Exploring strategies for the design of artificial transcription factors
Exploring strategies for the design of artificial transcription factors
复制标题
DOI：
10.1074/jbc.m406809200
复制
发表时间：
2005-02-04
影响因子：
4.8
通讯作者：
Barbas, CF
中科院分区：
文献类型：
--
作者：
Gräslund, T;Li, XL;Barbas, CF
人工转录因子可以被工程化以与特定DNA序列相互作用，从而调节细胞内的内源基因表达。实施这种方法的一个重要障碍是选择适当的DNA序列进行靶向。我们推断，一个好的靶位点应该位于染色质中，在那里它可以被DNA结合蛋白所接近，并且应该在已知的基因转录调节因子附近。在这里，我们探索了这些标准的有效性，以指导我们选择潜在的调节γ-珠蛋白表达。设计了几种基于锌指的转录激活因子以靶向γ-珠蛋白启动子的-117-位附近的位点。该区域邻近已知的和潜在的天然转录因子的结合位点。设计并研究了三种转录因子，确定了有效的转录激活因子ggl-VP 64-RA。该转录因子能够直接与γ-珠蛋白启动子相互作用，并以选择性方式上调报告基因构建体以及内源基因的表达。将gg 1-VP 64-RA表达载体转染或逆转录病毒递送该转录因子到红白血病细胞系K562中导致胎儿血红蛋白增加。表达gg 1-vp 64-HA的细胞的γ-珠蛋白含量显示出比天然K562细胞系高16倍的胎儿血红蛋白水平。这些转录激活因子构成珠蛋白基因座的一类新的调节因子，其可适用于治疗由该基因座中的突变引起的疾病，例如镰状细胞病和地中海贫血病。
Artificial transcription factors can be engineered to interact with specific DNA sequences to modulate endogenous gene expression within cells. A significant hurdle to implementation of this approach is the selection of the appropriate DNA sequence for targeting. We reasoned that a good target site should be located in chromatin, where it is accessible to DNA-binding proteins, and it should be, in the close vicinity of known transcriptional regulators of the gene. Here we have explored the efficacy of these criteria to guide our selection of potential regulators of gamma-globin expression. Several zinc finger-based transcriptional activators were designed to target the sites proximal to the -117-position of the gamma-globin promoter. This region is proximal to the binding sites of known and potential natural transcription factors. Design and study of three transcription factors identified the potent transcriptional activator, ggl-VP64-RA. This transcription factor was able to interact directly with the gamma-globin promoter and up-regulate expression of reporter gene constructs as well as the endogenous gene in a selective manner. Transfection of a gg1-VP64-RA expression vector or retroviral delivery of this transcription factor into the erythroleukemia cell line K562 resulted in an increase of fetal hemoglobin. The gamma-globin content of cells expressing gg1-vp64-HA showed up to 16-fold higher levels of fetal hemoglobin than the native K562 cell line. These transcriptional activators constitute a novel class of regulators of the globin locus that may be suitable for treatment of diseases arising from mutations in this locus such as sickle cell disease and thalassemic diseases.