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• W2017907154 abstract "CD30, as a member of the tumor necrosis factor (TNF. receptor family, is expressed on the surface of activated lymphoid cells. CD30 overexpression is a characteristic of lymphoproliferative diseases such as Hodgkin's/non-Hodgkin's lymphomas, embryonal carcinoma, and a number of Th2-associated diseases. The CD30 gene has been mapped to a region of the murine genome that is involved in susceptibility to systemic lupus erythematosus. Functionally, CD30 may play a role in the deletion of autoreactive T cells. We were interested in determining the molecular nature of CD30 overexpression. Sequence comparison has revealed significant identity between the TATA-less human and murine CD30 promoters; they share a number of common consensus binding motifs. Transfection assays identified three regions of transcriptional importance; the region between position −1.2 kb and −336 bp, containing a CCAT microsatellite sequence, a conserved Sp1 site at positions −43 to −38, and a downstream promoter element (DPE) at positions +24 to +29. EMSA and DNase I footprinting showed specific DNA-protein interactions of the CD30 promoter with the Sp1 site and the CCAT repeat region. The DPE element was shown to be essential for start site selection. We conclude that the conserved Sp1 site at −43 to −38 is associated with maximum reporter gene activity, the DPE element is required for start site selection, and the CCAT tetranucleotide repeats act to repress transcription. We also have shown that the microsatellite is multiallelic, when we screened a random healthy population. Further studies are required to determine whether microsatellite instability in the repressor predisposes susceptible individuals to CD30 overexpression. CD30, as a member of the tumor necrosis factor (TNF. receptor family, is expressed on the surface of activated lymphoid cells. CD30 overexpression is a characteristic of lymphoproliferative diseases such as Hodgkin's/non-Hodgkin's lymphomas, embryonal carcinoma, and a number of Th2-associated diseases. The CD30 gene has been mapped to a region of the murine genome that is involved in susceptibility to systemic lupus erythematosus. Functionally, CD30 may play a role in the deletion of autoreactive T cells. We were interested in determining the molecular nature of CD30 overexpression. Sequence comparison has revealed significant identity between the TATA-less human and murine CD30 promoters; they share a number of common consensus binding motifs. Transfection assays identified three regions of transcriptional importance; the region between position −1.2 kb and −336 bp, containing a CCAT microsatellite sequence, a conserved Sp1 site at positions −43 to −38, and a downstream promoter element (DPE) at positions +24 to +29. EMSA and DNase I footprinting showed specific DNA-protein interactions of the CD30 promoter with the Sp1 site and the CCAT repeat region. The DPE element was shown to be essential for start site selection. We conclude that the conserved Sp1 site at −43 to −38 is associated with maximum reporter gene activity, the DPE element is required for start site selection, and the CCAT tetranucleotide repeats act to repress transcription. We also have shown that the microsatellite is multiallelic, when we screened a random healthy population. Further studies are required to determine whether microsatellite instability in the repressor predisposes susceptible individuals to CD30 overexpression. CD30 is a member of the tumor necrosis factor (TNF) receptor family that is expressed on the surface of activated lymphoid cells, particularly activated peripheral blood B and T cells, and a small population of cells in the perifollicular area of hyperplastic lymph nodes and tonsils.1Andreesen R Osterholz J Lohr GW Bross KJ A Hodgkin cell-specific antigen is expressed on a subset of auto- and alloactivated T (helper) lymphoblasts.Blood. 1984; 63: 1299-1302Crossref PubMed Google Scholar, 2Ellis TM Simms PE Slivnick DJ Jack HM Fisher RI CD30 is a signal-transducing molecule that defines a subset of human activated CD45RO+ T cells.J Immunol. 1993; 151: 2380-2389PubMed Google Scholar, 3Froese P Lemke H Gerdes J Havsteen B Schwarting R Hansen H Stein H Biochemical characterization and biosynthesis of the Ki-1 antigen in Hodgkin-derived and virus-transformed human B and T lymphoid cell lines.J Immunol. 1987; 139: 2081-2087PubMed Google Scholar, 4Hecht TT Longo DL Cossman J Bolen JB Hsu SM Israel M Fischer RI Production and characterization of a monoclonal antibody that binds Reed-Sternberg cells.J Immunol. 1985; 134: 4231-4236PubMed Google Scholar, 5Schwarting R Gerdes J Durkop H Falini B Pileri S Stein H BER-H2: a new anti-Ki-1 (CD30) monoclonal antibody directed at a formol-resistant epitope.Blood. 1989; 74: 1678-1689Crossref PubMed Google Scholar, 6Stein H Mason DY Gerdes J O'Connor N Wainscoat J Pallesen G Gatter K Falini B Delsol G Lemke H The expression of the Hodgkin's disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells.Blood. 1985; 66: 848-858Crossref PubMed Google Scholar The cellular distribution of CD30 suggests that, like other family members, CD30 is involved in the regulation of immune processes. The exact role CD30 plays is still under investigation. However, signaling through CD30 has been shown to produce both proliferative and apoptotic effects, depending on the cell type expressing the protein.7Bowen MA Olsen KJ Cheng L Avila D Podack ER Functional effects of CD30 on a large granular lymphoma cell line, YT. Inhibition of cytotoxicity, regulation of CD28 and IL-2R, and induction of homotypic aggregation.J Immunol. 1993; 151: 5896-5906PubMed Google Scholar, 8Bowen MA Lee RK Miragliotta G Nam SY Podack ER Structure and expression of murine CD30 and its role in cytokine production.J Immunol. 1996; 156: 442-449PubMed Google Scholar, 9Lee SY Kandala G Liou ML Liou HC Choi Y CD30/TNF receptor-associated factor interaction: NF-kappa B activation and binding specificity.Proc Natl Acad Sci USA. 1996; 93: 9699-9703Crossref PubMed Scopus (162) Google Scholar, 10Gruss HJ Boiani N Williams DE Armitage RJ Smith CA Goodwin RG Pleiotropic effects of the CD30 ligand on CD30-expressing cells and lymphoma cell lines.Blood. 1994; 83: 2045-2056Crossref PubMed Google Scholar, 11Smith CA Gruss HJ Davis T Andrson D Farrah T Baker E Sutherland GR Brannan CI Copeland NG Jenkins NA CD30 antigen, a marker for Hodgkin's lymphoma, is a receptor whose ligand defines an emerging family of cytokines with homology to TNF.Cell. 1993; 73: 1349-1360Abstract Full Text PDF PubMed Scopus (518) Google Scholar Cross-linking of CD30 molecules results in an influx of intracellular Ca2+ and induction of NFκB activity consistent with the positive effect of CD30 on proliferation.2Ellis TM Simms PE Slivnick DJ Jack HM Fisher RI CD30 is a signal-transducing molecule that defines a subset of human activated CD45RO+ T cells.J Immunol. 1993; 151: 2380-2389PubMed Google Scholar, 9Lee SY Kandala G Liou ML Liou HC Choi Y CD30/TNF receptor-associated factor interaction: NF-kappa B activation and binding specificity.Proc Natl Acad Sci USA. 1996; 93: 9699-9703Crossref PubMed Scopus (162) Google Scholar, 12Ansieau S Scheffrahn I Mosialos G Brand H Duyster J Kaye K Harada J Dougall B Hubinger G Kieff E Herrmann F Leutz A Gruss HJ Tumor necrosis factor receptor-associated factor (TRAF)-1, TRAF-2, and TRAF-3 interact in vivo with the CD30 cytoplasmic domain; TRAF-2 mediates CD30-induced nuclear factor kappa B activation.Proc Natl Acad Sci USA. 1996; 93: 14053-14058Crossref PubMed Scopus (72) Google Scholar, 13Duckett CS Gedrich RW Gilfillan MC Thompson CB Induction of nuclear factor kappaB by the CD30 receptor is mediated by TRAF1 and TRAF2.Mol Cell Biol. 1997; 17: 1535-1542Crossref PubMed Google Scholar, 14Tsitsikov EN Wright DA Geha RS CD30 induction of human immunodeficiency virus gene transcription is mediated by TRAF2.Proc Natl Acad Sci USA. 1997; 94: 1390-1395Crossref PubMed Scopus (29) Google Scholar CD30 has been implicated in the control of differentiation and/or selection events within the thymus. CD30-deficient mice have been found to contain elevated numbers of thymocytes and exhibit defects in negative selection, suggesting a role for CD30 in the deletion of autoreactive T cells.15Amakawa R Hakem A Kundig TM Matsuyama T Simard JJ Timms E Wakeham A Mittruecker HW Griesser H Takimoto H Schmitts R Shahinian A Ohashi P Penninger JM Mak TW Impaired negative selection of T cells in Hodgkin's disease antigen CD30-deficient mice.Cell. 1996; 84: 551-562Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar These findings have been confirmed in transgenic mice overexpressing CD30, in which overexpression enhances the deletion of CD4+/CD8+ thymocytes and so may act as a costimulatory molecule in thymic negative selection.16Chiarle R Podda A Prolla G Podack ER Thorbecke GJ Inghirami G CD30 overexpression enhances negative selection in the thymus and mediates programmed cell death via a Bcl-2-sensitive pathway.J Immunol. 1999; 163: 194-205PubMed Google Scholar CD30 was originally identified as a surface marker on Hodgkin's and Reed-Sternberg (H-RS)3Froese P Lemke H Gerdes J Havsteen B Schwarting R Hansen H Stein H Biochemical characterization and biosynthesis of the Ki-1 antigen in Hodgkin-derived and virus-transformed human B and T lymphoid cell lines.J Immunol. 1987; 139: 2081-2087PubMed Google Scholar cells, the neoplastic component of Hodgkin's disease.17Schwab U Stein H Gerdes J Lemke H Kirchner H Schaadt M Diehl V Production of a monoclonal antibody specific for Hodgkin and Sternberg-Reed cells of Hodgkin's disease and a subset of normal lymphoid cells.Nature. 1982; 299: 65-67Crossref PubMed Scopus (748) Google Scholar, 18Stein H Gerdes J Schwab U Lemke H Mason DY Ziegler A Schienle W Diehl V Identification of Hodgkin and Sternberg-Reed cells as a unique cell type derived from a newly-detected small-cell population.Int J Cancer. 1982; 30: 445-459Crossref PubMed Scopus (326) Google Scholar Diagnosis of Hodgkin's disease is based on the presence of a small population of these H-RS cells surrounded by a cellular infiltrate composed of normal immunoreactive lymphocytes, neutrophils, eosinophils, histiocytes, plasma, and stromal cells.10Gruss HJ Boiani N Williams DE Armitage RJ Smith CA Goodwin RG Pleiotropic effects of the CD30 ligand on CD30-expressing cells and lymphoma cell lines.Blood. 1994; 83: 2045-2056Crossref PubMed Google Scholar, 19de Bruin PC Gruss HJ van der Valk P Willemze R Meijer CJ CD30 expression in normal and neoplastic lymphoid tissue: biological aspects and clinical implications.Leukemia. 1995; 9: 1620-1627PubMed Google Scholar The exact cellular origin of these H-RS cells remains to be determined, although all of these cell types have been implicated.20Drexler HG Amlot PL Minowada J Hodgkin's disease-derived cell lines—conflicting clues for the origin of Hodgkin's disease?.Leukemia. 1987; 1: 629-637PubMed Google Scholar, 21Hsu SM Hsu PL The nature of Reed-Sternberg cells: phenotype, genotype, and other properties.Crit Rev Oncog. 1994; 5: 213-245Crossref PubMed Google Scholar Overexpression of CD30 is characteristic of Hodgkin's disease, where the interaction of CD30 with its ligand CD153 appears to be involved in the regulation of cell-cell interactions, particularly between the H-RS cells and the surrounding lymphoid cells.22Gruss HJ Pinto A Duyster J Poppema S Herrmann F Hodgkin's disease: a tumor with disturbed immunological pathways.Immunol Today. 1997; 18: 156-163Abstract Full Text PDF PubMed Scopus (127) Google Scholar Hence it has been suggested that loss of regulatory control may lead to the observed overexpression of CD30 and contribute to the progression of the disease state. In the later stages of Hodgkin's disease, the membrane form of CD30 is proteolytically cleaved, and high serum levels of the soluble form of CD30 can be detected, making it a good clinical marker for disease progression.23Hansen HP Kisseleva T Kobarg J Horn-Lohrens O Havsteen B Lemke H A zinc metalloproteinase is responsible for the release of CD30 on human tumor cell lines.Int J Cancer. 1995; 63: 750-756Crossref PubMed Scopus (61) Google Scholar, 24Nawrocki JF Kirsten ES Fisher RI Biochemical and structural properties of a Hodgkin's disease-related membrane protein.J Immunol. 1988; 141: 672-680PubMed Google Scholar The soluble form of CD30 may function by competing with surface CD30 for the binding of CD153 (CD30 ligand), thus preventing signaling through the membrane form of CD30, blocking normal intracellular signaling processes, as seen with TNF.25Seckinger P Isaaz S Dayer JM A human inhibitor of tumor necrosis factor alpha.J Exp Med. 1988; 167: 1511-1516Crossref PubMed Scopus (225) Google Scholar Interestingly, CD30 overexpression is also a characteristic of other lymphoproliferative disorders, such as non-Hodgkin's lymphoma, as well as embryonal carcinoma and a number of Th2-associated diseases.26Bengtsson A Holm L Back O Fransson J Scheynius A Elevated serum levels of soluble CD30 in patients with atopic dermatitis (AD).Clin Exp Immunol. 1997; 109: 533-537Crossref PubMed Scopus (65) Google Scholar, 27Biswas P Smith CA Goletti D Hardy EC Jackson RW Fauci AS Cross-linking of CD30 induces HIV expression in chronically infected T cells.Immunity. 1995; 2: 587-596Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 28Chilosi M Facchetti F Notarangelo LD Romagnani S Del Prete G Almerigogna F De Carli M Pizzolo G CD30 cell expression and abnormal soluble CD30 serum accumulation in Omenn's syndrome: evidence for a T helper 2-mediated condition.Eur J Immunol. 1996; 26: 329-334Crossref PubMed Scopus (111) Google Scholar, 29Del Prete G Maggi E Pizzolo G Romagnani S CD30, Th2 cytokines and HIV infection: a complex and fascinating link.Immunol Today. 1995; 16: 76-80Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 30Latza U Foss HD Durkop H Eitelbach F Dieckmann KP Loy V Unger M Pizzolo G Stein H CD30 antigen in embryonal carcinoma and embryogenesis and release of the soluble molecule.Am J Pathol. 1995; 146: 463-471PubMed Google Scholar, 31Nadali G Vinante F Stein H Todeschini G Teccho C Morosato L Chilosi M Menestrina F Kinney MC Greer JP Serum levels of the soluble form of CD30 molecule as a tumor marker in CD30+ anaplastic large-cell lymphoma.J Clin Oncol. 1995; 13: 1355-1360PubMed Google Scholar, 32Romagnani S Annunziato F Manetti R Almerigogna F Biagiotti R Giudizi MG Ravina A Gianno V Tomasevic L Maggi E Role for CD30 in HIV expression.Immunol Lett. 1996; 51: 83-88Crossref PubMed Scopus (15) Google Scholar Recently it has been shown that CD30 signaling limits the proliferative potential of autoreactive CD8 effector T cells and protects against autoimmune diabetes.33Kurts C Carbone FR Krummel MF Koch KM Miller JF Heath WR Signalling through CD30 protects against autoimmune diabetes mediated by CD8 T cells.Nature. 1999; 398: 341-344Crossref PubMed Scopus (104) Google Scholar It is possible that the observed alterations in the normal expression of CD30 may result in the progression of these CD30-positive diseases through autocrine or paracrine signaling mechanisms. The mechanisms involved in up-regulation of CD30 expression have yet to be investigated. As a first step toward identifying factors involved in regulating CD30 expression we have recently described the isolation and characterization of the 5′ region of the human and murine CD30 genes.34Croager EJ Muir TM Abraham LJ Analysis of the human and mouse promoter region of the non-Hodgkin's lymphoma-associated CD30 gene.J Interferon Cytokine Res. 1998; 18: 915-920PubMed Google Scholar Sequence comparison revealed significant identity between the mouse and human CD30 promoters. Both genes belong to an expanding group that lack consensus TATA and CAAT boxes, an increasingly common feature among members of the TNF receptor family.35Cheng J Liu C Koopman WJ Mountz JD Characterization of human Fas gene. Exon/intron organization and promoter region.J Immunol. 1995; 154: 1239-1245PubMed Google Scholar, 36Kemper O Wallach D Cloning and partial characterization of the promoter for the human p55 tumor necrosis factor (TNF) receptor.Gene. 1993; 134: 209-216Crossref PubMed Scopus (39) Google Scholar, 37Santee SM Owen-Schaub LB Human tumor necrosis factor receptor p75/80 (CD120b) gene structure and promoter characterization.J Biol Chem. 1996; 271: 21151-21159Crossref PubMed Scopus (140) Google Scholar, 38Yoo J Stone RT Kappes SM Toldo SS Fries R Beattie CW Genomic organization and chromosomal mapping of the bovine Fas/APO-1 gene.DNA Cell Biol. 1996; 15: 377-385Crossref PubMed Scopus (10) Google Scholar The human and murine CD30 promoters share a number of common consensus transcription factor binding motifs that may be involved in the regulation of gene expression. In particular, our previous study revealed the presence of conserved Sp1 and initiator elements,34Croager EJ Muir TM Abraham LJ Analysis of the human and mouse promoter region of the non-Hodgkin's lymphoma-associated CD30 gene.J Interferon Cytokine Res. 1998; 18: 915-920PubMed Google Scholar both of which have been implicated in positioning transcriptional machinery in genes that lack a TATA box.39Carcamo J Buckbinder L Reinberg D The initiator directs the assembly of a transcription factor IID-dependent transcription complex.Proc Natl Acad Sci USA. 1991; 88: 8052-8056Crossref PubMed Scopus (119) Google Scholar, 40Smale ST Schmidt MC Berk AJ Baltimore D Transcriptional activation by Sp1 as directed through TATA or initiator: specific requirement for mammalian transcription factor IID.Proc Natl Acad Sci USA. 1990; 87: 4509-4513Crossref PubMed Scopus (353) Google Scholar, 41Smale ST Baltimore D The “initiator” as a transcription control element.Cell. 1989; 57: 103-113Abstract Full Text PDF PubMed Scopus (1153) Google Scholar, 42Zenzie-Gregory B Khachi A Garraway IP Smale ST Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence.Mol Cell Biol. 1993; 13: 3841-3849Crossref PubMed Scopus (92) Google Scholar In this report we present the results of functional studies carried out to investigate these conserved regions in the human CD30 promoter. We have identified a number of functionally important regulatory regions, including an Sp1 element in the minimal promoter and a downstream promoter element (DPE) within the initiator, which is required for start site selection. In addition, the discovery of a polymorphic, upstream tetranucleotide repeat that binds proteins acting to repress transcriptional activity of the CD30 promoter, may implicate this region in the dysregulation of CD30 expression in neoplastic cells. Cultured cell lines were obtained from the American Type Culture Collection (Bethesda, MD). All cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mmol/L l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. Jurkat T cells were transfected by electroporation, using a BioRad (Hercules, CA) Gene Pulser at 240 V, 960 μF. Approximately 4 × 106 cells in a final volume of 400 μl were cotransfected with 10 μg of each test construct and 300 ng of control plasmid pRL-TK. Cells were cultured for 32 hours after transfection in 5 ml complete media before preparation of cellular extracts. Cells were harvested, washed twice in phosphate-buffered saline, and lysed in 300 μl of passive lysis buffer (Promega Corp., Madison, WI). Promoter activity of constructs and controls was measured using the Dual Luciferase Assay kit (Promega), following the manufacturer's instructions. Total RNA was isolated from Jurkat T cells transfected with either the −336 CD30/luciferase construct or the DPE downstream mutant, using the RNasol B method (Geneworks, Adelaide, Australia). A fragment of the CD30 promoter from −83 to +167 plus 153 bp (bp) of the luciferase gene derived from the WT CD30/luciferase construct was subcloned into pGEM-3ZF+ (Promega), linearized with an enzyme distal to the promoter, and used as a template for the T7 MAXI Script in Vitro Transcription Kit (Ambion, Austin, TX) to generate an antisense riboprobe. RNase protection assays were performed on 50 μg total RNA, using the Hybspeed RPA kit (Ambion). Protected fragments were analyzed on a 6. denaturing polyacrylamide/urea gel. Gels were dried and exposed to X-ray film. A 3.7-kb CD30 DNA fragment spanning the region from approximately −3500 to +199 relative to the major transcription start site was generated by polymerase chain reaction (PCR) from a plasmid containing the human CD30 promoter. This fragment was directionally cloned, via SacI and HindIII sites in the primers, into the polylinker of pGL3-Basic (Promega) to generate the wild-type CD30 reporter construct. The Quick-Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used to introduce KpnI or SacI restriction sites at different positions within the wild-type construct to generate a series of mutant constructs. Mutated plasmids were subsequently digested with SacI or KpnI and religated to generate deletion constructs. The microsatellite region of the CD30 promoter (∼400 bp) was isolated from the CD30 promoter by digestion with KpnI and cloned upstream from the SV40 promoter in the plasmid pGL3-Promoter (Promega) to generate a heterologous promoter construct. All DNA used in transfections was prepared using the Qiafilter plasmid maxi kit (Qiagen GmbH, Hilden, Germany), and all constructs were checked by sequencing. Nuclear extracts were prepared according to the method of Li et al.43Li YC Ross J Scheppler JA Franza Jr, BR An in vitro transcription analysis of early responses of the human immunodeficiency virus type 1 long terminal repeat to different transcriptional activators.Mol Cell Biol. 1991; 11: 1883-1893Crossref PubMed Scopus (76) Google Scholar For DNase I footprinting, a fragment of the CD30 gene spanning the region from −311 to +152 bp was generated by PCR and labeled with [γ-32P]ATP, using T4 DNA polymerase (Promega). Approximately 10,000 cpm of the labeled DNA fragment was incubated with 25 μg nuclear extract or 5 μg BSA and 1 μg poly(dI-dC) for 20 minutes on ice. Samples were treated with increasing units of DNase I (0.02–0.04 U with BSA and 2–10 U with nuclear extract) at room temperature for 2 minutes. Samples were analyzed using a 6% denaturing polyacrylamide/urea gel. For comparison a G/A ladder of the same end-labeled DNA fragment was generated by Maxam-Gilbert chemistry. Wild-type and mutant double-stranded oligonucleotides representing −57 to −22 of the CD30 promoter or the microsatellite region [(CCAT)5CACCTTATGCAT(CCAT)2' were synthesized for electrophoretic mobility shift assay (EMSA. with a single 5′ G overhang to facilitate end-labeling with [α-32P]dCTP. Nuclear proteins were preincubated on ice with 1 μg poly(dI-dC) in a binding buffer (4. Ficoll, 20 mmol/L HEPES (pH 7.9), 1 mmol/L EDTA, 1 mmol/L DTT, 50 mmol/L KCl, 0.8 μg ssDNA) to give a final reaction volume of 20 μl. For competition assays a 50-fold molar excess of specific or nonspecific competitor, as indicated, was incubated with nuclear extract for 10 minutes on ice before the addition of 32P-labeled oligonucleotide. Nuclear proteins were then incubated with 32P-labeled oligonucleotide (0.32 pmol) for 30 minutes on ice. For supershift assays anti-Sp1 or anti-Sp3 antibody was added after 30 minutes and incubated on ice for 1 hour. Products were analyzed by electrophoresis through a 6% polyacrylamide gel at 150 V. DNA was obtained by mouthwash from seven randomly chosen individuals essentially as described, but with the use of 10% Chelex (Biorad) instead of EDTA.44Lench N Stanier P Williamson R Simple non-invasive method to obtain DNA for gene analysis.Lancet. 1988; 1: 1356-1358Abstract PubMed Scopus (264) Google Scholar An aliquot (1/20th of the total) of DNA was used in a 50-ml PCR reaction with 25 pmol each of the microsatellite flanking primers 30MSF (5′-ACCCATTTACCCACTCACCTGC-3′. and 30MSR (5′-CAACTGGCCTAGGGAGACTGC-3′). The reactions contained 2.0 mmol/L MgCl2, 1× PCR buffer (Promega, Madison, WI), 2 mmol/L dNTPs, and 1 U Taq polymerase (Promega). Reactions were treated as follows: 94°C for 5 minutes; 29 cycles of 94°C for 30s, 60°C for 30s, 72°C for 1 minute; and 72°C for 10 minutes. PCR products were analyzed on 6% nondenaturing polyacrylamide gel electrophoresis (PAGE). To identify DNA elements that control the regulation of CD30 transcription, we subcloned the 5′ flanking region of the human CD30 gene from −3.5 kb to +199 bp into the luciferase expression vector pGL3-Basic. Restriction sites were introduced into the core recognition sequences contained in the full-length CD30/luciferase construct (−3.5 kb) by site-directed mutagenesis to generate mutant/deletion derivatives for transient transfection assays in Jurkat T cells. Potential transcription factor binding sites chosen for mutation included two Sp1 sites, two ETS sites, and an MZF site. The region of the CD30 promoter containing the Sp1 site (−43 to −38) has been previously shown to be highly conserved between human and murine CD30 promoter sequences. Preliminary transfection results also indicate that the region between −90 and −25 is important in driving transcription.34Croager EJ Muir TM Abraham LJ Analysis of the human and mouse promoter region of the non-Hodgkin's lymphoma-associated CD30 gene.J Interferon Cytokine Res. 1998; 18: 915-920PubMed Google Scholar Analysis of transient transfection results showed that the full-length −3.5-kb construct was able to direct low-level expression of the luciferase reporter in Jurkat T cells as illustrated in Figure 1. Deletion of DNA sequences between −3.5 kb to −1.2 kb had no effect on promoter activity. However, deletion of the sequence between −1.2 kb and −336 resulted in a 3.5-fold increase in promoter activity (Figure 1A). This result suggests that the region contained strong repressor activity. Further sequential deletion of sequences between −336 and −90 had little effect on promoter activity. Deletion of the region between −90 and −40 had a dramatic effect on promoter activity and resulted in a decrease in activity to the level of the full-length construct and confirmed the presence of strong positive regulatory elements in the immediate upstream region containing the Sp1 site. Results generated from transfection of mutant constructs in Jurkat T cells (Figure 1B) indicated that mutations within the upstream ETS sites (−238 to −233 and −158 to −153) had little effect on expression. However, mutation of the highly conserved Sp1 site at position −43 to −38 had a profound effect on transcription (Figure 1B) and completely abolished activity to the level seen with the luciferase vector alone (data not shown), suggesting that proteins binding within this region are essential for CD30 expression. This result is consistent with data generated from analysis of the 5′ CD30 deletion series, where deletion of this potential Sp1 site essentially abolishes high-level transcription. To define DNA-protein interactions in the region surrounding the conserved Sp1 site, we performed in vitro DNase I footprint assays, using a fragment from −311 to +152 of the CD30 promoter as a probe. Three major regions of protection were identified in the presence of Jurkat T-cell nuclear extract. These regions encompassed nucleotides −159 to −149, −142 to −128, and −44 to −28 (Figure 2A). The region spanning −44 to −28 contains the conserved Sp1 site, which appears to be essential for high-level expression of the −3.5-kb CD30/luciferase construct. The two upstream regions (−164 to −148 and −144 to −131) both contain sites mutated in the transfection experiments; however, the mutations did not appear to alter basal transcription levels (Figure 1B). A double-stranded oligonucleotide probe, representing the protected region from −44 to −28 of the CD30 promoter, was used in EMSA to analyze the interaction of this region with Jurkat T-cell nuclear proteins. Six major complexes (a–f) were formed between the probe and nuclear proteins (Figure 2B, lane 1). All of these complexes could be competed away by using a 50-fold molar excess of unlabeled probe (Figure 2B, lane 2). Complexes a and b could be completely competed away by using a 50-fold molar excess of consensus Sp1 GC and GT box sequences, suggesting that complexes a and b recognized Sp1-binding sequences (Figure 2B, lanes 3 and 4). An unrelated GC-rich oligonucleotide sequence (NI) was able to compete for complex d and f binding but was less efficient at competing away complexes a–c (Figure 2B, lane 5). To determine whether members of the Sp family of transcription factors were involved in formation of complexes a and b, the DNA protein complexes, formed during the binding reaction, were incubated in the presence of either anti-Sp1 or anti-Sp3 antibodies. Results indicated that complexes a and b were supershifted with the Sp1 antibody, suggesting that Sp1 formed part of these complexes (Figure 2B, lane 6). No shift was seen with the Sp3 antibody (lane 7), indicating that the complexes a and b were antigenically related to Sp1 specifically. To further characterize the nature of complexes c–e, mutated versions of the EMSA oligonucleotide were designed and used to identify the relative binding position of the observed complexes. Three mutated oligonucleotides were generated (M1, M2, M3) and used as probes or competitors in EMSAs with Jurkat T-cell extracts (Figure 3). As predicted, mutation of the consensus Sp1 binding site in M2 affected bind" @default.
• W2017907154 created "2016-06-24" @default.
• W2017907154 creator A5045382368 @default.
• W2017907154 creator A5047156111 @default.
• W2017907154 creator A5088340341 @default.
• W2017907154 date "2000-05-01" @default.
• W2017907154 modified "2023-09-27" @default.
• W2017907154 title "Involvement of Sp1 and Microsatellite Repressor Sequences in the Transcriptional Control of the Human CD30 Gene" @default.
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