Neuronal Epigenomic Changes in Neurodevelopment and Disease

描述（由申请人提供）：已知早期生活经验和环境条件，特别是发生在大脑可塑性增强期的那些条件，可以促进独立于DNA密码变化的生理和行为的长期变化。越来越多的证据表明，表观基因组过程在这些表观遗传现象中起着重要作用。特别是，来自各种来源的最新数据表明，DNA甲基化的经验依赖性变化可以通过对神经元基因表达的持续影响对神经功能产生长期影响。然而，这些修饰发生在体内高度异质神经组织中相对较少的细胞中，这一事实限制了现有基因组方法对这些修饰的研究，并使潜在分子机制的研究变得非常复杂。我们建议采取双管齐下的办法开始解决这些问题。首先，我们将采用还原论的方法来研究经验驱动的DNA甲基化变化，采用分离的神经元培养系统，该系统显示活动诱导的DNA甲基化变化，并且在该系统中，大量细胞可以通过强大的刺激同步激活。此外，为了补充和解决这种还原论方法固有的局限性，我们还开发了一种通用的遗传策略，可以从体内定义的细胞类型中特异性地分离染色质，从而可以使用大规模平行测序技术分析特定神经细胞群体中因早期生活经历而引起的DNA甲基化变化。因此，我们建议：1)利用分离的神经元培养系统来表征神经元活动诱导的DNA甲基化变化；2)在体内研究经验驱动的行为和生理变化与神经元表观基因组的长期相关性。我们希望所提出的实验将为神经元表观基因组修饰的分析建立新的方法，促进我们对发育中的中枢神经系统DNA甲基化调控的理解，并最终为这些机制对神经发育、认知行为和疾病的重要性提供新的见解。

DESCRIPTION (provided by applicant): Early-life experiential and environmental conditions, particularly those occurring during heightened periods of brain plasticity, are known to promote long-term changes in physiology and behavior that act independently of changes in the DNA code. Accumulating evidence suggests an important role for epigenomic processes in these epigenetic phenomena. In particular, recent data from a variety of sources suggest that experience-dependent changes in DNA methylation can have a long-lasting impact on neural function through sustained effects on neuronal gene expression. However, the fact that these modifications occur in vivo in a relatively small number of cells within highly heterogeneous neural tissue limits the study of these modifications with existing genomic approaches and greatly complicates investigation of the underlying molecular mechanisms. We propose a two-pronged approach to begin to address these issues. First, we will pursue a reductionist approach to the study of experience-driven changes in DNA methylation, employing a dissociated neuronal culture system that shows activity-induced changes in DNA methylation and in which a large number of cells can be synchronously activated with robust stimuli. Moreover, to complement and address limitations inherent in this reductionist approach, we have also developed a general genetic strategy to specifically isolate chromatin from defined cell types in vivo, enabling the analysis of DNA methylation changes induced in specific neuronal cell populations in response to early-life experiences using massively parallel sequencing techniques. Thus, we propose: 1) To employ a dissociated neuronal culture system to characterize neuronal activity-induced changes in DNA methylation, and 2) To investigate long-lasting neuronal epigenomic correlates to experience-driven behavioral and physiological changes in vivo. It is our hope that the proposed experiments will establish new approaches for the analysis of neuronal epigenomic modifications, advance our understanding of the regulation of DNA methylation in the developing central nervous system, and ultimately provide new insights into the importance of these mechanisms for neurodevelopment, cognitive behavior, and disease.