Unique DNA binding specificity of Oct-3/4 and its ability to maintain ES in pluripotent state

胚胎干细胞具有无所不能的特性，可以分化为任何类型的细胞，并且已经证明胚胎八聚体因子Oct-3/4在维持胚胎干细胞特异性方面起着核心作用。由于Oct-1和Oct-6蛋白存在于ES细胞中，这一事实表明Oct-3/4蛋白，而不是其他Octamer因子，具有一些独特的生化特性，这些特性与细胞的这种生物学特性有关。在这个项目中，我做了几个实验，以了解这个Oct-3/4特定的生化特征是什么。首先，通过使用由Oct-3/4和Oct-6组成的一系列嵌合蛋白，我绘制了参与维持胚胎干细胞多能性的Oct-3/4结构域。这些分析表明，连接体部分和POU特异性结构域的一个特定氨基酸是必需的，但其他部分如转录激活结构域可以被Oct-6蛋白的相应部分所取代。与野生型Oct-3/4相比，胚胎干细胞携带更多的上述嵌合蛋白表达未分化的标记物，如Rex-1和UTF1。此外，我们证实这些细胞保留了胚胎干细胞最重要的特性之一，即能够转化为任何不同类型的外胚层、中胚层和内胚层细胞。我还系统地测定了八聚体因子和上述嵌合蛋白的DNA结合特异性。这些分析鉴定出9个不同的核苷酸序列，它们可以作为Oct-3/4的特异性或优先结合位点。此外，我还研究了Oct-3/4独特的DNA结合特异性与其维持胚胎干细胞多能状态的能力有关的可能性。这些分析确实揭示了与特定序列5‘-ACTAGCAT-3’结合的能力与保持胚胎干细胞处于未分化状态的能力之间存在着完美的相关性。有趣的是，该序列存在于UTF1调控区，并在支持ES细胞中UTF1表达中发挥核心作用。从这些结果中，我得出Oct-3/4下游基因可以分为两组，一组在其调控区域携带一致的八聚体序列，另一组在其增强子中携带上述变异的八聚体序列，而胚胎干细胞对Oct-3/4的关键需求是由于该蛋白具有支持第二组Oct-3/4下游基因表达的独特能力。少

ES cells have omnipotent property which can be differentiated to any types of cells and it has been shown that the embryonic Octamer factor, Oct-3/4 plays a central role in maintaining this ES cell specific property. As Oct-1 and Oct-6 proteins are present in ES cells, this fact indicates that Oct-3/4 protein, but not other Octamer factors, has some unique biochemical characteristics which is involved in this biological property of the cells. In this project, I have performed several experiments in order to know what this Oct-3/4 specific biochemical characteristics is. At first, I have mapped the Oct-3/4 domains involved in maintaining pluripotent property of ES cells by using a series of chimeric proteins composed of Oct-3/4 and Oct-6. These analyses revealed that the linker portion and one particular amino acid in POU specific domain are essential, but other portions such as transcriptional activation domains can be replaced by corresponding portions of Oct-6 protein. ES cells beari … More ng the above chimeric protein instead of the wild-type Oct-3/4 express undifferentiated markers such as Rex-1 and UTF1. Furthermore, we confirmed that these cells retains one of the most important properties of ES cells, i.e., the ability to convert to any different types of ectodermal, mesodermal, and endodermal cells.I have also systematically determined DNA binding specificity of Octamer factors as well as the above chimeric proteins by gel shift assay. These analyses led to the identification of 9 different nucleotide sequences which are able to serve as specific or preferential binding sites for Oct-3/4. Furthermore, I have pursued the possibility that this unique DNA binding specificity of the Oct-3/4 is related to its ability to maintain ES cells in pluripotent state. These analyses indeed reveal that there is perfect correlation between the ability to bind to one particular sequence, 5'-ACTAGCAT-3' and that to keep ES cells in undifferentiated state. Interestingly, this sequence is the one which is present in the UTF1 regulatory region and plays central role in supporting UTF1 expression in ES cells.From these results, I concluded that Oct-3/4 downstream genes can be classified into 2 different groups, i.e., one bearing consensus Octamer sequences in their regulatory regions and the other carrying the above variant Octamer sequences in their enhancers and crucial requirement of Oct-3/4 in ES cells is due to its unique ability of this protein to support expression of second group of Oct-3/4 downstream genes. Less