Gene Regulation in the Immune System

 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-启动炎症和定制免疫反应。模式识别受体（PRRs）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 IRF 3、IRF 5和IRF 7是响应病毒和细胞内病原体的PRR信号传导的核心。尽管有共同的DNA结合特异性和激活模式，IRF 3，IRF 5和IRF 7（以下简称IRF 3/5/7）在宿主防御中发挥重叠但不同的作用。关于IRF 3/5/7激活上游的信号事件的差异，我们知道得很多;然而，关于IRF 3/5/7如何在DNA水平上区分它们各自的靶基因以引起不同的生物学结果，我们知之甚少 并针对病原体调整免疫反应。 该提案旨在利用基因组规模技术和数据集的进步，以解决管理病原体检测的基因调控网络中的IRF 3/5/7顺式调控特异性问题。蛋白结合微阵列（PBM）将用于表征IRF 3/5/7二聚体的DNA结合，解决我们目前模型中的缺陷。为了研究IRF 3/5/7结合差异如何导致调控差异，PBM结合数据将与染色质免疫沉淀（ChIP）和基因表达数据集整合。将使用基于细胞的报告基因试验（Aim 1）对假设进行检验。为了检查NF-κ B B（IRF 3/5/7的已知辅因子）的作用，我们将（1）使用ChIP-seq检查野生型和NF-κ B B敲低细胞中的IRF结合，和（2）使用PBM检查IRF-NF-κ B B复合物的DNA结合特异性（目的2）。我们假设IRF和NF-κ B B之间的差异相互作用将有助于它们各自的生物学作用。为了揭示可能导致调控差异的新IRF辅因子，我们将使用计算和蛋白质组学相结合的方法。计算基序分析将应用于ChIP-seq数据集，以识别与IRF结合相关的基序（和同源因子）。传统的分离技术与质谱联用（质谱）将用于从细胞裂解物中鉴定与DNA寡核苷酸上的IRF因子共结合的新因子（目的3）。 这些研究有望为IRF 3/5/7不同功能的机制提供重要见解。描述IRF 3/5/7功能中辅因子相互作用的作用和范围将为深入了解宿主防御反应中信号通路的相互依赖性提供帮助，并可能揭示炎症治疗干预的新机会。更广泛地说，该提案旨在阐明PRR-IRF 3/5/7信号传导特异性，以期建立更完整的宿主防御模型，可以解决诸如感染病原体之间的拮抗作用以及感染与系统性红斑狼疮等自身免疫性疾病之间的关系等现象。（SLE），这与PRR-IRF 5/7信号传导的改变密切相关。

 DESCRIPTION (provided by applicant): The body's pathogen detection system is critical to host defense - initiating inflammation and tailoring the immune response. Pattern recognition receptors (PRRs) 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 PRR 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 overlapping yet distinct roles in host defense. Much is known regarding differences in the signaling events upstream of IRF3/5/7 activation; however, little is known about how IRF3/5/7 discriminate their individual target genes at the DNA level to elicit different biological outcomes and tailor the immune response to pathogens. This proposal aims to capitalize upon advances in genome-scale techniques and datasets to address the issue of IRF3/5/7 cis-regulatory specificity in the gene regulatory network governing pathogen detection. Protein binding microarrays (PBMs) will be used to characterize the DNA binding of IRF3/5/7 dimers, addressing deficiencies in our current models. To examine how IRF3/5/7 binding differences contribute to regulatory differences, PBM binding data will be integrated with chromatin immunoprecipitation (ChIP) and gene expression datasets. Hypotheses will be tested using cell-based reporter assays (Aim 1). To examine the role of NF-B - a known cofactor of IRF3/5/7 - we will (1) use ChIP-seq to examine IRF binding in wild- type and NF-B knockdown cells, and (2) use PBMs to examine DNA binding specificity of IRF- NF-B complexes (Aim 2). We hypothesize that differential interactions between the IRFs and NF-B will contribute to their individual biological roles. To uncover novel IRF cofactors that might contribute to regulatory differences, we will use combined computational and proteomics approaches. Computational motif analysis will be applied to ChIP-seq datasets to identify motifs (and cognate factors) associated with IRF binding. Traditional separation techniques coupled with mass spectrometry (mass-spec) will be used to identify novel factors from cell lysates that co-bind with IRF factors on DNA oligonucleotides (Aim 3). These studies are expected to provide critical insights into the mechanisms underlying different IRF3/5/7 functions. Delineating the role and scope of cofactor interactions in IRF3/5/7 function will provide insight into the interdependence of signaling pathways in the host defense response, and may reveal new opportunities for therapeutic intervention in inflammation. More broadly, this proposal aims to clarify PRR-IRF3/5/7 signaling specificity with a view to a more complete model of host defense that can address phenomena such as antagonism between infecting pathogens, and the relation between infection and autoimmune diseases such as systemic lupus erythematosus (SLE), which is strongly associated with altered PRR-IRF5/7 signaling.