Roles of Oct4 dimers during mouse germline development

POU转录因子Oct 4是哺乳动物生殖系发育和胚胎干细胞多能性的关键调节因子，因此是控制与哺乳动物生殖相关的基因表达的最重要因素之一。 Oct 4蛋白水平的细微变化决定了这些最早期干细胞沿沿着三种不同谱系的分化。 最近描述的成体干细胞的可塑性引发了对Oct 4的新的和进一步的兴趣，因为它也可能参与这些细胞重新获得多能性的过程。 二聚体相互作用在将细胞内和细胞间信号转导为生物功能中通常是至关重要的。 不同的POU二聚体构象表现出不同的活性。 这项工作的目的是了解Oct 4二聚体在小鼠生殖系发育和胚胎干细胞潜能中的作用。 研究Oct 4的异常构象提供了对胚胎丢失和不育的深入了解。本质上，所提出的研究将为两种不同的Oct 4二聚体建立牢固的结构-功能关系。Oct 4蛋白在识别同源DNA元件方面表现出令人难以置信的多样性。 其DNA结合结构域POU结构域通过由柔性接头区连接的两个结构独立的亚结构域赋予这种特征。 为了在种系发育期间以精确的方式调节Oct 4功能，将基于与DNA复合的两种构象的分子结构来定义两种Oct 4二聚体构象所必需的确定性蛋白质-蛋白质相互作用（目的1）。 通过将点突变引入内源性Oct 4基因并分析发育中小鼠胚胎的表型，确定Oct 4二聚体形成在种系发育和干细胞潜能中的作用（目的2）。 为了充分评估两种二聚体的功能，将阐明调节Oct 4 POU二聚体活性的方式。 这些包括Oct 4的分子内和分子间相互作用（目的3）。 使用最近在实验室中开发的从胚胎干细胞衍生生殖细胞的强大体外系统，将完全表征两种二聚体在生殖细胞形成中的作用（目的4）。 由此，可以在限定的培养条件下探索引起多能性和种系确定的细胞机制。 这些研究将允许在体外和体内操作干细胞潜能和生殖细胞谱系。预期的结果将有相关的适用性干细胞和生殖生物学，并导致更好地了解人类生殖。

The POU transcription factor Oct4 is a key regulator of mammalian germline development and pluripotency of embryonic stem cells and is thus one of the most important factors in controlling the expression of genes associated with mammalian reproduction. Subtle changes in Oct4 protein levels determine differentiation of these earliest stem cells along three different lineages. The recently described plasticity of adult stem cells has sparked new and further interest in Oct4, as it may also be involved in the process by which these cells regain pluripotency. Dimer interactions are often crucial in transducing inter- and intracellular signals into biological function. Different POU dimer conformations exhibit distinct activities. The objective of the proposed work is to understand the role of Oct4 dimers in mouse germline development and embryonic stem cell potency. Studying aberrant conformations of Oct4 provide insight into embryo loss and infertility. Essentially, the proposed research will establish a firm structure - function relationship for two different Oct4 dimers. The Oct4 protein exhibits incredible diversity in the recognition of cognate DNA elements. Its DNA-binding domain, the POU domain, imparts this feature through its two structurally independent subdomains connected by a flexible linker region. To modulate Oct4 function in a precise manner during germline development, the definitive protein-protein interactions necessary for both Oct4 dimer conformations will be defined based on the molecular structure of both conformations in complex with DNA (aim 1). The role of Oct4 dimer formation in germline development and stem cell potency will be established by introducing point mutations into the endogenous Oct4 gene and by analyzing the resulting phenotypes in the developing mouse embryo (aim 2). To fully assess the function of both dimers the manner by which Oct4 POU dimer activity is regulated will be unraveled. These include intra- and inter-molecular interactions of Oct4 (aim 3). Using a powerful in vitro system recently developed in the laboratory to derive germ cells from embryonic stem cells, the role of both dimers in germ cell formation will be completely characterized, (aim 4). By this, the cellular mechanisms causing pluripotency and germline determination can be explored in defined culture conditions. These studies will allow stem cell potency and the germ cell lineage to be manipulated both in vitro and in vivo. The anticipated results will have relevant applicability to stem cell and germline biology and lead to a better understanding of human reproduction.