ERα与Sp1相互作用激活LRP16启动子转录活性

Transcription Activation of LRP16 Promoter by ERα and Sp1 Interactions

根据已报道的LRP1 6启动子序列 (2 6kb) ,采用PCR反应获得 6个启动子 5′删除突变体 ,分别插入pGL3 Basic载体 ,构建 6种 5′缺失报告基因表达载体 (pS1 ～pS6) .分别与ERα真核表达载体共转染MCF 7细胞 .用双荧光素酶报告系统测定荧光素酶活性 ,以明确LRP1 6基因上游启动子区域中的雌激素反应序列 .结果显示 ,pS1 ～pS6均有雌二醇反应性 .进而对pS5的 3′端缺失分析发现LRP1 6基因翻译起始位点上游 - 2 1 4至 - 2 5 1位置的序列具有雌激素应答 ,序列分析发现该片段序列中包含了一个供转录因子Sp1结合的GC富含位点和一个ERα反应元件的半位点 (1 2ERE Sp1 ) ,进一步突变分析显示这两个元件均为雌激素反应性所必需 .以含这两个顺式元件的序列 (- 2 5 3bp至 - 2 2 4bp)作为探针 ,超级迁移凝胶电泳试验结果表明了ERα和Sp1蛋白均可以和探针结合 .研究发现了雌激素上调LRP1 6基因表达的一个增强子元件— 1 2ERE Sp1 ,ERα和Sp1蛋白需要与DNA结合形成复合体 。

To investigate the mechanism of the up regulative effect of LRP 16 mRNA by estrogen receptor α(ERα), the PCR primers were designed based on the DNA sequence of LRP 16 promoter (2.6 kb) reported, and 6 various 5′ deleted mutation fragments were amplified, then inserted into pGL3 Basic vector to make luciferase reporter constructs(pS1 to pS6). Various luciferase reporters were co transfected into MCF 7 cells with ERα expression plasmid, The luciferase activity was determined by dual luciferase report assay. The results showed that E2 induced reporter gene activity in pS1 to pS 6 cotransfection groups. The results of 3′ terminus deletion analysis of pS 5 demonstrated that sequences from -214 bp to - 251 bp of LRP 16 gene 5′ flanking region, which contained an estrogen responsive element (ERE) half site and a Sp1 binding GC rich region, were required for E2 action. Further mutation analysis demonstrated that the two motifs were essential for E2 responsiveness. Using sequence from -253 to -224 as probe, gel mobility shift assay was applied and the results confirmed that both ERα and Sp1 were required for hormone induced transactivation which involved in both ERα and Sp1 directly binding to DNA. An 1/2ERE/Sp1 cis element model for E2 transactivation in LRP 16 gene promoter region was identified, and the interaction between ERα and Sp1 proteins in transcription factors /DNA complex was required for E2 transactivation.