Proteasomal regulation of proliferation in the C. elegans germ line

干细胞对于多细胞生物体的正常发育和组织动态平衡是必不可少的。它们能够完成这些功能，部分原因是它们产生自我更新和分化的子代细胞的独特能力。分化的子代细胞形成所需的组织，而自我更新的子代细胞保留其干细胞特性并维持干细胞种群。我实验室的总体计划是了解干细胞行为是如何控制的。我们使用线虫生殖系作为模型，它在每个性腺臂的远端维持干细胞群，使蠕虫在其一生中产生数千个配子。某些蛋白质(促增殖蛋白)的联合表达为干细胞提供了其身份和自我更新的能力；因此，这些蛋白质的特征将为了解干细胞的行为和功能提供重要的洞察。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。这项建议中描述的研究的目标是描述蛋白酶体降解如何有助于干细胞自我更新的调节。我们已经进行了RNAi筛选，并鉴定了E3-泛素连接酶的五个底物识别亚单位(SRSS)，它们参与控制干细胞的自我更新。我们将首先确定其中一个SRS，RFP-1，是如何控制MRG-1染色域蛋白的积累的，我们确定MRG-1是干细胞增殖的促进剂。我们的基因分析还表明，RFP-1不是唯一针对MRG-1进行降解的SRs。因此，我们将确定其他SRS，并确定它们如何将其活动与RFP-1协调起来。然后，我们将对我们在最初的RNAi筛查中确定的其他四个SRS进行遗传分析，以确定它们各自可以控制干细胞功能的哪些方面。接下来，我们将识别和表征每个SRs的蛋白质靶标，并确认蛋白质降解有助于它们的积累模式。最后，我们将进行基因筛查，作为一种独立的方法来识别促进增殖的蛋白质。鉴定这些蛋白质，并了解它们的活性如何受到蛋白质降解的控制，将为干细胞行为如何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.