Proteasomal regulation of proliferation in the C. elegans germ line

干细胞对多细胞生物的正常发育和组织稳态至关重要。它们之所以能够实现这些功能，部分原因在于它们具有产生自我更新和分化子细胞的独特能力。分化的子细胞形成所需的组织，而自我更新的子细胞保留其干细胞特性并维持干细胞群。我实验室的总体项目是了解干细胞的行为是如何被控制的。我们使用秀丽隐杆线虫生殖系作为模型，它在每个性腺臂的远端维持一个干细胞群，使蠕虫在其一生中产生数千个配子。某些蛋白质（促进增殖的蛋白质）的联合表达提供了干细胞的身份和自我更新的能力；因此，这些蛋白质的表征将为干细胞的行为和功能提供重要的见解。我们发现蛋白酶体降解是控制增殖促进蛋白积累的关键机制。本研究的目标是描述蛋白酶体降解如何促进干细胞自我更新的调节。我们已经进行了RNAi筛选，并鉴定了参与控制干细胞自我更新的e3 -泛素连接酶的五个底物识别亚基（srs）。我们将首先描述这些srs之一RFP-1如何控制MRG-1染色体结构域蛋白的积累，我们将其确定为干细胞增殖的启动子。我们的遗传分析也表明RFP-1不可能是唯一针对MRG-1降解的SRS。因此，我们将识别这些其他srs，并确定它们如何与RFP-1协调其活性。然后，我们将对我们在最初的RNAi筛选中发现的其他四个srs进行遗传分析，以确定干细胞功能的哪些方面可以由它们各自控制。接下来，我们将识别和表征每个SRS的蛋白质靶点，并确认蛋白质降解有助于它们的积累模式。最后，我们将进行基因筛选作为一种独立的方法来识别增殖促进蛋白。鉴定这些蛋白质，并了解它们的活性是如何由蛋白质降解控制的，将为了解干细胞行为是如何被控制提供重要的见解。

Stem cells are essential for proper development and tissue homeostasis in multicellular organisms. They are able to fulfill these functions due, in part, to their unique ability to produce both self-renewing and differentiating daughter cells. Differentiating daughter cells form the desired tissue, while self-renewing daughter cells retain their stem cell character and maintain the stem cell population. The overall program in my lab is to understand how stem cell behaviour is controlled. We use the C. elegans germ line as a model, which maintains a stem cell population in the distal end of each gonad arm, allowing the worm to produce thousands of gametes throughout its life. The combined expression of certain proteins (proliferation-promoting proteins) provides stem cells with their identity and ability to self-renew; therefore, characterization of these proteins will provide significant insight into stem cell behaviour and function. We identified proteasomal degradation as a key mechanism in controlling the accumulation of proliferation-promoting proteins. The goal of the research described in this proposal is to characterize how proteasomal degradation contributes to the regulation of stem cell self-renewal. We have already conducted an RNAi screen and identified five substrate recognition subunits (SRSs) of E3-ubiquitin ligases that participate in controlling stem cell self-renewal. We will first characterize how one of these SRSs, RFP-1, controls the accumulation of the MRG-1 chromodomain protein, which we identified as a promoter of stem cell proliferation. Our genetic analysis has also demonstrated that RFP-1 cannot be the only SRS that targets MRG-1 for degradation. Therefore, we will identify these other SRSs and determine how they coordinate their activity with RFP-1. We will then perform genetic analyses of the other four SRSs that we identified in our initial RNAi screen to identify what aspect of stem cell function could be controlled by each of them. Next, we will identify and characterize the protein targets of each SRS, and confirm that protein degradation contributes to their pattern of accumulation. Finally, we will conduct a genetic screen as an independent approach to identify proliferation-promoting proteins. Identification of these proteins, and gaining an understanding of how their activity is controlled by protein degradation, will provide significant insights into how stem cell behaviour is controlled.