A SELECTIVE TRANSCRIPTIONAL INDUCTION SYSTEM FOR MAMMALIAN-CELLS BASED ON GA14-ESTROGEN RECEPTOR FUSION PROTEINS
A SELECTIVE TRANSCRIPTIONAL INDUCTION SYSTEM FOR MAMMALIAN-CELLS BASED ON GA14-ESTROGEN RECEPTOR FUSION PROTEINS
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DOI：
10.1073/pnas.90.5.1657
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发表时间：
1993-03-01
影响因子：
11.1
通讯作者：
BUSSLINGER, M
中科院分区：
文献类型：
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作者：
BRASELMANN, S;GRANINGER, P;BUSSLINGER, M
大多数哺乳动物细胞既不表达Gal4样活性，也不表达内源性雌激素受体，因此雌激素对它们来说是一个惰性信号。出于这两个原因，我们开发了一个基于雌激素调节转录因子Gal-ER的选择性诱导系统。Gal-ER由酵母Gal4蛋白的DNA结合域与人雌激素受体的激素结合域融合而成，因此在哺乳动物细胞中应该在Gal4反应启动子的控制下对转基因基因进行排他性调控。对该诱导系统进行了两个主要的改进。首先，构建了一个人工合成的Gal4反应启动子，该启动子由4个Gal4结合位点、一个反向CCAAT元件、一个TATA盒和腺病毒主要的晚期起始区组成。该启动子在缺乏Gal-ER的情况下表现出极低的基础活性，而在配体激活的Gal-ER存在的情况下表现出高度的诱导性。其次，通过将疱疹病毒蛋白VP16的强激活结构域融合到其C末端，使转录因子GalER变得更有效，更不容易受到细胞类型特异性变异的影响。作为对雌激素的反应，Gal-ER-VP16在瞬时转染的NIH3T3和P19细胞中诱导Gal4反应启动子至少100倍。表达整合的Gal-ER和Gal4反应的FOS基因的大鼠成纤维细胞系以严格的雌激素依赖方式转化。外源Fos基因在添加雌激素后1-2小时内迅速诱导达到最高水平。Fos活性的升高反过来刺激了内源性fra-1基因的转录。这些数据表明，Gal-ER诱导系统作为调节异源基因的强大遗传开关，特别是在哺乳动物细胞中识别Fos靶标是有效的。
Most mammalian cells neither express any Gal4-like activity nor endogenous estrogen receptor, thus rendering estrogen an inert signal for them. For these two reasons we have developed a selective induction system based on the estrogen-regulable transcription factor Gal-ER. Gal-ER consists of the DNA-binding domain of the yeast Gal4 protein fused to the hormone-binding domain of the human estrogen receptor and hence should exclusively regulate a transfected gene under the control of a Gal4-responsive promoter in mammalian cells. Two major improvements of this induction system were made. First, a synthetic Gal4-responsive promoter was constructed which consisted of four Gal4-binding sites, an inverted CCAAT element, a TATA box, and the adenovirus major late initiation region. This promoter showed extremely low basal activity in the absence and high inducibility in the presence of ligand-activated Gal-ER. Second, the transcription factor Gal-ER was rendered more potent and less susceptible to cell type-specific variation by fusing the strong activating domain of the herpesvirus protein VP16 onto its C terminus. In response to estrogen, Gal-ER-VP16 induced the Gal4-responsive promoter at least 100-fold in transiently transfected NIH 3T3 and P19 cells. Rat fibroblast cell lines expressing integrated Gal-ER and Gal4-responsive fos genes were transformed in a strictly estrogen-dependent manner. The exogenous fos gene was rapidly induced to maximal levels within 1-2 hr of estrogen addition. Elevated Fos activity in turn stimulated transcription of the endogenous fra-1 gene. These data demonstrate the utility of the Gal-ER induction system as a powerful genetic switch for regulating heterologous genes and, in particular, for identifying Fos targets in mammalian cells.