Age-dependent levels of 5-methyl-, 5-hydroxymethyl-, and 5-formylcytosine in human and mouse brain tissues.

The absolute levels of 5-hydroxymethylcytosine (hmC) and 5-methylcytosine (mC) in human brain tissues at various ages were determined. Additionally, absolute levels of 5-formylcytosine (fC) in adult individuals and cytosine modification levels in sorted neurons were quantified. These data were compared with age-related fC, hmC, and mC levels in mouse brain samples.

A rapid screening system evaluates novel inhibitors of DNA methylation and suggests F-box proteins as potential therapeutic targets for high-risk neuroblastoma.

After extensive research on radiochemotherapy, 5-year survival rates of children with high risk neuroblastoma still do not exceed 50%, owing to adverse side-effects exemplified by doxorubicin-induced cardiomyopathy. A promising new approach is the combination of conventional therapies with specific modulation of cell signaling pathways promoting therapeutic resistance, such as inhibition of aberrant kinase activity or re-expression of silenced tumor suppressor genes by means of chromatin remodeling. In this regard, we established a system that allows to identify potential drug targets as well as to validate respective candidate inhibitors in high-risk neuroblastoma model cell lines.

Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA.

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs).

Targeted mutagenesis results in an activation of DNA methyltransferase 1 and confirms an autoinhibitory role of its RFTS domain.

The N-terminal regulatory part of DNA methyltransferase 1 (Dnmt1) contains a replication foci targeting sequence (RFTS) domain, which is involved in the recruitment of Dnmt1 to replication forks. The RFTS domain has been observed in a crystal structure to bind to the catalytic domain of the enzyme and block its catalytic centre. Removal of the RFTS domain led to activation of Dnmt1, thus suggesting an autoinhibitory role of this domain.

Deamination, oxidation, and C-C bond cleavage reactivity of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine.

Three new cytosine derived DNA modifications, 5-hydroxymethyl-2'-deoxycytidine (hmdC), 5-formyl-2'-deoxycytidine (fdC) and 5-carboxy-2'-deoxycytidine (cadC) were recently discovered in mammalian DNA, particularly in stem cell DNA. Their function is currently not clear, but it is assumed that in stem cells they might be intermediates of an active demethylation process. This process may involve base excision repair, C-C bond cleaving reactions or deamination of hmdC to 5-hydroxymethyl-2'-deoxyuridine (hmdU).

Synthesis of 5-hydroxymethyl-, 5-formyl-, and 5-carboxycytidine-triphosphates and their incorporation into oligonucleotides by polymerase chain reaction.

The synthesis of the triphosphates of 5-hydroxymethyl-, 5-formyl-, and 5-carboxycytidine and the incorporation of these building blocks into long DNA fragments using the polymerase chain reaction (PCR) are reported. In this way DNA fragments containing multiple hmC, fC, and caC nucleobases are readily accessible.

Mechanism and stem-cell activity of 5-carboxycytosine decarboxylation determined by isotope tracing.

Eraserhead: Stem cells seem to erase epigenetic information by decarboxylation of the newly discovered epigenetic base 5-carboxycytosine (caC; see picture). This reaction is likely to involve a nucleophilic attack of the C5-C6 double bond.

Low values of 5-hydroxymethylcytosine (5hmC), the "sixth base," are associated with anaplasia in human brain tumors.

5-Methylcytosine (5 mC) in genomic DNA has important epigenetic functions in embryonic development and tumor biology. 5-Hydroxymethylcytosine (5 hmC) is generated from 5 mC by the action of the TET (Ten-Eleven-Translocation) enzymes and may be an intermediate to further oxidation and finally demethylation of 5 mC. We have used immunohistochemistry (IHC) and isotope-based liquid chromatography mass spectrometry (LC-MS) to investigate the presence and distribution of 5 hmC in human brain and brain tumors.