hmC as a putative intermediate in the demethylation process? 106

process?

There are several possibilities of the potential role of hmC. It has been described that methyl-CpG binding protein 2 (MeCP2) is unable to bind to the corresponding sequences when mCs were converted to hmCs in CpG sequences.[259] Here the question arises, if hmC is an intermediate in the active demethylation process. A recent study has shown that MTases are able to deformylate hmC in in vitro experiments.[260] Oxidation of the hydroxymethyl group to a formyl group would yield 5-formylcytosine (fC) which could expel formic acid and react to dC. Another possibility would be further oxidation of hmC or fC to yield 5-carboxylcytosine (caC) which possesses a carboxy group and would enable quick decarboxylation to regenerate dC (Figure 54)[261] Nature's most proficient enzyme orotate decarboxylase catalyzes a similar reaction in which orotate (6-carboxyuracil) is decarboxylated to uracil.[262-263] Similar oxidation and decarboxylation reactions are known for thymine in the pyrimidine salvage pathway of certain eukaryotes.[263-265] _{Another potential}

active demethylation pathway is the excision of hmC by DNA glycosylases.[266] _{hmC could be}

converted by deamination with an activation-induced deaminase (AID) analog to yield the uracil derivative hmU.[18] This uridine derivative is known to be a substrate for the base excision repair (BER) enzyme SMUG1.[267-268] The deoxynucleotide hmC might be a possible intermediate in BER as well, because in vitro experiments in extracts from calf thymus have shown BER activity for hmC.[269]

Figure 54: Depiction of the known cytosine modifications mC and hmC and the putative oxidative “demethylation” intermediates fC and caC. The base excision repair (BER) pathway is a second possible demethylation pathway via the intermediate hmU.

In order to test the idea that hmC is an intermediate of a possible oxidative demethylation pathway, we used our HPLC-MS method to detect the presence of fC, caC, and hmU in different tissues. The three putative intermediates were synthesized by M. Münzel and used in this thesis to determine their chromatographic and mass spectrometric properties. Therefore, the same HPLC gradient used for hmC quantification was employed, which already provides ideal separation of all five modified DNA nucleosides (Figure 55). The difference of each modified nucleoside also allows unambiguously assignments by their different molecular weights (Figure 55).

Figure 55: HPLC chromatogram and nucleoside specific mass spectra of the modified DNA nucleosides hmC, and mC with the putative demethylation intermediates caC, hmU, and fC. Nucleosides are ordered to their retention time.

Knowing the retention times of the putative nucleosides all previous recorded LC-MS data were screened for these nucleosides. We could however not detect any caC, hmU or fC in the quantification experiments described above, in which we used 10 µg DNA (Figure 48 and Figure 49). Therefore, the amount of hydrolyzed DNA was enriched up to 16 times. We hydrolyzed 716 samples of one tissue in parallel to ensure quantitative digestion and combined all samples afterwards which contain in total 70160 µg DNA. The nucleoside mixture was taken up in 100 µL ddH2O, d2-hmC was spiked and the sample analyzed via

HPLC-MS. Importantly, the column was not overload or the entrance of the mass spectrometer capped. Thus, highly accurate mass spectrometric data were obtained. Also no memory effect was observed in blank runs after these LC-MS measurements.

As representative tissues for high, average, and low hmC content, olfactory bulb, retina, cerebellum, kidney, and liver were chosen. Despite higher DNA concentrations, we could not detect any of the three putative intermediates fC, caC, or hmU. The exact concentration of hmC in the analyzed samples was determined and even in the olfactory bulb with 342 pmol hmC non of these three nucleosides was detected. The detection limit was found to be in the

low picomolar range (Figure 56) and all investigated compounds proved to be stable during enzymatic hydrolysis, with minor instability of caC. Nevertheless, if these nucleosides were present, we would be able to detect these derivatives even in traces 350700 times less abundant than hmC. Thus if present, fC does not reach levels above 7·10-4% of all nucleosides or 0.3% of all hmC. With other words our results exclude that caC is present in genomic DNA with more than 3.5 caCs in 105 nucleosides.

Figure 56: Detected values of the potential hmC demethylation intermediates in olfactory bulb as an example. A) Detection limits determined with synthetic nucleoside samples. B) Detection limits in digested DNA nucleoside samples. The red line indicates the detection limits of the modified nucleosides. The detection limit for hmC is 1.5 pmol.

The data presented in this thesis provide new insights into the distribution of the modification hmC in mammalian tissue. We showed that hmC is present in every cell type in the mammalian body and its distribution is tissue dependent, ranging from from 0.03%0.7%. This allowed us to classify tissues in three different groups with tissues from the CNS containing the highest amounts of hmC with up to 20-fold more compared to other tissues. Additionally, substantial further oxidation of hmC to fC or to caC or deamination of hmC to give hmU can be excluded. Nevertheless, the absence of these two putative pathways cannot fully be ruled out, but the data indicate that the unavoidable intermediates do not accumulate to any significant level. Either these reactions do not occur on large scale or the investigated intermediates are so short lived that they are not released from the enzymatic complex.[254]