Enhancer element at the 3'-flanking region controls transcriptional response to hypoxia in the human erythropoietin gene.
Enhancer element at the 3'-flanking region controls transcriptional response to hypoxia in the human erythropoietin gene.
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DOI：
10.1016/s0021-9258(18)98438-3
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发表时间：
1991-08
期刊：
影响因子：
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通讯作者：
Ingrid A Beck;S. Ramirez;R. Weinmann;Jaime Caros
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文献类型：
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作者：
Ingrid A Beck;S. Ramirez;R. Weinmann;Jaime Caros
促红细胞生成素基因表达在缺氧条件下被极大地刺激。促红细胞生成素基因的激活似乎主要在基因转录水平上受到调节。为了研究参与对缺氧反应的顺式作用元件，通过从人促红细胞生成素基因的外显子II到V的内部缺失构建了一个迷你基因，并用于在促红细胞生成素产生的Hep 3B细胞系中的瞬时转染测定。最初发现，缺氧反应存在于含有400个碱基对（bp）的5 '侧翼区和600个bp的3'侧翼区的促红细胞生成素片段中。缺失分析表明，当从微型基因构建体中去除5 '侧翼序列、外显子和内含子I以及外显子V的高度保守区域时，对缺氧反应没有显著影响。然而，去除含有基因3'末端和3'侧翼序列的片段完全消除了缺氧反应性。将上述片段重新插入到微型基因的5'端上游恢复了对缺氧的反应。使用杂合促红细胞生成素-氯霉素-乙酰转移酶构建体的进一步分析允许将增强子样元件定位在人促红细胞生成素基因的多聚腺苷酸化位点下游约120 bp的3 ′-侧翼区中。激活这些序列的位置和方向的独立性和刺激15倍的转录的促红细胞生成素基因在缺氧。
Erythropoietin gene expression is greatly stimulated under conditions of hypoxia. The activation of the erythropoietin gene appears regulated primarily at the level of gene transcription. To study cis-acting elements involved in the response to hypoxia a mini-gene was constructed by an internal deletion from exon II to V of the human erythropoietin gene and used in transient transfection assays in the erythropoietin producing Hep 3B cell line. It was initially found that hypoxia responsiveness was present in an erythropoietin fragment containing 400 base pairs (bp) of 5'-flanking and 600 bp of 3'-flanking regions. Deletion analysis showed no significant effect on the response to hypoxia when highly conserved regions of 5'-flanking sequence, exon and intron I, and exon V were removed from the mini-gene construct. However, removal of a fragment containing the 3' end of the gene and 3'-flanking sequences completely eliminated hypoxia responsiveness. Reinsertion of the above fragment upstream of the 5' end of the mini-gene restored the response to hypoxia. Further analysis using hybrid erythropoietin-chloramphenicol-acetyltransferase constructs allowed the localization of enhancer-like element(s) in the 3'-flanking region, approximately 120 bp downstream of the polyadenylation site of the human erythropoietin gene. Activation by these sequences were position- and orientation-independent and stimulated 15-fold transcription of the erythropoietin gene in response to hypoxia.