Tissue-specific variation in DNA methylation levels along human chromosome 1

DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters. Here, we use a methylation-profiling strategy that is predominantly responsive to methylation differences outside of CpG islands. The method compares the yield from two samples of size-selected fragments generated by a methylation-sensitive restriction enzyme. We profile eight different normal tissues relative to spleen for each of two human donors using a custom array of genomic clones covering the euchromatic portion of human chromosome 1 and representing 8% of the human genome. We observe gross regional differences in methylation states across chromosome 1 between tissues from the same individual, with the most striking differences detected in the comparison of cerebellum and spleen. Profiles of the same tissue from different donors are strikingly similar, as are the profiles of different lobes of the brain. Using DNMT1-deficient cells as a test case, we show that much finer-scale fluctuations in relative fragment yield can be detected when the methylation profiling is performed using a high-resolution oligomer array for chromosome 1. The varied patterns of methylation differences detected between tissues by this method reinforce the potential functional significance of regional differences in methylation levels outside of CpG islands. DNA digested with methylation-sensitive restriction enzyme (HpaII) and size-selected. Eleven human tissue types studied, most from two human individuals, each in triplicate, and one cell line. Two control hybrizations consisting of DNA prepared independently from the same tissue.

DNA甲基化（DNA methylation）是一类关键的表观遗传修饰（epigenetic modification），在调控基因表达（gene expression）与抑制异常转录（spurious transcription）中发挥核心作用。当前，针对不同组织开展基因组扫描以筛选差异甲基化位点的多数方法，均聚焦于CpG岛（CpG islands）内的CpG位点甲基化——这类CpG位点富集区域通常与基因启动子（gene promoters）存在关联。
本研究采用一种甲基化谱分析（methylation-profiling）策略，该策略主要响应CpG岛外区域的甲基化差异。该方法通过比较甲基化敏感限制性内切酶（methylation-sensitive restriction enzyme）酶切产生的、经片段大小筛选的两类样本的信号产出量。
我们使用一套覆盖人类1号染色体常染色质（euchromatic）区域、代表人类基因组8%序列的定制化基因组克隆阵列，针对两名人类供体的八种不同正常组织，分别以脾脏作为参照进行甲基化谱分析。
我们观察到，同一个体的不同组织间，1号染色体上存在显著的区域甲基化状态差异；其中小脑与脾脏的对照样本中检测到的差异最为突出。
来自不同供体的同一组织的甲基化谱高度相似，大脑不同脑叶的谱图亦呈现高度一致性。
以DNMT1缺陷细胞（DNMT1-deficient cells）作为测试模型，我们证实，当使用针对1号染色体的高分辨率寡核苷酸阵列（high-resolution oligomer array）开展甲基化谱分析时，可检测到片段相对产出量的更精细尺度波动。
本方法在不同组织间检测到的多样化甲基化差异模式，进一步强化了CpG岛外区域甲基化水平的区域差异具备潜在功能重要性这一结论。
实验流程采用甲基化敏感限制性内切酶HpaII对DNA进行酶切，并经片段大小筛选。本研究共分析11种人体组织类型，其中多数样本来自两名人类个体，每个样本均设置三次重复；此外还包含1种细胞系。同时设置两组对照杂交实验，所用DNA均来自同一组织的独立制备样本。