Profiling DNA Methylation and Hydroxymethylation at Retrotransposable Elements
Profiling DNA Methylation and Hydroxymethylation at Retrotransposable Elements
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DOI：
10.1007/978-1-4939-3372-3_24
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发表时间：
2016-01-01
期刊：
影响因子：
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通讯作者：
Branco, Miguel R.
中科院分区：
文献类型：
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作者：
de la Rica, Lorenzo;Stanley, Jatinder S.;Branco, Miguel R.
DNA甲基化是控制哺乳动物反转录转座因子转录活性的关键表观遗传修饰。它氧化为DNA羟甲基化与DNA去甲基化和反转录转座子的再激活有关。在这里，我们详细描述了三种方法来测量DNA甲基化和羟甲基化在特定的基因组靶点的协议：glucMS-qPCR，和两种测序方法（焦磷酸测序和高通量测序）分析亚硫酸氢盐和氧化亚硫酸氢盐修饰的DNA。所有这三种技术都提供了单碱基分辨率下甲基化和羟甲基化水平的绝对测量。讨论了方法之间的差异，主要是关于吞吐量和目标覆盖范围。这些构成了我们实验室用于准确调查逆转录转座因子表观遗传学的核心技术。
DNA methylation is a key epigenetic modification controlling the transcriptional activity of mammalian retrotransposable elements. Its oxidation to DNA hydroxymethylation has been linked to DNA demethylation and reactivation of retrotransposons. Here we describe in detail protocols for three methods to measure DNA methylation and hydroxymethylation at specific genomic targets: glucMS-qPCR, and two sequencing approaches (pyrosequencing and high-throughput sequencing) for analyzing bisulfite- and oxidative bisulfite-modified DNA. All three techniques provide absolute measurements of methylation and hydroxymethylation levels at single-base resolution. Differences between the methods are discussed, mainly with respect to throughput and target coverage. These constitute the core techniques that are used in our laboratory for accurately surveying the epigenetics of retrotransposable elements.