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Am J Physiol Renal Physiol 294: F281-F290, 2008. First published November 7, 2007; doi:10.1152/ajprenal.00370.2007
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Tuberin regulates the DNA repair enzyme OGG1

Samy L. Habib,1,2 Daniel J. Riley,1,2 Lenin Mahimainathan,1 Basant Bhandari,1 Goutam Ghosh Choudhury,1,2 and Hanna E. Abboud1,2

1George O'Brien Kidney Research Center, Department of Medicine, University of Texas Health Science Center, and 2South Texas Veterans Healthcare System, Geriatric Research, Education, and Clinical Center, San Antonio, Texas

Submitted 7 August 2007 ; accepted in final form 7 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The tuberous sclerosis complex (TSC) is caused by defects in one of two tumor suppressor genes, TSC-1 or TSC-2. The TSC-2 gene encodes tuberin, a protein involved in the pathogenesis of kidney tumors, both angiomyolipomas and renal cell carcinomas. We investigated a potential role for tuberin in regulating a key DNA repair pathway. Downregulation of tuberin in human renal epithelial cells using siRNA resulted in a marked decrease in the abundance of the 8-oxoG-DNA glycosylase (OGG1). Mouse embryonic fibroblasts deficient in tuberin (TSC2–/– and TSC2+/–) also had markedly decreased OGG1 mRNA and protein expression, as well as undetectable OGG1 activity accompanied by accumulation of 8-oxodG. Gel shift analyses and chromatin immunoprecipatation identified the transcription factor NF-YA as a regulator of OGG1 activity. The binding of NF-YA to the OGG1 promoter was significantly reduced in TSC2–/– compared with TSC2+/+ cells. Introduction of TSC2 cDNA into the tuberin-deficient cells restored NF-YA and OGG1 expression. Transcriptional activity of the OGG1 promoter was also decreased in tuberin-null cells. In addition, mutation of both CAAT boxes, the sites to which NF-YA binds, completely inhibits OGG1 promoter activity. These data provide the first evidence that tuberin regulates a specific DNA repair enzyme, OGG1. This regulation may be important in the pathogenesis of kidney tumors in patients with TSC.

TUBEROUS SCLEROSIS COMPLEX (TSC) is a genetic disorder associated with benign tumors in many organs, including angiomyolipomas in the kidneys. More rarely it is associated with renal cell carcinoma (RCC) (30, 32). Mutation in Von Hippel-Lindau (VHL) gene, on the other hand, often causes RCC (8). TSC affects about 1 million individuals worldwide, with an estimated prevalence of up to one in 6,000 newborns (26). Loss of heterozygosity (LOH) at the TSC2 locus has been detected in TSC-associated RCC, as well as in sporadic forms of RCC (1, 24, 25). The precise mechanism by which tuberin deficiency predisposes to kidney tumors is not known.

The TSC2 gene encodes a protein called tuberin (35). Tuberin is a structurally complex protein, containing several functional domains (2, 17, 21). It normally exists in an active state and forms a heterodimeric complex with hamartin, the protein encoded by the TSC1 gene. Tuberin can be inactivated by several mechanisms including changes in subcellular localization, dissociation from hamartin and other regulatory proteins, or degradation of the hamartin-tuberin complex (23, 29, 33). Deficiency or inactivation of tuberin is associated with human malignancies including RCC (22).

Tumors develop and/or progress through the accumulation of multiple gene mutations, the sum of which results in cell transformation and proliferation (14). Many of these mutations occur as a result of irreparable or incompletely repaired genomic DNA, which is constantly subject to assault from intrinsic and environmental insults. Oxidized forms of DNA in particular are produced in mammalian cells as a byproduct of normal metabolism or in response to exogenous sources of reactive oxygen species (ROS). 8-Oxo-deoxyguanine (8-oxodG) is one of the major base lesions formed after oxidative damage to DNA (11). 8-OxodG pairs with adenine during DNA synthesis, increasing G:C to T:A transversions (16). 8-OxodG in DNA is repaired primarily via the DNA base excision repair pathway. The gene encoding the DNA repair enzyme that recognizes and excises 8-oxodG is 8-oxoG-DNA glycosylase (OGG1) (5, 28).

Deficiency of OGG1 in yeast or its homolog formamidopyrimidine-DNA glycosylase in bacteria results in a spontaneous mutator phenotype (12). The steady-state levels of 8-oxodG are significantly higher in the livers of OGG1–/– mice compared with wild-type animals (19). The OGG1 gene is somatically mutated in some cancer cells and is highly polymorphic among humans (7, 8, 31). LOH at the OGG1 allele, located on chromosome 3p25, was found in 85% of 99 human kidney clear cell carcinoma samples, identifying loss of OGG1 function as a possible consequence of multistep carcinogenesis in the kidney (3). OGG1 expression is regulated by the transcription factor NF-YA, which specifically recognizes a CAAT box motif in the consensus sequence of the OGG1 promoter. NF-YA is induced in response to DNA damage by alkylating agents (20). To date, however, little information is known about the cellular and molecular mechanisms that regulate OGG1. The constitutive expression of OGG1 protein in kidneys of rats heterozygots for tuberin is decreased compared with wild-type rats (13). The present study was conducted to investigate whether tuberin directly modulates OGG1 repair and DNA damage pathway(s) and to explore potential mechanisms involved.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Mouse embryonic fibroblasts (MEFs) derived from TSC2–/–, TSC2+/–, and TSC2+/+ embryos were generously provided by Dr. D. J. Kwiatkowski (Harvard Medical School). The cells were grown in DMEM supplemented with 10% FBS. Human embryonic kidney epithelial cells (HEK293) were obtained from American Type Culture Collection (Manassas, VA) and maintained in DMEM with 10% FBS. All cell lines were grown at 37°C in a humidified atmosphere of 5% CO2.

Downregulation of tuberin by siRNA. HEK293 cells were grown in six-well plates. Before transfection, cells (30–50% confluent) were washed with PBS and media were replaced with 800 µl of OPTI-MEM I (Invitrogen). In parallel, 4 µl of oligofectamine (Invitrogen) were combined with 11 µl of OPTI-MEM I and incubated at room temperature for 10 min. SMART selected siRNA duplexes with "UU" overhangs and 5' phosphate on the antisense strand were obtained in a kit from Dharmacon. The siRNA specific for TSC2 was a mixture of four pooled duplexes. According to the manufacturer, these siRNA efficiently blocks tuberin expression by 70%. An amount of 1.5 µg of the indicated duplex was diluted into 180 µl of OPTI-MEM I, added to the Oligofectamine/OPTI-MEM I mixture, and incubated at room temperature for 20 min. The siRNA complexes were then added to the cells. After incubation for 3–4 h in a 5% CO2 incubator, 1 ml of fresh medium was added to a final serum concentration of 10%. Forty-eight hours after transfection, cells were harvested for Western blot or RT-PCR analysis. The control construct used in parallel experiments contains four pooled, nonspecific siRNA duplexes provided by Dharmacon (18). Under these experimental conditions, we routinely obtain more than 70% downregulation of tuberin (Fig. 1A).


Figure 1
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  		Fig. 1. Downregulation of tuberin expression in human renal epithelial cells results in decrease in 8-oxoG-DNA glycosylase (OGG1) expression. A: immunoblot analysis of tuberin and OGG1 in human embryonic kidney epithelial cells (HEK293) cells 48 h after transfection with siRNA directed against tuberous sclerosis complex (TSC2). Actin was used as a loading control. B: total RNA from cells with TSC2-specific siRNA or pooled, nonspecific siRNA (control) was extracted and subjected to RT-PCR with the corresponding specific primer for each gene (OGG1 or actin). Products were separated on agarose gel electrophoresis and visualized by ethidium bromide staining under UV light. A and B, right: intensity of each band was quantified by densitometry. Histograms represent means ± SE (n = 3). Significant differences from cells transfected with the TSC2-specific siRNA are indicated by **P < 0.01.

 
Protein extraction and immunoblot analysis. Cell lysates were prepared using lysis buffer (1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing the protease inhibitors phenylmethylsulfonyl fluoride (10 mg/ml), leupeptin (10 mg/ml), and aprotinin (20 mg/ml). Cell lysates were centrifuged at 14,000 g for 30 min at 4°C. Protein concentration was determined with the Bradford assay (6) using bovine serum albumin as a standard. Protein (100 µg) was subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes at a constant voltage of 200 V for 1–1.5 h. PVDF membranes were blocked in 5% nonfat dried milk in TBS-0.1% Tween buffer [25 mM Tris·HCl, 0.2 mM NaCl, 0.1% Tween 20 (vol/vol), pH 7.6] (TBS-T) for 1 h. Membranes were incubated with the respective primary antibodies overnight at 4°C. Rabbit polyclonal antibody raised against human OGG1 protein was generously provided by Dr. S. Mitra (University of Texas Medical Branch at Galveston, Texas). This antibody also recognizes mouse OGG1. Goat anti-NF-YA was purchased from Santa Cruz Biotechnology and β-actin antibody from Oncogene Research Products. All primary antibodies were prepared at 1:1,000 dilutions in TBST. Membranes were washed 5x with TBST and then incubated with an appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. An enhanced chemiluminescence kit (Amersham) was used to identify protein expression. Membranes were stripped with 0.2 M NaOH for 10 min each, blocked with 5% milk for 1 h, and then incubated with the respective primary and secondary antibodies, as described earlier. Expression of each protein was quantified by densitometry using National Institutes of Health Image 1.62 software.

mRNA analysis by RT-PCR analysis. RNA was extracted from exponentially growing MEFs or HEK293 cells using RNA Isolation Solvent (Tel-Test). RNA was quantified by spectrophotometer at 260 nm, and its integrity was tested by formaldehyde/agarose gel electrophoresis. First-strand synthesis of cDNA was carried in 20 µl total reaction volume as follows: 1 µl oligo12–18, 2 µg total RNA, 1 µl of 10 mM dNTP mix (10 mM each dATP, dGTP, dCTP, and dTTP), and 12 µl distilled water. The mixture was heated at 65°C for 5 min and then quickly chilled on ice. The contents of the tube were collected by brief centrifugation and then 4 µl 5x first-strand buffer, 2 µl 0.1 M DTT, and 1 µl RNaseOUT Recombinant Ribonuclease Inhibitor (40 U/µl). The mixture was incubated at 42°C for 2 min and then 1 µl of SUPERSCRIPT II (200 U) was added and mixed by pipetting. The reaction mixture was incubated for 50 min at 42°C and then was inactivated by heating at 94°C for 2 min. PCR reaction was carried out in a final volume of 50 µl as follows: 5 µl 10x PCR buffer, 1.5 µl 50 mM MgCl2, 1 µl 10 mM dNTP, 0.4 µl Taq DNA polymerase (5 U/µl), 2 µl cDNA (from first-strand reaction), 1 µl each of OGG1 upstream/reverse primers (5'-AACATTGCTCGCATCACTGGC/5'-GATGTCCACAGGCACAGCCTG), and 38.1 µl autoclaved distilled water. OGG1 was amplified using a thermocycler (Biometra) programmed for 25 cycles [95°C (5 min), 95°C (1 min), 52°C (50 s), 72°C (1 min), and 72°C (10 min)]. The amplified product was 356-bp long. For β-actin, an internal control of amplification, upstream/reverse primers were 5'-GATGACCCAGATCATGTTTGAGA/5'-CTGTAGGCATTTCTGGAGATA synthesizing a 528-bp product. The β-actin sequence was amplified for 25 cycles [95°C (3 min), 95°C (40 s), 52°C (50 s), 72°C (50 s), and 72°C (10 min)]. The PCR products were separated on 2% agarose gels. PCR products were analyzed on ethidium bromide-stained gels. The yield was integrated for each sample on an image analyzer and the ratio of OGG1 to actin was then calculated.

Analysis of OGG1 enzyme activity. OGG1 activity was measured as described previously (34). MEF cells were suspended in 0.25 ml lysis buffer (20 mM Tris·HCl, pH 8.0, 1 mM EDTA, 250 mM NaCl, 0.8 µg/ml antipain, 0.8 mg/ml leupeptin, 0.8 µg/ml aprotinin). The cell suspension was sonicated for 8 s at 4°C with pulses of 1 s each between 10-s intervals. After centrifugation (12,000 rpm) at 4°C for 15 min, the supernatant was recovered and protein content was determined by the Bradford method (6). A 24-mer oligonucleotide containing the oxidized G base (R&D Systems) was labeled at its 5' end using [32P]ATP and T4 polynucleotide kinase. The 32P-labeled strand was hybridized with the complementary oligonucleotide by incubation at 90°C for 10 min followed by slow cooling to RT. The assay mixture (20 µl final volume) contained 50 fmol of 32P-labeled DNA duplex and cell extracts (50 µg of protein) in 1x REC buffer (10 mM HEPES-KOH, pH 7.4, 100 mM KCl, 10 nM EDTA, and 0.1 mg/ml BSA). Serial dilutions of purified OGG1 enzyme (0.125, 0.25, 0.37, and 0.5 U) were used as positive control. The reactions were performed at 37°C for 1 h. Ten microliters of 3x alkali loading buffer (300 mM NaOH, 97% formamide, and 0.2% bromophenol blue) were added, and the samples were heated at 95°C for 10 min and then quickly cooled to 2–8°C. The cleavage products were resolved by 20% denaturing PAGE in the presence of 7 M urea.

8-OxodG assay. DNA was isolated from the MEF cells and detection of dG and 8-oxodG was performed on DNA hydrolyzed with nuclease P1 and alkaline phosphatase as previously described and validated (13). Aliquots (90 µl) of DNA hydrolysates were injected onto a Partisil 5-µm ODS-3 reverse-phase analytical column for HPLC analysis with the eluate monitored with a UV photodiode array (Shimadzo, SPD M10A) and electrochemical (EC) detectors (ESA Coul Array). Authentic standards of 8-oxodG and dG were analyzed along with every batch of samples. Salmon sperm DNA (5–50 µg) was used as a positive control for DNA digestion reactions. Standard curves for dG and 8-oxodG were prepared and quantitation was performed by linear regression analyses. Data were expressed as picomoles of 8-oxodG/dG x 10–5 in 90 µl of DNA hydrolysate.

EMSAs. Nuclear proteins were extracted from TSC2–/– and TSC2+/+ cells using nuclear and cytoplasmic extraction kits (Pierce, IL). The protein concentration of the nuclear extracts was determined using the Bradford method (6). EMSA binding reactions were performed for 20 min in a final volume of 20 µl at room temperature containing 5 µg of the nuclear extract, 20 fmol of the end-labeled double-stranded 48-bp oligonucleotide covering the region of the hOGG1 promoter from –110 to –61 (control), and 1 µl of poly (dI–dC)·(dI–dC). Mutated, inverted CAAT motif (mutant) was incubated with the nuclear extracts isolated from both cell lines. The super shift assays were performed by preincubating nuclear extracts with 5 µl of polyclonal antibodies against NF-YA (Santa Cruz Biotechnology). Anti-REHB antibody (Santa Cruz Biotechnology) was used as a control antibody to confirm the binding specificity of NF-YA to the hOGG1 promoter. The reaction was carried out at room temperature for 30 min before adding the radiolabeled probe. Competition assays were performed in the presence of a 100-fold excess of the unlabeled oligonucleotide. The complexes were resolved using a 5% nondenaturing polyacrylamide gel. The gels were dried and exposed overnight at –70°C.

Chromatin immunoprecipitation assay. The assay was performed using a chromatin immunoprecipitation (ChIP) kit (Active Motif). Briefly, DNA and proteins from HEK293 cells grown in 100-mm dishes were cross-linked using 1% formaldehyde. After being washed with PBS, cells were resuspended in lysis buffer containing the same protease inhibitor cocktail used for Western blotting. DNA was digested for 5 min into small fragments of 100 to 500 bp by an enzymatic method. The supernatant was collected and precleared using protein G-agarose beads. The recovered supernatant was incubated with either anti-NF-YA antibody or an isotype control IgG overnight followed by incubation with protein G-agarose beads for 2 h. The beads were washed with buffers provided in the kit. The immunoprecipitated DNA was eluted from the beads with 1% SDS and 1 M NaHCO3 solution followed by reverse cross linking using 5 M NaCl and RNase A at 65°C overnight. After digestion with proteinase K, DNA was purified on a column method. PCR was performed on the purified DNA using hOGG1 promoter-specific primers forward (–209/–228) (5'-CCA GAT GGA ACT CGT TAG CG-3') and reverse (–9/–29) (5'-CAG ACC ACA GCA CCA CCG GAA-3').

Adenovirus infection. Adenovirus vector expressing wild-type TSC2 (Ad-TSC2) was kindly provided by Dr. D. J. Noonan (University of Kentucky) (10). HEK293 cells were grown to 70–80% confluency and then infected with Ad-TSC2. Viral stocks were prepared and tittered using the serial dilution technique as described in the Adeno-X Expression Systems User Manual (Clontech Laboratories). Tuberin-null and heterozygous MEF cells were grown to 60–70% confluency in six-well plates. Infection of TSC2-null and heterozygous MEF cells with 20 multiplicity of infection (MOI) showed appreciable expression of TSC2 protein. An adenovirus vector expressing green fluorescence protein (Ad GFP) was used as a control. Forty-eight hours postinfection, the cells were harvested for immunoblotting. We used this time point in our study as it produced vector-derived tuberin in appreciable amount.

Transcriptional activity of OGG1 promoter. A reporter plasmid containing OGG1 promoter with luciferase reporter gene (courtesy of Dr. P. Radicella) (5) was used to determine the transcriptional activity of OGG1 promoter. A plasmid containing β-galatosidase reporter was used as control. Plasmids were transfected into MEF cells using the LipofectAMINE and Plus Reagent method (Invitrogen, Life Technologies). In addition, wild-type and tuberin-null cells were infected with a recombinant adenovirus expressing tuberin (Ad-TSC2). An otherwise identical adenovirus expressing β-glatctosidease (Ad β-GAL) was used as a control. The cells were again infected with MOI = 20. MEF cells (60–70% confluence) were grown in six-well plates. Before transfection, cells were washed with PBS and media were replaced with 800 µl of OPTI-MEM I (Invitrogen). Precomplex of the DNA with Plus Reagent in Opti-MEM was mixed and incubated at room temperature for 15 min. LipofectAMINE was added to the complex of DNA and Plus reagent and incubated for 15 min at room temperature. DNA and Plus reagent-LipofectAMINE complexes were added to each well and incubated at 37°C with 5% CO2. After incubation for 3–4 h, 1 ml of fresh media with 20% serum was added to a final concentration of 10%. Forty-eight hours after transfection, cells were harvested for luciferase and β-galatosidase assay. Cells were washed with PBS and lysed in 0.4 ml of lysis buffer. Luciferase activity was determined using the Luciferase Reporter Assay System by a luminometer according to the manufacturer's instructions (Promega) and normalized by β-galatosidase activity.

Mutations in the inverted CAAT box of the OGG1 promoter. Plasmids containing mutations at inverted CAAT box sites in the OGG1 promoter were constructed using PCR followed by cloning of PCR products into a pGL3-basic vector (Promega). Briefly, two forward primers containing a KpnI site at 5' and sequences –106 to +74 of the OGG1 promoter with mutations were synthesized: NF-YA (no mutation): 5'-CCC GGG TAC CTG ATT TCT CTT TGG CGC CTC C-3', and NF-YA-Mut: 5'-CCC GGG TAC CTG ATT TCT CTT TGG CGC CTC CTA CCT CCT CCT CGG TTT GGC TAC CTC T-3'. A reverse primer containing a HindIII site at 3' sequences corresponding to the pGL3 basic vector was also synthesized: 5'-CAT GGT GGC TTT ACC AAC AGT ACC GGA ATG-3'. PCR reactions were performed using pR143 clone (4) as the template DNA with an appropriate set of primers, Taq polymerase, and dNTPs, as described previously. The PCR products were digested with KpnI and HindIII and ligated into pGL3 basic vector using T4 ligase followed by transformation of DH5{alpha} cells (Invitrogen Life Technologies). The mutations in the constructs were confirmed by DNA sequencing. The reporter plasmids with the OGG1 promoter driving firefly luciferase were used to determine the transcriptional activity of OGG1 promoter with and without mutations at inverted CAAT regions. Plasmids were transfected into MEF cells using the LipofectAMINE and Plus Reagent method (Invitrogen, Life Technologies) as described above.

Statistics. Data are presented as means ± SE. Statistical differences were determined using ANOVA followed by Student Dunnett's (Exp. vs. Control) test using 1 trial analysis. P values <0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Downregulation of TSC2 results in decreased OGG1 expression. To determine the effect of tuberin on OGG1 expression, expression of tuberin was downregulated using siRNA directed against the TSC2 gene in human renal epithelial cells. The cells transfected with duplex siRNA oligonucleotide complementary to TSC2 had decreased tuberin protein expression compared with cells transfected with nonspecific siRNA duplexes as control oligonucleotides (Fig. 1A). Downregulation of tuberin resulted in decreased in OGG1 protein expression. OGG1 mRNA expression was also decreased in cells transfected with siRNA against TSC2 (Fig. 1B).

OGG1 mRNA and protein expression in tuberin-deficient cells. To determine whether complete genetic deficiency of tuberin is similarly associated with loss of OGG1 expression, lysates of MEFs expressing different copy numbers of TSC2 were analyzed. Tuberin expression was more than twofold higher in TSC2+/+ compared with TSC2+/– cells and was undetectable in TSC2–/– cells (Fig. 2A). Deficiency of tuberin in null and heterozygous cells was associated with a gene copy number-dependent decrease in OGG1 expression compared with wild-type cells, indicating that tuberin is an upstream regulator of OGG1 protein and gene expression (Fig. 2A). RT-PCR from TSC2 null cells similarly showed a twofold decrease in the abundance of OGG1 mRNA compared with wild-type cells (Fig. 2B). A similar decrease in OGG1 mRNA expression was observed in the heterozygous compared with wild-type cells (Fig. 2B).


Figure 2
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  		Fig. 2. Deficiency of tuberin results in decreased OGG1 mRNA and protein expression. A: tuberin and OGG1 expression in TSC2–/–, TSC2+/–, and TSC2+/+ cells. Cells were maintained in medium containing 10% fetal bovine serum until confluent. Cell lysates were prepared and protein extracts (100 µg) were loaded onto a 7% SDS-polyacrylamide gels and transfered to PVDF membrane. The membrane was incubated with antibodies specific for OGG1, followed by different, specific, horseradish peroxidase-conjugated secondary antibodies. The proteins were visualized with ECL. B: RT-PCR on RNA prepared from wild-type and tuberin-deficient cells was performed using 1 µg RNA from each sample to synthesize first- and second-strand DNA. PCR products were analyzed on an ethidium bromide-stained gel. A and B, right: intensity of each band was quantified by densitometry. Histograms represent means ± SE (n = 3). Significant differences from wild-type cells are indicated by *P < 0.05 and **P < 0.01.

 
Deficiency of tuberin inhibits OGG1 activity and increases accumulation of 8-oxodG. To determine whether decreases in mRNA and protein expression were reflected in changes in OGG1 enzymatic activity, such activity was measured and compared in wild-type and tuberin-deficient cells. DNA glycosylase activity of the OGG1 enzyme was assayed as the cleavage of an 8-oxoG-containing oligomer, which releases oxidized guanine base from a 32P-labeled 21 oligonuleotide (32). Unlike the activity seen in the wild-type cells, there was no detectable OGG1 activity in the null or heterozygous cells (Fig. 3, A and B). Since OGG1 is the major DNA base excision repair enzyme that recognizes and excises 8-oxodG, we next determined directly whether changes in OGG1 abundance influence the accumulation of 8-oxodG. DNA was isolated from TSC2–/–, TSC2+/–, and TSC2+/+ cells and 8-oxodG levels were analyzed by HPLC. 8-OxodG levels were significantly higher in TSC2–/– and TSC2+/– cells compared with TSC2+/+ cells (Fig. 3C).


Figure 3
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  		Fig. 3. Lack of OGG1 activity and increased 8-oxodG in tuberin-deficient cells. A: 21-mer containing an 8-oxoG lesion was labeled at its 5'-end using [32P]ATP and incubated with cell extracts from null, heterozygous, and wild-type mouse embryonic fibroblasts (MEFs). Oligonucleotide cleavage products were analyzed on DNA sequencing gels and subjected to autoradiography. Different dilutions of human OGG1 enzyme (E) or buffer alone (S) were analyzed as positive and negative controls, respectively. Top arrow indicates the 21-mer of 8-oxodG as a substrate and bottom arrow is the DNA cleavage product (13-mer). B: gel shown is representative of multiple similar experiments. C: DNA was extracted and digested with nuclease P1. The detection of dG and 8-oxodG was performed by HPLC-EC analysis. Authentic standards of 8-oxodG and dG were analyzed simultaneously. Data were expressed as pmol 8-oxodG/dG x 10–5 in 90 µl of DNA hydrolysate. Significant difference from wild-type cells is indicated by *P < 0.05.

 
Regulation of OGG1 by NF-YA. The decrease in OGG1 protein expression is associated with decreased OGG1 mRNA levels in tuberin-deficient cells (Fig. 2B), suggesting decreased transcription or destabilization of the mRNA. To further investigate the mechanism by which tuberin deficiency regulates OGG1 expression, EMSAs were performed to determine the capacity of NF-YA to bind to the OGG1 promoter in the TSC2-null and wild-type MEFs. Binding of NF-YA in MEFs was analyzed using labeled oligonucleotide corresponding to the OGG1 promoter region between –110 and –61, which contains two inverted CAAT boxes (Fig. 4A). Stronger protein binding to OGG1 promoter-specific DNA complexes was seen in nuclear extracts from wild-type cells compared with those from TSC2-null cells (Fig. 4B). The band representing the protein-DNA complexes competed effectively by unlabeled oligonucleotide in 100-fold excess (Fig. 4B). This band was absent when the nuclear extracts were incubated with a mutant oligonucleotide containing T for A base substitutions in the two inverted CAAT motifs (Fig. 4C). To confirm the specificity of the protein-DNA interaction, the cell extracts were also preincubated with an antibody recognizing NF-YA or with a nonspecific antibody. A portion of the DNA-protein complexes was supershifted in the presence of the NF-YA antibodies, but not by the nonspecific antibody, indicating that NF-YA is indeed a component of these complexes (Fig. 4D). The partial shift suggests the involvement of other transcription factor(s) in regulation of OGG1 promoter.


Figure 4
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  		Fig. 4. OGG1 promoter region containing inverted CAAT motifs binds to NF-YA by electrophoretic mobility shift. A: intact 48-bp double-strand end-labeled oligonucleotides (wild-type) and mutant containing the inverted CAAT sites spanning the region –110 to –61 (48 bp) of the OGG1 promoter. B: oligonucleotides were incubated with nuclear extracts and protein-DNA complexes were visualized by autoradiography. Competition was performed in the presence of a 20-fold excess of unlabeled oligonucleotides. C: control or mutant oligonucleotides were incubated with the nuclear extracts from both cell lines as in A. Lane 4 was moved from its original position on the gel. D: portion of the DNA-NF-YA protein complexes was supershifted when NF-YA antibodies were added to the incubation reactions. The density of the original band was also decreased in both cell lines. Nonspecific IgG did not cause any shifts in the original band. The results shown are representative of 3 experiments. E: ChIP assay: HEK293 cells were fixed and lysed, and DNA was digested using an enzymatic method. Immunoprecipitation was performed using an NF-YA antibody or a nonspecific goat IgG. The protein DNA complexes were denatured, and DNA was purified followed by PCR using OGG1 promoter primers. PCR was performed on the input (clarified chromatin from genomic DNA) from 293 cells, as well as on the DNA immunoprecipitated using anti-NF-YA and nonspecific goat IgG. PCR products were analyzed by gel electrophoresis and the size of the specific PCR-amplified OGG1 promoter fragment (220 bp) was determined using the DNA markers (M).

 
ChIP assay. To confirm further that NF-YA directly binds the inverted CAAT box region in the human OGG1 promoter, we performed a ChIP assay using HEK293 cell extracts. The anti-NF-YA antibody, but not the control IgG antibody, precipitated the OGG1 promoter fragment (220 bp) containing binding sites for inverted CAAT regions (Fig. 4E). These data show that NF-YA does indeed bind to the OGG1 promoter at CAAT site or sites located in the region –9/–228.

Introduction of TSC2 cDNA into the tuberin-deficient cells restored NF-YA and OGG1 expression. We next examined whether complete genetic deficiency of tuberin is similarly associated with deficiency of NF-YA expression. We examined the basal levels of NF-YA in MEF cells expressing different copy numbers of TSC2. Deficiency of tuberin in null and heterozygous cells was associated with decreased NF-YA expression compared with wild-type cells (Fig. 5A). Infection of TSC2-null and heterozygous cells with Ad-TSC2 restored the wild-type pattern of NF-YA and OGG1 expression, suggesting that tuberin regulates OGG1 at least through the transcription factor, NF-YA (Fig. 5, B and C).


Figure 5
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  		Fig. 5. Decreased NF-YA expression in tuberin-deficient cells is restored by reintroduction of wild-type tuberin. A: NF-YA expression in TSC2–/–, TSC2+/–, and TSC2+/+ cells was measured by Western blot using NF-YA antibody. Significant differences from wild-type cells are indicated by *P < 0.01. B and C: tuberin-null and heterozygous cells were grown on a 6-well plate and infected with an adenovirus expressing tuberin (Ad-TSC2). An otherwise identical adenovirus expressing β-GAL was used as a control. Cell lysates were used to measure tuberin, NF-YA, and OGG1 expression by Western blot. Actin expression serves as a loading control.

 
Transcriptional activity of the OGG1 promoter. To determine whether decreased transcription is a mechanism for the decrease in OGG1 mRNA, transcriptional activity of the OGG1 promoter was measured in MEFs transfected with a heterologous OGG1-promoter luciferase construct. Transcriptional activity from the OGG1 promoter was threefold lower in null and heterozygous cells compared with wild-type cells (Fig. 6B). In addition, both wild-type and tuberin-null cells infected with adenovirus expressing tuberin (Ad-TSC2) showed higher transcriptional activity from the OGG1 promoter compared with uninfected cells. The increase in OGG1 promoter activity in tuberin-deficient cells reexpressing tuberin via the recombinant adenovirus was threefold compared with uninfected cells and comparable to the activity in uninfected wild-type cells (Fig. 6C).


Figure 6
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  		Fig. 6. Decreased transcription from an OGG1-luciferase promoter in tuberin-deficient cells is restored by reintroduction of wild-type tuberin. A and B: reporter plasmid containing the OGG1 promoter driving expression of the luciferase and β-galactosidase reporter genes was transfected into the MEF cells using LipofectAMINE and Plus Reagent. Forty-eight hours after transfection, cells were washed and lysed. Luciferase activity was determined and normalized by β-gal activity. C: wild-type and tuberin-null cells were infected with an adenovirus expressing tuberin (Ad-TSC2) or adenovirus expressing β-GAL as a control. A reporter plasmid containing the OGG1 promoter was also transfected into these cells. Luciferase activity was determined using the Luciferase Reporter Assay System as described in MATERIALS AND METHODS. Histograms represent means ± SE from 4 experiments. Significant differences from wild-type cells are indicated by **P < 0.01.

 
Mutation in CAAT boxes inhibits the activity of OGG1 promoter. The constructed reporter plasmids contain within the OGG1 promoter two inverted CAAT boxes (+74 to –106 nt), to which NF-YA is purported to bind to drive the expression of the luciferase reporter gene (Fig. 7A). Mutations in both inverted CAAT boxes (–93 to –98 and –67 to –71, Mut 3) completely abolished the luciferase activity of OGG1. These results strongly suggest that the CAAT sites at which NF-YA binds are required for transcription of OGG1.


Figure 7
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  		Fig. 7. Mutation in the CAAT box of NF-YA inhibits the activity of the OGG1 promoter in wild-type and TSC2-deficient cells. A and B: reporter plasmids containing the OGG1 promoter with or without mutations in the inverted CAAT boxes (+74 to –106 nt) that drive the expression of the luciferase reporter gene were transfected into MEFs. Forty-eight hours after transfection, luciferase activity was determined using the Luciferase Reporter Assay System. pGL3 basic vector was used as a control to normalize luciferase activity (1-fold). Histograms represent means ± SE from 4 to 8 experiments.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study reports that tuberin regulates the expression and activity of the DNA excision repair enzyme OGG1 and that tuberin exerts this effect at least partially through the transcription factor, NF-YA. The data demonstrate that tuberin deficiency is associated with downregulation of OGG1 protein and mRNA, as well as with marked decrease of OGG1 activity. Downregulation of OGG1 is associated with increased accumulation of 8-oxodG. These data indicate a novel role for tuberin in the regulation of a major DNA repair pathway and provide a potential mechanism by which TSC2 mutations and tuberin deficiency predispose to the genesis and progression of kidney tumors.

Several approaches were utilized to conclusively demonstrate that tuberin regulates OGG1. First, downregulation of tuberin in renal epithelial cells by siRNA against TSC2 was associated with decrease in OGG1 mRNA and protein. Second, in MEFs cells harboring one or two mutant TSC2 alleles, the expression of OGG1 mRNA and protein was decreased and associated with a loss of OGG1 activity. Third, introduction of TSC2 cDNA into the tuberin-deficient cells restored NF-YA and OGG1 expression. The decrease of OGG1 in cells deficient for tuberin indicates that tuberin is upstream of OGG1. In mammalian cells, the base excision pathway initiated by OGG1 represents the main defense against the mutagenic effects of 8-oxodG. Indeed, the loss of OGG1 activity in our studies resulted in the accumulation of significant amounts of 8-oxodG, indicating that loss of tuberin is biologically significant. Treatment of rats, heterozygous for TSC2, with an oxidative DNA damaging agent increases 8-oxodG formation (13), but the accumulation of 8-oxodG in tuberin-deficient cells in our studies occurred in the absence of exposure to exogenous oxidants. 8-OxodG induces mutation via misincorporation of DNA bases present in the unrepaired DNA adducts, or by slippage of DNA polymerase during replicative bypass.

The decrease in OGG1 mRNA in tuberin-deficient cells suggests that decreased transcription is one potential mechanism responsible for downregulation of OGG1 protein. NF-YA has recently been shown to regulate the transcription of OGG1 in response to alkylating agents (20). NF-YA binds to the OGG1 promoter and activates OGG1 transcription. The OGG1 promoter contains two inverted CAAT boxes in the region between –121 and –61, which are important for binding of NF-YA to the consensus sequence of OGG1 promoter. Indeed, using EMSA, we found that NF-YA binding was significantly lower in tuberin-null cells compared with wild-type cells, since NF-YA protein-DNA complex formation was decreased in null cells compared with wild-type cells. Mutations in the two inverted CAAT motifs of the region between –121 and –61 of the OGG1 promoter completely eliminated the formation of DNA protein complexes. The specificity of the DNA protein complex was confirmed by demonstrating that the complex supershifted in the presence of anti-NF-YA antibodies. Such antibodies did not completely block nuclear proteins from binding to CAAT boxes or other sequences in the OGG1 promoter (Fig. 4D). Therefore, other transcription factors in addition to NF-YA probably also regulate OGG1 transcription. Other transcription factors could also bind to the promiscuous CAAT boxes. ChIP analysis further provided confirmation that NF-YA binds the OGG1 promoter at CAAT sites located in the –9 to –228 region. Collectively, the data indicate decreased binding of NF-YA to the OGG1 promoter in tuberin-deficient cells. Transcriptional activity using a heterologous OGG1-luciferase promoter in tuberin-deficient cells was also decreased compared with wild-type cells, clearly demonstrating that tuberin deficiency downregulates OGG1 through inhibition of its transcription.

In addition, mutation in the inverted CAAT boxes completely abolished the OGG1 promoter-dependent luciferase activity. These findings provide additional support suggesting that NF-YA is an important regulator of OGG1 transcription, especially in TSC2-deficient cells.

In summary, deficiency of tuberin is associated with decreased expression and loss of function of OGG1 in HEK and MEF cells. Impaired OGG1 repair enzyme activity results in the accumulation of 8-oxodG, suggesting that tuberin plays a significant role in protecting the cells from oxidative DNA damage. Partial or complete loss of tuberin is sufficient to downregulate OGG1, with consequent decreased transcription of OGG1. NF-YA is clearly an important regulator of the OGG1 promoter transcriptional activity. We propose that loss of OGG1 expression and function in tuberin-deficient cells results in the accumulation of mismatched DNA base lesions, a form of genomic instability that if left unrepaired promotes additional genetic alterations leading kidney tumor phenotype (Fig. 8). Further studies to identify the mechanisms by which tuberin deficiency regulates expression, localization, or activity of NF-YA and other transcription factors should help clarify how tuberin regulates DNA repair pathways involved in tumor formation.


Figure 8
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  		Fig. 8. Proposed model for the role of tuberin in the regulation of OGG1. In wild-type cells, the tuberin-hamartin complex allows damaged DNA to be repaired adequately. Loss-of-function mutation of even one, but more importantly two alleles of the TSC2 gene, inhibits OGG1 activity and reduces OGG1 abundance by a transcriptional mechanism, in part by inhibiting the abundance and binding of NY-FA to the OGG1 promoter. Decreased OGG1 expression and activity, in turn, lead to a mutator phenotype, with faulty repair of oxidized DNA lesions, and to the accumulation of 8-oxodG. Accumulation of mismatched DNA base lesions promotes additional genetic alterations and leads to kidney tumors.

 

   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by grants from The National Kidney Foundation of South and Central Texas, George M. O'Brien Kidney Center Development and Feasibility program, The American Diabetes Association and New Investigator Award from South Texas Veterans Healthcare System (to S. L. Habib), National Institutes of Health (NIH) Grants RO1-DK-61626 (to D. J. Riley) and R37-DK-33665 (to H. E. Abboud). G. G. Choudhury is supported by a VA Merit review, VA Career Scientist Award, and NIH Grant RO1-DK-50190.


   ACKNOWLEDGMENTS
 
The authors acknowledge Dr. D. J. Kwiatkowski at Harvard Medical School for providing the MEF cell lines, Dr. P. Radicella at Radiobiologie Moleculaire et Cellulaire for providing OGG1 promoter, and Dr. S. Mitra at the University of Texas M. D. Anderson and Sealy Center for Molecular Science (Galveston, TX) for providing the OGG1 antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. L. Habib, Dept. of Medicine-MSC 7882, The Univ. of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229 (e-mail: habib{at}uthscsa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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