Gene Regulation in the Immune System

 描述(申请人提供)：人体的病原体检测系统对宿主防御至关重要。Toll样受体(Toll-like Receptor，TLRs)结合病原体相关分子，激活转录因子网络，调节宿主防御基因。转录因子IRF3、IRF5和IRF7在TLR应答病毒和细胞内病原体的信号转导中起核心作用。尽管有共同的DNA结合特异性和激活模式，但IRF3、IRF5和IRF7(以下简称IRF3/5/7)在宿主防御中发挥着不同但重叠的作用。关于IRF3/5/7激活上游的信号事件的差异已知很多；然而，关于IRF3/5/7如何区分各自的靶基因以引起不同的生物学结果和针对病原体的免疫反应，人们知之甚少。这一建议整合了多种基因组水平的技术来研究IRF3/5/7在TLR信号通路中的特异性和差异作用机制。IRF3/5/7的全基因组结合将在人巨噬细胞中定位，以响应多个TLR信号的激活(TLR3的dsRNA模拟；TLR4的脂多糖(LPS)；TLR7的单链RNA模拟)。这将提供响应TLR信号的IRF3/5/7结合的第一个直接比较。为了将Tf结合与功能变化联系起来，染色质可及性和基因表达将分别通过DNase I超敏分析(DNase-seq)和RNA-seq进行监测。为了研究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IRF3/5/7二聚体的DNA结合。PBM结合数据将与其他基因组数据集整合，构建IRF3/5/7功能的全基因组模型。假说将使用基于细胞的报告分析进行检验(目标1)。为了研究已知的κ3/5/7的辅因子--核因子-κB的作用，我们检测了核因子结合、染色质可及性和基因表达，并使用外周血巨噬细胞来检测核因子-核因子-κB复合体的特异性。我们假设，IRF和NF-κB之间的不同相互作用将有助于它们各自的生物学作用(目标2)。为了研究巨噬细胞主要调控因子IRF8和PU.1的作用，我们将表征IRF8和PU.1的基因组结合，并将这些数据与IRF8敲除巨噬细胞中的IRF结合、染色质可及性和基因表达图谱相结合。为了解决蛋白质相互作用的作用，PBMS将被用来表征IRF3/5/7与IRF8：PU.1复合体的DNA结合。我们假设IRF3/5/7和IRF8：PU.1之间在特定调控元件上的协同作用以单核/巨噬细胞特异性的方式定制IRF特异性功能。预计这些研究将为管理IRF3/5/7特定功能的机制提供关键的见解。我们的目标是开发一个全面的IRF调控网络模型，该模型可以在不同的细胞环境中使用，并可能为自身免疫性疾病提供洞察，例如与IRF5/7上调相关的系统性红斑狼疮(SLE)。

 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.