A genetic approach to defining the Ttc21b interactome in mammalian ciliopathies

描述(申请人提供)：纤毛疾病是一系列疾病，由初级纤毛功能缺陷引起，影响1：800人。初生纤毛是以微管为基础的细胞器，几乎存在于所有细胞中，对许多分子通路的信号转导至关重要。纤毛病影响广泛的组织，包括神经系统、头面部组织、骨骼、肾脏、肺和消化器官。这些都表现为先天性和成人发病的缺陷。对纤毛病患者的遗传学研究表明，TTC21B(四肽重复结构域包含蛋白1B)是迄今为止发现的最常见的纤毛基因突变。除了与TTC21B缺失相关的缺陷外，反式纤毛基因突变还会导致纤毛疾病。我们最近发现，小鼠Ttc21b基因缺失会导致围产期死亡和器官发生缺陷。我们还注意到，这些表型中的一些取决于特定的近交系小鼠品系背景。TTC21B蛋白很大，具有许多蛋白质-蛋白质相互作用结构域，对纤毛的鞭毛内转运和调节信号转导起重要作用。所有这些数据共同引导我们得出一个中心假设，即TTC21B是脚手架和贩运活动的网络枢纽，对于正确的纤毛形式和功能是必不可少的。这项应用的目标是识别与Ttc21b(Ttc21b)相互作用的基因和蛋白质，并开始了解它们在细胞中如何相互作用。该项目的基本原理是，更全面地了解TTC21B是如何起作用的，可能会让人们对一系列纤毛疾病有更深入的了解。我们将针对这一假设并通过以下三个具体目标来实现这些目标：1)在B6和FVB小鼠品系中识别包含修改Ttc21b缺失/缺失表型的基因的染色体区域；2)使用正向遗传学方法识别与Ttc21b的新的遗传相互作用；以及3)研究与Ttc21b相互作用的基因的作用机制。第一个目标将利用QTL分析来确定调节菌株特定表型的基因座 参见Ttc21bNull/Null胚胎。第二个目标将采取一种向前遗传的ENU突变方法，以无偏见的方式确定与TTC21B的新相互作用。第三个目标将概括在人类中发现的或之前在老鼠中发现的相互作用。在验证了这些相互作用产生睫状体病变表型后(S)，我们将在体外和体内进行进一步的分析，以研究睫状体功能障碍的分子机制。这些研究将集中在纤毛运输和Shh信号转导方面。这个项目的意义在于，这些研究将极大地提高我们对TTC21B在初级纤毛中的作用以及为什么功能紊乱导致纤毛疾病的理解。这些研究将填补一个重要的 我们在知识上的差距，并确定可能的治疗干预领域。这些进展并不是TTC21B所特有的，但很可能在很大程度上适用于初级纤毛生物学感兴趣的多个领域。本项目的创新之处在于应用无偏倚基因技术来鉴定Ttc21b相互作用组，并结合固体分子研究来确定其潜在机制(S)。

DESCRIPTION (provided by applicant): Ciliopathies are a spectrum of diseases resulting from defects in primary cilia function affecting 1:800 people. Primary cilia are microtubule based organelles found on almost all cells and crucial for proper signal transduction of a number of molecular pathways. Ciliopathies affect a wide range of tissues including the nervous system, craniofacial tissues, skeleton, kidneys, lungs and digestive organs. These manifest as both congenital and adult-onset defects. Genetic studies of ciliopathy patients show TTC21B (tetratricopeptide repeat domain-containing protein1B) is the most commonly mutated cilia gene identified to date. In addition to defects associated with loss of just TTC21B, mutations in trans with a number of other ciliary genes lead to ciliopathies. We have recently shown loss of Ttc21b in the mouse leads to perinatal lethality and organogenesis defects. We also note some of these phenotypes are dependent on the specific inbred mouse strain background. The TTC21B protein is large with many protein-protein interaction domains and important for intraflagellar transport and regulating signal transduction in the cilium. All of these data together lead us to the central hypothesis that TTC21B serves as a network hub for scaffolding and trafficking activities essential for proper cilia form and function. The goal of this application is identify genes and proteins interacting with Ttc21b: the Ttc21b "interactome," and begin to understand how these interact in the cell. The rationale for the project is that a more complete understanding of how TTC21B acts is likely to give insight to a range of ciliopathies. We will address this hypothesis and achieve these goals with the following three specific aims: 1) identify chromosomal regions containing genes modifying the Ttc21bnull/null phenotype in the B6 and FVB mouse strains, 2) identify novel genetic interactions with Ttc21b using a forward genetic approach, and 3) study functional mechanisms of genes interacting with Ttc21b. The first aim will utilize a QTL analysis to identify loci regulating the strain specific phenotypes we see in Ttc21bnull/null embryos. The second aim will take a forward genetic, ENU mutagenesis approach to identify novel interactions with TTC21B in an unbiased manner. The third aim will recapitulate interactions identified in humans or previously identified in mouse. After verifying these interactions yield ciliopathy phenotype(s), we will perform further analyses in vitro and in vivo to study the molecular mechanisms of ciliary dysfunction. These studies will focus on ciliary trafficking and Shh signal transduction. The significance of this project is that these studies wil together dramatically increase our understanding of how TTC21B acts within the primary cilium and why perturbation of function leads to ciliopathic disease. These studies will fill an important gap in our knowledge and identify possible areas for therapeutic intervention. These advances are not specific to TTC21B but are likely going to be largely applicable to multiple areas of primary cilia biology inter- est. The innovation of this project lies in the application of unbiase genetic techniques to identify the Ttc21b interactome in close concert with solid molecular studies to determine the underlying mechanism(s).