核受体及核受体辅活化子对芳香族化酶基因转录调控的研究

Transcriptional regulation of aromatase gene by nuclear receptors and a new family of nuclear receptor coactivators, PNRCs (Proline-rich nuclear receptor coactivators)

目的 研究核受体及核受体辅活化子对芳香族化酶基因转录的调控。方法 酵母单、双杂合筛选、蛋白质 蛋白质相互作用分析、DNA突变、报告基因转染功能分析、迁移率改变及足迹分析等技术。结果 ①鉴定了主导芳香族化酶基因在乳腺癌细胞中表达的启动子I.3和启动子Ⅱ的确切位置以及对这两个启动子起调节作用的沉默子 (Silencer)S1和cAMP效应要素 (CREaro)等顺式作用元件。②分离鉴定了能与类固醇衍生因子 1 (SterodogenicFactor1 ,SF1 )相互作用并参与芳香族化酶基因转录调控的转录因子 ,其中近 5 0 %的克隆编码两种新的富含脯氨酸的核受体辅调节蛋白质 ,命名为PNRC(Proline richNuclearReceptorCoactivators)。功能分析显示PNRC通过与SF1或ERR1相互作用 ,进一步增强SF1或ERR1对芳香族化酶基因启动子 1 .3的转录激活作用。③缺失突变及定点突变分析证明含SH3结合模体的 2 3个氨基酸残基区域是PNRC分子与核受体的相互作用位点。结论 我们鉴定了主导芳香族化酶基因在乳腺癌细胞中表达的启动子及调控序列 ,克隆了与DNA调控序列结合的蛋白质和通过蛋白质相互作用参与芳香族化酶基因转录调控的转录因子———一种新型的核受体辅活化子PNRC。

Objective To study the transcriptional regulation of the aromatase gene by nuclear receptors and nuclear receptor coactivators. Methods Yeast one hybrid and yeast two hybrid screenings, protein protein interaction assays, mammalian cell transfections and CAT assays, DNA deletion, mobility shift assay and DNase1 footprinting, et al. Results ① we have identified promoters 1.3 and Ⅱ, two major promoters driving aromatase expression in breast cancer cells, and two regulatory elements including a silencer S1 and a cAMP responsive element, CREaro. ②we have searched for co regulatory proteins interacting with these proteins using bovine SF1 as the bait in a yeast two hybrid screening of a human mammary gland cDNA expression library. About 50% of the positive clones obtained from this screening encode two novel nuclear receptor coactivators, named PNRCs (Proline rich Nuclear Receptor Coactivators). PNRCs interacted with the orphan receptors SF1 and ERRα1 in a ligand independent manner. These proteins function as a coactivator to enhance the transcriptional activation towards promoter 1.3 of the human aromatase gene mediated by SF1 and ERRα 1.③ Results from a mutagenesis study demonstrated that a 23 amino acid sequence region containing a SH3 binding motif is crucial for PNRCs to interact with the nuclear receptors. Conclusion we have identified promoters driving aromatase expression in breast cancer cells and their two regulatory elements ,and have cloned proteins binding to these regulatory elements and a new family of nuclear receptor coactivators, PNRCs, which act as transcriptional factors taking part in transcriptional regulation of aromatase gene by protein protein interaction .