TY - JOUR TI - Finding near-optimal Bayesian experimental designs via Genetic algorithms AU - Hamada, M. AU - Martz, H.F. AU - Reese, C.S. AU - Wilson, A.G. T2 - American Statistician AB - This article shows how a genetic algorithm can be used to find near-optimal Bayesia nexperimental designs for regression models. The design criterion considered is the expected Shannon information gain of the posterior distribution obtained from performing a given experiment compared with the prior distribution. Genetic algorithms are described and then applied to experimental design. The methodology is then illustrated with a wide range of examples: linear and nonlinear regression, single and multiple factors, and normal and Bernoulli distributed experimental data. DA - 2001/// PY - 2001/// DO - 10.1198/000313001317098121 VL - 55 IS - 3 SP - 175-181 UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-0035620460&partnerID=MN8TOARS KW - expected information gain KW - logistic regression KW - linear and nonlinear regression KW - multifactor designs KW - Shannon information ER - TY - JOUR TI - Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter - Basal transcription is mediated by Sp1 and Sp3 AU - Baek, SJ AU - Horowitz, JM AU - Eling, TE T2 - JOURNAL OF BIOLOGICAL CHEMISTRY AB - Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is known to be associated with anti-tumorigenic activity and belongs to the transforming growth factor-β superfamily. In the present study, we cloned the promoter region (−3500 to +41) and investigated the transcriptional regulatory mechanisms of the basal expression of the human NAG-1 gene. Several potential transcription factor-binding sites in this region were identified. Based on the results from clones of nested deletions, the construct between −133 and +41 base pairs contains three Sp1-binding sites (Sp1-A, Sp1-B, and Sp1-C), which confer basal transcription specific activity of NAG-1 expression. When the Sp1-C site was mutated (GG to TT), a 60–80% decrease in promoter activity was observed in HCT-116 cells. Gel shift, co-transfection, and chromatin immunoprecipitation assays showed that the Sp transcription factors bind to the Sp1-binding sites and transactivate NAG-1 expression. In addition, chicken ovalbumin upstream promoter-transcription factor 1 can interact with the C-terminal region of Sp1 and Sp3 proteins and induce NAG-1 promoter activity through Sp1 and Sp3 transcription factors. These results identify the critical regulatory regions for the human NAG-1 basal promoter. Furthermore, the results suggest that the level of expression of the NAG-1 gene will depend on the availability of Sp proteins and on co-factors such as chicken ovalbumin upstream promoter-transcription factor 1. Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is known to be associated with anti-tumorigenic activity and belongs to the transforming growth factor-β superfamily. In the present study, we cloned the promoter region (−3500 to +41) and investigated the transcriptional regulatory mechanisms of the basal expression of the human NAG-1 gene. Several potential transcription factor-binding sites in this region were identified. Based on the results from clones of nested deletions, the construct between −133 and +41 base pairs contains three Sp1-binding sites (Sp1-A, Sp1-B, and Sp1-C), which confer basal transcription specific activity of NAG-1 expression. When the Sp1-C site was mutated (GG to TT), a 60–80% decrease in promoter activity was observed in HCT-116 cells. Gel shift, co-transfection, and chromatin immunoprecipitation assays showed that the Sp transcription factors bind to the Sp1-binding sites and transactivate NAG-1 expression. In addition, chicken ovalbumin upstream promoter-transcription factor 1 can interact with the C-terminal region of Sp1 and Sp3 proteins and induce NAG-1 promoter activity through Sp1 and Sp3 transcription factors. These results identify the critical regulatory regions for the human NAG-1 basal promoter. Furthermore, the results suggest that the level of expression of the NAG-1 gene will depend on the availability of Sp proteins and on co-factors such as chicken ovalbumin upstream promoter-transcription factor 1. transforming growth factor-β nonsteroidal antiinflammatory drug-activated gene-1 base pair polymerase chain reaction electrophoretic mobility shift assay chicken ovalbumin upstream promoter-transcription factor 1 growth and differentiation factor kilobase(s) glutathioneS-transferase The TGF-β1 superfamily genes play roles in adult and embryonic growth and development, in inflammation, and in repair including angiogenesis (1Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1730) Google Scholar). This superfamily includes bone morphogenetic proteins, cartilage-derived morphogenetic proteins, Mullerrian inhibiting substance, activins, inhibins, growth and differentiation factors (GDFs), and TGF-β (1Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1730) Google Scholar). Multiple lines of evidence suggest that the TGF-β signaling pathway is a potent tumor suppressor of human colorectal carcinogenesis (2Markowitz S. Roberts A. Cytokine Growth Factor Rev. 1996; 7: 93-102Crossref PubMed Scopus (396) Google Scholar). In addition, overexpression of TGF-β in arterial endothelium results in apoptosis (3Schulick A.H. Taylor A.J. Zuo W. Qiu C.B. Dong G. Woodward R.N. Agah R. Roberts A.B. Virmani R. Dichek D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6983-6988Crossref PubMed Scopus (158) Google Scholar) and alteration of the TGF-β signal transduction pathway results in a reduction of tumorigenecity (4Zhu Y. Richardson J.A. Parada L.F. Graff J.M. Cell. 1998; 94: 703-714Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 5Wang J. Sun L. Myeroff L. Wang X. Gentry L.E. Yang J. Liang J. Zborowska E. Markowitz S. Willson J.K. Brattain M.G. J. Biol. Chem. 1995; 270: 22044-22049Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). Thus, signaling and transcriptional regulation of TGF-β play an important role in tumor development. Several promoters for members of the TGF-β superfamily have been described. The TGF-β isoforms exhibit great diversity in their promoter structure (6Malipiero U. Holler M. Werner U. Fontana A. Biochem. Biophys. Res. Commun. 1990; 171: 1145-1151Crossref PubMed Scopus (30) Google Scholar), whereas the promoter organization of the bone morphogenetic proteins appears to be highly conserved (7Gitelman S.E. Kobrin M. Lee A. Fet V. Lyons K. Hogan B.L.M. Derynck R. Mamm. Genome. 1997; 8: 212-214Crossref PubMed Scopus (16) Google Scholar). NAG-1 was identified as a pro-apoptotic and anti-tumorigenic protein (8Baek S.J. Kim K.S. Nixon J.B. Wilson L.C. Eling T.E. Mol. Pharmacol. 2001; 59: 901-908Crossref PubMed Scopus (358) Google Scholar). The human cDNA has been cloned by six different groups (also known as MIC-1, PDF, GDF-15, PLAB, and PTGFB) and encodes a secreted protein with homology to members of the TGF-β superfamily (8Baek S.J. Kim K.S. Nixon J.B. Wilson L.C. Eling T.E. Mol. Pharmacol. 2001; 59: 901-908Crossref PubMed Scopus (358) Google Scholar, 9Bootcov M.R. Bauskin A.R. Valenzuela S.M. Moore A.G. Bansal M. He X.Y. Zhang H.P. Donnellan M. Mahler S. Pryor K. Walsh B.J. Nicholson R.C. Fairlie W.D. Por S.B. Robbins J.M. Breit S.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11514-11519Crossref PubMed Scopus (880) Google Scholar, 10Lawton L.N. Bonaldo M.F. Jelenc P.C. Qiu L. Baumes S.A. Marcelino R.A. Jesus G.M. Wellington S. Knowles J.A. Warburton D. Brown S. Bento-Soares M. Gene (Amst.). 1997; 203: 17-26Crossref PubMed Scopus (152) Google Scholar, 11Paralkar V.M. Vail A.L. Grasser W.A. Brown T.A. Xu H. Vukicevic S. Ke H.Z. Qi H. Owen T.A. Thompson D.D. J. Biol. Chem. 1998; 273: 13760-13767Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 12Yokoyama-Kobayashi M. Saeki M. Sekine S. Kato S. J. Biochem. (Tokyo). 1997; 122: 622-626Crossref PubMed Scopus (95) Google Scholar, 13Hromas R. Hufford M. Sutton J. Xu D. Li Y. Lu L. Biochim. Biophys. Acta. 1997; 1354: 40-44Crossref PubMed Scopus (195) Google Scholar). Moreover, NAG-1 expression is up-regulated in human colorectal cancer cells by several nonsteroidal anti-inflammatory drugs that are known to have anti-tumorigenic and pro-apoptotic activities (8Baek S.J. Kim K.S. Nixon J.B. Wilson L.C. Eling T.E. Mol. Pharmacol. 2001; 59: 901-908Crossref PubMed Scopus (358) Google Scholar). It is also induced by the tumor suppressor gene p53 (14Li P.X. Wong J. Ayed A. Ngo D. Brade A.M. Arrowsmith C. Austin R.C. Klamut H.J. J. Biol. Chem. 2000; 275: 20127-20135Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 15Tan M. Wang Y. Guan K. Sun Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 109-114Crossref PubMed Scopus (230) Google Scholar). Although abundant expression of NAG-1 is present in the placenta and prostate, significant expression is also observed in colon and kidney (11Paralkar V.M. Vail A.L. Grasser W.A. Brown T.A. Xu H. Vukicevic S. Ke H.Z. Qi H. Owen T.A. Thompson D.D. J. Biol. Chem. 1998; 273: 13760-13767Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). At present, no information on the tissue-specific and/or basal transcriptional regulation is available. A recent publication reporting the partial promoter sequences of the human NAG-1 (also known as PTGFB) gene provide little information into the regulatory mechanisms of NAG-1 expression (10Lawton L.N. Bonaldo M.F. Jelenc P.C. Qiu L. Baumes S.A. Marcelino R.A. Jesus G.M. Wellington S. Knowles J.A. Warburton D. Brown S. Bento-Soares M. Gene (Amst.). 1997; 203: 17-26Crossref PubMed Scopus (152) Google Scholar). Therefore, further characterization of the NAG-1 promoter is required to elucidate the mechanisms for regulation of anti-tumorigenic TGF-β family proteins. In the present study, we isolated and characterized the NAG-1 promoter region and identified several cis-acting elements. In the proximal region of the NAG-1 promoter, Sp1-binding sites regulate basal NAG-1 expression. In addition, we have shown a difference between Sp1 and Sp3, with regards to the binding of Sp1 sites as well as to transactivation of NAG-1 gene. We also show that Sp1 and/or Sp3 proteins interact with COUP-TF1 transcription factor to transactivate NAG-1 promoter activity. These data provide a link between Sp1 transcription factors and anti-tumorigenic protein expression. Recombinant bacteriophage clones were isolated by the plaque hybridization method (16Benton W.D. Davis R.W. Science. 1977; 196: 180-182Crossref PubMed Scopus (2924) Google Scholar) from human genomic chromosome-19 specific library (American Type Culture Collection, Manassas, VA). The library was screened using a DNA probe containing 966 bp of the NAG-1 promoter, which was labeled by random priming (Ambion, Austin, TX) in the presence of [α-32P]dCTP (PerkinElmer Life Sciences; 3,000 Ci/mmol). After three rounds of screening, large scale phage DNA was prepared according to the procedures of Helms (1987) using DEAE-cellulose chromatography. One of the positive clones (λNAG61) was purified and identified to contain the 9-kb NAG-1 promoter and the full-length NAG-1 gene. The insert of λNAG61 clone was digested with EcoRI restriction enzyme and subcloned into plasmid vector (pBlueScript II) for sequencing analysis. In addition, the 3.5-kb SmaI fragment containing the NAG-1 promoter was cloned into pGLBasic3 luciferase reporter vector (Promega, Madison, WI) and assayed for luciferase activity. A −3500 to +41-bp human NAG-1 promoter construct (pNAG3500/LUC) was generated as follows. λNAG61 clone was digested with SmaI restriction enzyme, and the 3.5-kb fragment was isolated and ligated into pGLBasic3 luciferase vector (Promega) digested with SmaI. The deletion clones were generated from pNAG3500/LUC using ExoIII nuclease. COUP-TF1 in expression vector (pRSCOUP-TF1) and in pGEM7 vector (pGEM7-COUP-TF1) were generously provided by Dr. Tsai (Baylor College of Medicine, Houston, TX). Sp1 (pCMV4-Sp1flu) and Sp3 (pCMV4-Sp3flu) in expression vectors were described previously (17Udvadia A.J. Templeton D.J. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3953-3957Crossref PubMed Scopus (198) Google Scholar, 18Udvadia A.J. Rogers K.T. Higgins P.D. Murata Y. Martin K.H. Humphrey P.A. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3265-3269Crossref PubMed Scopus (187) Google Scholar). HNF-4α in an expression vector (pCMV-HNF4α) was generated by reverse transcriptase-PCR from HCT-116 cells using two primers: sense, 5′-CGGAGACGGACAAAGTCCGGGGAC-3′, and antisense, 5′-GTCCCCGGACTTTGTCCGTCTCCG-3′. The Sp1-binding sites (Sp1-A, Sp1-B, and Sp1-C) were deleted or mutated using the Quick Change site-mutagenesis kit (Stratagene). For the deletion of Sp1-A or Sp1-BC sites on the −1086, −474, and −133 promoter regions, the following primers were used: ΔSP1-A sense, 5′-CCCCCTAAATACACCCCCAGACTGTGGTCATTGG-3′; ΔSP1-A antisense, 5′-AAACACTCCAATGACCACAGTCTGGGGGTGTATTTAG-3′; ΔSP1-BC sense, 5′-ACTCTGCAGGCAGGGGGAGGAAGACGGACAAAG-3′; and ΔSP1-BC antisense, 5′-CCCCGGACTTTGTCCGTCTTCCTCCCCCTGCC-3′. For the point mutation of Sp1-BC sites on the −133 promoter region, the following primers were used: mut1 sense, 5′-GGAGTT CGGGACTGAGCAGGCGGAGACGGA-3′; mut1 antisense, 5′-TCCGTCTCCGCCTGCTCAGTCCCGAACTCC-3′; mut2 sense, 5′-GGAGGGCGGGACTGAGCATT CGGAGACGGA-3′; mut2 antisense, 5′-TCCGTCTCCGAATGCTCAGTCCCGCCCTCC-3′; mut12 sense, 5′-GGAGTT CGGGACTGAGCATT CGGAGACGGA-3′; and mut12 antisense, 5′-TCCGTCTCCGAATGCTCAGTCCCGAACTCC-3′. Site-specific mutations (underlined) were confirmed by DNA sequencing. HCT-116 cells were plated in 6-well plates at 2 × 105cells/well in McCoy's 5A medium supplemented with 10% fetal bovine serum. After growth for 16 h, plasmid mixtures containing 1 μg of NAG-1 promoter linked to luciferase and 0.1 μg of pRL-TK (Promega) were transfected by LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's protocol. After 48 h transfection, the cells were harvested in 1× luciferase lysis buffer, and luciferase activity was determined and normalized to the pRL-TK luciferase activity using a dual luciferase assay kit (Promega). Nuclear extracts were prepared as described previously (19Kim Y. Fischer S.M. J. Biol. Chem. 1998; 273: 27686-27694Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) with the following modification. In detail, exponentially growing cells were washed with cold phosphate-buffered saline. The cells were pelleted in a microcentrifuge tube for 10 s and incubated in two packed cell volumes of buffer A (10 mmHEPES, pH 8.0, 0.5% Nonidet P-40, 1.5 mmMgCl2, 10 mm KCl, 0.5 mmdithiothreitol, and 200 mm sucrose) for 5 min at 4 °C with flicking of the tube. The crude nuclei were collected by microcentrifugation for 15 s, and the pellets were rinsed with buffer A. After centrifugation, the pellets were resuspended in one packed cell volume of buffer B (20 mm HEPES, pH 7.9, 1.5 mm MgCl2, 420 mm NaCl, 0.2 mm EDTA, and 1.0 mm dithiothreitol) and incubated on a rocking platform for 30 min at 4 °C. The crude nuclear extracts were clarified by microcentrifugation for 5 min, and the supernatants were diluted 1:1 with buffer C (20 mmHEPES, pH 7.9, 100 mm KCl, 0.2 mm EDTA, 20% glycerol, and 1 mm dithiothreitol). Protease inhibitor mixture (Sigma; catalog number P8340) was added to each type of buffer. Nuclear extracts were frozen in liquid nitrogen and kept at −80 °C until use. For the gel shift assay, double-stranded oligonucleotides (Life Technologies, Inc.) were end-labeled with [γ-32P]ATP by T4 polynucleotide kinase (New England Biolabs). Assays were performed by incubating 4 μg of nuclear extracts in the binding buffer (Promega) containing 200,000 cpm of labeled probe for 20 min at room temperature. To assure the specific binding of transcription factors to the probe, the probe was chased by 1-, 10-, and 50-fold molar excesses of cold wild type or mutant oligonucleotide. For the supershift experiments, antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were incubated with nuclear extracts on ice for 30 min before adding to the binding reaction. The samples were then electrophoresed on 5% nondenaturing polyacrylamide gels with 0.5× TBE, and the gels were dried and subjected to autoradiography. Exponentially growing HCT-116 cells in 150 mm plates were fixed by the addition of 1% formaldehyde to the medium for 10 min. The cells were scraped and collected by centrifugation. After washing with phosphate-buffered saline, the cell pellets were resuspended in 300 μl of lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris, pH 8.1) containing proteinase inhibitor (Sigma). The cells were then sonicated 10 times for 10 s each time, and the lysates were cleared by centrifugation and diluted in immunoprecipitation buffer (0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 140 mm NaCl) containing proteinase inhibitor. Chromatin solution was precleared for 1 h at 4 °C on protein A-Sepharose 4B beads, and 20 μg of sonicated salmon sperm DNA, 50 μg of bovine serum albumin, and 10 μg of Sp1 or Sp3 antibody (Santa Cruz, CA) were added for overnight. The beads were washed four times with 1 ml of washing buffer (0.1% Triton X-100, 20 mm Tris, pH 8.0, 150 mm NaCl, 2 mm EDTA) and eluted by three successive 5-min incubations with 150 μl of elution buffer (1% SDS, 100 mmNaHCO3). After combining the three eluted solutions, 1 μl of RNase (10 mg/ml) was added, and NaCl was adjusted to 0.3m. Cross-links were reversed by heating at 65 °C for 4 h, and DNA was extracted by adding 10 μl of 2 mTris, pH 6.8, 10 μl of 0.5 m EDTA, 2 μl of proteinase K (20 mg/ml) at 45 °C for 2 h. After phenol/chlorform extraction and ethanol precipitation, the pellet was resuspended in 50 μl of H2O. For the PCR reaction (25 cycles), 4 μl of DNA and 0.1 μg of each primer were added into the PCR mixture solution (Sigma). After electrophoresis, the gel was transferred onto Nylon membrane and subjected to Sourthern analysis using32P-labeled oligonucleotide (5′-GGAGGGCGGGACTGAGCAGGCGGA-3′) as a probe. The GST-Sp1 clones were described previously (20Murata Y. Kim H.G. Rogers K.T. Udvadia A.J. Horowitz J.M. J. Biol. Chem. 1994; 269: 20674-20681Abstract Full Text PDF PubMed Google Scholar). The full-length Sp3 was amplified from pBSK-Sp3/flu (21Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar) by use of PCR and 5′ primer (5′-GGGGGATCCGCCACCATGAATTCCGGGCCATCGCCG-3′) and 3′ primer (5′-GGAATTCCTCCATTGTCTCATTTCCAG-3′). Amplified Sp3 product was subcloned into pCR-Blunt-TOPO (Invitrogen, CA) and subsequently subcloned into pGEX-2TK (Amersham Pharmacia Biotech) at theBamHI and EcoRI sites to create pGEX-Sp3-ALL. The pGEX-Sp3-B/C/Zn/D clone was generated by subcloning directly from pCR-M2/flu (21Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar) into the BamHI site of pGEX-2TK. The pGEX-Sp3-A/B was generated by cleaving pBSKSp3/flu (21Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar) withBamHI and BpmI. A 1300-bp DNA fragment was then isolated, treated with mung bean nuclease to create blunt ends, and subcloned into the SmaI site of pGEX-2TK. COUP-TF1 coding sequence was transcribed with Sp6 polymerase, followed by translation with rabbit reticulocyte and [35S]methionine. Fusion proteins were synthesized inEscherichia coli BL21 (Stratagene). Expression was induced by isopropthiogalactopyranoside (0.1 mm), and whole cell extracts were prepared by sonication and centrifugation. GST or GST fusion proteins were incubated for 30 min at 4 °C with glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) in NETN buffer (20 mm Tris, pH 8.0, 100 mm NaCl, 1 mm EDTA, and 0.5% Nonidet P-40). Subsequently, the beads were washed four times with NETN buffer and purified. GST alone or GST fusion proteins were run on SDS-polyacrylamide gel electrophoresis to determine the concentrations of these proteins after purification. Equal amounts of GST or GST fusion proteins were incubated with35S-labeled COUP-TF1 that had been produced in a rabbitin vitro translation kit (Promega) in NETN buffer. Finally, the beads were washed five times with NETN buffer and analyzed on 12% SDS-polyacrylamide gel electrophoresis. The gel was dried and exposed to x-ray film. The human NAG-1 gene has been mapped to chromosome 19p12–13.1 using fluorescence in situhybridization (10Lawton L.N. Bonaldo M.F. Jelenc P.C. Qiu L. Baumes S.A. Marcelino R.A. Jesus G.M. Wellington S. Knowles J.A. Warburton D. Brown S. Bento-Soares M. Gene (Amst.). 1997; 203: 17-26Crossref PubMed Scopus (152) Google Scholar). To clone and investigate transcriptional regulation of the NAG-1 gene, human chromosome 19-specific library constructed in λ charon 40 (American Type Culture Collection number 57766) was screened with a radiolabeled 966-bp fragment corresponding to the 5′ end of the human NAG-1 gene (PTGFB). One million plaques were screened, and eight positive clones were isolated and subjected to plaque purification. Southern blot and sequencing analysis indicated that one clone, λNAG61, contained the full-length of NAG-1 including two exons and a 9-kb 5′-flanking region (Fig. 1). The 9-kb promoter region was subcloned into a plasmid vector, sequenced on both strands by standard sequencing methods, and deposited into GenBank (accession number AF305420). Alternatively, the BAC clone (BAC182K4) containing NAG-1 gene was purchased from Research Genetics and digested with several restriction enzymes, and the NAG-1 gene was identified by Southern blot analysis (data not shown). Putative cis-acting elements were examined in the human NAG-1 sequences within the 3.5-kb promoter using the TESS (www.cbil.upenn.edu/cgi-bin/tess/tess33?_if=1&RQ= WELCOME) and TFsearch (www.cbrc.jp/research/db/TFSEARCH.html) programs. Numerous potential cis-acting elements were identified including but not limited to binding sites for Sp1, AP-1 (activator protein), AP-2, GR (glucocorticoid receptor), NF-κB, H4TF-1 (H4 histone gene-inducing element), HiNF-A (cell cycle-regulated human H1 histone gene), c-Myc, and MIG-1 (GC-box binding zinc finger protein) (Fig. 2). At this point, it was unclear which of these putative elements played a role in transcriptional regulation of NAG-1. However, Sp1-binding sites in the proximal promoter region of NAG-1 were interesting, because many TGF-β family members are regulated by Sp1 transcription factors (22Feng Z.M. Bardin C.W. Chen C.L. Mol. Endocrinol. 1989; 3: 939-948Crossref PubMed Scopus (59) Google Scholar, 23Kim Y. Ratziu V. Choi S.G. Lalazar A. Theiss G. Dang Q. Kim S.J. Friedman S.L. J. Biol. Chem. 1998; 273: 33750-33758Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 24Lafyatis R. Lechleider R. Kim S.J. Jakowlew S. Roberts A.B. Sporn M.B. J. Biol. Chem. 1990; 265: 19128-19136Abstract Full Text PDF PubMed Google Scholar). During evolution, the important transcriptional binding sites are conserved between species. Therefore, the human and mouse NAG-1 promoters were compared with the conserved cis-acting elements. Mouse NAG-1 gene (also known as mouse GDF-15) has been reported (25Hsiao E.C. Koniaris L.G. Zimmers-Koniaris T. Sebald S.M. Huynh T.V. Lee S.J. Mol. Cell. Biol. 2000; 20: 3742-3751Crossref PubMed Scopus (225) Google Scholar). Sequence comparison between the human and mouse NAG-1 promoter in the ∼700-bp region revealed a 39% homology with considerable gapping (Fig. 3). Thus, significant homology is not apparent in the NAG-1 promoter between human and mouse, implying that human and mouse NAG-1 may be regulated in different ways. However, the major potential transcriptional binding sites such as TATA, Sp1, AP-1, and Nkx-2 in the human and mouse NAG-1 promoters are present in the same sequential order, indicating the significance of these sites on NAG-1 expression.Figure 3Alignment of the ∼700-bp region of human and mouse NAG-1 promoters by the GCG program. The top strand represents human NAG-1 promoter, whereas the bottom strand represents mouse NAG-1 promoter (also known as GDF-15; GenBankTM accession number AJ011967). Theunderlined sequences represent several specific sequences with high homology between the two promoters.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To evaluate the importance of cis-acting elements in conferring basal NAG-1 expression, the 3.5-kbSmaI fragment was released from λNAG61 (Fig. 1) and ligated to pGLBasic3 luciferase vector (pNAG3500/LUC). In addition, we identified and confirmed a transcription initiation site using primer extension and 5′-rapid amplification of cDNA end experiments (data not shown), which is consistent with a previous report showing putative transcription initiation site (10Lawton L.N. Bonaldo M.F. Jelenc P.C. Qiu L. Baumes S.A. Marcelino R.A. Jesus G.M. Wellington S. Knowles J.A. Warburton D. Brown S. Bento-Soares M. Gene (Amst.). 1997; 203: 17-26Crossref PubMed Scopus (152) Google Scholar). Deletion analysis of the 3.5-kb promoter sequence was performed using ExoIII nuclease digestion to generate the deletion clones pNAG1739/LUC, pNAG1086/LUC, pNAG474/LUC, and pNAG133/LUC. The properly deleted DNA fragment without introducing any mutations was confirmed by sequencing the junction sequences. Each construct was transfected into HCT-116 human colorectal cancer cells, which have been reported to express endogenous NAG-1 (8Baek S.J. Kim K.S. Nixon J.B. Wilson L.C. Eling T.E. Mol. Pharmacol. 2001; 59: 901-908Crossref PubMed Scopus (358) Google Scholar). As an internal control, the plasmid pRL-TK (Promega) was used for adjusting transfection efficiency. As shown in Fig.4, the results obtained from the luciferase studies demonstrated a large increase in luciferase activity between constructs pNAG474/LUC and pNAG1086/LUC. In contrast, there was a decrease in luciferase activity between pNAG1086/LUC and pNAG1739/LUC. A similar decrease was found between pNAG1739/LUC and pNAG3500/LUC. These data suggest that there is a positive regulator between −474 and −1086. As a negative control, pGLBasic3 promoterless vector was also transfected into HCT-116 cells and resulted in no significant luciferase activity. The construct pNAG133/LUC showed around 17-fold induction of luciferase activity compared with pGLBasic3, suggesting that this region may contain basal transcription machinery. The promoter region within −133 bp was further investigated to find any specific cis-acting elements conferring basal expression. From the sequencing analysis, we found three potential Sp1 binding sites (Sp1-A, Sp1-B, and Sp1-C) (Fig. 5), and one of them (Sp1-C) is conserved in the mouse promoter (Fig. 3). In addition, many TGF-β superfamily genes are regulated by Sp1 transcription factors, which are usually associated with basal promoter activity. Therefore, the proximal region containing Sp1 sites were further examined to elucidate basal promoter activity. First, three Sp1 sites (Sp1-A, Sp1-B, and Sp1-C) were deleted to see whether these sites were important for basal transcription of NAG-1. As shown in Fig.5 A, the transfection of constructs with deleted Sp1-A sites resulted in ∼50% reduction of luciferase activity compared with the appropriate wild type constructs, pNAG1086/LUC, pNAG474/LUC, and pNAG133/LUC, respectively. In contrast, the constructs with deleted Sp1-BC sites resulted in much greater reduction (∼60–80%) compared with wild type constructs. Interestingly, the pNAG1086 construct showed the highest luciferase activity, and the deletion of Sp1-BC sites resulted in significant reduction of luciferase activity, indicating that Sp1-BC sites are crucial sites to regulate the basal level of NAG-1 promoter activity. It also suggests that a positive regulator is located in the region between −474 and −1086, which may independently work to the Sp1-BC site. Further examination was performed using point mutation. Sp1-BC sites were point-mutated using a site-directed mutagenesis kit. In the Sp1-BC sites, GG was mutated to TT in either one site or both sites (Fig. 5 B). The point mutation in Sp1-B site resulted in only ∼30% reduction of luciferase activity, whereas the mutation of either the Sp1-C site or both sites showed ∼70% reduction of luciferase activity. This suggests that the Sp1-C site is more crucial than Sp1-B site in terms of basal NAG-1 expression (Fig. 5 B). The point mutation of the Sp1-C site resulted in almost the same reduction as the deletion mutation as shown in Fig.5 A. Because the deletion and point mutation of Sp1 sites (Sp1-B and Sp1-C) resulted in a reduction of luciferase activity, the Sp1 sites in this promoter should bind the Sp family of transcription factors. First, gel shift assays were performed to address whether the HCT-116 cells contained binding activities for these regions. Sp isoforms (Sp1, Sp2, and Sp3) are expressed in HCT-116 cells as measured by Western analysis (data not shown). The results using nuclear extracts of HCT-116 cells and a probe corresponding to the Sp1-BC element at positions −73 to −44 (Fig.6 A, top panel) show multiple DNA-protein complexes with a mobility (Fig. 6 A,bottom panel, arrows α, β, andγ), similar to the previous reports using Sp1 consensus oligonucleotides (21Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar, 26Suske G. Gene (Amst.). 1999; 238: 291-300Crossref PubMed Scopus (985) Google Scholar). These bands represent a specific protein binding to the Sp1 sequence elements, because complex formation was diminished by the addition of 10 or 50 molar excess of nonradiolabeled identical competitor, but not by addition of the identical oligonucleotide in which the Sp1-BC sites were point-mutated (Fig.6 A). Furthermore, to determine which base pairs are important for the binding of these complexes, we performed mutational analysis of the Sp1 sites. The mutations that we generated changed selected pairs of guanidine to pairs of thymidine residues (Fig.6 A, top panel). An oligonucleotide containing mut12 was unable to compete for the binding of any of the complexes at 50-fold molar excess (Fig. 6 A, compare lane 5with lane 8). Interestingly, incubation with mut1 oligonucleotide did not fully compete with labeled oligonucleotide (Fig. 6 A, lane 6), indicating that Sp1-B site has probably ∼50% activity with regard to DNA binding. In contrast, incubation with mut2 oligonucleotide competes in bands α and β but not band γ, indicating that at least one protein preferentially binds to the Sp1-C site. To confirm that Sp1 binds to these sites, we performed a gel shift assay in the presence of Sp family antibodies to demonstrate supershifting. We also examined COUP-TF1 and HNF-4α antibodies for binding to this site. The DA - 2001/9/7/ PY - 2001/9/7/ DO - 10.1074/jbc.M101814200 VL - 276 IS - 36 SP - 33384-33392 SN - 0021-9258 ER - TY - JOUR TI - Alcatel - North Carolina State University Virtual Laboratory for End-to-End Quality of Service Engineering AU - Labbe, T. AU - Mohammed, A. AU - Streck, J. P. AU - Vouk, M. A. T2 - Alcatel Telecommunications Review DA - 2001/// PY - 2001/// IS - 3 SP - 227-231 ER -