Discovery of novel regulatory territories in the TNF/LT locus

TNF/LT 基因座中新调控区域的发现

• 批准号: 10408494
• 负责人: ANNE GOLDFELD
• 金额: $ 44.25万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10408494
• 关键词: 2019-nCoV; Acute; Architecture; Area; Arthritis; Autoimmune Diseases; CCCTC-binding factor; Cell Line; Cells; ChIP-seq; Chromatin; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Communicable Diseases; Data; Data Set; Disease; Disease Outcome; Disease model; Distal; Elements; Enhancers; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genomic Segment; Genomics; Goals; Hi-C; Human; IL6 gene; Immune; Immune Response Genes; Inbred BALB C Mice; Infection; Innate Immune Response; Interferon Type II; Knowledge; LTA gene; LTB gene; Ligands; Mediating; Molecular Conformation; Mouse Strains; Mus; Mycobacterium tuberculosis; Pathology; Phylogenetic Analysis; RNA; RNA Viruses; Regulation; Regulator Genes; Regulatory Element; Role; Sepsis; Site; Stimulus; T-Lymphocyte; TNF gene; Testing; Therapeutic; Three-dimensional analysis; Untranslated RNA; XCL1 gene; cell type; genomic locus; insight; macrophage; monocyte; next generation sequencing; nonhuman primate; novel; programs; recruit

Our goal is to understand the mechanisms of cell type- and stimulus-specific regulation of the human TNF gene and the TNF/LT locus genes (LTA and LTB) in T cells and monocytes/macrophages and to identify genomic regions that could potentially be targeted in TNF-driven disease states. Using unbiased next generation sequencing (NGS) approaches and CRISPR editing of human cells and mice, we will identify and elucidate function of transcriptional regulatory elements that modulate TNF, LTA, and LTB gene expression in T cells and monocytes/macrophages during activation and differentiation conditions and infectious challenges. Our preliminary studies using the NGS approaches of stranded RNA-, ATAC-, and HINT-seq reveal multiple novel highly conserved non-coding elements that transcribe eRNA in a cell type- specific manner in naïve T cells and in human monocytes/macrophages. They also show the cell type- specific hHS-8 enhancer that controls IFN-γ priming in monocytes/macrophages and enhances TNF and LTA in activated T cells we previously described. Our first goal will be to define the transcriptional territories and potential intrachromosomal interactions between the novel elements and hHS-8 with the TNF, LTA, and LTB genes. We will use ChIP-seq to determine the recruitment of the architectural protein CTCF, which mediates chromatin conformation, and the enrichment of H3K27Ac and H3K24Me, which are associated with enhancers. To select high potential regulatory areas this data will also be evaluated by a phylogenetic analysis of the TNF/LT locus in non-human primates to define highly conserved regions that predict regulatory function. These studies will guide our 3-dimensional analysis of locus architecture with Hi-C and CRISPR deletion of potential regulatory elements in cell lines and primary cells to establish their function. These studies then will provide a powerful framework and data set from which to interrogate these sites and new regulatory elements we will uncover in our analyses of (i) different states of human T cell and macrophage differentiation stimulated with TCR ligands or LPS and/or IFN-γ, respectively; (ii) the TNF/LT locus in primary T cells and BMDM from C57BL/6 and Balb/c mouse strains to evaluate concordance between the regulation of the murine and human TNF/LT loci as a baseline for performing studies in CRISPR-edited mice and testing the role of elements in acute (sepsis) and chronic (arthritis) TNF-mediated disease models. We will also characterize the role of distal elements that regulate TNF and the IL-6 gene expression, which shares regulatory similarities with TNF during infection with M. tuberculosis (MTb) or RNA viruses (Sendai and SARS-CoV-2), to elucidate broader gene expression programs. We anticipate that these studies will lead to a new understanding of how the TNF/LT genes are coordinately regulated, provide fundamental insights into gene regulation and the role of distal elements, and provide potential genomic targets to regulate TNF in a cell type-and inducer-specific manner in disease states.

我们的目标是了解细胞类型和特定于刺激的调节的机制 T细胞和单核细胞/巨噬细胞中的人类TNF基因和TNF/LT基因座基因（LTA和LTB）以及 确定可能针对TNF驱动疾病状态的基因组区域。使用公正 下一代测序（NGS）方法和人类细胞和小鼠的CRISPR编辑，我们将 识别和阐明调节TNF，LTA和LTB基因的转录调节元件的功能 在激活和分化条件下，T细胞和单核细胞/巨噬细胞的表达以及 传染性挑战。我们使用滞留的RNA，ATAC-和 hint-seq揭示了多个新颖的高度配置的非编码元素 幼稚T细胞和人类单核细胞/巨噬细胞中的特定方式。他们还显示细胞类型 - 特定的HHS-8增强子控制单核细胞/巨噬细胞中的IFN-γ启动并增强TNF和 我们先前描述的活化T细胞中的LTA。我们的第一个目标是定义转录领土 新元素与HHS-8与TNF，LTA和 LTB基因。我们将使用Chip-Seq确定建筑蛋白CTCF的募集，该蛋白CTCF 介导染色质构象，以及H3K27AC和H3K24ME的富集 与增强剂。为了选择高潜在的调节区域，该数据也将通过系统发育进行评估 非人类隐私中TNF/LT基因座的分析，以定义预测高度组成的区域 调节功能。这些研究将指导我们使用HI-C和HI-C和 CRISPR删除细胞系和原代细胞中潜在调节元件以建立其功能。 然后，这些研究将提供一个强大的框架和数据集，以询问这些站点和 我们将在（i）人类T细胞的不同状态和 TCR配体或LPS和/或IFN-γ刺激的巨噬细胞分化； （ii）TNF/LT 初级T细胞中的基因座和来自C57BL/6和BALB/C小鼠菌株的BMDM以评估一致性 在对鼠的调节和人类TNF/LT基因座的调节之间，作为进行研究的基线 CRISPR编辑的小鼠并测试元素在急性（败血症）和慢性（关节炎）TNF介导的作用 疾病模型。我们还将表征调节TNF和IL-6基因的远端元素的作用 表达，在结核分枝杆菌（MTB）或 RNA病毒（Sendai和SARS-COV-2），以阐明更广泛的基因表达程序。我们期待 这些研究将导致对TNF/LT基因如何协调调节的新了解， 提供对基因调节和远端元素作用的基本见解，并提供潜力 基因组靶标在疾病状​​态下以细胞类型和诱导的方式调节TNF。