Epigenetic control of apoptotic susceptibility

描述（申请人提供）：神经元凋亡在大脑发育中起着重要作用，并在各种神经退行性疾病中导致神经元损失。遗传学研究确定了细胞凋亡发生和发展的分子机制。在这一过程中起关键作用的是caspase家族的成员。Caspase-3似乎是神经元凋亡的主要效应因子。虽然凋亡是发育中的中枢神经系统的共同特征，但在正常生理条件下，成熟哺乳动物的大脑并不典型，最近的报道表明，在大脑成熟过程中，神经元对损伤诱导的凋亡的易感性也显著下降。我们的数据将凋亡潜能的发育减少与caspase-3基因的转录抑制联系起来。在此背景下，我们的目标是确定成熟大脑中该基因沉默的基本机制。我们鉴定、克隆和测序了大鼠caspase-3基因启动子，检测了其调控元件的功能，并鉴定了相应的人类caspase-3基因启动子。对调节caspase-3转录的关键转录因子的表达谱分析未发现其在脑成熟过程中的表达发生显著变化，提示caspase-3表达的发育调控可能涉及其他机制。大鼠和人caspase-3基因的启动子区域在相应的主要转录起始位点周围有共同的密集CpG岛。这些区域包括对caspase-3转录至关重要的几个Sp1结合位点。具有此类启动子结构的基因的转录起始抑制通常与GpG岛高甲基化的表观遗传调控有关，最近的一些研究表明DNA甲基化在基因表达的发育调控中起作用。本提案的目的是评估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，该基因基本调控区域的甲基化导致其在神经细胞中的表达下调，从而导致大脑发育过程中凋亡电位的抑制。我们提出了两个具体目标：(1)评估大鼠脑和原代神经元培养中caspase-3基因启动子的DNA甲基化程度和相关组蛋白修饰与发育年龄的关系；(2)检测DNA甲基化对原代神经元caspase-3基因表达和凋亡电位的影响。这项研究可能会发现一种新的内源性神经保护机制，这可能对改善现有治疗某些神经退行性疾病的治疗策略产生重要影响，例如围产期缺血和脑外伤，其中caspase依赖性凋亡对神经元损失的贡献非常显著。

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.