Checkpoints of TNF Gene Regulation

TNF基因调控的检查点

• 批准号: 9109364
• 负责人: ANNE GOLDFELD
• 金额: $ 42.48万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9109364
• 关键词: Acetylation; Acetylesterase; Acute; Adverse effects; Autoimmune Diseases; Binding; Binding Sites; CRISPR/Cas technology; Cells; Chromatin; Chromatin Loop; Clustered Regularly Interspaced Short Palindromic Repeats; Communicable Diseases; Control Locus; Cyclosporine; DNA Sequence; DNA-Protein Interaction; Data; Data Element; Deoxyribonuclease I; Deoxyribonucleases; Digestion; Disease; Distal; EP300 gene; Elements; Enhancers; Epigenetic Process; Event; Exposure to; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Enhancer Element; Genetic Transcription; Goals; Histones; Human; Human Cell Line; Hypersensitivity; IL12B gene; IRF1 gene; Immune; Immune response; Indium; Infection Control; Interferons; Interleukin-12; Interleukin-6; LTB gene; Laboratories; Lead; Ligands; Link; Lipopolysaccharides; Macrophage Activation; Mediating; Mediator of activation protein; Methylation; Modification; Molecular; Mus; Mycobacterium tuberculosis; Myeloid Cells; Nature; Nucleic Acid Regulatory Sequences; Pathology; Plague; Play; Proteins; RNA; Recruitment Activity; Regulation; Regulatory Element; Role; Sepsis; Septic Shock; Site; Specificity; Stimulus; Syndrome; T-Cell Activation; T-Lymphocyte; TNF gene; Testing; Therapeutic; Transcription Initiation Site; Tumor Necrosis Factor-Beta; base; cell type; chromatin remodeling; cytokine; demethylation; design; gene induction; hemodynamics; histone acetyltransferase; insight; latent infection; lymphotoxin beta; macrophage; monocyte; novel; nuclease; promoter; protein expression; protein protein interaction; public health relevance; research study; response; transcription factor

 DESCRIPTION (provided by applicant): Tumor necrosis factor (TNF) is located in a gene dense locus with the lymphotoxin (LT) α and β genes. The gene is highly induced in monocytes/macrophages by lipopolysaccharide (LPS) and its expression is further enhanced by pre-exposure to interferon (IFN)-γ, or IFN-γ priming. The tightly linked LT α and LTβ genes are expressed in activated T lymphocytes along with TNF, but not in monocytic cells. Consistent with this, we have found that the TNF/LT locus undergoes distinct chromatin remodeling events in primary human monocytic cells and T cells, and the chromatin signatures detected in the two cell types are distinct. However, in both cell types a distal DNase Hypersensitivity (DH) site ~8 kB upstream of the TNF transcription start site functions as an enhancer during both IFN-γ priming of LPS-induced human TNF transcription and in TNF expression in activated T cells. In primary human macrophages after IFN-γ treatment, hHS-8 becomes more accessible to DNase I digestion, displays increased levels of histone H3K27me3, and binds the transcription factor IRF1. Upon LPS stimulation of IFN-γ-primed cells, the H3K27me3-specific demethylase JMJD3 and the acetylase p300 are recruited to hHS-8 and acetylated H3K27 levels significantly increase while H3K27me3 levels decrease, and eRNA is transcribed. Specific targeting the hHS-8 IRF1 binding site in THP-1 cells in its chromatin context using a catalytically inactive `dead' Cas9 linked to the KRAB repressive domain, abolishes IFN-γ augmentation of LPS-induced TNF while LPS induction of the gene is unaffected. By contrast in activated primary T cells, NFATp is recruited to hHS-8, the site is decorated with H3K27Ac, and hHS-8 eRNA is produced. eRNA transcription is blocked by cyclosporine A treatment, further underscoring the importance of NFATp at this site. Furthermore, TNF levels are significantly reduced in HUT-78 hHS-8 NFATp targeted cells. LT α and LTβ gene regulation was also significantly decreased in these HUT-78 hHS-8 NFATp cells indicating that hHS-8 may be a locus control element in T cells and make intrachromosomal contacst with the newly identified LT regulatory elements. Based on these findings, we hypothesize that in activated macrophages, sequential H3K27 methylation, demethylation and acetylation events, poise and then trigger enhancer activity, respectively. We will test this hypothesis, and we will also investigate whether the IL-6 and IL12-B genes that are also primed in activated macrophages, are similarly regulated. We will test the hypothesis that IRF1 and NFATp function as `pioneer factors' and together with eRNA mediate hHS-8- promoter DNA looping and triggering of enhancer activity. We will also test the hypothesis that newly identified elements near the LT genes interact with hHS-8 in both dCas9/CRISPR edited human cell lines and in CRSPR mice we created. We expect to gain fundamental information about cell type- and stimulus-specific long-range enhancer function in general and to identify novel targets for potential modulation of TNF gene expression.

 描述(申请人提供)：肿瘤坏死因子与淋巴毒素(LT)α和β基因位于一个基因密集的区域。该基因在单核/巨噬细胞中被脂多糖高度诱导，并通过预先暴露于干扰素-γ或干扰素-γ预刺激进一步增强其表达。紧密连锁的LTα和LTβ基因是 与肿瘤坏死因子一起在活化的T淋巴细胞中表达，但在单核细胞中不表达。与此一致，我们发现在原代人类单核细胞和T细胞中，肿瘤坏死因子/LT基因座经历了不同的染色质重塑事件，并且在这两种细胞类型中检测到的染色质特征是不同的。然而，在这两种细胞中，肿瘤坏死因子转录起始点上游~8kB的远端DNA酶高敏感性位点在干扰素-γ启动脂多糖诱导的人肿瘤坏死因子转录和激活的T细胞表达肿瘤坏死因子的过程中都起到了增强子的作用。在原代人巨噬细胞中，经干扰素-γ处理后，HHS-8更容易被DNaseI消化，显示组蛋白H3K27me3水平增加，并结合转录因子IRF1。当脂多糖刺激干扰素-γ激活的细胞时，H3K27me3特异的脱甲基酶JMJD3和乙酰化酶p300被招募到HHS-8，乙酰化的H3K27水平显著增加，而H3K27me3水平下降，Erna被转录。在THP-1细胞的染色质环境中，使用与KRAb抑制结构域相连的催化失活的‘Dead’Cas9特异性靶向HHS-8IRF1结合部位，取消干扰素-γ对内毒素诱导的肿瘤坏死因子的增强作用，而内毒素对该基因的诱导不受影响。相比之下，在激活的原代T细胞中，NFATp被招募到HHS-8，用H3K27Ac修饰位点，产生HHS-8 Erna。Erna转录被环孢素A处理阻断，进一步强调了NFATp在这个位点的重要性。此外，在HUT-78 HHS-8 NFATP靶向细胞中，肿瘤坏死因子水平显著降低。在这些HUT-78HHS-8NFATP细胞中，LTα和LTβ基因的调控也明显降低，提示HHS-8可能是T细胞中的一个位点调控元件，并与新发现的LT调节元件形成染色体内接触。基于这些发现，我们假设在激活的巨噬细胞中，顺序的H3K27甲基化、去甲基化和乙酰化事件分别稳定并随后触发增强子活性。我们将验证这一假设，我们还将调查也在激活的巨噬细胞中启动的IL-6和IL-12-B基因是否受到类似的调控。我们将检验IRF1和NFATp作为“先锋因子”发挥作用的假设，并与Erna一起介导HHS-8启动子DNA环和增强子活性的触发。我们还将测试假设，即在dCas9/CRISPR编辑的人类细胞系和我们创建的CRSPR小鼠中，新发现的LT基因附近的元件与HHS-8相互作用。我们希望获得关于细胞类型和刺激特异性长程增强子功能的基本信息，并确定潜在调控肿瘤坏死因子基因表达的新靶点。