Epigenetic Control of Mucosal IRF1/IFN-III Antiviral Response by Enhancer-like Promoter and its Coding lncRNA

增强子样启动子及其编码lncRNA对粘膜IRF1/IFN-III抗病毒反应的表观遗传控制

• 批准号: 10495267
• 负责人: Allan R. Brasier
• 金额: $ 19.78万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10495267
• 关键词: Abbreviations; Address; Adult; Affinity Chromatography; Age; Allergens; Allergic; Antiviral Response; Binding; Biological Assay; CRISPR interference; CRISPR/Cas technology; Cells; Child; Chromatin; Chromatin Loop; Chromatin Structure; Chronic; Code; Complex; DNA Polymerase II; Detection; Enhancers; Epigenetic Process; Epithelial Cells; Epithelium; Euchromatin; Excision; Exhibits; Extrinsic asthma; Feedback; Gene Activation; Genes; Genetic Transcription; Genome; Goals; Host Defense; Human; IRF1 gene; IRF3 gene; Immunity; Impairment; Inflammation; Inflammatory Response; Interferons; Literature; Location; Measures; Mediating; Mediator; Mesenchymal; Microbiology; Modification; Molecular Conformation; Mucosal Immunity; Mucous Membrane; Natural Immunity; Nature; Nuclear; Pathway interactions; Patients; Pattern; Phosphorylation; Play; Poly I-C; Positioning Attribute; Production; Proteomics; Qualifying; RNA; Race; Recurrence; Reporting; Respiratory Syncytial Virus Infections; Respiratory System; Respiratory syncytial virus; Rhinovirus; Ribonucleoproteins; Role; Running; Serotyping; Signal Transduction; Techniques; Testing; Transcription Elongation; Transcription Repressor; Untranslated RNA; Upstream Enhancer; Viral; Viral Respiratory Tract Infection; Virus; Virus Diseases; Virus Replication; Work; airway epithelium; airway remodeling; allergic airway disease; antiviral immunity; asthma exacerbation; asthmatic; chromatin immunoprecipitation; chromosome conformation capture; cytokine; design; experimental study; gain of function; genome editing; genome-wide; in vivo; loss of function; negative elongation factor; next generation sequencing; novel; pathogenic virus; prevent; programs; promoter; recruit; respiratory infection virus; response; transcription factor

PROJECT SUMMARY/ABSTRACT Viral respiratory infections are the most common causes of exacerbations in children and adults with allergic asthma. Because pathogenic viruses initially replicate in the mucosa, the epithelium plays a key role in mounting innate inflammatory responses. Upon detection of replicating virus, the interferon regulatory factor-1 (IRF1) is highly induced, producing type III interferon (IFNL), a key mediator of mucosal innate immunity. We have discovered that the IRF1 gene is regulated by a previously unknown upstream enhancer-like promoter (Epromoter) whose activity is induced by viral infection and silenced by cell-state changes produced by allergic asthma. In preliminary studies, we find that the potential IRF1 Epromoter displays enhancer activity of virus- inducible IRF1 transcription in primary human small airway epithelial cells (hSAECs). The IRF1 Epromoter transcribes an unannotated IRF1 antisense upstream (AU) long noncoding RNA, IRF1-AU. Expression of the 5.8 kb IRF1-AU is inducible by viral infection and changed by mesenchymal transition. In this R21 application, we will investigate the hypothesis that IRF1 Epromoter is a cell-state regulated epigenetic regulator of mucosal IRF1-IFNL response. We will dissect the relative contributions of IRF1 Epromoter as the enhancer of IRF1 and the promoter of IRF1-AU on viral inducible IRF1-IFNL response in two hypotheses: 1. The IRF1- Epromoter maintains the IRF1-IFNL pathway in a highly inducible state by direct (looping) interactions with the proximal IRF1 promoter. We will test the presence of direct looping interactions by chromatin conformation capture (3C/4C) studies using KRAB-dCas9 silencing or CRISPR/Cas9 excision of IRF1- Epromoter. We will measure effects on the accessibility of IRF1 proximal promoter by quantifying binding of IRF3 and NFκB transcription factors, recruitment of transcriptional elongation machinery and accumulation of euchromatin marks by chromatin immunoprecipitation (XChIP) assays. Studies are also designed to examine the effects of cell-state transitions. 2. The inducible IRF1-AU is a ribonucleo-protein complex that facilitates transcriptional elongation of IRF1. We will modulate IRF1-AU using gain- and loss-of-function approaches and challenge wild type and mesenchymal transitioned hSAECs to rhinovirus. Changes in chromatin looping, recruitment of IRF3/NFκB and transcriptional elongation will be measured. Using established affinity purification (AP)-LC-MS, we will identify the composition of the IRF1-AU ribonucleoprotein complex. We are uniquely qualified to conduct these studies based on our track record of CRISPR/Cas9 genome targeting, XChIP and discovery proteomics. Upon completion of these studies, we will have elucidated a novel mechanism for epigenetic control of mucosal IRF1-IFNL production that will position us for a R01 level project. This project would establish the role of the IRF1 Epromoter and IRF1-AU in innate anti-viral immunity in vivo, and to identify how cell-state changes produced by allergic asthma modify its composition and function.

项目概要/摘要 病毒性呼吸道感染是过敏儿童和成人病情恶化的最常见原因 由于致病病毒最初在粘膜中复制，因此上皮在安装过程中起着关键作用。 在检测到复制病毒后，干扰素调节因子-1 (IRF1) 会被激活。 高度诱导，产生 III 型干扰素 (IFNL)，这是粘膜先天免疫的关键介质。 发现IRF1基因受到先前未知的上游增强子样启动子的调节 （Epromoter），其活性由病毒感染诱导，并因过敏引起的细胞状态变化而沉默 在初步研究中，我们发现潜在的 IRF1 Epromoter 显示出病毒的增强子活性。 原代人小气道上皮细胞 (hSAEC) 中的可诱导 IRF1 转录。 转录未注释的 IRF1 反义上游 (AU) 长非编码 RNA，IRF1-AU 的表达。 5.8 kb IRF1-AU 可通过病毒感染诱导并通过间质转化而改变。在该 R21 应用中， 我们将研究 IRF1 Epromoter 是细胞状态调节的表观遗传调节因子的假设 我们将剖析 IRF1 Epromoter 作为 IRF1-IFNL 增强子的相对贡献。 IRF1 和 IRF1-AU 启动子对病毒诱导 IRF1-IFNL 反应的影响有两种假设： 1. IRF1- Epromoter 通过直接（循环）相互作用将 IRF1-IFNL 通路维持在高度诱导状态 我们将测试染色质是否存在直接环相互作用。 使用 KRAB-dCas9 沉默或 CRISPR/Cas9 切除 IRF1 进行构象捕获 (3C/4C) 研究 我们将通过量化 IRF1 近端启动子的结合来测量对 IRF1 近端启动子可及性的影响。 IRF3 和 NFκB 转录因子、转录延伸机制的募集和积累 研究还旨在通过染色质免疫沉淀 (XChIP) 检测来检查常染色质标记。 2. 诱导型 IRF1-AU 是一种核糖核蛋白复合物，可促进细胞状态转变。 我们将使用功能获得和丧失的方法来调节 IRF1-AU。 并攻击野生型和间充质转化的 hSAEC 至鼻病毒染色质环的变化， 将使用已建立的亲和纯化来测量 IRF3/NFκB 的募集和转录延伸。 (AP)-LC-MS，我们将鉴定 IRF1-AU 核糖核蛋白复合物的组成，我们是独一无二的。 基于我们在 CRISPR/Cas9 基因组靶向、XChIP 和 完成这些研究后，我们将阐明一种新的机制。 粘膜 IRF1-IFNL 产生的表观遗传控制将使我们进入 R01 级项目。 将确定 IRF1 Epromoter 和 IRF1-AU 在体内先天抗病毒免疫中的作用，并鉴定 过敏性哮喘引起的细胞状态变化如何改变其组成和功能。