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

果蝇肠干细胞中主转录因子的遗传共同调控

基本信息

批准号：
9980960
负责人：
Mariano A Loza Coll
金额：
$ 14.5万
依托单位：
CALIFORNIA STATE UNIVERSITY NORTHRIDGE
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-05 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9980960
关键词：
Address Adult Affect Behavior Bioinformatics Biological Models Biology Cell Therapy Cell physiology Cells ChIP-seq Communities Data Databases Deposition Development Disease Down-Regulation Drosophila genus Drosophila melanogaster Ensure Epidermal Growth Factor Receptor Experimental Genetics Experimental Models Follow-Up Studies Gastrointestinal Diseases Gene Expression Genes Genetic Genetic Databases Genetic Models Genetic Transcription Genets Homeostasis Hormones Human Genetics Individual Infection Injury Intestinal Diseases Intestines Maintenance Malignant Neoplasms Mediating Messenger RNA Midgut Molecular Monitor Nutrient Organ Organism Pathway interactions Phenotype Process Proteins RNA RNA Interference Regulation Regulator Genes Reporter Research Research Project Grants Reverse Transcription Role Sister System Technology Testing Tissues Transgenes Validation Work adult stem cell base cell type comparative data integration data mining falls fighting fly genetic approach genome wide screen in silico in vivo in vivo Model intestinal homeostasis knock-down novel progenitor programs public repository response self-renewal stem stem cell homeostasis stem cells transcription factor

项目摘要

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.

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与新的细胞结合，通过其中包含的成体干细胞的分裂。成体干细胞分裂不对称地，产生一个自己的新副本（自我更新）和另一个最终将 “分化”成具有专门功能的细胞（例如，吸收营养，抵抗感染，产生激素，等等）。虽然在确定一些所谓的“主调节基因”方面取得了很大进展，控制干细胞在自我更新或分化之间的决定，我们知道的相对较少。在这个过程中由主调节器触发的特定遗传程序。这是尽管许多努力，通过全基因组筛选技术确定主调节器的目标，这些技术经常落入由于缺乏进一步验证而停用。在这里，我提出了一个研究项目，同时解决这两个问题问题，通过问.“我们能更有效地识别关键的干细胞基因，通过整合公共列表，在互补但不相关的研究中获得的推定的主调节器目标？”解决上述问题，我建议使用果蝇（果蝇）中肠作为体内实验模型。以前的研究已经产生了三个已知的主调节因子的推定靶点的独立列表，果蝇肠道中的干细胞（Escargot，Capicua和Stat 92 E）。我们用生物信息学来比较这些列表，并确定了仅由一个或所有三个主调节器靶向的基因，我们分别称之为“单调节”或“共调节”。在本建议的目标1中，我们将禁止在肠祖细胞中使用RNAi介导的蛋白质敲低的主要调节剂，并验证单-和基于通过逆转录确定的其表达变化的共调节候选物定量PCR（RT-qPCR）。在目标2中，我们将比较对维持、增殖率和在RNAi介导的敲低单或共转录后肠祖细胞的分化潜力在目标1中验证的受监管候选人。这种方法将使我们能够测试共同调节的假设，基因对于肠干细胞的生物学比单调节的对应物更经常地是关键的。最后，我们的初步观察表明，抑制Esg功能导致Stat 92 E减少，活动，在这里，我建议测试生物信息学预测的监管之间的联系Esg和 Stat 92 E是通过将另一个遗传和蛋白质相互作用数据库整合到。如果如果成功，该项目将：a）利用果蝇作为主要遗传学模型系统，了解和治疗胃肠道疾病的研究线索; B）提供概念验证，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。