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.
