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

摘要 胞嘧啶甲基化和组蛋白修饰是对转座元件有影响的表观基因组标记 (TE)、基因转录和异染色质形成。而主要出现在CG-二核苷酸中 背景，脑细胞中的DNA甲基化包含几乎等量的非CG甲基化(MCH)。妇幼保健 在神经元中积累，并与转录抑制相关，这一时期与 突触发生和神经元成熟。胚胎CG甲基化模式也在 从出生到出生后第二周这段时间。DNA甲基转移酶DNMT3A高度 在这一时期在大脑中表达。这项申请的初步数据表明，这种酶是 在围产期负责MC在神经元中的积累。有条件的基因敲除小鼠 DNMT3A在胚胎晚期(~E15， 由Neurod6-Cre驱动)。与早期胚胎动物寿命缩短的结果相反 缺失(由巢蛋白-Cre驱动)，或当缺失发生在出生后第二周后缺乏表型 (由CamK2a-Cre驱动)，由NeuroD6驱动的DNMT3A-KO(pyrDnmt3a-KO)动物没有出生后MC 积累，显著改变了基因表达，并在行为上发生了明显的变化 而不会改变寿命。这些结果支持这样的假设，即MC的积累和图案化 神经元在发育过程中需要对DNMT3A活性进行精确的调节。基于这些发现， 提示围产期MC的积累可能在时间和空间上是必需的。 基因调控是突触正常发育和回路形成所必需的。这一假设将得到检验。 通过描绘大脑发育过程中依赖DNMT3A的MC积累的动力学，通过 甲基化模式的破坏、转录失调和组蛋白修饰的特征 在皮质和海马区锥体和抑制性神经元中携带DNMT3A缺失的动物(目的 1)。为了了解DNMT3A在出生后皮质发育过程中的激活机制，本建议 将在第一次和第二次出生之间的发育过渡期间确定其结合伙伴 DNMT3A免疫复合体的质谱学研究。它还将评估以下方面的要求 这些DNMT3A的结合伙伴通过在培养细胞和 使用病毒传递系统的动物(AIM2)。最后，本提案将描述DNMT3A的影响 神经元发育和突触发生的缺失(Aim3)。

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).