Genetic co-regulation by master transcription factors in Drosophila intestinal stem cells

项目摘要 我们的许多器官保持健康和功能，因为它们取代了因损伤或疾病而失去的细胞 通过包含在其中的成体干细胞的分裂，产生新的细胞。成体干细胞分裂 不对称地，生成自身的新副本(自我更新)和最终将 “分化”成具有特殊功能的细胞(例如，吸收营养、抵抗感染、产生激素、 等)。虽然在识别一些所谓的“主要调控基因”方面取得了很大进展，但 控制干细胞是自我更新还是分化的决定，我们对此知之甚少 在这一过程中，由主调节器触发的特定遗传程序。尽管付出了许多努力，但这还是要做的 通过全基因组筛选技术识别主要调节器的靶标，这些技术经常落在 由于缺乏进一步的验证而停用。在这里，我提出一个研究项目，同时解决这两个问题 问题，通过询问…“我们是否可以通过整合关键干细胞基因的公开名单来更有效地识别 在互补性但无关的研究中获得的推定的主要监管目标？要解决上述问题 问题：我建议用果蝇(果蝇)中肠作为活体实验模型。 之前的研究已经为三个已知的主要监管机构生成了假设目标的独立列表 果蝇肠道中的干细胞(ESCARGET，Capicua和Stat92E)。我们使用生物信息学来比较 这些清单，并确定了假定只有一个或所有三个主调节器靶向的基因，这些基因 我们分别将其称为“单一监管”或“共同监管”。在本提案的目标1中，我们将抑制每个 掌握在肠道祖细胞中使用RNAi介导的蛋白敲除的调节因子，并验证单核和 根据逆转录确定的它们表达的变化共同调节候选基因 定量聚合酶链式反应(RT-qPCR)。在目标2中，我们将比较它们对维持、增殖率和 RNAi介导的单基因或共基因敲除后肠道祖细胞的分化潜能 受监管的候选对象在AIM 1中得到验证。这种方法将允许我们测试共同监管的假设 对于肠道干细胞的生物学来说，基因往往比单一调控的基因更关键。 最后，我们的初步观察表明，抑制ESG功能会导致Stat92E的减少 活性，在这里我建议测试关于ESG和调控之间联系的生物信息学预测 Stat92E是通过将另一个遗传和蛋白质相互作用数据数据库整合到。如果 这个项目的成功将：a)利用果蝇作为主要的遗传学模型系统来产生一系列新的 了解和治疗胃肠道疾病的研究线索；b)提供概念证明 鼓励和指导使用目前存放在公共数据库中的有价值的信息 很少被研究界使用。

Project Summary Many of our organs remain healthy and functional because they replace cells that are lost to injury or disease with new ones, through divisions of adult stem cells contained within them. Adult stem cells divide asymmetrically, generating a new copy of themselves (self-renew) and another cell that will eventually “differentiate” into a cell with specialized function (e.g. absorb nutrients, fight infections, produce a hormone, etc). While there has been great progress in identifying some of the so-called “master regulator genes” that control a stem cell’s decision between self-renewal or differentiation, we know comparatively much less about the specific genetic programs triggered by master regulators in the process. This is in spite of the many efforts to identify targets of master regulators through genome-wide screening technologies, which frequently fall in disuse due to a lack of further validation. Here I propose a research project that simultaneously addresses both problems, by asking… “Can we more efficiently identify critical stem cell genes by integrating public lists of putative master regulator targets obtained in complementary but unrelated studies?” To address the above question, I propose to use the Drosophila melanogaster (fruit fly) midgut as an in vivo experimental model. Previous studies have generated independent lists of putative targets for three known master regulators of stem cells in the Drosophila intestine (Escargot, Capicua and Stat92E). We used bioinformatics to compare these lists, and identified genes that are putatively targeted by only one or all three master regulators, which we refer to as “mono-“ or “co-regulated”, respectively. In Aim 1 of this proposal, we will inhibit each of the master regulators using RNAi-mediated protein knockdown in intestinal progenitors, and validate mono- and co-regulated candidates based on changes to their expression as determined by reverse transcription quantitative PCR (RT-qPCR). In Aim 2, we will compare the effects on the maintenance, proliferation rate and differentiation potential of intestinal progenitors following the RNAi-mediated knockdown of mono- or co- regulated candidates validated in Aim 1. This approach will allow us to test the hypothesis that co-regulated genes are more frequently critical for the biology of the intestinal stem cells than mono-regulated counterparts. Finally, our preliminary observations have shown that inhibiting Esg function leads to a reduction in Stat92E activity, and here I propose to test bioinformatic predictions about regulatory connections between Esg and Stat92E generated by incorporating yet another database of genetic and protein interaction data in. If successful, this project will: a) exploit Drosophila as a prime genetics model system to generate a host of new research leads for understanding and treating gastrointestinal diseases; b) provide a proof of concept to encourage and guide the use of valuable information that is currently deposited in public databases and yet rarely used by the research community.