Brasier A R, Tate J E, Ron D, Habener J F
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Boston.
Mol Endocrinol. 1989 Jun;3(6):1022-34. doi: 10.1210/mend-3-6-1022.
Angiotensinogen is the glycoprotein precursor of angiotensin II, an octapeptide hormone important for the regulation of blood pressure and volume homeostasis. The gene encoding angiotensinogen is expressed in liver and several other tissues, providing a model gene for understanding the role of cis-acting DNA control elements and trans-acting factors in tissue-type specific gene expression. To identify DNA control elements in the rat angiotensinogen gene we prepared an array of fusion genes consisting of either 5' or 3'-deleted sequences of the 5'-flanking region of the gene linked to a firefly luciferase reporter gene and analyzed the relative cellular specificity of expression of these genes after their introduction into hepato-carcinoma cells (Hep G2) that do express and placental cells (JEG-3) that do not express the endogenous angiotensinogen gene. Six transcriptionally active elements were found within 688 base pairs of 5'-flanking DNA. The interactions of DNA binding proteins with these elements was demonstrated by their specific protection to digestion with DNase I in the presence of liver cell extracts. The orientation and spatial requirements for transcription of two of the elements were analyzed further by the construction and expression of synthetic oligonucleotide cassettes incorporating the sequences of these elements when linked to a homologous (angiotensinogen) or a heterologous Simian virus 40 promoter and enhancer. One element located between 60 and 108 base pairs from the start of gene transcription functioned either as a silencer or an enhancer of transcription (SOAP box element), depending upon the distance from the angiotensinogen and viral gene promoters. Moreover, the SOAP box element demonstrated enhancer activity in JEG-3 cells when linked to the Simian virus 40 early promoter. An oligonucleotide mutation of the SOAP box element interfered with protein binding in a gel mobility shift assay and this mutant was transcriptionally inactive in both homologous and heterologous promoters. These observations indicate that expression of the angiotensinogen gene in liver cells is coordinately regulated by multiple cis-acting elements that interact with DNA binding proteins.
血管紧张素原是血管紧张素II的糖蛋白前体，血管紧张素II是一种八肽激素，对血压调节和容量稳态至关重要。编码血管紧张素原的基因在肝脏和其他几种组织中表达，为理解顺式作用DNA控制元件和反式作用因子在组织类型特异性基因表达中的作用提供了一个模型基因。为了鉴定大鼠血管紧张素原基因中的DNA控制元件，我们制备了一系列融合基因，这些融合基因由该基因5'侧翼区的5'或3'缺失序列与萤火虫荧光素酶报告基因相连组成，并在将这些基因导入表达内源性血管紧张素原基因的肝癌细胞（Hep G2）和不表达该基因的胎盘细胞（JEG-3）后，分析这些基因表达的相对细胞特异性。在5'侧翼DNA的688个碱基对内发现了6个转录活性元件。在肝细胞提取物存在的情况下，DNA结合蛋白与这些元件的相互作用通过它们对DNase I消化的特异性保护得以证明。当与同源（血管紧张素原）或异源猿猴病毒40启动子和增强子相连时，通过构建和表达包含这些元件序列的合成寡核苷酸盒，进一步分析了其中两个元件转录的方向和空间要求。一个位于距基因转录起始点60至108个碱基对之间的元件，根据其与血管紧张素原和病毒基因启动子的距离，可作为转录沉默子或增强子（SOAP盒元件）发挥作用。此外，当与猿猴病毒40早期启动子相连时，SOAP盒元件在JEG-3细胞中表现出增强子活性。在凝胶迁移率变动分析中，SOAP盒元件的寡核苷酸突变干扰了蛋白结合，并且该突变体在同源和异源启动子中均无转录活性。这些观察结果表明，肝细胞中血管紧张素原基因的表达由与DNA结合蛋白相互作用的多个顺式作用元件协同调节。
