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• W2065860483 abstract "Integrin-associated protein (IAP or CD47) is expressed in a variety of tissues, including the nervous system and immune system. To understand how cells control the expression of the IAP gene, we cloned the 5′-proximal region of the human IAP gene and investigated IAP promoter activity by transient transfection. RT-PCR confirmed the expression of IAP transcripts in human neuroblastoma IMR-32 and hepatoma HepG2 cells. Deletion analysis identified a core promoter of the human IAP gene located between nucleotide positions –232 and –12 relative to the translation initiation codon in these two cell lines. Site-directed mutagenesis and gel electrophoretic mobility shift assay identified a α-Pal/NRF-1 binding element within the IAP core promoter. Supershift assays using the α-Pal/NRF-1 antiserum confirmed the binding of this transcription factor on the α-Pal/NRF-1 site. Overexpression of the DNA binding domain of α-Pal/NRF-1 in cells enhanced DNA-α-Pal/NRF-1 binding in vitro. Furthermore, overexpression of full-length α-Pal/NRF-1 significantly enhanced IAP promoter activity while overexpression of dominant-negative mutant reduced promoter activity both in the cultured human cell lines and primary mouse cortical cells. These results revealed that α-Pal/NRF-1 is an essential transcription factor in the regulation of human IAP gene expression. Integrin-associated protein (IAP or CD47) is expressed in a variety of tissues, including the nervous system and immune system. To understand how cells control the expression of the IAP gene, we cloned the 5′-proximal region of the human IAP gene and investigated IAP promoter activity by transient transfection. RT-PCR confirmed the expression of IAP transcripts in human neuroblastoma IMR-32 and hepatoma HepG2 cells. Deletion analysis identified a core promoter of the human IAP gene located between nucleotide positions –232 and –12 relative to the translation initiation codon in these two cell lines. Site-directed mutagenesis and gel electrophoretic mobility shift assay identified a α-Pal/NRF-1 binding element within the IAP core promoter. Supershift assays using the α-Pal/NRF-1 antiserum confirmed the binding of this transcription factor on the α-Pal/NRF-1 site. Overexpression of the DNA binding domain of α-Pal/NRF-1 in cells enhanced DNA-α-Pal/NRF-1 binding in vitro. Furthermore, overexpression of full-length α-Pal/NRF-1 significantly enhanced IAP promoter activity while overexpression of dominant-negative mutant reduced promoter activity both in the cultured human cell lines and primary mouse cortical cells. These results revealed that α-Pal/NRF-1 is an essential transcription factor in the regulation of human IAP gene expression. Integrin-associated protein (IAP), 1The abbreviations used are: IAP, integrin-associated protein; NRF-1, nuclear respiratory factor 1; CREB, cAMP response element-binding protein; EMSA, electrophoretic mobility shift assay. also designated as CD47, is a multifunctional membrane protein that is expressed widely in the nervous system, immune system and many other tissues (1Brown E. Hopper L. Ho T. Gresham H. J. Cell Biol. 1990; 111: 2785-2794Crossref PubMed Scopus (315) Google Scholar, 2Lindberg F.P. Gresham H.D. Schwarz E. Brown E.J. J. Cell Biol. 1993; 123: 485-496Crossref PubMed Scopus (302) Google Scholar). In the adult rat central nervous system, IAP was related to memory formation of an aversive learning task (3Huang A.M. Wang H.L. Tang Y.P. Lee E.H.Y. J. Neurosci. 1998; 18: 4305-4313Crossref PubMed Google Scholar). In good memory rats trained in the inhibitory avoidance learning paradigm, the mRNA level of IAP in the hippocampus was increased. Injection of antisense oligonucleotides or the monoclonal antibody of IAP into the rat hippocampus impaired memory formation (3Huang A.M. Wang H.L. Tang Y.P. Lee E.H.Y. J. Neurosci. 1998; 18: 4305-4313Crossref PubMed Google Scholar, 4Chang H.P. Ma Y.L. Wan F.J. Tsai L.Y. Lindberg F.P. Lee E.H.Y. Neuroscience. 2001; 102: 289-296Crossref PubMed Scopus (29) Google Scholar). IAP-deficient mice showed deficits in memory retention in a similar behavioral paradigm (5Chang H.P. Lindberg F.P. Wang H.L. Huang A.M. Lee E.H.Y. Learn. Mem. 1999; 6: 448-457Crossref PubMed Scopus (59) Google Scholar). In the peripheral tissues, IAP was first discovered as a cell surface protein associated with integrin αvβ3 and it was involved in the enhancement of neutrophil adhesion, chemotaxis, and phagocytosis triggered by an extracellular matrix (1Brown E. Hopper L. Ho T. Gresham H. J. Cell Biol. 1990; 111: 2785-2794Crossref PubMed Scopus (315) Google Scholar, 6Gresham H.D. Goodwin J.L. Allen P.M. Anderson D.C. Brown E.J. J. Cell Biol. 1989; 108: 1935-1943Crossref PubMed Scopus (148) Google Scholar, 7Senior R.M. Gresham H.D. Griffin G.L. Brown E.J. Chung A.E. J. Clin. Investig. 1992; 90: 2251-2257Crossref PubMed Scopus (109) Google Scholar). IAP is also a functional component of several processes, including the transepithelial migration of neutrophils (8Parkos C.A. Colgan S.P. Liang T.W. Nusrat A. Bacarra A.E. Carnes D.K. Madara J.L. J. Cell Biol. 1996; 132: 437-450Crossref PubMed Scopus (181) Google Scholar), chemotaxis of endothelial cells and smooth muscle cells (9Gao A.-G. Lindberg F.P. Finn M.B. Blystone S.D. Brown E.J. Frazier W.A. J. Biol. Chem. 1996; 271: 21-24Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 10Wang X.-Q. Frazier W.A. Mol. Biol. Cell. 1998; 9: 865-874Crossref PubMed Scopus (139) Google Scholar), spreading and aggregation of platelets (11Chung J. Gao A.-G. Frazier W.A. J. Biol. Chem. 1997; 272: 14740-14746Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), and modulation of T-cell activation (12Ticchioni M. Deckert M. Mary F. Bernard G. Brown E.J. Bernard A. J. Immunol. 1997; 158: 677-684PubMed Google Scholar, 13Waclavicek M. Majdic O. Stulnig T. Berger M. Baumruker T. Knapp W. Pickl W.F. J. Immunol. 1997; 159: 5345-5354PubMed Google Scholar). Moreover, IAP functions as a self-check marker on red blood cells to prevent their clearance by macrophages (14Oldenborg P.-A. Zheleznyak A. Fang Y.-F. Lagenaur C.F. Gresham H.D. Lindberg F.P. Science. 2000; 288: 2051-2054Crossref PubMed Scopus (1281) Google Scholar). IAP mRNA and protein sequences are conserved among humans, mice, and rats (3Huang A.M. Wang H.L. Tang Y.P. Lee E.H.Y. J. Neurosci. 1998; 18: 4305-4313Crossref PubMed Google Scholar, 15Reinhold M.I. Lindberg F.P. Plas D. Reynolds S. Peters M.G. Brown E.J. J. Cell Sci. 1995; 108: 3419-3425Crossref PubMed Google Scholar), suggesting that it is evolutionally important to biological functions. IAP mRNA has five alternative splicing forms. These forms are also conserved in evolution (3Huang A.M. Wang H.L. Tang Y.P. Lee E.H.Y. J. Neurosci. 1998; 18: 4305-4313Crossref PubMed Google Scholar, 15Reinhold M.I. Lindberg F.P. Plas D. Reynolds S. Peters M.G. Brown E.J. J. Cell Sci. 1995; 108: 3419-3425Crossref PubMed Google Scholar, 16Schickel J. Stahn K. Zimmer K.-P. Sudbrak R. Størm T.M. Dürst M. Kiehntopf M. Deufel T. Biochem. Cell Biol. 2002; 80: 169-176Crossref PubMed Scopus (3) Google Scholar, 17Shahein Y.E.A. de Andrés D.F. Pérez de la Lastra J.M. Immunology. 2002; 106: 564-576Crossref PubMed Scopus (10) Google Scholar). In humans and mice, different forms of IAP mRNA were expressed at varying levels in different tissues; macrophage and endothelial cells expressed predominantly form 2 mRNA and brain tissues expressed predominantly form 4. Why different tissues express different levels and forms of IAP mRNA and how the IAP gene is regulated are, however, unknown. To answer these questions, we investigated the regulation of IAP gene promoter in human neuroblastoma and hepatoma cell lines by using luciferase reporter and gel electrophoretic mobility shift assays. We found that α-Pal/NRF-1 in the core promoter region is a positive regulator of the human IAP gene. Cell Culture—Human neuroblastoma IMR-32 (CCRC 60014) and hepatoma HepG2 (CCRC 60048) cell lines were purchased from Culture Collection and Research Center, Food Industry and Development Institute, Hsinchu, Taiwan. Cells were grown in minimum essential medium Eagle with Earle's salt base (Sigma) supplemented with 10% fetal bovine serum (HyClone, Logan, UT) in a humidified atmosphere containing 5% CO2 at 37 °C. RNA Isolation and Reverse Transcription (RT)-PCR—Total RNA was isolated from the cultured cells using TRIzol reagent (Invitrogen). RT-PCR was performed as described previously (3Huang A.M. Wang H.L. Tang Y.P. Lee E.H.Y. J. Neurosci. 1998; 18: 4305-4313Crossref PubMed Google Scholar). Briefly, total RNA (2 μg) was reverse-transcribed into cDNA in 20 μl of 1× first strand buffer containing 0.5 μg of oligo(dT) as a primer, 500 μm dNTP, and 200 units of SuperScript II (Invitrogen). PCR was performed in 20 μl of 1× PCR buffer containing 2 μl of RT products, 1 unit of AmpliTaq DNA polymerase (Roche Applied Science), 200 μm dNTP, 1.5 mm MgCl2, 0.5 μm [35S]dATP (Amersham Biosciences), and 0.4 μm primer pair. We used the primer pair that can distinguish the alternative splicing forms of IAP mRNA, Hiap14: 5′-TAA CCT CCT TCG TCA TTG CC and Hiap15: 5′-CGT AAG GGT CTC ATA GGT G. The PCR parameters were 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 30 s for 30 cycles, followed by a final elongation at 72 °C for 7 min. PCR products were analyzed on a 6% polyacrylamide-urea gel (acrylamide/bisacrylamide 19:1, 8 m urea in 1 × Tris borate-EDTA buffer). The gel was finally dried and analyzed by autoradiography. The image of cDNA bands was scanned by the Scan-Jet 4C scanner (Hewlett Packard). The optical densities of cDNA bands were quantified with the one-dimensional advanced Universal Software (American Applied Biotechnology, Fullerton, CA). Plasmids—pGL3-Basic and pRL-TK luciferase reporter vectors (Promega, Madison, WI) were used for IAP promoter reporter assays. A 7.7-kb human genomic clone (pIAP38) containing exon 1 of the IAP gene and 5′-upstream region was kindly provided by Dr. F. P. Lindberg. A plasmid, pBSII-445, was generated by inserting the 445-bp SacII fragment from pIAP38 into the pBlueScript II (SK–) vector (Stratagene, La Jolla, CA). The SacI/XhoI fragment from pBSII-445 was then inserted into the pCRII vector (Invitrogen) to create pHIAP445 for subsequent constructions. The reporter construct pGL3–272 was generated by inserting the XmaI/XhoI fragment from pHIAP445 into the pGL3-Basic vector. Different restriction enzyme-digested fragments from pIAP38 were then ligated into pGL3–272 to create a series of human IAP promoter constructs, including pGL3–1554, pGL3–730, and pGL3–456. Another series of human IAP promoter constructs containing shorter fragments than the insert in pGL3–272, including pGL3–232, pGL3–218, pGL3–209, pGL3–198, pGL3–191, pGL3–159, and pGL3–92, were similarly made by using differential forward primers on the IAP promoter with the KpnI site at the 5′-end and a common reverse primer (GLprimer2) on pGL3-Basic in the backbone of pGL3–272 for PCR to obtain the shorter fragments, which included differentially truncated IAP promoter regions and a common vector sequence containing the HindIII site. These fragments were then digested by KpnI and HindIII and inserted into pGL3-Basic. Promoter constructs containing nucleotide substitutions in the sequence motifs of Sp1 and α-Pal/NRF-1 were individually generated by PCR amplification with primer pairs spanning the mutant nucleotides according to the protocol of site-directed mutagenesis by overlap extension (18Aiyar A. Xiang Y. Leis J. Methods Mol. Biol. 1996; 57: 177-191PubMed Google Scholar). The plasmids pGL3–232m1, pGL3–232m2, and pGL3–232m3 were constructed in the backbone of pGL3–232 using primer pairs containing the introduced mutations. The Sp1 site GGGGCGGGGC was mutated into GTTGCTTGGC in the plasmid of pGL3–232m1. The α-Pal/NRF-1 element TGCGCGTGCGCG was mutated into TTTGCGTGCGCG, and TGCGCGTTTGCG in the plasmid of pGL3–232m2 and pGL3–232m3, respectively. Transfection and Dual-luciferase Assay—IMR-32 and HepG2 cells (1.5 × 105) were plated in each well of six-well plates. Transient transfection was carried out by the calcium phosphate precipitation method (19Jordan M. Schallhorn A. Wurm F.M. Nucleic Acids Res. 1996; 24: 596-601Crossref PubMed Scopus (732) Google Scholar). The plasmid pRL-TK was cotransfected to normalize the transfection efficiency. After 12 h of transfection, the medium was changed, and the cells were incubated at 37 °C for 24 or 48 h. The cells were washed in phosphate-buffered saline (137 mm sodium chloride, 2.7 mm potassium chloride, 10 mm dibasic sodium phosphate, and 2 mm monobasic potassium phosphate) and the lysates were prepared by scraping the cells from plates in the presence of 1× passive lysis buffer (Promega). Luciferase assays were performed by using Dual-Luciferase Assay System (Promega) and a Sirius luminometer (Berthold Detection System, Pforzheim, Germany). Preparation of Nuclear Extracts—IMR-32 and HepG2 cells were plated onto 6- or 10-cm cultured dishes and incubated for 2 days. The cells were washed with 2 ml of phosphate-buffered saline and collected in 1 ml of phosphate-buffered saline. The cells were centrifuged at 2,000 × g for 2 min, and the supernatant was discarded. The cell pellet was incubated in 400 μl of buffer A (10 mm HEPES (pH 7.9), 1.5 mm magnesium chloride, 10 mm potassium chloride, 0.5 mm phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol, 2 μg/ml leupeptin, 10 μg/ml aprotinin, 50 mm sodium fluoride, and 1 mm sodium orthovanadate) on ice for 10 min and then gently shaken for 10 s. The pellet of the crude nuclei was collected by centrifugation at 12,000 × g for 10 s. The pellet was resuspended in 100 μl of buffer C (20 mm HEPES (pH 7.9), 25% glycerol, 420 mm sodium chloride, 1.5 mm magnesium chloride, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol, 2 μg/ml leupeptin, 10 μg/ml aprotinin, 50 mm sodium fluoride, and 1 mm sodium orthovanadate) by vortex for 15 s, and then incubated on ice for 20 min. After centrifugation at 12,000 × g for 2 min, the supernatant containing the nuclear proteins was collected, quantified with BCA Protein Assay Reagent (Pierce), and stored at –70 °C in aliquots. Gel Electrophoretic Mobility Shift Assays (EMSA)—The EMSA used the following oligonucleotides: IAP α-Pal/NRF-1 (f), 5′-GAG TGC GCG TGC GCG GCT CT-3′; IAP α-Pal/NRF-1 (r), 3′-TCA CGC GCA CGC GCC GAG AG-5′; IAP α-Pal/NRF-1 mutation (f), 5′-GAG Ttt GCG Ttt GCG GCT CT-3′; IAP α-Pal/NRF-1 mutation (r), 3′-TCA aaC GCA aaC GCC GAG AG-5′; consensus α-Pal/NRF-1 (f), 5′-TTC TTT TGC GCA CGC GCT T-3; consensus α-Pal/NRF-1 (r), 3′-AAG AAA ACG CGT GCG CGA AGA ATC-5′; consensus α-Pal/NRF-1 mutation (f), 5′-TTC TTT TGt aaA CGa atT T-3′; consensus α-Pal/NRF-1 mutation (r), 3′-AAG AAA ACA ttT GCt tAA AGA ATC-5; consensus Sp1 (f), 5′-GTT GCG GGG CGG GGC CGA GTG-3′; consensus Sp1 (r), 3′-AAC GCC CCG CCC CGG CTC ACG-5′; consensus E2F-1 (f), 5′-TGC AAT TTC GCG CCA AAC TTG-3′; and consensus E2F-1 (r), 3′-GTT AAA GCG CGG TTT GAA C-5′. 30 pmol of each of the forward and reverse oligonucleotides placed in a volume of 23 μlof1× Klenow (DNA polymerase) buffer were heated at 94 °C for 2 min and annealed at room temperature for 30 min. The annealed double-stranded oligonucleotides were end-labeled by a fill-in reaction using DNA polymerase (Klenow) (Promega). One unit of the DNA polymerase (Klenow) and 40 μCi of [α-32P]dCTP (PerkinElmer Life Sciences) were added into the annealed oligonucleotides and the mixture was incubated at 30 °C for 15 min. The labeled oligonucleotides were purified by Sephadex G-50 columns (Amersham Biosciences). Cold double-stranded oligonucleotides were used as competitors. The DNA binding reaction was conducted at 4 °C for 30 min in a mixture containing 3 μg of nuclear extract, 10 mm Tris-Cl (pH 7.5), 50 mm sodium chloride, 0.5 mm dithiothreitol, 0.5 mm EDTA, 1 mm magnesium chloride, 4% glycerol, 0.05 μg poly(dI-dC)·poly(dI-dC) (Amersham Biosciences) and 2 × 104 cpm of 32P-labeled double-stranded oligonucleotides. In supershift assays, antibodies were incubated with the reaction mixture at 4 °C for 30 min before the addition of the IAP α-Pal/NRF-1 probes. The anti-NRF-1 goat polyclonal antiserum was kindly provided by Dr. Richard C. Scarpulla. The Sp1 and E2F antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-Myc antibody was from Invitrogen. The normal goat serum was from Vector Laboratories (Burlingame, CA). Samples were analyzed on a 4% polyacrylamide gel (acrylamide/bisacrylamide 29:1 in 0.5 × Tris borate-EDTA buffer) at 10 V/cm for 2.5 h. The gel was dried and analyzed by autoradiography. Construction and Overexpression of α-Pal/NRF-1—The cDNAs encoding the full-length and dominant-negative mutant of α-Pal/NRF-1 were constructed. The dominant-negative mutant consisted of the first N-terminal 304 residues of α-Pal/NRF-1, which contained the proposed DNA binding and nuclear localization domains and lacking the activation domain (20Virbasius C.A Virbasius J.V. Scarpulla R.C. Genes Dev. 1993; 7: 2431-2445Crossref PubMed Scopus (278) Google Scholar, 21Gómez-Cuadrado A. Martín M. Noël M. Ruiz-Carrillo A. Mol. Cell. Biol. 1995; 15: 6670-6685Crossref PubMed Google Scholar). Four α-Pal/NRF-1 cDNA fragments were obtained by RT-PCR. The primer pair AN-5 (5′-TTAAGCTT GCG CAG CCG CTC TGA GGA A) and AN-7 (5′-GACTCGAG CAC TGT TCC AAT GTC ACC AC) and primer pair AN-5 and AN-11 (5′-GACTCGAG TCA CTG TGA TGG TAC AAG ATG AGC) were used for the untagged full-length and dominant-negative α-Pal/NRF-1, respectively. Primer pair AN-5 and AN-6 (5′-GACTCGAG TCA CTG TTC CAA TGT CAC CA) and primer pair AN-5 and AN-10 (5′-GACTCGAG GTC TGT GAT GGT ACA AGA TGA G) were used for the Myc-tagged full-length and dominant-negative α-Pal/NRF-1, respectively. The underlined region indicates the restriction enzyme sites. These fragments were digested by HindIII and XhoI and inserted into pcDNA3.1 (B+) vector (Invitrogen). HepG2 cells (8 × 105) were placed onto 6-cm dishes for overexpression of α-Pal/NRF-1 using the same procedure for transient transfection of the reporter plasmids. After transient transfection, the cells were incubated for 48 h and harvested for nuclear protein extraction. Primary Cortical Culture—Primary cortical cells were prepared according to the protocol from Dichter (22Dichter M.A. Brain Res. 1978; 149: 279-293Crossref PubMed Scopus (406) Google Scholar). In brief, pregnant ICR mice, 15-days postconception, were anesthetized with pentobarbital, and embryos were removed. The cortices were removed and collected in MEM. The tissue was triturated three times with a fire-polished Pasteur pipette. Dissociated cells were plated onto 6-well plates coated with 0.09 mg/ml poly-l-lysine and grown in MEM supplemented with 10% fetal bovine serum (MEM 10), 50 units/ml penicillin, and streptomycin. After 24 h, plating media were replaced with MEM 10 and cells were treated with 3 μm of cytosine arabinoside. Twenty-four hours later, the medium was replaced with MEM 10 again, and cells were cultured for another 48 h for the transfection experiments. Statistics—The relative activity of different reporter constructs was compared. Statistical analysis was performed by unpaired Student's t test for pairwise comparisons. A p value <0.05 was regarded as significant. Expression of Integrin-associated Protein Gene in Human IMR-32 and HepG2 Cells—To use IMR-32 and HepG2 cells to study the IAP gene promoter, we examined firstly the expression of IAP transcripts in these cells by RT-PCR. We used primers that can detect alternative splicing forms of IAP mRNAs. As shown in Fig. 1A, both form 1 and form 2 IAP mRNAs were expressed in these two cell lines at a similar level. The major form of IAP mRNA in IMR-32 was form 4. But form 4 is not expressed in HepG2 cells at all. Total IAP transcripts were quantified. The level of total IAP transcripts expressed in IMR-32 cells was ∼3-fold of that expressed in HepG2 cells (Fig. 1B). These results confirmed the expression of the human IAP gene in these two cell lines. Determination of IAP Promoter Activity in IMR-32 and HepG2 Cells—To define the boundaries of a minimal IAP promoter region and identify cis elements that regulate the expression of IAP, we generated a series of 5′-IAP promoter deletion constructs and transfected them into IMR-32 and HepG2 cells. All plasmid constructs were defined relative to the translation initiation codon (Fig. 2A). The reporter constructs were cotransfected into IMR-32 and HepG2 cells with an internal control Renilla luciferase vector. The firefly luciferase activity of each reporter was normalized with the internal control to correct transfection efficiency. Results were represented as a fold-increase in activity with respect to that of the pGL3-Basic vector (Fig. 2, B and C) or the relative activity compared with that of the –232 construct (Fig. 4).Fig. 4Identification of functional cis elements in the IAP core promoter. Shorter promoter fragments were made by PCR (the –218, –209, and –198 constructs). Mutant constructs were made by site-directed mutagenesis (the –232m1, m2, and m3 constructs). The putative Sp1 and α-Pal/NRF-1 sites are indicated. Independent constructs and pRL-TK plasmids were cotransfected into IMR-32 cells using the same protocol as shown in Fig. 2. The promoter activity was expressed with respect to the –232 construct. *, p < 0.05; ***, p < 0.001; unpaired Student's t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In IMR-32 cells, the shortest reporter that still retained the basal promoter activity was the –232 construct, whereas deletion for another 41 bp (the –191 construct), 73 bp (the –159 construct), or 140 bp (the –92 construct) resulted in markedly loss of reporter activity. Addition of 40 bp to the –232 construct generated the –272 construct and stimulated the reporter activity by ∼25%. In the construct containing additional 184 bp (the –456 construct), the promoter activity was not increased. Interestingly, the reporter activity decreased to about the basal proximal promoter activity in the construct containing additional 274 bp (the –730 construct) and in the longest construct (the –1554 construct) (Fig. 2B). A similar pattern of promoter activity was observed when the reporter constructs were transfected into HepG2 cells, but with three exceptions (Fig. 2C). First, overall promoter strength relative to pGL3-Basic was much lower in HepG2 cells, about one-sixth to one-fourth compared with IMR-32 cells. Second, the constructs of –272 and –456 produced maximal activity in IMR-32 cells; in HepG2 cells, however, the construct of –730 produced maximal activity. Third, there were negative regulators located between –457 and –730 in IMR-32 cells; in HepG2 cells, however, there were negative regulators located between –730 and –1554. Results suggested a core promoter of the human IAP gene located between –232 and –12 upstream of the translation initiation codon in both IMR-32 and HepG2 cells. The sequence from –272 to ATG was searched for homology to previously described regulatory elements in several databases. There are many putative binding sites for transcription factor in this region (Fig. 3), including activating enhancer binding protein 2 (AP-2), Myc-associated zinc finger protein (Maz), cyclic-AMP responsive element-binding protein (CREB), transcription factor Sp1, E2 promoter binding factor (E2F), and α-Pal/nuclear respiratory factor 1 (α-Pal/NRF-1). Identification of cis-Elements in the Core Promoter of the IAP Gene—To determine more precisely the core promoter of the IAP gene, we generated several shorter or point mutation constructs. When 14 and 23 bp were deleted from the 5′-end of the –232 construct to generate the constructs of –218 and –209, respectively, the promoter activity of these two constructs was identical to that of the –232 construct in IMR-32 cells. This indicated that the sequence between –232 and –209 played no significant role in IAP gene expression under the current experimental conditions. When 34 bp were deleted from the 5′-end of the –232 construct to generate the –198 construct; however, the promoter activity was markedly reduced by 90% in IMR-32 cells, indicating that the sequence between –209 and –198 might be required for the IAP promoter activity (Fig. 4). As shown in Fig. 3, the region from –232 to –198 consists of a GC-rich sequence that includes the putative Sp1 and α-Pal/NRF-1 sites. Point mutations were introduced into these sites in IMR-32 cells to determine whether these sites were necessary for IAP promoter activity. When four bases of the Sp1 site were substituted with four T residues to generate the –232m1 construct, no significant effect on the IAP promoter activity was observed (Fig. 4), indicating that Sp1 site in this region was not required for the IAP promoter activity under this condition. The putative α-Pal/NRF-1 site in the IAP promoter is a 12-base tandem-repeat sequence, TGCGCGTGCGCG. When two bases in each of the repeat sequence were replaced by two T residues to generate the –232m2 and –232m3 constructs respectively, an 80% and a 90% drop in promoter activity was observed (Fig. 4). These results suggested that the consensus α-Pal/NRF-1 sequence, but not the consensus Sp1 sequence, is a functional regulatory element in the IAP promoter in IMR-32 cells. α-Pal/NRF-1 Is a Transcription Factor Regulating the IAP Promoter Activity—To demonstrate that the consensus α-Pal/NRF-1 sequence was functional, i.e. that there were endogenous nuclear proteins binding to this region, we performed the EMSA experiment. Nuclear extracts from IMR-32 cells were combined with 32P-fill-in-labeled double-stranded oligonucleotides in vitro. A major band of DNA-protein complex was found in all lanes when the nuclear extracts were incubated with the wild-type IAP α-Pal/NRF-1 probes (Fig. 5A, lane 3 and lanes 5–12), but not with the mutant IAP α-Pal/NRF-1 probes (Fig. 5A, lane 4). No band was found when nuclear extracts were not added into the probes (Fig. 5A, lanes 1 and 2). Competition analysis using a 10- or 60-fold molar excess of unlabeled probes was used to characterize the factor, which specifically binds to this sequence. As expected, the addition of a 10- or 60-fold molar excess of published wild-type consensus α-Pal/NRF-1 element reduced the intensity of these complexes (Fig. 5A, lanes 5 and 6), whereas the addition of the mutant consensus α-Pal/NRF-1 sequence did not (Fig. 5A, lanes 7 and 8). Results suggested that α-Pal/NRF-1 proteins might bind to the IAP α-Pal/NRF-1 element. However, the α-Pal/NRF-1 site is GC-rich and might therefore interact with factors other than α-Pal/NRF-1, such as Sp1 and E2F. We therefore used the unlabeled consensus Sp1 and E2F sequence for the competition experiment. The intensity of the migrating bands was not significantly reduced (Fig. 5A, lanes 9–12). Supershift assays using the anti-α-Pal/NRF-1 antiserum were used to further confirm the binding of the α-Pal/NRF-1 on its DNA element. The migrating bands were weakened when increasing amounts of α-Pal/NRF-1 antiserum were added and supershifted bands appeared (Fig. 5B, lanes 3–5). However, the Sp1 or E2F antibody did not generate any supershifted band (Fig. 5B, lanes 6 and 7), neither did the normal goat serum (Fig. 5B, lane 8). The EMSA experiments also revealed that the DNA binding activity of α-Pal/NRF-1 in IMR-32 cells (Fig. 5C, lanes 1–3) was much higher than that in HepG2 cells (Fig. 5C, lanes 4–6). The oligonucleotide probes and competitors used in the EMSA experiments were shown in Fig. 5D. These results strongly suggested that α-Pal/NRF-1 but not Sp1 or E2F binds to the IAP α-Pal/NRF-1 site. To further confirm that α-Pal/NRF-1 is the major transcription factor that binds to the IAP α-Pal/NRF-1 element, plasmids encoding the full-length or dominant-negative mutant of α-Pal/NRF-1 with or without a Myc tag were transiently transfected into HepG2 cells. The addition of a Myc tag in the C-terminal of α-Pal/NRF-1 is useful for the supershift assay in the EMSA experiments. In HepG2 cells, overexpression of the full-length α-Pal/NRF-1 (Fig. 6A, lanes 4–6) and Myc-tagged α-Pal/NRF-1 (Fig. 6B, lanes 3–5) enhanced the binding of DNA-protein complex in a dose-dependent manner as compared with the mock controls (Fig. 6A, lane 3 and 6B, lane 2). Overexpression of the dominant-negative mutant of α-Pal/NRF-1, which contains only the N-terminal DNA binding domain, did not affect endogenous DNA-protein binding in HepG2 cells but generated an additional band of DNA-protein complex with a smaller molecular weight. The DNA binding activity was strongly enhanced by the overexpression of the dominant-negative mutant (Fig. 6, A and B, lanes 7–9). This discrepancy may be related to the higher transfection efficiency of the plasmid that contained the dominant-negative mutant. In supershift assays, the band of DNA-protein complex containing the α-Pal/NRF-1-myc fusion protein was supershifted when monoclonal anti-Myc antibody was used (Fig. 6B, lane 6). The band of DNA-protein complex containing the dominant-negative mutant of α-Pal/NRF-1 fused with the Myc protein fragment was also completely supershifted by the monoclonal anti-Myc antibody (Fig. 6B, lane 10). These data strongly suggested that α-Pal/NRF-1 binds to this region (–204 to –193) of the human IAP gene promoter in vitro. If the α-Pal/NRF-1 protein binds to the α-Pal/NRF-1 element in the IAP promoter in vitro, it should functionally regulate the promoter activity of the IAP gene in vivo. To test this possibility, the full-length or dominant-negative α-Pal/" @default.
• W2065860483 created "2016-06-24" @default.
• W2065860483 creator A5049253275 @default.
• W2065860483 creator A5064285602 @default.
• W2065860483 date "2004-04-01" @default.
• W2065860483 modified "2023-10-16" @default.
• W2065860483 title "α-Pal/NRF-1 Regulates the Promoter of the Human Integrin-associated Protein/CD47 Gene" @default.
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