Dissecting interactions across gene regulatory layers in single cells

剖析单细胞基因调控层之间的相互作用

• 批准号: 10386536
• 负责人: Adam Norris
• 金额: $ 2.78万
• 依托单位: SOUTHERN METHODIST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10386536
• 关键词: Affect; Alleles; Alternative Splicing; Animals; Biological Phenomena; Biological Process; CRISPR/Cas technology; Caenorhabditis elegans; Cells; Disease; Ensure; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genome; Height; Individual; Malignant Neoplasms; Methods; Modification; Nematoda; Phenotype; Predisposition; Protein Isoforms; RNA Processing; RNA Splicing; RNA-Binding Proteins; Regulation; Regulator Genes; Reporter; Time; Translations; cell type; combinatorial; forward genetics; in vivo; mutant; novel; reverse genetics; transcription factor

ABSTRACT Biological processes are controlled by multiple genes working in concert to achieve a given function. This phenomenon is apparent in genetic interactions, defined as a phenotype observed in a double mutant not easily explained by the phenotypes in the respective single mutants. While genetic interactions have long been recognized as important drivers of animal phenotypes, it has not been possible to perform genetic interaction analysis in animals in a systematic, null allele, reverse-genetics fashion. This is a critical gap, because understanding healthy and disease states in animals requires an appreciation of how multiple genes coordinately affect a given phenotype. To overcome this gap, we have developed a CRISPR/Cas9 toolkit that enables targeted genome modification and subsequent genetic interaction analysis in the nematode worm Caenorhabditis elegans, thus enabling for the first time systematic targeted genetic interaction profiling in animals. We will focus on genetic interactions among factors regulating gene expression. Proper gene expression is controlled by multiple layers of regulation (e.g. transcription, RNA processing, translation) but little is known about how these layers are coordinated at the level of single cells. The first direction of the lab therefore is to profile genetic interactions between different layers of gene expression, specifically focusing on transcription factors (TFs) and RNA binding proteins (RBPs). Double mutant combinations with unexpected phenotypes will be the entry point to mechanistic understanding of how combinations of TFs and RBPs coordinately control gene expression. The second direction of the lab will be to understand the regulation of alternative splicing at the single cell level by combinations of TFs and RBPs. Individual cell types can be defined by the presence of TFs and the resulting gene expression patterns, but can also be further refined by the presence of splicing factors and the resulting isoforms expressed. We have created a large number of in vivo splicing reporters in C. elegans and found extensive alternative splicing at the single cell level. Using a combination of forward and reverse genetics we have identified a number of splicing factors, as well as a surprising number of TFs, important for specific alternative splicing regimes at the single cell level. We now plan to investigate the mechanisms by which these factors combine to control splicing at the single cell level, as well as the functional consequences of such splicing. Together these directions will represent a key advance in our understanding of combinatorial action of gene regulatory factors and how they coordinately ensure proper gene expression.

抽象的 生物过程由多种基因共同控制以实现给定功能。这 现象在遗传相互作用中显而易见，被定义为在双突变体中观察到的表型 在各个单个突变体中的表型很容易解释。而遗传相互作用长期很长 被认为是动物表型的重要驱动因素，不可能执行遗传 动物的相互作用分析以系统的无效等位基因，反遗传学方式。这是一个关键的差距， 因为了解动物的健康和疾病状态需要欣赏多个基因 协调影响给定的表型。为了克服这一差距，我们开发了一个CRISPR/CAS9工具包 在线虫蠕虫中启用有针对性的基因组修饰和随后的遗传相互作用分析 秀丽隐杆线虫，因此首次在系统的有针对性的遗传相互作用分析中实现 动物。我们将重点关注调节基因表达的因素之间的遗传相互作用。适当的基因 表达受多层调节（例如转录，RNA处理，翻译）的控制，但 关于这些层如何在单细胞水平上协调的知识知之甚少。实验室的第一个方向 因此，是要在基因表达的不同层之间概述遗传相互作用，专门针对 转录因子（TFS）和RNA结合蛋白（RBP）。意外的双重突变组合 表型将是对TFS和RBPS组合的机械理解的切入点 协同控制基因表达。实验室的第二个方向是了解 通过TFS和RBP的组合在单细胞水平上进行替代剪接。单个单元类型可以是 由TF的存在和产生的基因表达模式定义，但也可以通过 剪接因子的存在和所得的同工型表达。我们创建了大量 秀丽隐杆线虫中的体内剪接记者，并在单细胞水平上发现了广泛的替代剪接。使用 正向和反向遗传学的组合我们已经确定了许多剪接因子以及一个 TF的惊人数量，对于单个单元格的特定替代剪接状态很重要。我们现在 计划研究这些因素结合以控制单细胞水平的剪接的机制， 以及这种剪接的功能后果。这些指示共同代表一个关键 进步我们对基因调节因素的组合作用以及它们如何协调一致 确保适当的基因表达。