Li W W, Hsiung Y, Wong V, Galvin K, Zhou Y, Shi Y, Lee A S
Department of Biochemistry and Molecular Biology, Norris Cancer Center, University of Southern California School of Medicine, Los Angeles 90033, USA.
Mol Cell Biol. 1997 Jan;17(1):61-8. doi: 10.1128/MCB.17.1.61.
The highly conserved grp78 core promoter element plays an important role in the induction of grp78 under diverse stress signals. Previous studies have established a functional region in the 3' half of the core (stress-inducible change region [SICR]) which exhibits stress-inducible changes in stressed nuclei. The human transcription factor YY1 is shown to bind the SICR and transactivate the core element under stress conditions. Here we report that expression library screening with the core element has identified two new core binding proteins, YB-1 and dbpA. Both proteins belong to the Y-box family of proteins characterized by an evolutionarily conserved DNA binding motif, the cold shock domain (CSD). In contrast to YY1, which binds only double-stranded SICR, the Y-box/CSD proteins much prefer the lower strand of the SICR. The Y-box proteins can repress the inducibility of the grp78 core element mediated by treatment of cells with A23187, thapsigargin, and tunicamycin. In gel shift assays, YY1 binding to the core element is inhibited by either YB-1 or dbpA. A yeast interaction trap screen using LexA-YY1 as a bait and a HeLa cell cDNA-acid patch fusion library identified YB-1 as a YY1-interacting protein. In cotransfection experiments, the Y-box proteins antagonize the YY1-mediated enhancement of transcription directed by the grp78 core in stressed cells. Thus, the CSD proteins may be part of the stress signal transduction mechanism in the mammalian system.
高度保守的grp78核心启动子元件在多种应激信号诱导grp78表达过程中发挥重要作用。以往研究已在核心区域的3'半段确定了一个功能区（应激诱导变化区域[SICR]），该区域在应激细胞核中呈现应激诱导变化。研究表明，人类转录因子YY1在应激条件下可结合SICR并激活核心元件。在此，我们报告用核心元件进行表达文库筛选已鉴定出两种新的核心结合蛋白，YB-1和dbpA。这两种蛋白均属于Y-盒蛋白家族，其特征是具有进化上保守的DNA结合基序——冷休克结构域（CSD）。与仅结合双链SICR的YY1不同，Y-盒/CSD蛋白更倾向于结合SICR的下链。Y-盒蛋白可抑制用A23187、毒胡萝卜素和衣霉素处理细胞所介导的grp78核心元件的诱导性。在凝胶迁移实验中，YB-1或dbpA均可抑制YY1与核心元件的结合。以LexA-YY1为诱饵和HeLa细胞cDNA-酸性补丁融合文库进行的酵母相互作用陷阱筛选鉴定出YB-1是一种与YY1相互作用的蛋白。在共转染实验中，Y-盒蛋白可拮抗应激细胞中YY1介导的由grp78核心驱动的转录增强。因此，CSD蛋白可能是哺乳动物系统应激信号转导机制的一部分。
