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易感性中发挥机械作用 认知衰退和老年性神经退行性疾病的进展，如阿尔茨海默氏症。 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的差异影响 表达取决于修饰(MC/HMC)、上下文CG/非CG(也称为CH)和基因组 地点。理解表观遗传机制在脑老化和DNA中的作用的障碍 尤其是，在特定细胞中，缺乏定量准确的全基因组数据 类型。在不知道随年龄变化的改变的特定基因组位置的情况下， 不可能设计合理的、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和Mc的全基因组数据 在CG和CH环境中，特定细胞类型的HMC在整个生命周期中来自两性。要解决这一关键问题 问题我们已经开发了特定细胞类型的、三苯氧胺诱导的Cre转基因NuTRAP模型以允许 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，特定于细胞类型 全基因组氧化性亚硫酸氢盐测序将检测MC/HMC随增龄的海马区变化 (WGoxBS)在小胶质细胞、星形胶质细胞和神经元中。配对的表观基因组数据和转录组数据 动物将被用来：1)以细胞类型特定的方式用‘表观遗传时钟’评估衰老；2)确定 改变的修饰模式在与年龄相关的基因表达变化中的作用，3)决定 基因组调节区的差异修饰，以及4)确定表观基因组的基因组位置 正在编辑。在先前的研究中，我们已经确定与年龄相关的DNA修饰改变可以通过以下方法来预防 卡路里限制。在目标2中，我们将测试异型慢性异型共生是否可以逆转年龄相关的变化。 曾经存在于相同的CRE诱导的NuTRAP模型中。使用所有公共可用数据的唯一数据库， 经过注释的人类甲基化数据，我们将在人类身上验证我们的发现的各个方面。在《目标3》中，我们将 研究将MC/HMC的变化定向到特定基因组区域的靶向机制。这些研究 将允许通过改变的DNA修饰模式确定关键的基因组区域，这些DNA修饰模式可以 在未来的干预研究中被操纵。研究的最终目标是发展临床 针对表观基因组的干预措施，以在衰老过程中维持大脑功能并防止与年龄相关的 神经退行性变。

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.