Ho Shuk-mei, Tang Wan-yee
Department of Environmental Health, College of Medicine, University of Cincinnati, Cincinnati, OH, USA.
Reprod Toxicol. 2007 Apr-May;23(3):267-82. doi: 10.1016/j.reprotox.2007.01.004. Epub 2007 Jan 19.
Epigenetic changes are heritable modifications that do not involve alterations in the primary DNA sequence. They regulate crucial cellular functions such as genome stability, X-chromosome inactivation, and gene imprinting. Epidemiological and experimental observations now suggest that such changes may also explain the fetal basis of adult diseases such as cancer, obesity, diabetes, cardiovascular disorders, neurological diseases, and behavioral modifications. The main molecular events known to initiate and sustain epigenetic modifications are histone modification and DNA methylation. This review specifically focuses on existing and emerging technologies used in studying DNA methylation, which occurs primarily at CpG dinucleotides in the genome. These include standard exploratory tools used for global profiling of DNA methylation and targeted gene investigation: methylation sensitive restriction fingerprinting (MSRF), restriction landmark genomic scanning (RLGS), methylation CpG island amplification-representational difference analysis (MCA-RDA), differential methylation hybridization (DMH), and cDNA microarrays combined with treatment with demethylating agents and inhibitors of histone deacetylase. The basic operating principals, resource requirements, applications, and benefits and limitations of each methodology are discussed. Validation methodologies and functional assays needed to establish the role of a CpG-rich sequence in regulating the expression of a target or candidate gene are outlined. These include in silico database searches, methylation status studies (bisulfite genomic sequencing, COBRA, MS-PCR, MS-SSCP), gene expression studies, and promoter activity analyses. Our intention is to give readers a starting point for choosing methodologies and to suggest a workflow to follow during their investigations. We believe studies of epigenetic changes such as DNA methylation hold great promise in understanding the early origins of adult diseases and in advancing their diagnosis, prevention, and treatment.
表观遗传变化是可遗传的修饰，不涉及初级DNA序列的改变。它们调节关键的细胞功能，如基因组稳定性、X染色体失活和基因印记。流行病学和实验观察表明，这些变化也可能解释成人疾病如癌症、肥胖症、糖尿病、心血管疾病、神经疾病和行为改变的胎儿基础。已知启动和维持表观遗传修饰的主要分子事件是组蛋白修饰和DNA甲基化。本综述特别关注用于研究DNA甲基化的现有技术和新兴技术，DNA甲基化主要发生在基因组中的CpG二核苷酸处。这些技术包括用于DNA甲基化全局分析和靶向基因研究的标准探索工具：甲基化敏感限制性指纹图谱（MSRF）、限制性地标基因组扫描（RLGS）、甲基化CpG岛扩增-代表性差异分析（MCA-RDA）、差异甲基化杂交（DMH）以及与去甲基化剂和组蛋白脱乙酰酶抑制剂联合处理的cDNA微阵列。讨论了每种方法的基本操作原理、资源需求、应用以及优缺点。概述了确定富含CpG序列在调节靶基因或候选基因表达中的作用所需的验证方法和功能测定。这些方法包括计算机数据库搜索、甲基化状态研究（亚硫酸氢盐基因组测序、COBRA、甲基化特异性PCR、甲基化特异性单链构象多态性）、基因表达研究和启动子活性分析。我们的目的是为读者提供选择方法的起点，并建议在研究过程中遵循的工作流程。我们相信，对DNA甲基化等表观遗传变化的研究在理解成人疾病的早期起源以及推进其诊断、预防和治疗方面具有巨大潜力。
