Checkpoints of TNF Gene Regulation

TNF基因调控的检查点

• 批准号: 7028018
• 负责人: ANNE GOLDFELD
• 金额: $ 35.04万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 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 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7028018
• 关键词: RNA interference; Retroviridae; binding sites; bioinformatics; biological signal transduction; cell line; chromatin; chromatin immunoprecipitation; deoxyribonuclease I; gene induction /repression; genetic enhancer element; genetic promoter element; genetic regulation; genetic transcription; helper T lymphocyte; human tissue; immunogenetics; interferon gamma; interleukin 10; laboratory mouse; macrophage; microarray technology; nuclear factor kappa beta; nucleic acid sequence; transfection /expression vector; tumor necrosis factor alpha

DESCRIPTION (provided by applicant): Tumor necrosis factor (TNF) is a key mediator of inflammatory and immune responses produced by many cells including T and B lymphocytes, macrophages and dendritic cells. It is a critical cytokine for eradication of intracellular pathogens such as Mycobacterium tuberculosis (MTb), but uncontrolled can lead to severe disorders such as septic shock, cerebral malaria and autoimmunity. This is illustrated by the efficacy of anti-TNF antibody treatment of rheumatoid arthritis, inflammatory bowel disease and other inflammatory pathologies. TNF is a tightly regulated gene at the level of transcription. Our research has shown that a distinct enhanceosome is recruited to the TNF promoter when macrophage cell lines are stimulated with lipopolysaccharide (LPS) or MTb, distinct from the enhanceosomes recruited to the promoter upon stimulation of T cell lines with virus or calcium. Furthermore, genetic approaches and DNAse I hypersensitivity (DH) analysis in cell lines have identified regulatory elements involved in inducer and cell type specific regulation of the gene outside of the promoter in other non-coding sequences. Based on these findings, our hypothesis is that through a dynamic process of enhanceosome recruitment to the TNF promoter, which contains shared binding sites for distinct activators, together with remodeling of chromatin, the TNF gene achieves both the flexibility and specificity required for its regulation. We will test this hypothesis in primary murine T cells and macrophages in Aims 1 and 2 through the characterization of the chromatin environment of the TNF locus in these cells under different conditions, and by determining the transcription factors and signaling molecules involved in TNF gene expression. In Aim 3, we will examine the chromatin environment and transcription factors involved in the modulation of TNF gene expression by interferon-gamma (IFN-gamma) and interleukin-10 (IL-10). The checkpoints of TNF gene expression will be elucidated using a combination of approaches including: (i) chromatin remodeling by a combination of DH analysis and bioinformatic approaches; (ii) the use of specific inhibitors, RNAi knockdown or mice deficient for candidate transcription factor/signaling molecules; and/or retroviral vectors expressing candidate transcription factors or signalling molecules; (iii) the recruitment of specific TNF enhancer complexes to regulatory elements or the TNF promoter by ChIP analysis of primary cells under different conditions. These experiments will allow us to link the recruitment of specific TNF enhancer complexes with distinct adaptor proteins and subsequent signal transduction pathways. The overarching goals of this proposal are: 1) the identification of specific transcriptional targets allowing for cell and inducer directed therapeutic manipulation of TNF in inflammatory and infectious diseases, and 2) the further elucidation of the mechanisms of control and specificity of eukaryotic gene transcription.

描述(申请人提供)：肿瘤坏死因子是炎症和免疫反应的关键介质，由许多细胞产生，包括T和B淋巴细胞、巨噬细胞和树突状细胞。它是根除结核分枝杆菌(MTB)等细胞内病原体的关键细胞因子，但如果不加以控制，可能会导致感染性休克、脑型疟疾和自身免疫等严重疾病。抗肿瘤坏死因子抗体对类风湿性关节炎、炎症性肠病和其他炎性病理的治疗效果说明了这一点。肿瘤坏死因子是一个在转录水平上受到严格调控的基因。我们的研究表明，当巨噬细胞系被脂多糖(LPS)或MTB刺激时，肿瘤坏死因子启动子被招募到一个明显的增强体，这与病毒或钙刺激T细胞系时被招募到启动子的增强体不同。此外，遗传学方法和细胞系中的DNase I超敏分析已经在其他非编码序列中发现了与启动子以外的基因的诱导物和细胞类型特异性调控有关的调控元件。基于这些发现，我们的假设是，通过一个动态的增强体募集到肿瘤坏死因子启动子的过程，其中包含不同激活剂的共同结合部位，以及染色质的重塑，肿瘤坏死因子基因实现了其调节所需的灵活性和特异性。我们将在AIMS 1和AIMS 2中的原代小鼠T细胞和巨噬细胞中验证这一假设，方法是在不同条件下表征这些细胞中肿瘤坏死因子基因座的染色质环境，并确定参与肿瘤坏死因子基因表达的转录因子和信号分子。在目标3中，我们将研究染色质环境和转录因子参与干扰素-γ和白介素10对肿瘤坏死因子基因表达的调节。肿瘤坏死因子基因表达的检查点将使用多种方法相结合的方法来阐明，包括：(I)通过水解酶分析和生物信息学方法相结合的染色质重塑；(Ii)使用特定的抑制剂、RNAi敲除或缺乏候选转录因子/信号分子的小鼠；和/或表达候选转录因子或信号分子的逆转录病毒载体；(Iii)在不同条件下通过原代细胞的芯片分析将特定的肿瘤坏死因子增强子复合体招募到调控元件或肿瘤坏死因子启动子。这些实验将使我们能够将特定的肿瘤坏死因子增强子复合体的招募与不同的接头蛋白和随后的信号转导途径联系起来。这一建议的主要目标是：1)识别特定的转录靶点，允许细胞和诱导剂指导炎症和感染性疾病中的肿瘤坏死因子的治疗操作；2)进一步阐明真核基因转录的控制和特异性机制。