Tet enzymes oxidize 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate, respectively, to dC over the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. However, whether such direct C-C bond cleavage reactions at fdC and cadC occur in vivo remains an unanswered question. Here we report the incorporation of synthetic isotope- and (R)-2'-fluorine-labeled dC and fdC derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC-MS/MS provided quantitative data about the formed reaction products. The data show that the labeled fdC probe is efficiently converted into the corresponding labeled dC, most likely after its incorporation into the genome. Therefore, we conclude that fdC undergoes C-C bond cleavage in stem cells, leading to the direct re-installation of unmodified dC.

Abstract:Until recently,i tw as believed that the genomes of higher organisms contain, in addition to the four canonical DNAb ases,o nly 5-methyl-dC (m 5 dC) as am odified base to control epigenetic processes.I nr ecent years,t his view has changed dramatically with the discovery of 5-hydroxymethyldC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC) in DNAfrom stem cells and brain tissue.N 6 -methyldeoxyadenosine (m 6 dA) is the most recent base reported to be present in the genome of various eukaryotic organisms.T his base,t ogether with N 4 -methyldeoxycytidine (m 4 dC), was first reported to be ac omponent of bacterial genomes.I nt his work, we investigated the levels and distribution of these potentially epigenetically relevant DNAb ases by using an ovel ultrasensitive UHPLC-MS method. We further report quantitative data for m 5 dC,h mdC,f dC,a nd cadC,b ut we were unable to detect either m 4 dC or m 6 dA in DNAisolated from mouse embryonic stem cells or brain and liver tissue,w hichc alls into question their epigenetic relevance.The genetic material of living organisms is constructed from the four canonical nucleobases dA, dC,d G, and dT,w hich establish the sequence information that, in multicellular organisms,i ss tored in the nucleus of every cell ( Figure 1). In addition to the canonical bases,the methylated dC base 5-methyldeoxycytidine (m 5 dC) is frequently found.[1] The presence or absence of this base in specific promoter segments determines whether the gene is actively transcribed or silenced.[1] Thec ell-type-specific distribution of m 5 dC thus determines the identity of ag iven cell. Recently,5 -hydroxymethyldeoxycytidine (hmdC) was found as as ixth base of the genetic system [2,3] and in 2011, 5-formyldeoxycytidine (fdC) [4,5] and 5-carboxydeoxycytidine (cadC) [5,6] were also discovered, particularly in DNAisolated from stem cells,but also in brain DNA. It is currently believed that fdC and cadC are intermediates in an active demethylation process that allows cells to change the methylation pattern and hence the activity state of specific genes. [7,8] ForfdC,separate epigenetic functions are also envisaged. [9] While the genomes of bacteria are known to also contain N 4 -methyldeoxycytidine (m 4 dC) [10] and N 6 -methyldeoxyadenosine (m 6 dA), [11] attempts to detect these bases in the DNA of higher organisms have failed until recently. [12][13][14][15] m 6 dA has now been found in algae (0.4 mol %m 6 dA/A), [12] fruit flies (0.001 %-0.07 %m 6 dA/A), [14] and C. elegans (0.01 %-0.4 % m 6 dA/A), [13] and its presence has even been reported in the DNAo fv ertebrates (0.00009 %i nX. laevis [16] and 0.00019-0.003 %o fd Ai nm urine cells and tissue [17] ). These discoveries,e specially concerning the DNAo fv ertebrates,h ave spurred aw orldwide research interest in unraveling the function of these new bases in human genomic DNA. [18][19][20] In this study,w ed eveloped an ultrasensitive triple quadrupole mass spectrometry (QQQ-MS) method, which in combination with ultra-high-pressure chromatograph...

5-Formyl-dC (fdC) and 5-carboxy-dC (cadC) are newly discovered bases in the mammalian genome that are supposed to be substrates for base excision repair (BER) in the framework of active demethylation. The bases are recognized by the monofunctional thymine DNA glycosylase (Tdg), which cleaves the glycosidic bond of the bases to give potentially harmful abasic sites (AP-sites). Because of the turnover of fdC and cadC during cell state transitions, it is an open question to what extent such harmful AP-sites may accumulate during these processes. Here, we report the development of a new reagent that in combination with mass spectrometry (MS) allows us to quantify the levels of AP-sites. This combination also allowed the quantification of β-elimination (βE) products, which are repair intermediates of bifunctional DNA glycosylases. In combination with feeding of isotopically labeled nucleosides, we were able to trace the intermediates back to their original nucleobases. We show that, while the steady-state levels of fdC and cadC are substantially increased in Tdg-deficient cells, those of both AP- and βE-sites are unaltered. The levels of the detected BER intermediates are 1 and 2 orders of magnitude lower than those of cadC and fdC, respectively. Thus, neither the presence of fdC nor that of cadC in stem cells leads to the accumulation of harmful AP- and βE-site intermediates.

Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known and the underlying pathways are poorly understood. Here we use metabolic labelling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic methylcytidine (mdC), hydroxymethylcytidine (hmdC) and formylcytidine (fdC) across mouse pluripotent cell states.High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on Tet-mediated mdC oxidation, we unveiled an additional mdC oxidationindependent turnover process based on DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage committed cells. Thus, in pluripotent cells active mdC turnover involves both mdC oxidation-dependent and -independent processes. .