Mass spectrometry-coupled hypermorphic functional genomics

描述（由申请人提供）：功能基因组学的首要目标是全局定义所有基因组位点的功能-直接将基因型与表型联系起来。一个成功的功能基因组筛选需要技术来特异性地抑制或激活基因组中的每个基因。microrna的发现以及miRNA加工机制可以被劫持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。最近发现并利用近单倍体人类细胞进行功能基因组筛选，进一步增强了哺乳动物功能丧失基因组注释的能力。相比之下，基因组规模的功能获得技术尚未完全实现。尽管正在取得进展，但人类开放阅读框架克隆集合的不完全性以及建立一个阵列表达就绪库的高昂成本，极大地限制了它在大规模应用中的应用

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.