Smith Alexander E F, Farzaneh Farzin, Ford Kevin G
Department of Haematological and Molecular Medicine, Guy's, King's and St. Thomas' School of Medicine, The Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK.
Biol Chem. 2005 Feb;386(2):95-9. doi: 10.1515/BC.2005.012.
In order to demonstrate that an existing zinc-finger protein can be simply modified to enhance DNA binding and sequence discrimination in both episomal and chromatin contexts using existing zinc-finger DNA recognition code data, and without recourse to phage display and selection strategies, we have examined the consequences of a single zinc-finger extension to a synthetic three-zinc-finger VP16 fusion protein, on transcriptional activation from model target promoters harbouring the zinc-finger binding sequences. We report a nearly 10-fold enhanced transcriptional activation by the four-zinc-finger VP16 fusion protein relative to the progenitor three-finger VP16 protein in transient assays and a greater than five-fold enhancement in stable reporter-gene expression assays. A marked decrease in transcriptional activation was evident for the four-zinc-finger derivative from mutated regulatory regions compared to the progenitor protein, as a result of recognition site-size extension. This discriminatory effect was shown to be protein concentration-dependent. These observations suggest that four-zinc-finger proteins are stable functional motifs that can be a significant improvement over the progenitor three-zinc-finger protein, both in terms of specificity and the ability to target transcriptional function to promoters, and that single zinc-finger extension can therefore have a significant impact on DNA zinc-finger protein interactions. This is a simple route for modifying or enhancing the binding properties of existing synthetic zinc-finger-based transcription factors and may be particularly suited for the modification of endogenous zinc-finger transcription factors for promoter biasing applications.
为了证明利用现有的锌指DNA识别密码数据，无需借助噬菌体展示和筛选策略，就可以简单地修饰现有的锌指蛋白，以增强其在游离型和染色质环境中的DNA结合能力和序列识别能力，我们研究了在一个合成的三锌指VP16融合蛋白上单一锌指延伸，对含有锌指结合序列的模型靶启动子转录激活的影响。我们报告，在瞬时分析中，相对于原始的三指VP16蛋白，四锌指VP16融合蛋白的转录激活增强了近10倍，在稳定的报告基因表达分析中增强了超过5倍。由于识别位点大小的延伸，与原始蛋白相比，来自突变调控区域的四锌指衍生物的转录激活明显降低。这种区分效应显示为蛋白质浓度依赖性。这些观察结果表明，四锌指蛋白是稳定的功能基序，在特异性以及将转录功能靶向启动子的能力方面，相对于原始的三锌指蛋白可能有显著改进，因此单一锌指延伸可能对DNA与锌指蛋白的相互作用有显著影响。这是一种修饰或增强现有基于合成锌指的转录因子结合特性的简单途径，可能特别适合用于修饰内源性锌指转录因子以用于启动子偏向应用。
