Xanthoudakis S, Cohen L, Hiscott J
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada.
J Biol Chem. 1989 Jan 15;264(2):1139-45.
High efficiency nuclear protein extracts were prepared from uninduced and recombinant interferon (IFN-alpha 2-primed Sendai virus-induced HeLa S3 cells and analyzed for DNA binding proteins specific for the human interferon-beta regulatory element (IRE). Analysis of protein-DNA interactions by a gel electrophoresis DNA binding assay resolved three complexes (designated A, B, and C) specific for the IRE probe (-79 to -36) in uninduced and induced cells. Competition studies using greater than a 100-fold molar excess of unlabeled DNA fragments representing different IFN-beta promoter sequences localized the DNA binding domain to the region -79 to -64 (P1 probe), a region previously shown to be essential for virus-inducible gene expression. Other adjacent IFN-beta segments including the negative regulatory element (-65 to -30) as well as the SV40 enhancer, c-fos serum-responsive element, and IFN-alpha 1 promoter fragment (-131 to -71) were unable to compete for the IRE binding protein. The 16-base pair enhancer core (P1 probe) specifically bound similar amounts of protein from uninduced and induced extracts; a synthetic tetrahexamer, AAGTGA, competed efficiently for the binding to the P1 region. Competition studies indicated that adjacent upstream IFN-beta DNA sequences -94 to -78 (P5) also contained a sequence motif capable of binding the P1 protein. In vitro transcription of the IFN-beta template in the HeLa nuclear extracts was partially inhibited when increasing amounts of competitor IRE fragment were added to the reaction. These results demonstrate that multiple DNA sequence motifs within the IFN-beta regulatory element interact to bind transcription regulatory proteins which are present in normal and virus-infected HeLa cells.
从未诱导的以及重组干扰素（IFN-α2 预处理的仙台病毒诱导的 HeLa S3 细胞）中制备高效核蛋白提取物，并分析其与人干扰素-β调节元件（IRE）特异性结合的 DNA 结合蛋白。通过凝胶电泳 DNA 结合试验分析蛋白质 - DNA 相互作用，在未诱导和诱导的细胞中分辨出三种与 IRE 探针（-79 至 -36）特异性结合的复合物（分别命名为 A、B 和 C）。使用摩尔过量超过 100 倍的未标记 DNA 片段进行竞争研究，这些片段代表不同的 IFN-β启动子序列，结果将 DNA 结合结构域定位到 -79 至 -64 区域（P1 探针），该区域先前已被证明对病毒诱导型基因表达至关重要。其他相邻的 IFN-β片段，包括负调节元件（-65 至 -30）以及 SV40 增强子、c-fos 血清反应元件和 IFN-α1 启动子片段（-131 至 -71），均无法竞争 IRE 结合蛋白。16 个碱基对的增强子核心（P1 探针）特异性结合来自未诱导和诱导提取物的相似量的蛋白质；一种合成的四聚体 AAGTGA 能有效竞争与 P1 区域的结合。竞争研究表明，相邻的上游 IFN-βDNA 序列 -94 至 -78（P5）也包含一个能够结合 P1 蛋白的序列基序。当向反应中加入越来越多的竞争性 IRE 片段时，HeLa 核提取物中 IFN-β模板的体外转录受到部分抑制。这些结果表明，IFN-β调节元件内的多个 DNA 序列基序相互作用，以结合正常和病毒感染的 HeLa 细胞中存在的转录调节蛋白。
