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

项目总结/摘要 细胞的基因调控程序反映并在很大程度上决定细胞状态，并且是一个受到严格调控的， 动态系统在免疫系统中，免疫过程中需要基因调控的快速变化， 免疫反应，以及在不同免疫细胞类型的特化和分化期间。事实上， 免疫细胞中最重要的调节蛋白是转录因子，它们指定调节状态 一个细胞。多基因组分析评估基因调控的不同方面，并代表一个重要的工具， 为现代生物学研究和生物医学而设。目前，多种不同的基因组测定具有不同的 读出通常组合使用以产生全面的细胞基因调控谱， 成本、时间、技术专长和样品要求增加。在这里，我们建议开发一个单一的 针对低样本输入优化的多模式检测，将生成全面的基因调控信息 传统上仅可能使用多个平行测定。我们将优化micro-PRO-seq（μPRO-seq）检测， 下一代测序方法，其将产生三种不同的全基因组读数： 通过检测新生RNA分子实现基因合成的全面和定量测量;（ii） 定量检测全基因组的活性增强子，基因组中的调节元件， 通过结合转录因子的基因合成;和，（iii）鉴定以平衡状态存在的所有基因， 代表了一种新的基因调控模式，使基因对激活反应迅速。这 数据的组合具有广泛的适用性，特别适用于动态细胞类型，如原发性免疫细胞， 细胞通过仅检测新合成的新生RNA，μPRO-seq生成了一个更准确的快照， 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的读数 处于分化状态。随着临床相关性的增加，现代免疫学研究的主要目标是： 鉴定负责免疫应答和分化的特异性转录因子， 无数种不同的免疫细胞类型μPRO-seq高度响应这一需求，因为它直接识别 活性增强子，其与大多数转录因子的已建立的序列结合基序一起， 从而有效地鉴定调节对免疫细胞命运至关重要的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作为样品保留测定，（ii） 使用从男性和女性分离的一组不同的人T细胞亚群建立μPRO-seq的效用 （iii）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。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出的样品保留测定，其组合产生无与伦比的 全面描述免疫基因调节状态。

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.