相连的最小腺病毒主要晚期启动子在HepG2和CaCo-2细胞中的强度增强14.5至18.5倍。对启动子/增强子簇进行体外诱变表明，调控元件I（-736至-714）3'端存在的HRE发生突变会导致增强子活性丧失，载脂蛋白C-III增强子的调控元件F至J中一个或多个发生点突变或缺失会使其活性显著降低。增强子活性还需要近端载脂蛋白A-I启动子的HRE。载脂蛋白C-III增强子还可恢复因CCAAT/增强子结合蛋白结合位点突变而失活的近端载脂蛋白A-I和载脂蛋白B启动子的活性，这表明C/EBP可能不参与启动子/增强子簇的协同激活。这些发现表明，载脂蛋白C-III启动子的调控元件F至J作为一个通用的模块化增强子，可增强包含HRE的近端启动子的强度。在HepG2细胞中的这种增强作用可由与近端和远端HRE结合的HNF-4或其他核激素受体与与载脂蛋白C-III增强子结合的SP1或其他因子之间的协同相互作用来解释。在CaCo-2细胞中实现最佳活性以及该区域作为肠增强子发挥功能可能还需要其他因子。
