Ets-1 and Runx2 Regulate Transcription of a Metastatic Gene, Osteopontin, in Murine Colorectal Cancer Cells
2006; Elsevier BV; Volume: 281; Issue: 28 Linguagem: Inglês
10.1074/jbc.m511962200
ISSN1083-351X
AutoresPhilip Y. Wai, Zhiyong Mi, Chengjiang Gao, Hongtao Guo, Carlos E. Marroquin, Paul C. Kuo,
Tópico(s)Oral and Maxillofacial Pathology
ResumoOsteopontin (OPN) is a sialic acid-rich phosphoprotein secreted by a wide variety of cancers. We have shown previously that OPN is necessary for mediating hepatic metastasis in CT26 colorectal cancer cells. Although a variety of stimuli can induce OPN, the molecular mechanisms that regulate OPN gene transcription in colorectal cancer are unknown. We hypothesized that cis- and trans-regulatory elements determine OPN transcription in CT26 cells. OPN transcription was analyzed in CT26 cancer cells and compared with YAMC (young adult mouse colon) epithelial cells. Clonal deletion analysis of OPN promoter-luciferase constructs identified cis-regulatory regions. A specific promoter region, nucleotide (nt) –107 to –174, demonstrated a >8.0-fold increase in luciferase activity in CT26 compared with YAMC. Gel-shift assays sublocalized two cis-regulatory regions, nt –101 to –123 and nt –121 to –145, which specifically bind CT26 nuclear proteins. Competition with unlabeled mutant oligonucleotides revealed that the regions nt –115 to –118 and nt –129 to –134 were essential for protein binding. Subsequent supershift and chromatin immunoprecipitation assays confirmed the corresponding nuclear proteins to be Ets-1 and Runx2. Functional relevance was demonstrated through mutations in the Ets-1 and Runx2 consensus binding sites resulting in >60% decrease in OPN transcription. Ets-1 and Runx2 protein expression in CT26 was ablated using antisense oligonucleotides and resulted in a >7-fold decrease in OPN protein expression. Ets-1 and Runx2 are critical transcriptional regulators of OPN expression in CT26 colorectal cancer cells. Suppression of these transcription factors results in significant down-regulation of the OPN metastasis protein. Osteopontin (OPN) is a sialic acid-rich phosphoprotein secreted by a wide variety of cancers. We have shown previously that OPN is necessary for mediating hepatic metastasis in CT26 colorectal cancer cells. Although a variety of stimuli can induce OPN, the molecular mechanisms that regulate OPN gene transcription in colorectal cancer are unknown. We hypothesized that cis- and trans-regulatory elements determine OPN transcription in CT26 cells. OPN transcription was analyzed in CT26 cancer cells and compared with YAMC (young adult mouse colon) epithelial cells. Clonal deletion analysis of OPN promoter-luciferase constructs identified cis-regulatory regions. A specific promoter region, nucleotide (nt) –107 to –174, demonstrated a >8.0-fold increase in luciferase activity in CT26 compared with YAMC. Gel-shift assays sublocalized two cis-regulatory regions, nt –101 to –123 and nt –121 to –145, which specifically bind CT26 nuclear proteins. Competition with unlabeled mutant oligonucleotides revealed that the regions nt –115 to –118 and nt –129 to –134 were essential for protein binding. Subsequent supershift and chromatin immunoprecipitation assays confirmed the corresponding nuclear proteins to be Ets-1 and Runx2. Functional relevance was demonstrated through mutations in the Ets-1 and Runx2 consensus binding sites resulting in >60% decrease in OPN transcription. Ets-1 and Runx2 protein expression in CT26 was ablated using antisense oligonucleotides and resulted in a >7-fold decrease in OPN protein expression. Ets-1 and Runx2 are critical transcriptional regulators of OPN expression in CT26 colorectal cancer cells. Suppression of these transcription factors results in significant down-regulation of the OPN metastasis protein. Colorectal cancer continues to be the second leading cause of cancer-related deaths in the United States (1Jemal A. Murray T. Ward E. Samuels A. Tiwari R.C. Ghafoor A. Feuer E.J. Thun M.J. CA-Cancer J. Clin. 2005; 55: 10-30Crossref PubMed Scopus (5576) Google Scholar, 2Parker S.L. Tong T. Bolden S. Wingo P.A. CA-Cancer J. Clin. 1996; 46: 5-27Crossref PubMed Scopus (1971) Google Scholar). Many patients with advanced or recurrent disease develop distant metastases and fail locoregional therapy. The molecular mechanisms underlying tumor metastasis are not completely understood, but recent evidence implicates osteopontin (OPN) 2The abbreviations used are: OPN, osteopontin; YAMC, young adult mouse colon cells; Ets, E26 transformation-specific sequence; RNAi, RNA interference; C/EBP, CCAATT/enhancer-binding protein; VEGF, vascular endothelial growth factor; nt, nucleotide(s); NE, nuclear extract; MMP, matrix metalloproteinase; ChIP, chromatin immunoprecipitation; siRNA, short interfering RNA; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; AML, acute myeloid leukemia. as a key regulator of cancer cell metastasis. OPN, an extracellular matrix protein secreted by a wide variety of cancers, functionally enables tumor progression (3Wai P.Y. Kuo P.C. J. Surg. Res. 2004; 121: 228-241Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 4Denhardt D.T. Guo X. FASEB J. 1993; 7: 1475-1482Crossref PubMed Scopus (1010) Google Scholar, 5Craig A.M. Denhardt D.T. Gene. 1991; 100: 163-171Crossref PubMed Scopus (154) Google Scholar, 6Denhardt D.T. Noda M. J. Cell. Biochem. Suppl. 1998; 30–31: 92-102Crossref PubMed Google Scholar, 7O'Regan A. Berman J.S. Int. J. Exp. Pathol. 2000; 81: 373-390Crossref PubMed Scopus (317) Google Scholar, 8Weber G.F. Biochim. Biophys. Acta. 2001; 1552: 61-85Crossref PubMed Scopus (130) Google Scholar). The secreted phosphoprotein binds the αvβ integrin and CD44 families of receptors to propagate cellular signals (8Weber G.F. Biochim. Biophys. Acta. 2001; 1552: 61-85Crossref PubMed Scopus (130) Google Scholar, 9Goodison S. Urquidi V. Tarin D. Mol. 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In colorectal cancer, gene profiling studies have identified a positive correlation between advanced or metastatic colon tumors and abundant OPN expression (15Agrawal D. Chen T. Irby R. Quackenbush J. Chambers A.F. Szabo M. Cantor A. Coppola D. Yeatman T.J. J. Natl. Cancer Inst. 2002; 94: 513-521Crossref PubMed Scopus (367) Google Scholar, 16Yeatman T.J. Chambers A.F. Clin. Exp. Metastasis. 2003; 20: 85-90Crossref PubMed Scopus (83) Google Scholar). We have previously shown that stable, RNAi-mediated down-regulation of OPN expression in CT26 colon adenocarcinoma cells results in suppression of matrix metalloproteinase-2 (MMP-2) expression, decreased in vitro tumor cell motility and invasiveness, and attenuation of functional in vivo hepatic metastasis (17Wai P.Y. Mi Z. Guo H. Sarraf-Yazdi S. Gao C. Wei J. Marroquin C.E. Clary B. Kuo P.C. Carcinogenesis. 2005; 26: 741-751Crossref PubMed Scopus (92) Google Scholar). Together, these data suggest that OPN is necessary for CT26 colorectal metastasis. Subsequent studies in our laboratory have confirmed that OPN protein expression is highly up-regulated in metastatic CT26 but absent in nonmetastatic cell lines such as YAMC. In this study, we investigated the molecular determinants that regulate OPN expression in CT26 colorectal tumor cells. CT26, an undifferentiated murine colon adenocarcinoma cell line, produces aggressive pulmonary and hepatic metastases in murine models (18Corbett T.H. Griswold Jr., D.P. Roberts B.J. Peckham J.C. Schabel Jr., F.M. Cancer Res. 1975; 35: 2434-2439PubMed Google Scholar, 19Ikubo A. Aoki Y. Nagai E. Suzuki T. Clin. Exp. Metastasis. 1999; 17: 849-855Crossref PubMed Scopus (6) Google Scholar), and constitutively expresses OPN at high levels. We hypothesized that specific cis-regulatory domains and trans-factors control OPN expression in CT26 colorectal cancer. Using transient transfection and clonal deletion analysis of OPN promoter-luciferase constructs, we identified cis-regulatory regions within the OPN promoter in CT26 cells versus YAMC control. Gel-shift assays sublocalized 20–30-base pair regions in this cis-domain that could form DNA-nuclear protein complexes. Competition with unlabeled mutant probes characterized the nucleotide sequence of the essential nuclear factor binding sites. A TRANSFAC data base query and antibody-supershift assays identified the corresponding trans-regulatory nuclear proteins as Ets-1 and Runx2. Chromatin immunoprecipitation (ChIP) assays evaluated the in vivo binding capacity of these targets to the OPN promoter. Consensus binding site mutations in subcloned OPN promoter-luciferase constructs demonstrated a significant decrease in transcriptional activity. Antisense oligonucleotides to Ets-1 and Runx2 determined the functional effect of decreased expression of these trans-regulatory nuclear proteins in CT26 cells. Together, these data suggest that expression of the tumor metastasis protein, OPN, requires Ets-1 and Runx2. Cell Culture—CT26 murine colon carcinoma cells were grown as monolayer cultures in DMEM-10% fetal bovine serum (FBS) (Invitrogen) supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin. Cells were maintained in a 37 °C incubator with 5% CO2-humidified air. YAMC cells were provided as a gift from Dr. Robert Whitehead (Vanderbilt University Medical Center, Nashville, TN). YAMC is a conditionally immortalized murine colonic epithelial cell line that expresses SV40-large-T-antigen when stimulated by interferon-γ at 33 °C. SV40-large-T-antigen is heat labile and degraded at 37 °C. YAMC cells were grown in monolayer in RPMI 1640 (Invitrogen) supplemented with 5% FBS, 1 μg/ml insulin, 5 IU/ml mouse interferon-λ, 100 IU/ml penicillin, and 100 μg/ml streptomycin in a 33 °C incubator with 5% CO2 in humidified air. The utilization by established investigators of the metastatic and nonmetastatic companion cell lines CT26 and YAMC in studying colon cancer is well described in the literature (20Whitehead R.H. VanEeden P.E. Noble M.D. Ataliotis P. Jat P.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 587-591Crossref PubMed Scopus (338) Google Scholar, 21Pozzi A. Yan X. Macias-Perez I. Wei S. Hata A.N. Breyer R.M. Morrow J.D. Capdevila J.H. J. Biol. Chem. 2004; 279: 29797-29804Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Western Blot Analysis—Total cell lysates were prepared and analyzed by SDS-PAGE as described previously (22Gao C. Guo H. Wei J. Mi Z. Wai P. Kuo P.C. J. Biol. Chem. 2004; 279: 11236-11243Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Briefly, 35 μg of protein/lane was resolved on 4–20% polyacrylamide gels (Gradipore Inc., Hawthorne, NY) and transferred to polyvinylidene membranes (Amersham Biosciences). The primary antibodies used were: anti-OPN (R&D Systems, Minneapolis, MN), anti-glyceraldehyde-3-phosphate dehydrogenase (Ambion, Austin, TX), anti-PEBP2α (Santa Cruz Biotechnology, Santa Cruz, CA), anti-aml-3 (Calbiochem). Secondary antibodies used were: donkey anti-goat IgG-horseradish peroxidase (Santa Cruz Biotechnology), donkey anti-mouse IgG-horseradish peroxidase (Santa Cruz Biotechnology), or goat anti-rabbit (Santa Cruz Biotechnology). Immunodetection was performed using horseradish peroxidase-based SuperSignal chemiluminescent substrate (Pierce). For quantification, the bands were scanned into an AlphaImager 3400 (Alpha Innotech, San Leandro, CA) and normalized by dividing the measured density of protein bands by the density of glyceraldehyde-3-phosphate dehydrogenase control bands from corresponding cell lysates. Plasmid Constructs—5′-Deletion fragments of the OPN promoter subcloned into pXP2 plasmid encoding luciferase were gifts from Dr. D. Denhardt (Rutgers University). Deletion constructs were constructed by PCR from the following primers, and the fragments were subsequently cloned into pGL3-basic luciferase reporter plasmid: OPN –69 (–69 to +79), OPN –88 (–88 to +79), OPN –107 (–107 to +79), OPN –120 (–120 to +79), OPN –134 (–134 to +79), OPN –160 (–160 to +79), OPN –174 (–174 to +79), OPN –209 (–209 to +79), OPN –258 (–258 to +79), OPN –472 (–472 to +79), OPN –512 (–512 to +79), OPN –670 (–670 to +79), OPN –777 (–777 to +79), and OPN –1467 (–1467 to +79). Consensus binding site mutations corresponding to Ets-1 (nt –120 to –114; 5′-GAGGAAG-3′ to 5′-ACTTTTA-3′), Runx2 (nt –136 to –129; 5′-AACCACAA-3′ to 5′-GGACAATTT-3′) and an ETS-1/Runx2 double mutant were constructed using QuikChange® II site-directed mutagenesis kit (Stratagene, La Jolla, CA), with the OPN –512 wild type fragment as template, and used in transient transfection assays. Mutational accuracy was verified using DNA sequencing and restriction enzyme digestion. Transient Transfection and Activity Assay—Preliminary experiments using pGL3-β-galactosidase were performed to determine that transfection efficiency was comparable between the two cell lines, CT26 and YAMC. DNA transfections of CT26 and YAMC cells were carried out in 12-well plates using Lipofectamine 2000 (Invitrogen). 24 h prior to transfection, cells were harvested using trypsin and plated at a density of 2 × 105 cells/well in 12-well plates in DMEM-10% FBS without antibiotics. 2 μg of plasmid DNA diluted in Opti-MEM I (Invitrogen) and 2 μl of Lipofectamine 2000 diluted in Opti-MEM I were combined and incubated for 20 min at room temperature. These reagents were incubated with the previously plated cells for 4 h at 37°C in a CO2 incubator. After 4 h, the transfection medium was then replaced with DMEM-10% FBS. 0.1 μg of pRL-SV40-Renilla, which contains a full-length Renilla reniformi luciferase gene under the control of a constitutive promoter was added to each well as a control for transfection efficiency between different wells. 24 h after transfection, the cells were harvested in 150 μl of reporter lysis buffer (Promega), and dual luciferase reporter assays were performed by following the manufacturer's directions. 20 μl of lysate was used for measurement in a luminometer (Turner Designs TD-20/20, Sunnyvale, CA). The results were reported as relative luciferase activity, which represents a ratio of luciferase firefly activity/Renilla activity. The data represent the mean ± S.D. of three different experiments repeated in triplicate. Nuclear Extract Preparation—Monolayers of CT26 cells were washed with phosphate-buffered saline and harvested by scraping into cold phosphate-buffered saline. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mm HEPES, pH 7.9, 10 mm KCL, 0.1 mm EDTA, 0.1 mm EGTA, 1.0 mm dithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride; then 10% Nonidet P-40 was added and vortexed briefly. The nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mm HEPES, pH 7.9, 0.4 mm NaCl, 1.0 mm EDTA, 1.0 mm EGTA, 1.0 mm dithiothreitol, and 1.0 mm phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14,000 rpm, and the supernatant containing the nuclear proteins was stored at –80 C until use. Gel-shift and Supershift Assays—The assays were performed using CT26 and YAMC nuclear extracts. In competitive binding assays, unlabeled oligonucleotides were added at 100 m excess. Supershift assays were performed by the addition of 1 μl of polyclonal antibody directed against mouse Ets-1 (sc-111 X, Santa Cruz Biotechnology) and Runx2 (S-19 X, Santa Cruz Biotechnology; AML-3, Calbiochem). The oligonucleotides used in gel-shift assays were as follows (underlined for emphasis): probe A, nt –123 to –101 (5′-CCA GAG GAG GAA GTG TAG GAG CAG GT-3′); probe B, nt –145 to –121 (5′-TTT TTT TTT AAC CAC AAA ACC AGA G-3′); and probe C, nt –165 to –140 (5′-TGT TTC CTT TTC TTC CTT TTT TTT TT-3′). Each probe was prepared by end-labeling the double-stranded oligonucleotides with [32P]ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega, Madison, WI), followed by G-50 column purification (Biomax, Odenton, MD). The reactions were resolved on a 6% nondenaturing acrylamide gel in 1× Tris borate-EDTA buffer. All olignonucleotides used in the gel-shift assays were HPLC (high pressure liquid chromatography) grade. 20-bp oligonucleotides were synthesized to contain sequential 2–3-nt mutations (Fig. 3, C and D) and used as competitors in gel-shift assays to identify essential protein binding sites. Probe A mutants containing the nt –118 to –115 (5-GGAA-3′ to 5′-TTTT-3′) binding site mutation or the Ets-1 consensus binding site mutation (5′-gagACTTTTAtgtaggagcaggt-3′) were constructed (Integrated DNA Technologies, Coralville, IA), labeled using [32P]ATP as described above, and used in gel-shift assays. Probe B mutants containing the nt –134 to –129 (5-CCACAA-3′ to 5′-ACATTT-3′) binding site mutation or the Runx2 consensus binding site mutation (5′-ttttGGACATTTaaccagaggaggaagtgta-3′) were constructed (Integrated DNA Technologies), labeled using [32P]ATP as described above, and used in gel-shift assays. Transcription Factor Data Base Analysis—The nucleotide sequence corresponding to the cis-regulatory domain in the OPN promoter was cross-referenced with the TRANSFAC transcription factor data base to identify consensus binding sites of known transcription factors. Candidate transcription factors were screened for using antibody-supershift analysis in the gel-shift assays, as described above, to confirm the presence of the factor in our oligonucleotide-protein complexes. Chromatin Immunoprecipitation Assay—Chromatin isolated from CT26 and YAMC cells were fixed and immunoprecipitated using the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer's instructions. The purified chromatin was immunoprecipitated using 10 μg of anti-AML-3, anti-PEBP2α, anti-Ets-1, or IgG isotype. After DNA purification, the presence of the selected DNA sequence was assessed by PCR. The PCR product was 265 bp in length. The PCR program was: 94 °C for 4 min followed by 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 45 s, for a total of 28 cycles, and then 72 °C for 7 min. PCR products were resolved in 10% acrylamide gels. The average size of the sonicated DNA fragments subjected to immunoprecipitation was 300 bp as determined by ethidium bromide gel electrophoresis. The ChIP assay utilized PCR primers 5′-CCTTTCATCCCCACTGATGT-3′ and 5′-TGAGGTTTTTGCCACTACCC-3′. Antisense Oligonucleotide Design and Assay—Sense and antisense oligonucleotides were designed according to GenBank™ sequences X53953 (Ets-1: sense, 5′-AGCCAACCCTACCTACCCAG-3′; antisense, 5′-TGGGTAGGTAGGGTTGGCT-3′) and NM 009820 (Runx2: sense, 5′-GCCACCACTCACTACCACAC-3′; antisense, 5′-GTGTGGTAGTGAGTGGTGGC-3′) to inhibit the expression of Ets-1 and Runx2. Transfection of sense or antisense to Ets-1 or Runx2 and cotransfection of Ets-1/Runx2 sense or antisense into CT26 cells was performed using Lipofectamine 2000, as described above. After 48 h, cells were collected for analysis of OPN protein expression by Western blotting. Ets-1 and Runx2 siRNA and Assays—We utilized an RNAi-mediated approach to develop cell lines that could be utilized in an in vivo model of metastasis. Ets-1 and Runx2 siRNA (sc-35346, sc-37146; Santa Cruz Biotechnology) were used at a concentration of 10 μm for transient transfection of CT26 cells as described above. CT26 were transfected using mismatch siRNA or Ets-1/Runx2 siRNA. After 24 h, cells were transfected again using the corresponding siRNA. After 72 h from the initial transfection, cell lysates were collected and analyzed using Western blotting to confirm the extent of Ets-1, Runx2, and OPN protein expression. Subsequently, cells were assayed for viability by trypan blue exclusion. Stably down-regulated cell lines would then be developed for use in a previously established model of in vivo experimental metastasis (17Wai P.Y. Mi Z. Guo H. Sarraf-Yazdi S. Gao C. Wei J. Marroquin C.E. Clary B. Kuo P.C. Carcinogenesis. 2005; 26: 741-751Crossref PubMed Scopus (92) Google Scholar). Statistical Analysis—Data are expressed as mean ± S.D. Statistical analysis was performed using SigmaStat, version 3 (Systat Software, Point Richmond, CA). Individual comparisons were made with Student's t test. The criterion for significance was p < 0.05 for all comparisons. Transient Transfection Analysis of OPN Promoter Deletion Constructs—To localize potential cis-acting elements in the OPN promoter, deletion constructs were analyzed in CT26 colon cancer cells using transient transfection (Fig. 1). Serial deletion constructs demonstrated a significant 3-fold increase in luciferase activity between nt –88 and –258 in CT26 cells (Fig. 1A). Transient transfection of these deletion constructs in YAMC, a nonmetastatic colonic epithelial cell line, did not demonstrate any increased luciferase activity (Fig. 1A). CT26 demonstrated an ∼8-fold increased luciferase activity in comparison with YAMC. Deletion fragments containing nucleotides upstream from –258 did not confer any additional, significant increase in luciferase activity in CT26 cells (Fig. 1A). Using further serial deletion constructs, this area of increased OPN promoter activity was further localized to the length of the promoter from nt –107 to –160 (Fig. 1B). Fragment –120 and –134 demonstrated an incremental 3.8-fold and a 6.4-fold increase in luciferase activity in comparison with control. These results suggest that more than one enhancer may reside in the nt –107 to –160 cis-regulatory domain of the OPN promoter. Gel-shift Analysis—To determine whether trans-activating nuclear factors may regulate the nt –107 to –160 region of the OPN promoter, gel-shift analysis was performed using [32P]ATP-labeled fragments (∼20 to 30 nt) that span this region of interest (Fig. 2). Nuclear proteins were isolated from CT26 cells and incubated with three overlapping, labeled oligonucleotide probes: probe A (nt –101 to –123); probe B (nt –121 to –145); and probe C (nt –140 to –165), as described under “Experimental Procedures.” Nuclear proteins bound to probe A (Fig. 2, lane 4) and probe B (Fig. 2, lane 5) but not to probe C. Three gel-shift complexes (E1, E2, and E3) were present with probe A, whereas two distinct complexes (R1 and R2) were seen with probe B. Gel-shift complexes E1, E2, and E3 and R1 were extinguished in the presence of 50-fold excess unlabeled probe A or B, respectively (Fig. 2, lanes 7 and 8), but persisted in the presence of a 50-fold excess of nonspecific unlabeled competitor (Fig. 2, lanes 10 and 11). These gel-shift complexes corresponding to E1, E2, E3, and R1 suggest that potential trans-regulatory nuclear proteins are bound to the OPN promoter in the region of nt –101 to –145 in CT26 cells. The binding sites were further characterized by serial mutations of the OPN promoter between nt –101 to –123 in probe A (Fig. 3A) and between nt –121 to –145 in probe B (Fig. 3C). These mutated sequences were then used as excess unlabeled competitors in gel-shift assays (Fig. 3, B and D). Mutation of nucleotides –115 to –118 in probe A resulted in persistence of complexes E1, E2, and E3 (Fig. 3B, lanes 8 and 9), whereas mutations of nt –101 to –114 and nt –119 to –123 resulted in ablation of complexes E1, E2, and E3 in competition gel-shift assays (Fig. 3B, lanes 1–7, 10, and 11). These data suggest that nt –115 to –118 (AAGG) represents an essential binding site for the nuclear proteins corresponding to the E1, E2, and E3 complexes. Mutation of nt –129 to –134 in probe B resulted in persistence of complexes R1 and R2 (Fig. 3D, lanes 5–7), whereas mutations of nt –121 to –128 and –135 to –145 resulted in ablation of R1 in competition gel-shift assays (Fig. 3D, lanes 1–4 and 8–12). These data suggest that nt –129 to –134 (AACACC) represents an essential binding site for the nuclear proteins corresponding to the R1 complex. Together, these data from the probe A and B serial deletions indicate that two potential enhancer binding sites (nt –115 to –118 and nt –129 to –134) reside within the cis-regulatory region nt –101 to –145. Identification of trans-Regulatory Proteins and Confirmation of DNA-Protein Binding—To identify the nuclear factors that bind our cis-element of interest, we searched the TRANSFAC data base for consensus binding sites of known transcription factors within the nt –101 to –145 segment (Fig. 4). The region nt –115 to –118 matched with the known consensus binding site of Ets (Fig. 4A), whereas the region nt –129 to –134 matched with Runx2, SBF-1 (spliceosome-binding factor-1), and C/EBP (Fig. 4B). We selected these nuclear proteins as candidates for specific binding in supershift and ChIP assays. Gelshift assays were repeated in the presence of Ets-1 antibody (Fig. 5A, lanes 7 and 8) and Runx2 antibodies (Fig. 5B, lanes 7–8 and 10–11) with the previously described DNA probes A and B. In the presence of antibody against Ets-1, complexes E1, E2, and E3 were diminished, and a specific supershift band was formed (Fig. 5A, lanes 7 and 8). In the presence of antibody against Runx2 (S-19 X), complex R1 was diminished with formation of a supershift band (Fig. 5B, lanes 7 and 8). This effect was more pronounced with the use of α-aml-3, a different antibody raised against Runx2, with the formation of an intense supershift band (Fig. 5B, lanes 10 and 11). No shift was detected using IgG isotype, and no complexes were formed between the antibodies and DNA probe in the absence of nuclear extract (Fig. 5, A, lane 5, and B, lanes 6 and 9). No supershift complexes were formed using antibodies to SBF-1 and C/EBP (data not shown). ChIP assays were then performed to confirm the in vivo binding of Ets-1 and Runx2 to this portion of the OPN promoter (Fig. 5C). Control reactions using no antibody or IgG isotype did not exhibit specific immunoprecipitation, whereas reactions containing antibodies to Ets-1 or Runx2 demonstrated specific binding to this region of the OPN promoter. Chromatin from YAMC cells did not result in specific Ets-1 or Runx2 antibody-mediated immunoprecipitation (Fig. 5D). Together, these data suggest that Ets-1 and Runx2 bind specifically to enhancer domains in the cis-regulatory region nt –101 to –145 of the OPN promoter in CT26 cells.FIGURE 5Identification of the trans-regulatory protein and confirmation of binding. A, gel-shift assays using probe A were repeated in the presence of antibody to Ets-1. Supershift bands (SS) were detected with increasing concentration of anti-Ets-1 in lane 7 (μg/μl) and lane 8 (μg/μl). Probe A + NE + IgG isotype (lane 6), probe A + anti-Ets-1 in the absence of NE (lane 5), free probe A (lane 1), probe A + NE (lane 2), probe A + NE + specific competitor (SC)(lane 3), and probe A + NE + nonspecific competitor (NC) (lane 4) were used as controls. This blot is representative of three experiments. B, gel-shift assays using probe B were repeated in the presence of antibodies to Runx2 (S-19 X and α-aml-3). Supershift bands were detected with increasing concentration of S-19 X and α-aml-3 in lanes 7 and 8 and lanes 10 and 11, respectively. Probe B + NE + IgG isotype (lane 5), probe B + S-19 X in the absence of NE (lane 6), probe B + α-aml-3 in the absence of NE (lane 9), free probe B (lane 1), probe B + NE (lane 2), probe B + NE + specific competitor (lane 3), and probe B + NE + nonspecific competitor (lane 4) were used as controls. This blot is representative of three experiments. C, ChIP analysis of Ets-1 and Runx2 binding. Chromatin from CT26 cells was fixed and immunoprecipitated using the ChIP assay kit as recommended by the manufacturer (Upstate Biotechnology). The purified chromatin was immunoprecipitated using 10 μg of anti-Ets-1, SC-19 X, anti-aml-3, corresponding IgG isotype, or no antibody (no Ab) as control. The input fraction corresponded to 0.1% of the chromatin solution before immunoprecipitation. After DNA purification, the presence of the selected DNA sequence was assessed by PCR. The blot is representative of three experiments. D, chromatin from YAMC cells was fixed and immunoprecipitated using the ChIP assay kit as recommended by the manufacturer (Upstate Biotechnology). The purified chromatin was immunoprecipitated using 10 μg of α-Ets-1, SC-19 X, α-aml-3, the corresponding IgG isotype, or no antibody as control. The input fraction corresponded to 0.1% of the chromatin solution before immunoprecipitation. After DNA purification, the presence of the selected DNA sequence was assessed by PCR. The blot is representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Functional Analysis of ETS-1 and Runx2 on DNA Binding, OPN Transcription, and Protein Expression—To determine whether trans-activation of Ets-1 and Runx2 is critical to the transcription of OPN in CT26 cells, we constructed OPN promoter sequences containing mutant binding sites for Ets-1 and Runx2 and analyzed them in gel-shift and luciferase-promoter assays. Mutation of nt –115 to –118 from AAGG to TTTT resulted in ablation of the E1 and E3 complexes with significant reduction in the binding of E2 in gel-shift assays (Fig. 6, lane 2). This effect was duplicated by mutating the consensus binding site for Ets-1 with complete ablation of complexes E1, E2 and E3 (Fig. 6, lane 3). Both mutation of nt –129 to –13
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