Semenza G L, Wang G L
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Mol Cell Biol. 1992 Dec;12(12):5447-54. doi: 10.1128/mcb.12.12.5447-5454.1992.
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 nt 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.
我们从人促红细胞生成素基因3'侧翼序列中鉴定出一段50个核苷酸的增强子，当将其克隆到猿猴病毒40启动子-氯霉素乙酰转移酶报告基因的3'端并在Hep3B细胞中瞬时表达时，该增强子可介导缺氧诱导的七倍转录诱导。该序列的核苷酸（nt）1至33以两个串联拷贝存在时可介导报告基因表达的七倍诱导，而以单个拷贝存在时为三倍诱导，这表明nt 34至50结合了一种可放大诱导信号的因子。DNA酶I足迹分析表明一种组成型核因子与nt 26至48结合。诱变研究表明，nt 4至12和19至23对诱导至关重要，因为这两个位点的替换都会消除缺氧诱导的表达。电泳迁移率变动分析鉴定出一种核因子，它与跨越nt 1至18的探针结合，但不与含有消除增强子功能突变的探针结合。因子结合是由缺氧诱导的，并且其诱导对放线菌酮处理敏感。因此，我们定义了一个功能上由三部分组成的、50 nt的缺氧诱导增强子，它结合了几种核因子，其中一种是通过从头合成蛋白质由缺氧诱导产生的。
