TNF Alpha In Inflammation

描述（由申请人提供）：TNF α的过度表达几乎总是有害的。小鼠中的过度表达与全身性自身免疫和器官特异性炎症变化相关。在患有多种炎症性疾病的人中观察到过度表达。为了防止过度表达的有害后果，TNF α似乎使用转录沉默和翻译沉默。目前提出的假设是，TNF α的转录抑制是通过DNA甲基化、组蛋白脱乙酰化和核定位效应的组合介导的。在单核细胞中，这些转录抑制策略被假设为受成熟刺激的调节。我们的初步数据表明，进行性组蛋白乙酰化伴随着单核细胞分化，并且增强的组蛋白乙酰化可以允许非表达细胞表达TNF α。这些数据有力地表明，组蛋白乙酰化在单核细胞成熟时建立转录能力中起重要作用。目前尚不清楚组蛋白乙酰化是否会影响DNA甲基化，反之亦然。该提案将解决DNA甲基化、组蛋白乙酰化和常染色质位置是变量的假设，这些变量独立调节并支配TNF α基因座的转录能力。为了确定这三个变量的作用，我们将首先分析控制TNF α基因座核定位的因素。DNA甲基化和组蛋白乙酰化的成熟、刺激和操作将作为调节核定位的变量进行评价。DNA甲基化在调节TNF α表达中的作用将在第二个目标中进行检查。亚硫酸氢盐分析将用于定义不同成熟阶段单核细胞中的甲基化模式，并使用特异性抑制剂检查DNA甲基化和组蛋白乙酰化之间的关系。在第三个目标中，染色质免疫沉淀测定将用于鉴定存在或不存在于无活性和活性启动子上的转录因子。通过在前一个应用周期中所做的工作确定的候选阻遏蛋白GCF 2将被检查对转录、组蛋白乙酰化和DNA甲基化的影响。总的来说，该建议将定义TNF α位点的潜在抑制机制。

DESCRIPTION (provided by applicant): Over-expression of TNF alpha is almost always deleterious. Over-expression in mice is associated with both systemic autoimmunity and organ-specific inflammatory changes. Over-expression is seen in humans with a wide range of inflammatory disorders. To prevent the deleterious consequences of over-expression, TNF alpha appears to use both transcriptional silencing and translational silencing. The hypothesis addressed in the current proposal is that transcriptional repression of TNF alpha is mediated through a combination of DNA methylation, histone deacetylation and nuclear localization effects. In monocytes, these transcriptional repression strategies are hypothesized to be regulated by maturational stimuli. Our preliminary data demonstrates that progressive histone acetylation accompanies monocyte differentiation and that enforced histone acetylation can allow a non-expressing cell to express TNF alpha. These data strongly suggest that histone acetylation plays an important role in establishing transcriptional competency as monocytes mature. It is unknown at this time whether histone acetylation affects DNA methylation or vice versa. This proposal will address the hypothesis that DNA methylation, histone acetylation, and location with euchromatin are variables, which are regulated independently and govern the competence of the TNF alpha locus for transcription. To define the roles of these three variables, we will first analyze factors that govern the nuclear localization of the TNF alpha locus. Maturation, stimulation, and manipulation of DNA methylation and histone acetylation will be evaluated as variables regulating nuclear localization. The role of DNA methylation in the regulation of TNF alpha expression will be examined in the second aim. Bisulfite analysis will be used to define methylation patterns in monocytes at different maturational stages and the relationship between DNA methylation and histone acetylation examined using specific inhibitors. In the third aim, chromatin immunoprecipitation assays will be used to identify the presence of absence of transcription factors residing on inactive and active promoters. A candidate repressor protein, GCF2, identified through the work done in the previous application cycle will be examined for effects on transcription, histone acetylation, and DNA methylation. Overall, this proposal will define the mechanisms underlying repression of the TNF alpha locus.