Gene Regulation in the Immune System

免疫系统中的基因调控

• 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
• 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

 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。TF IRF3、IRF5和IRF7是响应病毒和细胞内病原体的TLR信号传导的中心。尽管有共同的DNA结合特异性和激活模式，IRF3，IRF5和IRF7（以下简称IRF3/5/7）在宿主防御中发挥不同但重叠的作用。关于IRF3/5/7激活上游信号事件的差异，我们知道得很多;然而，关于IRF3/5/7如何区分它们各自的靶基因以引发不同的生物学结果并定制对病原体的免疫应答，我们知之甚少。 该提案整合了多种基因组规模的技术，以研究IRF3/5/7在TLR信号通路中的特异性机制和差异作用，该信号通路对宿主防御至关重要。响应于多种TLR信号激活（TLR 3的dsRNA模拟物; TLR 4的脂多糖（LPS）;和TLR 7的ssRNA模拟物），将在人巨噬细胞中绘制IRF 3/5/7的全基因组结合。这将提供IRF3/5/7结合响应TLR信号传导的第一个直接比较。为了使TF结合与功能变化相关，将分别通过DNA酶I超敏反应分析（DNase-seq）和RNA-seq监测染色质可及性和基因表达。为了解决DNA结合在IRF 3/5/7特异性功能中的作用，将使用蛋白质结合微阵列（PBM）来表征IRF 3/5/7二聚体的DNA结合。PBM结合数据将与其他基因组数据集整合，以构建IRF3/5/7功能的全基因组模型。将使用基于细胞的报告基因试验（Aim 1）对假设进行检验。为了检测NF-κ B（一种已知的IRF 3/5/7辅因子）的作用，我们检测了NF-κ B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检测IRF-NF-κ B复合物的DNA结合特异性。我们假设IRF和NF-κ B之间的差异相互作用将有助于其各自的生物学作用（目的2）。为了检查巨噬细胞主调节因子IRF8和PU.1的作用，我们将表征IRF8和PU.1的基因组结合，并将该数据与IRF结合、染色质可及性和IRF8敲除巨噬细胞中的基因表达的图谱整合。为了解决蛋白质相互作用的作用，PBM将用于表征IRF3/5/7与IRF8：PU.1复合物的DNA结合。我们假设IRF3/5/7和IRF8：PU.1在特定调控元件处的协同相互作用以单核细胞/巨噬细胞特异性方式调节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。我们的目标是开发一个全面的IRF调控网络模型，该模型可用于不同的细胞环境，并可提供对自身免疫性疾病（如系统性红斑狼疮（SLE））的深入了解，这些疾病与IRF 5/7的上调相关。