Sex divergence and cell specificity of age-related hippocampal DNA modifications

摘要 中枢神经系统中的表观遗传过程可能在易感性和 认知能力下降和与年龄相关的神经退行性疾病的进展，如阿尔茨海默氏症。DNA 修饰，主要是胞嘧啶碱基甲基化和羟甲基化（分别为mC和hmC）， DNA可及性和基因调控/表达的基本调节因子，对基因具有不同的影响 表达取决于修饰（mC/hmC）、背景CG/非CG（也称为CH）和基因组 位置.阻碍理解表观遗传机制在脑老化和DNA中的作用的障碍 特别是修改，一直缺乏定量准确的，在特定细胞的全基因组数据， 类型如果不知道随着衰老而改变的修饰的特定基因组位置， 不可能设计出合理的、机械的研究来揭示表观遗传的功能效应， 重新配置。因此，该领域的关键下一步是生成mC的全基因组数据， hmC在CG和CH背景下的特定细胞类型，从两种性别的整个生命周期。处理这个关键 我们已经开发了细胞类型特异性、他莫昔芬诱导型Cre转基因NuTRAP模型， 从小胶质细胞、星形胶质细胞和神经元中分离核酸（DNA和RNA）。在目标1中，细胞类型特异性 通过全基因组氧化亚硫酸氢盐测序检查海马mC/hmC随衰老的变化 （WGoxBS）在小胶质细胞、星形胶质细胞和神经元中的表达。配对的表观基因组学和转录组学数据来自相同的 动物将用于：1）以细胞类型特异性方式用“表观遗传时钟”评估衰老，2）确定 改变的修饰模式在基因表达中与年龄相关的变化中的作用，3）确定 基因组调控区的差异修饰，以及4）鉴定表观基因组的基因座 编辑.在先前的研究中，我们已经确定，与年龄相关的DNA修饰变化可以通过以下方式预防： 热量限制。在目标2中，我们将测试异时共生是否可以逆转与年龄相关的变化 一旦存在于相同的Cre诱导的NuTRAP模型中。利用一个独特的数据库， 注释的人类甲基化数据，我们将验证我们在人类中的发现的方面。在目标3中， 研究将mC/hmC的变化导向特定基因组区域的靶向机制。这些研究 将允许确定具有改变的DNA修饰模式的关键基因组区域， 在未来的干预性研究中进行操作。研究的最终目的是发展临床 针对表观基因组的干预措施，以维持大脑功能与衰老和预防年龄相关的 神经变性

Abstract Epigenetic processes in the central nervous system may play a mechanistic role in susceptibility to and progression of cognitive decline and age-related neurodegenerative diseases, such as Alzheimer's. DNA modifications, principally cytosine base methylation and hydroxymethylation (mC and hmC respectively), are fundamental regulators of DNA accessibility and gene regulation/expression with differential effects on gene expression depending on the modification (mC/hmC), context CG/non-CG (also known as CH), and genomic location. A barrier to progress in understanding the role of epigenetic mechanisms in brain aging and DNA modifications in particular, has been the lack of quantitatively accurate, genome-wide data in specific cell types. Without the knowledge of the specific genomic locations of altered modifications with aging it is impossible to design well-rationalized, mechanistic studies that unravel the functional effects of epigenetic reconfiguration. Therefore, the critical next step for the field is to generate this genome-wide data of mC and hmC in CG and CH contexts in specific cell types from both sexes across the lifespan. To address this critical issue we have developed cell-type specific, tamoxifen-inducible Cre, transgenic NuTRAP models to allow isolation of nucleic acids (DNA & RNA) from microglia, astrocytes, and neurons. In Aim 1, cell type-specific hippocampal changes in mC/hmC with aging will be examined by whole genome oxidative bisulfite sequencing (WGoxBS) in microglia, astrocytes, and neurons. Paired epigenomic and transcriptomic data from the same animals will be used to: 1) assess aging with `epigenetic clocks' in a cell-type specific fashion, 2) determine the role of altered modification patterns in age-related changes in gene expression, 3) determine enrichment of differential modifications in regulatory regions of the genome, and 4) identify genomic loci for epigenome editing. In prior studies we have determined that age-related DNA modification changes can be prevented by caloric restriction. In Aim 2 we will test whether heterochronic parabiosis can reverse age-related changes once extant in the same Cre-inducible, NuTRAP models. Using a unique database of all publicly available, annotated human methylation data we will validate aspects of our findings in humans. In Aim 3 we will examine the targeting mechanisms that direct changes in mC/hmC to specific genomic regions. These studies will allow the determination of critical genomic regions with altered DNA modification patterns that can be manipulated in future interventional studies. The ultimate goal of the research is to develop clinical interventions that target the epigenome to maintain brain function with aging and prevent age-related neurodegeneration.