Tetracycline-regulated gene expression mediated by a novel chimeric repressor that recruits histone deacetylases in mammalian cells
Tetracycline-regulated gene expression mediated by a novel chimeric repressor that recruits histone deacetylases in mammalian cells
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DOI：
10.1074/jbc.m106924200
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发表时间：
2001-11-30
影响因子：
4.8
通讯作者：
He, TC
中科院分区：
文献类型：
--
作者：
Jiang, W;Zhou, L;He, TC
调控基因表达将提供重要的平台，从中可以研究基因功能，并可能开发更安全的基因治疗手段。组蛋白去乙酰化酶最近被证明在调节基因表达中起重要作用。在这里，我们研究了是否可以通过使用一种新的嵌合阻遏物，招募组蛋白脱乙酰酶的四环素反应启动子，实现更严格的控制表达。通过将四环素阻遏物（TetR）与人Mad1的mSin3相互作用结构域融合，设计了这种嵌合阻遏物，并显示出以高亲和力结合tetO（2）元件，并且其结合被强力霉素有效地消除。嵌合阻遏物显示直接与历史脱乙酰酶复合物的mSin3相互作用。通过使用含有嵌合阻遏物表达盒和四环素应答启动子的单个载体，进一步简化了该诱导型系统。当瞬时引入哺乳动物细胞中时，嵌合阻遏物系统表现出比TetR对照显著更低的荧光素酶活性基础水平（高达25倍）。当稳定转染到HEK 293细胞中时，嵌合阻遏物系统显示出以多西环素剂量和时间依赖性方式对绿色荧光蛋白表达施加严格控制。因此，这种新的嵌合阻遏物提供了一种更严格地调控基因表达的有效手段，简化的诱导系统可用于广泛的基础和临床研究。
Regulated gene expression will provide important platforms from which gene functions can be investigated and safer means of gene therapy may be developed. Histone deacetylases have recently been shown to play an important role in regulating gene expression. Here we investigated whether a more tightly controlled expression could be achieved by using a novel chimeric repressor that recruits histone deacetylases to a tetracycline-responsive promoter. This chimeric repressor was engineered by fusing the tetracycline repressor (TetR) with an mSin3-interacting domain of human Mad1 and was shown to bind the tetO(2) element with high affinity, and its binding was efficiently abrogated by doxycycline. The chimeric repressor was shown to directly interact with mSin3 of the historic deacetylase complex. This inducible system was further simplified by using a single vector that contained both a chimeric repressor expression cassette and a tetracycline-responsive promoter. When transiently introduced into mammalian cells, the chimeric repressor system exhibited a significantly lower basal level of luciferase activity (up to 25-fold) than that of the TetR control. When stably transfected into HEK 293 cells, the chimeric repressor system was shown to exert a tight control of green fluorescent protein expression in a doxycycline dose- and time-dependent fashion. Therefore, this novel chimeric repressor provides an effective means for more tightly regulated gene expression, and the simplified inducible system may be used for a broad range of basic and clinical studies.