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• W2007284190 abstract "To elucidate the mechanisms underlining α3(V) collagen chain expression, we performed an initial analysis of the structure and function of the core promoter of the human COL5A3 gene. The core promoter, which lacks a typical TATA motif and has a high GC content, was defined within the –129 bp immediately upstream from the major transcription start site by transient transfection experiments. In this region, we identified four DNA-protein complexes, named A, B, C, and D, by a combination of DNase I footprinting and electrophoretic mobility shift assays. Electrophoretic mobility shift assays using mutant oligonucleotide revealed that the complexes A, B, C, and D bind to –122 to –117, the –101 to –96, the –83 to –78, and the –68 to –57 bp, respectively. The competition assays using consensus oligonucleotides and supershift assays with specific antibodies showed that complex A consists of CBF/NF-Y. In a chromatin immunoprecipitation assay, CBF/NF-Y protein directly bound to this region, in vivo. Functional analysis showed that CBF/NF-Y activated the gene, whereas the proteins of complexes B and C repressed its activity. Furthermore, overexpression of a mutant form of the CBF-B/NF-YA subunit, which forms CBF/NF-Y with CBF-A/NF-YB and CBF-C/NF-YC subunits, inhibited promoter activity. To elucidate the mechanisms underlining α3(V) collagen chain expression, we performed an initial analysis of the structure and function of the core promoter of the human COL5A3 gene. The core promoter, which lacks a typical TATA motif and has a high GC content, was defined within the –129 bp immediately upstream from the major transcription start site by transient transfection experiments. In this region, we identified four DNA-protein complexes, named A, B, C, and D, by a combination of DNase I footprinting and electrophoretic mobility shift assays. Electrophoretic mobility shift assays using mutant oligonucleotide revealed that the complexes A, B, C, and D bind to –122 to –117, the –101 to –96, the –83 to –78, and the –68 to –57 bp, respectively. The competition assays using consensus oligonucleotides and supershift assays with specific antibodies showed that complex A consists of CBF/NF-Y. In a chromatin immunoprecipitation assay, CBF/NF-Y protein directly bound to this region, in vivo. Functional analysis showed that CBF/NF-Y activated the gene, whereas the proteins of complexes B and C repressed its activity. Furthermore, overexpression of a mutant form of the CBF-B/NF-YA subunit, which forms CBF/NF-Y with CBF-A/NF-YB and CBF-C/NF-YC subunits, inhibited promoter activity. Vertebrate collagens, a large family of extracellular proteins, are critically important for the formation and function of virtually every organ system (1Vuorio E. de Crombregghe B. Annu. Rev. Biochem. 1990; 59: 837-872Crossref PubMed Scopus (392) Google Scholar). Among them, fibrillar collagen, which includes five different molecular types I, II, III, V, and XI, participates in the formation of fibrils with molecules packed in quarter-staggered arrays (2van der Rest M. Garrone R. FASEB J. 1991; 5: 2814-2823Crossref PubMed Scopus (994) Google Scholar, 3Brown J.C. Timpl R. Int. Arch. Allergy Immunol. 1995; 107: 484-490Crossref PubMed Scopus (125) Google Scholar). The fibrillar collagens are divided into major types (I–III) and minor types (V and XI) based on their relative expression levels. Minor fibrillar collagen types V and XI are incorporated into the fibrils of the much more abundant collagen types I and II, respectively, and act as regulators of the sizes and shapes of the resultant heterotypic fibrils (4Adachi E. Hayashi T. Connect. Tissue Res. 1986; 14: 257-266Crossref PubMed Scopus (143) Google Scholar, 5Mendler M. Eich-Bender S.G. Vaughn L. Winterhalter K.H. Bruckner P. J. Cell Biol. 1989; 108: 191-197Crossref PubMed Scopus (405) Google Scholar, 6Linsenmayer T.F. Gibney E. Igoe F. Gordon M.K. Fitch J.M. Fessler L.I. Birk D.E. J. Cell Biol. 1993; 121: 1181-1189Crossref PubMed Scopus (247) Google Scholar, 7Andrikopoulos K. Liu X. Keene D.R. Jaenisch R. Ramirez F. Nat. Genet. 1995; 9: 31-39Crossref PubMed Scopus (189) Google Scholar). The collagen molecules are either homotrimers with α chains or heterotrimers with two or three different α chains. The predominant molecular form of type V is the heterotrimer [α1(V)]2α2(V) and is expressed in most tissues (8Burgeson R.E. El Adli F.A. Kaitila I.I. Hollister D.W. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2579-2583Crossref PubMed Scopus (306) Google Scholar). Other forms of type V collagen include the [α1(V)]3 homotrimer that is synthesized in cultures of hamster lung cells (9Haralson M.A. Michell W.M. Rhodes R.K. Kresina T.F. Gay R. Miller E.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5206-5210Crossref PubMed Scopus (65) Google Scholar) and the α1(V)α2(V)α3(V) heterotrimer that can be extracted from the placenta (10Sage H. Bornstein P. Biochemistry. 1979; 18: 3815-3822Crossref PubMed Scopus (151) Google Scholar, 11Brown R.A. Shuttleworth C.A. Weiss J.B. Biochem. Biophys. Res. Commun. 1978; 71: 1167-1174Google Scholar, 12Rhodes R.K. Miller E.J. Collagen Relat. Res. 1981; 1: 337-343Crossref PubMed Scopus (45) Google Scholar). Cross-type heterotrimer composed of α2(V) and α1(XI) chains is present in the rhabdomyosarcoma cell line A204 and bovine vitreous (13Kleman J.-P. Hartmann D.J. Ramirez F. van der Rest M. Eur. J. Biochem. 1992; 210: 329-335Crossref PubMed Scopus (67) Google Scholar, 14Mayne R. Brewton R.G. Mayne P.M. Baker J.R. J. Biol. Chem. 1993; 268: 9381-9386Abstract Full Text PDF PubMed Google Scholar). These findings suggest that type V and type XI chains constitute a single collagen type in which different combinations of chains associate in a tissue-specific manner. Recently, a fourth chain, α4(V), expressed in rat Schwann cells was reported (15Chernousov M.A. Rothblum K. Tyler W.A. Stahl R.C. Carey D.J. J. Biol. Chem. 2000; 275: 28208-28215Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). This α chain, which can form molecules with α1(V) and α2(V) chains, seems to be the counterpart of the mouse and human α3(V) chain (16Imamura Y. Scott I.C. Greenspan D.S. J. Biol. Chem. 2000; 275: 8749-8759Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Type V collagen, which is widely expressed spatially and temporally, is expressed in the mouse embryo as early as 11 days post-coitum (17Wu Y.-L. Sumiyoshi H. Khaleduzzaman M. Ninomiya Y. Yoshioka H. Biochim. Biophys. Acta. 1998; 1397: 275-284Crossref PubMed Scopus (10) Google Scholar). Altered production of type V collagen is associated with some connective tissue pathology, such as inflammation, some forms of cancer, and atherosclerosis (18Narayanan A. Engel S. Page R.C. Collagen Relat. Res. 1983; 3: 323-334Crossref PubMed Scopus (36) Google Scholar, 19Barsky S.H. Rao C.N. Grotendorst G.R. Liotta L.A. Am. J. Pathol. 1982; 108: 276-283PubMed Google Scholar, 20Ooshima A. Science. 1981; 213: 666-668Crossref PubMed Scopus (113) Google Scholar). Characterization of the cis-acting elements and trans-acting nuclear factors that modulate correct patterns of gene expression is necessary for understanding physiological and pathological conditions. Among the collagen genes, the transcriptional regulation of type I collagen has been most extensively studied, showing common features of the proximal promoter and tissue-specific enhancer (21Ramirez F. Di Liberto M. FASEB J. 1990; 4: 16116-16123Crossref Scopus (88) Google Scholar, 22Slack J.L. Liska D.J. Bornstein P. Am. J. Med. Genet. 1993; 45: 140-151Crossref PubMed Scopus (110) Google Scholar). With regard to minor collagen, Penkov et al. (23Penkov D. Tanaka S. Rocco G.D. Berthelsen J. Blasi F. Ramirez F. J. Biol. Chem. 2000; 275: 16681-16689Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) identified the nuclear factors binding to the proximal promoter of COL5A2. Their results indicate that members of the homeotic complex and TALE class of homeoproteins, PBX1/2, PREP1, and HOXB1, were involved in regulation of COL5A2. B-myb, a member of the myb gene family, indirectly repressed the COL5A2 gene promoter by interacting with the positive factor that binds to the first exon (24Kypreos K.E. Marhamati D.J. Sonenshein G.E. Matrix Biol. 1999; 18: 275-285Crossref PubMed Scopus (11) Google Scholar). Lee and Greenspan (25Lee S. Greenspan D.S. Biochem. J. 1995; 310: 15-22Crossref PubMed Scopus (16) Google Scholar) suggested that the GAGA boxes in the promoter and the first exon of the COL5A1 gene affect the transcription. We have recently characterized the proximal promoter of the COL11A1 (26Matsuo N. Wang Y-H. Sumiyoshi H. Sakata-Takatani K. Nagato H. Sakai K. Sakurai M. Yoshioka H. J. Biol. Chem. 2003; 278: 32763-32770Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and Col5a1 genes (27Sakata-Takatani K. Matsuo N. Sumiyoshi H. Tsuda T. Yoshioka H. Matrix Biol. 2004; 23: 87-99Crossref PubMed Scopus (13) Google Scholar). In these studies, we demonstrated that the CCAAT binding factor, CBF/NF-Y, bound to the CCAAT motif in the proximal promoter and is involved in activation of the genes. Recently, human and mouse full-length pro-α3(V) sequences were provided (16Imamura Y. Scott I.C. Greenspan D.S. J. Biol. Chem. 2000; 275: 8749-8759Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). These were shown to be closely related to the pro-α1(V) chain. In situ hybridization of mouse embryo detected its expression in epimysial sheaths of developing muscles and within nascent ligaments adjacent to forming bones and in joints, where it probably forms molecules with α1(V) and α2(V). In the present study, we cloned the human promoter DNA fragment and characterized the core promoter of the α3(V) collagen gene. DNase I footprinting and DNA binding assays have demonstrated four nuclear protein binding sites in the promoter region. Cell transfection experiments showed that one works as an activator and two as repressors. Finally, functional analysis combined with oligonucleotide competition and supershift assays showed that CBF/NF-Y acts as a transcriptional activator of COL5A3. Cells and Cell Culture Conditions—Most of the cell lines used in this study were purchased from the American Type Culture Collection (ATCC) and the Japanese Collection of Research Bioresources (JCRB) cell banks. A204 cells (human embryonal rhabdomyosarcoma cell line, ATCC HTB-82), HT-1080 cells (human fibrosarcoma cell line, ATCC CCL-121), U251MG cells (human glioblastoma cell line, JCRB, IFO50288), IMR32 cells (human neuroblastoma cell line, ATCC CCL-1277), and A498 cells (human epithelial cell line, ATCC HB-44) were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum (Sanko Junyaku, Tokyo, Japan) at 37 °C in a 5% CO2/air environment. Jurkat cells (human T cell line, ATCC TIB-152) and YST-1 cells, a primary cell line of human Schwannoma cells (kindly provided by Dr. Y. Nagashima, Yokohama City University School of Medicine), were cultured in RPMI 1640 medium containing 10% fetal bovine serum in the same environment. Isolation of RNA—Total RNA was isolated from cultured cells using Isogen (Nippon Gene Co. Ltd., Tokyo, Japan) according to the manufacturer's instructions. RNA was quantified by optical density (A260) and stored at –80 °C until used. Reverse Transcription-Polymerase Chain Reaction (RT 1The abbreviations used are: RT, reverse transcription; CBF/NF-Y, CCAAT-binding factor; RACE, rapid amplification of cDNA ends; EMSA, electrophoretic mobility shift assay; CHIP, chromatin immunoprecipitation assay; PBS, phosphate-buffered saline; ss, single-stranded; FP, footprint; wt, wild-type; CEBP, cAMP element-binding protein.-PCR)—The expression of COL5A3 mRNA in various cell lines was investigated by RT-PCR. Three micrograms of total RNA was reverse transcribed by random hexamer priming using SuperScript II reverse transcriptase (Invitrogen). The single-stranded cDNA was amplified by PCR using COL5A3-specific primer pairs and glyceraldehide-3-phosphate dehydrogenase primers. The primers are listed in Supplemental Materials Table S1. PCR was carried out for 30 cycles using a step cycle of 94 °C for 30 s, 55 °C for 20 s, 72 °C for 30 s, followed by 72 °C for 7 min. The PCR products were analyzed by electrophoresis on a 4% ethidium bromide-stained agarose gel. The amplified fragment was eluted from the gel and subcloned into the pGEM-T Easy vector (Promega) for sequencing. Screening Human BAC Library and Cloning of Genomic DNA Fragment—A human genomic BAC library was screened to obtain the COL5A3 genomic fragment using the PCR protocol supplied by the manufacturer (Genome System, Inc., St. Louis, MO). The primers specific for the COL5A3 used in the PCR-based screening are listed in Supplemental Materials Table S1. The DNA from the PCR-positive BAC clone was purified and the sequences at both ends were determined by a direct sequencing method using Sp6 and T7 primers specific for the BAC cloning vector arms. The sequences were compared with those in the EMBO/GenBank™ data base. To obtain a smaller DNA fragment that contains the promoter region of COL5A3, the DNA was digested with ApaI because this restriction site was located just upstream of the initiating ATG codon. The resulting fragments were subcloned into the pBluescript (SK+) vector. The sublibrary was screened by colony hybridization. Rapid Amplification of 5′-cDNA Ends (5′-RACE)—The 5′-RACE experiment was carried out according to the method described by Frohman et al. (28Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Crossref PubMed Scopus (4337) Google Scholar). Briefly, 10 μg of total RNA from A204 or YST-1 cells was reverse transcribed using the COL5A3-specific primer, RACE1 or RACE3 (Fig. 2C). Homopolymeric dC was added to the 3′-end of the first strand cDNA using terminal deoxynucleotidyl transferase in the presence of dCTP. The dC-tailed cDNA was amplified with PCR using a poly(dG) containing the 5′ adapter primer and a 3′ COL5A3-specific primer, RACE2 or RACE4, which was located just upstream of RACE1 or RACE3, respectively (Fig. 2C). PCR was carried out for 35 cycles using a step cycle of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min, followed by 72 °C for 8 min. The 5′-RACE products were purified by electrophoresis on a 4% ethidium bromide-stained agarose gel, and then subcloned into the pGEM-T Easy vector for sequencing. In Vitro Transcription and RNase Protection Assay—RNase protection assays were carried out to confirm the transcription initiation sites. A 252-bp genomic fragment, containing putative transcription initiation sites from the data of the 5′-RACE experiment, was generated by PCR. This PCR product was subcloned into pGEM-T Easy vector, followed by digestion with EcoRI, and subcloned into the EcoRI site of pBluescript (SK+) vector. After linearization with BamHI, the antisense riboprobe was synthesized using the in vitro transcription kit, MAXIscript (Ambion, Austin, TX), according to the manufacturer's instructions. This riboprobe was examined by 5% polyacrylamide gel containing 8 m urea, and the full-length riboprobe was recovered from the gel. RNase protection analysis was performed using a ribonuclease protection assay kit, RPAIII (Ambion), following the manufacturer's protocol. Briefly, 20 μg of total RNA isolated from A204 or YST-1 cells and 8 × 104 cpm of riboprobe were dissolved in 10 μl of hybridization buffer and incubated overnight at 42 °C. After digestion with the RNase A/T1 mixture, protected RNA fragments were separated on a 6% polyacrylamide gel containing 8 m urea. As a size marker, 32P-end-labeled ϕX174 DNA/HaeIII marker was loaded onto the same gel. After the run had finished, the gel was transferred onto 3MM paper (Whatman) and dried under vacuum. The dried gel was visualized by autoradiography using a Bio Imaging Analyzer FLA-5000 (Fuji Film, Tokyo, Japan). Construction of Chimeric Plasmids—The ApaI genomic fragment corresponding to the region from –1785 to +112 bp of the COL5A3 was blunt-ended with T4 DNA polymerase and subcloned into the SmaI site of pGL3-Basic vector (Promega). To generate the 5′ stepwise deletion constructs, PCR procedures were applied. PCR was performed using sets of oligonucleotide primers that are SacI site-linked 5′ and HindIII site-linked 3′ primers specific for the COL5A3 sequence and the pGL3–1785/+109 plasmid as a template. These PCR products were subcloned into the pGEM-T Easy vector, followed by digestion with SacI and HindIII, and subcloned into the SacI/HindIII site of pGL3-Basic vector. Internal deletion and substitution mutation constructs were generated by site-directed mutagenesis using the pGL3–310/+109 plasmid as a template. The primers used in the PCR amplification are listed in Supplemental Materials Table S1. The PCR products were digested with endonuclease, followed by self-ligation. All mutagenesis plasmids were digested with SacI and HindIII and re-cloned into the SacI/HindIII site of pGL3-Basic vector. Construction of the dominant negative CBF-B/NF-YA was generated by RT-PCR as previously described (26Matsuo N. Wang Y-H. Sumiyoshi H. Sakata-Takatani K. Nagato H. Sakai K. Sakurai M. Yoshioka H. J. Biol. Chem. 2003; 278: 32763-32770Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 29Mantovani 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). Transient Transfection and Luciferase Assays—Transient transfection experiments were carried out using A204 and YST-1 cells. The cells were plated at a density of 2 × 105 per 35-mm dish ∼18 h before transfection. For transient transfection, 1 μg of plasmid DNA was transfected into these cells by using the LipofectAMINE Plus reagent system (Invitrogen). Plasmid pRL-TK vector (Promega) was always cotransfected as an internal control for transfection efficiency. After an additional cultivation for 48 h, the transfected cells were harvested, lysed, centrifuged to pellet the debris, and subjected to luciferase assay. Luciferase activities were measured as chemiluminescence in a luminometer (Lumat LB 9507, PerkinElmer Life Sciences) using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. The cotransfection experiments with a mutant type of CBF-B/NF-YA subunit expression vector were performed as previously described (26Matsuo N. Wang Y-H. Sumiyoshi H. Sakata-Takatani K. Nagato H. Sakai K. Sakurai M. Yoshioka H. J. Biol. Chem. 2003; 278: 32763-32770Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 27Sakata-Takatani K. Matsuo N. Sumiyoshi H. Tsuda T. Yoshioka H. Matrix Biol. 2004; 23: 87-99Crossref PubMed Scopus (13) Google Scholar). All transfections were repeated in triplicate and the results were expressed as the mean ± S.D. of three independent experiments. Preparation of Nuclear Extracts—Nuclear extracts from A204 and YST-1 cells were prepared according to the method of Dignam et al. (30Dignam J.D. Martin P.L. Shastry B.S. Roeder R.G. Methods Enzymol. 1983; 101: 582-598Crossref PubMed Scopus (745) Google Scholar). Briefly, cells, 1 × 108, were scraped in ice-cold phosphate-buffered saline (PBS), centrifuged for 5 min at 1,500 × g, and washed with PBS before being recentrifuged. The pellets were resuspended in buffer (10 mm HEPES, pH 7.8, 10 mm KCl, 0.1 mm EDTA, 0.1% Nonidet P-40), incubated on ice for 10 min, and homogenized. Nuclei were pelleted by centrifugation at 3,000 × g for 10 min at 4 °C, followed by resuspension in buffer (50 mm HEPES, pH 7.8, 420 mm KCl, 5 mm MgCl2, 0.1 mm EDTA, 20% glycerol) and mixed by rotation at 4 °C for 1 h. After centrifugation at 24,000 × g for 30 min at 4 °C, the supernatants were collected and stored at –80 °C until used. The protein concentration of the nuclear extracts was determined by Bradford assay (Bio-Rad) using bovine serum albumin as a standard. DNase I Footprinting Assay—Probes for DNase I footprinting were generated by PCR amplification using sets of oligonucleotides that are SacI site-linked 5′ and HindIII site-linked 3′ primers specific for COL5A3, and all PCR products were subcloned into the pGEM-T Easy vector. These plasmids were digested with HindIII and radiolabeled with [α-32P]dCTP using Klenow fragment to fill in the HindIII overhanging sites. After digestion with SacI, these probes were examined by a 4.5% non-denaturing polyacrylamide gel and 3′ end-labeled probes were recovered from the gel. DNase I footprinting was performed according to the protocol described by Galas and Schmitz (31Galas D.J. Schmitz A. Nucleic Acids Res. 1978; 5: 3157-3170Crossref PubMed Scopus (1334) Google Scholar) with some modifications. Briefly, the 3′ end-labeled probe (50,000 cpm) was incubated at 25 °C for 30 min with 50 μg of nuclear extracts (or bovine serum albumin as control) in a 100-μl reaction mixture containing 20 mm Tris-HCl (pH 7.9), 3 mm MgCl2, 100 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 5 μg of bovine serum albumin, and 4 μg of poly(dI-dC). After the addition of 5 units of DNase I (or 0.1 unit with bovine serum albumin as control) to the mixture, the incubation was continued for 5 min at 25 °C. The reaction was stopped by adding 100 μl of a stop buffer (200 mm sodium acetate, 20 mm EDTA, 1% SDS, 10 μg of yeast tRNA), and DNA fragments were subsequently extracted with phenol/chloroform and precipitated with ethanol before being loaded onto a 6% polyacrylamide gel containing 8 m urea. After electrophoresis, the gel was transferred onto 3 MM paper, dried, and exposed to a BioImaging Analyzer FLA-5000. Electrophoretic Mobility Shift Assay (EMSA)—Wild-type and mutant probes used for EMSA were generated by PCR using each set of HindIII site-linked primers, and all PCR products were subcloned into the pGEM-T Easy vector. All plasmids were digested with HindIII and the digested fragments were radiolabeled with [α-32P]dCTP using Klenow fragment to fill in the HindIII overhanging sites. The binding reaction was carried out for 30 min at 25 °C in 25 μl of binding buffer (50 mm HEPES, pH 7.8, 250 mm KCl, 25 mm MgCl2, 5 mm EDTA, 50% glycerol) containing 20,000–30,000 cpm of labeled probe, 3 μg of poly(dI-dC), and 5–15 μg of nuclear extracts. For the competition assays, double strand oligonucleotides containing a consensus CBF, CBF mutant, NF-1, CEBP, GATA-1, c-Myb, and MEF-1 binding site were generated by annealing equimolar complementary oligonucleotides. The consensus sequences are shown in Fig. 7A. For the supershift assay, anti-CBF-A/NF-YB, anti-CBF-B/NF-YA, anti-CBF-C/NF-YC, anti-NF-1, anti-CEBP, and anti-GATA-1 polyclonal antibodies and preimmune goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were purchased. For these interference assays, 5–100-fold molar excess of unlabeled competitor or 2 μg of antibody was added to the reaction mixture for 1 h at 4 °C before the addition of radiolabeled probe. The DNA-protein complexes were separated on a 4.5% non-denaturing polyacrylamide gel in a 0.25× Tris borate electrophoresis buffer at 200 V. Following completion of running, the gel was transferred onto 3MM paper and dried under vacuum. The dried gel was visualized by autoradiography using a BioImaging Analyzer FLA-5000. Chromatin Immunoprecipitation (CHIP) Assay—CHIP assays were performed using a chromatin immunoprecipitation assay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer's protocol. Briefly, A204 and YST-1 cells were inoculated at a density of 5 × 106 per 100-mm dish. The cells were fixed in a final concentration of 1% formaldehyde for 10 min at room temperature. After washing twice with PBS, cells were removed from dishes in PBS containing 1 mm EDTA, and harvested by centrifugation at 1,000 × g for 1 min at 4 °C. The cell pellets were resuspended in SDS lysis buffer, incubated for 10 min at 4 °C, and sonicated four times for 10 s yielding DNA fragments 200–1000 bp in size. After centrifugation, the supernatant was diluted in CHIP dilution buffer, precleared with salmon sperm DNA/protein A-agarose slurry, and immunoprecipitated with the indicated antibodies overnight at 4 °C. Immunocomplexes were captured on the ssDNA/protein A-agarose slurry, and washed with low salt wash buffer, high salt wash buffer, and LiCl wash buffer and finally washed twice with TE buffer. The immunocomplexes were eluted by incubation for 15 min at 25 °C with 200 μl of elution buffer (1% SDS, 100 mm NaHCO3, 1 mm dithiothreitol). To reverse the cross-linking of DNA, the elutes were treated with 8 μlof5 m NaCl and incubated for 4 h at 65 °C, followed by treatment with proteinase K for 1 h at 45 °C. The DNA fragments were extracted with phenol/chloroform and precipitated with ethanol. PCR were carried out for 40 cycles using a step cycle of 94 °C for 30 s, 55 °C for 20 s, 72 °C for 30 s, followed by 72 °C for 8 min. The PCR products were analyzed by electrophoresis on a 4% ethidium bromide-stained agarose gel. DNA Sequencing—Nucleotide sequences were determined by automated DNA sequencing (ABI PRISM 310 Genetic Analyzer, Applied Biosystems, Foster, CA) using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems). RNA Expression of COL5A3 in Cultured Cells—Initially, we examined the expression of the COL5A3 in various culture cells using the RT-PCR technique. As shown in Fig. 1, the predicted band of 295 bp was observed in YST-1 (strong) and A204 (weak) cells. The PCR products were verified by nucleotide sequence to confirm the specificity of the amplified COL5A3 sequence. By contrast, the band was not detected in HT1080, Jurkat, U251MG, IMR32, and A498 cells. From these results, we decided to use A204 and YST-1 cell lines for the analysis of the COL5A3 promoter. Structural Analysis of the 5′ Portion of COL5A3—To obtain the COL5A3 genomic fragment, we screened a human genomic BAC library by PCR, and isolated one positive clone, 218M16. Compared with the EMBO/GenBank™ data base, DNA sequences of both ends of the clone were identical with those of two clones, AC008742 and AC020931, in chromosome 19 of Homo sapiens. From the data base, this BAC genomic fragment, which was ∼141 kb in size, contained the promoter region and both exons and introns 1–32 of COL5A3 (Fig. 2A). To subclone a DNA fragment containing the promoter region, the BAC genomic fragment was digested with ApaI endonuclease and subcloned into the pBluescript (SK+) vector. The sublibrary was screened by colony hybridization and the insert DNA of the positive clone was sequenced using COL5A3-specific or vector-specific primers. As a result, a 1897-bp genomic fragment containing the 5′-flanking region of COL5A3 was isolated (Fig. 2B). To determine the transcription initiation sites of COL5A3, 5′-RACE experiments and RNase protection assays were performed. The 5′-RACE was carried out using total RNA derived from A204 and YST-1 cells. Sixteen independent clones, eight clones from A204 cells and eight clones from YST-1 cells, were selected and sequenced. As shown in Fig. 2C, the 5′-ends of the COL5A3 cDNA were located at 113 to 119 bp upstream from the initiating ATG codon, and 13 of the 16 RACE products were generated from the same position, which suggested that the major transcription initiation site is located 119 bp upstream from the initiating ATG codon. On the basis of these data, we also performed RNase protection assays to confirm the transcription initiation sites. In this experiment, we used a 357-nucleotide riboprobe spanning nucleotides containing the putative transcription initiation site, and at least two specific protected bands were observed in both A204 and YST-1 cells (Fig. 2D). The protected bands were about 115 and 100 nucleotides in size, and the larger protected band corresponded to the results of the 5′-RACE. Therefore, we concluded that COL5A3 contains at least two transcription initiation sites and that the major transcription initiation site is located 119 bp upstream from the initiating ATG codon. The COL5A3 promoter also lacks a canonical TATA box and has a high GC content. Functional Analysis of the COL5A3 Promoter Region—To define the proximal regulatory regions in the COL5A3 promoter, a series of chimeric constructs containing progressive 5′ end and internal deletions linked to the luciferase gene were transfected into A204 and YST-1 cells (Fig. 3A), and luciferase assays were carried out. The longest construct, pGL3–1785/+109, derived from a 1.9-kb genomic fragment of the 5′-flanking region of COL5A3, had strong transcriptional activity. The activity of each construct was compared with this construct. As shown in Fig. 3B, deletion from –1785 to –129 bp had no significant change on luciferase activity; however, deletion to –40 bp resulted in a significantly lower luciferase activity compared with that of pGL3–1785/+109, and deletion to the +3 bp showed almost complete loss of luciferase activity in both A204 and YST-1 cells. In addition, the deletion mutant of pGL3–310/+109(del–129/+2), which is deleted between –129 and +2, also showed a significantly lower luciferase activity in both cells. These results indicate that the region between the –129 and +2 bp is important for basal transcriptional activity of the COL5A3 promoter. Identification of Nuclear Factor Binding Sites in the Core Promoter of the COL5A3—To identify transcription factor binding sites on the proximal promoter region of COL5A3, we initially performed DNase I footprinting. Two different probes, which contain the region from the –160 to –51 bp (Fig. 4A) and from the –96 to +30 bp (Fig. 4B), were used in this experiment. As shown in Fig. 4, A and B, two protected regions, FP (footprint) 1 in the –124 to –117 bp, and FP2 in the –69 to –63 bp regions, were" @default.
• W2007284190 created "2016-06-24" @default.
• W2007284190 creator A5024037872 @default.
• W2007284190 creator A5026995671 @default.
• W2007284190 creator A5059981236 @default.
• W2007284190 creator A5073224610 @default.
• W2007284190 creator A5073232782 @default.
• W2007284190 creator A5073355813 @default.
• W2007284190 date "2004-11-01" @default.
• W2007284190 modified "2023-09-28" @default.
• W2007284190 title "The Transcription Factor CCAAT-binding Factor CBF/NF-Y and Two Repressors Regulate the Core Promoter of the Human Pro-α3(V) Collagen Gene (COL5A3)" @default.
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