Checkpoints of TNF Gene Regulation

描述（由申请人提供）：肿瘤坏死因子（Tumor necrosis factor， TNF）是许多细胞（包括T淋巴细胞、B淋巴细胞、巨噬细胞和树突状细胞）产生的炎症和免疫反应的关键介质。它是消灭结核分枝杆菌（MTb）等细胞内病原体的关键细胞因子，但不受控制可导致感染性休克、脑疟疾和自身免疫等严重疾病。抗tnf抗体治疗类风湿关节炎、炎症性肠病和其他炎症性病理的疗效说明了这一点。TNF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，当巨噬细胞系受到脂多糖（LPS）或MTb刺激时，一个独特的增强体被募集到TNF启动子上，这与用病毒或钙刺激T细胞系时募集到启动子上的增强体不同。此外，遗传学方法和细胞系的DNAse I超敏性（DH）分析已经确定了在其他非编码序列中参与启动子外基因的诱导剂和细胞类型特异性调控的调控元件。基于这些发现，我们的假设是，通过一个动态的过程，增强体招募到TNF启动子，其中包含不同激活剂的共享结合位点，以及染色质的重塑，TNF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的原代小鼠T细胞和巨噬细胞中验证这一假设，通过表征这些细胞在不同条件下TNF位点的染色质环境，并通过确定参与TNF基因表达的转录因子和信号分子。在Aim 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通过以下几种方法进行阐明：(i)结合DH分析和生物信息学方法进行染色质重塑；（ii）使用特异性抑制剂、RNAi敲低或候选转录因子/信号分子缺失小鼠；表达候选转录因子或信号分子的逆转录病毒载体；（iii）通过对不同条件下原代细胞的ChIP分析，将特定的TNF增强子复合物募集到调节元件或TNF启动子上。这些实验将使我们能够将特定TNF增强子复合物的募集与不同的接头蛋白和随后的信号转导途径联系起来。本提案的总体目标是：1)确定特定的转录靶点，允许细胞和诱导剂在炎症和感染性疾病中指导TNF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。

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.