Characterization of hypoxia-responsive enhancer in the human erythropoietin gene shows presence of hypoxia-inducible 120-Kd nuclear DNA-binding protein in erythropoietin-producing and nonproducing cells
Characterization of hypoxia-responsive enhancer in the human erythropoietin gene shows presence of hypoxia-inducible 120-Kd nuclear DNA-binding protein in erythropoietin-producing and nonproducing cells
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DOI：
10.1182/blood.v82.3.704.704
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发表时间：
1993-08
期刊：
影响因子：
20.3
通讯作者：
I. Beck;R. Weinmann;J. Caro
中科院分区：
文献类型：
--
作者：
I. Beck;R. Weinmann;J. Caro
缺氧或钴时促红细胞生成素 (Epo) 的产生主要是通过 Epo 基因转录激活介导的。最近，在小鼠和人类 Epo 基因的 3' 侧翼区域发现了缺氧反应增强子。使用 Hep 3B 细胞中的功能分析，我们将最小增强子元件定义为从人类 Epo 基因 3' 侧翼区域的 Apa1 位点开始的 29 bp 片段。最小元素的诱变研究确定了完全增强子活性所必需的三个不同区域。电泳迁移率变动分析显示缺氧和/或钴诱导的核 DNA 结合蛋白的存在，该蛋白与增强子的活性位点之一结合。茴香霉素（一种有效的蛋白质合成抑制剂）消除了缺氧结合活性的诱导，这表明从头合成蛋白质对于激活过程是必要的。通过使用紫外光交联对 DNA 结合蛋白进行进一步表征，鉴定出一种分子量约为 120-Kd 的蛋白质，该蛋白质仅存在于缺氧提取物中。人们发现该蛋白存在于 Epo 产生细胞和非 Epo 产生细胞的缺氧核提取物中，表明它可能参与细胞对缺氧反应的更普遍的机制。
Erythropoietin (Epo) production in response to hypoxia or cobalt is primarily mediated by activation of transcription of the Epo gene. Recently an hypoxia responsive enhancer was identified in the 3′ flanking region of the mouse and human Epo genes. Using functional analysis in Hep 3B cells we define here the minimal enhancer element as a 29-bp segment starting at the Apa1 site in the 3′ flanking region of the human Epo gene. Mutagenesis studies of the minimal element identified three different areas that are necessary for full enhancer activity. Electrophoretic mobility shift assays show the presence of hypoxia- and/or cobalt-inducible nuclear DNA-binding proteins that bind to one of the active sites of the enhancer. Induction of hypoxia- binding activity was abolished by Anisomycin, a potent protein synthesis inhibitor, suggesting that de novo protein synthesis is necessary for the activation process. Further characterization of DNA- binding proteins by use of UV light crosslinking identified a protein of molecular weight of approximately 120-Kd that was present only in hypoxic extracts. This protein was found to be present in hypoxic nuclear extracts from both Epo-producing and non-Epo-producing cells, suggesting that it may be involved in a more generalized mechanism of cellular response to hypoxia.