Dissecting interactions across gene regulatory layers in single cells

摘要 生物过程是由多个基因共同控制的，以实现给定的功能。这 这种现象在遗传互作中很明显，定义为在双突变NOT中观察到的表型 很容易通过各自的单个突变体的表型来解释。虽然基因的相互作用有很长的时间 已经被认为是动物表型的重要驱动因素，但还不可能进行遗传 以系统、零等位基因、反向遗传学的方式对动物进行交互作用分析。这是一个关键的缺口， 因为了解动物的健康和疾病状态需要了解多个基因如何 协同影响一种特定的表型。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改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的靶向遗传相互作用分析 动物。我们将重点研究调控基因表达的因素之间的遗传相互作用。合适的基因 表达受多层调控(如转录、RNA加工、翻译)控制，但 关于这些层是如何在单细胞水平上协调的，我们知之甚少。实验室的第一个方向 因此，要描绘不同基因表达层之间的遗传相互作用，特别是关注 转录因子(TF)和RNA结合蛋白(RBPs)。具有意想不到的双重突变组合 表型将是从机械上理解TFS和RBPs如何组合的切入点 协调控制基因表达。劳顾会的第二个方向将是了解 通过转录因子和限制性商业惯例的组合在单细胞水平上进行选择性剪接。单个单元格类型可以是 由转录因子的存在和由此产生的基因表达模式定义，但也可以通过 剪接因子的存在和由此产生的异构体表达。我们已经创建了大量的In 体内剪接记者在线虫中发现了广泛的单细胞水平的选择性剪接。使用 正向和反向遗传学的组合我们已经确定了一些剪接因子，以及一个 数量惊人的转录因子，在单细胞水平上对特定的选择性剪接机制很重要。我们现在 计划研究这些因素结合在一起控制单细胞水平剪接的机制， 以及这种拼接的功能后果。这些方向加在一起将代表一个关键 基因调控因子的组合作用及其协同作用的研究进展 确保适当的基因表达。

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.