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: 10373575
• 负责人: Allan R. Brasier
• 金额: $ 25.28万
• 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 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10373575
• 关键词: Abbreviations; Address; Adult; Affinity Chromatography; Age; Allergens; Allergic; Antiviral Agents; 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; Epithelial Cells; 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 of activation protein; 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; RNA; 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; base; chromatin immunoprecipitation; chromosome conformation capture; cytokine; design; experimental study; 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.

项目摘要/摘要 病毒呼吸道感染是患有过敏反应的儿童和成人加重的最常见原因 哮喘。因为致病性病毒最初在粘膜中复制，所以上皮在安装中起关键作用 先天的炎症反应。在发现复制病毒后，干扰素调节因子1（IRF1）为 高度诱导的，产生III型干扰素（IFNL），这是粘膜先天免疫的关键介体。我们有 发现IRF1基因受到以前未知的上游增强子样启动子的调节 （e宣言者）其活性是由病毒感染诱导的，并被过敏反应所产生的细胞态变化沉默 哮喘。在初步研究中，我们发现潜在的IRF1 ePromoter显示了病毒的增强剂活性 原代人小气道上皮细胞（HSAEC）中的诱导IRF1转录。 IRF1 EPROMOTER 在上游（AU）长的非编码RNA，IRF1-AU转录未注释的IRF1反义反义。表达 5.8 kb IRF1-AU可通过病毒感染诱导，并通过间充质转变改变。在此R21应用程序中， 我们将调查IRF1 Epromoter是细胞态调节的表观遗传调节剂的假设 粘膜IRF1-IFNL响应。我们将剖析IRF1 Epromoter作为增强子的相对贡献 IRF1和IRF1-AU对病毒诱导IRF1-IFNL反应的启动子在两个假设中：1。IRF1- 通过直接（循环）相互作用 与代理IRF1启动子。我们将测试染色质直接循环相互作用的存在 使用KRAB-DCAS9沉默或CRISPR/CAS9 IRF1-的构象捕获（3C/4C）研究 ePromoter。我们将通过量化结合来衡量IRF1近端启动子的可及性的影响 IRF3和NFκB转录因子，转录伸长机制的募集和积累 染色质免疫沉淀（XCHIP）测定法。研究还旨在检查 细胞状态过渡的影响。 2。诱导的IRF1-AU是一种核糖核蛋白络合物，可促进 IRF1的转录伸长。我们将使用功能障碍方法来调节IRF1-AU 并挑战野生型和间充质过渡到鼻病毒。染色质循环的变化， 将测量IRF3/NFκB和转录伸长的募集。使用已建立的亲和力净化 （AP）-LC-MS，我们将确定IRF1-AU核糖核蛋白复合物的组成。我们是独特的 根据我们的CRISPR/CAS9基因组靶向，Xchip和 发现蛋白质组学。这些研究完成后，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为R01级项目定位。这个项目 将确定IRF1 ePromoter和irf1-au在体内先天抗病毒免疫中的作用，并确定 过敏性哮喘产生的细胞状态如何改变其组成和功能。