甲基化胞嘧啶与羟甲基化胞嘧啶在小鼠不同器官中的含量与全基因组分布规律

Abundance and genome-wide distribution patterns of 5-methylcytosine and 5-hydroxymethylcytosine in different mouse organs

目的检测小鼠主要脏器中甲基化胞嘧啶(5mC)和羟甲基化胞嘧啶(5hmC)的含量与全基因组分布规律。方法摘取正常雄性C57BL/6小鼠主要脏器并抽提基因组DNA。采用DNA点杂交法检测5mC和5hmC的含量,采用甲基化和羟甲基化DNA特异性抗体免疫沉淀富集小鼠心脏、肝脏、肾脏、肺脏、脑皮质与小脑基因组DNA中含5mC或5hmC的DNA片段,通过全基因组测序技术和生物信息学分析比较不同器官基因组DNA中5mC和5hmC的分布规律。结果点杂交检测结果显示,小鼠主要器官脑皮质、肾脏、心脏、肝脏的5mC含量的差异均无统计学意义(P值均>0.05),但脑皮质的5hmC含量显著高于其他组织(P值分别<0.05、0.01)。小鼠心脏、肝脏、肺脏、肾脏、脑皮质和小脑的甲基化和羟甲基化DNA特异性抗体免疫沉淀和全基因组测序结果显示,5mC主要分布于基因间区域,而5hmC多位于基因本体区域。虽然小鼠不同器官中5hmC含量不同,但在5hmC基因本体区域的分布曲线相似。结论不同于5mC,小鼠不同器官5hmC含量存在明显差别。5hmC在全基因组范围的分布特点也与5mC存在差异。

Objective To investigate the abundance and genome-wide distribution patterns of 5-methylcytosine （5mC） and 5-hydroxymethylcytosine （5hmC） in key organs of mouse. Methods Different organs of normal male C57 BL/6 mice were collected for genomic DNA extraction. The global levels of 5mC and 5hmC were quantified and compared by DNA dot blot assay. The DNA fragments enriched with 5mC or 5hmC were immunoprecipitated by specific antibodies and sequenced by next-generation sequencing （MeDIP-seq, hMeDIP- seq）. The DNA methylomes and hydroxymethylomes of different mouse organs （such as heart, liver, kidney, lung, and pallium） were compared by conventional bioinformatic methods. Results The dot blot assay showed that different mouse organs contained similar 5mC levels （all P 〉 0. 05）, but the level of 5hmC in pallium was significantly higher than those in the other tissues （P〈0. 05 or 0.01 ）. MeDIP-seq and hMeDIP-seq detection showed that 5hmC was particularly enriched in gene bodies, but 5mC was more selectively targeted to intergenic regions. The distribution curves of 5mC or 5hmC across gene bodies were very similar among different mouse organs. Conclusion Unlike 5mC, the global 5hmC levels are highly variable among different mouse organs. The genome-wide distribution of 5hmC shows distinguishing feature compared to that of 5mC.