The zinc finger repressor, ZBP-89, binds to the silencer element of the human vimentin gene and complexes with the transcriptional activator, Sp1
The zinc finger repressor, ZBP-89, binds to the silencer element of the human vimentin gene and complexes with the transcriptional activator, Sp1
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DOI：
10.1074/jbc.275.17.12879
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发表时间：
2000-04-28
影响因子：
4.8
通讯作者：
Zehner, ZE
中科院分区：
文献类型：
--
作者：
Wieczorek, E;Lin, ZL;Zehner, ZE
波形蛋白是真核细胞骨架的组成部分，属于中间丝蛋白家族。它表现出组织和发育特异性表达的复杂模式。它也是许多肿瘤细胞转移潜力的标志物。以前，人波形蛋白启动子被证明含有几个区域，用于结合正性和负性作用调节因子。到目前为止，在发育过程中关闭特定组织中波形蛋白合成的沉默元件还没有精确定位;其结合蛋白也不清楚。在体内DMS足迹连接介导的PCR划定的位置鸟嘌呤残基的波形蛋白的表达很重要。HeLa细胞中各种波形蛋白5 '端启动子序列及其突变体的瞬时转染测定精确地定义了两个调节元件，一个负元件和一个相邻的正作用元件。条带移动分析、UV交联和Southwestern印迹分析证实沉默元件特异性结合蛋白质。有证据表明ZBP-89是波形蛋白沉默元件的结合因子，ZBP-89是一种锌指Kruppel样阻遏蛋白。免疫共沉淀和DNA亲和层析证明：Sp1与ZBP-89异源二聚化时，绑定到沉默元件产生的DNA-蛋白质复合物的流动性是无法区分的HeLa细胞核提取物中显示的带移测定。
Vimentin is a component of the eukaryotic cytoskeleton belonging to the family of intermediate filament proteins. It exhibits a complex pattern of tissue- and development-specific expression. It is also a marker of the metastatic potential of many tumor cells. Previously, the human vimentin promoter was shown to contain several regions for the binding of positive and negative acting regulatory factors. Until now, the silencer element, which shuts down vimentin synthesis in selected tissues during development, was not precisely localized; nor was its binding protein known. In vivo DMS footprinting by ligation-mediated PCR delineated the position of guanine residues important to vimentin expression. Transient transfection assays in HeLa cells of various vimentin 5'-end promoter sequences and mutants thereof precisely defined two regulatory elements, a negative element and an adjoining positive acting element. Band shift assays, UV cross-linking, and Southwestern blot analysis confirm that the silencer element specifically binds a protein. Several lines of evidence show that ZBP-89, a zinc finger, Kruppel-like repressor protein is vimentin's silencer element binding factor. Co-immunoprecipitation and DNA affinity chromatography prove that: Sp1 heterodimerizes with ZBP-89 when bound to the silencer element to yield a DNA-protein complex whose mobility is indistinguishable from that displayed by HeLa nuclear extract in band shift assays.