Identification and characterization of a cAMP-responsive element in the region upstream from promoter 1.3 of the human aromatase gene.
Identification and characterization of a cAMP-responsive element in the region upstream from promoter 1.3 of the human aromatase gene.
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人类芳香酶基因启动子 1.3 上游区域 cAMP 响应元件的鉴定和表征。
DOI：
10.1006/abbi.1999.1454
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Chen,S
中科院分区：
文献类型：
--
作者：
Zhou,D;Chen,S
芳香酶将雄激素转化为雌激素。这种酶的表达是由多个组织特异性启动子驱动的，这些启动子是不同调控的。芳香化酶在乳腺癌及其周围脂肪细胞中的表达主要受启动子I.3和启动子II的调控，而在正常乳腺脂肪组织中的表达主要受启动子I.4的调控。与启动子II一样，启动子I.3被认为是cAMP驱动的启动子，之前的细胞培养实验证明了这一点。在本研究中，我们发现并鉴定了一个位于启动子1.3上游的cAMP反应元件(CREaro)。经缺失和突变分析，该阳性元件位于−66和−59bp之间，位于启动子1.3DNA转录起始点。突变分析表明，CREaro对启动子I.3的增强子活性高于CREpal。DNA迁移率改变分析表明，WS3TF乳腺肿瘤成纤维细胞和SK-BR-3乳腺癌细胞的核蛋白都与该CREaro结合。紫外光交联实验表明，成纤维细胞中主要结合蛋白的相对分子质量约为60 kDa，这与已知的Cre结合蛋白不同。有人认为CREB1在肿瘤成纤维细胞中不表达，因为使用抗CREB1抗体的Western印迹分析不能在这些细胞的核蛋白提取液中检测到CREB1。使用从SK-BR-3细胞中提取的核蛋白进行的DNA迁移率漂移分析表明，至少有两种蛋白质与CREaro结合，其中一种被鉴定为CREB1。这些研究为启动子I.3是cAMP反应启动子提供了直接证据。
Aromatase converts androgens to estrogens. The expression of this enzyme is driven by multiple tissue-specific promoters which are differentially regulated. Aromatase expression in breast cancer and the surrounding adipose cells is directed mainly by promoters I.3 and II, while its expression in the normal breast adipose tissue is driven by promoter I.4. Like promoter II, promoters I.3 is thought to be a cAMP-driven promoter, demonstrated previously by cell culture experiments. In the present study, we have identified and characterized a cAMP-responsive element (CREaro) upstream from promoter 1.3. This positive element, TGAAGTCA, between −66 and −59 bp relative to the transcriptional start site of promoter 1.3 was identified by DNA deletion and mutation analyses. The sequence of CREaro is one base different from the consensus CRE sequence (CREpal; TGACGTCA), and the mutational analysis revealed that CREaro had a higher enhancer activity to promoter I.3 than CREpal. Nuclear proteins from both WS3TF breast tumor fibroblasts and SK-BR-3 breast cancer cells bound to this CREaro, as demonstrated by DNA mobility shift assay. The molecular weight of the major binding protein in fibroblasts was determined to be approximately 60 kDa, as shown by UV crosslinking, which is different from those of known CRE-binding proteins. It is thought that CREB1 is not expressed in tumor fibroblasts because the Western blot analysis using anti-CREB1 antibody was not able to detect CREB1 in the nuclear protein extract from these cells. DNA mobility shift analysis using a nuclear protein extract from SK-BR-3 cells revealed that at least two proteins bound to the CREaro and that one of these proteins was identified to be CREB1. These studies provide direct evidence that promoter I.3 is a cAMP-responsive promoter.
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DOI：
--
发表时间：
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期刊：
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DOI：
10.1016/s0960-0760(97)80010-6
发表时间：
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期刊：
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影响因子：
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