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• W2034300738 abstract "Manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, is necessary for survival of aerobic life. Previously, we demonstrated that a Sp1-based promoter is essential for constitutive transcription and a NF-κB-based intronic enhancer is responsible for cytokine-mediated induction. Here we show that nucleophosmin (NPM), a RNA-binding protein, binds to an 11G single-stranded loop in the promoter region and serves to integrate the Sp1 and NF-κB responses. Disruption of the loop structure causes a reduction of both constitutive and inductive transcription due to loss of the binding motif for NPM. Interaction of NF-κB·NPM·Sp1 facilitated by binding of NPM to the loop structure in the promoter region appears to comprise the basic complex for the transcriptional stimulation. These results suggest a novel molecular mechanism for communication between the enhancer and the GC-rich promoter. Manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, is necessary for survival of aerobic life. Previously, we demonstrated that a Sp1-based promoter is essential for constitutive transcription and a NF-κB-based intronic enhancer is responsible for cytokine-mediated induction. Here we show that nucleophosmin (NPM), a RNA-binding protein, binds to an 11G single-stranded loop in the promoter region and serves to integrate the Sp1 and NF-κB responses. Disruption of the loop structure causes a reduction of both constitutive and inductive transcription due to loss of the binding motif for NPM. Interaction of NF-κB·NPM·Sp1 facilitated by binding of NPM to the loop structure in the promoter region appears to comprise the basic complex for the transcriptional stimulation. These results suggest a novel molecular mechanism for communication between the enhancer and the GC-rich promoter. MnSOD 2The abbreviations used are: MnSOD, manganese superoxide dismutase; NPM, nucleophosmin; I2E, second intronic element; DMS, dimethyl sulfate; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; TPA, 12-O-tetradecanoylphorbol-13-acetate; ChIP, chromatin immunoprecipitation; RT, reverse transcription; siRNA, post-transcriptional small interfering RNA.2The abbreviations used are: MnSOD, manganese superoxide dismutase; NPM, nucleophosmin; I2E, second intronic element; DMS, dimethyl sulfate; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; TPA, 12-O-tetradecanoylphorbol-13-acetate; ChIP, chromatin immunoprecipitation; RT, reverse transcription; siRNA, post-transcriptional small interfering RNA. is an antioxidant enzyme located in mitochondria and encoded by the nuclear sod2 gene, whose primary function is removal of superoxide radicals generated by the mitochondrial electron transport chain (1.Weisigger R.A. Fridovich I. J. Biol. Chem. 1973; 248: 4793-4796Abstract Full Text PDF PubMed Google Scholar, 2.Fridovich I. Science. 1978; 201: 875-880Crossref PubMed Scopus (2746) Google Scholar). It is well established that MnSOD is essential for survival of aerobic life, because it mitigates reactive oxygen species-mediated cytotoxicity. MnSOD knock-out mice exhibit dilated cardiomyopathy, neuronal injury, and neonatal lethality (3.Lebovitz R.M. Zhang H. Vogel H. Cartwright Jr., J. Dionne L. Lu N. Huang S. Chan P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9782-9787Crossref PubMed Scopus (824) Google Scholar, 4.Li Y. Huang T.T. Carlson E.J. Melov S. Ursell P.C. Olson J.L. Noble L.J. Yoshimura M.P. Berger C. Chan P.H. Wallace D.C. Epstein C.J. Nat. Genet. 1995; 11: 376-381Crossref PubMed Scopus (1437) Google Scholar). Transfection of MnSOD cDNA into cultured cells significantly reduces cell death initiated by cytotoxic agents such as tumor necrosis factor (TNF) (5.Wong G.H. Elwell J. Oberley L.W. Goeddel D.V. Cell. 1989; 58: 923-931Abstract Full Text PDF PubMed Scopus (761) Google Scholar), iron (6.Keller J.N. Kindy M.S. Holtsberg F.W. St Clair D.K. Yen H.C. Germeyer A. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar), nitric oxide (7.Gonzalez-Zulueta M. Ensz L.M. Mukhine G. Lebovitz R.M. Azacka R.M. Engelhardt J.F. Oberley L.W. Dawson V.L. Dawson T.M. J. Neurosci. 1998; 18: 2040-2055Crossref PubMed Google Scholar), alkalosis (8.Majima H.J. Oberley T.D. Furukawa K. Mattson M.P. Yen H.-C. Szweda L.I. St Clair D.K. J. Biol. Chem. 1998; 273: 8217-8224Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), and hypoxia (9.Kiningham K.K. Oberley T.D. Lin S. Mattingly C.A. St Clair D.K. FASEB J. 1999; 13: 1601-1610Crossref PubMed Scopus (91) Google Scholar). Expression of the human sod2 gene in mice protects against oxygen-induced lung injury (10.Wispe J.R. Arner B.B. Clark J.C. Dey C.R. Neuman J. Glasser S.W. Crapo J.D. Chang L.-Y. Whitsett J.A. J. Biol. Chem. 1992; 267: 23937-23941Abstract Full Text PDF PubMed Google Scholar), Adriamycin-induced cardiac toxicity (11.Yen H.-C. Oberley T.D. Vichitbandha S. Ho Y.-S. St Clair D.K. J. Clin. Invest. 1996; 98: 1253-1260Crossref PubMed Scopus (395) Google Scholar), and ischemic brain injury (6.Keller J.N. Kindy M.S. Holtsberg F.W. St Clair D.K. Yen H.C. Germeyer A. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar). In addition to the role of MnSOD as an anti-oxidant, numerous studies suggest that MnSOD also acts as a tumor suppressor (12.Oberley L.W. Biomed. Pharmacother. 2005; 59: 143-148Crossref PubMed Scopus (201) Google Scholar). MnSOD activity is reduced in many types of transformed cells, and it appears that loss of MnSOD activity may be a general characteristic of many cancers (13.Oberley L.W. Buettner G.B. Cancer Res. 1979; 39: 1141-1149PubMed Google Scholar). We previously identified specific mutations located in the promoter region of the sod2 gene from cancer cells, which repress promoter activity apparently due to modification of transcription factor binding motifs (14.Xu Y. Krishnan A. Wan X.S. Majima H. Yeh C.-C. Ludewig G. Kasarskis E.J. St Clair D.K. Oncogene. 1999; 18: 93-102Crossref PubMed Scopus (107) Google Scholar).The sod2 gene is highly conserved among many species, including human, bovine, rat, and mouse (15.Marlhens F. Nicole A. Sinet P.M. Biochem. Biophys. Res. Commun. 1985; 129: 300-305Crossref PubMed Scopus (47) Google Scholar). The proximal promoter region of the sod2 gene is characterized by an absence of TATA or CAAT boxes but exhibits multiple CG motifs containing binding sites for transcription factors Sp1 and AP-2 (16.Wan X.S. Devalaraja M.N. St Clair D.K. DNA Cell Biol. 1994; 13: 1127-1136Crossref PubMed Scopus (191) Google Scholar). Transcriptional analysis demonstrates that Sp1 is a key transcriptional activator of the sod2 gene, whereas AP-2 down-regulates the transcription by interfering with Sp1 activation (17.Yeh C.-C. Wan X.S. St. Calir D.K. DNA Cell Biol. 1998; 17: 921-930Crossref PubMed Scopus (44) Google Scholar, 18.Xu Y. Porntadavity S. St Clair D.K. Biochem. J. 2002; 362: 401-412Crossref PubMed Scopus (110) Google Scholar). In the family of antioxidant proteins, MnSOD is a member whose expression is rapidly up-regulated in response to oxidative stress. A number of studies have demonstrated that numerous stimuli activate MnSOD expression in various cells and tissues (5.Wong G.H. Elwell J. Oberley L.W. Goeddel D.V. Cell. 1989; 58: 923-931Abstract Full Text PDF PubMed Scopus (761) Google Scholar, 19.Xu Y. Kiningham K.K. Devalaraja M.N. Yeh C.-C. Majima H. Kasarskis E.J. St Clair D.K. DNA Cell Boil. 1999; 18: 709-722Crossref PubMed Scopus (202) Google Scholar). We and others identified an intronic NF-κB enhancer element in both the human and mouse sod2 gene that is responsible for induction of MnSOD by TNF-α and interleukin-1β (IL-1β) (19.Xu Y. Kiningham K.K. Devalaraja M.N. Yeh C.-C. Majima H. Kasarskis E.J. St Clair D.K. DNA Cell Boil. 1999; 18: 709-722Crossref PubMed Scopus (202) Google Scholar, 20.Jones S.P. Ping D. Boss J.M. Mol. Cell. Biol. 1997; 17: 6970-6981Crossref PubMed Scopus (212) Google Scholar). We also demonstrated that treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) induces expression of the human sod2 gene by specifically augmenting Sp1 binding to the promoter region (21.Porntadavity S. Xu Y. Kiningham K.K. Rangnekar V.M. Prachayasitikul V. St Clair D.K. DNA Cell Biol. 2001; 20: 473-481Crossref PubMed Scopus (36) Google Scholar) and that combined treatment with TPA and cytokines (TNF-α and IL-1β) further synergistically increases expression (22.Kiningham K.K. Xu Y. Daosukho C. Popova B. St Clair D.K. Biochem. J. 2001; 353: 147-156Crossref PubMed Scopus (100) Google Scholar). Recently, we identified NPM, a RNA-binding protein, as a cofactor with NF-κBin cytokine-mediated MnSOD induction (23.Dhar S.K. Lynn B.C. Daosukho C. St Clair D.K. J. Biol. Chem. 2004; 279: 28209-28219Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar).The study of transcriptional regulation of the sod2 gene has demonstrated that a Sp1-activated proximal promoter is sufficient for constitutive transcription and that a NF-κB-activated enhancer is necessary for maximal stimulation. However, the mechanism of interaction between the enhancer and the GC-rich promoter to regulate MnSOD expression has not been elucidated. In the present study, we delineate the mechanism for molecular interaction between the enhancer and the promoter. The presence of a single-stranded 11G-loop structure in the human sod2 promoter region was suggested by the in vivo susceptibility of this region to dimethyl sulfate (DMS), and the result was confirmed by digesting genomic DNA with single-stranded DNA-specific S1 nuclease. Deletional and mutational analyses show that formation of the 11G-loop in the promoter region is necessary for basal promoter activity. Co-transfection with NPM results in transcriptional activation dependent on the integrity of the promoter secondary structure. electrophoretic mobility shift assay, DNase I footprinting, and chromatin immunoprecipitation (ChIP) assays indicate that NPM directly binds to the upper strand 11G-loop structure but not to the lower strand or to double-stranded DNA. Transcriptional stimulation by combined TPA and cytokine treatment demonstrates that both NPM and the 11G-loop are also required for high level induction. The essential role of NPM for transcriptional regulation was confirmed by RNA interference. Analysis of protein interaction using a two-hybrid system shows formation of an NF-κB·NPM·Sp1 complex that is maintained by the association of NPM with the 11G-loop structure. ChIP assay further confirms protein complex formation, presumably bridging the enhancer and promoter regions via NPM anchored to the DNA loop. These data demonstrate the importance of NPM bound to the DNA loop structure in recruitment and interaction of transcription factors near the transcription start site and increase our understanding of the role of enhancer-promoter cooperation in transcriptional regulation of genes lacking TATA promoters.MATERIALS AND METHODSCell Transfection and Treatments−VA13, an SV40-transformed human lung fibroblast WI38 cell line, was obtained from the American Type Culture Collection and grown in the recommended media. For cell transfection, the cells were plated in 12-well culture plates at a density of 5 × 105 cells/well. After culturing overnight, luciferase reporter constructs were co-transfected into cells with a β-galactosidase expression construct using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 36 h, the cells were treated with 100 units/ml recombinant human TNF-α (R&D Systems, Inc.), 2 ng/ml recombinant human IL-1β (Endogen), 100 mm TPA (Sigma), or a combination of the three reagents (hereafter referred to as “TIT”). After 12 h, the cells were washed once with phosphate-buffered saline and lysed in Passive Lysis Buffer (Promega, Madison, WI). Activity of the luciferase reporter was measured using a luciferase assay kit (Promega) with a TD-20/20 luminometer. β-Galactosidase activity was measured using chlorophenol red-α-d-galactopyranoside monosodium (Roche Applied Science) as a colorimetric substrate. Transcription activity was estimated by β-galactosidase-normalized luciferase responses.For stable transfection, luciferase reporter constructs were linearized by digestion with XmnI and co-transfected with pSV2-NEO for selection of individual clones using the culture media containing 400 μg/ml Geneticin (Invitrogen). The luciferase assay was used to select stable transfected cell clones, which were confirmed by Southern hybridization. The procedure for Southern hybridization has been previously described (24.St Clair D.K. Holland J.C. Cancer Res. 1991; 51: 939-943PubMed Google Scholar). Briefly, genomic DNA isolated from each individual cell clone was digested with PstI, separated on 0.8% agarose gels, transferred to Nylon transfer membranes, and hybridized with a luciferase-specific probe. For preparation of the probe, a 400-bp fragment of the luciferase coding region was amplified by PCR and labeled with [α-32P]dCTP using the Random primers DNA labeling system (Invitrogen). Sequences of the PCR primers were: forward, 5′-CTGAATACAAATCACAGAAT-3′; reverse, 5′-GCAGACCAGTAGATCCAGAG-3′. The probe was purified by a Nick column (Amersham Biosciences). Southern blots were scanned by a using a Typhoon 8600 scanner (Amersham Biosciences).DMS Reactivity and S1 Nuclease Digestion−Susceptibility of the 11G-loop region to DMS in vivo was measured in VA13 cells using a procedure of ligation-mediated PCR as previously described (25.Granger S.W. Fan H. J. Virol. 1998; 72: 8961-8970Crossref PubMed Google Scholar). The cells were treated with a series of DMS dilutions, and genomic DNA was extracted from the treated cells. Genomic DNA extracted from DMS-untreated cells was subjected to A + G and C + T chemical sequencing using a Maxam-Gilbert DNA sequencing kit (Sigma). 2 μg of DNA was cleaved with 10% piperidine at 90 °C for 30 min, and the sample was dried using a speed vacuum. Piperidine was removed by ethanol precipitation twice. The cleaved DNA was extended by pfu DNA polymerase (Stratagene) with a gene-specific primer1 (P1, 5′-ATCTGCTGAAGCCCGCTGCCGAAGC-3′) and tagged with a linker by T4 DNA ligase (New England Biolabs). The ligated DNA fragments were amplified by AccuPrimer GC-Rich DNA polymerase (Invitrogen) using a linker primer with a gene-specific primer2 (P2, 5′-GCCGAAGCCACCACAGCCACGAGT-3′). The PCR products were extracted by phenol/chloroform and then precipitated by ethanol. The purified PCR products were labeled by the AccuPrimer GC-Rich DNA polymerase with a gene-specific primer3 (P3, 5′-TCCTGCGCCGCCCGCGGGCCTTAAGAAA-3′), which was prelabeled with 32P. The samples were separated on 6% (w/v) polyacrylamide sequencing gels and scanned by Typhoon 8600. To confirm the results from the detection of the 11G-loop in vivo, genomic DNA extracted from DMS-untreated VA13 cells was treated with S1 nuclease, a single-stranded nucleotide-specific nuclease. To determine the position of S1 nuclease cleavage, the S1 nuclease-treated DNA sample was ligated using a rapid DNA ligation kit containing T4 DNA ligase (Roche Applied Science) and amplified by PCR with a gene-specific primer set. The PCR products were observed on 2% agarose gel and purified by treating with ExoSAP-IT (USP Corp.). DNA sequencing for the PCR products was performed by Davis Sequencing Service. Furthermore, DNA folding analysis was performed to predict formation of the loop structure using the mfold web server (version 3.2).Expression Constructs−Previously, we constructed sod2 promoter reporter (P7) and enhancer-promoter reporter (I2E-P7) plasmids, as well as a plasmid with the mutated NF-κB binding site in the I2E region (NF-κB/M) (19.Xu Y. Kiningham K.K. Devalaraja M.N. Yeh C.-C. Majima H. Kasarskis E.J. St Clair D.K. DNA Cell Boil. 1999; 18: 709-722Crossref PubMed Scopus (202) Google Scholar). In this study, site-directed mutagenesis was used to generate constructs in which the 11G-loop was interrupted using several different strategies illustrated in Fig. 2A. Oligonucleotides containing deletions and mutations were cloned to replace the wild-type sequences between SmaI (–131) and PvuII (–61) sites. Sequences of mutation- and deletion-targeting oligonucleotides are described by upper strands as below: loop/Del, 5′-GGGGTTGGGCGCGGCGGGCGCGGGGCGGGGCCCCGC———–CGGGGCGGCGGTGCCCCTTGCGGCGCAG-3′ (break line shows the 11G sequence that was deleted); loop/Str, 5′-GGGGTTGGGCGCGGCGGGCGCGGGGCGGGGaCaGaGGGGGGGGGGGCGGGGCGGCGGTGCCCTTGCGGCGCAG-3′ (underline, the 11G sequence; small letters, the replaced bases); loop/Cancer, 5′-GGGGTTGGGCGCGGCCGGGCGCGGGGCGGGGCtCGCGGGGGGaGGGGGCGGGGCGGCGGTGCCCTTGCGGCGCAG-3′ (small letters, the mutations identified in cancer cells); and loop/Atl, 5′-GGGGTTGGGCGCGGCGGGCGCGGGGCGGGGaCaGaGGGGGGGGGGtCtGtGCGGCGGTGCCCTTGCGGCGCAG-3′. The constructs were confirmed by DNA sequencing. Expression constructs for human NF-κB p50, NF-κB p65, Sp1, and NPM were included in the co-transfection experiments. The p50, p65, and Sp1 constructs were gifts from colleagues (26.Courey A.J. Holtzman D.A. Jackson S.P. Tjian R. Cell. 1989; 59: 827-836Abstract Full Text PDF PubMed Scopus (389) Google Scholar, 27.Bours V. Burd P.R. Brown K. Villalobos J. Park S. Ryseck R.P. Bravo R. Kelly K. Siebenlist U. Mol. Cell. Biol. 1992; 12: 685-695Crossref PubMed Google Scholar, 28.Ruben S.M. Dillon P.J. Schreck R. Henkel T. Chen C.H. Maher M. Baeuerle P.A. Rosen C.A. Science. 1991; 251: 1490-1493Crossref PubMed Scopus (289) Google Scholar), and NPM (29.Strausberg R.L. Feingold E.A. Grouse L.H. Derge J.G. Klausner R.D. Collins F.S. Wagner L. Shenmen C.M. Schuler G.D. Altschul S.F. Zeeberg B. Buetow K.H. Schaefer C.F. Bhat N.K. Hopkins R.F. Jordan H. Moore T. Max S.I. Wang J. Hsieh F. Diatchenko L. Marusina K. Farmer A.A. Rubin G.M. Hong L. Stapleton M. Soares M.B. Bonaldo M.F. Casavant T.L. Scheetz T.E. Brownstein M.J. Usdin T.B. Toshiyuki S. Carninci P. Prange C. Raha S.S. Loquellano N.A. Peters G.J. Abramson R.D. Mullahy S.J. Bosak S.A. McEwan P.J. McKernan K.J. Malek J.A. Gunaratne P.H. Richards S. Worley K.C. Hale S. Garcia A.M. Gay L.J. Hulyk S.W. Villalon D.K. Muzny D.M. Sodergren E.J. Lu X. Gibbs R.A. Fahey J. Helton E. Ketteman M. Madan A. Rodrigues S. Sanchez A. Whiting M. Madan A. Young A.C. Shevchenko Y. Bouffard G.G. Blakesley R.W. Touchman J.W. Green E.D. Dickson M.C. Rodriguez A.C. Grimwood J. Schmutz J. Myers R.M. Butterfield Y.S. Krzywinski M.I. Skalska U. Smailus D.E. Schnerch A. Schein J.E. Jones S.J. Marra M.A. Mammalian Gene Collection Program Team Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16899-16903Crossref PubMed Scopus (1548) Google Scholar) was subcloned into a pcDNA vector by Invitrogen.RT-PCR−To quantify MnSOD expression, mRNA was isolated from 5 × 106 untreated or treated cells using a MicroFastTrack 2.0 mRNA isolation kit (Invitrogen). The mRNA served as a template to synthesize single-stranded cDNA by reverse transcription with poly-T primer (Invitrogen). After template digestion with RNase H (Invitrogen), the cDNA was amplified by TaqDNA polymerase (Promega) using specific primers for the human sod2 gene. Sequences were: forward, 5′-AGCATGTTGAGCCGGGCAGT-3′; reverse, 5′-AGGTTGTTCACGTAGGCCGC-3′. To normalize MnSOD RT-PCR products, a set of primers was used to amplify the human β-actin gene. The primers were: forward, 5′-TGATGATATCGCCGCGCTCGTCGT-3′; reverse, 5′-CACAGCCTGGATAGCAACGTACAT-3′.Immunoblotting Analysis−To quantify protein levels, total cellular protein was isolated. 50 μg of protein extracts from the cultured cells were fractionated on SDS-PAGE, 8% (w/v) polyacrylamide gels, and transferred to nitrocellulose membranes. MnSOD was quantified using a primary polyclonal MnSOD antibody (Upstate Biotech.) with a goat anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). NPM and β-actin were measured using primary monoclonal antibodies against NPM (NeoMarkers) and β-actin (Sigma) with a goat anti-mouse IgGhorseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). Human β-actin was used to normalize MnSOD and NPM. Immunoblots were visualized by an enhanced chemiluminescence detection system (ECL, Amersham Biosciences).Nuclear Extraction and Electrophoretic Mobility Shift Assay−10 μg of NPM/pcDNA or the pcDNA vector control were transfected into 5 × 106 VA13 cells. Nuclear proteins were extracted from the transfected cells as described previously (19.Xu Y. Kiningham K.K. Devalaraja M.N. Yeh C.-C. Majima H. Kasarskis E.J. St Clair D.K. DNA Cell Boil. 1999; 18: 709-722Crossref PubMed Scopus (202) Google Scholar). Purified NPM protein was purchased from Abnova Corp. A fragment (–110 to –77) containing the wild-type (loop/WT) or mutant-type sequences (loop/Str) was labeled with [γ-32P]ATP by T4 DNA kinase (New England Biolabs). The probes were purified on 20% (w/v) polyacrylamide gels and quantified by scintillation counting (Beckman). 10 μg of the nuclear extracts and 0.2 μg of NPM protein were incubated with 0.5 pm probes for 30 min on ice before separation on 4% (w/v) polyacrylamide gels in 0.5× Tris borate-EDTA (TBE) electrophoresis buffer. Specific binding was confirmed by probe competition reactions with 100-fold concentration of cold oligonucleotides containing loop/WT, loop/Str, loop/Cancer, or non-self control. To confirm the specific binding, 1 μg of NPM antibody was incubated on ice with 0.2 μg of NPM protein for 1 h prior to incubation with the probes. After electrophoresis, gels were dried and scanned by using a Typhoon 8600.DNase I Footprinting Analysis−A ClaI-HindIII fragment containing the promoter sequence (–210 to +1) was obtained by digesting P7/pGL3 and purified using a gel-purification kit (Qiagen). The fragment was labeled with [γ-32P]ATP by T4 DNA kinase and digested with BglII to generate double-stranded DNA probes with top-strand labeling. The labeled double-stranded DNA was boiled for 5 min to generate single-stranded probe labeled at the 5′-end. The double-stranded and single-stranded probes (1 pm) were incubated with 1 μg of Sp1, AP-2, and NPM proteins on ice for 10 min within a Core DNase I Footprinting System (Promega) followed by partial digestion with DNase I as described previously (18.Xu Y. Porntadavity S. St Clair D.K. Biochem. J. 2002; 362: 401-412Crossref PubMed Scopus (110) Google Scholar). The footprinting samples were separated on 6% (w/v) polyacrylamide sequencing gels and scanned by using a Typhoon 8600.RNA Interference−Post-transcriptional small interfering RNA (siRNA) was used to test the effect of NPM on transcriptional regulation. NPM siRNA (0.1 μm, Santa Cruz Biotechnology) was co-transfected with I2E-P7 constructs in each well of 12-well plates containing a low density of cells (105/well) in serum-reduced Opti-MEM medium (Invitrogen) and followed by TIT treatment. NPM was measured by Western blots, and the effects of NPM siRNA on regulation of transcription were measured by reporter responses.ChIP−ChIP assay was performed to investigate protein-DNA and protein-protein interactions involved in transcriptional regulation using a ChIP-IT system (Active Motif) according to the manufacturer’s protocol. PCR was used to quantify the enhancer and promoter regions. The sequences of the primer set for the enhancer region were: forward, 5′-CGGGGTTATGAAATTTGTTGAGTA-3′; reverse, 5′-CCACAAGTAAAGGACTGAAATTAA-3′. For amplifying the GC-rich sod2 promoter region, AccuPrimer GC-Rich DNA polymerase was used with a pair of primers for amplifying the (–154 to –6) fragment: forward, 5′-ACAGGCACGCAGGGCACCCCCGGGGTT-3′; reverse, 5′-TCCTGCGCCGCCCGCGGGCCTTAAGAAA-3′. An exon2 fragment was amplified as an untargeted control: forward, 5′-TGACCGGGCTGTGCTTTCTCG-3′; reverse, 5′-ACTGCCTCCCGCCGCTCAGCC-3′. The GAPDH promoter region pulled down by TFIIB antibody was amplified as an internal control using the following primers: forward, 5′-TACTAGCGGTTTTACGGGCG-3′; reverse, 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′. Chromatin pulled down by IgG served as a negative antibody control. In addition to DNA analysis for the ChIP products, Western blots were performed to quantify the associated proteins in the same ChIP preparations. The eluted complexes were separated on SDS-PAGE gels and blotted by polyclonal primary antibodies for NF-κB p50 or p65 and a monoclonal primary antibody for Sp1 (Santa Cruz Biotechnology).Two-hybrid Analysis−A mammalian hybridization system (Promega) was modified to detect protein-protein interactions. With exception of Sp1/pBD that was previously constructed (18.Xu Y. Porntadavity S. St Clair D.K. Biochem. J. 2002; 362: 401-412Crossref PubMed Scopus (110) Google Scholar), NF-κB p50, p65, and NPM were cloned in either pBIND or pACT within XbaI-NotI sites to tag them with either the Gal4-binding fusion domain or the VP16-activation fusion domain, respectively. Primers with XbaI and NotI sites at the 3′- and 5′-ends were designed to perform PCR cloning for the proteins of interest using a high-fidelity pfu Turbo DNA polymerase (Stratagene). Primers were designed according to gene information from NBCI: p50 (S76638), p65 (M62399), and NPM (BC016768). The resulting constructs were confirmed by DNA sequencing. Hybridization experiment was performed to investigate direct interaction between two proteins as described previously (18.Xu Y. Porntadavity S. St Clair D.K. Biochem. J. 2002; 362: 401-412Crossref PubMed Scopus (110) Google Scholar). The two-hybrid system was modified to examine the function of the third protein (NPM). To determine whether the loop structure is necessary for enhancer-promoter communication, single-stranded oligonucleotides with or without the 11G-loop structure were added to the modified two-hybridization system to compete for NPM binding. A 36-bp non-self oligonucleotide containing sequences of multiple cloning sites in the pGL3 vector was included as a negative control in the competition experiment.Statistical Analysis−Multiple independent cell transfection and reporter assays were performed. PCR products and Western blots were quantified using imaging quantitative software Quantity One (Bio-Rad). Comparisons between the different constructs or treatments were analyzed using one-way ANOVA and Tukey’s Multiple Comparison Test followed by data analysis with GraphPad Prism version 4.0. Differences at p < 0.01 were considered significant.RESULTSAn 11G-Loop in the Promoter Region Is Essential for the Constitutive Transcription of the Human sod2 Gene−Our previous studies demonstrated that the mutations found in the promoter region of the human sod2 gene in cancer cells repress constitutive transcription. We hypothesized that the mechanism involved the disruption of a putative single-stranded loop structure (14.Xu Y. Krishnan A. Wan X.S. Majima H. Yeh C.-C. Ludewig G. Kasarskis E.J. St Clair D.K. Oncogene. 1999; 18: 93-102Crossref PubMed Scopus (107) Google Scholar). To evaluate the presence of the loop structure in the promoter region in vivo, genomic DNA extracted from DMS-treated VA13 cells was cleaved by piperidine and screened by ligation-mediated PCR. As shown in Fig. 1A, the 11G single-stranded loop, identified by positions shown from chemical sequencing of genomic DNA, was susceptible to low concentrations of DMS. Furthermore, cleavage of genomic DNA by a single-stranded DNA-specific nuclease supports the presence of a single-stranded loop structure in the sod2 promoter. The S1 nuclease-cleaved fragments were ligated by T4 DNA ligase and amplified by PCR (Fig. 1B). The position of S1 nuclease cleavage was determined by sequencing the PCR products, which showed that a small fragment (–120 to –98) was removed by S1 nuclease (Fig. 1C). In addition, folding analysis for single-stranded DNA using mfold software predicted the loop structure with melting temperature (Tm) at 63.1 °C in 10 mm NaCl as illustrated in Fig. 1D.FIGURE 1Determination of an 11G single-stranded loop structure in the human sod2 promoter region. A, genomic DNA extracted from DMS-treated VA13 cells was cleaved by piperidine. Ligation-mediated PCR was used to screen the loop structure as illustrated at the right. As markers, genomic DNA extracted from DMS-untreated cells was chemically sequenced (C+T and A+G reactions). Sequence of the promoter region is labeled according to the results of chemical sequencing. Susceptibility of the 11G-loop to DMS is indicated by open arrows. B, 1 μg of genomic DNA was digested with 1 unit of S1 nuclease and followed by ligation using T4 DNA ligase. PCR was used to amplify the re-ligated promoter region. The size of PCR product amplified from the untreated control is indicated by the arrow. C, the PCR products in B were sequenced. Nucleotides removed by S1 nuclease are indicated. D, a putative 11G single-stranded loop structure. The sequence numbers shown in C and D are related to the transcription-initiation site (+1).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To elucidate the role of the 11G-loop in transcriptional regulation, we eliminated the loop structure using three different strategies as shown in Fig. 2A: deleting the 11G sequence (loop/Del), eliminating the 11G-loop by straightening the loop out using A/T bases to replace G/C bases (loop/Str), and replacing the wild-type sequence with the cancer-type sequence initially identified in the colon cancer cell line, HT29 (loop/Cancer). In addition, an alternative loop structure was constructed by reconstituting the loop through alternative base paring by changing G/C bases to A/T bases (loop/Alt). The promoter fra" @default.
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