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Mapping DNA methylation in hippocampal neurons through bisulfite sequencing

通过亚硫酸氢盐测序绘制海马神经元 DNA 甲基化图谱

基本信息

批准号：
8212245
负责人：
Guoping Fan
金额：
$ 19.25万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-15 至 2012-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8212245
关键词：
Address Adult Age Applications Grants Arabidopsis BDNF gene Behavior Binding Proteins Biological Models Brain Brain region Cell Differentiation process Cells Centromere Cytoplasmic Granules DNA DNA Methylation DNA Methyltransferase DNA Modification Methylases DNA Modification Process Development Discipline of Nursing Disease Epigenetic Process Face Family Fibroblasts Foundations Future Gene Expression Gene Expression Regulation Generations Genes Grant Hereditary Disease Hippocampal Formation Hippocampus (Brain)Human Human Genetics Immunologic Deficiency Syndromes Knock-out Lead Learning Limbic System Malignant Neoplasms Mammals Maps Memory Mental Retardation Methods Methyl-CpG-Binding Protein 2 Methylation Molecular Mus Mutant Strains Mice Mutation Nervous System Physiology Neuraxis Neuronal Differentiation Neuronal Plasticity Neurons Nucleotides Pattern Plants Play Public Health Regulation Research Project Grants Resolution Role Seizures Shotguns Solid Structure Syndrome System Techniques Testing Transgenic Mice Up-Regulation adult neurogenesis bisulfite brain cell cost dentate gyrus embryonic stem cell genome-wide histone modification human disease insight kainate long term memory member motor neuron function mouse model mutant nervous system disorder neural precursor cell neuron development neurophysiology neuropsychiatry next generation precursor cell promoter public health relevance

项目摘要

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.

描述（由申请人提供）：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关键机制，范围从细胞分化到神经元可塑性以及从学习和记忆到行为。表观基因组的失调可能导致各种神经精神疾病。为了阐明DNA甲基化在皮质和海马发育和功能中的作用，我们检测了DNA甲基转移酶（Dnmts，包括Dnmt 1、Dnmt 3a和Dnmt 3b）在发育和成年中枢神经系统（CNS）中的表达。有趣的是，我们发现Dnmt如Dnmt 1和Dnmt 3a在有丝分裂后的神经元中仍然高度表达。我们推测Dnmts在有丝分裂后CNS神经元中的表达是为了维持和调节DNA甲基化模式，其随后可以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，我们计划产生条件突变体与Cre/LoxP系统，其中Dnmt 1和Dnmt 3a都不存在于有丝分裂后皮层和海马神经元。这种独特的小鼠模型系统使我们能够在存在或不存在Dnmt 1和Dnmt 3a的情况下检查癫痫发作条件下的甲基化变化。在目标2中，我们将通过鸟枪硫酸氢盐测序（BS-Seq）绘制对照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缺陷海马神经元中DNA甲基化模式的潜在改变。这种BS-Seq方法已成功应用于在植物和人类细胞中以单核苷酸分辨率破译甲基化组。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化组。通过绘制DNA甲基化在正常和癫痫发作条件下的Dnmts的存在和不存在的CNS神经元中，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。我们的研究结果将为未来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。公共卫生相关性：这项资助旨在了解脑细胞中基因表达的调控机制-特别是检查神经细胞DNA中DNA修饰（即DNA甲基化）的模式。这种DNA修饰涉及基因表达的抑制，并且已知如果DNA甲基化模式异常，则可导致人类疾病，包括癌症和智力迟钝障碍。我们将使用高通量测序技术来鉴定发育中的脑细胞中的DNA甲基化模式，并研究DNA甲基化模式的扰动对脑发育和功能的影响，从而通过理解由于DNA甲基化扰动导致的精神发育迟滞障碍的病理机制来影响公共卫生。