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Carcinogenesis Advance Access originally published online on September 24, 2007
Carcinogenesis 2007 28(12):2552-2559; doi:10.1093/carcin/bgm214
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

CEBPG regulates ERCC5/XPG expression in human bronchial epithelial cells and this regulation is modified by E2F1/YY1 interactions

E.L. Crawford1,2,{dagger}, T. Blomquist1,2,{dagger}, D.N. Mullins1,2, Y. Yoon1, D.R. Hernandez1, M. Al-Bagdhadi1, J. Ruiz1, J. Hammersley1 and J.C. Willey1,2,*

1 Department of Medicine
2 Department of Pathology, The University of Toledo, Toledo, OH 43614, USA

* To whom correspondence should be addressed. Tel: +1 419 383 3541; Fax: +1 419 383-6244;Email: james.willey2{at}utoledo.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Marked inter-individual variation in lung cancer risk cannot be accounted for solely by cigarette smoke and other environmental exposures. Evidence suggests that variation in bronchial epithelial cell expression of key DNA repair genes plays a role. Variation in these genes correlates with variation in expression of CEBPG and E2F1 transcription factors. Here, we investigated the mechanistic basis for correlation of the DNA repair gene ERCC5 (previously known as XPG) with CEBPG and E2F1. CEBPG expression vector transfected into H23 or H460 cell lines up-regulated endogenous ERCC5 and also luciferase from a reporter construct containing 589 bp of ERCC5 5' regulatory region. A recognition site for CEBPG and a region containing sites for YY1 on the sense strand and E2F1 on the anti-sense strand participated in CEBPG up-regulation of ERCC5. CEBPG, E2F1 and YY1 binding to their respective sites were confirmed by electrophoretic mobility shift assay. Thus, we conclude that CEBPG regulates ERCC5 expression and this regulation is modified by E2F1/YY1 interactions. Several polymorphisms have been identified in these regions and, based on the data presented here, it is reasonable to hypothesize that they may contribute to risk for bronchogenic carcinoma.

Abbreviations: ACTB, β-actin; BC, bronchogenic carcinoma; BEC, bronchial epithelial cell; IS, internal standard; mRNA, messenger RNA; NE, nuclear extract; NT, native template; P-1, promoter-1; P-3, promoter-3; PCR, polymerase chain reaction; SMIS, standardized mixture of internal standard; StaRT–PCR, Standardized reverse transcription–polymerase chain reaction


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Bronchogenic carcinoma (BC) currently is the leading cause of cancer-related death in the USA, causing 28% of all cancer deaths (1). Although cigarette smoking is the major risk factor for BC, only ~10–15% of heavy smokers develop BC, consistent with marked inter-individual variation in risk (2). This variation is not completely explained by variation in exposure to smoke or environmental factors (3–5). Due to the employment of increasingly effective methods to reduce the prevalence of cigarette smoking, there are increasing numbers of ex-smokers in the population (6). Importantly, although the risk of BC decreases over time following smoking cessation, it never reaches the level observed among those who never smoked (7–9) and BC now occurs most commonly among ex-smokers (10,11). One important way to reduce the cost and suffering from BC among ex-smokers is to develop better biomarkers for identifying the 10–15% of heavy smokers at highest risk (12,13) so that they can be included in early detection and chemoprevention trials.

Prior studies strongly suggest that there is inter-individual variation in inherited BC risk factors (14–16). One clear heritable risk factor for certain cancers is suboptimal DNA repair gene capability (14,17,18). Multiple studies support the relationship between reduced overall DNA repair capacity and risk for BC (2,18–27). However, no single DNA repair gene variant is associated with highly penetrant familial inheritance of increased BC risk (14). These findings are consistent with a model in which many DNA repair genes contribute to protection of normal bronchial epithelial cells (BECs) from DNA damage such that an allele that reduces function of one gene causes only small overall reduction in DNA repair capacity, and in each individual with substantially reduced capacity, a different set of multiple different genes is responsible.

Evidence supporting the association of inter-individual variation in regulation of DNA repair genes with inter-individual variation in lung cancer risk was provided in our previous study in which there was correlation between transcript abundance of key DNA repair and antioxidant genes and CEBPG transcription factor in normal BECs of non-BC individuals (28). Importantly, this correlation was not present in normal BEC of BC individuals. E2F1 expression also was correlated with the expression of most of these DNA repair and antioxidant genes in normal BEC of non-BC individuals, but at a lower level than CEBPG, and the correlation was not significantly lower in BC individuals. Notably, the correlation of CEBPG or E2F1 with these genes was not observed with 12 other transcription factors assessed, including four other members of the CEBP family (CEBPA, CEBPB, CEBPD and CEBPE). These findings led us to hypothesize that polymorphisms in the regulatory regions of the DNA repair and antioxidant genes result in suboptimal regulation by CEBPG, leading to increased risk for BC.

The focus of this study is ERCC5. Although also known as XPG, the HUGO Gene Nomenclature Committee of ERCC5 is used for this and all other genes mentioned here. In the last few years, several studies have found an association between DNA sequence variations in the ERCC5-coding region and increased risk for BC (29,30). The reported concordance between cancer risk and coding region polymorphisms in these studies varies and results have been negative in some studies (31). This experience is similar to studies attempting to find association between BC risk and polymorphisms in coding regions of other DNA repair, antioxidant or xenobiotic metabolism genes (32). One reason given for poor concordance among these studies is that because the prior likelihood of association between particular polymorphisms and risk was not ascertained, there was high likelihood of false-positive results (17,33–38). Another potential reason for poor concordance may be lack of control for polymorphisms that are not in the coding region but yet have profound effect on gene expression and/or function. In this context, there is an increasing body of literature suggesting that polymorphisms within the non-coding regulatory regions of genes contribute to inter-individual variation in observed phenotypes (39–41).

ERCC5 was one of the DNA repair genes that were suboptimally regulated by CEBPG in BC individuals (28). In an effort to identify which non-coding regulatory regions of ERCC5 may contain sequence variations capable of altering the risk phenotype, we evaluated a 589 bp region of the ERCC5 promoter containing putative binding sites for multiple transcription factors including CEBPG, E2F1 and YY1.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 References
 
Primary sample acquisition
Adult patients undergoing diagnostic bronchoscopy at The University of Toledo Medical Center were approached to participate in this study. Informed consent was obtained according to the guidelines approved and monitored by the Institutional Review Board of The University of Toledo. Following diagnostic testing, grossly normal BEC samples from a non-diseased area of the airways were obtained via bronchoscopic bronchial brushing as reported previously (28). For BC individuals, samples were taken from the non-cancerous lung. Cells were recovered from the bronchial brush through gentle agitation in ice-cold 0.9% NaCl solution and then pelleted.

Cell culture and transfection
Human lung adenocarcinoma cells lines (H460 and H23) were obtained from National Center for Biotechnology Information and cultured using RPMI 1640 medium supplemented with 10% fetal bovine serum. One day prior to transfection, cells were trypsinized and seeded into 60 mm dishes at a predetermined density such that cells were 90–95% confluent at the time of transfection. Cells were transfected with Lipofectamine 2000 (20 µl per dish) and OptiMEM I medium (both from Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Cells were incubated with the liposome complexes for 6 h and then the media were replaced with RPMI 1640 + 10% fetal bovine serum. Cells were harvested after an additional 42 h of incubation.

Luciferase assay
Following transfection and 42 h of incubation, cells were trypsinized and counted. Equivalent cell numbers from each transfection were pelleted and re-suspended in 0.25 M Tris–Cl, pH 8.0, at a concentration of 104 cells per ml, lysed by 4–5 flash freeze/thaw cycles in dry ice/ethanol or 37°C water baths and centrifuged briefly to pellet cellular debris. Ten microliter aliquots were used for luminescence measurement using a Lumat LB 9507 tube luminometer (Berthold Technologies GmbH & Co KG, Bad Wildbad, Germany) and Luciferase Assay Reagent (Promega Corporation, Madison, WI).

Western blot analysis
Transfected cells were lysed in NP40 Cell Lysis Buffer from Invitrogen or 0.25 mM, pH 8, Tris–Cl buffer. Samples were then flash frozen in a dry ice/ethanol bath and flash thawed in a 37°C water bath three times. Total protein concentration was determined colorimetrically using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Ten micrograms of total protein was denatured and loaded on to a sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel (14% Tris–glycine 1.5 mm pore size) and electrophoresed for 1.5 h at 120 V. Purified CEBPG protein (Abnova Corp., Taipei, Taiwan) was used as a positive control. Transfer to the polyvinylidene difluoride membrane was achieved by electrophoresis for 2 h at 30 V at 4°C. Membranes then were washed, blocked and probed with 30 µg of CEBPG (Abnova Corp.) or 0.8 µg of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, Cambridge, MA) mouse monoclonal antibody diluted in 10 ml of 5% milk. Detection of CEBPG protein/Ab complexes was performed by hybridization to goat anti-mouse secondary antibody conjugated to horseradish peroxidase followed by electrochemiluminescence.

RNA extraction and reverse transcription
Primary or cultured cells were lysed in TriReagent. Total RNA was extracted according to the TriReagent Manufacturer Protocol (Molecular Research Center, Cincinnati, OH). Following extraction, messenger RNA (mRNA) samples were reverse transcribed using Moloney-Murine Leukemia Virus reverse transcriptase and an oligo dT primer as reported previously (28). mRNA samples from transfected cells were treated with RNase-free DNase (Ambion DNA-free kit, Austin, TX) one or two times to remove residual plasmid DNA prior to reverse transcription.

Transcript abundance measurement
Transcript abundance data were obtained by standardized reverse transcription–polymerase chain reaction (StaRT–PCR) (42,43). According to this method, each target gene and loading control gene was measured relative to a known amount of its respective internal standard (IS) within a standardized mixture of internal standards (SMISs). Each competitive template IS was 10–20% shorter in length than the target gene native template (NT) PCR product but both were amplified with the same efficiency by the same pair of primers. In these studies, β-actin (ACTB) was used as a loading control gene. For each PCR, each cDNA sample was diluted such that a 1 µl volume contained ~60 000 molecules of ACTB cDNA. The proper concentration was identified by PCR amplifying a serial dilution of cDNA in the presence of 1 µl of SMIS that contained 10–13 M ACTB IS (60 000 molecules). Prior to amplification, equal volumes of cDNA and SMIS were combined into a master mix along with the appropriate volume of RNase-free H2O, 3 mM MgCl2, 0.2 mM deoxyribonucleoside triphosphates and a minimum of 0.1 U Taq polymerase (Promega, Madison, WI). Each master mixture was prepared to contain sufficient cDNA and SMIS to measure each of the desired genes. This mixture was divided into tubes (or wells) containing primers for single genes. All PCRs were performed in a Rapidcycler (Idaho Technologies, Salt Lake City, Utah) for 35 cycles. All reactions were denatured for 5 s at 94°C, annealed for 10 s at 58°C and elongated for 15 s at 72°C. Following PCR amplification, the IS and NT for each gene were electrophoretically separated and quantified and number of NT molecules for each target gene and for ACTB was quantified by comparison with known number of molecules for each respective IS. Transcript abundance values were then calculated as target gene molecules per 106 ACTB molecules.

StaRT–PCR technology is licensed to Gene Express (Toledo, OH). Many of the reagents used in this study are available commercially and were obtained through Gene Express. StaRT–PCR reagents for each of the measured genes that were not commercially available, including primers and IS, were prepared according to previously described methods (42,43). Whenever possible, triplicate experiments were performed.

Vectors and constructs
Full-length CEBPG and E2F1 expression vectors in the mammalian expression vector pCMV-SPORT6 were purchased from Open Biosystems (Huntsville, AL). The negative control plasmid pCMV-SPORT6 was generated by excising the CEBPG insert through restriction digestion with AvaI (Fisher Scientific International, Hampton, NH). A full-length CEBPB clone in the pOTB7 vector was purchased from Open Biosystems, excised with EcoRI and XhoI enzymes (Invitrogen Corporation, Carlsbad, CA and Fisher Scientific International, respectively) and ligated into pCMV-SPORT6.

Various lengths of the ERCC5 promoter were directionally cloned into pGL2-Basic vector (Promega Corporation) in front of the luciferase reporter gene using the HindIII and XhoI restriction sites (Figure 1). Sites in the ERCC5 promoter were deleted using a combination of PCR methods described previously (44,45). The specific methods used are schematically presented in supplementary Figure 1 (available at Carcinogenesis Online). Point mutations were generated using the Higuchi method (44).


Figure 1
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  		Fig. 1. Analysis of ERCC5 regulatory region by transfecting various ERCC5-luciferase constructs into two Non-small cell lung cancer cell lines. Various constructs containing regions of the ERCC5 promoter were created as described in the Materials and Methods. The absolute position of transcript start site 2 (TSS2) on human chromosome 13 is 102296175 (ref|NT_009952.14|Hs13_10109). The position of the 5' end of each construct relative to transcript start site 2 is provided to the left of the construct. Positions of the putative binding sites relative to transcript start site 2 are as follows: VMYB = –144, CEBP(1) and ELK1(1) = –122, E2F(1) = –87, CEBP(2) = –63, EVI1 = –48, ELK1(2) and AREB6(1) = +3, E2F(2) and YY1 = +22, ELK1(3) and AREB6(2) = +84. The position of TSS1 is +246 relative to transcript start site 2. Relative light units (RLUs) for the endogenous effects are provided as a percentage of P-3 RLU. NA, not assessed and del, deleted.

 
All ligations were performed using T4 DNA ligase from the Invitrogen Corporation . All plasmids were sequenced and confirmed for correct sequence and orientation prior to use in further studies (University of Iowa DNA Facility, Iowa City, Iowa).

Transcription factor recognition site analysis
As reported previously (28), MatInspector Version 7.4.3 (Genomatix Software GmbH, Munich, Germany, http://www.genomatix.de/products/index.html) software was used to identify putative transcription factor recognition sites in the ERCC5 promoter. A full list of putative binding sites is provided in supplementary Table I (available at Carcinogenesis Online).

Preparation of nuclear extract
Nuclear extracts (NEs) were prepared from H460 cells as follows: ~9.3 x 107 cells were harvested via trypsinization, washed twice with ice-cold phosphate-buffered saline and centrifuged at low speed (700g, 4°C and 10 min) in a 1.5 ml microcentrifuge tube. Approximately 600 µl of packed cells were re-suspended in 1800 µl of NucBuster Extraction Reagent 1 (Novagen, San Diego, CA). The suspension was mixed for 15 s at highest speed with a Baxter S/P S8223 vortexer, incubated on ice for 5 min and vortexed again for 15 s. The suspension was centrifuged at 16 000g for 5 min at 4°C in a 1.5 µl microcentrifuge tube. The supernatant was discarded and the nuclear pellet was re-suspended in 12 µl of 100x Protease Inhibitor Cocktail, 12 µl of 100 mM dithiothreitol and 900 µl of NucBuster Extraction Reagent 2 (Novagen). The suspension again was vortexed, incubated on ice for 5 min and vortexed one last time. The suspension was centrifuged at 16 000g for 5 min at 4°C. The supernatant was aliquoted and stored in NucBuster Reagent 2 at –80°C. Total protein yield was measured spectrophotometrically on a Nano-Drop ND-1000 UV-VIS Spectrophotometer (Nanodrop Techonologies, Wilmington, DE).

Electrophoretic mobility shift assay
Three different oligonucleotides corresponding to sequences within the promoter region and 5' UTR of ERCC5 gene on human chromosome 13 (ref|NT_009952.14|Hs13_10109) were synthesized (Invitrogen Corporation, Frederick, MD) and used in electrophoretic mobility shift assay and supershift assays (supplementary Table II is available at Carcinogenesis Online). These included a 32 bp oligonucleotide [CEBP(2)] spanning nucleotide 16587778 to nucleotide 16587809 and a 30 bp oligonucleotide (ERCC5 E2F/YY1) spanning nucleotide 16587856 to nucleotide 16587885. Positive control oligonucleotides known to bind E2F (42 bp) or YY1 (27 bp) were also synthesized (46,47).

The double-stranded DNA fragments were labeled with [{gamma}-32P] deoxyadenosine triphosphate using 5' End Labeling Kit (Amersham Biosciences, Piscataway, NJ). The probes were purified using ProbeQuant G-50 Micro Columns (GE Healthcare, Piscataway, NJ) and serially diluted in 150 mM Sodium Tris EDTA to 50 fmol/µl of labeled double-stranded DNA probe. Twenty microliter volumes containing 0.9, 3.0, 9.0, 15.0 or 30.0 µg of H460 NE, 50.0 ng of E2F1 (Jena Bioscience, Jena, Germany) or 1 µg of CEBPG recombinant protein (Abnova Corp.) were mixed with 50 fmol of labeled probe (supplementary Table II is available at Carcinogenesis Online) in 6 mM Tris–HCl, pH 7.4, 60 mM NaCl, 1.2 mM MgCl2, 0.3 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 0.3 µg/µl bovine serum albumin and 3.6% glycerol and then incubated for 30 min on ice followed by 40 m at room temperature. For supershift reactions, 2 mg E2F1 (sc-251x) (Santa Cruz Biotechnology, Santa Cruz, CA), 2 mg of YY1 (sc-7341x) or 3 µg CEBPG antibody (Abnova Corp.) were added during the initial incubation on ice before labeled probe was added. Competition was performed with the addition of excess unlabeled E2F- or YY1-positive control probes during the initial incubation on ice before labeled probe was added. DNA/protein complexes were electrophoresed in a non-denaturing 6% polyacrylamide gel equilibrated with 0.5x Tris Borate EDTA at 4°C for 240 min at 180 V. After electrophoresis, gels were dried and autoradiographed.

Densitometry and image analysis
Briefly, autoradiographs were digitized using a UMAX professional high-resolution backlit flatbed image scanner. Relative densitometry of bands on the autoradiograms were measured using NIH ImageJ gel analysis suite combined with background subtraction and rolling ball radius set to 5000 (48). Subsequent data analysis was performed using Microsoft Excel, v2002.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 References
 
Effects of CEBPG or CEBPB expression vectors on ERCC5 promoter activity
Co-transfection of CEBPG XV5 expression vector with 3 µg of ERCC5 promoter-1 (P-1) luciferase construct into H23 caused 3- to 6-fold higher activation of the exogenous ERCC5 promoter than the empty pGL2-Basic vector (Figures 1 and 2). In contrast, co-transfection of CEBPB expression vector and P-1 vector did not lead to an increased luciferase activity (data not shown). Endogenous levels of ERCC5 mRNA also were significantly increased by transfection of CEBPG XV5 relative to transfection with pGL2-Basic (6500 mRNA/106 ACTB mRNA versus 3100 mRNA/106 ACTB mRNA, P = 0.0028). Transfection of CEBPG XV5 also led to an increased CEBPG protein as confirmed by western blot (Figure 3). Low levels of CEBPG protein seen in lysates not transfected with the CEBPG expression vector probably represent endogenous expression of the protein in H23 cells. The identity of faint larger bands observed in lanes transfected with CEBPG is presently not known but one possibility is post-translational modification of CEBPG protein.


Figure 2
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  		Fig. 2. Luciferase activity in H23 following co-transfection of CEBPG expression vector or empty vector control with ERCC5 P-1 luciferase construct. Different levels of CEBPG expression vector (7, 10 or 15 µg) or empty vector control (pCMV-SPORT6) were co-transfected with 3 µg of ERCC5 P-1 luciferase construct into H23 cells.

 


Figure 3
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  		Fig. 3. CEBPG protein expression in H23 cells transfected with CEBPG, CEBPB or both expression vectors. H23 cells transfected with CEBPG and/or CEBPB expression vectors or empty vector control (pCMV-SPORT6) were analyzed for the presence of CEBPG protein by western blot analysis. Ten micrograms of each cell lysate was loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis. CEBPG protein was expressed at an increased level in each lysate from cells transfected with the CEBPG expression vector but not in those transfected with pCMV-SPORT6 or CEBPB expression vector alone.

 
Evaluation of ERCC5 regulatory region by deletion analysis
Transfection of ERCC5-luciferase reporter constructs alone (without co-transfection of an expression vector) enabled analysis of the ERCC5 promoter regions primarily responsible for up- or down-regulation of this gene by endogenous factors. Background associated with the empty vector control (pGL2-Basic) was subtracted from all values. The P-1 ERCC5-Luciferase reporter construct contained a 589 bp segment of the ERCC5 regulatory region in which the 5' end is 540 bp upstream of the ERCC5.2 transcription start site. P-1 and constructs containing serial deletion of P-1 were tested for endogenous activity in cell lines with low (H23-380 mRNA/106 ACTB mRNA) or high (H460-2460 mRNA/106 ACTB mRNA) CEBPG transcript abundance (Figure 1). All results are presented relative to promoter-3 (P-3) due to the relatively high level of P-3 activity and because this construct was used to create additional deletion constructs. Compared with P-1, P-3 contained a deletion of a putative v-Myb site and distal CEBP [CEBP(1)] recognition site. Correspondingly, the luciferase values for P-1 were lower in H23 (40%) and H460 (30%) compared with P-3. The site responsible for down-regulation likely was v-Myb since deletion of this site but not CEBP(1) in P-2 resulted in about the same increase in activity. Little or no further change was observed with deletion of the more distal E2F(1) site in P-4. However, a marked reduction to 10% (H23) or 30% (H460) was observed in P-5 in which there was a deletion of the more proximal CEBP site [CEBP(2)] as well as putative sites for ELK1, AREB6 and EVI1.

In order to more closely define the role of the CEBP(2) site, it was selectively deleted from P-3 to create P-3delCEBP(2), u sing a combination of the previously described methods (44,45) and those developed as part of this study (supplementary Figure 1 is available at Carcinogenesis Online). CEBP(2) deletion was associated with a 30% (H23) or 40% (H460) reduction in luciferase activity relative to P-3. Deletion of the YY1/E2F sites in P-5 to create P-6 caused no further reduction in activity in either H23 or H460.

The possible role of EVI1, ELK1 and/or AREB6 in conferring the remaining activity in P-3delCEBP(2) was assessed by measuring transcript abundance of these three transcription factors in normal BEC (supplementary Table III is available at Carcinogenesis Online) (28). EVI1 was expressed at very low level in normal BEC (28), while ELK1 and AREB6 were expressed at high levels (supplementary Table III is available at Carcinogenesis Online).

Specific deletion of the ELK1 and AREB6 sites [P-3delELK1(2)/AREB6(1)] reduced endogenous transcriptional activity by 80% (H23) or 60% (H460) relative to P-3.

Specific deletion of the E2F/YY1 site in the P-3delE2F(2)/YY1 construct was associated with a 3-fold increase in luciferase expression in H460 and 4.3-fold increase in H23 (Figure 1), consistent with repressive effect of the E2F/YY1 site in these cell lines.

Co-transfection of CEBPG XV5 with various ERCC5 promoter regions into H23 (low endogenous CEBPG) or H460 (low endogenous CEBPG) was conducted to study which regions were essential for up-regulation by CEBPG. For these experiments, 15 µg of CEBPG XV5 was transfected, based on the results of experiments presented in Figure 2. Some degree of up-regulation was observed with each promoter region tested in both cell lines with the exception of P-3delELK1(2)/AREB6(1) and P-4.5 in H460 cells. In general, the level of induction was greater in H23. Induction of luciferase was observed even for P-3delCEBP(2) in which neither putative CEBP binding site was present.

mRNA expression and correlation in primary normal BEC
As reported previously, expression of ERCC5 mRNA was significantly correlated with CEBPG and E2F1 mRNA levels in normal BEC (28). This correlation held true in a set of 76 additional samples measured here (19 normal BEC from individuals with BC and 57 normal BEC from individuals without BC). Expression of five additional transcription factors for which putative binding sites were found in the region of ERCC5 promoter assessed here (AREB6, CEBPD, DDIT3, ELK1 and YY1) was measured in 20 normal BEC (10 non-cancer and 10 cancer). Average gene expression levels are provided in supplementary Table III (available at Carcinogenesis Online). No significant correlation was found between ERCC5 and any of these transcription factors among either the BC or non-BC subgroups or among the combined set of 20.

Electrophoretic mobility shift assay experiments
According to the Genomatix analysis (49), a putative CEBP consensus site, CEBP(2), exists immediately upstream from putative E2F/YY1 recognition sites. Based on ERCC5-luciferase experiments, removal of CEBP(2) decreased endogenous regulation of the ERCC5 promoter by 30–40%. To directly test whether CEBPG was specifically recruited to this consensus site, CEBPG recombinant protein was incubated with CEBP(2) probe from ERCC5 regulatory region. As is evident from Figure 4, CEBPG recombinant protein caused mobility shift of the CEBPG probe and antibody resulted in a supershift.


Figure 4
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  		Fig. 4. Electrophoretic mobility shift assay of CEBPG recombinant protein binding to CEBP(2) consensus probe. Recombinant CEBPG protein binds to the upstream CEBP(2) consensus site in the ERCC5 promoter. The lowest and middle arrows point to the two CEBPG-shifted bands in lane 2. The highest arrow points to the CEBPG antibody (Ab) supershifted upper band in lane 3.

 
In a previously reported study, E2F1 was the only transcription factor other than the bZIP family member CEBPG to be significantly correlated with ERCC5 (28). Genomatix analysis identified a putative YY1 binding site directly opposite to the E2F binding site. We hypothesized that titration of H460 NE against a 30 bp region spanning putative recognition sites for both E2F and YY1 would result in delay of protein complexes containing E2F1 and YY1 protein. Three predominant complexes were observed with the ERCC5 E2F/YY1 probe when incubated with 9 µg or more of H460 NE (Figures 5A and 6A; supplementary Figure 2 available at Carcinogenesis Online). However, band complex 2 was clearly visible following incubation with as little as 0.3 µg (data not shown). According to the densitometric analysis, as the concentration of H460 NE was increased, the intensity of band complex 2 increased significantly more rapidly than band complex 1 or 3.


Figure 5
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  		Fig. 5. Electrophoretic mobility shift assay of E2F1 binding to ERCC5 E2F/YY1 consensus probe. (A) As negative controls, the binding reaction mixture contained only E2F positive control probe (lane 1) or ERCC5 E2F/YY1 consensus sequence probe (lane 4). As positive controls, binding reaction mixtures were incubated with E2F positive control probe and E2F1 recombinant protein (RP) (lane 2) or ERCC5 E2F/YY1 probe and H460 NE (lane 5). Addition of 2 µg of E2F1 antibody (Ab) to both E2F1 recombinant protein with E2F positive control probe (lane 3) and H460 NE with ERCC5 E2F/YY1 consensus sequence probe (lane 6) resulted in supershift of E2F1 recombinant protein–E2F-positive control probe complex (lane 3) and alteration in the density of bands 1 and 2 for H460 NE-ERCC5 E2F/YY1 probe complex (lane 6). Additional competition experiments are presented in supplementary Figure 2 (available at Carcinogenesis Online). (B) Densitometric analysis of the effect of E2F1 antibody on the ratio of band complex 1:2 for ERCC5 E2F/YY1 probe from lanes 5 and 6.

 


Figure 6
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  		Fig. 6. Electrophoretic mobility shift assay of YY1 binding to ERCC5 E2F/YY1 consensus probe. (A) As negative controls, binding reaction mixtures contained only YY1 positive control probe (lane 1) or ERCC5 E2F/YY1 consensus sequence probe (lane 6). As positive controls, binding reaction mixtures were incubated with YY1 positive control probe (lanes 2 and 4) or ERCC5 E2F/YY1 probe (lane 7) and H460 NE. Addition of 2 µg of YY1 antibody (Ab) to both H460 NE with YY1 positive control probe (lanes 3 and 5) and H460 NE with ERCC5 E2F/YY1 consensus sequence probe (lane 9) resulted in supershift for only the H460 NE-YY1-positive control complex (lanes 3 and 5) and not H460 NE-ERCC5 E2F/YY1 complex (lane 9). Addition of both 25x cold YY1 positive control probe and 2 µg of YY1 antibody resulted in decreased intensity of bands 2 and 3 (lane 10). Additional competition experiments are presented in supplementary Figure 2 (available at Carcinogenesis Online). (B) Densitometry of the ratio of complex 1:2 revealed that complex 2 for ERCC5 E2F/YY1 probe is differentially affected by the addition of YY1 antibody with 25x cold YY1 control probe competitor (lane 10), and not 25x cold YY1 control probe competitor (lane 8) or YY1 antibody (lane 9) alone. A single electrophoretic mobility shift assay was performed.

 
To explore the specificity of the three band complexes to YY1 and/or E2F proteins, we performed competition experiments with unlabeled YY1 and E2F positive control probes. Both unlabeled control probes successfully competed out band complex 2 (supplementary Figure 2 is available at Carcinogenesis Online). Additionally, to a lesser degree, YY1 control probe competed out band complex 3 and E2F control probe competed out band complex 1 and other higher molecular weight bands (supplementary Figure 2 is available at Carcinogenesis Online).

E2F1 antibody caused a statistically significant (P < 0.001) increase from 1.0 to 1.75 in the band complex 1:2 density ratio for ERCC5 E2F/YY1 probe (Figure 5A and B) which indicated that E2F1 recognizes this region.

For YY1, the addition of cold-positive YY1 control probe to ERCC5 E2F/YY1 probe was associated with a small increase in the ratio of band complex 1:2 consistent with competitive blocking of band complex 2 (Figure 6A and B). In contrast, no apparent effect on band complex 1:2 was observed with YY1 antibody alone. However, the increase in band complex 1:2 effect was dramatically increased when both YY1 cold probe and antibody were present. Specifically, the increase in ratio was 4.8-fold for ERCC5 E2F/YY1 (Figure 6A and B).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 References
 
Based on previous studies, key DNA repair and antioxidant genes were correlated at the transcript abundance level in normal BEC of non-BC individuals (28). Computer-aided analysis identified six putative transcription factor family binding sites common among the correlated genes. Ten transcription factors potentially capable of binding at these sites and expressed in normal BEC were studied. Of these, only CEBPG and E2F1 were correlated with DNA repair genes in normal BEC of non-BC individuals and this correlation was significantly lower in normal BEC of BC individuals for CEBPG (28). These findings support the hypothesis that CEBPG up-regulates these key genes. In this study, we tested the hypothesis that CEBPG up-regulates the key DNA repair gene ERCC5. The data presented here support this hypothesis and provide initial understanding of the mechanisms responsible.

CEBPG is a truncated isoform of the CEBP family of transcription factors and can bind to DNA but not transactivate by itself. It participates in transcriptional regulation by forming heterodimers with other CEBP family members (50–53). Evidence from this study indicates that the CEBP recognition site participates in CEBPG regulation of ERCC5. Specifically, CEBPG protein binds to this site and expression of exogenous CEBPG protein leads to increased levels of endogenous ERCC5 transcript.

However, because deletion of CEBP(2) site did not result in a complete loss of regulation by CEBPG, this suggests that additional adjacent binding sites play a part in ERCC5 regulation, likely by way of protein–protein interactions. As reported here, deletion of a nearby putative Ets family member binding site [ELK1(2)/AREB6(1)] led to a significant reduction in ERCC5 promoter activity in response to both endogenous and exogenous CEBPG protein expression. We conclude that this ELK1/AREB6 recognition site contributes to CEBPG regulation of ERCC5, either through indirect protein–protein interactions or direct recognition of a non-canonical sequence motif not yet characterized. Heterodimer formation between CEBP family members and Ets family members has been reported to play a role in regulation of other genes (54–58), and it is reasonable to hypothesize that it may play a role in ERCC5 regulation.

In addition to the likely role of Ets family members, the data reported here support the role of E2F1 in CEBPG regulation of ERCC5. Specifically, removal of the E2F/YY1 binding site was associated with a >2-fold up-regulation of the ERCC5 promoter. We confirmed by electrophoretic mobility shift assay that both E2F1 and YY1 proteins bind to this region (Figures 5A and 6A; supplementary Figure 2 is available at Carcinogenesis Online). The varying response of complexes 1, 2 and 3 to antibody supershift and cold-positive control probe competition observed in our gel shift assays (Figures 5A and 6A; supplementary Figure 2 is available at Carcinogenesis Online) indicate that there are likely multiple complexes with varying compositions of E2F1, YY1 and possibly other factors. From our results, it is likely that complex 1 primarily consists of E2F1 protein, complex 3 primarily YY1 protein and complex 2 a combination of the two.

The results reported here suggest that binding of E2F1 and/or YY1 to their predicted recognition sites may modify CEBPG regulation of ERCC5. However, whether this modification is induction or repression may be in part determined by competitive binding between E2F1 and YY1, and certain conditions may determine whether the effect of E2F1 or YY1 dominates. For example, the previously reported weak but significant positive correlation between E2F1 and ERCC5 mRNA levels indicates that E2F1 up-regulates ERCC5 in normal BEC (28). In contrast, in the H460 and H23 carcinoma cell lines removal of the E2F/YY1 site resulted in up-regulation of ERCC5. Previous studies indicate that, in general, E2F1 up-regulates DNA repair genes (59,60) and YY1 is frequently implicated in repression of transcription (47).

Evidence reported here for a role of E2F1 in regulating ERCC5 is consistent with increasing evidence over the last 10 years that signals induced by DNA damage up-regulate E2F1 (61–65) and that E2F1 up-regulates transcription of key DNA repair genes (66). Following DNA damage, phosphorylation of E2F1 by both ataxia telangiectasia mutated (64) and Chk2 (65) stabilizes it and alters its binding affinity for its DNA recognition sites. This shifts E2F1 binding from sites in cell cycle regulatory genes to sites in other classes of genes, including apoptosis genes (67). There also is increasing indirect evidence that E2F1 regulates transcription of key DNA repair genes (59). Skin of mice with knockout of both E2F1 alleles have reduced overall nucleotide exicision repair capacity following ultraviolet B irradiation, whereas transgenic mice with up-regulated E2F1 have increased nucleotide exicision repair capacity (66). In a large survey study using ChIP analysis, it was determined that E2F1 bound to regulatory regions of many DNA repair genes (59). Interestingly, and consistent with the results reported here, others have reported the need for YY1 elements to be located in close approximation to putative E2F consensus sites for the gene of interest to maintain optimal regulation of transcription (68,69).

The approach used by Mullins et al. (28) followed by that reported here provides a means to identify functionally important non-coding regions of genes and increase the likelihood that particular variants in these regions are functionally active in gene regulation. This approach provides an appropriate means to respond to recent recommendations that inclusion of particular DNA variants in molecular epidemiological studies be based on clearly established functional relevance (33–35). In this specific case, the approach provides a practical mechanistic method to investigate inter-individual variation in regulation of ERCC5, a key DNA repair gene, and thus susceptibility to BC and possibly other diseases. Here, we provide evidence that three regions of the ERCC5 promoter contribute to transcription regulation. Several polymorphisms have been reported within these regions (Entrez SNP, http://www.ncbi.nlm.nih.gov/sites/entrez?db=Snp) and, based on the data presented here, there is reason to believe that they play a functional role in ERCC5 transcription and/or function. Combining epidemiologic analysis of functionally significant DNA variants in the ERCC5 regulatory region with analysis of variants in the ERCC5-coding region previously associated with BC risk likely will lead to better understanding of the association of ERCC5 functional activity with BC risk.

Ultimately, the most informative assessment of the association between DNA variants and BC risk probably will include analysis of multiple variants, highly likely to be associated with risk, located in both the regulatory and coding regions of multiple key DNA repair and antioxidant genes, including ERCC5.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary Figures 1 and 2 and Tables I–III can be found at http://carcin.oxfordjournals.org/


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
We would like to thank Dr Herman Schut for the generous donation of [{gamma}-32P] deoxyadenosine triphosphate and the members of Dr Kandace Williams' Group for their generous troubleshooting help with gel shift assays.

Conflicts of Interest Statement: J.C.W. and E.L.C. own significant equity interest in Gene Express, which produces and markets standardized reverse transcription–PCR reagents that were used in this study. In addition, J.C.W. acts as a consultant/independent contractor for Gene Express. All relationships between the authors and Gene Express are overseen and managed by The University of Toledo Conflict of Interest Committee.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received August 3, 2007; revised September 12, 2007; accepted September 15, 2007.


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