Epigenetic control of apoptotic susceptibility

描述（由申请人提供）：神经元凋亡在脑发育中起重要作用，并导致各种神经退行性疾病下的神经元丢失。遗传学研究建立了负责细胞凋亡的启动和进展的分子机制。在这个过程中的关键作用归因于caspase家族的成员。Caspase-3似乎是神经元凋亡的主要效应物。虽然是发育中的CNS的共同特征，但细胞凋亡在正常生理条件下对于成熟的哺乳动物脑是非典型的，并且最近的报告提供了证据，表明神经元对损伤诱导的细胞凋亡的易感性在脑成熟期间也显著下降。我们的数据链接的凋亡潜力的发展下降，半胱天冬酶-3基因的转录抑制。在此背景下，我们的目标是确定该基因在成熟大脑中沉默的基本机制。我们鉴定了大鼠caspase-3基因启动子，克隆并测序，检测了其调控元件的功能，并鉴定了相应的人caspase-3基因启动子。表达谱的基本转录因子，调节caspase-3的转录，并没有发现显着的变化，在大脑成熟过程中，他们的表达，这表明其他机制参与发育调节caspase-3的表达。大鼠和人caspase-3基因的启动子区在相应的主要转录起始位点周围具有共同的密集CpG岛。这些区域包括几个共识的Sp1的结合位点的caspase-3转录所必需的。具有这种启动子结构的基因的转录起始的抑制通常与通过GpG岛的超甲基化的表观遗传调节相关，并且最近的几项研究已经表明DNA甲基化在基因表达的发育调节中的作用。本研究的目的是评估caspase-3启动子的DNA甲基化在调节caspase依赖性细胞死亡的神经易感性中的作用。我们的工作假设是，该基因的重要调控区域的甲基化导致其在神经细胞中的表达下调，随后导致脑发育过程中细胞凋亡潜能的抑制。我们提出两个具体目标：（1）评估大鼠脑和原代神经元培养物中caspase-3基因启动子的DNA甲基化程度和相关的组蛋白修饰作为发育年龄的函数;和（2）检查DNA甲基化对原代神经元培养物中caspase-3基因表达和凋亡潜力的影响。这项研究可能会导致一种新的内源性神经保护机制，可能有重要的影响，改善现有的治疗策略，以治疗某些神经退行性疾病，如围产期缺血和脑外伤，其中的贡献半胱天冬酶依赖性细胞凋亡的神经元损失是非常显着的。

DESCRIPTION (provided by applicant): Neuronal apoptosis plays an essential role in brain development and contributes to neuronal loss under various neurodegenerative conditions. Genetic studies established molecular mechanisms responsible for initiation and progression of apoptosis. A critical role in this process was attributed to members of the caspase family. Caspase-3 appears to be a major effector in neuronal apoptosis. While being a common feature of developing CNS, apoptosis is atypical for mature mammalian brain under normal physiological conditions, and recent reports provide evidence that neuronal susceptibility to injury-induced apoptosis also markedly declines during brain maturation. Our data link the developmental decrease in apoptotic potential to transcriptional repression of the caspase-3 gene. With this background our objective was to identify essential basic mechanisms that underlie the silencing of this gene in mature brain. We have identified, cloned and sequenced the rat caspase-3 gene promoter, examined function of its regulatory elements, and identified the corresponding promoter of the human caspase-3 gene. Expression profiling of the essential transcription factors, which regulate caspase-3 transcription, did not reveal significant changes in their expression during brain maturation, suggesting that other mechanisms are involved in developmental regulation of caspase-3 expression. Promoter regions of rat and human caspase-3 genes have common dense CpG islands surrounding corresponding major transcription start sites. These regions include several consensus Sp1 binding sites essential for caspase-3 transcription. Inhibition of transcription initiation of genes with such promoter structures is generally associated with epigenetic regulation by hypermethylation of GpG islands, and a role for DNA methylation in developmental regulation of gene expression has been suggested by several recent studies. The goal of this proposal is to evaluate a role for DNA methylation of the caspase-3 promoter in regulation of neural susceptibility to caspase-dependent cell death. Our working hypothesis is that methylation of essential regulatory regions of this gene results in downregulation of its expression in neural cells, which subsequently leads to the repression of apoptotic potential during brain development. We propose two Specific Aims: (1) To evaluate an extent of DNA methylation of the caspase-3 gene promoter and associated histone modifications in rat brain and primary neuronal cultures as functions of developmental age; and (2) To examine the effect of DNA methylation on the caspase-3 gene expression and apoptotic potential in primary neuronal cultures. This study may result in identification of a novel endogenous neuroprotective mechanism that may have an important impact on improvement of existing therapeutic strategies to treat certain neurodegenerative conditions, such as perinatal ischemia and brain trauma, where the contribution of caspase-dependent apoptosis to neuronal loss is highly significant.