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Discovery of novel regulatory territories in the TNF/LT locus

TNF/LT 基因座中新调控区域的发现

基本信息

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
项目类别：
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

项目摘要

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