Critical assessment of DNA adenine methylation in brain cells from healthy aging and Alzheimer's disease

项目摘要/摘要 DNA甲基化参与了许多重要生物过程的表观遗传调控。盛行的 在真核生物中，DNA甲基化几乎只发生在胞嘧啶(5mC)的第五位。 随着多项研究报告N6病毒的存在，这一教条在过去五年中得到了修订。 甲基腺嘌呤(6 MA)跨真核生物，包括小鼠和人类基因组。一些研究发现 证据表明，6 mA在哺乳动物中发挥着不同的功能。一些研究也报告了 在某些神经细胞和社会应激下，6 mA水平升高，表明6 mA可能起着重要作用 在健康和疾病方面。然而，一些研究对哺乳动物体内6 mA的存在提出了质疑。 基因组，突出了混杂因素的多个来源。这场激烈的辩论创造了不同寻常的 表观遗传群体中的混乱。然而，数十项研究继续报告了约6 mA的新发现 人类基因组，包括在知名期刊上发表的一些论文。一些正在进行的研究已经设置了 旨在研究6 mA在脑细胞和脑部疾病中的功能作用，特别是在阿尔茨海默病中。 为了毫不含糊地评估人类基因组中6 mA的丰度和流行程度，必须 采用可靠、灵敏的6 mA映射方法。然而，过去和正在进行的研究大多采用 以抗体为基础的方法，与非特异性相关。虽然单分子实时 测序(smrt-seq)已被广泛用于在细菌中以碱基分辨率定位6 mA，这是我们最近的工作。 其他人发现，现有的方法对低6 mA的真核基因组不够灵敏 富足。为了解决这一根本技术差距，我们将以我们在SMRT；10年的经验为基础- SEQ将开发一种灵敏的6 mA检测新方法，并以中性的视角进行批判性检查 6 mA在人类基因组和阿尔茨海默病(AD)患者的脑组织中，初级年龄- 相关的紧张症(部分，另一种类型的痴呆症)和对照组。这个项目意义重大，非常及时。 因为(1)如果新方法支持人类基因组中6 mA的存在，它将提供持续的 以及未来用一种急需的工具来检测某些细胞类型和阿尔茨海默病的6 mA的研究；(2)如果 新的方法并不像报道的那样支持6 mA的显著水平，它将作为急需的直接 澄清当前辩论的证据。

PROJECT SUMMARY/ABSTRACT DNA methylation contributes to epigenetic regulation of many important biological processes. The prevailing dogma is that DNA methylation almost exclusively occurs at the fifth position of cytosine (5mC) in eukaryotes. This dogma has been revised in the past five years as multiple studies reported the existence of N6- methyladenine (6mA) across eukaryotes including the mouse and human genome. A few studies have found evidence that suggests the diverse functions 6mA plays in mammals. Some studies have also reported increased 6mA level in certain neuronal cells and upon social stress, suggesting 6mA may play important roles in health and diseases. However, several studies have challenged the presence of 6mA in mammalian genomes, highlighting multiple sources of confounding factors. The active debate has created unusual confusions in the epigenetic community. Yet, tens of studies continued to report new discoveries about 6mA in the human genome including some papers at high-profile journals. A few ongoing research studies have set out to examine the functional roles of 6mA in brain cells and brain disorders, especially in Alzheimer’s disease. To unambiguously assess the abundance and prevalence of 6mA in the human genome, it is imperative to employ a reliable and sensitive 6mA mapping method. However, past and ongoing studies mostly employ antibody-based methods, which are associated with non-specificity. Although Single Molecule Real-Time sequencing (SMRT-seq) has been widely used to map 6mA at base resolution in bacteria, recent work by us and others have found that existing methods are not sensitive enough for eukaryotic genomes with low 6mA abundance. To address this fundamental technological gap, we will build on our >10-yr experience in SMRT- seq to develop a new method for sensitive 6mA detection, and take a neutral perspective to critically examine 6mA in the human genome and in brain tissues from patients with Alzheimer’s disease (AD), Primary age- related tauopathy (PART, another type of dementia) and controls. This project is significant and very timely because (1) if the novel method supports the existence of 6mA in the human genome, it will provide ongoing and future studies with a much-needed tool to detect 6mA in certain cell types and Alzheimer’s diseases; (2) if the novel method does not support a significant level of 6mA as reported, it will serve as a much-needed direct evidence to clarify the current debate.