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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要细胞的基因调控程序反映并很大程度上决定了细胞的状态，是一个受到严格调控和调控的程序。在免疫系统中，免疫过程中需要基因调控的快速变化。事实上，在不同免疫细胞类型的规范和分化过程中。免疫细胞中最重要的调节蛋白是转录因子，它指定调节状态多个基因组分析评估基因调控的不同方面，是一种重要的工具。目前，多种不同的基因组检测具有不同的特点。读数通常组合使用以生成全面的细胞基因调控谱，从而产生成本、时间、技术专长和样品要求的增加。在这里我们建议开发一个单一的。针对低样本输入进行优化的多模式测定，将生成全面的基因调控信息传统上只能使用多个并行测定，我们将优化 micro-PRO-seq (μPRO-seq) 测定，下一代测序方法，将生成三种不同的全基因组读数：(i) 通过检测新生 RNA 分子实现基因合成的全面定量测量 (ii) 在全基因组范围内定量检测活性增强子，即基因组中指定的调控元件通过结合转录因子进行基因合成；以及（iii）识别处于平衡状态的所有基因，代表了一种新的基因调控模式，使基因能够对激活做出快速反应。数据组合具有广泛的适用性，特别适用于动态细胞类型，例如初级免疫通过仅检测新合成的新生 RNA，μPRO-seq 可以生成更准确的快照。比其他技术更积极地表达基因——读数可以更好地反映对刺激或变化的反应现代免疫学研究的一个主要目标是在分化状态下，其临床意义越来越大。鉴定负责免疫反应和分化的特定转录因子 μPRO-seq 对这种需求高度敏感，因为它可以直接识别。活性增强子，与大多数转录因子的既定序列结合基序一起，从而有效地识别调节免疫细胞命运核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 作为样本保留检测，(ii) 使用一组从男性和女性中分离的不同人类 T 细胞亚群建立 μPRO-seq 的实用性跨越不同年龄段，(iii) 开发信息学工具，最大限度地利用 μPRO-seq 数据免疫学研究和临床诊断的实现将产生强大的、多模式的、具有多个全基因组读数的样本保留测定，结合起来产生无与伦比的和全面描述免疫基因调控状态。