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). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.

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. For hmC, an initial steady increase is observed, which levels off with age to a final steady-state value of 1.2 % in human brain tissue. This level is nearly twice as high as in mouse cerebral cortex. In contrast, fC declines rapidly with age during early developmental stages, thus suggesting that while hmC is a stable epigenetic mark, fC is more likely an intermediate of active DNA demethylation during early brain development. The trends in global cytosine modification dynamics during the lifespan of an organism are conserved between humans and mice and show similar patterns in different organs.

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. Here, we destabilised the interaction of the RFTS domain with the catalytic domain by site-directed mutagenesis and purified the corresponding Dnmt1 variants. Our data show that these mutations resulted in an up to fourfold increase in Dnmt1 methylation activity in vitro. Activation of Dnmt1 was not accompanied by a change in its preference for methylation of hemimethylated CpG sites. We also show that the Dnmt1 E572R/D575R variant has a higher DNA methylation activity in human cells after transfection into HCT116 cells, which are hypomorphic for Dnmt1. Our findings strongly support the autoinhibitory role of the RFTS domain, and indicate that it contributes to the regulation of Dnmt1 activity in cells.

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). Here we report chemical studies that enlighten the chemical reactivity of the new cytosine nucleobases. We investigated their sensitivity toward oxidation and deamination and we studied the C-C bond cleaving reactivity of hmdC, fdC, and cadC in the absence and presence of thiols as biologically relevant (organo)catalysts. We show that hmdC is in comparison to mdC rapidly oxidized to fdC already in the presence of air. In contrast, deamination reactions were found to occur only to a minor extent. The C-C bond cleavage reactions require the presence of high concentration of thiols and are acid catalyzed. While hmdC dehydroxymethylates very slowly, fdC and especially cadC react considerably faster to dC. Thiols are active site residues in many DNA modifiying enzymes indicating that such enzymes could play a role in an alternative active DNA demethylation mechanism via deformylation of fdC or decarboxylation of cadC. Quantum-chemical calculations support the catalytic influence of a thiol on the C-C bond cleavage.

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. In the normal adult brain, IHC identified 61.5% 5 hmC positive cells in the cortex and 32.4% 5 hmC in white matter (WM) areas. In tumors, positive staining of cells ranged from 1.1% in glioblastomas (GBMs) (WHO Grade IV) to 8.9% in Grade I gliomas (pilocytic astrocytomas). In the normal adult human brain, LC-MS also showed highest values in cortical areas (1.17% 5 hmC/dG [deoxyguanosine]), in the cerebral WM we measured around 0.70% 5 hmC/dG. levels were related to tumor differentiation, ranging from lowest values of 0.078% 5 hmC/dG in GBMs (WHO Grade IV) to 0.24% 5 hmC/dG in WHO Grade II diffuse astrocytomas. 5 hmC measurements were unrelated to 5 mC values. We find that the number of 5 hmC positive cells and the amount of 5 hmC/dG in the genome that has been proposed to be related to pluripotency and lineage commitment in embryonic stem cells is also associated with brain tumor differentiation and anaplasia.

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.

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. Cell culture, drug exposure, shRNA-mediated knockdown and phenotype analysis are integrated into an efficient and versatile single well-based protocol. By utilizing this system, we assessed RG108, SGI-1027 and nanaomycin A, three novel DNA methyltransferase inhibitors that have not been tested in neuroblastoma cell lines so far, for their potential of synergistic anti-tumor activity in combination with doxorubicin. We found that, similarly to azacytidine, SGI-1027 and nanaomycin A mediate synergistic growth inhibition with doxorubicin independently of N-Myc status. However, they display high cytotoxicity but lack global DNA demethylation activity. Secondly, we conducted a lentiviral shRNA screen of F-box proteins, key regulators of protein stability, and identified Fbxw11/ß-TrCP2 as well as Fbxo5/Emi1 as potential therapeutic targets in neuroblastoma. These results complement existing studies and underline the reliability and versatility of our single well-based protocol.

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.