Braselmann S, Graninger P, Busslinger M
Research Institute of Molecular Pathology, Vienna, Austria.
Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1657-61. doi: 10.1073/pnas.90.5.1657.
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.
大多数哺乳动物细胞既不表达任何类似Gal4的活性，也不表达内源性雌激素受体，因此雌激素对它们来说是一种惰性信号。基于这两个原因，我们开发了一种基于雌激素可调节转录因子Gal-ER的选择性诱导系统。Gal-ER由酵母Gal4蛋白的DNA结合结构域与人雌激素受体的激素结合结构域融合而成，因此在哺乳动物细胞中，它应该仅在Gal4应答启动子的控制下调节转染基因。对该诱导系统进行了两项主要改进。首先，构建了一个合成的Gal4应答启动子，它由四个Gal4结合位点、一个反向CCAAT元件、一个TATA盒和腺病毒主要晚期起始区域组成。该启动子在无配体激活的Gal-ER时显示出极低的基础活性，而在有配体激活的Gal-ER时具有高诱导性。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Gal-ER更有效，且对细胞类型特异性变异更不敏感。在雌激素的作用下，Gal-ER-VP16在瞬时转染的NIH 3T3和P19细胞中诱导Gal4应答启动子至少100倍。表达整合的Gal-ER和Gal4应答fos基因的大鼠成纤维细胞系以严格的雌激素依赖性方式被转化。在添加雌激素后1-2小时内，外源fos基因迅速被诱导至最大水平。升高的Fos活性反过来刺激内源性fra-1基因的转录。这些数据证明了Gal-ER诱导系统作为一种强大的基因开关在调节异源基因方面的实用性，特别是在鉴定哺乳动物细胞中的Fos靶点方面。
