cHS4 insulator-mediated alleviation of promoter interference during cell-based expression of tandemly associated transgenes
cHS4 insulator-mediated alleviation of promoter interference during cell-based expression of tandemly associated transgenes
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DOI：
10.1016/j.jmb.2007.09.054
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发表时间：
2007-11-30
影响因子：
5.6
通讯作者：
Imamoto, Fumio
中科院分区：
文献类型：
--
作者：
Yahata, Kazuhide;Maeshima, Kazuhiro;Imamoto, Fumio
多个转基因在细胞或整个生物体中的表达是各种生物学功能的基础研究和潜在的临床应用如基因治疗的有力工具。作为用于此目的的模型系统，构建了多cDNA表达克隆，其在单个质粒上具有两个串联的荧光蛋白cDNA作为报告基因。当293细胞瞬时转染，下游基因显示显着较低的表达相比，上游的cDNA。通过在两个相邻cDNA之间的位点插入来自鸡β-珠蛋白基因座的cHS 4元件的绝缘子盒，这种转录干扰显著减轻。cHS 4的引入导致下游cDNA的表达水平急剧增加，确保串联转基因的表达水平相当。使用染色质免疫沉淀试验，我们证明了CTCF和USF 1招募组蛋白修饰复合物结合到cHS 4区域。通过siRNA消耗CTCF或USF 1导致减弱的作用的缓解。因此，我们的数据表明，CTCF和组蛋白修饰剂招募USF 1合作介导的抑制并列基因之间的转录干扰，大概是通过促进活性染色质构象的转基因。(c)2007爱思唯尔有限公司保留所有权利。
Expression of multiple transgenes in cells or whole organisms is a powerful tool for basic research of various biological functions and potentially for clinical applications such as gene therapy. As a model system for this purpose, multi-cDNA expression clones were constructed harboring two tandemly situated fluorescent protein cDNAs as reporter genes on a single plasmid. When 293 cells were transfected transiently, the downstream gene displayed significantly lower expression when compared with the upstream cDNA. Such transcriptional interference was markedly alleviated by inserting an insulator cassette of cHS4 elements derived from the chicken beta-globin locus at a site between two neighboring cDNAs. The introduction of cHS4 resulted in a drastic increase of the expression level of the downstream cDNA, ensuring comparable expression levels of the tandem transgenes. Using a chromatin immunoprecipitation assay, we demonstrated that CTCF and USF1 that recruit histone-modifying complexes are bound to the cHS4 region. Depletion of CTCF or USF1 by siRNA resulted in relief of the diminished effect. Our data thus indicate that CTCF and histone modifiers recruited by USF1 cooperatively mediate the suppression of transcriptional interference between apposed genes, presumably by facilitating active chromatin conformation over the transgenes. (c) 2007 Elsevier Ltd. All rights reserved.