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The Transcription Factor CCAAT-binding Factor CBF/NF-Y Regulates the Proximal Promoter Activity in the Human α1(XI) Collagen Gene (COL11A1)

2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês

10.1074/jbc.m305599200

1083-351X

Noritaka Matsuo, Yuhua Wang, Hideaki Sumiyoshi, Keiko Sakata-Takatani, Hitoshi Nagato, Kumiko Sakai, Mami Sakurai, Hidekatsu Yoshioka,

RNA Research and Splicing

We have characterized the proximal promoter region of the human COL11A1 gene. Transient transfection assays indicate that the segment from –199 to +1 is necessary for the activation of basal transcription. Electrophoretic mobility shift assays (EMSAs) demonstrated that the ATTGG sequence, within the –147 to –121 fragment, is critical to bind nuclear proteins in the proximal COL11A1 promoter. We demonstrated that the CCAAT binding factor (CBF/NF-Y) bound to this region using an interference assay with consensus oligonucleotides and a supershift assay with specific antibodies in an EMSA. In a chromatin immunoprecipitation assay and EMSA using DNA-affinity-purified proteins, CBF/NF-Y proteins directly bound this region in vitro and in vivo. We also showed that four tandem copies of the CBF/NF-Y-binding fragment produced higher transcriptional activity than one or two copies, whereas the absence of a CBF/NF-Y-binding fragment suppressed the COL11A1 promoter activity. Furthermore, overexpression of a dominant-negative CBF-B/NF-YA subunit significantly inhibited promoter activity in both transient and stable cells. These results indicate that the CBF/NF-Y proteins regulate the transcription of COL11A1 by directly binding to the ATTGG sequence in the proximal promoter region. We have characterized the proximal promoter region of the human COL11A1 gene. Transient transfection assays indicate that the segment from –199 to +1 is necessary for the activation of basal transcription. Electrophoretic mobility shift assays (EMSAs) demonstrated that the ATTGG sequence, within the –147 to –121 fragment, is critical to bind nuclear proteins in the proximal COL11A1 promoter. We demonstrated that the CCAAT binding factor (CBF/NF-Y) bound to this region using an interference assay with consensus oligonucleotides and a supershift assay with specific antibodies in an EMSA. In a chromatin immunoprecipitation assay and EMSA using DNA-affinity-purified proteins, CBF/NF-Y proteins directly bound this region in vitro and in vivo. We also showed that four tandem copies of the CBF/NF-Y-binding fragment produced higher transcriptional activity than one or two copies, whereas the absence of a CBF/NF-Y-binding fragment suppressed the COL11A1 promoter activity. Furthermore, overexpression of a dominant-negative CBF-B/NF-YA subunit significantly inhibited promoter activity in both transient and stable cells. These results indicate that the CBF/NF-Y proteins regulate the transcription of COL11A1 by directly binding to the ATTGG sequence in the proximal promoter region. The collagen superfamily, one of the extracellular matrix proteins, plays an important role, not only in stabilizing the tissues as structural components but also in regulating a variety of biological functions, such as development, differentiation, proliferation, and morphogenesis (1Olsen B.R. Ninomiya Y. Extracellular Matrix, Anchor and Adhesion Proteins. 2nd Ed. Oxford University Press, New York1999: 380-407Google Scholar, 2Vuorio E. de Crombrugghe B. Annu. Rev. Biochem. 1990; 59: 837-872Crossref PubMed Scopus (393) Google Scholar, 3Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1388) Google Scholar). Among them, types I, II, III, V, and XI collagens are included in the group of fibril-forming collagens, based on their structural and functional features, and are divided into two subgroups, major (I, II and III) and minor (V and XI) fibrillar collagen (4Fichard A. Kleman J.-P. Ruggiero F. Matrix Biol. 1994; 14: 515-531Crossref Scopus (168) Google Scholar) on the basis of their contents in tissues. Type XI collagen is a component of the collagen fibrillar network found in cartilage (5Burgeson R.E. Hollister D.W. Biochem. Biophys. Res. Commun. 1979; 87: 1124-1131Crossref PubMed Scopus (200) Google Scholar), and consists of three genetically distinct polypeptide chains: α1(XI), α2(XI), and α3(XI); the last of these is thought to be overglycosylated α1(II) chains (6Burgeson R.E. Hebda P. Morris N. Hollister D.W. J. Biol. Chem. 1982; 257: 7852-7856Abstract Full Text PDF PubMed Google Scholar). Although type XI collagen is a relatively minor collagen and is buried within the major collagen fibrils, it is important for the regulation of fibril diameter (7Mendler M. Eich-Bender S.G. Vaughan L. Winterhalter K.H. Bruckner P. J. Cell Biol. 1989; 108: 191-197Crossref PubMed Scopus (406) Google Scholar). Chondrodysplasia mice (cho), which do not synthesize α1(XI) chains, show irregular collagen fibrils in their cartilage (8Li Y. Lacerda D.A. Warman M.L. Beier D.R. Yoshioka H. Ninomiya Y. Oxford J.T. Morris N.P. Andrikopoulos K. Ramirez F. Wardell B.B. Lifferth G.D. Teuscher C. Woodward S.R. Taylor B.A. Seegmiller R.E. Olsen B.R. Cell. 1995; 80: 423-430Abstract Full Text PDF PubMed Scopus (303) Google Scholar). Contrary to the previous findings, the α1(XI) chain is not restricted to cartilage (9Bernard M. Yoshioka H. Rodriguez M. van der Rest M. Kimura I. Ninomiya Y. Olsen B.R. Ramirez F. J. Biol. Chem. 1988; 263: 17159-17166Abstract Full Text PDF PubMed Google Scholar, 10Niyibizi C. Eyre D.R. FEBS Lett. 1989; 242: 314-318Crossref PubMed Scopus (92) Google Scholar, 11Yoshioka H. Ramirez F. J. Biol. Chem. 1990; 265: 6423-6426Abstract Full Text PDF PubMed Google Scholar, 12Brown K.E. Lawrence R. Sonenshein G.E. J. Biol. Chem. 1991; 266: 23268-23273Abstract Full Text PDF PubMed Google Scholar, 13Nah H.D. Barembaum M. Upholt W.B. J. Biol. Chem. 1992; 267: 22581-22586Abstract Full Text PDF PubMed Google Scholar, 14Yoshioka H. Iyama K. Inoguchi K. Khaleduzzaman M. Ninomiya Y. Ramirez F. Dev. Dyn. 1995; 204: 41-47Crossref PubMed Scopus (60) Google Scholar), and it was demonstrated that the α1(XI) chain could form a heterotrimer with the α2(V) chain in a 2:1 ratio in non-cartilaginous cells and tissues (15Kleman J.P. Hartmann D.J. Ramirez F. van der Rest M. Eur. J. Biochem. 1992; 210: 329-335Crossref PubMed Scopus (67) Google Scholar, 16Mayne R. Brewton R.G. Mayne P.M. Baker J.R. J. Biol. Chem. 1993; 268: 9381-9386Abstract Full Text PDF PubMed Google Scholar). Although the precise function of this cross-type trimer remains unclear, the α1(XI) collagen gene is more broadly expressed than other collagen genes, showing that the α1(XI) collagen gene is the sole collagen gene to be expressed in both cartilaginous and non-cartilaginous tissues. This implies that the regulation of the α1(XI) collagen gene might be more complex than expected. We have previously reported the structural and functional features of the human α1(XI) collagen gene (COL11A1) promoter (17Yoshioka H. Greenwel P. Inoguchi K. Truter S. Inagaki Y. Ninomiya Y. Ramirez F. J. Biol. Chem. 1995; 270: 418-424Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). DNase I footprinting has mapped nine areas, FP1 to FP9, covering the region –541 to +1, where nuclear proteins probably bind. In characterizing the –541 to –199 segment, FP9-protein, which has homology to the GATA consensus motif and binds at –531 to –487, was recognized to be a distinct +100-kDa polypeptide (18Kinoshita A. Greenwel P. Tanaka S. Di Liberto M. Yoshioka H. Ramirez F. J. Biol. Chem. 1997; 272: 31777-31784Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). It was expressed in mesenchymal cells, and its level of binding activity was positively correlated with the degree of cell differentiation in osteoblastic and skeletal muscle cell lines, in vitro. At –395 to –379, the core sequence of FP7 exhibits some homology with that of FPB 1The abbreviations used are: FPB, Foot Printing B; EMSA, electrophoretic mobility shift assay; C/EBP, CCAAT/enhancer binding protein; CBF, CCAAT binding factor; wt, wild type; CTF, CCAAT transcription factor; NF-Y, nuclear factor for Y box. in COL5A2, and FP7-protein complexes can be competed with an excess of FPB oligonucleotides in an EMSA using nuclear extracts from the 1120 cell line (17Yoshioka H. Greenwel P. Inoguchi K. Truter S. Inagaki Y. Ninomiya Y. Ramirez F. J. Biol. Chem. 1995; 270: 418-424Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 19Truter S. Di Liberto M. Inagaki Y. Ramirez F. J. Biol. Chem. 1992; 267: 25389-25395Abstract Full Text PDF PubMed Google Scholar). Further studies using transfection experiments and EMSAs in A204 cells, however, indicated that the FPB-protein complexes identified were PBX, PREP and HOX proteins and therefore different from the FP7-binding proteins (20Penkov D. Tanaka S. Di Rocco G. Berthelsen J. Blasi F. Ramirez F. J. Biol. Chem. 2000; 275: 16681-16689Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In this study, we have characterized the region downstream of the –541 to –199 region in the human COL11A1 promoter. Transient transfection assays indicate that the segment from –199 to +1 is necessary for the activation of basal transcription, and EMSAs demonstrate that the ATTGG sequence within the –147 to –121 fragment is critical for binding nuclear proteins. Furthermore, we identify the CCAAT binding factor CBF/NF-Y as binding to this region. Cells and Cell Culture—Human rhabdomyosarcoma cell line A204 (17Yoshioka H. Greenwel P. Inoguchi K. Truter S. Inagaki Y. Ninomiya Y. Ramirez F. J. Biol. Chem. 1995; 270: 418-424Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 18Kinoshita A. Greenwel P. Tanaka S. Di Liberto M. Yoshioka H. Ramirez F. J. Biol. Chem. 1997; 272: 31777-31784Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) was used in this study. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heart-inactivated fetal bovine serum (Sanko Junyaku, Tokyo) at 37 °C in humidified 5% CO2/95% air. Construction of Chimeric Plasmids—Essentially, all COL11A1 promoter-luciferase gene constructs were derived from –1454 COL11A1/CAT plasmid that fused to the CAT gene as described previously (17Yoshioka H. Greenwel P. Inoguchi K. Truter S. Inagaki Y. Ninomiya Y. Ramirez F. J. Biol. Chem. 1995; 270: 418-424Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). To obtain various 5′ deletion constructs, these fragments were generated by PCR using sets of oligonucleotide primers, which are SacI site-linked 5′- and XhoI site-linked 3′-primers specific for the promoter sequence. These PCR products were cloned into the pGEM-T Easy vector (Promega, Madison, WI), followed by digestion with SacI and XhoI, and subcloned into the SacI/XhoI site of pGL3-Basic vector. The constructs that contained tandem fragments, PGL3(–199/–65)x2 and pGL3(–147/–121)x4, were chosen. Their DNA sizes were checked on agarose gel, and the direction of the constructs was confirmed by sequence. For internal deletion and mutation constructs, site-directed mutagenesis was apply (21Imai Y. Matsushima Y. Sugimura T. Terada M. Nucleic Acids Res. 1991; 19: 2785Crossref PubMed Scopus (314) Google Scholar). The constructs were derived from pGL3–541/+271 plasmid. For 6 base-substitution, we introduced EcoRI sequence, GAATTC, for substitution sites. The primers for 6 base-substitution constructs, pGL3–541E1, pGL3–541E2, pGL3–541E3, and pGL3–541E4, were as follows: E1: sense, 5′-gaattcTGGGTCTGACCCTCAGCCTG-3′; antisense, 5′-gaattcCGACTCTGGGGCGGCCCCAA-3′; E2: sense, 5′-gaattcCTCAGCCTGCTTGTCAGTTT-3′; antisense, 5′-gaattcACCCAATCACACGACTCTGG-3′; E3: sense, 5′-gaattcTGTGATTGGGTCTGACCCTC-3′; antisense, 5′-gaattcTGGGGCGGCCCCAAGCCCGC-3′; and E4: sense, 5′-gaattcCTGACCCTCAGCCTGCTTGT-3′; antisense, 5′-gaattcTCACACGACTCTGGGGCGG-3′ (the EcoRI sites for mutation are indicated by lowercase letters). PCR products were digested with EcoRI, followed by self-ligation. All mutagenesis plasmids were digested with SacI and XhoI and re-cloned into the SacI/XhoI site of pGL3-Basic vector. The primers for pGL3–541CBFm, pGL3-del–147/–121, and pGL3-del–199/–65 were as follows: for CBF mutant construct pGL3–541CBFm: sense, GaaATacGggCTGACCCTCAGCCTGCTT; antisense, ccCgtATttCACGACTCTGGGGCGGCCC (mutated nucleotides are indicated by lowercase letters); for deletion construct pGL3-del-147/–121: sense, 5′-AGCCTGCTTGTCAGTTTCGC-3′; antisense, 5′-GGGGCGGCCCCAAGCCCGCC-3′; for deletion construct pGL3-del–199/–65: sense, 5′-GGCGGAGGAGGGGGCTGCCC-3′; antisense, 5′-GAGCAGGCCCAGCCCACCAA-3′. Construct of the dominant-negative CBF-B/NF-YA was generated by reverse transcriptase PCR as described previously (22Mantovani R. Li X.-Y. Pessara U. van Huisjduijnen R.H. Benoist C. Mathis D. J. Biol. Chem. 1994; 269: 20340-20346Abstract Full Text PDF PubMed Google Scholar, 23Lok C.-N. Lang A.J. Mirski S.E.L. Cole S.P.C. Biochem. J. 2002; 368: 742-751Crossref Google Scholar). Two sets of primers were as follows: for 5′-CBF-B/NF-YAmut: sense, 5′-gtcgacGGAGGGACCATGGAGCAGTATA-3′; antisense, 5′-GCGGCCGCCTTCCGTGCCATGGCATGAC-3′, and for 3′-CBF-B/NF-YAmut: sense, 5′-GCGGCCGCAGGTGGACGATTTTTCTCTC-3′; antisense, 5′-tctagaGGGTTAGGACACTCGGATGATC-3′ (functional mutated nucleotides are indicated by lowercase letters.) These PCR products were cloned into the pGEM-T Easy vector and subcloned into the SalI/NotI site for 5′-CBF-B/NF-YA mut, followed by NotI/XbaI site for 3′-CBF-B/NF-YAmut into the empty vector. Finally, this dominant-negative form of CBF-B/NF-YA was cloned into pCXN2 mammalian expression vector (24Niwa H. Yamamura K. Miyazaki J. Gene. 1991; 108: 193-199Crossref PubMed Scopus (4616) Google Scholar). All constructs mentioned above were sequenced on an ABI 310 sequencer (Applied Biosystems) according to the manufacturer's protocol. Transfection and Luciferase Assays—The cells were inoculated at a density of 2 × 105 per 35-mm dish 24 h before transfection. For transient transfection, each of plasmid DNA was transfected into these cells by using the calcium phosphate precipitation method followed by a 15% glycerol shock for 60 s (25Chen C. Okayama H. Mol. Cell Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar). After an additional cultivation for 48 h, the transfected cells were harvested, lysed, centrifuged to pellet the debris and performed to luciferase assay as described below. For stable transfection, 1 μg of pGL3–199/+271 or pGL3(–147/–121)x4 luciferase construct with 50 ng of pMAM2-BSD selection vector were transfected into A204 cells by using the LipofectAMINE Plus reagent system (Invitrogen) and cultured for 48 h. These transfected cells were replated at a density of 1 × 103 per 100-mm dish with fresh medium containing 8 μg/ml of blasticidin (Funakoshi, Tokyo, Japan). After an additional cultivation for 2 weeks, the resistant colonies were isolated and further cultured in the present of blasticidin, followed by assayed the luciferase activity to select stable cell lines. Luciferase activities were assayed by the Dual-LuciferaseTM Reporter Assay System according to the manufacturer's protocol (Promega, Madison, WI) using a luminometer (Lumat L.D. 9507; PerkinElmer Life Sciences). Five micrograms of firefly luciferase reporter construct and/or dominant-negative CBF-B/NF-YA expression vector were cotransfected with 0.25 μg of pRL-TK Renilla reniformis luciferase expression vector as an internal control for transfection efficiency. The pGL3-Basic and pGL3-Control vectors were used for each experiment as negative and positive controls, respectively. Relative luciferase activities (percentages) of each construct were normalized against the activity of pGL3-Control vector and results were expressed as mean ± S.E. of three to five independent experiments. Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts were prepared according to Dignam et al. (26Dignam J.D. Martin P.L. Shastry B.S. Roeder R.G. Methods Enzymol. 1983; 101: 582-598Crossref PubMed Scopus (746) Google Scholar) with some modifications. All buffers contained the protease inhibitors leupeptin (2 μg/ml), aprotinin (2 μg/ml), pepstatin A (2 μg/ml), phenylmethylsulfonyl fluoride (0.5 mm), and dithiothreitol (1 mm). Cells (1 × 108) were resuspended in buffer (10 mm HEPES, pH 7.8, 10 mm KCl, 0.1 mm EDTA, and 0.1% Nonidet P-40), followed by incubation on ice for 10 min and homogenized. After centrifugation at 3000 rpm for 10 min, the cell pellets were resuspended in buffer (50 mm HEPES, pH 7.8, 420 mm KCl, 5 mm MgCl2, 0.1 mm EDTA, and 20% glycerol) and rotated at 4 °Cfor1h.The supernatants with nuclear proteins were recovered by centrifugation at 24,000 × g for 30 min, and protein concentration of the nuclear extracts was determined by the Bradford's colorimetric reagent (Bio-Rad) using bovine serum albumin as a standard. Wild-type and mutant probes derived from COL11A1 promoter fragment were generated by PCR using each set of HindIII site-linked primers, and all PCR products were cloned into the pGEM-T Easy vector (Promega). Both sense and antisense oligonucleotides of the mutant fragments covering –147 to –121, CBFwt, CBFmt, NF-1, C/EBP, and GATA consensus overhanging HindIII sites at the ends were synthesized (Proligo Japan KK, Kyoto, Japan), annealed to make double-stranded oligonucleotides, digested with HindIII, and cloned into the HindIII site of pBluescript SK vector. The CBFwt, CBFmt, NF-1, C/EBP, and GATA consensus sequences, based on the data from Santa Cruz Biotechnology, Inc., used as competitors, were as follows: CBFwt, 5′-AGACCGTACGTGATTGGTTAATCTCTT-3′; CBFmt5′-AGACCGTACGAAATACGGGAATCTCTT-3′ (mutated nucleotides are underlined); NF-Y, 5′-TTTTGGATTGAAGCCAATATGATAA-3′; C/EBP, 5′-TGCAGATTGCGCAATCTGCA-3′; and GATA, 5′-CACTTGATAACAGAAAGTGATAACTCT-3′. All plasmids were digested with HindIII and the digested fragments were radiolabeled with [α-32P]dCTP using Klenow fragment to fill in the HindIII overhang sites. For EMSA, the binding reaction was performed for 30 min at 25 °C in 25 μl of reaction buffer (50 mm HEPES, pH 7.8, 250 mm KCl, 25 mm MgCl2, 5 mm EDTA, and 50% glycerol) containing 20,000–30,000 cpm of labeled probe, 3 μg of poly(dI-dC), and 1–20 μg of nuclear extracts. For competitors and antibody interference assay, unlabeled probes or antibody was added to the reaction mixture for 1 h at 4 °C before the addition of [α-32P]dCTP labeled probe. These antibodies against CBF-A/NF-YB, CBF-B/NF-YB, CBF-C/NF-YC, C/EBP, NF-1, GATA-1, and pre-immune goat IgG were purchased from Santa Cruz Biotechnology. The DNA-protein complexes were separated on 4.5% nondenaturing polyacrylamide gel in 0.25× Tris-borate/EDTA with 2.5% glycerol at 4 °C and visualized by autoradiography using Bio Imaging Analyzer BAS-2000 (Fuji Film, Tokyo, Japan). Purification of DNA Binding Protein Using DNA Affinity Latex Beads—DNA affinity latex beads were prepared as described previously (27Inomata Y. Kawaguchi H. Hiramoto M. Wada Y. Handa H. Anal. Biochem. 1992; 206: 109-114Crossref PubMed Scopus (66) Google Scholar). To purify DNA binding proteins, the DNA-latex beads were added to the nuclear extracts and incubated for 24 h at 4 °C. After centrifugation at 15,000 rpm for 5 min, the beads were washed three times with 0.1× buffer A (50 mm Tris-HCl. pH 7.9, 20% glycerol, 0.1 m KCl, 1 mm EDTA, 1 mm dithiothreitol, and 0.1% Nonidet P-40) containing 0.1 m KCl. To elute the binding proteins from the DNA-latex beads, the beads were resuspended in 0.4× buffer A containing 0.4 m KCl and incubated for 15 min at 4 °C. After centrifugation at 15,000 rpm for 5 min, the supernatant containing DNA binding proteins was recovered and concentrated by microcon-10 (Millipore). Chromatin Immunoprecipitation Assay—Chromatin immunoprecipitation assays were performed using a chromatin immunoprecipitation assay kit (Upstate Biotech, Lake Placid, NY) according to the manufacturer's protocol. All solutions were contained with 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ml pepstatin A. A204 cells were inoculated at a density of 1 × 106 per 100-mm dish 16 h before formaldehyde cross-linking and chromatin immunoprecipitation. Cells were fixed with 1% formaldehyde for 15 min at 37 °C. After washing twice with PBS, cell pellets were resuspended in SDS lysis buffer, incubated for 10 min at 4 °C, and sonicated four times for 10 s using ultrasonic homogenizer VP-5S (TAITEC, Tokyo, Japan). After centrifugation, the supernatant was diluted in chromatin immunoprecipitation dilution buffer and then incubated overnight at 4 °C with anti-CBF-A/NF-YB, CBF-B/NF-YA, and CBF-C/NF-YC antiserum (Santa Cruz Biotechnology). Immune complexes were recovered by the addition of 60 μl of salmon sperm DNA/protein A-agarose slurry, followed by incubation for 2 h at 4 °C. After washing the beads with both low- and high-salt buffer, then LiCl buffer, and finally Tris/EDTA buffer, the immune complexes were eluted by incubation for 15 min at 25 °C with 200 μl of elution buffer (1% SDS, 100 mm NaHCO3, and 1 mm dithiothreitol). To reverse the cross-linking of DNA, the elutes were added to 8 μl of 5 m NaCl and incubated for 4 h at 65 °C, followed by treatment with proteinase K for 1 h at 45 °C. DNA was recovered by phenolchloroform extraction and ethanol precipitation, and then the pellets were resuspended in 50 μl of Tris/EDTA buffer. Quantitative PCR was carried out for 35 cycles using 5 μl of sample DNA solution, and PCR products were separated on 2% agarose gels in 1× Tris-acetate/EDTA. Deletion Analysis of the Human COL11A1 Promoter—To delineate the proximal regulatory regions in the human COL11A1 promoter, a series of chimeric constructs containing 5′-end deletions linked to the luciferase gene were generated, and then luciferase assays were carried out (Fig. 1A). The activity of each construct was compared with the longest construct, pGL3–291/+271. As shown in Fig. 1B, the promoter activity of pGL3–199/+271 was not significantly reduced compared with that of pGL3–291/+271. However, deletion to –88 produced a significant reduction in the transcriptional activity; furthermore, deletion to +1 produced activity similar to that of the pGL3-Basic negative control vector. This result indicates that the segment spanning the –199 to +1 region is important for basal transcriptional activity of the human COL11A1 promoter. Definition of the Nuclear Proteins Binding Site in the Proximal COL11A1 Promoter—On the basis of the aforementioned data, we examined the binding proteins that interact in this region. We prepared three overlapping oligonucleotides, GS1–GS3, covering the –199 to +1 region (Fig. 2A), and carried out EMSAs. As shown in Fig. 2B, 32P-labeled GS2 probe, covering –147 to –65, and GS3 probe, covering –199 to –121, bound nuclear proteins extracted from A204 cells in a dose-dependent manner. Both probes were found to form DNA-protein complexes with the same mobility in an EMSA. To determine whether GS2 and GS3 oligonucleotides bind the same proteins, these probes were used with each other in a competition assay. The 32P-labeled GS2-protein complexes could be competed away not only by the corresponding unlabeled probe (Fig. 2C, lane 4), but also by the unlabeled GS3 oligonucleotide (Fig. 2C, lane 5). Likewise, the 32P-labeled GS3-specific band also disappeared when either unlabeled GS3 or GS2 oligonucleotides were added as competitors (Fig. 2C, lanes 9 and 10); however, an excess of the unlabeled GS1 (–88 to +1) oligonucleotides did not compete for the binding of 32P-labeled GS2 and GS3 probes (Fig. 2C, lanes 3 and 8). To further dissect this protein-binding region, another three oligonucleotides covering the GS2 and GS3 regions were generated (Fig. 2A). Fig. 2D shows that 32P-labeled GS4 probe, covering –147 to –121, formed DNA-protein complexes in a dose-dependent manner. However, neither 32P-labeled GS5 nor GS6 probes, which do not possess the GS4 region, could bind protein. These results indicate that a 27-bp sequence from –147 to –121 in the proximal COL11A1 promoter region is necessary to bind nuclear proteins. We subsequently carried out EMSAs using substitution mutation probes within the 27-bp sequence to narrow down the critical region for binding (Fig. 3A). The 32P-labeled GSE1 and GSE4 probes, which contain 6 single bp substitutions, could not form DNA-protein complexes, whereas the 32P-labeled GSE2, GSE3, and GSwt probes bound protein in a dose-dependent manner. In addition, Fig. 3C shows that an excess of the unlabeled GSE2 and GSE3 probes inhibited the binding activity in a manner similar to that of GSwt, whereas both GSE1 and GSE4 could not compete with the 32P-labeled GSwt-specific band. To confirm a core-binding site, we further performed competition experiments using oligonucleotides with 2 single bp mutations. As shown in Fig. 3D, an excess of unlabeled GSM1, GSM2, GSM3, and GSM4 failed to inhibit the binding, whereas GSM5 and GSM6 blocked the binding of nuclear proteins (Fig. 3C). These results suggest that the GTGATTGG sequence within the 27-bp region was the critical binding site within the proximal COL11A1 promoter.Fig. 3Definition of the nuclear protein-binding region in the proximal COL11A1 promoter by EMSA. A, oligonucleotide sequences of various probes used for EMSA. GSwt is the sequence from –147 to –121 in the proximal COL11A1 promoter. In the mutated probes, identical and mutated nucleotides are indicated by dots and lowercase letters, respectively. B, binding assay using substituted mutation probes. 32P-labeled GSwt (lanes 1–3), GSE1 (lanes 4–6), GSE2 (lanes 7–9), GSE3 (lanes 10 –12), and GSE4 (lanes 13–15) were incubated with nuclear extracts from A204 cells and separated on a 4.5% nondenaturing polyacrylamide gel. C, interference assays using 6 single bp substituted mutation probes as competitors. 32P-labeled GSwt was incubated with nuclear extracts from A204 cells in the presence of competitors, GSwt (lane 3), GSE1 (lane 4), GSE2 (lane 5), GSE3 (lane 6), and GSE4 (lane 7). D, interference assays using 2 single bp substituted mutation probes as competitors. 32P-labeled GSwt was incubated with nuclear extract from A204 cells in the presence of competitors GSwt (lane 3) and GSM1-6 (lane 4–9).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Identification of DNA-Binding Proteins in the Proximal COL11A1 Promoter—To identify the nuclear binding proteins in the proximal COL11A1 promoter, we searched the computer data base and found four candidate transcription factors: CCAAT binding factor CBF/NF-Y, CCAAT transcription factor NF-1/CTF, CCAAT/enhancer binding protein C/EBP, and GATA (Fig. 4A). An excess of the consensus oligonucleotides of CBF/NF-Y inhibited the binding of the 32P-labeled GSwt probe (Fig. 4B, lane 2), whereas NF-1, C/EBF, GATA consensus, and CBFmt oligonucleotides could not abolish the binding activity observed (Fig. 4B, lanes 3–6). To further characterize the binding protein, we performed an interference assay using specific antibodies against CBF/NF-Y, NF-Y, C/EBP, and GATA1. As shown in Fig. 4C, the DNA-protein complex was only supershifted by anti-CBF antibodies, namely anti-CBF-A/NF-YB (Fig. 4C, lane 3), CBF-B/NF-YA (Fig. 4C, lane 4), and CBF-C/NF-YC (Fig. 4C, lane 5). Specific antibodies against the other proteins (Fig. 4C, lanes 6–8) and control IgG (Fig. 4C, lane 9) failed to inhibit the supershift (Fig. 4B). CBF/NF-Y Binds to the COL11A1 Proximal Promoter both in Vitro and in Vivo—To examine whether CBF/NF-Y binds directly to the COL11A1 proximal promoter, we performed two in vitro and in vivo binding experiments. For the in vitro binding analysis, four tandem copies of the –147 to –121 GS4 oligonucleotide were generated and immobilized on latex beads. These were then used for the purification of binding proteins from A204 cell nuclear extracts, and the purified protein was subsequently used in EMSA analyses. As shown in Fig. 5, the 32P-labeled GS4 probe bound to the affinity-purified proteins (lane 4) at levels similar to those of the crude extracts (lane 1), and the bands were supershifted by anti-CBF-A/NF-YB (lane 6), CBF-B/NF-YA (lane 7), and CBF-C/NF-YC (lane 8) antibodies. Furthermore, the affinity-purified proteins strongly bound to the CBF/NF-Y consensus oligonucleotides (lane 9) and were clearly supershifted by anti-CBF/NF-Y antibodies (lanes 10–12). Next, we performed a chromatin immunoprecipitation analysis to examine whether CBF/NF-Y binds to the COL11A1 proximal promoter in vivo. Protein-DNA complexes were immunoprecipitated with antibodies, the cross-links reversed, and the recovered DNA fragments were monitored by PCR using primers for the –291 to +1 region of the COL11A1 promoter. DNA fragments immunoprecipitated with polyclonal antibodies against CBF/NF-Y could be amplified by PCR using the indicated primers (Fig. 6, lanes 5 and 6) as well as the positive control (lane 2), whereas those immunoprecipitated with normal goat IgG (lane 4) or without antibody (lane 3) could not. These results indicate that CBF/NF-Y binds directly to the ATTGG region in the COL11A1 proximal promoter.Fig. 6CBF/NF-Y binds the ATTGG region in the COL11A1 proximal promoter, in vivo. Chromatin immunoprecipitation analysis was performed to monitor the binding of CBF/NF-Y to the COL11A1 proximal promoter, in vivo. Protein-DNA complexes were incubated with polyclonal antibodies against CBF-A/NF-YB, CBF-B/NF-YA, and CBF-C/NF-YC, and isolated by immunoprecipitation (lanes 5 and 6). Positive control was prepared before immunoprecipitation (lane 2) and negative controls were isolated by immunoprecipitation with normal goat IgG (lane 4) or without antibody (lane 3), respectively. All immunoprecipitated DNA fragments were analyzed by PCR with the indicated primers, and a 100-bp ladder was used as a molecular weight marker (lane 1).View Large Image Figure ViewerDownload Hi-res