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.

中文翻译：

---

人端粒酶催化亚基（hTERT）基因启动子的克隆以及永生化和癌细胞中转录激活所必需的近端核心启动子序列的鉴定。

端粒酶活化被认为是细胞永生化和癌变的关键步骤。在包含人类端粒酶的三个主要亚基中，人类端粒酶催化亚基（hTERT）已被证明是人类端粒酶酶活性的限速决定因素。然而，关于如何在人类细胞中调节hTERT的表达还知之甚少。为了鉴定控制hTERT基因表达的调控元件，克隆并鉴定了约3.5 kb的hTERT 5'侧翼序列。hTERT的启动子富含GC，缺少TATA和CAAT框。CapSite Hunting方法确定了已发布cDNA序列第一个核苷酸上游19 bp的转录起始位点。瞬时表达测定表明，hTERT的转录在癌细胞系中被显着激活，但在正常原代细胞中被抑制。使用成纤维细胞谱系在转化的各个阶段，我们发现转录发生在克服复制性衰老并表达端粒酶活性的菌株中。hTERT启动子的缺失分析确定了转录起始位点上游的181-bp核心启动子区域。凝胶位移分析揭示了与核心启动子结合的两个主要因素，即E盒（CACGTG）结合因子和Sp1。c-Myc的过表达导致核心启动子的转录活性显着增加。这些发现表明，hTERT的表达在转录机制中受到严格调节，