Rhodopsin targeted transcriptional silencing by DNA-binding
Rhodopsin targeted transcriptional silencing by DNA-binding
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DOI：
10.7554/elife/12242
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发表时间：
2016-03-14
期刊：
影响因子：
7.7
通讯作者：
Surace, Enrico Maria
中科院分区：
文献类型：
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作者：
Botta, Salvatore;Marrocco, Elena;Surace, Enrico Maria
转录因子（TF）通过其DNA结合结构域（DBD）和效应结构域（ED）的组合活性起作用，使得能够在基因组规模上协调基因表达。在这里，我们表明，在体内交付的工程DNA结合蛋白解偶联的阻遏结构域可以产生有效的和基因特异性的转录沉默。为了干扰Rhodopsin（RHO）功能获得性突变，我们设计了靶向RHOcis调节元件（CRE）的20个碱基对（bp）的ZF 6-DNA结合蛋白（ZF 6-DB），并证明了在光感受器中腺相关病毒（AAV）载体介导的表达后Rho特异性转录沉默。数据显示，20 bp长的基因组DNA序列是RHO表达所必需的，并且光感受器递送相应的同源合成反式作用因子ZF 6-DB而不具有典型艾德的内在转录抑制特性，阻断了Rho表达，而全基因组转录物扰动可忽略不计。这些数据支持DNA结合介导的沉默作为治疗功能获得性突变的新模式。
Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of function mutations.