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刺激特异性调节的机制 人 TNF 基因以及 T 细胞和单核细胞/巨噬细胞中的 TNF/LT 基因座基因（LTA 和 LTB） 使用公正的方法来识别 TNF 驱动的疾病状态中可能靶向的基因组区域。 下一代测序 (NGS) 方法以及人类细胞和小鼠的 CRISPR 编辑，我们将 识别并阐明调节 TNF、LTA 和 LTB 基因的转录调控元件的功能 在激活和分化条件下 T 细胞和单核细胞/巨噬细胞中的表达 我们使用搁浅 RNA、ATAC 和 NGS 方法进行的初步研究。 HINT-seq 揭示了多种新型高度保守的非编码元件，它们在细胞类型中转录 eRNA 幼稚 T 细胞和人类单核细胞/巨噬细胞中的特定方式它们还显示细胞类型。 特异性 hHS-8 增强剂，控制单核细胞/巨噬细胞中的 IFN-γ 启动并增强 TNF 和 我们之前描述的活化 T 细胞中的 LTA 是定义转录区域。 以及新元件和 hHS-8 与 TNF、LTA 和 我们将使用 ChIP-seq 来确定结构蛋白 CTCF 的招募，该蛋白 介导染色质构象以及 H3K27Ac 和 H3K24Me 的富集，这与相关 为了选择高潜力的监管区域，该数据还将通过系统发育进行评估。 分析非人类灵长类动物的 TNF/LT 基因座，以确定预测的高度保守区域 这些研究将指导我们使用 Hi-C 和 进行基因座结构的 3 维分析。 CRISPR 删除细胞系和原代细胞中的潜在调控元件以建立其功能。 这些研究将提供一个强大的框架和数据集，用于询问这些网站和 我们将在对 (i) 人类 T 细胞的不同状态和 分别用 TCR 配体或 LPS 和/或 IFN-γ 刺激巨噬细胞分化； C57BL/6 和 Balb/c 小鼠品系的原代 T 细胞和 BMDM 中的位点以评估一致性 小鼠和人类 TNF/LT 基因座的调节之间作为进行研究的基线 CRISPR 编辑小鼠并测试元件在急性（败血症）和慢性（关节炎）TNF 介导中的作用 我们还将描述调节 TNF 和 IL-6 基因的远端元件的作用。 表达，与结核分枝杆菌 (MTb) 感染期间的 TNF 具有相似的调节作用或 我们预计 RNA 病毒（仙台病毒和 SARS-CoV-2）将阐明更广泛的基因表达程序。 这些研究将带来对 TNF/LT 基因如何协调调节的新认识， 提供对基因调控和远端元件作用的基本见解，并提供潜在的 基因组靶点在疾病状态下以细胞类型和诱导物特异性方式调节 TNF。