Discovery of novel regulatory territories in the TNF/LT locus

我们的目标是了解细胞类型和刺激特异性调节的机制 人肿瘤坏死因子基因和T细胞和单核/巨噬细胞的肿瘤坏死因子/LT基因(LTA和LTB) 以确定在肿瘤坏死因子驱动的疾病状态下可能成为靶点的基因组区域。使用不偏不倚 下一代测序(NGS)方法和人类细胞和小鼠的CRISPR编辑，我们将 鉴定和阐明调节肿瘤坏死因子、LTA和LTB基因的转录调控元件的功能 T细胞和单核/巨噬细胞在激活和分化条件下的表达 具有传染性的挑战。我们用链状RNA-、ATAC-和 Hint-seq揭示了多个新的高度保守的非编码元件，它们在一种细胞类型- 在幼稚T细胞和人单核/巨噬细胞中的特异方式。它们还显示了细胞类型- 特异的hhs-8增强子，控制单核/巨噬细胞中干扰素-γ的启动，并增强肿瘤坏死因子和 激活的T细胞中的LTA，我们之前已经描述过了。我们的第一个目标将是定义转录区域 以及新元件和HHS-8与肿瘤坏死因子、LTA和 LTB基因。我们将使用CHIP-SEQ来确定建筑蛋白CTCF的招募，它 介导染色质构象，以及与之相关的H3K27Ac和H3K24Me的浓缩 使用增强剂。为了选择高潜力的调控区域，这一数据还将通过系统发育学进行评估 分析非人灵长类动物的肿瘤坏死因子/LT基因座以确定预测 监管职能。这些研究将指导我们用Hi-C和Hi-C对轨迹建筑进行三维分析 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，用于询问这些地点和 我们将在对人类T细胞不同状态的分析中发现新的调节元件 TcR配体或脂多糖和/或干扰素-γ刺激巨噬细胞分化；(Ii)肿瘤坏死因子/淋巴细胞 C57BL/6和Balb/c小鼠原代T细胞和BMDM基因座的一致性评价 以小鼠和人的肿瘤坏死因子/LT基因座的调节为基线进行的研究 CRISPR编辑的小鼠和测试元素在急性(脓毒症)和慢性(关节炎)肿瘤坏死因子介导中的作用 疾病模型。我们还将描述调节肿瘤坏死因子和IL-6基因的末端元件的作用 表达，在感染结核分枝杆菌(MTB)或 RNA病毒(仙台和SARS-CoV-2)，以阐明更广泛的基因表达程序。我们期待着 这些研究将导致对肿瘤坏死因子/LT基因是如何协调调控的新的理解， 提供对基因调控和末端元件的作用的基本见解，并提供潜在的 在疾病状态下以细胞类型和诱导物特异的方式调节肿瘤坏死因子的基因组靶点。

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.