A Single Comprehensive Assay for Gene Regulatory Profiling Optimized for Minimal Sample Input Requirements

针对最小样本输入要求进行优化的基因调控分析的单一综合分析

• 批准号: 10618150
• 负责人: ANDREW W GRIMSON
• 金额: $ 43.86万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-05 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10618150
• 关键词: ATAC-seq; Age; Binding; Biological Assay; Biology; Cells; Communities; DNA-Directed RNA Polymerase; Data; Data Set; Detection; Diagnosis; Dimensions; Enhancers; Female; Gene Expression; Gene Expression Regulation; Gene Order; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Goals; Human; Immune; Immune response; Immune system; Immunologics; Immunology; Individual; Liquid substance; Maps; Measures; Messenger RNA; Methodology; Methods; Modernization; Mus; Nucleotides; Output; Performance; Polymerase; Positioning Attribute; Protocols documentation; RNA; Reaction; Regulator Genes; Regulatory Element; Reproducibility; Research; Research Personnel; Resources; Rest; Running; Sampling; Scientific Advances and Accomplishments; Specific qualifier value; Stimulus; System; T-Lymphocyte Subsets; Targeted Research; Technical Expertise; Technology; Time; Validation; Variant; analysis pipeline; base; candidate identification; cell type; clinical diagnostics; clinically relevant; computerized tools; cost; cost effective; design; dynamic system; gene synthesis; genetic regulatory protein; genome-wide; improved; informatics tool; male; multimodality; multiple datasets; multiple omics; next generation sequencing; novel; personalized medicine; programs; promoter; recruit; response; tool; transcription factor; transcriptome sequencing; user-friendly

PROJECT SUMMARY / ABSTRACT The gene regulatory program of a cell reflects and largely determines cell state, and is a tightly regulated and dynamic system. In the immune system, rapid changes in gene regulation are required during immune responses, and also during specification and differentiation of different immune cell types. Indeed, many of the most important regulatory proteins in immune cells are transcription factors, which specify the regulatory state of a cell. Multiple genomic assays assess diverse aspects of gene regulation, and represent an important tool set for modern biology research and biomedicine. Currently, multiple different genomic assays with distinct readouts are often used in combination to generate comprehensive cell gene regulatory profiles, with a resulting increase in cost, time, technical expertise and sample requirements. Here we propose to develop a single multimodal assay optimized for low sample input, which will generate comprehensive gene regulatory information traditionally only possible using multiple parallel assays. We will optimize the micro-PRO-seq (μPRO-seq) assay, a next-generation sequencing methodology, which will generate three distinct genome-wide readouts: (i) a comprehensive and quantitative measure of gene synthesis, achieved by detecting nascent RNA molecules; (ii) quantitative detection genome-wide of active enhancers, the regulatory elements in the genome that specify gene synthesis by binding transcription factors; and, (iii) identification of all genes existing in a poised state, representing a novel mode of gene regulation that positions genes to respond rapidly to activation. This combination of data has broad applicability, with particular utility for dynamic cell types such as primary immune cells. By detecting only newly synthesized nascent RNAs, μPRO-seq generates a more accurate snapshot of actively expressed genes than other technologies – a readout that better reflects response to stimulus or change in differentiation state. A major goal of modern immunology research, with increasing clinical relevance, is the identification of the specific transcription factors responsible for immune responses and differentiation across myriad different immune cell types. μPRO-seq is highly responsive to this demand, in that it directly identifies active enhancers, which together with the established sequence binding motifs for most transcription factors, thereby efficiently identifies candidate transcription factors that regulate genes central to immunologic cell fate. The main objectives of this proposal are to (i) develop and optimize μPRO-seq as a sample-sparing assay, (ii) establish the utility of μPRO-seq using a panel of different human T cell subsets isolated from male and females across a range of ages, and (iii) develop informatics tools to maximize the utility of μPRO-seq data for immunology research and clinical diagnostics. Accomplishing our aims will produce a robust, multimodal, sample-sparing assay with multiple genome-wide readouts, which in combination produce an unparalleled and comprehensive delineation of immunologic gene regulatory status.

项目摘要 /摘要 细胞的基因调节程序反映并在很大程度上决定了细胞状态，并且是严格调节的，并且 动态系统。在免疫系统中，免疫期间需要快速变化基因调节 反应，以及在不同免疫物类型的规范和分化过程中。确实，许多 免疫细胞中最重要的调节蛋白是转录因子，该因子指定调节状态 细胞。多种基因组评估评估基因调节的不同方面，代表了重要的工具 为现代生物学研究和生物医学素设置。目前，多种不同的基因组测定 读数通常组合使用以生成全面的细胞基因调节曲线，从而产生 增加成本，时间，技术专长和样本要求。在这里，我们建议开发一个 针对低样本输入优化的多模式测定，该测定将产生全面的基因调节信息 传统上，只有使用多个平行测定才有可能。我们将优化微型Pro-seq（μpro-seq）测定法， 下一代测序方法，它将生成三个不同的全基因组读数：（i） 通过检测新生的RNA分子实现的基因合成的全面和定量测量； （ii） 活性增强子的定量检测基因组，基因组中指定的调节元素 通过结合转录因子的基因合成； （iii）鉴定所有以中毒状态存在的基因， 代表一种新型的基因调节模式，该模式定位基因以迅速反应激活。这 数据的组合具有广泛的适用性，以及针对动态细胞类型（例如原发性免疫）的效用 细胞。通过仅检测新综合的新生RNA，μPro-seq生成更准确的快照 主动表达的基因比其他技术 - 读数可以更好地反映对刺激或变化的反应 处于差异状态。随着临床相关性的增加，现代免疫学研究的主要目标是 识别负责免疫反应和分化的特定转录因子 无数不同的免疫菌株类型。 μpro-seq对此需求高度响应，因为它直接识别 活性增强子，以及大多数转录因子的已建立序列结合基序， 因此有效地确定了调节免疫细胞命运中心基因的候选转录因子。 该提案的主要目标是（i）开发和优化μPro-seq作为样品比较测定，（ii） 使用从雄性和女性分离的不同人类T细胞子集建立μPro-seq的实用性 在一系列年龄段以及（iii）开发信息工具以最大程度地提高μpro-seq数据的实用性 免疫学研究和临床诊断。实现我们的目标将产生强大的多模式， 带有多个全基因组读数的样品比较测定，结合产生无与伦比的读数 免疫基因调节状态的全面描述。