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Originally published In Press as doi:10.1074/jbc.M505526200 on August 22, 2005

J. Biol. Chem., Vol. 280, Issue 42, 35272-35280, October 21, 2005
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Induction of the Human Oxidized Base-specific DNA Glycosylase NEIL1 by Reactive Oxygen Species*{boxs}

Aditi Das{ddagger}, Tapas K. Hazra{ddagger}, Istvan Boldogh§, Sankar Mitra{ddagger}1, and Kishor K. Bhakat{ddagger}

From the {ddagger}Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, the §Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555

Received for publication, May 20, 2005 , and in revised form, August 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
NEIL1, a mammalian DNA glycosylase and ortholog of Escherichia coli Nei/Fpg, is involved in the repair of oxidatively damaged bases in mammalian cells. Exposure of HCT116 human colon carcinoma cells to reactive oxygen species, generated by glucose oxidase (GO), enhanced the levels of NEIL1 mRNA and polypeptide by 2–4-fold by 6 h after GO treatment. A similar oxidative stress-induced increase in human NEIL1 (hNEIL1) promoter-dependent luciferase expression in HCT116 cells indicates that reactive oxygen species activates NEIL1 transcription. The transcriptional start site of hNEIL1 was mapped, and the upstream promoter sequence was characterized via luciferase reporter assay. Two identical CRE/AP-1-binding sites were identified in the promoter that binds transcription factors c-Jun and CREB/ATF2. This binding was significantly enhanced in extracts of cells treated with GO. Furthermore, a simultaneous increase in the level of phosphorylated c-Jun suggests its involvement in up-regulating the NEIL1 promoter. Oxidative stress-induced activation of NEIL1 appears to be involved in the feedback regulation of cellular repair activity needed to handle an increase in the level of oxidative base damage.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
ROS,2 generated endogenously and as a result of environmental insult, induce oxidative DNA damage, which could affect the integrity of cellular genomes. Oxidative DNA damage, including a multitude of damaged bases, abasic (activator protein (AP)) sites, and DNA strand breaks, is believed to be responsible for sporadic mutations leading to cancer and other pathological as well as age-related syndromes (1–4). Most of the oxidative damage in DNA is repaired via the base excision repair pathway, in which oxidized and altered bases are excised by DNA glycosylases in the first step of repair (5). Base excision repair is highly conserved among all organisms ranging from Escherichia coli to humans. In E. coli, three oxidatively damaged base-specific glycosylases, endonuclease III (Nth), formamidopyrimidine-DNA glycosylase (Fpg), and endonuclease VIII (Nei), excise modified DNA bases with overlapping specificity. Based on structural similarity and reaction mechanism, these enzymes are classified in two families with Nth in one and Nei and Fpg in the other (6–8). In mammalian cells, only two DNA glycosylases, both belonging to the Nth family, were identified earlier for excision of oxidized bases. NTH1 removes mostly oxidized pyrimidines, e.g. thymine glycol, whereas 8-oxoguanine DNA glycosylase (OGG1), functionally similar to Fpg but mechanistically analogous to Nth, is most active with 8-oxoguanine and formamidopyrimidine-guanine substrates (9, 10). Most surprisingly, both NTH1- and OGG1-null mice are viable and show no major phenotype despite the known mutagenic and toxic effects of their substrate lesions (11, 12). These results suggested the presence of additional DNA glycosylases in mammals that could serve as back-up enzymes for NTH1 and OGG1.

We and subsequently several other groups identified and characterized two additional human DNA glycosylases specific for oxidatively damaged bases and named them NEIL1 and NEIL2 (13–17). Both of these enzymes belong to the Fpg/Nei family; NEIL1 and NEIL2 share weak sequence similarity but have common characteristics, including overlapping substrate ranges (15–19). However, NEIL1 differs from NEIL2 because of its S-phase-specific activation, whereas NEIL2 expression is independent of the cell cycle (13, 14). We have shown recently that both NEIL1 and NEIL2 possess an unusual ability to excise lesions from the DNA bubble or single-stranded structures that could not be carried out by OGG1 or NTH1 (20). This novel substrate structure preference of NEILs raises the possibility of their specialized in vivo functions for repair of oxidized bases in transient bubbles formed during replication and/or transcription.

Taken together, these results support the scenario that OGG1 and NTH1 are involved primarily in repair of oxidative damage formed in inactive sequences constituting the bulk of the genome. In contrast, NEIL1 and NEIL2 may be preferentially involved in repair of oxidized bases from the active sequences during replication or transcription, which should be more urgent than the repair of inactive sequences. In this report, we examined the possibility of modulation of NEIL1 activity, and we observed that human NEIL1 (hNEIL1) is activated by ROS. We then characterized the hNEIL1 promoter and identified a pair of CRE/AP-1 sequences involved in the oxidative stress response.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Chemicals—The human colorectal carcinoma line HCT116 (with wild type p53), a gift of B. Vogelstein (The Johns Hopkins University), was cultured in McCoy's 5A modified medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 µg/ml; Invitrogen). At 70% confluence, the cells were treated with glucose oxidase (GO; Roche Applied Science) at 100 ng/ml for 1 h, followed by washing with and incubation in fresh medium. This treatment did not affect cell viability, as judged by the rate of cell growth.

Measurement of Intracellular ROS Level—Changes in intracellular ROS level after GO treatment were determined as described previously (21). Briefly, the cells were suspended in phosphate-buffered saline and then treated with 5 µM 5-(and-6) carboxy-2', 7'-dichlorodihydrofluorescein diacetate (H2DCF-DA; Molecular Probes) for 15 min at 37 °C. In control experiments, we determined that cellular uptake of H2DCF-DA reached a plateau at about 18 min when 0.6% of DCF, the oxidized form of H2DCF, was released in the medium (data not shown). As a control, we also treated the cells with DCF, and we observed a small increase in intracellular fluorescence (<5%) after glucose oxidase treatment (data not shown). The change in DCF fluorescence of treated versus mock-treated cells was determined by flow cytometry (BD Biosciences FAC-Scan). The mean fluorescence for 12,000 cells, from three or more independent experiments, was analyzed, after correction for DCF extrusion in the medium, and expressed as ±S.E. (Fig. 1).

Western Analysis—Cells were washed with ice-cold phosphate-buffered saline, lysed on ice for 30 min in a lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture; Roche Applied Science), and then microcentrifuged. Western analysis of the supernatant was carried out with antibodies to hNEIL1 (Alpha Diagnostics, San Antonio, TX), CREB, ATF2, c-Jun, and phospho-c-Jun (Santa Cruz Biotechnology), using enhanced chemiluminescence assay (ECL kit; Amersham Biosciences); the signals were quantitated by densitometric scanning.

RT-PCR Analysis—RNA extraction was carried out with the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. Total RNA (2–5 µg) was reverse-transcribed using a Titan one-step RT-PCR kit (Roche Applied Science), followed by a limiting cycle amplification protocol using the following primer combinations: hNEIL1 forward, 5'-CGG CGG CTG CGT GGA GAA GTC-3', and reverse, 5'-GTC CCA GCG GCC GAA CCG GCG-3', designed to amplify a 300-bp region; GAPDH forward, 5'-GTG AAG GTC GGA GTC AAC-3', and reverse, 5'-GGTGAAGACGCCAGTGGA-3', designed to amplify a 294-bp region (total number of cycles: 24 for hNEIL1 and 20 for GAPDH). The products of the semiquantitative RT-PCR were resolved in a 1.8% agarose gel, stained with ethidium bromide, and photographed.

For quantitative, real time RT-PCR, one-step RT-PCR was performed with 100 ng of cellular RNA for both the target gene and an endogenous control in single-plex tubes using TaqMan one-step RT-PCR master mix reagent kit (P/N 4309169). The cycling parameters (for 40 cycles) in an ABI7000 thermal cycler were as follows: reverse transcription at 48 °C for 30 min, AmpliTaq activation 95 °C for 10 min, denaturation 95 °C for 15 s, and annealing/extension 60 °C for 1 min. Duplicate CT values were calculated using Microsoft Excel and using a comparative CT({Delta}{Delta}CT) method as described by the manufacturer (Applied Biosystems). The amount of target (2–{Delta}{Delta}CT) was calculated after normalization to the 18 S RNA as an endogenous reference, and relative to a calibrator (one of the experimental samples).

Electrophoretic Gel Mobility Shift Analysis (EMSA)—Nuclear extracts from HCT116 cells were prepared as described earlier (22). For EMSA, 5 µg of nuclear extract was incubated with 2 pmol of 5'-32P-labeled duplex oligonucleotides and 1 µg of poly(dI-dC)·(dI-dC) in a binding buffer containing 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, and 10 mM Tris-HCl, pH 7.5, for 30 min at room temperature, followed by electrophoresis in 6% nondenaturing polyacrylamide gel in 0.5 x Tris borate/EDTA (TBE) at 200 V for 1 h. The gels were dried and subjected to PhosphorImager analysis. Competition of binding was performed in the presence of a 50- or 100-fold excess of the unlabeled oligonucleotide and incubating for 20 min at room temperature before adding the probe. Supershift assays were performed by preincubating the assay mixture with 4 µg of antibody against CREB/ATF2 or c-Jun for 1 h at 4°C before the addition of 32P-labeled oligonucleotide duplex probe.

Genomic Cloning of the 5'-Upstream Region of hNEIL1—An NdeI/XhoI fragment of the hNEIL1 cDNA was used to screen a BAC DNA library (Resgen) under stringent conditions. Restriction mapping and Southern blot analysis of BAC clones identified 18-kb HindIII, 13-kb EcoRI/HindIII, and 12-kb BamHI/HindIII fragments that hybridized to the probe. The 13-kb EcoRI/HindIII fragment was further subcloned into pBluescript and sequenced. The sequences of the genomic clone were confirmed by matching with the published NEIL1 gene sequence (GenBankTM accession number AAH10876 [GenBank] ). Analysis of candidate cis-elements to identify relevant transcription factors was performed using the programs version 2.2. Parameters for MatInspector were set for 1.0 core similarity (a 4-nucleotide highly conserved sequence) and 0.85 matrix similarity, employing the vertebrate matrix group.

Primer Extension Analysis—An antisense primer, P2 (5'-GGCTGAGGCAGGAGAATC-3'), complementary to the 5'-untranslated region of the hNEIL1 cDNA, near the beginning of exon 1, was 5'-32P-labeled using [{gamma}-32P]ATP and T4 polynucleotide kinase and annealed to 50 µg of total RNA at 58 °C for 20 min. The primer extension was performed with the annealed RNA DNA duplex, using 5 units of avian myeloblastosis virus-reverse transcriptase (Promega) at 42 °C for 1 h in the supplied extension buffer, and the products were resolved in a 6% polyacrylamide sequencing gel. A control reaction was set up with a second antisense oligo P1, further downstream to the P2 sequence. Sequencing reactions, generated by Sanger's dideoxy termination method, using primer P2 annealed to the hNEIL1 plasmid, were run in parallel to determine the exact length of the primer extension products.

Plasmids, Transfection, and Luciferase Assay—A 2.1-kb fragment spanning the upstream sequence of hNEIL1, including part of the first exon, was subcloned from the 13-kb genomic clone at the XhoI/HindIII sites of the promoterless luciferase vector pGL2-basic (Promega), and the recombinant plasmid was named p(–2100) hNEIL1 luc. Starting with this plasmid, we generated several deletion constructs by PCR amplification, with sense primers representing different segments of the 5' regions of hNEIL1; the 5' deletion fragments were subsequently fused upstream of the luciferase gene in the expression vector. The sequences of sense primers to generate these reporter plasmid constructs are given in TABLE ONE. All of these were used in combination with a common antisense primer, 5'-CTT CCC GGG TTC AAG TGA TTC TCC TGC CTC-3'. Point mutations were generated in p(–1800/+40)NEIL1 luc with the QuikChange site-directed mutagenesis kit (Stratagene) per the manufacturer's instructions. The following oligo sets used are as follows (mutated nucleotides are underlined): distal CRE/AP-1 site, 5'-GGC AGG CAG ATC ACC TGA GGT TGG GAG TTT GAG ACC AGC-3' and 5'-GCT GGT CTC AAA CTC CCA ACC TCA GGT GAT CTG CCT GCC-3'; proximal CRE/AP-1 site, 5'-GGC GGG CAG ATT ACT TGA GG T TGG GAG TTC AAG ACC AGC-3' and 5'-GCT GGT CTT GAA CTC CCA ACC TCA AGT AAT CTG CCC GCC-3'. The nucleotide sequences of both deletion and mutant constructs were verified by sequencing. The HCT116 cells were transfected with the reporter plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The cells were harvested 36 h after transfection, and their extracts were assayed for luciferase activity using a dual-luciferase reporter assay system (Promega) as per the manufacturer's instructions. All experiments were performed at least in triplicate, and the data were normalized to co-transfected Renilla luciferase (control reporter) to correct for variation in transfection efficiency. Co-expression experiments were performed using expression vectors containing full-length cDNAs of the transcription factor c-Jun.


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  		TABLE ONE
Primer sequences for luciferase constructs

 
Statistical Analysis—In all experiments, including densitometric scans of Western blots or RT-PCR analysis and luciferase assays, results were calculated as the means (±S.D.) of three independent experiments. Statistical analyses were performed using one-way analysis of variance for comparisons of multiple groups. p values of 0.05 or less were deemed to be statistically significant. All statistical tests were done using SigmaStat software (Jandel Scientific).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Oxidative Stress Transiently Increases NEIL1 Protein and mRNA Levels in HCT116 Cells—To explore the effects of oxidative stress on NEIL1 expression, we treated HCT116 cells for 1 h with a subtoxic dose (100 ng/ml) of the ROS generator GO, and we then analyzed NEIL1 protein and mRNA levels at various times. GO treatment caused 2.4 ± 0.3-fold increase in the intracellular ROS level in these cells after 1 h (Fig. 1). Mock and GO-treated cells were harvested at 0, 3, 6, 9, 12, and 20 h. Western analysis of whole cell and more significantly nuclear extracts showed an increase in the NEIL1 protein level within 3 h of GO treatment, reaching up to 2–4-fold over the control at 6–9 h, and before returning to the base line by 20 h (Fig. 2). The OGG1 polypeptide level remained unchanged over a similar time course of GO treatment (Fig. 2A). The GO treatment similarly increased the NEIL1 mRNA level in HCT116 cells (up to 4-fold at 6–9 h), as analyzed by both semi-quantitative (Fig. 3A) and quantitative RT-PCR (Fig. 3B). The GO treatment was performed in parallel in MRC5, human primary fibroblasts. Real time RT-PCR analysis of RNA revealed a similar induction of the transcript level upon exposure to ROS (Fig. 3B).



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  		FIGURE 1.
Changes in intracellular ROS levels after GO treatment. A, HCT116 cells were mock-treated or treated with increasing amounts of GO. Cells were subsequently loaded with 5 µM of H2DCF-DA, and the change in DCF fluorescence was determined by flow cytometry as described under "Experimental Procedures." B, kinetics of fluorescence induction in HCT116 cells exposed to 100 ng/ml GO.

 



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  		FIGURE 2.
Induction of hNEIL1 in oxidatively stressed HCT116 cells. A, after treating HCT116 cells with GO (100 ng/ml), as described under "Experimental Procedures," total lysates of cells, harvested at various times, were analyzed for NEIL1 and OGG1 levels by Western blotting. {beta}-Actin was used as the loading control (left panel); the ratios of band intensities of hNEIL1 or hOGG1 to {beta}-actin are shown in the right panel. Results correspond to mean (± S.D.) from three separate experiments. B, left panel, Western analysis of nuclear extracts from GO-treated HCT116 cells; lamin B, loading control; right panel, graphical representation of these results; *, p < 0.05; **, p < 0.01.

 
Identification and Characterization of cis-Elements in the hNEIL1 Promoter—To analyze further the basis for ROS-induced activation of the NEIL1 gene, we characterized its cis-elements. We cloned a 13-kb EcoRI/HindIII genomic fragment containing the complete transcription unit of the NEIL1 gene into pBluescript, together with a significant part of the 5'-flanking region. Comparison of the genomic sequences of hNEIL1 to its cDNA showed that the transcribed sequence spans ~8kb and contains 10 exons. The transcriptional start site was determined by primer extension analysis; the longest product observed extended 40 bp from the primer, P2, indicating a G in the sense strand as the site most frequently used for transcription (Fig. 4). Primer P1 failed to show any primer extension product. The initiation codon of the NEIL1 polypeptide was located in the 2nd exon, exon 1 (1 kb) being the untranslated region. Transfac software analysis of a 2.1-kb fragment spanning upstream sequences, including part of the 1st exon, identified several putative cis-elements for transcription factors. It is interesting to note the presence of binding sites for several b-Zip family proteins, including c-Jun, CREB, and NF-E2/AP-1-related factors (Fig. 5A).



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  		FIGURE 3.
Activation of the hNEIL1 gene in response to oxidative stress. A, left panel, quantitation of hNEIL1 mRNA in GO-treated HCT116 cells by semiquantitative RT-PCR as in Fig. 2. GAPDH was used as an internal reference. Right panel, graphical representation of hNEIL1 induction normalized to GAPDH. B, left panel, quantitation of hNEIL-1 mRNA in MRC5 and HCT116 cells by real time PCR, normalized to endogenous 18 S RNA; right panel; graphical representation of these results; p < 0.05.

 



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  		FIGURE 4.
Mapping of NEIL1 transcriptional initiation site. A pair of oligodeoxynucleotides, complementary to 5'-untranslated region of the hNEIL1 cDNA, was annealed to 50 µg of total RNA from HCT116 cells and extended with avian myeloblastosis virus-reverse transcriptase in separate reactions as described under "Experimental Procedures." A, 1st lane, primer extension product with P1 oligo; 2nd lane, product with P2 oligo. M, marker. B, dideoxynucleotide sequence of NEIL1 5'-flanking DNA region primed with P2 oligo showing C, T, A, and G ladders. The arrow indicates transcription initiation site, corresponding to the P2 primer extension product.

 
To test for basal promoter activity of the upstream sequences, a NEIL1 promoter luciferase reporter construct p(–2100/+40)NEIL1 luc, containing NEIL1 genomic sequence –2100 to +40 bp, was used for transient expression in transfected HCT116 cells, as described under "Experimental Procedures." A series of nested 5' deletion mutants of the NEIL1 promoter was subsequently generated in pGL2-basic (as shown in TABLE ONE and Fig. 5B), and the luciferase activity was measured. Deletion of the region between –1800 and –900 bp caused a 2–3-fold decrease of luciferase activity. Additional deletion of the downstream sequence to –600 or –500 bp did not further decrease promoter activity, whereas deletion of the region spanning –300 and –100 bp resulted in another 3-fold decrease in promoter activity, indicating that this region is the major contributor to NEIL1 expression. Furthermore, the minimal promoter activity could be detected in the plasmid containing the –50/+40 sequences.

Oxidative Stress Transactivates the NEIL1 Gene Promoter—To identify the promoter region essential for transcriptional up-regulation of NEIL1 during oxidative stress, HCT116 cells were transfected with three different NEIL1 reporter constructs: p(–1200) NEIL1 luc (bearing both proximal and distal CRE/AP-1 elements); p(–900) NEIL1 luc (bearing only proximal CRE/AP-1); and p(–50) NEIL1 luc (lacking both CRE/AP-1 elements). The luciferase activity was measured in extracts of transfected cells after GO treatment (Fig. 6A). A >2-fold increase in luciferase activity was observed with the p(–1200) NEIL1 luc construct within 3 h after exposure to GO. This up-regulation reached its peak at 6 h (>3.5-fold) and returned to the basal level at 20 h. Similarly, an ~3-fold induction was observed with p(–900) NEIL1 luc after exposure to GO. However, the promoter-reporter construct p(–50) NEIL luc, containing the minimal promoter sequence, showed no up-regulation in luciferase activity under these conditions. These results indicate that the cis-acting elements responsible for oxidative stress-induced NEIL1 promoter activity are located 60–1200 bp 5' to the transcriptional start site.

Oxidative Stress Activates the NEIL1 Promoter via a Pair of Proximal and Distal CRE/AP-1 Sequences—Examination of the sequence within the –60- to –1200-bp region of the NEIL1 promoter revealed two copies of putative elements at positions –61 and –1018, both with sequence similarity to the palindromic consensus CRE (TGACGTCA) and AP-1 (TGACTCA). To test whether these CRE-like sites are required for oxidative stress-induced expression of NEIL1, we mutated these sites either individually or simultaneously and assayed the promoter activity, following GO treatment (Fig. 6B). Mutation of the proximal CRE (Mut1), showed an ~4-fold decrease in oxidative stress-induced promoter activation, whereas mutation of distal CRE/AP-1 (Mut2) showed a 2-fold decrease compared with the wild type. Moreover, mutation at both CRE/AP-1 sites (Mut3) further decreased the GO-induced promoter activation by ~6-fold. These observations suggest that the two CRE/AP-1 sites are independently involved in oxidative stress-mediated activation of the NEIL1 promoter.



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  		FIGURE 5.
5'-Upstream sequence of the hNEIL1 gene and characterization of its promoter activity. A, the nucleotide sequence of the 5'-flanking region and part of the first exon of the hNEIL1 gene. The numbering is based on the transcriptional initiation site (+1). Consensus sequences for binding to the regulatory factors are shown, with highlighted CRE/AP-1 sequences. The arrows mark the complementary strand of the oligonucleotide used for primer extension with P2. B, DNA fragments of the 5'-regulatory region were cloned upstream of luc-coding sequence, and their promoter activity was determined as described under "Experimental Procedures"; *, p < 0.05, **, p < 0.001.

 
Oxidative Stress Stimulates Binding of CREB/c-Jun to NEIL1 CRE/AP-1—To identify the transcription factors interacting with NEIL1-CRE/AP-1, we incubated nuclear extracts with 32P-labeled oligo duplexes of sequences corresponding to the proximal and distal NEIL1-CRE sites, respectively. As shown in Fig. 7, two closely spaced complexes were observed in both instances (indicated by arrows) that could be eliminated by addition of 100-fold molar excess of unlabeled NEIL1-specific or consensus AP-1 oligo. Lack of competition with a similar excess of mutated AP-1 oligonucleotides established the binding specificities of the proximal as well as distal NEIL1-CRE/AP-1 sequences. Similar results were obtained when the CRE consensus and mutant oligonucleotides were used as competitors in EMSA (data not shown). Additionally, no specific binding was observed with the mutated NEIL1-CRE/AP-1 oligo (Fig. 7, last lane in panel i).

We then investigated whether oxidative stress leading to activation of the NEIL1 promoter was associated with increased binding of the CRE/AP-1 complex to the cis-element. Treatment of HCT116 with GO resulted in a significant increase in the binding of the nuclear extract to the NEIL1 CRE/AP-1 (Fig. 7, A and B, panel ii), as early as 30 min after treatment, which returned to the basal level within an hour. To identify the proteins present in the binding complexes, supershift analyses were performed with antibodies specific for various members of the CREB/ATF and AP-1 families (Fig. 7, A and B, panel iii). Both anti-Jun (c-Jun/AP-1) and CREB-1 antibodies decreased the amount of the specific DNA-protein complexes. A pronounced supershifted complex was generated with the phospho-c-Jun antibody (KM1). Incubation of nuclear extracts with ATF1 antibody did not generate a supershifted band, whereas ATF2 antibody slightly decreased the level of the specific complex. With extracts of stressed cells, the CRE complex was completely supershifted with addition of phosphorylated c-Jun-specific antibody (KM1), suggesting that under oxidative stress the majority of c-Jun bound to the NEIL1-CRE was in the phosphorylated form. We have further concluded that NEIL1-CRE complex responsive to oxidative stress consists of c-Jun and CREB proteins.



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  		FIGURE 6.
Identification of CRE/AP-1-binding sites required for ROS-induced activation. A, luciferase activity after transfection with p(–1200) NEIL1 luc, p(–900) NEIL1 luc, and p (–50) NEIL1 luc plasmids and treatment with GO (100 ng/ml). *, #, p < 0.05; **, ##, p < 0.001. B, effect of mutation of CRE/AP-1 at residues –61 and –1018 of NEIL1 promoter. WT, wild type; Mut1, mutation at the proximal CRE; Mut2, mutation at the distal CRE; Mut3, mutations at both CREs; Luc, pGL2-basic. Other details are given under "Experimental Procedures"; *, p < 0.05; **, p < 0.001.

 
GO Treatment Stimulates Phosphorylation of c-Jun—We then investigated whether oxidative insult by GO treatment, which induced NEIL1 promoter activity and CRE protein binding, increased the levels of CREB, c-Jun, and ATF2 polypeptides. Western blot analysis of GO-treated cells showed that the CREB and ATF2 levels were slightly enhanced, but a marked increase in the level of phosphorylated c-Jun was observed within 30 min of exposure to GO (Fig. 8A). These results indicate that CRE/AP-1 activation was correlated with oxidative stress-induced up-regulation of NEIL1 expression.



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  		FIGURE 7.
Effects of ROS on the binding of c-Jun/CREB to CRE/AP-1 in the NEIL1 promoter. HCT116 nuclear extracts (2 µg) were incubated with 32P-labeled proximal (A) or distal (B) NEIL1 CRE/AP-1 oligo with molar excess of WT or mutant oligonucleotides for EMSA (panel i). I and II indicate specific complexes; the nonspecific complex III was not competed with NEIL1 CRE/AP-1 or consensus CRE/AP-1 oligo. Binding experiments were performed with nuclear extracts of GO-treated cells (panel ii). For supershift analysis (panel iii), the reaction mixtures were incubated with antibodies to c-Jun, CREB, ATF1, and ATF2; the supershifted (ss) complexes are indicated by arrows.

 
c-Jun Induces Both Basal and Oxidative Stress-induced Promoter Activity of NEIL1—The EMSA studies suggested that the oxidative stress-induced enhancement of the affinity of nuclear extracts for the NEIL1 promoter was due to activation of c-Jun. We directly tested this possibility by examining the effect of c-Jun overexpression on NEIL1 promoter activity. HCT116 cells were co-transfected with a c-Jun expression plasmid and p(–1800/+40)NEIL1 luc containing both copies of CRE/AP-1. c-Jun dose-dependent increase in luciferase activity was observed, and GO treatment further increased the activity by about 2-fold (Fig. 8B). Stimulation of activity due to c-Jun overexpression was significantly attenuated when both CRE/AP-1 were mutated in the NEIL1 promoter construct, Mut3 (Fig. 8B). This confirmed that promoter activation was mediated by the CREs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We observed oxidative stress-dependent activation of hNEIL using sublethal GO treatment for sustained ROS generation. An increase in the levels of both NEIL1 mRNA and polypeptide suggested that ROS enhances NEIL1 transcription. To elucidate the molecular basis of NEIL1 induction, we first characterized the hNEIL1 promoter. Functional analysis of nested deletions of the NEIL1 gene upstream of the coding region revealed that the basal promoter is localized in a 50-bp segment 5' to the transcription start site. However, robust promoter activity induced by the –1200 construct indicated the presence of major cis-elements in the distal region of the promoter. Analysis of the sequence spanning –1800 to +40 bp of the promoter suggested the presence of cis-elements specific for various members of the b-Zip family of transcription factors. The b-Zip proteins are characterized by the presence of a basic domain required for interaction with DNA and an adjacent leucine zipper domain that allows dimerization between the family members (24). We identified a pair of nearly identical DNA sequences that differ by a single nucleotide from the palindromic consensus CRE, a target for binding of the b-Zip proteins CREB and ATF2 (25–27). Additionally, this CRE-like sequence is similar to the AP-1 element (28), and so could serve as a target for the binding of c-Jun and or c-Fos transcription factors, which also belong to the b-Zip family. To identify the specific trans-acting elements, we performed EMSA with a NEIL1 CRE oligo and nuclear extracts. Supershift of the specific complex with anti-c-Jun antibody and decrease in binding of nuclear extract in the presence of anti-CREB and anti-ATF2 antibodies confirmed the presence of these proteins in the binding complex.



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  		FIGURE 8.
Oxidative stress-induced increase in c-Jun/CREB levels. A, left panel, nuclear extract (50 µg) of GO-treated HCT116 cells were used for Western analysis with antibodies against phospho-c-Jun (KM1), CREB, and ATF2. Lamin B was the loading control. Right panel, increase in CREB and ATF2 levels was quantified relative to the GO-untreated level. *, p < 0.05; **, p < 0.001; #, p < 0.05; ##, p < 0.001. B, HCT116 cells were co-transfected with WT NEIL1 luc (solid bars) or MutCRE NEIL1 luc (striped bars) and c-Jun expression plasmid; other details are described under "Experimental Procedures"; **, p < 0.001.

 
Further analysis of promoter reporter constructs containing both CRE/AP-1 sequences in the NEIL 5' region showed a significant increase in luciferase activity relative to that with a deletion construct containing only the proximal CRE/AP-1. Oxidative stress activated both, whereas the minimal promoter lacking the CREs was not activated by oxidative stress. Mutational analysis further showed the requirement of key bases in both CRE/AP-1 for response to oxidative stress. These findings confirm that GO induces hNEIL1 expression via transactivation of the NEIL1 promoter. To the best of our knowledge, this is the first evidence for a role of CRE/AP-1 in NEIL1 expression.

In addition to their role in the regulation of a variety of genes, the CRE/AP-1 family of transcription factors are among the most prominent regulators of the oxidative stress response. Furthermore, the consensus sequence recognized by the classic CRE/AP-1 family members, 5'-TGA(G/C)T(C/A)A-3', is similar to the antioxidant-response element core sequence 5'-GTGACNNNGC-3'. The presence of similar sequences in the 5' region of the NEIL1 promoter strongly suggests that NEIL1 induction upon cellular exposure to ROS is regulated by binding of the members of the CREB/c-Jun family of transcription factors similar to these antioxidant-response element-like upstream elements. Our studies also confirmed that c-Jun and the other related transcription factors are induced by GO treatment. Further studies will be needed to determine the mechanism by which GO promotes CREB/c-Jun binding in order to identify other members of the signaling pathway.

Several other mammalian repair genes are inducible by oxidative/genotoxic stress. The O6-methylguanine-DNA-methyltransferase (MGMT) gene is transcriptionally activated by different types of genotoxic agents, including alkylating drugs and ionizing radiation (29, 30). The MGMT promoter contains two AP-1-binding sites, which play a key role in MGMT basal regulation (31). However, the elements required for MGMT induction remain to be identified. DNA polymerase {beta} is induced by simple methylating agents, such as MNNG which is also mediated through a CREB (32). The polymerase {beta} expression level is also enhanced by oxidative treatments (33). Similarly expression of mammalian APE1 (AP endonuclease 1) was induced by oxidative stress (34, 35). The promoter of the human APE1 contains consensus sequences for binding NF-{kappa}B, CRE, and AP-1. Similar to our observation for NEIL1, transcriptional activation of the APE1 gene was shown to require CREB/c-Jun (36). The human OGG1 promoter was recently shown to contain a pair of inverted CCAAT motifs that are crucial to its induction by the DNA-alkylating agent methylmethane sulfonate (37). Furthermore, the transcription factor NF-YA was shown to interact with the methylmethane sulfonate regulatory elements. Earlier the yeast NTH1 gene was shown to contain multiple stress-response elements in its promoter (38). However, we have shown here that OGG1 was not activated by oxidative stress. The teleological basis for activation of OGG1 and NTH1 by nonoxidants is not clear.

In summary, our results demonstrate that ROS up-regulate hNEIL1 expression through activation of the transcription factors, CREB/c-Jun. An important question that remains to be answered is the biological role of hNEIL1 activation in response to oxidative stress. We hypothesize that endogenous ROS induce DNA damage at a basal, steady-state level in unstressed cells. However, exogenous oxidative stress, although increasing the genomic damage level, simultaneously activates the cellular response to repair the additional load of DNA damage. NEIL1 activation is a part of this coordinated cellular response. This scenario implies that NEIL1 or other proteins activated by oxidative stress are limiting in the repair pathway. It is possible that NEIL1 has a unique role among the oxidized base-specific mammalian DNA glycosylases, including OGG1 and NTH1, by preferentially repairing damage in active genes. Lack of OGG1 activation by ROS underscores the key difference in regulation of NEIL1 and OGG1. This is further supported by the observations that OGG1- or NTH1-null mouse fibroblasts are no more sensitive to ROS than the parental wild type cells (12, 39). In contrast, down-regulation of NEIL1 expression by small interfering RNA in mouse embryonic stem cells sensitizes these cells to {gamma}-irradiation (40). Thus the fine-tuning of NEIL1 expression, both in response to cell cycle or external oxidative stress, may be more critical than of OGG1 or NTH1. How such modulation of NEIL1 expression is regulated in the context of global response remains to be investigated.


   FOOTNOTES
 
* This work was supported by United States Public Health Service Grants RO1 CA81063 and PO1 CA92584 and NIEHS Center Grant ES06676 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Fig. S1. Back

1 To whom correspondence should be addressed: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.136 Medical Research Bldg., Rte. 1079, Galveston, TX 77555. Tel.: 409-772-1780; Fax: 409-747-8608; E-mail: samitra{at}utmb.edu.

2 The abbreviations used are: ROS, reactive oxygen species; AP-1, activator protein 1; ATF, activating transcription factor; CRE, cyclic AMP-response element; CREB, cAMP-response element-binding protein; EMSA, electrophoretic mobility shift analysis; GO, glucose oxidase; luc, luciferase; Mut, mutant; NE, nuclear extract; NEIL, Nei-like; NTH1, endonuclease III homolog 1; OGG1, 8-oxoguanine-DNA glycosylase1; WT, wild type; h, human; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H2DCF-DA, 5-(and-6) carboxy-2', 7'-dichlorodihydrofluorescein diacetate; RT, reverse transcription; Fpg, formamidopyrimidine-DNA glycosylase; oligo, oligonucleotide; MGMT, O6-methylguanine-DNA-methyltransferase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Tadahide Izumi (Louisiana State University Cancer Center, New Orleans) for valuable suggestions and also providing the c-Jun expression vector and Dr. David Konkel for critically reading the manuscript. We acknowledge Wanda Smith for expert secretarial assistance.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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