Regulation of stem cell fate by FOXO and RNA binding proteins

在理解如何调节发育途径以维持 干细胞在长时间的静止期或非分裂期具有多能性。FOXO系列的 转录因子是干细胞维持和静止的关键调节因子。然而， FOXO蛋白影响发育途径以控制细胞命运的机制很差 明白了。此应用程序利用线虫系统的强大功能来解决 单一FOXO同源基因daf-16调节保守的发育途径的机制 在静止状态下保持干细胞的多能性。为了模拟干细胞静止，我们将使用 静止的达尔幼虫阶段，在发育中期被采用，以应对不利的环境 条件。这种方法是创新的，因为线虫系统允许我们研究静止的， 体内单细胞分辨率的多潜能细胞，补充哺乳动物的研究。的长期目标是 这个实验室是为了破译在Dauer过程中促进多能性的机制。表皮缝隙细胞， 干细胞模型是多能的，并经历一种特有的自我更新的细胞分裂模式 每个幼虫阶段，直到成虫分化。在Dauer过程中，Seam细胞处于静止和活跃状态 这些机制保持了多功能性。初步数据证实，在DAUER期间，FOXO/DAF-16块 正向调节编码RNA结合蛋白的三个基因表达的成体细胞命运 (限制性商业惯例)。这些限制性商业惯例的直系物调节干细胞和祖细胞的增殖和功能。 苍蝇和哺乳动物。本申请的目的是解开FOXO/DAF-16 通过限制性商业惯例在静止的达尔阶段调节成体细胞的命运。三个具体目标是 建议实现这一目标。1)确定FOXO/DAF-16与限制性商业惯例的遗传关系。 功能丧失和功能获得实验将建立FOXO/daf-16对限制性商业惯例的调节，a 控制细胞命运的新机制。2)识别直接阻止成体细胞命运的RBP靶点 宁静。限制性商业惯例的直接信使核糖核酸靶点将通过iCLIP确定。功能测试将确定 哪些靶点参与调节Seam细胞的命运。这些实验将共同阐明 FOXO/DAF-16调控的限制性商业惯例与成体细胞命运的关系。3)剖析转录 成体细胞命运标记在静止期的调节。初步数据表明，在达乌尔期间， FOXO/DAF-16和三个限制性商业惯例阻断成体特异性基因转录报告的表达， 广泛用于标记成年细胞的命运。候选转录的启动子解剖和功能测试 这些因素将破译一个关键的成年细胞命运标记的静止期特异性调节。拟议中的工作 具有重要意义，因为它将阐明发育途径的调节是如何与 多能细胞中静止状态的调节。

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.