OGG1 is essential in oxidative stress induced DNA demethylation

    OGG1 is essential in oxidative stress induced DNA demethylation Xiaolong Zhou, Ziheng Zhuang, Wentao Wang, Lingfeng He, Huan Wu, Yan Cao, Feiyan Pan, Jing Zhao, Zhigang Hu, Chandra Sekhar, Zhigang Guo PII: DOI: Reference:

S0898-6568(16)30128-0 doi: 10.1016/j.cellsig.2016.05.021 CLS 8699

To appear in:

Cellular Signalling

Received date: Revised date: Accepted date:

30 December 2015 27 May 2016 27 May 2016

Please cite this article as: Xiaolong Zhou, Ziheng Zhuang, Wentao Wang, Lingfeng He, Huan Wu, Yan Cao, Feiyan Pan, Jing Zhao, Zhigang Hu, Chandra Sekhar, Zhigang Guo, OGG1 is essential in oxidative stress induced DNA demethylation, Cellular Signalling (2016), doi: 10.1016/j.cellsig.2016.05.021

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OGG1 is Essential in Oxidative Stress Induced DNA Demethylation

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Xiaolong Zhou1a, Ziheng Zhuang2,3a, Wentao Wang1a, Lingfeng He1a, Huan Wu1, Yan Cao1, Feiyan Pan1, Jing Zhao1, Zhigang Hu1, Chandra Sekhar1 and Zhigang Guo1*

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Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, 1 WenYuan Road, Nanjing, China 210023. 2 Changzhou No.7 people's hospital, Changzhou, China 213011. 3 School of Pharmaceutical Engineering and Life Sciences, Changzhou University, Changzhou, China 213011. a These authors contributed equally to this work *To whom correspondence and requests for materials should be addressed (e-mail:[email protected])

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Running title: OGG1 regulates DNA demethylation

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Abbreviations: 8-oxoguanine DNA glycosylase-1 (OGG1), 8-oxoguanine (8-oxoG), reactive oxygen species (ROS), α-ketoglutarate (α-KG), Methylated DNA immunoprecipitations (MeDIP), 5-methyl cytosine (5mC), 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC), 5-carboxylcytosine (5caC), base excision repair (BER), CpG dinucleotides (CpGs), Ethidium bromide (EtdBr), thymine DNA glycosylase (TDG), Uracil-DNA glycosylase (UNG).

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ACCEPTED MANUSCRIPT Abstract DNA demethylation is an essential cellular activity to regulate gene expression; however, the

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mechanism that triggers DNA demethylation remains unknown. Furthermore, DNA demethylation

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was recently demonstrated to be induced by oxidative stress without a clear molecular mechanism.

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In this manuscript, we demonstrated that 8-oxoguanine DNA glycosylase-1 (OGG1) is the essential

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protein involved in oxidative stress-induced DNA demethylation. Oxidative stress induced the formation of 8-oxoguanine (8-oxoG). We found that OGG1, the 8-oxoG binding protein, promotes

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DNA demethylation by interacting and recruiting TET1 to the 8-oxoG lesion. Downregulation of

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OGG1 makes cells resistant to oxidative stress-induced DNA demethylation, while over-expression

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of OGG1 renders cells susceptible to DNA demethylation by oxidative stress. These data not only

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illustrate the importance of base excision repair (BER) in DNA demethylation but also reveal how the DNA demethylation signal is transferred to downstream DNA demethylation enzymes. Key words: DNA demethylation, Oxidative stress, OGG1, TET1, 8-oxoG

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1. Introduction

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Oxidative stress refers to the cellular phenotype mainly generated by excessive production of reactive oxygen species (ROS) [1]. ROS are formed as a by-product of oxygen metabolism.

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Generally, ROS are classified into three categories: O2-, OH and H2O2. ROS can damage DNA,

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proteins and lipids [2]. In humans, ROS are involved in the pathophysiology of many diseases

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including cancer [3]. However, ROS are necessary for cell proliferation, survival and migration [4].

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Metabolic and environmental factors can increase production of ROS. Hydroxyl radicals cause base deletions, modifications, single/double strand break and chromosomal recombination [5]. Recent

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studies have indicated that oxidative stress can also alter DNA methylation levels [6, 7].

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DNA demethylation is an essential activity in cells to balance DNA methylation levels. In essence, DNA demethylation removes a methyl group from DNA nucleotides. Defects in DNA demethylation could lead to DNA hypermethylation and are closely associated with various diseases, such as cancer [8]. DNA demethylation can be classified into two categories: passive demethylation and active demethylation. The passive process occurs in newly synthesized DNA during replication. In contrast, active DNA demethylation occurs independently of DNA replication. The mechanisms involved in the passive DNA demethylation have been fairly well characterized. However, the mechanism(s) of active demethylation remain poorly understood. There are several proposed mechanisms of active DNA demethylation. Recently, the TET-TDG demethylation pathway has been proposed as a major mechanism in active DNA demethylation, TET proteins oxidize the methylcytosine, and normal cytosine is restored through thymine DNA glycosylase (TDG) [9-11]. 3

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However, it remains unknown how the DNA demethylation signal is relayed to TET proteins or how

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the TET proteins are recruited to DNA demethylation.

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8-oxoG is a major form of oxidized DNA damage and is used as a biomarker of oxidative damage

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[12-15]. Some studies imply 8-oxoG are transcription (epigenetic) markers [16]. Thymine glycol is

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another major form of DNA damage from oxidation [17, 18]. In adult somatic cells, DNA methylation

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typically occurs in the context of CpG dinucleotides (CpGs). Non-CpG methylation, such as CpT, CpA, or CpC, is prevalent in embryonic stem cells and neural cells [19-22]. 8-oxoG is mainly

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repaired by the DNA glycosylase OGG1 and preferentially by the short patch BER [23]. In mouse

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Ogg1-/- cells, no 8-oxoG glycosylase activity is detected by the oligonucleotide substrates [24].

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Additional DNA repair glycosylases include the NEIL family proteins that are also able to remove 8-oxoG [25]. Even though 8-oxoG is not a substrate for any of the NEIL glycosylases, the further oxidation products derived from 8-oxoG indeed are substrates for all three enzymes [26-29]. Recent studies have demonstrated that DNA demethylation could be induced by oxidative stress. Formation of 8-oxoG due to oxidative stress stimulates demethylation of adjacent 5-methyl cytosines (5mC) [30-34]. However, all of the specific mechanisms of oxidative stress-induced DNA demethylation remain unclear.

OGG1 was once regarded as a DNA repair system protein and was not involved in DNA demethylation. In this manuscript, we demonstrated that OGG1 is also essential for oxidative stress-induced DNA demethylation. We propose a novel DNA demethylation model involving OGG1: 4

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1. Oxidative stress leads to 8-oxoG formation; 2. OGG1 binds to 8-oxoG lesions; 3. OGG1 both

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interacts with and recruits TET1 to 8-oxoG lesions; 4. TET1 oxidizes 5mC adjacent to 8-oxoG.

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2. Materials and Methods

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2.1. Cell culture and treatments

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An Ogg1 null cell line was kindly supplied by Professor Istvan Boldogh (Sraly Center for Molecular Medicine, Texas). Ogg1 null cells, MCF-7 cells and HeLa cells were cultured in DMEM basic

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(GIBCO) containing 10% (v/v) foetal cattle Serum, 100 μg/ml penicillin/streptomycin mixtures at

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37℃ with 5% CO2. Immortalized human mammary epithelial (MCF-10A) cells were cultured in

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DMEM-F12 (GIBCO) containing 10% (v/v) foetal cattle Serum, 100 ng/ml Cholera toxin, 0.5 μg/ml

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hydrocortisone, 20 ng/ml recombinant human epidermal growth factor, 0.5 μg/ml fungi zone, 2 mM L-glutamine and 100 μg/ml penicillin/streptomycin mixtures at 37℃ with 5% CO2. Cells approximately over 90% confluence were treated with 500 μM H2O2 diluted in PBS for 30 min at 37℃ and were then allowed to recover in DMEM/DMEM-F12 for the indicated periods of time points before fixation or extraction.

2.2. Quantification of 8-oxoG, 5mC and 5-hydroxymethyl cytosine (5hmC) by dot blot analysis Purified genomic DNA was denatured at 95℃ for 10 min and chilled on ice for 5 min. 100 ng of the DNA was spotted onto a piece of Hybond-N+ membrane (RPN303B, Ge healthcare, UK) and then UV cross-linked at 70000 μJ/cm2 for 2 min. The membranes were then blocked with 1% BSA in PBS, and signals were detected using mouse monoclonal antibody for 5mC (ab10805, Abcam, USA), 5

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mouse monoclonal antibody for 8-oxoG (4354-MC-050, Trevigen, USA), mouse monoclonal

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antibody for 5hmC (ab178771, Abcam, USA) and a horseradish peroxidase-conjugated rabbit anti-mouse IgG second-antibody (ab6728, Abcam, USA). All antibodies have high specifity for their

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targets (Fig. S1).

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2.3. Plasmid

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Full-length mouse Ogg1 cDNA from MEF cells was inserted into the EcoR I and BamH I sites of the pcDNA3.1 (-) vector (Invitrogen), the resulting expression vector was denoted as pcDNA3.1-OGG1.

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The primers used to construct this plasmid are listed in the supplemental material (Table S1).

Primers

for

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2.4. Real-time quantitative PCR (qPCR) analysis OGG1

and

BACE1

were

synthesized

using

Primer

Bank

(pga.mgh.Harvard.edu/primerbank). Primers for glyceraldeyhyde 3-phosphate dehydrogenase (GAPDH) were used as an internal control. In this study, all of the amplification products had a single bright band of the correct size, and a melting-curve analysis for each band showed only one peak. Total RNA was extracted from cells using Trizol® Reagent (Invitrogen) according to the manufacturer’s protocol. The reverse transcription of total RNA (1 μg) was performed using a RevertAid™ RT reagent Kit (RR036A, Takara) in a 20 μl reaction volume according to the manufacturer. Primer information for the Real-time quantitative PCR is also available in the supplemental material (Table S1).

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2.5. Western blot analysis

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Cells were lysed with RIPA lysis buffer (P0013B, beyotime, China) and 1 mM PMSF (ST506, beyotime, China). Protein concentration of cell lysate was determined by the BCA method (Pierce,

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Rockford, USA). Ten micrograms of total protein per sample was loaded onto sodium dodecylsulfate

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polyacrylamide gel electrophoresis (SDS–PAGE) at 80 V for 3-4 h and transferred to PVDF

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membrane at 350 mA for 90 min (Version7, Roche, USA) using an electro-blotting method. After

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incubating in blocking buffer [PBST with 1% (w/v) BSA (A7030, Sigma)] for 1 h, membranes were incubated with rabbit polyclonal antibody for OGG1 (ab135940, Abcam, USA) or rabbit polyclonal

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antibody for TET1 (ab157004, Abcam, USA) at 4℃ for 12 h. After primary antibodies were used, the

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membranes were washed before Horseradish Peroxidase (HRP)-conjugated Goat anti-rabbit IgG

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second-antibody (sc-2030, Santa Cruz, USA) was added for 1 h at room temperature and washed again. The membranes were visualized with an ECL Western blot detection kit (NC15080, Thermo). The TBB5 (Cat#AM1031A, Abgent, China) protein level was also examined as an internal control. The chemiluminescence intensity of each protein band was quantified using the Image J program, and then protein levels were normalized by the amount of TBB5 protein.

2.6. Immunoprecipitation assay Cell extracts were diluted with IP buffer (50 mM Tris–HCl pH 8.0, 100 mM NaCl, 5 mM MgCl2, 1% NP-40). Antibodies were incubated with protein A/G agarose (SC2003, Santa Cruz, USA) in advance and then added to the diluted cell extract. After an overnight incubation, the beads were washed with IP buffer and the immunoprecipitated proteins were analysed by western blotting. The 7

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co-immunoprecipitation was performed with and without ethidium bromide (EtBr) according to the

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method of S. Giri [35]. Recombinant Flag-tagged human TET1 (hTET1) protein was purified from HEK293 cells transfected with (full-length) Flag-hTET1 plasmid. Purified recombinant hTET1 and

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hOGG1 (M0241, NEB, USA) proteins were incubated in IP buffer with or without 8-oxoG

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oligonucleotides (double strand) at 4 °C for overnight, followed by co-precipitation using anti-flag M2

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beads (M8823, Sigma, USA) or protein A/G agarose (SC2003, Santa Cruz, USA) using antibodies

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against Flag or OGG1 (sc-376935, Santa Cruz, USA) as indicated. The beads were washed five times using IP buffer and the immunoprecipitated proteins were analysed by western blotting.

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Normal rabbit IgG (sc-2027, Santa Cruz, USA) or normal mouse IgG (sc-2025, Santa Cruz, USA)

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was used as a negative control. The antibodies used were as follows: mouse monoclonal antibody

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for OGG1 (sc-376935, Santa Cruz, USA), mouse monoclonal antibody for Flag (ab49763, Abcam, USA), and rabbit polyclonal antibody for TET1 (ab157004, Abcam, USA). Antibodies were used in the amount of 3 μg per IP. Oligonucleotide information for the co-immunoprecipitation is available in the supplemental material (Table S2).

2.7. Immunofluorescence assay Cells were grown on coverslips. Next, cells on the coverslips were fixed with 4% paraformaldehyde at 4℃ for 10 h, then rinsed in PBS for 3 times every 5 min, incubated in 100% methanol for 10 min at -20°C, and then rinsed once in PBS for 5 min. After incubation in blocking buffer (PBS with 0.2% Triton X-100 and 1% BSA) for 2 h, the coverslips were incubated overnight at 4℃ with mouse monoclonal antibody for OGG1 (sc-376935, Santa Cruz, USA) and rabbit polyclonal antibody for 8

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TET1 (ab191698, Abcam, USA). Next, the coverslips were incubated with goat anti-mouse IgG 488

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(green) (A11001, Invitrogen, USA) and goat anti-rabbit IgG 594 (red) (A11012, Invitrogen, USA) for

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taken using a laser scanning confocal microscopy (Nikon).

Fluorescent images were

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1 h in the dark. After, the cell nuclei were stained with DAPI for 20 min.

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2.8. Chromatin immunoprecipitation assay

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Formaldehyde was added at a final concentration of 1% directly to media of MCF-10A cells. Fixation proceeded at room temperature for 10 min and was stopped by the addition of glycine to a final

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concentration of 0.125 M for 15 min. Cells were centrifuged and rinsed 3 times in cold PBS with 1

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mM PMSF. Then, cell nuclei were collected according to the manufacturer’s protocol, SimpleChIP

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Enzymatic CHIP Kit (#9002, Cell Signalling Technology, USA). Samples were sonicated on ice with an Ultrasonics sonicator at setting 5 for six 10 s pulses to an average chromatin length of approximately 300 to 800 bp. For the immunoprecipitation, rabbit polyclonal antibodies for TET1 (ab191734, Abcam, USA) and rabbit polyclonal antibodies for OGG1 (ab135940, Abcam, USA) were added in combination to the nuclear sonicate. After the immunoprecipitation, the IP was eluted and the DNA was recovered. DNA obtained from IP samples were quantified by real-time PCR and normalized to input DNA control samples. Primer information for the ChIP assay is available in the supplemental material (Table S1).

2.9. Methylated DNA immunoprecipitations assay (MeDIP) Genomic DNA was extracted by overnight proteinase K digestion in lysis buffer (50 mM Tris-HCl pH 9

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8.0, 10 mM EDTA pH 8.0, 0.5% SDS) prior to phenol-chloroform extraction, ethanol precipitation

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and RNaseA digestion. Genomic DNA was sonicated to produce DNA fragments ranging in size from 300 to 800 bp. Fragmented DNA (5 μg for MeDIP) was denatured for 10 minutes at 95°C and

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immunoprecipitated overnight at 4°C with 2 μg Mouse monoclonal antibody for 5mC (ab10805,

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Abcam, USA) in a final volume of 500 μl immunoprecipitation (IP) buffer (10 mM sodium phosphate

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pH 7.0, 140 mM NaCl, 0.5% Triton X-100). The mixture was incubated with 60 μl protein A/G agarose (SC2003, Santa Cruz, USA) for 2 h prior to washing all unbound fragments three times with

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1 ml IP buffer. Washed beads were then resuspended in 250 μl of lysis buffer and incubated with

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proteinase K for 2 h at 50°C. Immunoprecipitated DNA fragments were then purified using DNA

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purification columns (28104, QIAGEN, Germany) and eluting into 20 μl TE. For qPCR analysis, 10μl

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were diluted in 100 μl TE with each qPCR reaction using 2 μl of diluted DNA. DNA copies in immunoprecipitation samples were normalized to input DNA control samples. Primer information for MeDIP is available in the supplemental material (Table S1).

2.10. Statistics Data are presented as means ± SEM. Significant differences were analysed by paired student’s tests or one-way analysis of variance (ANOVA) using the SPSS software, version 16.0 (SPSS Inc., Chicago, IL, USA). P-values < 0.05 were considered to be statistically significant.

3. Results

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3.1. H2O2 induces global genomic DNA demethylation

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To verify whether oxidative stress could induce DNA demethylation, HeLa, MCF-7 and MCF-10A cells were treated with 500 μM H2O2 for 6 hours. Genomic DNA was extracted and methylation

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levels were measured by dot blot using anti-5mC antibody. The results showed that the amount of

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5mC decreased significantly after H2O2 treatment (Figs. 1A-F), indicating global DNA demethylation

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occurred due to oxidative stress.

3.2. H2O2-induced global genomic DNA demethylation is OGG1-dependent

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In mammalian cells, guanine can be oxidized to form 8-oxoG under oxidative stress. More

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interesting, 5mC is susceptible to oxidation and can convert to 5hmC, when adjacent to 8-oxoG

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[30-34]. Because OGG1 protein is specific for 8-oxoG binding, we therefore hypothesized OGG1 is involved in oxidative stress-induced DNA demethylation. Because we demonstrated that H2O2 caused demethylation in the previous section, we tested whether H 2O2 specifically induced generation of 8-oxoG. Indeed, cells had significantly higher levels of 8-oxoG after H2O2 treatment than the control cells, which suggests 8-oxoG is involved in DNA demethylation (Fig. 2A). To investigate the role of OGG1 in DNA demethylation, we knocked down OGG1 in MCF-10A cells by siRNA (Fig. 2C). While H2O2 induced DNA demethylation dramatically in control cells, H2O2-induced global genomic DNA demethylation is inhibited in OGG1 knockdown cells (Fig. 2E). These data indicate that OGG1 is involved in H2O2-induced DNA demethylation.

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To verify further the role of OGG1 in DNA demethylation induced by H2O2, we used a MEF cell line

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where Ogg1 is completely absent (Fig. 3A). While H2O2 treatment in WT MEF cells induces global DNA demethylation, DNA methylation did not decrease in Ogg1 null MEF cells (Figs. 3B and C).

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However, over-expression of exogenous OGG1 in Ogg1 null MEF cells (Fig. 3D) restored DNA

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demethylation after H2O2 treatment (Figs. 3E and F). These results imply that OGG1 is essential in

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H2O2 induced DNA demethylation.

3.3. TET1 is associated with H2O2-induced DNA demethylation and interacts with OGG1 in MCF10A

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cells

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The TET protein family are part of the main pathway for active DNA demethylation. The process that

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converts 5mC to 5hmC via TET proteins is considered the main mechanism for active DNA demethylation [36]. To test whether the TET pathway reacts to H2O2-induced DNA demethylation, we monitored the change of 5hmC levels after H2O2 treatment. As shown in Figure 4A, the 5hmc level increased in 3 hours after H2O2 treatment. These data demonstrate that an unknown TET protein is involved in H2O2-induced DNA demethylation. To identify which TET protein responds to the H2O2 treatment, we measured the ability of OGG1 to interact with each of the three TET proteins. Co-immunoprecipitation assays revealed that OGG1 only interacted with TET1 (Figs. 4C and D) and neither of the other two proteins (data not shown). To investigate whether OGG1 and TET1 interact

during

oxidative

stress,

cells

were

treated

with

H2O2 followed

by

another

co-immunoprecipitation assay. The co-immunoprecipitation result indicated that OGG1 and TET1 interacted even more during oxidative stress (Fig. 4E). To test if the interaction between OGG1 and 12

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TET1 is DNA dependent, EtBr was added to the cell lysate to disrupt the association between DNA

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and proteins. Addition of EtBr did not affect the interaction between OGG1 and TET1, which showed

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the OGG1/TET1 interaction is DNA-independent (Fig. 4F).

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To further confirm that OGG1 and TET1 interact physically, we performed pull down assays using

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purified OGG1 and TET1 proteins. The pull down assays showed OGG1 directly interacts with TET1

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(Figs. 4G and H). To confirm the previous results that OGG1 and TET1 interact directly (Figure 4F), 8-oxoG was added to the pull down assay buffer and did not affect the OGG1/TET1 interaction, (Figs. 4I and J). Furthermore, the 8-oxoG

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further suggesting this interaction is not DNA-dependent

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oligonucleotide still could pull down TET1 protein in the presence of OGG1. However, the 8-oxoG

results

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oligonucleotide could not pull down TET1 in the absence of OGG1 (Figure 4K). These pull down that

TET1

binds

8-oxoG

oligonucleotide

by

interacting

with

OGG1.

Immunofluorescence of TET1 and OGG1 in cells also shows how they interact by foci formation after oxidative stress (Fig. 4L). These immunofluorescence results imply that OGG1 recruits TET1 to 8-oxoG lesions and facilitates DNA demethylation of CpGs after H2O2 treatment.

3.4. OGG1 and TET1 are associated with H2O2 induced demethylation on CpG island of BACE1 promoter To determine how OGG1 targets a specific gene during H 2O2-induced DNA demethylation, we compared gene expression profiles from cells treated with or without H2O2. mRNA levels for BACE1 were significantly elevated after H2O2 treatment, which suggests that CpG islands in its promoters 13

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were targeted for DNA demethylation [37]. Therefore, BACE1 was the gene chosen to study the

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mechanism of DNA demethylation induced by H2O2. As shown in figure 5A, H2O2 treatment enhances BACE1 expression in control cells, but knock-down of OGG1 caused BACE1 expression

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to remain the same during H2O2 treatment. Elevation of BACE1 mRNA levels after H2O2 treatment

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could result from DNA demethylation at CpG islands in the BACE1 promoter region. To test the

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methylation status of CpG islands in the BACE1 promoter region, MeDIP was performed. MeDIP

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analysis showed that the 5mC levels of CpG islands in the BACE1 promoter were significantly decreased after H2O2 treatment in control cells. However, the in OGG1 knockdown cells remained

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unchanged (Fig. 5B). Because TET1 was shown earlier to interact with OGG1 in cells, we speculate

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that OGG1 may also coordinate with TET1 on the BACE1 gene promoter. To test this hypothesis of

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TET1 recruitment to the BACE1 promoter, H2O2 treatment could be used to signal OGG1 at the BACE1 promoter region and thereby recruit TET1. To determine whether OGG1 recruits TET1 to the BACE1 promoter, we performed CHIP assays to measure the presence of OGG1 and TET1 on BACE1 CpG islands. The CHIP assays indicated a significant increase of OGG1 and TET1 recruitment after H2O2 treatment (Fig. 5C). Furthermore, we performed CHIP assays in MEF WT and MEF Ogg1-/- cells to determine whether TET1 recruitment to BACE1 CpG islands depended upon OGG1. The absence of OGG1 indicated that TET1 recruitment to BACE1 CpG islands depends on OGG1 after H2O2 treatment (Fig. 5D). Taken together, these results suggest that OGG1 interacts with TET1 after H2O2 treatment causing DNA demethylation at CpG islands in the BACE1 promoter region to increase gene expression.

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4. Discussion

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In this study, we demonstrated that 8-oxoG and OGG1 are signal molecules for DNA demethylation via oxidative stress. Previous reports believed that 8-oxoG is a damaged base in need of repair by

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the BER pathway. However, we show that 8-oxoG also serves as a DNA demethylation signal to

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switch on the expression of genes responsible to relieve stress. Indeed, we observed that H 2O2

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generates 8-oxoG lesions (Fig. 2A) as well as subsequent DNA demethylation (Figs. 1A, C and E)

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and gene expression (Fig. 5A). A TET protein initially activates DNA demethylation enzymatically by converting 5mC to 5hmC. However, TET1 protein did not directly bind 8-oxoG (Fig. 4K). Instead,

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OGG1 binds 8-oxoG and recruits TET1 (Figs. 4C-L). Thus, we believe OGG1 is the bridge between

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the DNA demethylation signal (oxidative stress and 8-oxoG) and the initial demethylation step

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(TET1 protein) (Fig. 6). Our model is supported by other reports where 8-oxoG promotes demethylation of adjacent 5mC [30-34].

Though DNA demethylation is essential to regulation of gene expression, how DNA demethylation is triggered remains unclear. TET family proteins are known to be involved somehow in active DNA demethylation. TET proteins convert 5mC to 5hmC, 5-formylcytosine (5fC), or 5-carboxylcytosine (5caC) [9-11]. 5fC and 5caC are recognized and excised by TDG and replaced with unmodified cytosines through the DNA BER pathway [38]. TDG gene expression is low in mouse zygotes, and its function can be replaced by Uracil-DNA glycosylase (UNG2) [39]. Recently, mismatch repair was discovered to be involved in active DNA demethylation [40]. TET family proteins can also induce passive DNA demethylation. Over-expression of TET proteins in HEK293T cells induces cell 15

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proliferation predominantly by the passive DNA demethylation pathway [41]. Here, we found that

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H2O2 rapidly reduces global DNA methylation (Figs 1A, C and E). However, in BEAS-2B cells for long-term treatment, the level of global DNA methylation will be increased after exposed with H2O2

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for 3 days [42].

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8-oxoG is an abundant oxidized base lesion induced by environmental oxidative stress or various

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cellular oxidoreductases. OGG1 is the primary enzyme responsible for excision of 8-oxoG lesions. Some studies have suggested that OGG1 is recruited to open chromatin regions for 8-oxoG repair

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to occur after 3 h treatment with KBrO3 [43]. Ogg1 null mice have higher rates of cancer, which could

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be due to DNA demethylation [24]. Despite the primary function of OGG1 is to prevent mutagenesis,

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recent studies have shown that cytosolic OGG1 responds to excessive 8-oxoG and activates small GTPases and downstream signalling that change gene expression [44-47]. In MDA-231 cells, estrogen induces demethylation of H3K9me2 by activating LSD1 by recruiting OGG1 and topoisomerase II β to the bcl-2 and pS2 promoters to induce gene expression [48]. Formation of 8-oxoG in CpG sequences strongly inhibits methylation of adjacent cytosine residues [32, 34]. Therefore, OGG1 might serve as a transcriptional regulator of oxidative stress-induced DNA demethylation. Here, we find that OGG1 also responds to H2O2-induced DNA demethylation (Figs. 2E and 3E). DNA methylation of Ogg1 null MEF cells presented low sensitivity to oxidative stress (Fig. 3B). Others have demonstrated that NEIL enzymes also remove DNA 8-oxoG lesions, such as OGG1, but they do not directly bind and process oxidized methylcytosine. Neil2 alongside Tdg and Tet3 remove genomic 5fC and 5caC caused by oxidative DNA demethylation in early Xenopus 16

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embryos [49].

TET family proteins (TET1, TET3 and TET3) share a conserved cysteine-rich domain and

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significantly differ in their dioxygenase motif involved in binding Fe (II) and α-ketoglutarate (α-KG).

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TET1 is best understood in embryonic stem (ES) cells and responsible for ES cells’ self-renewal and

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differentiation. Little is known about the role of TET1 in differentiated cells. Over-expression of both

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mouse Tet1 and Tet2 catalytic domains in U2OS and HEK293 cells greatly reduces global 5mC levels, but over-expression of the mouse Tet3 catalytic domain in these cells has no effect on global

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5mC levels [50]. In contrast, over-expression of full-length TET1 has little effect on global genomic

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DNA methylation. The reason full-length TET1 has little effect on global genomic methylation is

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probably due to its CXXC binding domain that specifically targets TET1 to hypomethylated regions and strongly limits the 5hmC production capacity in HEK293T cells [51]. TET1 is mainly enriched in unmethylated CpG-rich genomic regions [52-54]; but stresses on the system such as during aging, in cancer or in inflammatory conditions, subtle DNA methylation changes are induced by TET1 [55-57]. Recent studies have suggested that TET1 regulates transcription in mouse embryonic stem cells (ESCs). TET1 also regulates gene transcription through interacting with O-linked N-acetylglucosamine transferase or recruiting PRC2 and Sin3a [53, 58, 59]. Thus, we suggested TET1 may be involved in oxidative stress-induced DNA demethylation and gene expression for stress relief In the present study, we have demonstrated OGG1 interacts with TET1 (Figs. 4C~L). Furthermore, the interaction between OGG1 and TET1 increases under oxidative stress (Fig. 4E); 8-oxoG lesions formed by the oxidative stress enhance recruitment of OGG1 and TET1 to the DNA 17

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8-oxoG lesions. We also found the interaction between OGG1 and TET1 is not DNA dependent

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(Figs. 4F, I and J). Specifically, TET1 is recruited to 8-oxoG sites via OGG1; but TET1 cannot bind 8-oxoG directly (Fig. 4K). To determine how OGG1 effects DNA demethylation, we found that OGG1

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causes specific DNA demethylation in response to oxidative stress at the promoter CpG island of

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the BACE1 gene (Figs. 5A and B), Despite the decreased global DNA methylation under oxidative

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stress, recent studies have shown that some antioxidant enzymes are silenced via promoter

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hypermethylation under oxidative stress [60, 61]. Furthermore, we showed that OGG1 recruits TET1 to cause DNA demethylation of the CpG islands of the BACE1 promoter under oxidative stress (Figs.

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5. Conclusions

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5C and D).

The data presented in this study indicate that the first enzyme in BER-pathway, OGG1, is also involved in oxidative stress-induced DNA demethylation. The tight correlation between 8-oxoG repair and demethylation of methyl-CpGs suggest a model in which oxidative stress recruits OGG1/TET1 complex proteins to 8-oxoG and facilitates conversion of adjacent 5mC to 5hmC, 5fC, 5caC, and finally restore the normal C. If the repair occurs at CpG islands, genes related to oxidative stress may also likely change expression.

Disclosure of Potential Conflicts of Interest The authors declare that there is no conflict of interest.

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Acknowledgements

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We thank Professor Istvan Boldogh for kindly supplying MEF WT and Ogg1 null cell lines. This work was supported by National Key Basic Research Program of China for young scientists

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(2013CB911600), The General Program (Key Program, Major Research Plan) of the National

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Natural Science Foundation of China (31271449), the Jiangsu Province Natural Science Fund for

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Distinguished Young Scholars (BK20130044), the State Key Program of Jiangsu Province Natural

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Science Foundation (BK20130061), the Research Fund for the Doctoral Program of Higher Education of China (RFDP) (20133207110005), the Program for New Century Excellent Talents in

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University of Ministry of Education of China (NCET-13-0868), the Jiangsu province natural science

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foundation projects (BK20141448), a Project Funded by the Priority Academic Program

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Development of Jiangsu Higher Education Institutions (20110101) and the Postdoctoral Fund in Jiangsu Province (1401040C).

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Figure Legends

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Fig. 1. H2O2 induces global genomic DNA demethylation in different cells. (A, C and E) MCF-10A, MCF-7 and HeLa cells were treated with 500 μM H2O2 for 6 hours. Total Genomic DNA was

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collected and 100 ng spotted onto the Hybond-N+ membrane. DNA methylation levels were

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determined on the dot blot using anti-5mC antibody. Genomic DNA from cells with PBS treatment

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was used as negative control. (B, D and F) Quantification of the left panel using Image J software.

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The mean value in the control group was set as 1. Graphs show means ± SEM (n = 3/group).

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Letters denote significant (P<0.05) differences between values.

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Fig. 2. Knock-down of OGG1 by siRNA inhibits DNA demethylation caused by H2O2 induced in

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MCF-10A cell. (A) MCF-10A cells were treated with 500 μM H2O2 for 30 min. Total Genomic DNA was collected and 100 ng was spotted onto the Hybond-N+ membrane, and the 8-oxoG levels in total genomic DNA was measured on the dot blot using

anti-8oxoG antibody. (B) Quantification of

the panel A using Image J software. The mean value in the control group was set as 1. (C) Knock-down of OGG1 in MCF-10A Cells by siRNA. Cells were transfected with OGG1 siRNA or control scrambled siRNA (scr siRNA). OGG1 protein levels were determined by anti-OGG1 antibody. TBB5 was used as an internal loading control. (D) Quantification results of panel C. The relative expression levels were normalized to TBB5. (E) Cells pre-treated with OGG1 siRNA or control scrambled siRNA were subjected to H2O2 treatment for 6 hours. Total Genomic DNA was collected and 100 ng was spotted onto the Hybond-N+ membrane for dot blot assay using anti-5mC antibody. (F) Quantification of panel E using Image J software. The mean value in the control group was set 20

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as 1. Graphs show means ± SEM (n = 3/group). Letters denote significant (P<0.05) differences

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between values.

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Fig. 3. OGG1 is essential for H2O2 induced DNA demethylation in MEF cells. (A) Verification of

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OGG1 levels in MEF WT and Ogg1-/- cells was measured using anti-OGG1 antibody. TBB5 was

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used as an internal control. (B) MEF WT or Ogg1-/- cells were treated with H2O2 or PBS for 6 hours.

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Total genomic DNA was collected and 100 ng was spotted onto the Hybond-N+ membrane. The methylation level was determined by anti-5mC antibody. (C) Quantification of panel B. (D) Verify the

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over-expression of exogenous OGG1 in MEF Ogg1-/- cells. pcDNA3.1 vectors with or without Ogg1

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gene insertion were transfected into MEF Ogg1-/- cells. The expression of exogenous OGG1 was

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confirmed by western blot. (E) MEF Ogg1-/- cells transfected with pcDNA3.1-OGG1 or control were treated with H2O2 for 6 hours. Total genomic DNA was collected and 100 ng was spotted onto the Hybond-N+ membrane and analysed by dot blot using anti-5mC antibody. (F) Quantification of panel E. In each C and F, the mean value in the control group was set as 1. Graphs show means ± SEM (n = 3/group). Letters denote significant (P<0.05) differences between values.

Fig. 4. TET1 interacts with OGG1 during H2O2 induced DNA demethylation. (A) Total Genomic DNA of MCF-10A cells was collected after treatment with H2O2, 100 ng was spotted onto the Hybond-N+ membrane and the 5hmC level was determined by anti-5hmC antibody. (B) Quantification of panel A. The mean value in the control group was set as 1. Graphs show means ± SEM (n = 3/group). Letters denote significant (P<0.05) differences between values. C and D, Interaction between 21

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OGG1 and Tet1 in MCF-10A cells. (C) Immunoprecipitated samples by OGG1 antibody was

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subjected to western blot using anti-TET1 antibody. (D) Immunoprecipitated samples by TET1 antibody was subjected to western blot using anti-OGG1 antibody. (E) Samples immunoprecipitated

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using OGG1 antibody with or without H2O2 was tested by western blot using anti-TET1 antibody. (F)

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Samples immunoprecipitated using OGG1 antibody with or without EtBr was tested by western blot

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using anti-TET1 antibody. Purified OGG1 and Flag-TET1 were incubated with antibodies against

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Flag (G) or OGG1 (H). The pull down samples were tested by western blot analysis using the antibodies indicated. (I) OGG1, Flag-TET1 and antibodies against Flag (I) or OGG1 (J) were

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subjected to pull down assays with or without 8-oxoG present. Pull down samples were analysed by

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western blot using antibodies indicated. (K) Biotin labelled 8-oxoG oligonucleotide was incubated

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with OOG1 and Flag-TET1 was pulled down using streptavidin beads. Samples were tested by western blot using antibodies indicated. Anti-OGG1 or anti-Flag antibodies. (L) Localization of OGG1 and TET1 in the nucleus of MCF-10A cells treated with H2O2 for 3 hours. A white bar in the image indicates 10 μm.

Fig. 5. OGG1 and TET1 are involved in promoter demethylation of CpG islands at BACE1 during H2O2 treatment. (A) H2O2 induced BACE1 expression is prevented by OGG1 knock down. MCF-10A cells transfected with control scrambled siRNA or OGG1 siRNA were treated with H2O2 or PBS for 12 hours. The BACE1 mRNA in MCF-10A cells were analysed by qRT-PCR. The mean value in the control group was set as 1. (B) The effect OGG1 on methylation levels of BACE1 CpG islands in MCF-10A cells upon H2O2 treatment for 6 hours. MeDIP was performed using digested chromatin 22

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from the treatment groups as indicated. Following immunoprecipitation with an anti-5mC antibody,

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enrichment of the 5mC-containing DNA sequences was quantified by real-time PCR. Relative amounts of the 5mC-containing DNA sequences compared to the BACE1 input in each group were

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calculated (n = 3/group). Normal mouse IgG was used as the negative control. (C) Chromatin

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immunoprecipitations were performed using digested chromatin from the H 2O2 (3 hours) or control

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treatment groups of MCF-10A cells. Following immunoprecipitation with an anti-OGG1 or TET1

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antibody, enrichment of the OGG1 or TET1-containing DNA sequence was quantified by real-time PCR. (D) Chromatin immunoprecipitations were performed using digested chromatin from the H2O2

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(3 hours) or control treatment groups of MEF WT and Ogg-/- cells. Following immunoprecipitation

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with an anti-Tet1 antibody, enrichment of the Tet1-containing DNA sequence was quantified by

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real-time PCR. In C and D, relative amounts of the OGG1 or TET1-containing DNA sequence compared to the BACE1 input in each group were calculated (n = 3/group). Histone H3 rabbit antibody, Human RPL30 Exon 3 primers or Mouse RPL30 Intron 2 primers were used as the positive control, normal rabbit IgG as the negative control. Graphs show means ± SEM. Letters denote significant (P<0.05) differences between values.

Fig. 6. Model of OGG1 associated DNA demethylation. Upon oxidative stress, guanidine is oxidized to form oxo-dG. OGG1 binds and recruits TET1 to the oxo-dG lesion. TET1 then initiates the DNA demethylation process of the adjacent cytosine. The oxidative stress-induced genes are expressed.

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References [1] W. Droge, R. Kinscherf, Aberrant insulin receptor signaling and amino acid homeostasis as a major cause of oxidative stress in aging, Antioxid Redox Signal, 10 (2008) 661-678.

PT

[2] T. Mohammadpour, M. Hosseini, A. Naderi, R. Karami, H.R. Sadeghnia, M. Soukhtanloo, F. Vafaee, Protection against brain tissues oxidative damage as a possible mechanism for the beneficial effects of Rosa damascena

RI

hydroalcoholic extract on scopolamine induced memory impairment in rats, Nutr Neurosci, 18 (2015) 329-336. [3] B. Halliwell, Oxidative stress and cancer: have we moved forward?, Biochem J, 401 (2007) 1-11.

SC

[4] D.A. Clopton, P. Saltman, Low-level oxidative stress causes cell-cycle specific arrest in cultured cells, Biochem Biophys Res Commun, 210 (1995) 189-196.

[5] M. Valko, M. Izakovic, M. Mazur, C.J. Rhodes, J. Telser, Role of oxygen radicals in DNA damage and cancer

NU

incidence, Mol Cell Biochem, 266 (2004) 37-56.

[6] K.V. Donkena, C.Y. Young, D.J. Tindall, Oxidative stress and DNA methylation in prostate cancer, Obstet Gynecol Int, 2010 (2010) 302051.

MA

[7] S.O. Lim, J.M. Gu, M.S. Kim, H.S. Kim, Y.N. Park, C.K. Park, J.W. Cho, Y.M. Park, G. Jung, Epigenetic changes induced by reactive oxygen species in hepatocellular carcinoma: methylation of the E-cadherin promoter, Gastroenterology, 135 (2008) 2128-2140, 2140 e2121-2128.

D

[8] M.M. Dawlaty, A. Breiling, T. Le, M.I. Barrasa, G. Raddatz, Q. Gao, B.E. Powell, A.W. Cheng, K.F. Faull, F. Lyko, R. Jaenisch, Loss of Tet enzymes compromises proper differentiation of embryonic stem cells, Dev Cell, 29 (2014)

TE

102-111.

[9] C. Dahl, K. Gronbaek, P. Guldberg, Advances in DNA methylation: 5-hydroxymethylcytosine revisited, Clin Chim Acta,

AC CE P

412 (2011) 831-836.

[10] Y.F. He, B.Z. Li, Z. Li, P. Liu, Y. Wang, Q. Tang, J. Ding, Y. Jia, Z. Chen, L. Li, Y. Sun, X. Li, Q. Dai, C.X. Song, K. Zhang, C. He, G.L. Xu, Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA, Science, 333 (2011) 1303-1307.

[11] M. Tahiliani, K.P. Koh, Y. Shen, W.A. Pastor, H. Bandukwala, Y. Brudno, S. Agarwal, L.M. Iyer, D.R. Liu, L. Aravind, A. Rao, Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1, Science, 324 (2009) 930-935.

[12] K.C. Cheng, D.S. Cahill, H. Kasai, S. Nishimura, L.A. Loeb, 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions, J Biol Chem, 267 (1992) 166-172. [13] M. Dizdaroglu, Formation of an 8-hydroxyguanine moiety in deoxyribonucleic acid on gamma-irradiation in aqueous solution, Biochemistry, 24 (1985) 4476-4481. [14] S. Shibutani, M. Takeshita, A.P. Grollman, Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG, Nature, 349 (1991) 431-434. [15] N.H. Zawia, D.K. Lahiri, F. Cardozo-Pelaez, Epigenetics, oxidative stress, and Alzheimer disease, Free Radic Biol Med, 46 (2009) 1241-1249. [16] E. Zarakowska, D. Gackowski, M. Foksinski, R. Olinski, Are 8-oxoguanine (8-oxoGua) and 5-hydroxymethyluracil (5-hmUra) oxidatively damaged DNA bases or transcription (epigenetic) marks?, Mutat Res Genet Toxicol Environ Mutagen, 764-765 (2014) 58-63. [17] R. Cathcart, E. Schwiers, R.L. Saul, B.N. Ames, Thymine glycol and thymidine glycol in human and rat urine: a possible assay for oxidative DNA damage, Proc Natl Acad Sci U S A, 81 (1984) 5633-5637. 24

ACCEPTED MANUSCRIPT [18] A.K. Basu, E.L. Loechler, S.A. Leadon, J.M. Essigmann, Genetic effects of thymine glycol: site-specific mutagenesis and molecular modeling studies, Proc Natl Acad Sci U S A, 86 (1989) 7677-7681. [19] J.E. Dodge, B.H. Ramsahoye, Z.G. Wo, M. Okano, E. Li, De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation, Gene, 289 (2002) 41-48.

PT

[20] T.R. Haines, D.I. Rodenhiser, P.J. Ainsworth, Allele-specific non-CpG methylation of the Nf1 gene during early mouse development, Dev Biol, 240 (2001) 585-598.

RI

[21] R. Lister, E.A. Mukamel, J.R. Nery, M. Urich, C.A. Puddifoot, N.D. Johnson, J. Lucero, Y. Huang, A.J. Dwork, M.D. Schultz, M. Yu, J. Tonti-Filippini, H. Heyn, S. Hu, J.C. Wu, A. Rao, M. Esteller, C. He, F.G. Haghighi, T.J. Sejnowski, M.M.

SC

Behrens, J.R. Ecker, Global epigenomic reconfiguration during mammalian brain development, Science, 341 (2013) 1237905.

[22] R. Lister, M. Pelizzola, R.H. Dowen, R.D. Hawkins, G. Hon, J. Tonti-Filippini, J.R. Nery, L. Lee, Z. Ye, Q.M. Ngo, L.

NU

Edsall, J. Antosiewicz-Bourget, R. Stewart, V. Ruotti, A.H. Millar, J.A. Thomson, B. Ren, J.R. Ecker, Human DNA methylomes at base resolution show widespread epigenomic differences, Nature, 462 (2009) 315-322. [23] P. Fortini, E. Parlanti, O.M. Sidorkina, J. Laval, E. Dogliotti, The type of DNA glycosylase determines the base

MA

excision repair pathway in mammalian cells, J Biol Chem, 274 (1999) 15230-15236. [24] A. Klungland, I. Rosewell, S. Hollenbach, E. Larsen, G. Daly, B. Epe, E. Seeberg, T. Lindahl, D.E. Barnes, Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage, Proc Natl Acad Sci

D

U S A, 96 (1999) 13300-13305.

[25] I. Morland, V. Rolseth, L. Luna, T. Rognes, M. Bjoras, E. Seeberg, Human DNA glycosylases of the bacterial

TE

Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA, Nucleic Acids Res, 30 (2002) 4926-4936.

AC CE P

[26] M. Liu, V. Bandaru, J.P. Bond, P. Jaruga, X. Zhao, P.P. Christov, C.J. Burrows, C.J. Rizzo, M. Dizdaroglu, S.S. Wallace, The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo, Proc Natl Acad Sci U S A, 107 (2010) 4925-4930.

[27] N. Krishnamurthy, X. Zhao, C.J. Burrows, S.S. David, Superior removal of hydantoin lesions relative to other oxidized bases by the human DNA glycosylase hNEIL1, Biochemistry, 47 (2008) 7137-7146. [28] M.K. Hailer, P.G. Slade, B.D. Martin, T.A. Rosenquist, K.D. Sugden, Recognition of the oxidized lesions spiroiminodihydantoin and guanidinohydantoin in DNA by the mammalian base excision repair glycosylases NEIL1 and NEIL2, DNA Repair (Amst), 4 (2005) 41-50.

[29] M. Liu, S. Doublie, S.S. Wallace, Neil3, the final frontier for the DNA glycosylases that recognize oxidative damage, Mutat Res, 743-744 (2013) 4-11. [30] T. Masuda, H. Shinoara, M. Kondo, Reactions of hydroxyl radicals with nucleic acid bases and the related compounds in gamma-irradiated aqueous solution, J Radiat Res, 16 (1975) 153-161. [31] K.N. Rogstad, Y.H. Jang, L.C. Sowers, W.A. Goddard, 3rd, First principles calculations of the pKa values and tautomers of isoguanine and xanthine, Chem Res Toxicol, 16 (2003) 1455-1462. [32] P.W. Turk, A. Laayoun, S.S. Smith, S.A. Weitzman, DNA adduct 8-hydroxyl-2'-deoxyguanosine (8-hydroxyguanine) affects function of human DNA methyltransferase, Carcinogenesis, 16 (1995) 1253-1255. [33] V. Valinluck, H.H. Tsai, D.K. Rogstad, A. Burdzy, A. Bird, L.C. Sowers, Oxidative damage to methyl-CpG sequences inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2), Nucleic Acids Res, 32 (2004) 4100-4108. [34] S.A. Weitzman, P.W. Turk, D.H. Milkowski, K. Kozlowski, Free radical adducts induce alterations in DNA cytosine 25

ACCEPTED MANUSCRIPT methylation, Proc Natl Acad Sci U S A, 91 (1994) 1261-1264. [35] S. Giri, V. Aggarwal, J. Pontis, Z. Shen, A. Chakraborty, A. Khan, C. Mizzen, K.V. Prasanth, S. Ait-Si-Ali, T. Ha, S.G. Prasanth, The preRC protein ORCA organizes heterochromatin by assembling histone H3 lysine 9 methyltransferases on chromatin, Elife, 4 (2015).

PT

[36] H. Wu, Y. Zhang, Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation, Genes Dev, 25 (2011) 2436-2452.

RI

[37] X. Gu, J. Sun, S. Li, X. Wu, L. Li, Oxidative stress induces DNA demethylation and histone acetylation in SH-SY5Y cells: potential epigenetic mechanisms in gene transcription in Abeta production, Neurobiol Aging, 34 (2013) 1069-1079.

SC

[38] A. Maiti, A.C. Drohat, Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites, J Biol Chem, 286 (2011) 35334-35338. [39] J.H. Xue, G.F. Xu, T.P. Gu, G.D. Chen, B.B. Han, Z.M. Xu, M. Bjoras, H.E. Krokan, G.L. Xu, Y.R. Du, Uracil-DNA

NU

Glycosylase UNG Promotes Tet-mediated DNA Demethylation, J Biol Chem, 291 (2016) 731-738. [40] I. Grin, A.A. Ishchenko, An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation, Nucleic Acids Res, (2016).

MA

[41] C. Jin, T. Qin, M.C. Barton, J. Jelinek, J.P. Issa, Minimal role of base excision repair in TET-induced global DNA demethylation in HEK293T cells, Epigenetics, 10 (2015) 1006-1013. [42] Y. Niu, T.L. DesMarais, Z. Tong, Y. Yao, M. Costa, Oxidative stress alters global histone modification and DNA

D

methylation, Free Radic Biol Med, 82 (2015) 22-28.

[43] R. Amouroux, A. Campalans, B. Epe, J.P. Radicella, Oxidative stress triggers the preferential assembly of base

TE

excision repair complexes on open chromatin regions, Nucleic Acids Res, 38 (2010) 2878-2890. [44] L. Aguilera-Aguirre, K. Hosoki, A. Bacsi, Z. Radak, S. Sur, M.L. Hegde, B. Tian, A. Saavedra-Molina, A.R. Brasier, X.

AC CE P

Ba, I. Boldogh, Whole transcriptome analysis reveals a role for OGG1-initiated DNA repair signaling in airway remodeling, Free Radic Biol Med, 89 (2015) 20-33. [45] I. Boldogh, G. Hajas, L. Aguilera-Aguirre, M.L. Hegde, Z. Radak, A. Bacsi, S. Sur, T.K. Hazra, S. Mitra, Activation of ras signaling pathway by 8-oxoguanine DNA glycosylase bound to its excision product, 8-oxoguanine, J Biol Chem, 287 (2012) 20769-20773.

[46] G. Hajas, A. Bacsi, L. Aguilera-Aguirre, M.L. Hegde, K.H. Tapas, S. Sur, Z. Radak, X. Ba, I. Boldogh, 8-Oxoguanine DNA glycosylase-1 links DNA repair to cellular signaling via the activation of the small GTPase Rac1, Free Radic Biol Med, 61 (2013) 384-394.

[47] J. Luo, K. Hosoki, A. Bacsi, Z. Radak, M.L. Hegde, S. Sur, T.K. Hazra, A.R. Brasier, X. Ba, I. Boldogh, 8-Oxoguanine DNA glycosylase-1-mediated DNA repair is associated with Rho GTPase activation and alpha-smooth muscle actin polymerization, Free Radic Biol Med, 73 (2014) 430-438. [48] B. Perillo, M.N. Ombra, A. Bertoni, C. Cuozzo, S. Sacchetti, A. Sasso, L. Chiariotti, A. Malorni, C. Abbondanza, E.V. Avvedimento, DNA oxidation as triggered by H3K9me2 demethylation drives estrogen-induced gene expression, Science, 319 (2008) 202-206. [49] L. Schomacher, D. Han, M.U. Musheev, K. Arab, S. Kienhofer, A. von Seggern, C. Niehrs, Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation, Nat Struct Mol Biol, 23 (2016) 116-124. [50] S. Ito, A.C. D'Alessio, O.V. Taranova, K. Hong, L.C. Sowers, Y. Zhang, Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification, Nature, 466 (2010) 1129-1133. [51] C. Jin, Y. Lu, J. Jelinek, S. Liang, M.R. Estecio, M.C. Barton, J.P. Issa, TET1 is a maintenance DNA demethylase that prevents methylation spreading in differentiated cells, Nucleic Acids Res, 42 (2014) 6956-6971. 26

ACCEPTED MANUSCRIPT [52] A.P. Bird, CpG-rich islands and the function of DNA methylation, Nature, 321 (1986) 209-213. [53] H. Wu, A.C. D'Alessio, S. Ito, K. Xia, Z. Wang, K. Cui, K. Zhao, Y.E. Sun, Y. Zhang, Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells, Nature, 473 (2011) 389-393. [54] Y. Xu, F. Wu, L. Tan, L. Kong, L. Xiong, J. Deng, A.J. Barbera, L. Zheng, H. Zhang, S. Huang, J. Min, T. Nicholson, T. hydroxylase in mouse embryonic stem cells, Mol Cell, 42 (2011) 451-464.

PT

Chen, G. Xu, Y. Shi, K. Zhang, Y.G. Shi, Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1

RI

[55] S.B. Baylin, J.G. Herman, J.R. Graff, P.M. Vertino, J.P. Issa, Alterations in DNA methylation: a fundamental aspect of neoplasia, Adv Cancer Res, 72 (1998) 141-196.

SC

[56] J.P. Issa, CpG-island methylation in aging and cancer, Curr Top Microbiol Immunol, 249 (2000) 101-118. [57] J.P. Issa, N. Ahuja, M. Toyota, M.P. Bronner, T.A. Brentnall, Accelerated age-related CpG island methylation in ulcerative colitis, Cancer Res, 61 (2001) 3573-3577.

NU

[58] K. Williams, J. Christensen, M.T. Pedersen, J.V. Johansen, P.A. Cloos, J. Rappsilber, K. Helin, TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity, Nature, 473 (2011) 343-348. [59] F.T. Shi, H. Kim, W. Lu, Q. He, D. Liu, M.A. Goodell, M. Wan, Z. Songyang, Ten-eleven translocation 1 (Tet1) is

MA

regulated by O-linked N-acetylglucosamine transferase (Ogt) for target gene repression in mouse embryonic stem cells, J Biol Chem, 288 (2013) 20776-20784.

[60] S. Majumder, K. Ghoshal, Z. Li, Y. Bo, S.T. Jacob, Silencing of metallothionein-I gene in mouse lymphosarcoma

D

cells by methylation, Oncogene, 18 (1999) 6287-6295.

[61] K. Ghoshal, S. Majumder, Z. Li, X. Dong, S.T. Jacob, Suppression of metallothionein gene expression in a rat

AC CE P

TE

hepatoma because of promoter-specific DNA methylation, J Biol Chem, 275 (2000) 539-547.

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ACCEPTED MANUSCRIPT Highlights: 1. OGG1 is involved in oxidative stress induced DNA demethylation.

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2. OGG1 binds and recruits TET1 to the 8-oxodG adjacent 5mC site.

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3. This manuscript illustrates the important roles of BER in DNA demethylation.

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