Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells.
Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells.
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发表时间：
1999-02
期刊：
影响因子：
11.2
通讯作者：
M. Takakura;S. Kyo;T. Kanaya;H. Hirano;J. Takeda;M. Yutsudo;M. Inoue
中科院分区：
文献类型：
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作者：
M. Takakura;S. Kyo;T. Kanaya;H. Hirano;J. Takeda;M. Yutsudo;M. Inoue
端粒酶的激活被认为是细胞永生化和癌变的关键步骤。在构成人类端粒酶的三个主要亚基中，人类端粒酶催化亚单位(HTERT)被证明是人类端粒酶活性的限速决定因素。然而，关于hTERT在人类细胞中的表达是如何调控的，目前还知之甚少。为了确定控制hTERT基因表达的调控元件，克隆并鉴定了约3.5kb的hTERT基因5‘侧翼序列。HTERT启动子富含GC，缺乏TATA和CAAT盒。CapSite Hunting方法确定了已发表的cdna序列第一个核苷酸上游的19个核苷酸的转录起始点。瞬时表达分析表明，hTERT转录在癌细胞系中被显著激活，而在正常原代细胞中被抑制。利用转化不同阶段的成纤维细胞谱系，我们发现在克服复制衰老并表达端粒酶活性的菌株中发生了转录。HTERT启动子缺失分析表明，hTERT核心启动子位于转录起始点上游181个碱基的区域。凝胶漂移分析揭示了与核心启动子结合的两个主要因子：E盒(CACGTG)结合因子和Sp1。C-Myc的过表达导致核心启动子转录活性显著增强。这些发现表明hTERT的表达在转录机制中受到严格的调控，并且hTERT的反式激活需要包含E盒和Sp1位点的近端核心启动子。
Telomerase activation is thought to be a critical step in cellular immortalization and carcinogenesis. Of the three major subunits comprising human telomerase, human telomerase catalytic subunit (hTERT) has been shown to be a rate-limiting determinant of the enzymatic activity of human telomerase. However, little is known concerning how expression of hTERT is regulated in human cells. To identify the regulatory elements controlling hTERT gene expression, approximately 3.5 kb of the 5'-flanking sequence of hTERT was cloned and characterized. The promoter of hTERT was GC rich and lacked both TATA and CAAT boxes. The CapSite Hunting method identified transcription start site 19 bp upstream of the first nucleotide of the published cDNA sequence. Transient expression assays revealed that transcription of hTERT was significantly activated in cancer cell lines but repressed in normal primary cells. Using the fibroblast lineage at various stages of transformation, we found that transcription occurred in strains that had overcome replicative senescence and expressed telomerase activity. Deletion analysis of hTERT promoter identified the 181-bp core promoter region upstream of the transcription start site. Gel shift analysis revealed two major factors binding to core promoter, an E box (CACGTG) binding factor and Sp1. Overexpression of c-Myc resulted in a significant increase in transcriptional activity of the core promoter. These findings suggest that hTERT expression is strictly regulated at the transcription machinery, and that the proximal core promoter containing an E box and Sp1 sites is required for transactivation of hTERT.