Adaptation of the targeted capture Methyl-Seq platform for the mouse genome identifies novel tissue-specific DNA methylation patterns of genes involved in neurodevelopment

Methyl-Seq was recently developed as a targeted approach to assess DNA methylation (DNAm) at a genome-wide level in human. We adapted it for mouse and sought to examine DNAm differences across liver and 2 brain regions: cortex and hippocampus. A custom hybridization array was designed to isolate 99 Mb of CpG islands, shores, shelves, and regulatory elements in the mouse genome. This was followed by bisulfite conversion and sequencing on the Illumina HiSeq2000. The majority of differentially methylated cytosines (DMCs) were present at greater than expected frequency in introns, intergenic regions, near CpG islands, and transcriptional enhancers. Liver-specific enhancers were observed to be methylated in cortex, while cortex specific enhancers were methylated in the liver. Interestingly, commonly shared enhancers were differentially methylated between the liver and cortex. Gene ontology and pathway analysis showed that genes that were hypomethylated in the cortex and hippocampus were enriched for neuronal components and neuronal function. In contrast, genes that were hypomethylated in the liver were enriched for cellular components important for liver function. Bisulfite-pyrosequencing validation of 75 DMCs from 19 different loci showed a correlation of r = 0.87 with Methyl-Seq data. We also identified genes involved in neurodevelopment that were not previously reported to be differentially methylated across brain regions. This platform constitutes a valuable tool for future genome-wide studies involving mouse models of disease.

Methyl-Seq是近期开发的一种靶向检测人类全基因组水平DNA甲基化（DNA methylation, DNAm）的方法。本研究将其适配至小鼠模型，旨在探究肝脏与两个脑区——大脑皮层及海马体——之间的DNA甲基化差异。我们设计了定制化杂交芯片，用于分离小鼠基因组中99 Mb的CpG岛（CpG island）、CpG岸（CpG shore）、CpG架（CpG shelf）及调控元件，随后进行亚硫酸氢盐转化（bisulfite conversion），并在Illumina HiSeq2000平台上完成测序。绝大多数差异甲基化胞嘧啶（differentially methylated cytosines, DMCs）在内含子、基因间区、CpG岛附近及转录增强子区域的出现频率高于预期。研究发现，肝脏特异性增强子在大脑皮层中呈现甲基化状态，而大脑皮层特异性增强子则在肝脏中发生甲基化。值得注意的是，肝脏与大脑皮层共有的增强子在二者之间呈现差异甲基化模式。基因本体（Gene Ontology, GO）富集分析与通路分析结果显示，在大脑皮层及海马体中呈低甲基化状态的基因，显著富集于神经元组分及神经元功能相关通路；与之相反，在肝脏中呈低甲基化状态的基因，则显著富集于与肝脏功能密切相关的细胞组分相关通路。针对19个不同基因座上的75个DMCs进行亚硫酸氢盐焦磷酸测序（Bisulfite-pyrosequencing）验证，结果显示其与Methyl-Seq数据的相关系数r=0.87。本研究还鉴定出一批参与神经发育过程的基因，此前尚无研究报道这些基因在不同脑区之间存在差异甲基化现象。该平台为未来基于疾病小鼠模型的全基因组水平研究提供了极具价值的研究工具。