Mapping DNA methylation in hippocampal neurons through bisulfite sequencing

描述(由申请人提供)：通过DNA甲基化和组蛋白修饰进行的表观遗传基因调控已被证明是神经系统发育和功能的关键机制，从细胞分化到神经元可塑性，从学习和记忆到行为。表观基因组的放松调控可能会导致各种神经精神障碍。为了探讨DNA甲基化在大脑皮质和海马区发育和功能中的作用，我们检测了DNA甲基化转移酶(DNMT，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DNMT如DNMT1和DNMT3A在有丝分裂后神经元中仍有高表达。我们假设DNMTs在有丝分裂后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，在目标1中，我们计划用Cre/loxP系统产生条件突变，在该系统中，DNMT1和DNMT3A在有丝分裂后的皮质和海马神经元中都缺失。这个独特的小鼠模型系统使我们能够在存在或不存在DNMT1和DNMT3A的情况下检查癫痫发作条件下甲基化的变化。在目标2中，我们将通过鸟枪式硫酸氢测序(BS-Seq)定位控制和癫痫状态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甲基化模式的潜在变化。这种BS-Seq方法已经成功地应用于在植物和人类细胞中以单核苷酸分辨率破译甲基组。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存在和不存在时中枢神经系统神经元的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的作用。我们的发现将为未来了解异常甲基化在神经疾病中的作用奠定坚实的基础。 公共卫生相关性：这项拨款建议了解脑细胞中基因表达的调节机制-具体地说，研究神经细胞DNA中DNA修饰(即DNA甲基化)的模式。这种DNA修饰与抑制基因表达有关，已知如果DNA甲基化模式异常，可能会导致包括癌症和智力低下在内的人类疾病。我们将使用高通量测序技术来识别发育中的脑细胞中的DNA甲基化模式，并研究DNA甲基化模式扰动对大脑发育和功能的影响，从而为了解DNA甲基化扰动引起的智力低下障碍的病理机制铺平道路，从而影响公众健康。

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.