A NUCLEAR FACTOR INDUCED BY HYPOXIA VIA DENOVO PROTEIN-SYNTHESIS BINDS TO THE HUMAN ERYTHROPOIETIN GENE ENHANCER AT A SITE REQUIRED FOR TRANSCRIPTIONAL ACTIVATION
A NUCLEAR FACTOR INDUCED BY HYPOXIA VIA DENOVO PROTEIN-SYNTHESIS BINDS TO THE HUMAN ERYTHROPOIETIN GENE ENHANCER AT A SITE REQUIRED FOR TRANSCRIPTIONAL ACTIVATION
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DOI：
10.1128/mcb.12.12.5447
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发表时间：
1992-12-01
影响因子：
5.3
通讯作者：
WANG, GL
中科院分区：
文献类型：
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作者：
SEMENZA, GL;WANG, GL
我们已经从人促红细胞生成素基因3 '侧翼序列中鉴定出一个50个核苷酸的增强子，当将其克隆到猿病毒40启动子-氯霉素乙酰转移酶报告基因的3'端并在Hep 3B细胞中瞬时表达时，该增强子可以介导对缺氧的7倍转录诱导。当以两个串联拷贝存在时，该序列的核苷酸（nt）1至33介导报告基因表达的七倍诱导，与以单个拷贝存在时的三倍诱导相比，这表明nt 34至50结合放大诱导信号的因子。DNA酶I足迹法证明了组成性核因子与nt 26至48的结合。诱变研究表明，nt 4至12和19至23是诱导所必需的，因为在任一位点的取代消除了缺氧诱导的表达。电泳迁移率变动分析鉴定了与跨越nt 1至18的探针结合但不与含有消除增强子功能的突变的探针结合的核因子。缺氧诱导因子结合，其诱导对放线菌酮处理敏感。因此，我们已经定义了一个功能三方，50-nt缺氧诱导增强子结合几个核因子，其中之一是通过从头蛋白质合成缺氧诱导。
We have identified a 50-nucleotide enhancer from the human erythropoietin gene 3'-flanking sequence which can mediate a sevenfold transcriptional induction in response to hypoxia when cloned 3' to a simian virus 40 promoter-chloramphenicol acetyltransferase reporter gene and transiently expressed in Hep3B cells. Nucleotides (nt) 1 to 33 of this sequence mediate sevenfold induction of reporter gene expression when present in two tandem copies compared with threefold induction when present in a single copy, suggesting that nt 34 to 50 bind a factor which amplifies the induction signal. DNase I footprinting demonstrated binding of a constitutive nuclear factor to nt 26 to 48. Mutagenesis studies revealed that nt 4 to 12 and 19 to 23 are essential for induction, as substitutions at either site eliminated hypoxia-induced expression, Electrophoretic mobility shift assays identified a nuclear factor which bound to a probe spanning ut 1 to 18 but not to a probe containing a mutation which eliminated enhancer function. Factor binding was induced by hypoxia, and its induction was sensitive to cycloheximide treatment. We have thus defined a functionally tripartite, 50-nt hypoxia-inducible enhancer which binds several nuclear factors, one of which is induced by hypoxia via de novo protein synthesis.