Defining the Epigenetic Architecture Associated with Early-Life Stress

描述（由申请人提供）：心理健康障碍的遗传基础是高度复杂的，涉及风险基因、环境和经验因素之间的多方面相互作用。众所周知，不良的早期生活事件显著地增加了以后生活中精神疾病的易感性。然而，环境因素与神经系统遗传程序相互作用的表观遗传机制仍然知之甚少。这部分是由于神经细胞类型的复杂异质性和现有技术的局限性。在此，我们提出从概念和技术上创新的方法来研究早期生活压力诱导的表观遗传修饰，如DNA甲基化和染色质组织。我们计划通过直接测序甲基化DNA和直接检测甲基- CpG结合蛋白（MBPs）与甲基化DNA的动态关联来绘制应激诱导的DNA甲基化变化。为了实现这一目标，我们将产生两种转基因小鼠系：一种系以空间和时间可控的方式表达生物素连接酶（BirA）和GFP；而另一株则携带一个内源性MBP，该MBP被标记为生物素化信号序列。在这两种小鼠系的后代中，MBP将在一定数量的gfp阳性神经元中被特异性生物素化。在对这些转基因小鼠进行实验处理后，将通过高通量测序MeDIP-seq和bioMBP-ChIP-seq绘制特定神经元群体的全基因组DNA甲基化位点。此外，将通过系统质谱bioMBP-ChIP-MS/MS对每个MBP相关的染色质复合物进行表征，以研究表观遗传修饰的分子机制。通过基因组学和蛋白质组学相结合的方法，我们希望能够深入了解早期生活压力与易感基因相互作用并赋予精神疾病风险的表观遗传机制。我们提出的研究也将有助于更好地了解精神健康障碍的潜在原因，并为改进诊断和干预提供必要的基础。

DESCRIPTION (provided by applicant): The genetic underpinnings of mental health disorders are highly complex, involving multifaceted interactions between risk genes, the environment, and experiential factors. It is well known that adverse early life events confer significantly greater susceptibility to psychiatric conditions in later life. However, the epigenetic mechanisms by which environmental factors interact with genetic programs in the nervous system remain poorly understood. This is partially due to the complex heterogeneity of neuronal cell types and the limitations of existing techniques. Here we propose to investigate the epigenetic modifications such as DNA methylation and chromatin organization induced by early-life stress with conceptually and technically innovative approaches. We plan to map the stress-induced DNA methylation changes by directly sequencing the methylated DNA and by directly examining the dynamic association of methyl- CpG binding proteins (MBPs) with methylated DNA. To achieve this, we will generate two transgenic mouse lines: one line expresses biotin ligase (BirA) and GFP in a spatially and temporally controlled manner; while the other line carries an endogenous MBP tagged with a biotinylation signal sequence. In the resulting progeny of these two mouse lines, MBP will be specifically biotinylated in a defined population of GFP-positive neurons. Following experimental treatment of these transgenic mice, the genome-wide DNA methylation loci in specific neuronal populations will be mapped by high throughput sequencing MeDIP-seq and bioMBP-ChIP-seq. In addition, the chromatin complexes associated with each MBP will be characterized by systematic mass spectrometry bioMBP-ChIP-MS/MS to investigate the molecular mechanisms underlying epigenetic modifications. With the combined genomic and proteomic approaches, we hope to gain an insight into the epigenetic mechanisms through which early-life stress interacts with susceptibility genes and confers risks to mental illness. Our proposed study will also allow greater understanding of the underlying causes of mental health disorders and provide the necessary foundation for improved diagnosis and interventions. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to develop an innovative strategy to investigate the epigenetic mechanisms by which environmental factors such as early life stress interact with genetics, and how these interactions increase the risk of mental illness. We plan to generate novel genetically modified mouse lines to tag methyl-CpG binding proteins specifically in a defined population of neurons. We will then investigate the epigenetic changes associated with environmental cues in the brain with both genomic and proteomic approaches. The proposed studies will be of significance not only in understanding the epigenetic control of experience-dependent brain development, but also in understanding the molecular and cellular basis of mental disorders. It is tempting to argue that our research may identify potential molecular, cellular, and circuit targets to intervene and/or prevent mental illness.