Wieczorek E, Lin Z, Perkins E B, Law D J, Merchant J L, Zehner Z E
Department of Biochemistry and Molecular Biophysics and the Massey Cancer Center, Medical College of Virginia Campus/Virginia Commonwealth University, Richmond, Virginia 23298, USA.
J Biol Chem. 2000 Apr 28;275(17):12879-88. doi: 10.1074/jbc.275.17.12879.
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.
波形蛋白是真核细胞骨架的一个组成部分，属于中间丝蛋白家族。它呈现出一种复杂的组织和发育特异性表达模式。它也是许多肿瘤细胞转移潜能的标志物。此前，已表明人类波形蛋白启动子包含几个用于结合正性和负性作用调节因子的区域。直到现在，在发育过程中关闭特定组织中波形蛋白合成的沉默元件尚未被精确定位，其结合蛋白也不为人知。通过连接介导的PCR进行的体内二甲基亚砜足迹分析确定了对波形蛋白表达重要的鸟嘌呤残基的位置。在HeLa细胞中对各种波形蛋白5'端启动子序列及其突变体进行的瞬时转染分析精确地确定了两个调节元件，一个负性元件和一个相邻的正性作用元件。凝胶迁移实验、紫外线交联和蛋白质印迹分析证实沉默元件特异性结合一种蛋白质。多项证据表明，锌指、类 Kruppel 抑制蛋白ZBP-89是波形蛋白的沉默元件结合因子。免疫共沉淀和DNA亲和层析证明，当Sp1与沉默元件结合时，它会与ZBP-89形成异二聚体，产生一种DNA-蛋白质复合物，其迁移率与凝胶迁移实验中HeLa细胞核提取物显示的迁移率无法区分。
