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Mass spectrometry-coupled hypermorphic functional genomics

质谱耦合超态功能基因组学

基本信息

批准号：
8917142
负责人：
Michael Benjamin Major
金额：
$ 16.44万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8917142
关键词：
Acetylation Adult Affinity Affinity Chromatography Algorithms Automobile Driving Binding Bioinformatics Biological Biological Assay Biotin Cell Survival Cells Collection Copy Number Polymorphism Coupled DNA Sequence Alteration Data Detection Development EP300 gene Employment Engineering Event Future Gene Expression Gene Mutation Gene Silencing Gene Targeting Genes Genetic Transcription Genome Genomics Genotype Goals Grant Guns Haploidy Health Homeostasis Human Human Cell Line Human Characteristics Human Genome Laboratory Research Libraries Ligase Malignant Neoplasms Mass Spectrum Analysis MicroRNAs Modification Molecular Monitor Mutagenesis Mutation Nature Oncogenic Open Reading Frames Pathway interactions Phenotype Positioning Attribute Post-Translational Protein Processing Printing Process Proteins RNA Interference RNA Splicing Regulatory Pathway Reporter Research Research Personnel Signal Pathway Signal Transduction Signal Transduction Pathway Technology Testing Tissues Transcriptional Activation Tretinoin Viral WNT Signaling Pathway Work base cancer genomics cell behavior cost cost effective data reduction empowered foot functional genomics gain of function genome annotation genome-wide improved interest loss of function notch protein novel promoter research study screening success tool transcription factor

项目摘要

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.

描述（申请人提供）：功能基因组学的首要目标是全面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。成功的功能基因组学筛选需要特异性抑制或激活基因组中每个基因的技术。microRNA的发现和认识到miRNA加工机制可以被劫持用于序列特异性基因沉默（RNAi技术）创建了用于基因组注释的高通量功能丧失方法。最近发现和使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。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，但人类开放式阅读框架克隆集合的不完整性以及建立阵列式表达就绪库的高昂成本极大地限制了其在广大领域的应用大多数学术研究实验室。这项资助的主要目标是实现一个高通量、具有成本效益和全基因组功能获得性筛选平台。我们建议使用基于质谱的方法来检测蛋白质过表达的“足迹”随机诱变屏幕。除了揭示当过度表达时导致给定表型的基因之外，这种方法还阐明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β-连环蛋白依赖性WNT信号通路的新型激活剂。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扩展性，普遍适用性和低成本。这项建议有两个主要目标。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善质谱法对过度表达蛋白的检测。其次，使用筛选平台和一组新的通路特异性转录报告基因，我们将鉴定和验证以下通路的激活剂：WNT/b-连环蛋白、视黄酸、Notch、NF κ B、TGF β和NRF 2。由此产生的数据有望在蛋白质水平上揭示功能获得基因型-表型关系。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制研究提供数据减少策略。