Ambrosetti D C, Schöler H R, Dailey L, Basilico C
Department of Microbiology, New York University School of Medicine, New York, New York 10016, USA.
J Biol Chem. 2000 Jul 28;275(30):23387-97. doi: 10.1074/jbc.M000932200.
Fibroblast growth factor (FGF)-4 gene expression in the inner cell mass of the blastocyst and in EC cells requires the combined activity of two transcriptional regulators, Sox2 and Oct-3, which bind to adjacent sites on the FGF-4 enhancer DNA and synergistically activate transcription. Sox2 and Oct-3 bind cooperatively to the enhancer DNA through their DNA-binding, high mobility group and POU domains, respectively. These two domains, however, are not sufficient to activate transcription. We have analyzed a number of Sox2 and Oct-3 deletion mutants to identify the domains within each protein that contribute to the activity of the Sox2 x Oct-3 complex. Within Oct-3, we have identified two activation domains, the N-terminal AD1 and the C-terminal AD2, that play a role in the activity of the Sox2 x Oct-3 complex. AD1 also displays transcriptional activation functions in the absence of Sox2 while AD2 function was only detected within the Sox2 x Oct-3 complex. In Sox2, we have identified three activation domains within its C terminus: R1, R2, and R3. R1 and R2 can potentiate weak activation by Sox2 in the absence of Oct-3 but their deletion has no effect on the Sox2 x Oct-3 complex. In contrast, R3 function is only observed when Sox2 is complexed with Oct-3. In addition, analysis of Oct-1/Oct-3 chimeras indicates that the Oct-3 homeodomain also plays a critical role in the formation of a functional Sox2 x Oct-3 complex. Our results are consistent with a model in which the synergistic action of Sox2 and Oct-3 results from two major processes. Cooperative binding of the factors to the enhancer DNA, mediated by their binding domains, stably tethers each factor to DNA and increases the activity of intrinsic activation domains within each protein. Protein-protein and protein-DNA interactions then may lead to reciprocal conformational changes that expose latent activation domains within each protein. These findings define a mechanism that may also be utilized by other Sox x POU protein complexes in gene activation.
成纤维细胞生长因子（FGF）-4基因在囊胚内细胞团和胚胎癌细胞中的表达需要两种转录调节因子Sox2和Oct-3的共同作用，它们与FGF-4增强子DNA上的相邻位点结合并协同激活转录。Sox2和Oct-3分别通过其DNA结合、高迁移率族和POU结构域与增强子DNA协同结合。然而，这两个结构域不足以激活转录。我们分析了许多Sox2和Oct-3缺失突变体，以确定每种蛋白质中有助于Sox2×Oct-3复合物活性的结构域。在Oct-3中，我们确定了两个激活结构域，即N端的AD1和C端的AD2，它们在Sox2×Oct-3复合物的活性中起作用。在没有Sox2的情况下，AD1也显示出转录激活功能，而AD2功能仅在Sox2×Oct-3复合物中检测到。在Sox2中，我们在其C端确定了三个激活结构域：R1、R2和R3。在没有Oct-3的情况下，R1和R2可以增强Sox2的弱激活作用，但它们的缺失对Sox2×Oct-3复合物没有影响。相反，只有当Sox2与Oct-3复合时才能观察到R3功能。此外，对Oct-1/Oct-3嵌合体的分析表明，Oct-3同源结构域在功能性Sox2×Oct-3复合物的形成中也起着关键作用。我们的结果与一个模型一致，在该模型中，Sox2和Oct-3的协同作用源于两个主要过程。因子通过其结合结构域与增强子DNA的协同结合，将每个因子稳定地束缚在DNA上，并增加每种蛋白质中内在激活结构域的活性。然后，蛋白质-蛋白质和蛋白质-DNA相互作用可能导致相互的构象变化，从而暴露每种蛋白质中的潜在激活结构域。这些发现定义了一种机制，其他Sox×POU蛋白复合物在基因激活中也可能利用这种机制。
