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，特别是在大脑可塑性增强期间发生的那些条件，会促进生理和行为的长期变化，这些变化独立于DNA密码的变化。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发挥着重要作用。特别是，最近来自各种来源的数据表明，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，事实上，这些修改发生在体内的细胞数量相对较少的高度异质性的神经组织内限制了这些修改与现有的基因组方法的研究，并大大复杂化调查的基础分子机制。我们建议采取双管齐下的办法，开始处理这些问题。首先，我们将追求一种还原论的方法来研究DNA甲基化的经验驱动的变化，采用解离的神经元培养系统，显示活动诱导的DNA甲基化的变化，其中大量的细胞可以被同步激活与强大的刺激。此外，为了补充和解决这种简化方法中固有的局限性，我们还开发了一种通用的遗传策略，以特异性地从体内定义的细胞类型中分离染色质，从而能够使用大规模平行测序技术分析特定神经元细胞群中诱导的DNA甲基化变化。因此，我们建议：1）采用分离的神经元培养系统来表征神经元活动诱导的DNA甲基化的变化，以及2）研究持久的神经元表观基因组与体内经验驱动的行为和生理变化的相关性。我们希望，拟议的实验将建立分析神经元表观基因组修饰的新方法，推进我们对发育中的中枢神经系统中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.