Stover D M, Zehner Z E
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia/Virginia Commonwealth University, Richmond 23298.
Mol Cell Biol. 1992 May;12(5):2230-40. doi: 10.1128/mcb.12.5.2230-2240.1992.
Vimentin is a tissue-specific, developmentally regulated member of the intermediate filament protein family normally expressed in cells of mesenchymal origin. Transcription factors which recognize specific cis-acting elements of the chicken gene include Sp-1 and the 95-kDa silencer protein which binds to a 40-bp silencer element at -608 (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we have identified a region upstream of the silencer element which restores gene activity. This region has been further delineated into two functional subelements of 75 and 260 bp. In transient transfection assays, the 75-bp element overrides the silencer effect of pStkCAT by 100%, while the 260-bp element is about half as active. Neither element affects gene activity when the silencer element is absent. Therefore, these elements do not function as enhancers, but they may serve only to override the silencer element and therefore can be viewed as antisilencers. In addition, the 75-bp element binds a specific 140-kDa protein, as determined by gel mobility shift assays and Southwestern (DNA-protein) blots, the binding site of which has been delineated to a 10- to 17-bp element by DNase I protection experiments. During myogenesis, a direct correlation can be made between the binding efficiency of the 140-kDa protein, the silencer protein, and gene activity in vivo. Genes known to contain a functional silencer element also contain at least one antisilencer element, as determined by sequence identity. Therefore, we have identified an antisilencer element and protein important in the developmental regulation of vimentin gene expression which may be involved in the regulation of other genes.
波形蛋白是中间丝蛋白家族中一种组织特异性、受发育调控的成员，通常在间充质来源的细胞中表达。识别鸡基因特定顺式作用元件的转录因子包括Sp-1和95-kDa沉默蛋白，后者与位于-608处的一个40-bp沉默元件结合（F. X. Farrell、C. M. Sax和Z. E. Zehner，《分子细胞生物学》10:2349 - 2358，1990）。在本研究中，我们鉴定出了沉默元件上游的一个区域，该区域可恢复基因活性。这个区域已进一步划分为两个功能亚元件，分别为75 bp和260 bp。在瞬时转染实验中，75-bp元件可100% 克服pStkCAT的沉默效应，而260-bp元件的活性约为其一半。当沉默元件不存在时，这两个元件均不影响基因活性。因此，这些元件并非作为增强子发挥作用，而可能仅用于克服沉默元件的作用，所以可被视为抗沉默子。此外，通过凝胶迁移率变动分析和蛋白质印迹法（DNA-蛋白质印迹）确定，75-bp元件可结合一种特定的140-kDa蛋白，通过DNase I保护实验已将其结合位点划定为一个10至17-bp的元件。在肌发生过程中，140-kDa蛋白、沉默蛋白的结合效率与体内基因活性之间存在直接关联。通过序列同一性确定，已知含有功能性沉默元件的基因也至少含有一个抗沉默子元件。因此，我们鉴定出了一个在波形蛋白基因表达的发育调控中起重要作用的抗沉默子元件和蛋白，其可能参与其他基因的调控。
