Lund Caren V, Blancafort Pilar, Popkov Mikhail, Barbas Carlos F
The Skaggs Institute for Chemical Biology and Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
J Mol Biol. 2004 Jul 9;340(3):599-613. doi: 10.1016/j.jmb.2004.04.057.
Regulation of endogenous gene expression has been achieved using synthetic zinc finger proteins fused to activation or repression domains, zinc finger transcription factors (TFZFs). Two key aspects of selective gene regulation using TFZFs are the accessibility of a zinc finger protein to its target DNA sequence and the interaction of the fused activation or repression domain with endogenous proteins. Previous work has shown that predicting a biologically active binding site at which a TF(ZF) can control gene expression is not always straightforward. Here, we used a library of preassembled three-finger zinc finger proteins (ZFPs) displayed on filamentous phage, and selected for ZFPs that bound along a 1.4 kb promoter fragment of the human ErbB-2 gene. Following affinity selection by phage display, 13 ZFPs were isolated and sequenced. Transcription factors were prepared by fusion of the zinc finger proteins with a VP64 activation domain or a KRAB repression domain and the transcriptional control imposed by these TFZFs was evaluated using luciferase reporter assays. Endogenous gene regulation activity was studied following retroviral delivery into A431 cells. Additional ZFP characterization included DNaseI footprinting to evaluate the integrity of each predicted protein:DNA interaction. The most promising TFZFs able to both up-regulate and down-regulate ErbB-2 expression were extended to six-finger proteins. The increased affinity and refined specificity demonstrated by the six-finger proteins provided reliable transcriptional control. As a result of studies with the six-finger proteins, the specific region of the promoter most accessible to transcriptional control by VP64-ZFP and KRAB-ZFP fusion proteins was elucidated and confirmed by DNaseI footprinting, flow cytometric analysis and immunofluorescence. The ZFP phage display library strategy disclosed here, coupled with the growing availability of genome sequencing information, provides a route to identifying gene-regulating TFZFs without the prerequisite of well-defined promoter elements.
通过将合成锌指蛋白与激活或抑制结构域融合，即锌指转录因子（TFZFs），实现了对内源基因表达的调控。使用TFZFs进行选择性基因调控的两个关键方面是锌指蛋白对其靶DNA序列的可及性以及融合的激活或抑制结构域与内源蛋白的相互作用。先前的研究表明，预测TF(ZF)能够控制基因表达的生物活性结合位点并非总是那么简单直接。在这里，我们使用了展示在丝状噬菌体上的预组装三指锌指蛋白（ZFPs）文库，并筛选出与人ErbB-2基因1.4 kb启动子片段结合的ZFPs。通过噬菌体展示进行亲和选择后，分离并测序了13个ZFP。通过将锌指蛋白与VP64激活结构域或KRAB抑制结构域融合来制备转录因子，并使用荧光素酶报告基因检测来评估这些TFZFs施加的转录控制。在将逆转录病毒导入A431细胞后，研究了内源基因调控活性。对ZFP的进一步表征包括DNaseI足迹分析，以评估每个预测的蛋白质：DNA相互作用的完整性。能够上调和下调ErbB-2表达的最有前景的TFZFs扩展为六指蛋白。六指蛋白表现出的亲和力增加和特异性提高提供了可靠的转录控制。通过对六指蛋白的研究，阐明了VP64-ZFP和KRAB-ZFP融合蛋白对转录控制最易接近的启动子特定区域，并通过DNaseI足迹分析、流式细胞术分析和免疫荧光进行了确认。本文公开的ZFP噬菌体展示文库策略，再加上越来越容易获得的基因组测序信息，提供了一条无需明确的启动子元件前提即可鉴定基因调控TFZFs的途径。
