Gene Regulation in the Immune System

免疫系统中的基因调控

基本信息

批准号：
9198482
负责人：
TREVOR W SIGGERS
金额：
$ 37.01万
依托单位：
BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-01-01 至 2020-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9198482
关键词：
Address Affect Autoimmune Diseases Bacteria Bacterial Infections Binding Binding Proteins Binding Sites Biological Biological Assay Cells Chromatin Complex DNA DNA Binding DNA Sequence Data Data Analyses Data Set Deoxyribonuclease I Dependency Dimerization Double-Stranded RNA Elements Event Family member Gene Expression Gene Expression Regulation Gene Targeting Genes Genomics Goals High-Throughput Nucleotide Sequencing Host Defense Human Hypersensitivity IRF3 gene Immune response Immune system Individual Infection Invaded Knock-out Link Lipopolysaccharides Lupus Maps Measures Mediating Modeling Monitor Outcome Pathogen detection Pattern Proteins Receptor Signaling Recruitment Activity Regulatory Element Reporter Research Role Shapes Signal Pathway Signal Transduction Site Specificity System Systemic Lupus Erythematosus TLR3 gene TLR4 gene TLR7 gene TNFRSF5 gene Techniques Testing Toll-like receptors Up-Regulation Virus Virus Diseases Work base chromatin immunoprecipitation cofactor defense response dimer experimental study fighting genome-wide insight knock-down macrophage monocyte p65 pathogen programs promoter public health relevance receptor binding response transcription factor transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): The body's pathogen detection system is critical to host defense. Toll-like receptors (TLRs) bind pathogen- associated molecules and activate a network of transcription factors (TFs) that regulate host defense genes. The TFs IRF3, IRF5 and IRF7 are central to TLR signaling in response to viruses and intracellular pathogens. Despite common DNA binding specificities and activation patterns, IRF3, IRF5 and IRF7 (hereafter IRF3/5/7) perform distinct yet overlapping roles in host defense. Much is known regarding differences in the signaling events upstream of IRF3/5/7 activation; however, little is known regarding how IRF3/5/7 discriminate their individual target genes to elicit different biological outcomes and tailor the immune response to pathogens. This proposal integrates multiple genome-scale techniques to examine the mechanisms of specificity and the differential role of IRF3/5/7 in the TLR signaling pathways central to host defense. Genome-wide binding of IRF3/5/7 will be mapped in human macrophages in response to multiple TLR signals activating (dsRNA mimic for TLR3; lipopolysaccharide(LPS) for TLR4; and ssRNA mimic for TLR7). This will provide the first direct comparison of IRF3/5/7 binding in response to TLR signaling. To relate TF binding with functional changes, chromatin accessibility and gene expression will be monitored by DNase I hypersensitivity analysis (DNase- seq) and RNA-seq, respectively. To address the role of DNA binding in IRF3/5/7-specific function, protein- binding microarrays (PBMs) will be used to characterize the DNA binding of IRF3/5/7 dimers. PBM binding data will be integrated with the other genomic datasets to construct a genome-wide model of IRF3/5/7 function. Hypotheses will be tested using cell-based reporter assays (Aim 1). To examine the role of NF-κB - a known cofactor of IRF3/5/7 - we examine IRF binding, chromatin accessibility and gene expression in NF-κB knockout macrophages, and use PBMs to examine DNA binding specificity of IRF-NF-κB complexes. We hypothesize that differential interactions between the IRFs and NF-κB will contribute to their individual biological roles (Aim 2). To examine the role o macrophage master regulators IRF8 and PU.1, we will characterize the genomic binding of IRF8 and PU.1, and integrate this data with maps of IRF binding, chromatin accessibility and gene expression in IRF8 knockout macrophages. To address the role of protein interactions, PBMs will be used to characterize the DNA binding of IRF3/5/7 with the IRF8:PU.1 complex. We hypothesize that cooperative interactions between IRF3/5/7 and IRF8:PU.1 at specific regulatory elements function to tailor IRF-specific functions in a monocyte/macrophage-specific manner. These studies are expected to provide critical insights into the mechanisms that govern IRF3/5/7-specific functions. Our goal is to develop a comprehensive model of the IRF regulatory network that can be used in different cellular contexts, and which may provide insight into autoimmune diseases, such as systemic lupus erythematosus (SLE), which are associated with upregulation of IRF5/7.

描述（由申请人提供）：身体的病原体检测系统对于宿主防御至关重要，Toll 样受体 (TLR) 结合病原体相关分子并激活调节宿主防御基因的转录因子 (TF) 网络。 IRF5 和 IRF7 是响应病毒和细胞内病原体的 TLR 信号传导的核心，尽管 IRF3、IRF5 和 IRF7（以下简称）具有共同的 DNA 结合特异性和激活模式。 IRF3/5/7) 在宿主防御中发挥不同但重叠的作用，但对于 IRF3/5/7 激活上游信号事件的差异知之甚少；该提案整合了多种基因组规模的技术，以检查 IRF3/5/7 在宿主全基因组防御核心的 TLR 信号通路中的特异性机制和差异作用。的绑定IRF3/5/7 将响应多个 TLR 信号激活而在人类巨噬细胞中进行定位（TLR3 的 dsRNA 模拟物；TLR4 的脂多糖 (LPS)；TLR7 的 ssRNA 模拟物），这将提供 IRF3/5/ 的首次直接比较。 7 响应 TLR 信号传导的结合为了将 TF 结合与功能变化、染色质可及性和基因表达联系起来，将通过 DNase I 超敏反应进行监测。分别分析 (DNase-seq) 和 RNA-seq 为了解决 DNA 结合在 IRF3/5/7 特异性功能中的作用，将使用蛋白质结合微阵列 (PBM) 来表征 IRF3/5/的 DNA 结合。 7 二聚体结合数据将与其他基因组数据集整合，构建 IRF3/5/7 功能的全基因组模型。将使用基于细胞的报告基因检测来测试假设（目标 1）。检查 NF-κB（IRF3/5/7 的已知辅助因子）的作用 - 我们检查 NF-κB 敲除巨噬细胞中的 IRF 结合、染色质可及性和基因表达，并使用 PBM 来检查 IRF-NF-κB 复合物的 DNA 结合特异性我们追求 IRF 和 NF-κB 之间的差异相互作用将有助于它们各自的生物学作用（目标 2）检查巨噬细胞主调节因子 IRF8 和 NF-κB 的作用。 PU.1，我们将表征 IRF8 和 PU.1 的基因组结合，并将这些数据与 IRF 结合、染色质可及性和 IRF8 敲除巨噬细胞中的基因表达图谱整合。我们表征了 IRF3/5/7 与 IRF8:PU.1 复合物的 DNA 结合，捕获了 IRF3/5/7 和 IRF8:PU.1 在特定调控元件功能上的协同相互作用。以单核细胞/巨噬细胞特异性方式发挥 IRF 特异性功能，这些研究有望为 IRF3/5/7 特异性功能的调控机制提供重要见解。可用于不同的细胞环境，并且可以深入了解自身免疫性疾病，例如与 IRF5/7 上调相关的系统性红斑狼疮 (SLE)。