The role of DNA methylation dynamics and patterning in postmitotic neuronal-maturation

DNA 甲基化动力学和模式在有丝分裂后神经元成熟中的作用

• 批准号: 9285686
• 负责人: M MARGARITA BEHRENS
• 金额: $ 72.05万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-16 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9285686
• 关键词: Adolescent; Affect; Age; Animals; Behavior; Behavioral; Binding; Birth; Brain; Cell Nucleus; Characteristics; Chromatin; Complement; Complex; CpG dinucleotide; Cultured Cells; Cytosine; DNA Methylation; DNA Modification Methylases; DNA Transposable Elements; DNMT3a; Data; Defect; Deposition; Development; Disease; Embryo; Enzymes; Female; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genomic DNA; Genomics; Heterochromatin; Hippocampus (Brain); In Vitro; Knockout Mice; Longevity; Mass Spectrum Analysis; Mediating; Methylation; Mitotic; Modification; Molecular; Morphology; Mus; Neurons; Nuclear Extract; Pattern; Perinatal; Phenotype; Physiological; Process; Proteins; Regulation; Role; Synapses; Synaptic Transmission; System; Testing; Time; Transcriptional Regulation; Viral; Viral Vector; Wild Type Mouse; base; brain cell; cell type; chromatin modification; critical period; epigenomics; excitatory neuron; experimental study; gene repression; hippocampal pyramidal neuron; histone modification; in vivo; inhibitory neuron; knock-down; male; methylation pattern; methylome; nestin protein; neurochemistry; neuron development; postnatal; promoter; public health relevance; small hairpin RNA; study characteristics; synaptogenesis; transcriptome; transcriptome sequencing

Abstract Cytosine methylation and histone modification are epigenomic marks with effects on transposable elements (TE), transcription of genes and heterochromatin formation. While occurring mainly in a CG-dinucleotide context, DNA methylation in brain cells contains nearly an equal amount of non-CG methylation (mCH). mCH accumulates in neurons and correlates with transcriptional repression at a period coinciding with synaptogenesis and neuronal maturation. Embryonic CG-methylation patterns also change dramatically during the period between birth and the second postnatal week. The DNA methyltransferase Dnmt3a is highly expressed in brain during this period. Preliminary data in this application suggests that this enzyme is responsible for the accumulation of mC in neurons during the perinatal period. A conditional knockout mouse was created, in which deletion of Dnmt3a in pyramidal neurons occurs during the late embryonic period (~E15, driven by Neurod6-Cre). Contrary to results showing a shortened lifespan in animals with earlier embryonic deletion (driven by Nestin-Cre), or lack of phenotype when the deletion occurs past the second postnatal week (driven by CamK2a-Cre), NeuroD6-driven Dnmt3a-KO (pyrDnmt3a-KO) animals show no postnatal mC accumulation, have significantly altered gene expression, and develop pronounced changes in behavior without changes in lifespan. These results support the hypothesis that mC accumulation and patterning in neurons requires precise regulation of Dnmt3a activity during neuronal development. Based on these findings, it is proposed that mC accumulation during the perinatal period may be essential for the spatial and temporal gene regulation required for proper synapse development and circuit formation. This hypothesis will be tested by delineating the dynamics of Dnmt3a-dependent mC accumulation during brain development, by characterizing the disruptions in methylation patterns, transcriptional dysregulation and histone modifications in animals carrying a deletion of Dnmt3a in pyramidal and inhibitory neurons from cortex and hippocampus (Aim 1). To understand the mechanisms of activation of Dnmt3a during postnatal cortical development, this proposal will identify its binding-partners during the developmental transition between the first and second postnatal week in neurons using mass spectrometry of Dnmt3a immunocomplexes. It will also assess the requirement of these binding partners for Dnmt3a function by transcriptional knockdown experiments in cultured cells and animals using a viral deliver system (Aim2). Finally, this proposal will characterize the effects of Dnmt3a deletion on neuron development and synaptogenesis (Aim3).

摘要 胞嘧啶甲基化和组蛋白修饰是影响转座因子的表观基因组标记 (TE)基因转录和异染色质形成。虽然主要存在于CG-二核苷酸中， 脑细胞中的DNA甲基化包含几乎等量的非CG甲基化（mCH）。MCH 在神经元中积累，并与转录抑制相关， 突触发生和神经元成熟。胚胎CG-甲基化模式也发生了显着变化， 从出生到产后第二周的这段时间。DNA甲基转移酶Dnmt3a是高度 在这段时间里，大脑中的本申请的初步数据表明，这种酶是 负责围产期神经元中mC的积累。条件性基因敲除小鼠 其中锥体神经元中Dnmt 3a的缺失发生在胚胎晚期（~E15， 由Neurod6-Cre驱动）。与早期胚胎发育的动物寿命缩短的结果相反， 缺失（由Nestin-Cre驱动），或在出生后第二周发生缺失时缺乏表型 （由CamK2a-Cre驱动），NeuroD6驱动的Dnmt3a-KO（pyrDnmt3a-KO）动物显示无出生后mC 积累，显著改变基因表达，并在行为上发生明显变化 而不会改变寿命。这些结果支持以下假设： 在神经元发育过程中，神经元需要精确调节Dnmt3a活性。根据这些发现， 建议围产期mC积累可能是必要的空间和时间 突触发育和电路形成所需的基因调控。这一假设将得到检验 通过描绘脑发育过程中Dnmt3a依赖性mC积累的动力学， 表征甲基化模式的破坏、转录失调和组蛋白修饰 在来自皮层和海马的锥体和抑制性神经元中携带Dnmt3a缺失的动物（Aim 1）。为了了解Dnmt3a在出生后皮层发育过程中的激活机制，本研究建议： 将在第一和第二次产后的发育过渡期间确定其结合伙伴 使用Dnmt3a免疫复合物的质谱法在神经元中的一周。它还将评估以下方面的需要： 这些Dnmt 3a的结合配偶体通过在培养细胞中的转录敲低实验发挥功能， 使用病毒递送系统（Aim2）的动物。最后，该提案将描述Dnmt 3a的影响， 缺失对神经元发育和突触发生的影响（Aim3）。