Chemical control of protein stability to analyze germline cell fate

描述（由申请人提供）：秀丽隐杆线虫（C. elegans）生殖系为研究调节干细胞维持和分化为精子或卵母细胞的分子控制提供了模型。GLP-1/ notch信号和FBF/PUF RNA结合蛋白都有助于维持干细胞和促进有丝分裂细胞分裂。fog1 /CPEB和fog3 /Tob蛋白调控生殖细胞向精子的分化。脊椎动物Tob被认为是一种具有抗增殖活性的转录调节剂和RNA调节剂。然而，Tob蛋白的作用机制尚不清楚。这些关键的调节因子是参与细胞增殖、干细胞维持和分化的基因网络的一部分。研究这些调节器如何在一个简单的模型系统中工作，在这个模型系统中可以探索关键问题，这将有助于了解它们如何在保守的脊椎动物中起作用。具体目的：1)开发一种化学方法来操纵秀丽隐杆线虫的蛋白质稳定性。2)通过化学方法调控关键生殖系干细胞调控因子（fog3、FBF-1和LAG-2）的存在或缺失，研究其对生殖细胞命运的控制。研究设计：我将一项新技术应用于秀丽隐杆线虫，在体内有条件地稳定转基因蛋白。当一个不稳定结构域（DD）与一个感兴趣的蛋白质融合时，它会导致融合蛋白的快速降解。然而，这种融合蛋白可以用一种叫做Shield1的小分子处理后稳定并发挥功能。通过将转基因置于组织特定的调控序列下，我将能够以空间和时间的方式控制融合蛋白的表达。fog3， FBF-1和LAG-2分别是干细胞分化和维持的保守调节因子，我计划使用这项技术来回答关于它们在生殖系中的功能的关键问题，这些问题是现有方法无法解决的。健康相关性：这些研究将有助于我们理解干细胞维持和分化的调控。由于生殖系干细胞维持的调控涉及从秀丽隐杆线虫到哺乳动物的遗传调控，我们的研究将有助于揭示这些基因如何控制干细胞生长并使其保持未分化状态的细节。研究使细胞保持有丝分裂生长阶段而不是使它们分化的基本成分，对于我们理解癌症的进展和治疗方法至关重要。

DESCRIPTION (provided by applicant): The Caenorhabditis elegans (C. elegans) germ line provides a model for the investigation of molecular controls regulating stem cell maintenance and differentiation into sperm or oocyte. Both GLP-1/Notches signaling as well as FBF/PUF RNA binding proteins contribute to maintain stem cells and promote mitotic cell division. FOG-1/CPEB and FOG-3/Tob proteins regulate the specification of germ cells into sperm. Vertebrate Tob has been suggested to be both a transcriptional regulator as well as an RNA regulator with antiproliferative activity. However, the mechanism by which Tob proteins function is poorly understood. These key regulators are part of a network of genes involved in cellular proliferation, stem cell maintenance and differentiation. Investigating how these regulators work in a simple model system where key questions can be explored will provide insight into how they may work among their conserved vertebrate counterparts. Specific Aims: 1) to develop a chemical method for manipulation of protein stability in C. elegans. 2) To chemically manipulate the presence or absence of key germline stem cell regulators (FOG-3, FBF-1, and LAG-2) and investigate their control of germ cell fate. Study Design: I have applied a new technology to C. elegans for conditionally stabilizing transgenic proteins in vivo. When a Destablization Domain (DD) is fused to a protein of interest, it causes rapid degradation of the fusion protein. However, this fusion protein can be stabilized and rendered functional when treated with a small molecule called Shield1. By placing transgenes under tissue specific regulatory sequences I will be able to control expression of fusion proteins in a spatial and temporal manner. FOG-3, FBF-1, and LAG-2 are conserved regulators of stem cell differentiation and maintenance, respectively, and I plan to use this technology to answer key questions about their function in the germ line that have not been possible with existing approaches. Health Relevance: These studies will contribute to our understanding of the regulation of stem cell maintenance and differentiation. Since the regulation of germ line stem cell maintenance involves genetic regulators which are conserved form C. elegans to mammals, our studies will help to uncover details of how these genes govern stem cell growth and keep them in an undifferentiated state. Investigating the basic components that keep cells in a mitotic growth phase versus causing them to differentiate is essential in our understanding of cancer progression and methods of treatment. PUBLIC HEALTH RELEVANCE: The regulators of stem cell self renewal being studied in this proposal are important for maintaining the balance between cell growth and differentiation. When disrupted, this balance between growth and differentiation can lead to devastating diseases, such as cancer. Studying a powerful genetic animal system like the worm C. elegans will reveal new mammalian signaling components. Combined with the potential of the worm for use in drug screens, it is clear the benefits of using C. elegans to contribute to the advancement of cancer research.