Transcriptional analysis of human survivin gene expression
Transcriptional analysis of human survivin gene expression
复制标题
DOI：
10.1042/0264-6021:3440305
复制
发表时间：
1999-12-01
影响因子：
4.1
通讯作者：
Altieri, DC
中科院分区：
文献类型：
--
作者：
Li, FZ;Altieri, DC
维持组织和器官的体内平衡依赖于控制细胞凋亡（程序性细胞死亡）的基因的调节表达。在这项研究中，我们已经调查了生存素基因的基础转录要求，IAP（细胞凋亡抑制剂）显着上调癌症。对人生存素基因5'侧翼区的分析表明，存在一个TATA-less启动子，该启动子含有一个类似于250 nt的典型CpG岛、三个细胞周期依赖元件、一个细胞周期同源区和许多Spl位点。用甲基化敏感和不敏感的限制性内切酶消化的人类基因组DNA的基于PCR的分析表明，CpG岛在正常和肿瘤组织中都未甲基化。引物延伸和S1核酸酶图谱的人生存素基因确定了两个主要的转录起始位点在位置-72和-57/-61从起始ATG。用截短的或嵌套的生存素启动子-荧光素酶构建体转染宫颈癌HeLa细胞，揭示了增强子和阻遏子序列的存在，并鉴定了人生存素基因近端~ 230 nt内的最小启动子区域。人存活素启动子的无偏诱变分析显示，靶向位置-171和-151处的Spl序列消除了类似于63- 82%的基础转录活性。DNA寡核苷酸电泳迁移率变动分析证实了存活素Spl序列和HeLa细胞提取物之间的DNA-蛋白质复合物的形成，在反应中通过存活素Spl位点的诱变而废除。这些发现确定了survirin基因表达的基本转录要求。
The preservation of tissue and organ homoeostasis depends on the regulated expression of genes controlling apoptosis (programmed cell death). In this study, we have investigated the basal transcriptional requirements of the survivin gene, an IAP (inhibitor of apoptosis) prominently up-regulated in cancer. Analysis of the 5' flanking region of the human survivin gene revealed the presence of a TATA-less promoter containing a canonical CpG island of similar to 250 nt, three cell cycle dependent elements, one cell cycle homology region and numerous Spl sites. PCR-based analysis of human genomic DNA, digested with methylation-sensitive and -insensitive restriction enzymes, indicated that the CpG island was unmethylated in both normal and neoplastic tissues. Primer extension and S1 nuclease mapping of the human survivin gene identified two main transcription start sites at position -72 and within -57/-61 from the initiating ATG. Transfection of cervical carcinoma HeLa cells with truncated or nested survivin promoter-luciferase constructs revealed the presence of both enhancer and repressor sequences and identified a minimal promoter region within the proximal -230 nt of the human survivin gene. Unbiased mutagenesis analysis of the human survivin promoter revealed that targeting the Spl sequences at position -171 and -151 abolished basal transcriptional activity by similar to 63-82 %. Electrophoretic mobility-shift assay with DNA oligonucleotides confirmed formation of a DNA-protein complex between the survivin Spl sequences and HeLa cell extracts in a reaction abolished by mutagenesis of the survivin Spl sites. These findings identify the basal transcriptional requirements of survirin gene expression.