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.

Mind over matter: LC‐MS has allowed the amount of the post‐replicatively formed DNA base 5‐hydroxymethylcytosine (see picture; left) to be quantified in brain tissue. The nucleoside is most abundant in areas that are associated with higher cognitive functions, and its content in mouse hippocampi seems to increase with age. The new method enables hydroxymethylcytosine to be quantified with unprecedented accuracy.

5-Methylcytosine (5mC) in genomic DNA has important epigenetic functions in embryonic development and tumor biology. 5-Hydroxymethylcytosine (5hmC) is generated from 5mC by the action of the TET (Ten-Eleven-Translocation) enzymes and may be an intermediate to further oxidation and finally demethylation of 5mC. We have used immunohistochemistry (IHC) and isotope-based liquid chromatography mass spectrometry (LC-MS) to investigate the presence and distribution of 5hmC in human brain and brain tumors. In the normal adult brain, IHC identified 61.5% 5hmC positive cells in the cortex and 32.4% 5hmC 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% 5hmC/dG [deoxyguanosine]), in the cerebral WM we measured around 0.70% 5hmC/dG. 5hmC levels were related to tumor differentiation, ranging from lowest values of 0.078% 5hmC/dG in GBMs (WHO Grade IV) to 0.24% 5hmC/dG in WHO Grade II diffuse astrocytomas. 5hmC measurements were unrelated to 5mC values. We find that the number of 5hmC positive cells and the amount of 5hmC/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.Epigenetics, the study of mechanisms that control gene expression (in a potentially heritable way), may be the most rapidly expanding field in tumor biology. On a molecular level, (i) DNA methylation, i.e., covalent modification of cytosine bases resulting in 5-methylcytosine (5mC), (ii) histone modifications and (iii) nucleosome positioning are regarded as the driving epigenetic mechanisms. They are fundamental to the regulation of many cellular processes, including gene and micro RNA expression, DNA-protein interactions, suppression of transposable element mobility, cellular differentiation, embryogenesis, X-chromosome inactivation and genomic imprinting.In tumor biology, DNA methylation is the best-studied epigenetic change. Epigenetic silencing of O 6 -methylguanine DNA-methyltransferase (MGMT) 1 has been described as a strong predictive factor of treatment response to chemotherapy with alkylating agents of glioblastoma (GBM) and anaplastic astrocytoma (AA). 2 Methylation of CpG islands in the MGMT promoter with ensuing repression of MGMT transcriptional activity is generally viewed as the cause for this correlation. 3 Oxidation of 5mC leading to 5-hydroxymethylcytosine (5hmC) has been identified as a new epigenetic phenomenon in mouse Purkinje cells. 4 Three possible modes of action of 5hmC were discussed. (i) It might influence chromatin structure and local transcriptional activity by recruiting selective 5hmC-binding proteins or excluding 5mC-binding proteins.(ii) Conversion of 5mC to 5hmC might facilitate passive DNA demethylation by excluding the maintenance DNMT1, which recognizes 5hmC poorly. (iii) 5hmC may...

5-Hydroxymethylcytosine (hmC) was recently discovered as a new constituent of mammalian DNA. Besides 5-methylcytosine (mC), it is the only other modified base in higher organisms. The discovery is of enormous importance because it shows that the methylation of cytosines to imprint epigenetic information is not a final chemical step that leads to gene silencing but that further chemistry occurs at the methyl group that might have regulatory function. Recent progress in hmC detection--most notably LC-MS and glucosyltransferase assays--helped to decipher the precise distribution of hmC in the body. This led to the surprising finding that, in contrast to constant mC levels, the hmC levels are strongly tissue-specific. The highest values of hmC are found in the central nervous system. It was furthermore discovered that hmC is involved in regulating the pluripotency of stem cells and that it is connected to the processes of cellular development and carcinogenesis. Evidence is currently accumulating that hmC may not exclusively be an intermediate of an active demethylation process, but that it functions instead as an important epigenetic marker.

Onchocerciasis, also known as “river blindness”, is a neglected tropical disease infecting millions of people mainly in Africa and the Middle East but also in South America and Central America. Disease infectivity initiates from the filarial parasitic nematode Onchocerca volvulus , which is transmitted by the blackfly vector Simulium sp. carrying infectious third-stage larvae. Ivermectin has controlled transmission of microfilariae, with an African Program elimination target date of 2025. However, there is currently no point-of-care diagnostic that can distinguish the burden of infection—including active and/or past infection—and enable the elimination program to be effectively monitored. Here, we describe how liquid chromatography-MS–based urine metabolome analysis can be exploited for the identification of a unique biomarker, N -acetyltyramine- O ,β-glucuronide (NATOG), a neurotransmitter-derived secretion metabolite from O. volvulus . The regulation of this tyramine neurotransmitter was found to be linked to patient disease infection, including the controversial antibiotic doxycycline treatment that has been shown to both sterilize and kill adult female worms. Further clues to its regulation have been elucidated through biosynthetic pathway determination within the nematode and its human host. Our results demonstrate that NATOG tracks O. volvulus metabolism in both worms and humans, and thus can be considered a host-specific biomarker for onchocerciasis progression. Liquid chromatography-MS–based urine metabolome analysis discovery of NATOG not only has broad implications for a noninvasive host-specific onchocerciasis diagnostic but provides a basis for the metabolome mining of other neglected tropical diseases for the discovery of distinct biomarkers and monitoring of disease progression.