Gene expression, Epigenetics and Bioinformatics Core

基因表达、表观遗传学和生物信息学核心

• 批准号: 10591868
• 负责人: Matthew Eugene Pipkin
• 金额: $ 3.87万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10591868
• 关键词: Gene; expression; Epigenetics; Bioinformatics; Core

PROJECT SUMMARY / ABSTRACT Core C (Pipkin) The overarching goal of Core C is to provide standardized, uniform and innovative next generation sequencing (NGS) and computational approaches to address the genome-scale experiments proposed in Projects 1-3. The individual Projects goals are to analyze pooled in vivo screens, gene expression, transcription and chromatin structure and to discern the basic molecular regulation of T and B cell mediated immunity. Core C will facilitate these objectives by using NGS approaches to analyze (1) pooled in vivo gene loss-of-function screens by sequencing, (2) expression of chromatin-associated RNA (nascent RNA), and mature mRNA from limiting ex vivo cell numbers, and single cells (scRNA-seq) using RNA-seq; (3) chromatin accessibility via the assay for transposase-accessible chromatin followed by sequencing (ATAC-seq), and nucleosome organization (MNase- seq and BEM-seq); and (4) CRF and TF binding to near base-pair resolution after chromatin immunoprecipitation from small numbers of primary lymphocytes using ChIP-exo (Aims 1 and 2). Core C has demonstrated experience in developing and applying all of these approaches. In addition, Core C has established a centralized, interlinked and documented framework for the storage, analysis and sharing of these large datasets between Projects 1-3 (Aim 3), and will apply available algorithms in creative arrangements that comprise computational approaches to identify and define operational cis-regulatory regions based on chromatin accessibility, nucleosome organization and histone modifications, and to infer utilized TF binding site motifs within these regions and predict their cognate TFs. Furthermore, these observations will be correlated with transcriptional activity (nascent RNA expression) and RNA Pol II activity (ChIP-seq) and TF and CRF binding events (ChIP- exo), and overall gene expression (mRNA) in the context of gene-perturbations to clarify functionally how gene regulatory networks drive T cell differentiation and function during immune responses in vivo.

项目概要/摘要 核心C（皮普金） Core C的总体目标是提供标准化、统一和创新的下一代测序 （NGS）和计算方法来解决项目 1-3 中提出的基因组规模实验。这 各个项目的目标是分析汇总的体内筛选、基因表达、转录和染色质 结构并辨别 T 细胞和 B 细胞介导的免疫的基本分子调节。核心C将促进 通过使用 NGS 方法分析 (1) 汇集的体内基因功能丧失筛选来实现这些目标 测序，(2) 染色质相关 RNA（新生 RNA）和限制性外源成熟 mRNA 的表达 使用 RNA-seq 的体内细胞数和单细胞 (scRNA-seq)； (3) 通过检测染色质可及性 转座酶可及的染色质随后进行测序 (ATAC-seq) 和核小体组织 (MNase- seq 和 BEM-seq）； (4) 染色质免疫沉淀后 CRF 和 TF 与近碱基对分辨率结合 使用 ChIP-exo 从少量原代淋巴细胞中分离（目标 1 和 2）。核心C已经证明 开发和应用所有这些方法的经验。此外，Core C还建立了一个集中的、 用于存储、分析和共享这些大型数据集的相互链接和记录的框架 项目 1-3（目标 3），并将在包括计算在内的创造性安排中应用可用的算法 基于染色质可及性识别和定义可操作顺式调控区域的方法， 核小体组织和组蛋白修饰，并推断这些中使用的 TF 结合位点基序 区域并预测它们的同源 TF。此外，这些观察结果将与转录相关 活性（新生 RNA 表达）和 RNA Pol II 活性 (ChIP-seq) 以及 TF 和 CRF 结合事件 (ChIP- exo）和基因扰动背景下的整体基因表达（mRNA），以阐明基因在功能上的作用 调节网络在体内免疫反应过程中驱动 T 细胞分化和功能。