Regulation of stem cell fate by FOXO and RNA binding proteins

在理解发育途径是如何调节以维持 干细胞在长时间静止或不分裂期间的多能性。FOXO家族 转录因子是干细胞维持和静止的关键调节因子。但 FOXO蛋白影响发育途径以控制细胞命运的机制很差， 明白这个应用程序充分利用了C语言的强大功能。elegans系统来解决 单一FOXO直系同源物daf-16调节保守发育途径的机制， 在静止期间保持干细胞的多能性。为了模拟干细胞静止，我们将使用 静止的dauer幼虫阶段，在发育过程中对不利的环境作出反应 条件这种方法是创新的，因为C。elegans系统允许我们研究静止的， 多能细胞在体内单细胞分辨率，补充哺乳动物研究。的长期目标 这个实验室的目的是破译在Dauer过程中促进多能性的机制。表皮缝细胞 干细胞模型是多能，并在 每一个幼虫阶段，直到成年分化。在dauer过程中，缝细胞有静止和活跃两种状态 机制维持多能性。初步数据确定，在Dauer期间，FOXO/DAF-16区块 通过积极调节编码RNA结合蛋白的三个基因的表达来调节成体细胞的命运 （限制性商业惯例）。这些RBP的直系同源物调节干细胞和祖细胞的增殖和功能， 苍蝇和哺乳动物。本申请的目的是揭示FOXO/daf-16 在静止期通过RBP调节成体细胞的命运。三个具体目标是 为实现这一目标而提出。1)确定FOXO/daf-16与RBPs之间的遗传关系。 功能丧失和功能获得实验将确定FOXO/daf-16对RBP的调节， 控制细胞命运的新机制。2)确定阻断成年细胞命运的直接RBP靶点， 安静将通过iCLIP鉴定RBP的直接mRNA靶点。功能测试将确定 这些靶点参与Seam细胞命运的调节。这些实验将共同阐明 FOXO/daf-16调节的RBP与成体细胞命运之间的联系。3)剖析转录 在静止期间调节成体细胞命运标记物。初步数据表明在道尔期间， FOXO/daf-16和三种RBP阻断成人特异性基因的转录报告基因的表达， 广泛用于标记成年细胞的命运。启动子切割和候选转录的功能测试 因子将破译一个关键的成体细胞命运标记的静止特异性调节。拟议工作 是重要的，因为它将阐明如何调节发育途径是协调的， 调节多能细胞的静止状态。

A fundamental gap exists in the understanding of how developmental pathways are regulated to maintain stem cell multipotency during extended periods of quiescence, or non-division. The FOXO family of transcription factors are key regulators of stem cell maintenance and quiescence. However, the mechanisms by which FOXO proteins impact developmental pathways to control cell fate are poorly understood. This application capitalizes on the power of the C. elegans system to address the mechanisms by which the single FOXO ortholog, daf-16, regulates conserved developmental pathways to preserve stem cell multipotency during quiescence. To model stem cell quiescence, we will use the quiescent dauer larva stage, adopted midway through development in response to adverse environmental conditions. This approach is innovative because the C. elegans system allows us to study quiescent, multipotent cells in vivo at single cell resolution, complementing mammalian studies. The long-term goal of this lab is to decipher the mechanisms that promote multipotency during dauer. Epidermal seam cells, the stem cell model, are multipotent and undergo a characteristic pattern of self-renewing cell divisions at each larval stage until differentiating at adulthood. During dauer, seam cells are quiescent and active mechanisms maintain multipotency. Preliminary data establish that during dauer, FOXO/daf-16 blocks adult cell fate by positively regulating the expression of three genes that encode RNA-binding proteins (RBPs). The orthologs of these RBPs regulate the proliferation and function of stem and progenitor cells in flies and mammals. The objective of this application is to unravel the mechanisms by which FOXO/daf-16 acts via RBPs to regulate adult cell fate during the quiescent dauer stage. Three specific aims are proposed to meet this objective. 1) Determine the genetic relationship between FOXO/daf-16 and RBPs. Loss-of-function and gain-of-function experiments will establish the regulation of RBPs by FOXO/daf-16, a novel mechanism to control cell fate. 2) Identify direct RBP targets that block adult cell fate during quiescence. Direct mRNA targets of RBPs will be identified by iCLIP. Functional testing will determine which targets are involved in the regulation of seam cell fate. Together these experiments will elucidate the connection between FOXO/daf-16-regulated RBPs and adult cell fate. 3) Dissect the transcriptional regulation of an adult cell fate marker during quiescence. Preliminary data establish that during dauer, FOXO/daf-16 and the three RBPs block expression of a transcriptional reporter of an adult-specific gene, widely used to mark adult cell fate. Promoter dissection and functional testing of candidate transcription factors will decipher the quiescence-specific regulation of a key adult cell fate marker. The proposed work is significant because it will illuminate how the regulation of developmental pathways is coordinated with the regulation of quiescence in multipotent cells.