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

TLDR

5‑Hydroxymethylcytosine (hmC) is a recently identified sixth base in mammalian DNA, whose function is unknown but is known to derive from 5‑methylcytosine (mC), suggesting it may be an intermediate in active demethylation involving further oxidation to formyl or carboxyl groups followed by deformylation or decarboxylation. The study aims to precisely quantify hmC levels across mouse tissues and to search for putative demethylation intermediates fC, caC, and hmU. This was achieved using an ultra‑sensitive isotope‑based LC‑MS method. The data indicate that an active oxidative demethylation pathway is unlikely, and that hmC is present in all tissues and cell types, with the highest concentrations in neuronal cells of the CNS.

Abstract

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.