Planteamiento del problema y delimitación

The TET proteins were found to oxidize 5-mC to 5-hmC and further to 5-fC and 5-caC (Tahiliani et al. 2009; He et al. 2011; Ito et al. 2011). LCMSMS analysis of global 5-fC and 5-caC levels in Tdg knockout and TdgΔcat cells revealed 2-9 fold enrichments compared to wildtype cells, which is in agreement

with TDG being the primary DNA glycosylase responsible for the excision of 5-fC and 5-caC. However, as these higher oxidized 5-mC derivatives are not maintained by Dnmt1 (Valinluck and Sowers 2007; Inoue et al. 2011) and, thus, diluted by DNA replication, it is striking that such a high level of 5-fC and 5-caC accumulate in TDG-deficient cells. We therefore asked whether 5-fC and 5-caC might arise spontaneously through non-enzymatic oxidation of 5-hmC and, thus, if oxidative stress might have an impact on epigenetic stability.

We treated Tdg-/- _{ES cells complemented with wildtype Tdg or Tdg}

Δcat with H2O2 to induce oxidative

stress and subsequently performed immunofluorescence staining with a specific anti 5-caC antibody. Surprisingly, we found 5-caC to localize in distinct foci throughout the nucleus in absence of oxidative damage and independent of cell cycle stage, irrespective of the TDG status. These foci did not co- localizing with focal PCNA during S-phase (Fig. 4-04a), indicating that they do not associate with progressing replication forks. As 5-caC localization in ES cells has not been described so far, neither by immunofluorescence staining nor by genome wide mapping, it remains to be verified that 5-caC indeed clusters in foci. However, 15min of incubation in H2O2 appeared to increase the number of

foci in a dose dependent manner in wildtype cells but not in TDGΔcat (Fig. 4-04b), suggesting that the

latter is either refractory to H2O2-induced generation of 5-caC or that the already elevated levels of

5-caC in TDGΔcat represent a ground state that allows no further induction of 5-caC without erratic

effects. The wider range of foci numbers observed in TDGΔcat might indicate the latter.

We next measured the global levels of 5-mC, 5-hmC, 5-fC, 5-caC and - as a control - 8-oxoG by LCMSMS. 15min of H2O2 exposure plus 15min of recovery increased the global 8-oxoG levels

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Interestingly, 5-mC levels were decreased and 5-hmC, 5-fC and 5-caC levels were increased by H2O2 in

wildtype cells, suggesting that the application of oxidative stress indeed oxidized 5-mC to 5-hmC and further. However, the H2O2 treatment appeared ineffective in TdgΔcat and knockout cells, as evident

from the unchanged levels of 8-oxoG. The variable efficacy of the treatment might be caused by the instability of H2O2. Surprisingly though, the effect observed in wildtype cells appeared to be reversed

in TdgΔcat and knockout cells suggesting that the treatment converted 5-hmC, 5-fC and 5-caC in a way

not detectable in our approach.

Fig. 4-04: Non-enzymatic generation of 5-fC and 5-caC. a) Immunofluorescence staining of 5-caC and PCNA in

wildtype ES cells mock treated in PBS for 15min. DAPI was used to stain DNA. b) Number of 5-caC foci per cell (n=7-12), visually assessed in z-stacks acquired on a confocal microscope (Leica SP5). Floating bars indicate median plus range. Statistical analysis by two-way Anova with Bonferroni post-test. ** p<0.001. c) LCMSMS quantification of the indicated modified bases in the genome of wildtype (wt), TDGΔcat (Δcat)and knockout (ko)

cells. Bars indicate number per 106 unmodified bases (A, C, T and G).

a)

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These results were obtained in a single experiment the reproduction of which failed due to the erratic effects of H2O2 on the induction of oxidative damage. Still, my preliminary results indicate that

higher oxidized 5-mC derivatives could indeed arise non-enzymatically, suggesting that oxidative stress, besides inducing genetic mutations, might also destabilize cell programming. The question how oxidative damage affects 5-mC and its derivatives thus remains an important one and will have to be readdressed in a more robust set-up. Other reagents which are more stable, i.e. menadione, might prove more suitable to study the generation of 5-fC and 5-caC under oxidative stress.

Methods:

ES cell culture. Essentially as described in Appendix III. Cells were cultured in 2i medium prior to treatment with 100 μM  (if  not  indicated  otherwise)  H2O2 in PBS or just PBS (mock control) for 15 min.

Cells were left for 15 min in culture medium to recover followed by further processing.

Immunocytochemistry. Essentially as described in Appendix II. Cells were fixed in -20°C cold methanol for 5 min on ice and then rehydrated for 2x 15 min in PBS. After permeabilization with 0.2% TritonX- 100/PBS for 5 min on ice, cells were rinsed with PBS and DNA was depurinated with 2 N HCl for 20 min. Cells were washed with PBS and neutralized by two 10 min washes in 100mM Tris-HCl pH 8. After blocking with blocking solution (BS; 2% BSA, 0.05% Tween20, PBS) for 1 h at room temperature, cells were incubated with the primary antibodies (anti 5-caC 1:500, Active Motif, Belgium; anti-PCNA FITC 1:400, Leinco Technologies, Missouri USA) in BS over night at 4°C. Three 10 min washes with BS were followed by incubation with secondary antibody (goat anti rabbit Alexa 594 1:500, Invitrogen – Life Technologies Switzerland) in BS for 1 h. After two 10 min washes in BS and one in PBS, DNA was stained   with   5   μg/mL   DAPI.   Excess   DAPI   was   removed   by   two   5 min washes in PBS followed by mounting in Vectashield (Vector Labs, UK). Z-stacks were acquired on a Leica SP5 with 405 nm, 488nm and 594 nm laser lines (Leica, Switzerland).

LCMSMS. As described in Appendix III. Conditions for 8-oxo(dG) were as for C-modifications. Mass spectrometry detection of 8-oxo(dG) was performed using an MDS Sciex API5000 triple quadrupole (Applied Biosystems – Life Technologies, Norway) operating in positive electrospray ionization mode for the mass transition 284.1/168.2.

Contribution: I cultured the ES cells and performed the treatment with H2O2. Moreover, I established

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of 5-caC foci number. I further prepared genomic DNA from treated and mock treated cells for LCMSMS analysis.