This methylation occurs predominantly in CpG islands (areas with high occurrence of the CG motif) of promoters. mdC is introduced by specialized DNA methyltransferase (DNMT) enzymes.
In 2009, study showed 1,2 the discovery of 5-hydroxymethyl-2’-deoxyCytidine (hmdC), a novel dC modification in Purkinje neurons and embryonic stem cells. Later, a third report found this modification to be strongly enriched in brain tissues associated with higher cognitive functions.3 This new dC modification is generated by the action of a-ketoglutarate dependent TET enzymes (ten eleven translocation), which oxidizes mdC to hmdC. This finding stimulated discussion about active demethylation pathways that could occur, e.g., via base excision repair (BER), with the help of specialized DNA glycosylases. Alternatively, one could envision a process in which the hydroxymethyl group of hmdC is further oxidized to a formyl or carboxyl functionality followed by elimination of either formic acid or carbon dioxide4,5 .
A number of recent publications provides data that support both pathways. It was discovered that hmdC could be deaminated by activation-induced deaminase (AID) enzymes to provide 5-hydroxymethyl-2’-deoxyUridine (hmdU). This compound was shown to be excised by the SMUG-1 DNA glycosylase6 . After initial failure to detect any further oxidized hmdC derivatives in somatic tissues4, newly developed mass spectrometric technologies, in combination with the available reference compounds, finally enabled researchers to gather strong support for the putative oxidative demethylation pathway. These methods and standards enabled the discovery of 5-formyl-2’-deoxyCytidine (fdC) in differentiating embryonic stem cells.7 Recently, a similar technology also led to the discovery of 5-carboxyl-2’-deoxyCytidine (cdC)8,9, but the amount of fdC and cdC measured differs largely in all three reports.
Research is currently ongoing to unravel the true levels and fate of these further oxidized dC bases in somatic tissues and in different stem cells. Even along the oxidative pathway, base excision processes have been proposed to play a major role with two reports showing that thymidine DNA glycosylase (TDG) accepts both fdC and cdC as substrates.8,10 A possible oxidative demethylation pathway would clearly rely on the existence of a dedicated decarboxylase that is able to convert cdC back into dC. Such an intriguing decarboxylation would enable nature to remove the 5-methyl group in mdC without introducing DNA strand-breaks that accompany any BER based base removal.
Bio-Synthesis has has supported epigenetic resarch by providing the synthesis of oligonucletoides containing all the new cytosine analogues: hmdC, fdC and cdC. These modified base can be incorporate during synthesis at any positions using conventional solid-phase oligonucleotide synthesis chemistry but repalcing the standard cytosine DNA base with mehtylated DNA cytosine base.
- 5-hydroxymethyl-dC [hmdC]
- 5-hydroxymethyl-dC II [hmdCII]
- 5-carboxy-dC [cdC]
- 5-Formyl-dC [fdC]
The first generation hmdC phosphoramidite was fairly very well accepted but requires fairly harsh synthesis conditions. Therefore, a second generation building block (5-Hydroxymethyl-dC II) developed by Carell and co-workers that is compatible with UltraMild deprotection has been introduced.6 5-Formyl-dC and 5-carboxy-dC may find uses in research into DNA damage and repair processes.
References:
- S. Kriaucionis, and N. Heintz, Science, 2009, 324, 929-30.
- M. Tahiliani, et al., Science, 2009, 324, 930-935.
- M. Münzel, et al., Angewandte Chemie-International Edition, 2010, 49, 5375-5377.
- D. Globisch, et al., PLoS One, 2010, 5, e15367.
- S.C. Wu, and Y. Zhang, Nat Rev Mol Cell Biol, 2010, 11, 607-20.
- M. Münzel, D. Globisch, C. Trindler, and T. Carell, Org Lett, 2010, 12, 5671-3.
- M. Münzel, et al., Improved Synthesis and Evaluation of Oligonucleotides Containing 5-Hydroxymethylcytosine, 5-Formylcytosine and 5-Carboxylcytosine. In Chemistry - A European Journal, 2011, in press.