Genomic structure and characterization of the 5′-flanking region of the human ghrelin gene
Genomic structure and characterization of the 5′-flanking region of the human ghrelin gene
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DOI：
10.1210/en.2003-1718
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发表时间：
2004-09-01
期刊：
影响因子：
4.8
通讯作者：
Nakao, K
中科院分区：
文献类型：
--
作者：
Kanamoto, N;Akamizu, T;Nakao, K
Ghrelin 是 GH 促分泌素受体的内源性配体，可诱导 GH 分泌、食物摄入和正能量平衡。尽管生长素释放肽表现出多种激素作用，但调节生长素释放肽表达和分泌的机制仍不清楚。为了了解人类生长素释放肽基因表达的调控，我们检查了人类生长素释放肽基因 5' 侧翼区域约 5,000 bp 的基因组结构。我们对 cDNA 末端进行快速扩增以估计转录起始位点，表明人类生长素释放肽基因内有两个转录起始位点。两个转录本在人胃中均等表达，而较长的转录本主要在人甲状腺髓样癌（TT）细胞系中表达。使用含有该基因 5' 侧翼区域的启动子-报告基因构建体进行的功能分析表明，位于 -349 至 -193 区域内的序列对于 TT 细胞中的人生长素释放肽启动子功能是必需的。该区域内存在一些反式激活调节蛋白的共有序列，包括 E-box 位点。该位点的破坏降低了启动子活性的 40%。启动子的上游区域有两个额外的推定 E-box 位点，定点诱变表明这些位点也参与启动子激活。电泳迁移率变动分析表明上游刺激因子特异性地与这些 E-box 元件结合。这些结果表明上游刺激因子转录因子在调节人生长素释放肽表达中具有潜在作用。
Ghrelin, an endogenous ligand for the GH secretagogue receptor, induces GH secretion, food intake, and positive energy balance. Although ghrelin exhibits a variety of hormonal actions, the mechanisms regulating ghrelin expression and secretion remain unclear. To understand regulation of human ghrelin gene expression, we examined the genomic structure of approximately 5,000 bp of the 5'-flanking region of the human ghrelin gene. We performed rapid amplification of cDNA ends to estimate transcriptional start sites, indicating that there are two transcriptional initiation sites within the human ghrelin gene. Both transcripts were equally expressed in the human stomach, whereas the longer transcript was mainly expressed in a human medullary thyroid carcinoma (TT) cell line. Functional analysis using promoter-reporter constructs containing the 5'-flanking region of the gene indicated that the sequence residing within the -349 to -193 region is necessary for human ghrelin promoter function in TT cells. Within this region existed several consensus sequences for a number of transactivating regulatory proteins, including an E-box site. Destruction of this site decreased to 40% of the promoter activity. The upstream region of the promoter has two additional putative E-box sites, and site-directed mutagenesis suggested that these are also involved in promoter activation. Electrophoretic mobility shift assays demonstrated that the upstream stimulatory factor specifically bound to these E-box elements. These results suggest a potential role for upstream stimulatory factor transcription factors in the regulation of human ghrelin expression.