Paik Y K, Chang D J, Reardon C A, Walker M D, Taxman E, Taylor J M
Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiovascular Research Institute, San Francisco, California 94140-0608.
J Biol Chem. 1988 Sep 15;263(26):13340-9.
Multiple cis-acting regulatory elements have been mapped within a 1-kilobase fragment spanning nucleotides -651 through +356 of the human apolipoprotein E gene using a transient expression system based on the chloramphenicol acetyltransferase gene as well as DNase I footprinting techniques. A 651-base pair 5'-flanking region of the human apolipoprotein E gene was capable of directing chloramphenicol acetyltransferase gene expression over a 48-fold range among the various cultured cell lines tested. Deletion analysis of this 651-base pair upstream region linked to either the chloramphenicol acetyltransferase gene or the intact apolipoprotein E structural sequences revealed at least three regulatory domains within the proximal 383 nucleotides. One of these domains contained a GC box transcriptional control element. Further analysis demonstrated that the other two domains contained enhancer-like activity. These enhancer-like elements were located within the nucleotides spanning -366 to -246 and -193 to -124. A third enhancer element was identified in the first intron, within nucleotides +44 to +262. Changing the distance of the three enhancer elements from the transcription start site and reversing their orientation did not significantly alter their effects on transcription rates. However, enhancer activity was influenced by the promoter and cell line that were used. DNase I footprinting assays showed that specific sequences within two of these elements (-193 to -124 and +44 to +262) bind proteins in nuclear extracts from HepG2 and Chinese hamster ovary cells. A protein footprint also was identified for a GC box element at nucleotides -59 to -45. Thus, control of apolipoprotein E gene expression is the result of a complex interaction of several different regulatory elements.
利用基于氯霉素乙酰转移酶基因的瞬时表达系统以及DNA酶I足迹技术，已在跨越人类载脂蛋白E基因核苷酸-651至+356的1千碱基片段内定位了多个顺式作用调节元件。人类载脂蛋白E基因的651碱基对5'侧翼区域能够在测试的各种培养细胞系中指导氯霉素乙酰转移酶基因在48倍的范围内表达。对与氯霉素乙酰转移酶基因或完整的载脂蛋白E结构序列相连的这651碱基对上游区域进行缺失分析，发现在近端383个核苷酸内至少有三个调节结构域。其中一个结构域含有一个GC盒转录控制元件。进一步分析表明，另外两个结构域具有增强子样活性。这些增强子样元件位于核苷酸-366至-246和-193至-124之间。在第一个内含子中，核苷酸+44至+262之间鉴定出第三个增强子元件。改变这三个增强子元件与转录起始位点的距离并颠倒它们的方向，并没有显著改变它们对转录速率的影响。然而，增强子活性受所用启动子和细胞系的影响。DNA酶I足迹分析表明，这些元件中的两个（-193至-124和+44至+262）内的特定序列与来自HepG2和中国仓鼠卵巢细胞的核提取物中的蛋白质结合。在核苷酸-59至-45处的一个GC盒元件也鉴定出一个蛋白质足迹。因此，载脂蛋白E基因表达的调控是几种不同调节元件复杂相互作用的结果。
