Yang C, Zhou D, Chen S
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Cancer Res. 1998 Dec 15;58(24):5695-700.
We have previously identified a silencer element (S1) that is situated between promoters I.3 and II of the human aromatase gene and that down-regulates the action of these promoters. We recently applied the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library for genes encoding the proteins binding to the silencer region. Most proteins identified from this approach belong to the nuclear receptor superfamily. Fifty % of the positive clones encode for ERR alpha-1, and other positive clones include EAR-2, EAR-3 (COUP-TF1), RAR gamma, and p120E4F. Because ERR alpha-1 was found to be the major protein interacting with S1, we decided to examine the regulatory action of ERR alpha-1 on promoter I.3 of the human aromatase gene. Using a reporter plasmid that includes the aromatase genomic fragment containing promoter I.3 and S1, ERR alpha-1 was found to have a positive regulatory function in breast cancer SK-BR-3 cells. Gel mobility shift assays have confirmed that ERR alpha-1 binds to S1 in a dose-dependent manner, and DNase I footprinting analysis has revealed that ERR alpha-1 binds to a region, 5'-AAGGTCAGAAAT-3', which is within S1 and between 96 and 107 bp relative to the transcriptional start site of promoter I.3. In addition, despite the fact that the nuclear receptor SF1 was shown previously to bind to the same site and to mediate a cAMP response in ovary, our yeast one-hybrid screening did not find any SF-1 clones. Gel mobility shift assays further revealed that SF-1 can bind to the silencer element with an affinity comparable with ERR alpha-1. Because our reverse transcription-PCR analysis was not able to detect SF1 mRNA in breast cancer tissue or in SK-BR-3 cells, it is thought that SF1 protein is not expressed in breast cancer tissue. Two ERR alpha-1 RNA variants with differences at the 5'-end have been reported. Our reverse transcription-PCR analysis identified the shorter variant in 28 of 32 breast tumor specimens and the longer variant in only 1 specimen. In addition, the shorter variant was detected in breast cancer SK-BR-3 cells as well as in a breast tumor fibroblast line WS3TF. The results suggest that ERR alpha-1 is one of the nuclear proteins interacting with S1 in breast cancer tissue. It is thought that the silencer element in the human aromatase gene may function differently in different tissues because of distinct expression patterns of transcription factors.
我们之前已鉴定出一个沉默子元件（S1），它位于人类芳香化酶基因的启动子I.3和II之间，可下调这些启动子的活性。我们最近应用酵母单杂交方法，从人乳腺组织杂交cDNA表达文库中筛选编码与该沉默子区域结合蛋白的基因。通过这种方法鉴定出的大多数蛋白质属于核受体超家族。50%的阳性克隆编码ERRα-1，其他阳性克隆包括EAR-2、EAR-3（COUP-TF1）、RARγ和p120E4F。由于发现ERRα-1是与S1相互作用的主要蛋白质，我们决定研究ERRα-1对人类芳香化酶基因启动子I.3的调控作用。使用一个包含含有启动子I.3和S1的芳香化酶基因组片段的报告质粒，发现ERRα-1在乳腺癌SK-BR-3细胞中具有正调控功能。凝胶迁移率变动分析证实ERRα-1以剂量依赖方式与S1结合，DNase I足迹分析表明ERRα-1结合到一个区域5'-AAGGTCAGAAAT-3'，该区域在S1内，相对于启动子I.3的转录起始位点位于96至107 bp之间。此外，尽管之前显示核受体SF1可结合到相同位点并在卵巢中介导cAMP反应，但我们的酵母单杂交筛选未发现任何SF-1克隆。凝胶迁移率变动分析进一步表明SF-1能以与ERRα-1相当的亲和力结合到沉默子元件。由于我们的逆转录PCR分析未能在乳腺癌组织或SK-BR-3细胞中检测到SF1 mRNA，推测SF1蛋白在乳腺癌组织中不表达。已报道有两种在5'-端存在差异的ERRα-1 RNA变体。我们的逆转录PCR分析在32个乳腺肿瘤标本中的28个中鉴定出较短的变体，仅在1个标本中鉴定出较长的变体。此外，在乳腺癌SK-BR-3细胞以及乳腺肿瘤成纤维细胞系WS3TF中也检测到了较短的变体。结果表明ERRα-1是乳腺癌组织中与S1相互作用的核蛋白之一。推测由于转录因子的不同表达模式，人类芳香化酶基因中的沉默子元件在不同组织中可能发挥不同的功能。
