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

Oct-3/4 独特的 DNA 结合特异性及其维持 ES 多能状态的能力

• 批准号: 13670131
• 负责人: OKUDA Akihiko
• 金额: $ 2.3万
• 依托单位: Saitama Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670131/
• 关键词: ES cells; Octamer factor; Oct-3; 4; pluripotency; undifferentiated states; POU-ドメイン

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

ES细胞具有全能性，可以分化为任何类型的细胞，并且已经显示胚胎八聚体因子Oct-3/4在维持这种ES细胞特异性特性中起核心作用。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，而不是其他八聚体因子，具有一些独特的生化特性，这涉及细胞的这种生物学特性。在这个项目中，我已经进行了几个实验，以了解这个Oct-3/4特定的生化特征是什么。首先，我利用一系列由Oct-3/4和Oct-6组成的嵌合蛋白，定位了维持ES细胞多能性的Oct-3/4结构域。这些分析表明，POU特异性结构域中的接头部分和一个特定的氨基酸是必需的，但其他部分如转录激活结构域可以被Oct-6蛋白的相应部分取代。胚胎干细胞 ...更多信息 用上述嵌合蛋白代替野生型Oct-3/4表达未分化标记物，如雷克斯-1和UTF 1。此外，我们证实这些细胞保留了ES细胞最重要的特性之一，即，转化为任何不同类型的外胚层、中胚层和内胚层细胞的能力。我还通过凝胶迁移试验系统地测定了八聚体因子以及上述嵌合蛋白的DNA结合特异性。这些分析导致鉴定出9种不同的核苷酸序列，其能够作为Oct-3/4的特异性或优先结合位点。此外，我一直在寻找Oct-3/4的这种独特的DNA结合特异性与其维持ES细胞处于多能状态的能力有关的可能性。这些分析确实揭示了结合一个特定序列5 '-ACTAGCAT-3'的能力与保持ES细胞处于未分化状态的能力之间存在完美的相关性。有趣的是，这个序列是存在于UTF 1调控区的序列，并且在支持UTF 1在ES细胞中的表达中起核心作用。一个在其调节区中携带共有八聚体序列，另一个在其增强子中携带上述变体八聚体序列，并且ES细胞中对Oct-3/4的关键需求是由于该蛋白质支持第二组Oct-3/4下游基因表达的独特能力。少

项目成果

Masazumi Nishimoto: "The embryonic Octamer factor 3/4 displays distinct DNA binding specificity from those of other Octamer factors"Biochem.Biophys.Res.Commun.. 302. 581-586 (2003)

Masazumi Nishimoto：“胚胎八聚体因子 3/4 显示出与其他八聚体因子不同的 DNA 结合特异性”Biochem.Biophys.Res.Commun.. 302. 581-586 (2003)

Nishimoto, M., Miyagi, S., Katayanagi, T., Tomioka, M., Muramatsu, M., and Okuda, A.: "The embryonic Octamer factor 3/4 displays distinct DNA binding specificity from those of other Octamer factors"Biochem. Biophys. Res. Commun.. 302. 581-586 (2003)

Nishimoto, M.、Miyagi, S.、Katayanagi, T.、Tomioka, M.、Muramatsu, M. 和 Okuda, A.：“胚胎八聚体因子 3/4 显示出与其他八聚体因子不同的 DNA 结合特异性

Nishimoto Masazumi: "Structural analysis of the UTF1 gene encoding a transcriptional coactivator expressed in pluripotent embryonic stem cells"Biochem. Biophys. Res. Commun.. 285. 945-953 (2001)

Nishimoto Masazumi：“编码多能胚胎干细胞中表达的转录共激活因子的 UTF1 基因的结构分析”Biochem。

Tomioka, M., Nishimoto, M., Miyagi, S., Katayanagi, T., Fukui, N., Niwa, H., Muramatsu, M., and Okuda, A.: "Identification of Sox-2 regulatory region which is under the control of Oct-3/4-Sox-2 complex"Nucleic Acids Res. 30. 3202-3213 (2002)

Tomioka, M.、Nishimoto, M.、Miyagi, S.、Katayanagi, T.、Fukui, N.、Niwa, H.、Muramatsu, M. 和 Okuda, A.：“Sox-2 调控区的识别

Mizuho Tomioka: "Identification of Sox-2 regulatory region which is under the control of Oct-3/4-Sox-2 complex"Nucleic Acids Research. 30. 3202-3213 (2002)

Mizuho Tomioka：“Oct-3/4-Sox-2 复合体控制下的 Sox-2 调节区的鉴定”核酸研究。