Dissecting interactions across gene regulatory layers in single cells

摘要 生物过程由多个基因共同控制，以实现给定的功能。这 在遗传相互作用中，这种现象是明显的，定义为在双突变体中观察到的表型， 很容易解释的表型在各自的单一突变体。虽然基因的相互作用 被认为是动物表型的重要驱动因素，但还不可能进行遗传学研究。 以系统的、无效等位基因的、反向遗传学的方式在动物中进行相互作用分析。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种基因是如何 协同影响给定的表型。为了克服这一差距，我们开发了一个CRISPR/Cas9工具包， 能够在线虫中进行靶向基因组修饰和随后的遗传相互作用分析， 秀丽隐杆线虫，从而使首次系统的有针对性的遗传相互作用分析， 动物我们将集中在基因表达调控因子之间的遗传相互作用。合适的基因 表达受多层调控（例如转录、RNA加工、翻译）控制，但 关于这些层如何在单个细胞的水平上协调的知之甚少。实验室的第一个方向 因此，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， 表型将是从机理上理解TF和RBP组合如何 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可变剪接。单个细胞类型可以是 通过TF的存在和产生的基因表达模式来定义，但也可以通过以下方法进一步细化： 剪接因子的存在和表达的所得同种型。我们创造了大量的 体内剪接报告基因在C.并在单细胞水平上发现了广泛的选择性剪接。使用 结合正向和反向遗传学，我们已经确定了一些剪接因子，以及一个 在单个细胞水平上，这对于特定的选择性剪接机制是重要的。我们现在 计划研究这些因子联合收割机在单细胞水平控制剪接的机制， 以及这种剪接的功能结果。这些方向合在一起就代表了一把钥匙 我们对基因调控因子的组合作用以及它们如何协调作用的理解取得了进展 确保基因正确表达。

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.