Toward controlling gene expression at will:: Specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks
Toward controlling gene expression at will:: Specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks
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DOI：
10.1073/pnas.95.25.14628
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发表时间：
1998-12-08
影响因子：
11.1
通讯作者：
Barbas, CF
中科院分区：
文献类型：
--
作者：
Beerli, RR;Segal, DJ;Barbas, CF
为了建立一个通用的系统控制基因的表达，我们已经研究了新的多指锌指蛋白，识别延伸的DNA序列的构建方法。在其他地方，我们已经描述了识别64个成员锌指字母表的5 '-GNN-3'子集的序列的锌指结构域的产生。在这里，我们报告使用这些结构域作为模块化的构建模块的多指蛋白质特异性识别9或18 bp的序列。本发明开发了一种快速PCR组装方法，该方法与该预定义的锌指结构域集合一起，提供了容易获得的1700万种结合18-bp DNA位点的5 '-（GNN）（6）-3'家族的新蛋白质。为了检验该策略在基因控制中的功效，选择人erbB-2基因作为模型。通过与Kruppel相关盒（KRAB）、ERD或SID阻遏物结构域融合，特异性识别该基因5 '非翻译区中18-bp序列的多指蛋白被转化为转录阻遏物。通过与单纯疱疹病毒VP 16激活结构域或与VP 16的最小激活结构域的四聚体重复（称为VP 64）融合来产生转录激活因子。我们证明，基因阻遏和激活可以通过将设计的蛋白质靶向到基因转录区域内的单个位点来实现。我们预计，基因特异性转录调节因子的类型在这里描述的基因治疗，功能基因组学，和转基因生物的产生将发现不同的应用。
To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5'-GNN-3' subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9- or 18-bp sequences. A rapid PCR assembly method,vas developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5'-(GNN)(6)-3' family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5'-untranslated region of this gene was converted into a transcriptional repressor by fusion with Kruppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16's minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that gene-specific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.