Durante, M.

Herrlitz, M.

Leifke, A. L.

Natale, F.

Taucher-Scholz, G.

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Formation of 5hmC following exposure to Ionizing Radiation ∗M. Herrlitz1,2, F. Natale1,2, A.L. Leifke1, M. Durante1,2,3, and G. Taucher-Scholz1,31GSI, Darmstadt, Germany; 2FIAS, Frankfurt Institute for Advanced Studies, Germany; 3TUD, Darmstadt, GermanyEukaryotic DNA methylation (DNAm), which is theaddition of a methyl group on cytosine bases (C), is anepigenetic mechanism involved in several biological pro-cesses, including replication, transcription, and carcino-genesis. Besides methylcytosine (5mC), hydroxymethyl-cytosine (5hmC) is another modified C moiety. 5hmCis formed by the enzymatic activity of TET (ten eleventranslocation) enzymes oxidizing 5mC to 5hmC and fur-ther to formyl- and carboxyl-cytosine, the latter being fi-nally replaced by C via base excision repair [1]. Conver-sion of 5mC to 5hmC could change the DNA binding affin-ity of several proteins and thus influence regulation of genetranscription [2]. Previous works reported that ionizing ra-diation (IR) and DNA double strand break (DSB) repairprocesses are able to induce changes of DNAm levels [3,4, 5]. However, these studies were performed several pop-ulation doublings after IR exposure and could not rule outreplication-dependent DNAm changes. Whether the lattermay take place also at short times after exposure to IR re-mains controversial. Increased DNA methylase levels atlater times (≥ 24 hours) after IR exposure have been re-cently reported [6]. These findings suggest that increasedDNAm levels would not occur until DNA repair is accom-plished. Based on such observation, we hypothesize that IRmay rather reduce DNAm levels at earlier times by trigger-ing enzymatic activities regulating such process, e.g. TETenzymes. Therefore, we examined DNAm changes withinone replication cycle time frame after IR (≤ 24 hours), fo-cusing on DNA demethylation. Namely, we investigated5hmC formation as an intermediate of DNA demethylationand whether this modification was involved in the DSB re-pair. We investigated human fibroblasts 30 minutes afterirradiation with carbon ions and observed 5hmC formationalong the ion trajectory. Both fluorescence microscopy im-ages and intensity profiles indicate that 5hmC signal colo-calizes with γH2AX (DSBs marker) (Fig. 1). Similar re-sults were obtained upon irradiation with X-rays (Fig. 2A).Colocalization was statistically verified using intensity cor-relation analysis (ICA) [7]. Briefly, PDM (Product of theDifferences from the Mean) values were calculated for eachpixel in both channels. If the signals were positively corre-lated, this would result in a positive PDM value and, thus,in a positive correlation. Our results show that most PDMvalues are positive, displaying a moderate positive corre-lation between 5hmC and γH2AX at DSBs (RPearson =0.425, Fig. 2B).∗Supported by Beilstein Stiftung (NanoL project). Work is part ofHGS HIRe and partially funded by BMBF grant 02NUK001A.A BFigure 1: AG1522D cells were irradiated with carbon ions(LET: 170 keV/μm; fluence: 3x106 p/cm2) under lowangle, fixed after 30 minutes and stained against 5hmC(green) and γH2AX (red). DNA was stained with DAPI(blue). A) Micrograph showing 5hmC (green) formationat DSBs sites indicated by γH2AX (red). White line: iontrajectory; scale bar = 5 μm. B) Fluorescence intensity pro-files showing colocalization of 5hmC (green) and γH2AX(red). DNA is illustrated by the blue line.A BFigure 2: AG1522D human fibroblasts were irradiated with0.5 Gy X-rays, fixed after 30 minutes and stained as in Fig.1. A) 5hmC (green) accumulates at DSBs, indicated byγH2AX staining (red). Scale bar = 10 μm. B) ICA graphof γH2AX signal. The horizontal line indicates mean PFIvalues. With increasing PFI, PDM values increase.Our results indicate that 5hmC is formed at DSB sitescolocalizing with γH2AX, after both heavy ions and X-rays irradiation. Such observation suggests a possible in-volvement of 5hmC in the DSB repair. We are currentlyinvestigating the colocalization of 5hmC and γH2AX overtime, during DNA repair, performing kinetics of foci for-mation in a time frame of ≤ 24 hours after irradiation.References[1] Ito et al. (2011) Science, 333, p. 1300 ff[2] Spruijt et al. (2013) Cell, 152, p. 1146 ff[3] Goetz et al. (2011) Radiation Research, 175, p. 575 ff[4] Cuozzo et al. (2007) PLoSGenet, 3, p. 1144 ff[5] O’Hagan et al. (2008) PLoSGenet, 4, e1000155[6] Chaudhry et al. (2012) DNA Cell Biol, 31, p. 908 ff[7] Li et al. (2004) J Neurosci, 24, p. 4070 ffBIOPHYSICS-04 GSI SCIENTIFIC REPORT 2013206 doi:10.15120/GR-2014-1-BIOPHYSICS-04

Formation of 5hmC following exposure to Ionizing Radiation

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Formation of 5hmC following exposure to Ionizing Radiation

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