Ambrosetti D C, Basilico C, Dailey L
Department of Microbiology and Kaplan Cancer Center, New York University School of Medicine, New York 10016, USA.
Mol Cell Biol. 1997 Nov;17(11):6321-9. doi: 10.1128/MCB.17.11.6321.
Octamer binding and Sox factors are thought to play important roles in development by potentiating the transcriptional activation of specific gene subsets. The proteins within these factor families are related by the presence of highly conserved DNA binding domains, the octamer binding protein POU domain or the Sox factors HMG domain. We have previously shown that fibroblast growth factor 4 (FGF-4) gene expression in embryonal carcinoma cells requires a synergistic interaction between Oct-3 and Sox2 on the FGF-4 enhancer. Sox2 and Oct-3 bind to adjacent sites within this enhancer to form a ternary protein-DNA complex (Oct-3*) whose assembly correlates with enhancer activity. We now demonstrate that increasing the distance between the octamer and Sox binding sites by base pair insertion results in a loss of enhancer function. Significantly, those enhancer "spacing mutants" which failed to activate transcription were also compromised in their ability to form the Oct* complexes even though they could still bind both Sox2 and the octamer binding proteins, suggesting that a direct interaction between Sox2 and Oct-3 is necessary for enhancer function. Consistent with this hypothesis, Oct-3 and Sox2 can participate in a direct protein-protein interaction in vitro in the absence of DNA, and both this interaction and assembly of the ternary Oct* complexes require only the octamer protein POU and Sox2 HMG domains. Assembly of the ternary complex by these two protein domains occurs in a cooperative manner on FGF-4 enhancer DNA, and the loss of this cooperative interaction contributes to the defect in Oct-3* formation observed for the enhancer spacing mutants. These observations indicate that Oct-3* assembly results from protein-protein interactions between the domains of Sox2 and Oct-3 that mediate their binding to DNA, but it also requires a specific arrangement of the binding sites within the FGF-4 enhancer DNA. Thus, these results define one parameter that is fundamental to synergistic activation by Sox2 and Oct-3 and further emphasize the critical role of enhancer DNA sequences in the proper assembly of functional activation complexes.
八聚体结合因子和Sox因子被认为通过增强特定基因亚群的转录激活在发育过程中发挥重要作用。这些因子家族中的蛋白质通过存在高度保守的DNA结合结构域相关联，即八聚体结合蛋白POU结构域或Sox因子HMG结构域。我们之前已经表明，胚胎癌细胞中纤维母细胞生长因子4（FGF - 4）基因的表达需要Oct - 3和Sox2在FGF - 4增强子上的协同相互作用。Sox2和Oct - 3结合到该增强子内的相邻位点，形成三元蛋白质 - DNA复合物（Oct - 3*），其组装与增强子活性相关。我们现在证明，通过碱基对插入增加八聚体和Sox结合位点之间的距离会导致增强子功能丧失。值得注意的是，那些未能激活转录的增强子“间隔突变体”在形成Oct复合物的能力上也受到损害，尽管它们仍然可以结合Sox2和八聚体结合蛋白，这表明Sox2和Oct - 3之间的直接相互作用对于增强子功能是必需的。与该假设一致，Oct - 3和Sox2在没有DNA的情况下可以在体外参与直接的蛋白质 - 蛋白质相互作用，并且这种相互作用以及三元Oct复合物的组装仅需要八聚体蛋白POU和Sox2 HMG结构域。这两个蛋白质结构域在FGF - 4增强子DNA上以协同方式组装三元复合物，并且这种协同相互作用的丧失导致了在增强子间隔突变体中观察到的Oct - 3形成缺陷。这些观察结果表明，Oct - 3的组装是由Sox2和Oct - 3结构域之间的蛋白质 - 蛋白质相互作用介导的，这些相互作用介导它们与DNA的结合，但它也需要FGF - 4增强子DNA内结合位点的特定排列。因此，这些结果定义了Sox2和Oct - 3协同激活所必需的一个基本参数，并进一步强调了增强子DNA序列在功能性激活复合物正确组装中的关键作用。
