Tissue Distribution of 5-Hydroxymethylcytosine and Search for Active Demethylation Intermediates

5–Hydroxymethylcytosine (hmC) was recently detected as the sixth base in mammalian tissue at so far controversial levels. The function of the modified base is currently unknown, but it is certain that the base is generated from 5-methylcytosine (mC). This fuels the hypothesis that it represents an intermediate of an active demethylation process, which could involve further oxidation of the hydroxymethyl group to a formyl or carboxyl group followed by either deformylation or decarboxylation. Here, we use an ultra-sensitive and accurate isotope based LC-MS method to precisely determine the levels of hmC in various mouse tissues and we searched for 5–formylcytosine (fC), 5-carboxylcytosine (caC), and 5–hydroxymethyluracil (hmU) as putative active demethylation intermediates. Our data suggest that an active oxidative mC demethylation pathway is unlikely to occur. Additionally, we show using HPLC-MS analysis and immunohistochemistry that hmC is present in all tissues and cell types with highest concentrations in neuronal cells of the CNS.5-羟甲基胞嘧啶（5–Hydroxymethylcytosine，hmC）作为哺乳动物基因组中的第六种碱基，近期在哺乳动物组织中被检测到，但其含量水平迄今仍存在争议。目前这种修饰碱基的生物学功能尚未明确，但可以确定其由5-甲基胞嘧啶（5-methylcytosine，mC）转化生成。这一发现催生了相关假说，即hmC是活性去甲基化过程的中间产物，该过程可能通过将羟甲基进一步氧化为甲酰基或羧基，随后再经脱甲酰基或脱羧基反应完成。本研究采用基于同位素标记的超灵敏精准液相色谱-质谱联用法（LC-MS），精准定量了多种小鼠组织中的hmC含量，并同时检测了5-甲酰基胞嘧啶（5–formylcytosine，fC）、5-羧基胞嘧啶（5-carboxylcytosine，caC）以及5-羟甲基尿嘧啶（5–hydroxymethyluracil，hmU）这几种潜在的活性去甲基化中间产物。本研究数据表明，活性氧化性mC去甲基化通路极有可能并不存在。此外，本研究通过高效液相色谱-质谱联用法（HPLC-MS）与免疫组织化学实验证实，hmC广泛存在于所有组织与细胞类型中，其中以中枢神经系统（CNS）神经元内的含量最高。