Checkpoints of TNF Gene Regulation

描述（由申请人提供）：肿瘤坏死因子（TNF）的表达失调与多种疾病状态有关，包括哮喘、过敏、关节炎、心血管疾病、炎症性肠病、糖尿病、湿疹、狼疮、几种不同形式的癌症和多种感染性疾病，包括败血症、结核病（TB）和HIV-1。虽然TNF转录是由感染、应激和炎症期间与细胞激活相关的广泛刺激诱导的，但我们实验室的工作表明，不同细胞类型和不同刺激对TNF基因调控的机制是不同的。这种精致的细胞类型和刺激特异性转录是由一个高度保守、紧凑和模块化的近端启动子区域指导的，在这个区域中，不同的转录因子组合成更高阶的二级结构或增强体。最近，我们已经证明这种特异性延伸到TNF位点的染色质环境，其中保守的功能遗传区域与细胞类型和刺激特异性dna酶超敏位点（HSSs）、染色体内相互作用以及与核基质的相互作用相关。识别潜在的治疗靶点来控制特定细胞类型中TNF的表达，并对特定刺激做出反应，这是目前以非特异性、系统性方式起作用的TNF抑制剂的一个非常有吸引力的替代方案。本提案将测试的第一个假设是，TNF位点的细胞类型和刺激特异性染色质环境是控制原代人细胞中TNF基因转录的关键调节因子。我们将描述人类T细胞和巨噬细胞中TNF位点的染色质环境特征，包括与这些细胞受刺激和未受刺激状态相关的组成型和瞬态dna酶I hss的位置、组蛋白修饰、蛋白质- dna接触和核小体定位。我们的研究将检查多种生理相关刺激对T细胞和单核细胞的影响。本提案将测试的第二个假设是，在控制TNF基因表达的过程中，特定因素在TNF位点建立高阶染色质相互作用并影响DNA拓扑结构。我们将确定NFATp在T细胞中激活依赖性染色体内相互作用形成中的作用。通过将野生型和突变型TNF/LT基因座整合到细胞系中，我们将测试特异性HSSs对TNF基因转录的影响。这些细胞类型特异性hss包含与拓扑异构酶II相互作用的基质附着区域，以及参与z型到b型DNA转化的潜在区域。这些hss可能通过抵消两侧基因的转录诱导的超卷曲来促进TNF基因的转录。因此，这些研究不仅将阐明控制TNF基因调控的新机制，可能对真核基因转录有更广泛的影响，而且还将描述控制TNF表达的潜在临床检查点。

DESCRIPTION (provided by applicant): Dysregulated expression of tumor necrosis factor (TNF) has been implicated in multiple disease states, including asthma, allergy, arthritis, cardiovascular diseases, inflammatory bowel disease, diabetes, eczema, lupus, several different forms of cancer, and multiple infectious diseases including sepsis, tuberculosis (TB), and HIV-1. While TNF transcription is induced by a wide range of stimuli associated with cellular activation during infection, stress, and inflammation, work in our laboratory has shown that the mechanisms of TNF gene regulation are distinct for different cell types and for different stimuli. This exquisite cell-type and stimulus-specific transcription is directed by a highly conserved, compact, and modular proximal promoter region, where distinct sets of transcription factors assemble into higher order secondary structures, or enhanceosomes. More recently, we have shown that this specificity extends to the chromatin environment of the TNF locus, where conserved functional genetic regions are associated with cell type- and stimulus-specific DNase hypersensitivity sites (HSSs), intrachromosomal interactions, and interactions with the nuclear matrix. The identification of potential therapeutic targets to control TNF expression in specific cell types and in response to specific stimuli presents a highly attractive alternative to current TNF inhibitors that function in a non-specific, systemic fashion. The first hypothesis that will be tested in this proposal is that the cell type- and stimulus-specific chromatin environment of the TNF locus is a critical regulator in the control of TNF gene transcription in primary human cells. We will characterize the features of the chromatin environment of the TNF locus in human T cells and macrophages, including the location of constitutive and transient DNase I HSSs, histone modifications, protein-DNA contacts, and nucleosome positioning, associated with the stimulated and unstimulated state of these cells. Our studies will examine the impact of multiple physiologically relevant stimuli in T cells and monocytes. The second hypothesis that will be tested in this proposal is that specific factors establish higher-order chromatin interactions and impact DNA topology at the TNF locus in the control of TNF gene expression. We will determine the role of NFATp in the formation of activation-dependent intracrhomosomal interactions in T cells. Through integration of wild-type and mutant TNF/LT loci into cell lines, we will test the impact of specific HSSs upon TNF gene transcription. These cells type-specific HSSs contain matrix attachment regions that interact with topoisomerase II as well as a potential region involved in the transition of Z-to-B-form DNA. These HSSs potentially promote TNF gene transcription by counteracting transcription-induced supercoiling by flanking genes. Thus, these studies will not only elucidate novel mechanisms in the control of TNF gene regulation that may have broader implications for eukaryotic gene transcription, but also delineate potential clinical checkpoints for the control of TNF expression. PUBLIC HEALTH RELEVANCE: Too much or too little tumor necrosis factor (TNF) protein influences a variety of diseases, including asthma, allergy, arthritis, sepsis, tuberculosis, and AIDS. Although current blockers of TNF have been used effectively in certain autoimmune diseases, these therapies are limited because of their lack of specificity and ensuing complications. By focusing our studies on the initial steps involved in the activation of the TNF gene in different kinds of cells, we hope to discover new therapeutic targets that allow for highly specific control of TNF protein levels.