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TNF基因和T细胞和单核细胞/巨噬细胞中的TNF/LT基因座基因（LTA和LTB）， 以确定可能在TNF驱动的疾病状态中潜在靶向的基因组区域。使用无偏 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TNF、LTA和LTB基因转录调节元件的功能 在活化和分化条件期间T细胞和单核细胞/巨噬细胞中的表达， 传染性挑战我们的初步研究使用NGS方法的单链RNA-，ATAC-， HINT-seq揭示了在细胞类型中转录eRNA的多个新的高度保守的非编码元件 在幼稚T细胞和人单核细胞/巨噬细胞中的特异性方式。它们还显示了细胞类型- 特异性hHS-8增强子，其控制单核细胞/巨噬细胞中IFN-γ引发并增强TNF和 我们之前描述了活化T细胞中的LTA。我们的第一个目标是确定转录区域 以及新元件和hHS-8与TNF、LTA和 LTB基因。我们将使用ChIP-seq来确定结构蛋白CTCF的募集， 介导染色质构象，以及H3 K27 Ac和H3 K24 Me的富集，这与 用增强剂为了选择高潜力的调控区域，还将通过系统发育分析评估该数据。 分析非人灵长类动物中的TNF/LT基因座，以确定预测 调节功能。这些研究将指导我们用Hi-C和 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。 然后，这些研究将提供一个强大的框架和数据集，从其中询问这些网站， 我们将在我们对（i）人类T细胞的不同状态， 分别用TCR配体或LPS和/或IFN-γ刺激的巨噬细胞分化;（ii）TNF/LT 在来自C57 BL/6和Balb/c小鼠品系的原代T细胞和BMDM中的基因座中进行分析，以评估一致性 作为进行以下研究的基线的鼠和人TNF/LT基因座的调节之间的差异： CRISPR编辑的小鼠和测试元件在急性（脓毒症）和慢性（关节炎）TNF介导的炎症中的作用 疾病模型我们还将描述调节TNF和IL-6基因的远端元件的作用 表达，其在感染M.结核病（MTb）或 RNA病毒（仙台和SARS-CoV-2），以阐明更广泛的基因表达计划。我们预计 这些研究将导致对TNF/LT基因如何协调调节的新理解， 为基因调控和远端元件的作用提供了基本的见解，并提供了潜在的 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。

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.