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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：
10159213
负责人：
ANDREW W GRIMSON
金额：
$ 43.68万
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/10159213
关键词：
ATAC-seq Age Binding Biological Assay Biology Cells Communities DNA-Directed RNA Polymerase Data Data Set Detection Diagnosis Diagnostic Enhancers Female Foundations 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 Medical 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 Thromboplastin Time Validation Variant analysis pipeline base 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.

项目摘要/摘要