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。小鼠的过度表达与全身自身免疫和器官特异性炎症变化有关。过度表达见于多种炎症性疾病患者。为了防止过度表达的有害后果，TNF α似乎同时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、组蛋白去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是由成熟刺激调节的。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，并且强制组蛋白乙酰化可以使非表达细胞表达TNF α。这些数据强烈表明，组蛋白乙酰化在单核细胞成熟时转录能力的建立中起着重要作用。目前尚不清楚组蛋白乙酰化是否影响DNA甲基化，反之亦然。该建议将解决DNA甲基化，组蛋白乙酰化和与常染色质的位置是变量的假设，这些变量是独立调节的，并控制TNF α位点的转录能力。为了定义这三个变量的作用，我们将首先分析控制TNF α位点核定位的因素。成熟、刺激和操纵DNA甲基化和组蛋白乙酰化将被评估为调节核定位的变量。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研究。亚硫酸氢盐分析将用于确定不同成熟阶段单核细胞的甲基化模式，并使用特定抑制剂检测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驻留在非活性启动子和活性启动子上的转录因子的存在和缺失。通过前一个应用周期的工作确定的候选抑制蛋白GCF2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，这一建议将明确抑制TNF α基因座的机制。

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.