Kochmanski Joseph, Savonen Candace, Bernstein Alison I
Department of Translational Neuroscience, College of Human Medicine, Michigan State University, Grand Rapids, MI, United States.
Front Genet. 2019 Sep 10;10:801. doi: 10.3389/fgene.2019.00801. eCollection 2019.
Epigenetic marks operate at multiple chromosomal levels to regulate gene expression, from direct covalent modification of DNA to three-dimensional chromosomal structure. Research has shown that 5-methylcytosine (5-mC) and its oxidized form, 5-hydroxymethylcytosine (5-hmC), are stable epigenetic marks with distinct genomic distributions and separate regulatory functions. In addition, recent data indicate that 5-hmC plays a critical regulatory role in the mammalian brain, emphasizing the importance of considering this alternative DNA modification in the context of neuroepigenetics. Traditional bisulfite (BS) treatment-based methods to measure the methylome are not able to distinguish between 5-mC and 5-hmC, meaning much of the existing literature does not differentiate these two DNA modifications. Recently developed methods, including Tet-assisted bisulfite treatment and oxidative bisulfite treatment, allow for differentiation of 5-hmC and/or 5-mC levels at base-pair resolution when combined with next-generation sequencing or methylation arrays. Despite these technological advances, there remains a lack of clarity regarding the appropriate statistical methods for integration of 5-mC and 5-hmC data. As a result, it can be difficult to determine the effects of an experimental treatment on 5-mC and 5-hmC dynamics. Here, we propose a statistical approach involving mixed effects to simultaneously model paired 5-mC and 5-hmC data as repeated measures. We tested this approach using publicly available BS/oxidative bisulfite-450K array data and showed that our new approach detected far more CpG probes with paired changes in 5-mC and 5-hmC by Alzheimer's disease status (n = 14,183 probes) compared with the overlapping differential probes generated from separate models for each epigenetic mark (n = 68). Of note, all 68 of the overlapping probe IDs from the separate models were also significant in our new modeling approach, supporting the sensitivity of our new analysis method. Using the proposed approach, it will be possible to determine the effects of an experimental treatment on both 5-mC and 5-hmC at the base-pair level.
表观遗传标记在多个染色体水平发挥作用以调控基因表达，从DNA的直接共价修饰到三维染色体结构。研究表明，5-甲基胞嘧啶（5-mC）及其氧化形式5-羟甲基胞嘧啶（5-hmC）是具有独特基因组分布和不同调控功能的稳定表观遗传标记。此外，最近的数据表明5-hmC在哺乳动物大脑中起关键调控作用，这凸显了在神经表观遗传学背景下考虑这种替代性DNA修饰的重要性。基于传统亚硫酸氢盐（BS）处理的甲基化组测量方法无法区分5-mC和5-hmC，这意味着现有许多文献并未区分这两种DNA修饰。最近开发的方法，包括Tet辅助亚硫酸氢盐处理和氧化亚硫酸氢盐处理，与下一代测序或甲基化阵列结合时，能够在碱基对分辨率下区分5-hmC和/或5-mC水平。尽管有这些技术进步，但对于整合5-mC和5-hmC数据的适当统计方法仍缺乏清晰认识。因此，很难确定实验处理对5-mC和5-hmC动态的影响。在此，我们提出一种涉及混合效应的统计方法，将配对的5-mC和5-hmC数据作为重复测量进行同时建模。我们使用公开可用的BS/氧化亚硫酸氢盐-450K阵列数据测试了这种方法，结果表明与针对每个表观遗传标记的单独模型生成的重叠差异探针（n = 68）相比，我们的新方法检测到更多因阿尔茨海默病状态而在5-mC和5-hmC中出现配对变化的CpG探针（n = 14,183个探针）。值得注意的是，来自单独模型的所有68个重叠探针ID在我们的新建模方法中也具有显著性，这支持了我们新分析方法的敏感性。使用所提出的方法，将有可能在碱基对水平确定实验处理对5-mC和5-hmC两者的影响。
