Böhm S K, Gum J R, Erickson R H, Hicks J W, Kim Y S
Gastrointestinal Research Laboratory (151M2), Department of Veterans Affairs Medical Center, San Francisco, CA, USA.
Biochem J. 1995 Nov 1;311 ( Pt 3)(Pt 3):835-43. doi: 10.1042/bj3110835.
The dipeptidyl peptidase IV gene encodes a plasma-membrane exopeptidase that is highly expressed in small intestine, lung and kidney. In order to better understand the mechanisms responsible for this tissue-specific expression we cloned, sequenced and functionally characterized the 5'-flanking region of the human dipeptidyl peptidase IV gene. The first 500 bases of the 5'-flanking sequence constituted an unmethylated CpG island, contained several Sp1-binding sites and lacked a consensus TATA box, all characteristics of gene promoters lacking tissue-specific expression. RNase-protection analysis using both small intestinal and Caco2 cell RNA indicated that the dipeptidyl peptidase IV transcript was initiated from no fewer than six major and 12 minor start sites. The 5'-flanking sequence also exhibited functional promoter activity in transient transfection experiments. Here, various lengths of the sequence were cloned upstream of a luciferase gene and introduced into cultured cells using lipofectin. A region located between bases -150 and -109 relative to the start of translation was found to be important for high-level promoter activity in both Caco2 and HepG2 cells. Moreover, Caco2 cells and HepG2 cells, which express high levels of dipeptidyl peptidase IV activity, exhibited much higher normalized luciferase activity after transfection than did 3T3, Jurkat or COS-7 cells, which have low enzyme levels. Sodium butyrate was found to increase both enzyme activity and normalized luciferase in HepG2 cells. Thus the dipeptidyl peptidase IV promoter possesses the ability to initiate transcription in a tissue-specific fashion in spite of having the sequence characteristics of a housekeeping gene promoter.
二肽基肽酶IV基因编码一种在小肠、肺和肾脏中高度表达的质膜外肽酶。为了更好地理解这种组织特异性表达的机制，我们克隆、测序并对人二肽基肽酶IV基因的5'侧翼区进行了功能表征。5'侧翼序列的前500个碱基构成了一个未甲基化的CpG岛，包含几个Sp1结合位点且缺乏共有TATA盒，这些都是缺乏组织特异性表达的基因启动子的特征。使用小肠和Caco2细胞RNA进行的核糖核酸酶保护分析表明，二肽基肽酶IV转录本至少从六个主要起始位点和十二个次要起始位点起始。在瞬时转染实验中，5'侧翼序列也表现出功能性启动子活性。在此，将不同长度的该序列克隆到荧光素酶基因的上游，并使用脂质体转染到培养细胞中。发现相对于翻译起始位点，位于碱基-150至-109之间的区域对于Caco2和HepG2细胞中的高水平启动子活性很重要。此外，表达高水平二肽基肽酶IV活性的Caco2细胞和HepG2细胞在转染后表现出比酶水平低的3T3、Jurkat或COS-7细胞更高的标准化荧光素酶活性。发现丁酸钠可增加HepG2细胞中的酶活性和标准化荧光素酶活性。因此，尽管二肽基肽酶IV启动子具有管家基因启动子的序列特征，但它仍具有以组织特异性方式起始转录的能力。
