Mass spectrometry-coupled hypermorphic functional genomics

描述(由申请人提供)：功能基因组学的总体目标是在全球范围内定义所有基因组座位的功能--直接将基因型与表型联系起来。一个成功的功能基因组学筛查需要有专门抑制或激活基因组中每个基因的技术。MicroRNAs的发现和miRNA处理机制可能被劫持以用于序列特异性基因沉默(RNAi技术)的认识为基因组注释创造了一种高通量的功能丧失方法。最近近单倍体人类细胞的发现和用于功能基因组筛选的使用进一步增强了哺乳动物功能丧失的基因组注释。相比之下，基因组规模的功能获得技术还没有完全实现。尽管正在取得进展，但人类开放阅读框架克隆收集的不完全性质，以及建立排列的表达就绪文库通常令人望而却步的成本，极大地限制了其在 大多数学术研究实验室。这笔赠款的主要目标是实现一个高通量、高成本效益和全基因组功能增益筛选平台。我们建议使用一种基于质谱学的方法来检测在“足迹”随机突变筛选中过度表达的蛋白质。除了揭示当过度表达时导致给定表型的基因外，这种方法还揭示了蛋白质相互作用网络和蛋白质翻译后修饰。在概念验证实验中，我们发现并验证了依赖于b-连环蛋白的WNT信号通路的新激活物。这项工作支持了发现平台的变革潜力，部分原因是展示了可伸缩性、普遍适用性和低成本。这项提议有两个主要目标。首先，虽然我们的初步筛选是成功的，但还需要额外的分子工程来改进质谱学对过度表达蛋白质的检测。其次，利用筛选平台和一组新的通路特异性转录记者，我们将鉴定和验证以下通路的激活因子：WNT/b-连环蛋白、维甲酸、Notch、NFkB、TGFb和NRF2。由此产生的数据有望揭示蛋白质水平上的功能增益、基因型和表型之间的关系。我们将把我们的数据与癌症衍生的基因组拷贝数改变相结合，为未来具有翻译前景的机械性研究提供数据缩减策略。

DESCRIPTION (provided by applicant): The over-arching goal of functional genomics is to globally define the function of all genomic loci-to directly connect genotype with phenotype. A successful functional genomics screen requires technology to specifically inhibit or activate each gene in the genome. The discovery of microRNAs and the realization that the miRNA processing machinery could be hijacked for sequence-specific gene silencing (RNAi technologies) created a high-throughput loss-of-function approach for genome annotation. The recent discovery and employment of near-haploid human cells for functional genomic screening further empowers mammalian loss-of-function genome annotation. In contrast, genome-scale gain-of-function technologies have yet to be fully realized. Although progress is being made, the incomplete nature of human open reading frame clone collections and the often prohibitive costs of establishing an arrayed expression-ready library dramatically limit its application for the vast majority of academic research laboratories. The principle goal of this grant is to realize a high throughput, cost-effective and genome-wide gain-of-function screening platform. We propose to use a mass spectrometry-based approach to detect proteins over-expressed in a 'foot-printed' random mutagenesis screen. In addition to revealing the genes that when over-expressed result in a given phenotype, this approach illuminates protein interaction networks and protein post translational modifications. In proof-of-concept experiments, we discovered and validated novel activators of the b-catenin dependent WNT signaling pathway. This work supports the transformative potential of the discovery platform, in part by demonstrating scalability, general applicability and low cost. This proposal has two primary goals. First, although our preliminary screens were successful, additional molecular engineering is needed to improve detection of the over-expressed protein by mass spectrometry. Second, using the screening platform and a new panel of pathway-specific transcriptional reporters, we will identify and validate activators of th following pathways: WNT/ b-catenin, Retinoic Acid, Notch, NFkB, TGFb and NRF2. The resulting data promises to reveal gain-of-function genotype- phenotype relationships at the protein-level. We will integrate our data with cancer-derived genomic copy number alterations to provide a data reduction strategy for future mechanistic studies with translational promise.