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: 10428588
• 负责人: Adam Norris
• 金额: $ 36.01万
• 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/10428588
• 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 生物过程由多个基因控制，这些基因协同工作以实现给定的功能。这 现象在遗传相互作用中很明显，定义为在双突变体中观察到的表型 很容易通过各个单突变体的表型来解释。尽管基因相互作用由来已久 被认为是动物表型的重要驱动因素，但目前还不可能进行遗传研究 以系统、无效等位基因、反向遗传学方式对动物进行相互作用分析。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了解多个基因如何 协调地影响给定的表型。为了克服这一差距，我们开发了 CRISPR/Cas9 工具包 在线虫中实现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，从而首次实现了系统性靶向遗传相互作用分析 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 表达受到多层调控（例如转录、RNA 加工、翻译）的控制，但是 人们对这些层如何在单细胞水平上协调知之甚少。实验室第一方向 因此，要分析不同基因表达层之间的遗传相互作用，特别关注 转录因子 (TF) 和 RNA 结合蛋白 (RBP)。双突变体组合带来意想不到的效果 表型将成为从机制上理解 TF 和 RBP 组合如何发挥作用的切入点 协调控制基因表达。实验室的第二个方向是了解 通过 TF 和 RBP 的组合在单细胞水平上进行选择性剪接。单个细胞类型可以是 由 TF 的存在和由此产生的基因表达模式定义，但也可以通过以下方式进一步细化 剪接因子的存在和所表达的亚型。我们创造了大量的 在线虫体内剪接报告基因，发现单细胞水平上广泛的选择性剪接。使用 结合正向和反向遗传学，我们已经确定了许多剪接因子，以及 转录因子的数量惊人，对于单细胞水平的特定选择性剪接机制很重要。我们现在 计划研究这些因素结合起来控制单细胞水平剪接的机制， 以及这种拼接的功能后果。这些方向共同构成了一个关键 加深我们对基因调控因子的组合作用以及它们如何协调的理解 确保基因正确表达。