Checkpoints of TNF Gene Regulation

描述(申请人提供)：肿瘤坏死因子(TNF)的异常表达与多种疾病状态有关，包括哮喘、过敏、关节炎、心血管疾病、炎症性肠道疾病、糖尿病、湿疹、狼疮、几种不同形式的癌症，以及包括败血症、结核病(TB)和HIV-1在内的多种感染性疾病。虽然在感染、应激和炎症过程中，与细胞激活相关的一系列刺激可以诱导肿瘤坏死因子的转录，但我们实验室的工作表明，对于不同的细胞类型和不同的刺激，肿瘤坏死因子基因的调控机制是不同的。这种精致的细胞型和刺激特异性转录受高度保守、紧凑和模块化的近端启动子区域的指导，在该区域，不同的转录因子组装成更高级别的二级结构或增强体。最近，我们发现这种特异性延伸到了肿瘤坏死因子基因座的染色质环境中，在那里保守的功能遗传区与细胞类型和刺激特异性DNA酶高敏感性位点(HSS)、染色体内相互作用以及与核基质的相互作用有关。识别潜在的治疗靶点来控制特定细胞类型中的肿瘤坏死因子的表达和对特定刺激的反应，是目前以非特异性、全身性方式发挥作用的肿瘤坏死因子抑制剂的一个极具吸引力的替代方案。将在这一建议中检验的第一个假设是，肿瘤坏死因子基因座的细胞类型和刺激特异性染色质环境是控制原代人类细胞中肿瘤坏死因子基因转录的关键调节因子。我们将描述人类T细胞和巨噬细胞中肿瘤坏死因子基因座染色质环境的特征，包括与这些细胞的刺激和非刺激状态相关的构成和瞬时DNase I HSS的位置、组蛋白修饰、蛋白质-DNA接触和核小体定位。我们的研究将检验多种生理相关刺激对T细胞和单核细胞的影响。第二个假设将在这个提议中被检验，那就是特定的因素建立了更高级别的染色质相互作用，并在控制肿瘤坏死因子基因表达的过程中影响肿瘤坏死因子基因座的DNA拓扑。我们将确定NFATp在T细胞激活依赖的体内相互作用形成中的作用。通过将野生型和突变型的TNF/LT基因座整合到细胞系中，我们将测试特定的HSS对肿瘤坏死因子基因转录的影响。这些细胞类型特异的HSS包含与拓扑异构酶II相互作用的基质附着区，以及参与Z-型到B-型DNA转换的潜在区域。这些HSS可能通过抑制转录诱导的基因侧翼超螺旋来促进肿瘤坏死因子基因的转录。因此，这些研究不仅将阐明控制肿瘤坏死因子基因调控的新机制，这些机制可能对真核基因转录具有更广泛的意义，而且还将勾勒出控制肿瘤坏死因子表达的潜在临床检查点。 公共卫生相关性：肿瘤坏死因子(TNF)蛋白过多或过少会影响多种疾病，包括哮喘、过敏、关节炎、败血症、结核病和艾滋病。虽然目前的肿瘤坏死因子阻滞剂已经有效地用于某些自身免疫性疾病，但由于缺乏特异性和随之而来的并发症，这些治疗方法是有限的。通过将我们的研究重点放在不同类型细胞中涉及的肿瘤坏死因子基因激活的最初步骤上，我们希望发现新的治疗靶点，允许高度特异性地控制肿瘤坏死因子蛋白水平。

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.