Yamazaki J, Jelinek J, Hisamoto S, Tsukamoto A, Inaba M
Laboratory of Molecular Medicine, Graduate School of Veterinary Medicine, Hokkaido University, Japan.
Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia, PA 19140, USA.
Vet J. 2018 Jan;231:48-54. doi: 10.1016/j.tvjl.2017.11.007. Epub 2017 Nov 22.
DNA methylation is the conversion of cytosine to 5-methylcytosine, leading to changes in the interactions between DNA and proteins. Methylation of cytosine-guanine (CpG) islands (CGIs) is associated with gene expression silencing of the involved promoter. Although studies focussing on global changes or a few single loci in DNA methylation have been performed in dogs with certain diseases, genome-wide analysis of DNA methylation is required to prospectively identify specific regions with DNA methylation change. The hypothesis of this study was that next-generation sequencing with methylation-specific signatures created by sequential digestion of genomic DNA with SmaI and XmaI enzymes can provide quantitative information on methylation levels. Using blood from healthy dogs and cells obtained from canine lymphoma cell lines, approximately 100,000CpG sites across the dog genome were analysed with the novel method established in this study. CpG sites in CGIs broadly were shown to be either methylated or unmethylated in normal blood, while CpG sites not within CpG islands (NCGIs) were largely methylated. Thousands of CpG sites in lymphoma cell lines were found to gain methylation at normally unmethylated CGI sites and lose methylation at normally methylated NCGI sites. These hypermethylated CpG sites are located at promoter regions of hundreds of genes, such as TWIST2 and TLX3. In addition, genes annotated with 'Homeobox' and 'DNA-binding' characteristics have hypermethylated CpG sites in their promoter CGIs. Genome-wide quantitative DNA methylation analysis is a sensitive method that is likely to be suitable for studies of DNA methylation changes in cancer, as well as other common diseases in dogs.
DNA甲基化是指胞嘧啶转化为5-甲基胞嘧啶，从而导致DNA与蛋白质之间相互作用的改变。胞嘧啶-鸟嘌呤（CpG）岛的甲基化与相关启动子的基因表达沉默有关。尽管已经对患有某些疾病的犬进行了聚焦于DNA甲基化全局变化或少数单个位点的研究，但仍需要对DNA甲基化进行全基因组分析，以前瞻性地识别出DNA甲基化发生变化的特定区域。本研究的假设是，通过用SmaI和XmaI酶对基因组DNA进行顺序消化而创建的具有甲基化特异性特征的下一代测序能够提供甲基化水平的定量信息。使用健康犬的血液和从犬淋巴瘤细胞系获得的细胞，采用本研究建立的新方法对犬基因组中约100,000个CpG位点进行了分析。在正常血液中，CpG岛中的CpG位点广泛显示为甲基化或未甲基化，而不在CpG岛内的CpG位点（非CpG岛，NCGIs）大多为甲基化。在淋巴瘤细胞系中发现，数千个CpG位点在正常未甲基化的CpG岛位点获得甲基化，而在正常甲基化的非CpG岛位点失去甲基化。这些高甲基化的CpG位点位于数百个基因的启动子区域，如TWIST2和TLX3。此外，具有“同源盒”和“DNA结合”特征的基因在其启动子CpG岛中具有高甲基化的CpG位点。全基因组定量DNA甲基化分析是一种灵敏的方法，可能适用于犬癌症以及其他常见疾病中DNA甲基化变化的研究。
