Mapping DNA methylation in hippocampal neurons through bisulfite sequencing

描述（申请人提供）：通过DNA甲基化和组蛋白修饰的表观遗传基因调控已被证明是神经系统发育和功能的重要机制，从细胞分化到神经元可塑性，从学习记忆到行为。表观基因组的失调可能导致各种神经精神疾病。为了研究DNA甲基化在皮层和海马发育和功能中的作用，我们研究了DNA甲基化转移酶（Dnmts，包括Dnmt1、Dnmt3a和Dnmt3b）在发育中和成人中枢神经系统（CNS）中的表达。有趣的是，我们发现Dnmt1和Dnmt3a等Dnmts在有丝分裂后神经元中仍然高度表达。我们假设Dnmts在有丝分裂后的中枢神经系统神经元中的表达是维持和调节DNA甲基化模式，从而调节神经元基因表达的长期变化。为了验证这一假设，在Aim 1中，我们计划用Cre/LoxP系统生成条件突变体，其中有丝分裂后皮层和海马神经元中Dnmt1和Dnmt3a都缺失。这种独特的小鼠模型系统使我们能够在Dnmt1和Dnmt3a存在或不存在的情况下检查癫痫发作时甲基化的变化。在Aim 2中，我们将通过霰弹枪硫酸氢测序（BS-Seq）绘制控制和癫痫发作条件下海马齿状回神经元的全基因组甲基化模式，并确定dnmt缺陷海马神经元DNA甲基化模式的潜在改变。这种BS-Seq方法已经成功地应用于植物和人类细胞中单核苷酸分辨率的甲基组破译。随着下一代和第三代测序仪的出现，我们将以非常合理的成本获得哺乳动物海马神经元中的甲基组。通过绘制正常和癫痫状态下Dnmts存在和不存在的CNS神经元DNA甲基化图谱，我们将深入了解Dnmts和DNA甲基化在有丝分裂后神经元中的作用。我们的发现将为进一步了解异常甲基化在神经系统疾病中的作用奠定坚实的基础。

DESCRIPTION (provided by applicant): Epigenetic gene regulation through DNA methylation and histone modifications has been shown to be a crucial mechanism for the development and function of the nervous system, ranging from cell differentiation to neuronal plasticity and from learning & memory to behavior. The deregulation of the epigenome could lead to various neuropsychiatric disorders. To address the role of DNA methylation in the development and function of cortex and hippocampus, we have examined expression of DNA methyltransferases (Dnmts including Dnmt1, Dnmt3a, and Dnmt3b) in the developing and adult central nervous system (CNS). Intriguingly, we found that Dnmts such as Dnmt1 and Dnmt3a are still highly expressed in postmitotic neurons. We hypothesize that expression of Dnmts in postmitotic CNS neurons is to maintain and modulate DNA methylation patterns, which can subsequently regulate long-term changes of neuronal gene expression. To test this hypothesis, in Aim 1, we plan to generate conditional mutants with the Cre/LoxP system in which both Dnmt1 and Dnmt3a are absent in postmitotic cortical and hippocampal neurons. This unique mouse model system allows us to examine methylation changes under seizure condition in the presence or absence of both Dnmt1 and Dnmt3a. In Aim 2, we will map genome-wide methylation patterns in hippocampal dentate gyrus neurons in control and seizure conditions through shotgun bisulfate sequencing (BS-Seq) and determine the potential alteration of DNA methylation patterns in Dnmt-deficient hippocampal neurons. This BS-Seq approach has been successfully applied to decipher methylomes at single nucleotide resolution in plants and human cells. With the advent of next-generation and third generation sequencers, we would obtain methylomes in mammalian hippocampal neurons at a very reasonable cost. By mapping DNA methylation in CNS neurons under normal and seizure conditions in the presence and absence of Dnmts, we will gain insight into the role of Dnmts and DNA methylation in postmitotic neurons. Our findings will lay a solid foundation for future study to understand the involvement of abnormal methylation in neurological disorders. PUBLIC HEALTH RELEVANCE: This grant proposes to understand regulatory mechanisms of gene expression in brain cells - specifically, to examine the pattern of DNA modification (namely DNA methylation) in the DNA of nerve cells. This DNA modification is involved in the inhibition of gene expression and it is known that if DNA methylation pattern is abnormal, it can lead to human diseases including cancer and mental retardation disorders. We will use high throughput sequencing technique to identify DNA methylation patterns in the developing brain cells and examine the consequence of the perturbation of DNA methylation patterns on brain development and function, thus impacting public health by paving the way for understanding pathological mechanisms of mental retardation disorders due to the perturbation of DNA methylation.