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

项目摘要/摘要 呼吸道病毒感染是导致儿童和成人过敏性疾病恶化的最常见原因 哮喘。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关键作用。 先天的炎症反应。在检测到复制病毒时，干扰素调节因子-1(IRF1)是 高度诱导产生III型干扰素(IFNL)，这是粘膜天然免疫的关键介质。我们有 发现IRF1基因受一个未知的上游增强子样启动子调控 (启动子)，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 哮喘。在初步研究中，我们发现潜在的IRF1启动子具有病毒的增强活性- 原代人小气道上皮细胞(HSAECs)可诱导IRF1转录。IRF1启动子 转录一个未注释的IRF1反义上游(AU)长的非编码RNA，IRF1-AU。的表达 5.8kb的IRF1-AU可被病毒感染诱导，并可通过间充质转化而改变。在此R21应用程序中， 我们将研究IRF1 E启动子是细胞状态调节的表观遗传调节因子的假设。 黏膜IRF1-IFNL反应。我们将剖析IRF1启动子作为基因增强子的相对贡献。 IRF1和IRF1-AU启动子对病毒诱导的IRF1-IFNL反应的两个假设：1.IRF1- E启动子通过直接(环)相互作用将IRF1-IFNL途径维持在高度可诱导的状态 与IRF1启动子近端连接。我们将通过染色质测试直接循环相互作用的存在 使用KRAB-dCas9沉默或CRISPR/Cas9切除IRF1-的构象捕获(3C/4C)研究 发起人。我们将通过量化IRF1近端启动子的结合来衡量对IRF1近端启动子可及性的影响 IRF3和NFκB转录因子，转录延长机制的招募和转录延长机制的积累 常染色质标记采用染色质免疫沉淀(XChIP)法。研究的目的也是为了检查 细胞状态转换的影响。2.可诱导的IRF1-AU是一种核糖核蛋白复合体，可促进 IRF1的转录延伸。我们将使用增益和损耗函数方法来调制IRF1-AU 攻击野生型和间充质向鼻病毒转化的hSAECs。染色质环路的变化， 将测量irf3/nfκB的招募和转录延伸。使用已建立的亲和纯化 (AP)-LC-MS，我们将确定IRF1-AU核糖核蛋白复合体的组成。我们独一无二 有资格根据我们在CRISPR/Cas9基因组靶向、XChIP和 发现蛋白质组学。在这些研究完成后，我们将阐明一种新的机制，以 对粘膜IRF1-IFNL生产的表观遗传控制，这将使我们为R01级项目定位。这个项目 将确定IRF1启动子和IRF1-AU在体内先天抗病毒免疫中的作用，并鉴定 过敏性哮喘产生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.