Measurements of DNA Methylation at Seven Loci in Various Tissues of CD1 Mice

In humans, considerable variation in methylation at single loci and repetitive elements in various cells and tissues is observed. Recently, several inter- and intra-tissue correlations for DNA methylation have been reported. To investigate the extent and reproducibility of such correlations, we investigated inter- and intra-tissue methylation correlations among seven different loci in 9 different tissues in a population of 100 healthy seven-week-old CD1 outbred mice. We used a highly quantitative approach to measure methylation levels to high accuracy at two single loci in the alpha-actin and myosine light chain promoters, at three differentially methylated regions of the Peg3, Snrpn and Lit1 genes associated with imprinted loci, and at two repetitive elements in the Line-1 and IAP-LTR genes in the various tissues. In this population of mice, methylation at several loci was sex-associated and intra-tissue correlations among the studied loci were observed for brain and spleen. Inter-tissue correlations were rarely observed. To investigate method-dependent experimental variability, we re-analyzed the same spleen and tongue samples using SIRPH and pyrosequencing methods and reconfirmed intra-tissue correlations for spleen and sex-associated correlations for DNA methylation for tongue. When we repeated DNA methylation measurements for a second mouse population raised under similar conditions three months later, we did not detect sex-associated or intra-tissues correlations. Additional studies that examine large numbers of loci may be required to further understand the factors that influence stability of DNA methylation.

在人类中，已观测到不同细胞与组织内的单基因座（locus，复数形式loci）及重复元件（repetitive elements）的甲基化水平存在显著差异。近期已有多项研究报道了DNA甲基化（DNA methylation）的组织间与组织内关联特征。为探究此类关联的范围与可重复性，我们以100只健康7周龄远交系CD1小鼠（CD1 outbred mice）为研究对象，对9种不同组织中的7个基因座开展检测，以分析其甲基化水平的组织间与组织内关联。我们采用高精度定量方法，对各类组织中的三类位点进行甲基化水平精准测定：一是α-肌动蛋白（alpha-actin）与肌球蛋白轻链（myosine light chain）启动子区域的2个单基因座；二是与印记基因座（imprinted loci）相关的Peg3、Snrpn及Lit1基因的3个差异甲基化区域；三是Line-1与IAP-LTR基因的2个重复元件。在该小鼠群体中，多个基因座的甲基化水平存在性别关联，且在脑与脾脏中观测到了研究位点间的组织内关联；而组织间关联则极少被发现。为探究方法依赖的实验变异情况，我们采用SIRPH与焦磷酸测序（pyrosequencing）两种方法，对同一批脾脏与舌组织样本进行重新分析，再次验证了脾脏的组织内关联，以及舌组织甲基化水平的性别关联。当我们在三个月后，以饲养条件一致的第二组小鼠群体重复DNA甲基化检测时，未观测到性别关联或组织内关联。未来或需开展针对大量基因座的相关研究，以进一步解析影响DNA甲基化稳定性的各类因素。