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

项目摘要/摘要 细胞的基因调控程序反映并在很大程度上决定细胞的状态，是一个受到严格调控和 动态系统。在免疫系统中，在免疫过程中需要迅速改变基因调控。 在不同免疫细胞类型的特化和分化过程中也是如此。事实上，许多人 免疫细胞中最重要的调节蛋白是转录因子，它规定了调节状态。 一间牢房。多种基因组分析评估基因调控的不同方面，并代表着一个重要的工具 为现代生物研究和生物医学做好准备。目前，多种不同的基因组分析具有不同的 读数通常被组合使用来生成全面的细胞基因调控谱，结果是 增加成本、时间、技术专长和样品要求。在这里，我们建议开发一种单一的 多模式分析针对低样本输入进行了优化，将生成全面的基因调控信息 传统上，只能使用多个并行化验。我们将优化微量PRO-SEQ(μPRO-SEQ)检测， 下一代测序方法，它将产生三个不同的全基因组读数：(I)a 通过检测新生的RNA分子实现对基因合成的全面和定量测量； 全基因组范围的活性增强子的定量检测，基因组中的调节元件指定 通过结合转录因子进行基因合成；以及，(Iii)鉴定以稳定状态存在的所有基因， 代表了一种新的基因调控模式，使基因定位以对激活做出快速反应。这 数据组合具有广泛的适用性，特别适用于动态细胞类型，如初级免疫 细胞。通过只检测新合成的新生RNA，μPRO-SEQ生成更准确的 比其他技术更活跃地表达基因-更好地反映对刺激或变化的反应 处于分化状态。随着临床相关性的提高，现代免疫学研究的一个主要目标是 识别与免疫反应和分化有关的特定转录因子 无数种不同的免疫细胞类型。μPRO-SEQ高度响应了这一需求，因为它直接识别 活性增强子，与大多数转录因子已建立的序列结合基序一起， 从而有效地识别对免疫细胞命运至关重要的基因调控的候选转录因子。 这项建议的主要目标是(I)开发和优化μPRO-SEQ作为样本保留分析，(Ii) 利用从男性和女性分离的一组不同的人类T细胞亚群建立μPRO-SEQ的实用性 以及(3)开发信息学工具，以最大限度地利用μPRO-SEQ数据 免疫学研究和临床诊断学。实现我们的目标将产生一个强大的、多模式的、 具有多个全基因组读数的样本保留分析，结合起来产生了无与伦比的 全面勾画免疫基因调控状态。

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.