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

项目概要/摘要 病毒性呼吸道感染是儿童和成人过敏性鼻炎患者病情加重的最常见原因。 哮喘由于致病性病毒最初在粘膜中复制，因此上皮在安装中起关键作用。 先天性炎症反应在检测到复制型病毒时，干扰素调节因子-1（IRF 1） 高度诱导，产生III型干扰素（IFNL），粘膜先天免疫的关键介质。我们有 发现IRF 1基因受一个以前未知的上游增强子样启动子调控 （E启动子），其活性由病毒感染诱导，并由过敏性疾病引起的细胞状态变化沉默。 哮喘在初步研究中，我们发现潜在的IRF 1 E启动子显示病毒增强子活性， 原代人小气道上皮细胞（hSAEC）中的诱导型IRF 1转录。IRF 1 E启动子 转录未注释的IRF 1反义上游（Au）长非编码RNA，IRF 1-Au。表达 5.8 kb IRF 1-Au可被病毒感染诱导，并通过间充质转化而改变。在此R21应用中， 我们将研究IRF 1 E启动子是一种细胞状态调节的表观遗传调节因子的假设， 粘膜IRF 1-IFNL反应。我们将分析IRF 1 E启动子作为增强子的相对贡献， IRF 1和IRF 1-Au的启动子对病毒诱导的IRF 1-IFNL应答的影响存在两种假设：1. IRF1- Epromoter通过直接（成环）相互作用维持IRF 1-IFNL通路处于高度诱导状态 近端IRF 1启动子。我们将通过染色质测试直接成环相互作用的存在 使用KRAB-dCas 9沉默或CRISPR/Cas9切除IRF 1 - 100的构象捕获（3C/4C）研究。 Epromoter。我们将通过定量结合，测量对IRF 1近端启动子可及性的影响。 IRF 3和NFκB转录因子，转录延伸机制的募集和 染色质免疫沉淀（XChIP）分析的常染色质标记。研究还旨在检查 细胞状态转换的影响。2.诱导型IRF 1-Au是一种核糖核蛋白复合物， IRF 1的转录延伸。我们将使用功能获得和功能丧失方法调节IRF 1-Au 并用鼻病毒攻击野生型和间充质转化的hSAEC。染色质环的变化， 将测量IRF 3/NFκB的募集和转录延伸。使用已建立的亲和纯化 （AP）-LC-MS，我们将鉴定IRF 1-Au核糖核蛋白复合物的组成。我们是唯一 根据我们在CRISPR/Cas9基因组靶向、XChIP和 发现蛋白质组学。完成这些研究后，我们将阐明一种新的机制， 粘膜IRF 1-IFNL产生的表观遗传控制，这将使我们能够进行R 01级项目。这个项目 将建立IRF 1 E启动子和IRF 1-Au在体内先天性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。

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.