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活化相关的广泛刺激诱导的，但我们实验室的工作表明，TNF基因调控的机制对于不同的细胞类型和不同的刺激是不同的。这种精致的细胞类型和刺激特异性转录由高度保守、紧凑和模块化的近端启动子区域指导，其中不同的转录因子集合组装成更高级的二级结构或增强体。最近，我们已经表明，这种特异性延伸到染色质环境的TNF基因座，其中保守的功能遗传区域与细胞类型和刺激特异性DNA酶超敏反应位点（HSS），染色体内的相互作用，并与核基质的相互作用。鉴定潜在的治疗靶点以控制特定细胞类型中的TNF表达并响应于特定刺激，这为目前以非特异性、全身性方式发挥作用的TNF抑制剂提供了非常有吸引力的替代方案。 将在本提案中检验的第一个假设是，TNF基因座的细胞类型和刺激特异性染色质环境是原代人细胞中TNF基因转录控制的关键调节因子。我们将表征人T细胞和巨噬细胞中TNF位点的染色质环境的特征，包括组成性和瞬时DNA酶I HSS的位置、组蛋白修饰、蛋白质-DNA接触和核小体定位，这些与这些细胞的刺激和未刺激状态相关。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。第二个假设，将在本提案中进行测试的是，特定的因素建立高阶染色质相互作用和影响DNA拓扑结构的TNF基因表达的控制在TNF基因座。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，我们将测试特定HSS对TNF基因转录的影响。这些细胞类型特异性HSS含有与拓扑异构酶II相互作用的基质附着区以及参与Z型至B型DNA转变的潜在区域。这些HSSs可能通过侧翼基因抵消转录诱导的超螺旋来促进TNF基因的转录。因此，这些研究不仅将阐明新的机制，在控制TNF基因的调控，可能有更广泛的意义，真核基因转录，但也划定潜在的临床检查点的控制TNF的表达。 公共卫生相关性：过多或过少的肿瘤坏死因子（TNF）蛋白会影响多种疾病，包括哮喘、过敏、关节炎、败血症、结核病和艾滋病。虽然目前的TNF阻断剂已被有效地用于某些自身免疫性疾病，这些治疗是有限的，因为它们缺乏特异性和随之而来的并发症。通过将我们的研究集中在不同类型细胞中TNF基因激活的初始步骤上，我们希望发现新的治疗靶点，从而高度特异性地控制TNF蛋白水平。

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.