Checkpoints of TNF Gene Regulation

TNF基因调控的检查点

• 批准号: 7227536
• 负责人: ANNE GOLDFELD
• 金额: $ 34.02万
• 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/7227536
• 关键词: Adaptor Signaling Protein; Antibodies; Autoimmune Process; Autoimmunity; B-Lymphocytes; Binding; Binding Sites; Bioinformatics; CD4 Positive T Lymphocytes; Calcium; Cell Count; Cell Line; Cells; Cerebral Malaria; Cessation of life; Chromatin; Communicable Diseases; Complex; Condition; Cytokine Gene; DNA; Dendritic Cells; Deoxyribonuclease I; Disease; Distal; Enhancers; Environment; Functional RNA; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Goals; Helper-Inducer T-Lymphocyte; Hypersensitivity; Immune response; In Vitro; Inflammatory; Inflammatory Bowel Diseases; Interferon Type II; Interferons; Interleukin-10; Lead; Link; Lipopolysaccharides; Mediating; Mediator of activation protein; Microarray Analysis; Modeling; Molecular; Mus; Mycobacterium tuberculosis; Pathology; Phase; Pliability; Process; Production; Proteins; RNA Interference; Recruitment Activity; Regulation; Regulatory Element; Research; Resistance; Retroviral Vector; Rheumatoid Arthritis; Role; Septic Shock; Signal Pathway; Signal Transduction Pathway; Signaling Molecule; Site; Specificity; Staphylococcal Enterotoxin B; Stimulus; T-Lymphocyte; TNF gene; Testing; Therapeutic; Transcriptional Regulation; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Virus; base; cell type; chromatin remodeling; cytokine; gene induction; human TNF protein; human disease; in vivo; inhibitor/antagonist; macrophage; novel; pathogen; promoter; release of sequestered calcium ion into cytoplasm; research study; transcription factor

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（由申请人提供）：肿瘤坏死因子（TNF）是由许多细胞（包括T和B淋巴细胞、巨噬细胞和树突细胞）产生的炎症和免疫应答的关键介质。它是根除细胞内病原体如结核分枝杆菌（MTb）的关键细胞因子，但不加控制可导致严重疾病，如败血性休克、脑型疟疾和自身免疫。这通过抗TNF抗体治疗类风湿性关节炎、炎性肠病和其它炎性病理的功效来说明。TNF是一种在转录水平上受到严格调控的基因。我们的研究已经表明，当巨噬细胞系用脂多糖（LPS）或MTb刺激时，一种独特的增强体被募集到TNF启动子，这与用病毒或钙刺激T细胞系时募集到启动子的增强体不同。此外，在细胞系中的遗传方法和DNA酶I超敏性（DH）分析已经鉴定了在其它非编码序列中的启动子之外的基因的诱导物和细胞类型特异性调节中涉及的调节元件。基于这些发现，我们的假设是，通过增强体招聘的TNF启动子，其中包含不同的激活剂，与染色质重塑共享的结合位点的动态过程中，TNF基因实现其调节所需的灵活性和特异性。我们将在目标1和2中的原代鼠T细胞和巨噬细胞中检验这一假设，方法是在不同条件下表征这些细胞中TNF基因座的染色质环境，并确定TNF基因表达所涉及的转录因子和信号分子。在目标3中，我们将研究参与干扰素-γ（IFN-γ）和白细胞介素-10（IL-10）调节TNF基因表达的染色质环境和转录因子。TNF基因表达的检查点将使用方法的组合来阐明，所述方法包括：（i）通过DH分析和生物信息学方法的组合的染色质重塑;（ii）使用特异性抑制剂、RNAi敲低或候选转录因子/信号传导分子缺陷的小鼠;和/或表达候选转录因子或信号传导分子的逆转录病毒载体;（iii）通过在不同条件下对原代细胞进行ChIP分析，将特异性TNF增强子复合物募集至调节元件或TNF启动子。这些实验将使我们能够将特异性TNF增强子复合物的募集与不同的衔接蛋白和随后的信号转导途径联系起来。该提案的总体目标是：1）鉴定特异性转录靶点，从而允许在炎症和感染性疾病中对TNF进行细胞和诱导剂定向的治疗操作，以及2）进一步阐明真核基因转录的控制和特异性机制。