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• W2036745364 abstract "The bovine leukemia virus (BLV) promoter is located in its 5′-long terminal repeat and is composed of the U3, R, and U5 regions. BLV transcription is regulated bycis-acting elements located in the U3 region, including three 21-bp enhancers required for transactivation of the BLV promoter by the virus-encoded transactivator TaxBLV. In addition to the U3 cis-acting elements, both the R and U5 regions contain stimulatory sequences. To date, no transcription factor-binding site has been identified in the R region. Here sequence analysis of this region revealed the presence of a potential E box motif (5′-CACGTG-3′). By competition and supershift gel shift assays, we demonstrated that the basic helix-loop-helix transcription factors USF1 and USF2 specifically interacted with this R region E box motif. Mutations abolishing upstream stimulatory factor (USF) binding caused a reproducible decrease in basal or Tax-activated BLV promoter-driven gene expression in transient transfection assays of B-lymphoid cell lines. Cotransfection experiments showed that the USF1 and USF2a transactivators were able to act through the BLV R region E box. Taken together, these results physically and functionally characterize a USF-binding site in the R region of BLV. This E box motif located downstream of the transcription start site constitutes a new positive regulatory element involved in the transcriptional activity of the BLV promoter and could play an important role in virus replication. The bovine leukemia virus (BLV) promoter is located in its 5′-long terminal repeat and is composed of the U3, R, and U5 regions. BLV transcription is regulated bycis-acting elements located in the U3 region, including three 21-bp enhancers required for transactivation of the BLV promoter by the virus-encoded transactivator TaxBLV. In addition to the U3 cis-acting elements, both the R and U5 regions contain stimulatory sequences. To date, no transcription factor-binding site has been identified in the R region. Here sequence analysis of this region revealed the presence of a potential E box motif (5′-CACGTG-3′). By competition and supershift gel shift assays, we demonstrated that the basic helix-loop-helix transcription factors USF1 and USF2 specifically interacted with this R region E box motif. Mutations abolishing upstream stimulatory factor (USF) binding caused a reproducible decrease in basal or Tax-activated BLV promoter-driven gene expression in transient transfection assays of B-lymphoid cell lines. Cotransfection experiments showed that the USF1 and USF2a transactivators were able to act through the BLV R region E box. Taken together, these results physically and functionally characterize a USF-binding site in the R region of BLV. This E box motif located downstream of the transcription start site constitutes a new positive regulatory element involved in the transcriptional activity of the BLV promoter and could play an important role in virus replication. bovine leukemia virus electrophoretic mobility shift assay cAMP-responsive element CRE-binding protein activating transcription factor-1 and -2 upstream stimulatory factor nuclear factor κB interferon regulatory factor nucleotide long terminal repeat human T-lymphotropic virus human immunodeficiency virus, type 1 Tax-responsive element helix-loop-helix basic HLH leucine zipper phorbol myristate acetate downstream activator sequence peripheral blood mononuclear cells Epstein-Barr virus calcium/calmodulin-dependent protein kinase IV thymidine kinase wild-type glyceraldehyde-3-phosphate dehydrogenase luciferase Bovine leukemia virus (BLV)1 is a B-lymphotropic oncogenic retrovirus that infects cattle and is associated with enzootic bovine leukosis, a neoplastic proliferation of B-cells (1Burny A. Cleuter Y. Kettmann R. Mammerickx M. Marbaix G. Portetelle D. Van den B.A. Willems L. Thomas R. Cancer Surv. 1987; 6: 139-159PubMed Google Scholar, 2Burny A. Bruck C. Chantrenne H. Cleuter Y. Dekegel D. Ghysdael J. Kettman R. Leclercq M. Leunen J. Mammerickx M. Portetelle D. Klein G. Viral Oncology. Raven Press, Ltd., New York1980: 231-289Google Scholar, 3Burny A. Willems L. Callebaut I. Adam E. Cludts I. Dequiedt F. Droogmans L. Grimonpont C. Kerkhofs P. Mammerickx M. Portetelle D. Van den Broeke A. Kettman R. Minson A.C. Neil J.C. McRae M.A. Bovine Leukemia Virus: Biology and Mode of Transformation. Cambridge University Press, Cambridge, UK1994: 213-234Google Scholar, 4Kettmann R. Burny A. Callebaut I. Droogmans L. Mammerickx M. Willems L. Portetelle D. Levy J.A. Bovine Leukemia Virus. Plenum Publishing Corp., New York1994: 39-81Google Scholar, 5Willems L. Burny A. Dangoisse O. Collete D. Dequiedt F. Gatot J.S. Kerkhofs P. Lefèbvre L. Merezak C. Portetelle D. Twizere J.C. Kettmann R. Curr. Top. Virol. 1999; 1: 139-167Google Scholar, 6Willems L. Burny A. Collete D. Dangoisse O. Dequiedt F. Gatot J.S. Kerkhofs P. Lefebvre L. Merezak C. Peremans T. Portetelle D. Twizere J.C. Kettmann R. AIDS Res. Hum. Retroviruses. 2000; 16: 1787-1795Crossref PubMed Scopus (63) Google Scholar). BLV is closely related to human T-lymphotropic viruses HTLV-I and -II. The majority of BLV-infected cattle are asymptomatic carriers of the virus. Only about 30% of BLV-infected animals develop a preneoplastic condition termed persistent lymphocytosis, with 2–5% developing B-cell leukemia and/or lymphoma after a long latency period. The virus can be experimentally transmitted to sheep, in which it causes similar pathologies, providing a helpful model to understand BLV and HTLV-induced leukemogenesis. BLV infection is characterized by viral latency in the large majority of infected cells and by the absence of viremia. These features are thought to be due to the transcriptional repression of viral expression in vivo (7Kettmann R. Cleuter Y. Mammerickx M. Meunier-Rotival M. Bernardi G. Burny A. Chantrenne H. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2577-2581Crossref PubMed Scopus (89) Google Scholar, 8Van den Broeke A. Cleuter Y. Chen G. Portetelle D. Mammerickx M. Zagury D. Fouchard M Coulombel L. Kettman R. Burny A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9263-9567Crossref PubMed Scopus (43) Google Scholar, 9Gupta P. Ferrer J.F. Science. 1982; 215: 405-407Crossref PubMed Google Scholar). The latency is likely to be a viral strategy to escape the host immune response and allow tumor development. BLV transcription initiates at the unique promoter located in the 5′-long terminal repeat (5′-LTR) of the BLV genome. The 5′-LTR is composed of the U3, R, and U5 regions and transcription initiates at the U3-R junction. The U3 region contains the main sites that regulate viral transcription (Fig. 1) as follows: the promoter CAAT and TATA boxes (10Derse D. Casey J.W. Science. 1986; 231: 1437-1440Crossref PubMed Google Scholar, 11Katoh I. Yoshinaka Y. Ikawa Y. EMBO J. 1989; 8: 497-503Crossref PubMed Scopus (57) Google Scholar), a glucocorticoid-responsive element (12Bloom J.C. Kenyon S.J. Gabuzda T.G. Blood. 1979; 53: 899-912Crossref PubMed Google Scholar, 13Bloom J.C. Ganjam V.K. Gabuzda T.G. Cancer Res. 1980; 40: 2240-2244PubMed Google Scholar, 14Niermann G.L. Buehring G.C. Virology. 1997; 239: 249-258Crossref PubMed Scopus (34) Google Scholar, 15Xiao J. Buehring G.C. J. Virol. 1998; 72: 5994-6003Crossref PubMed Google Scholar), and a large segment protected in DNase footprinting assays containing NF-κB-related sites (16Brooks P.A. Nyborg J.K. Cockerell G.L. J. Virol. 1995; 69: 6005-6009Crossref PubMed Google Scholar, 17Brooks P.A. Cockerell G.L. Nyborg J.K. Virology. 1998; 243: 94-98Crossref PubMed Scopus (19) Google Scholar). Among the most important sites are three copies of an imperfectly conserved 21-bp sequence harboring in the middle a common 8-bp core sequence known as the cAMP-responsive element (CRE). At least three proteins, CRE-binding protein (CREB) and activating transcription factors-1 and -2 (ATF-1 and ATF-2), bind to these 21-bp enhancers in bovine B-lymphocytes, and the amount of generated complex correlates with the level of viral expression (18Adam E. Kerkhofs P. Mammerickx M. Kettmann R. Burny A. Droogmans L. Willems L. J. Virol. 1994; 68: 5845-5853Crossref PubMed Google Scholar, 19Adam E. Kerkhofs P. Mammerickx M. Burny A. Kettman R. Willems L. J. Virol. 1996; 70: 1990-1999Crossref PubMed Google Scholar). The 21-bp enhancers are also called Tax-responsive elements (TxREs) because transactivation of the BLV LTR by the virus-encoded transcriptional activator TaxBLV requires these enhancers. Because there is no evidence for direct binding of TaxBLV to DNA (20Derse D. J. Virol. 1987; 61: 2462-2471Crossref PubMed Google Scholar), it has been proposed that TaxBLV activation of transcription could be mediated, as reported for the HTLV system, through increased binding of the cellular proteins CREB, ATF-1, and ATF-2 (and possibly other factors yet to be identified) to the TxREs. Each of the 21-bp enhancers also contains an E box-homologous motif overlapping the CRE (Fig. 1). From 5′ to 3′, these E boxes are referred to as E-box1, E-box2 and E-box3, respectively (5Willems L. Burny A. Dangoisse O. Collete D. Dequiedt F. Gatot J.S. Kerkhofs P. Lefèbvre L. Merezak C. Portetelle D. Twizere J.C. Kettmann R. Curr. Top. Virol. 1999; 1: 139-167Google Scholar, 21Unk I. Kiss-Toth E. Boros I. Nucleic Acids Res. 1994; 22: 4872-4875Crossref PubMed Google Scholar). In addition to these cis-acting elements all situated in U3, we and others (10Derse D. Casey J.W. Science. 1986; 231: 1437-1440Crossref PubMed Google Scholar, 22Kiermer V. Van Lint C. Briclet D. Vanhulle C. Kettmann R. Verdin E. Burny A. Droogmans L. J. Virol. 1998; 72: 5526-5534Crossref PubMed Google Scholar, 23Kiss-Toth E. Unk I. Biochem. Biophys. Res. Commun. 1994; 202: 1553-1561Crossref PubMed Scopus (13) Google Scholar) have previously demonstrated that both the R and U5 regions of the BLV LTR, located downstream from the transcription start site, contain additional regulatory sequences stimulating BLV gene expression. An interferon regulatory factor (IRF)-binding site in U5 interacts with IRF-1 and IRF-2 and stimulates viral gene expression in the absence of TaxBLV (22Kiermer V. Van Lint C. Briclet D. Vanhulle C. Kettmann R. Verdin E. Burny A. Droogmans L. J. Virol. 1998; 72: 5526-5534Crossref PubMed Google Scholar). The presence of a 64-bp downstream activator sequence (DAS) at the 3′ end of the R region has been reported (10Derse D. Casey J.W. Science. 1986; 231: 1437-1440Crossref PubMed Google Scholar, 23Kiss-Toth E. Unk I. Biochem. Biophys. Res. Commun. 1994; 202: 1553-1561Crossref PubMed Scopus (13) Google Scholar) (Fig. 1). To date however, no transcription factor-binding site has been identified in the R region. In this report, the sequence analysis of the BLV R region revealed the presence of a potential E box site (hereafter referred to as E-box4), 5′-CACGTG-3′ (nt +173 to +178), that conforms to the core hexanucleotide consensus sequence of an E box motif, CANNTG. E box sites bind proteins that belong to the basic helix-loop-helix (bHLH) family of transcription factors, including c-Myc, Max, USF, or TFE3 (reviewed in Refs. 24Littlewood T.D. Evan G.I. Protein Profile. 1995; 2: 621-702PubMed Google Scholar, 25Atchley W.R. Fitch W.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5172-5176Crossref PubMed Scopus (449) Google Scholar, 26Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Crossref PubMed Scopus (1266) Google Scholar). bHLH proteins contain a basic (b) DNA binding domain and a helix-loop-helix (HLH) dimerization domain; the latter allows these proteins to interact and to form homo- and/or heterodimers. In the case of bHLH-ZIP-related proteins, the bHLH domain is contiguous with a second dimerization domain, a leucine zipper (ZIP), characterized by heptad repeats of leucines that occur immediately C-terminal to the bHLH motif. Precise DNA-binding site selection by individual bHLH family members is determined both by the central dinucleotide contained in the core hexamer sequence and by the flanking nucleotides (27Beckmann H. Kadesch T. Genes Dev. 1991; 5: 1057-1066Crossref PubMed Google Scholar, 28Cai M. Davis R.W. Cell. 1990; 61: 437-446Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 29Carr C.S. Sharp P.A. Mol. Cell. Biol. 1990; 10: 4384-4388Crossref PubMed Scopus (177) Google Scholar, 30Carthew R.W. Chodosh L.A. Sharp P.A. Cell. 1985; 43: 439-448Abstract Full Text PDF PubMed Scopus (394) Google Scholar, 31Corneliussen B. Thornell A. Hallberg B. Grundstrom T. J. Virol. 1991; 65: 6084-6093Crossref PubMed Google Scholar, 32Davis R.L. Cheng P.F. Lassar A.B. Weintraub H. Cell. 1990; 60: 733-746Abstract Full Text PDF PubMed Scopus (516) Google Scholar, 33Gregor P.D. Sawadogo M. Roeder R.G. Genes Dev. 1990; 4: 1730-1740Crossref PubMed Google Scholar, 34Mellor J. Jiang W. Funk M. Rathjen J. Barnes C.A. Hinz T. Hegemann J.H. Philippsen P. EMBO J. 1990; 9: 4017-4026Crossref PubMed Scopus (147) Google Scholar, 35Murre C. McCaw P.S. Baltimore D. Cell. 1989; 56: 777-783Abstract Full Text PDF PubMed Scopus (1768) Google Scholar, 36Murre C. McCaw P.S. Vaessin H. Caudy M. Jan L.Y. Jan Y.N. Cabrera C.V. Buskin J.N. Hauschka S.D. Lassar A.B. Cell. 1989; 58: 537-544Abstract Full Text PDF PubMed Scopus (1256) Google Scholar). Some of these transcription factors can act as either transactivators or repressors of gene expression, depending on the gene promoter or on their dimerization partner (33Gregor P.D. Sawadogo M. Roeder R.G. Genes Dev. 1990; 4: 1730-1740Crossref PubMed Google Scholar,37Girardet C. Walker W.H. Habener J.F. Mol. Endocrinol. 1996; 10: 879-891PubMed Google Scholar, 38Howcroft T.K. Richardson J.C. Singer D.S. EMBO J. 1993; 12: 3163-3169Crossref PubMed Scopus (51) Google Scholar, 39Jones N. Cell. 1990; 61: 9-11Abstract Full Text PDF PubMed Scopus (209) Google Scholar, 40Kim J.B. Spotts G.D. Halvorsen Y.D. Shih H.M. Ellenberger T. Towle H.C. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 2582-2588Crossref PubMed Google Scholar). The objective of the present study was to characterize physically and functionally the E-box4 motif located in the BLV R region. We performed competition and supershift gel shift assays with nuclear extracts prepared either from peripheral blood mononuclear cells (PBMCs) derived from a BLV-infected sheep or nuclear extracts from the human B-lymphoid cell line Raji. We demonstrated that the bHLH transcription factors USF1 and USF2 specifically interacted with the E-box4. A 2-bp mutation (central CG to TA) and another 2-bp mutation (3′ TG to GA) abrogated USF binding. To assess the transcriptional regulatory function of the E-box4, we tested the effect of these 2-bp mutations by transient transfection of B-lymphoid cell lines in the context of an LTR-luciferase reporter construct in presence or absence of a TaxBLV expression vector. Both mutations caused a reproducible 25% decrease in LTR-driven basal gene expression, indicating a positive functional role of the E-box4 motif in R. Ectopically expressed USF1 and USF2a proteins had an E-box4-dependent stimulatory effect on both the homologous BLV promoter and a heterologous thymidine kinase (TK) promoter containing multiple upstream E-box4 motifs. Mutation in the E-box4 impaired the responsiveness of the BLV promoter to TaxBLVbut not to other activators known to up-regulate BLV expression (overexpression of CREB2, calcium/calmodulin-dependent protein kinase IV (CaMKIV), or CREB2/CaMKIV and treatment with PMA/ionomycin). Moreover, mutation in the E-box4 in combination with a mutation in the IRF site in U5 decreased the LTR basal activity more than 2-fold. The identification of the E-box4 motif represents the first transcription factor-binding site reported in the R region of BLV. The animal used in this study was a BLV-seropositive adult sheep (M298) affected with persistent lymphocytosis, presenting a persistently elevated lymphocyte count and an inverted B/T-lymphocyte ratio. This sheep was housed at the Veterinary and Agrochemical Research Center (Uccle, Belgium). Blood samples were collected by jugular venipuncture, mixed with EDTA as an anticoagulant, and centrifuged at 1750 × g for 25 min at room temperature. The PBMCs were then isolated by Percoll gradient centrifugation (density 1.129 g/ml; Amersham Biosciences) and washed twice in phosphate-buffered saline containing 0.075% EDTA, with centrifugation steps at 450 × g for 10 min at room temperature. The cells were washed with phosphate-buffered saline (centrifugations at 200 × g for 10 min at room temperature) until the supernatant became clear. The Raji and Daudi cell lines are human B-lymphoid EBV-positive cell lines derived from Burkitt's lymphomas. The human epithelial HeLa cell line is derived from a cervical carcinoma and is transformed by human papilloma virus type 18. All media, sera (Myoclone Superplus), and supplements were from Invitrogen. Raji cells were grown in RPMI 1640-Glutamax I medium supplemented with 10% fetal bovine serum, 50 units of penicillin/ml, and 50 μg of streptomycin/ml. Daudi cells were maintained in RPMI 1640-Glutamax I medium with 10% fetal bovine serum, 10 mmHEPES buffer, 1 mm sodium pyruvate, 50 units of penicillin/ml, and 50 μg of streptomycin/ml. HeLa cells were cultured in Dulbecco's modified Eagle's-Glutamax I medium containing 5% fetal bovine serum, 50 units of penicillin/ml, and 50 μg of streptomycin/ml. All cells were grown at 37 °C in an atmosphere of 5% CO2. The BLV LTR used in this study is the 344 provirus described by Sagata et al. (41Sagata N. Yasunaga T. Tsuzuku-Kawamura J. Ohishi K. Ogawa Y. Ikawa Y. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 677-681Crossref PubMed Scopus (354) Google Scholar). The first nucleotide of R and the last nucleotide of U3 are considered +1 and −1, respectively. To construct the luciferase reporter plasmid pLTRwt-luc, we used PCR to amplify the BLV promoter region from a 344 wild-type (wt) plasmid (a kind gift from Dr. Luc Willems).SmaI sites were introduced into the 5′ and 3′ PCR primers, and the SmaI-restricted PCR fragment was cloned in pGL3-BASIC (Promega) digested with SmaI. The 5′ primer oligonucleotide corresponding to nt −211 to −182 contained an addedSmaI site (in bold) at the 5′ end (5′-TCCCCCGGGTnt−211GTATGAAAGATCATGCCGGCCTAGGCGCC-3′). The 3′ primer oligonucleotide corresponding to nt +291 to +320 contained an added SmaI site (in bold) at the 5′end (5′-TCCCCCGGGTnt+320GTTTGCCGGTCTCTCCTGGCCGCTAGAGG-3′). Amplification reaction was conducted with 100 ng of template plasmid DNA as specified in the protocol provided with the high fidelity Pfu DNA polymerase (Stratagene, La Jolla, CA) with a DNA thermal cycler 480 (PerkinElmer Life Sciences). The resulting plasmid was designated pLTRwt-luc. This construct was used as a substrate for mutagenesis by the QuickChange Site-directed Mutagenesis method (Stratagene). Different mutations were generated with the following pairs of mutagenic oligonucleotide primers (mutations are highlighted in bold and E box or IRF motifs are underlined on the coding strand primer): CV192/193 (E-box4-mutA), 5′-CCTCTGACCGTCTCCATATGGACTCTCTCTC-3′; CV262/265 (E-box4-mutB), 5′-ACCGTCTCCACGGAGACTCTCT-3′; CV311/312 (IRFmut), 5′-GTTTCCTGTCTTACAGTCTGTGTCTCGCGGCCCGCG-3′; CV194/195 (Eboxes1,2-mutA), 5′-GACAGAGACGTCATATGCCAGAAAAGCTGGTGACGGCATATGGTGGCTAGAATCC-3′; CV281/282 (Eboxes1,2mutB), 5′-GACAGAGACGTCAGCGACCAGAAAAGCTGGTGACGGCAGCGAGTGGCTAGAATCC-3′; CV196/197 (E-box3-mutA), 5′-GAGCTGCTGACCTCATATGCTGATAAAACAATAA-3′; and CV283/284 (E-box3-mutB), 5′-GAGCTGCTGACCTCACCGACTGATAAAACAATAA-3. Mutated constructs were fully resequenced after identification by cycle sequencing using the Thermosequenase DNA sequencing kit (Amersham Biosciences). Three mutated plasmids were designated pLTR(E-box4-mutA)-luc, pLTR(E-box4-mutB)-luc, and pLTR(IRF-mut)-luc. Four constructs containing combinations of mutations were also generated by site-directed mutagenesis (Stratagene) using simultaneously two or three of the mutagenic oligonucleotide pairs described above. These plasmids were generated by combining CV194/195-CV196/197, CV281/282-CV283/284, CV192/193-CV194/195-CV196/197, and CV262/265-CV281/282-CV283/284 and were designated pLTR(E-box1,2,3-mutA)-luc, pLTR(E-box1,2,3-mutB)-luc, pLTR(E-box1,2,3,4-mutA)-luc, and pLTR(E-box1,2,3,4-mutB)-luc, respectively. In addition, pLTR(E-box4-mutA)-luc and pLTR(E-box4-mutB)-luc were used as substrates for site-directed mutagenesis using the mutagenic oligonucleotide primers CV311/312, thereby generating pLTR(E-box4-mutA/IRFmut)-luc and pLTR(E-box4-mutB/IRFmut)-luc, respectively. The pTK-luc reporter plasmid contains the herpes simplex virus thymidine kinase (TK) minimal promoter and was generated by subcloning the SalI (position 417)-XhoI (position 597) fragment from pBLCAT2 (42Luckow B. Schutz G. Nucleic Acids Res. 1987; 155490Crossref PubMed Scopus (1396) Google Scholar) into the XhoI-restricted pGL2-BASIC reporter plasmid (Promega). The p(E-box4)3TK-luc and p(E-box4)7TK-luc were generated by inserting three direct repeats in the forward orientation and seven direct repeats in the reverse orientation, respectively, of an oligonucleotide with the sequence 5′-ACCGTCTCCACGTGGACTCTCT-3′ (the BLV E-box4 motif is underlined) into SmaI-digested pTK-luc. Similarly, the two mutated versions, p(E-box4-mutA)3TK-luc and p(E-box4-mutB)3TK-luc, were generated by inserting three direct repeats in the forward orientation of an oligonucleotide with the sequence 5′-ACCGTCTCCATATGGACTCTCT-3′ and 5′-ACCGTCTCCACGGAGACTCTCT-3′ (mutations are highlighted in bold), respectively, intoSmaI-digested pTK-luc. In all these constructs, the multimerized wild-type or mutated E box motifs are thus positioned upstream of the TK promoter. To construct pGEM-LTRBLV used in RNase protection analysis, a 201-bp fragment containing the BLV 5′-LTR from position −118 to +83 was generated by PCR amplification of pLTRwt-luc. XbaI andPstI restriction sites were introduced into the 5′ and 3′ PCR primers, respectively, and theXbaI-PstI-restricted PCR fragment was cloned in pGEM-3Zf(+) vector (Promega) digested with XbaI andPstI. The 5′ primer oligonucleotide corresponding to nt −118 to −87 contained an added XbaI site (inbold) at the 5′ end (5′-CGCTCTAGAGnt-118GCTAGAATCCCCGTACCTCCCCAACTTCCCC-3′). The 3′ primer oligonucleotide corresponding to nt +55 to +83 contained an added PstI site (in bold) at the 5′ end (5′-GGTTTTCTGCAGCnt+83GGATAGCCGACCAGAAGGTCTCGGGAGC-3′). To construct the expression plasmid for ovine c-Myc, we used PCR to amplify the coding region of the ovine c-myc cDNA described in Kiermer et al. (43Kiermer V. Dequiedt F. Masengo R. Cleuter Y. Briclet D. Ciesiolka M. Van den B.A. Willems L. Kettmann R. Burny A. Droogmans L. DNA Seq. 1997; 7: 235-238Crossref PubMed Scopus (2) Google Scholar). HindIII andBamHI restriction sites were introduced into the 5′ and 3′ PCR primers, respectively, and theHindIII-BamHI-restricted PCR fragment was cloned in pcDNA3.1 (+/−) vector (Invitrogen) digested withHindIII and BamHI. The 5′ primer oligonucleotide corresponding to nt 203–226 (according to the ovine c-Myc cDNA sequence, GenBankTM accession number Z68501) contained an added HindIII site (in bold) at the 5′ end (5′-GCCCAAGCTTAnt203CCGCGATGCCCCTCAACGTCAGC-3′). The 3′ primer oligonucleotide corresponding to nt 1571–1598 contained an added BamHI site (in bold) at the 5′ end (5′-GCCGGATCCTnt1598CCTCACTTTCCCTTAGTAACAAATGAG-3′). The resulting plasmid was designated pcDNA3-cMyc. The eukaryotic expression vectors pSG-TAXBLV and pSG-CREB2 (44Willems L. Kettmann R. Chen G. Portetelle D. Burny A. Derse D. J. Virol. 1992; 66: 766-772Crossref PubMed Google Scholar) were gifts from Drs. Luc Willems and Richard Kettmann. The expression plasmid pCaMKIV was a gift from Dr. Anthony Means (45Sun Z. Means R.L. LeMagueresse B. Means A.R. J. Biol. Chem. 1995; 270: 29507-29514Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The USF1 and USF2a expression vectors (kindly provided by Drs. A. Kahn and M. Raymondjean) contained the human USF1 and USF2a cDNAs cloned in the pCR3 expression vector parent plasmid (Invitrogen) and were described previously (46Lefrancois-Martinez A.M. Martinez A. Antoine B. Raymondjean M. Kahn A. J. Biol. Chem. 1995; 270: 2640-2643Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Raji cells were transfected by using the DEAE-dextran procedure as described previously (47Van Lint C. Ghysdael J. Paras Jr., P. Burny A. Verdin E. J. Virol. 1994; 68: 2632-2648Crossref PubMed Google Scholar). Briefly, cells were harvested at density of 106/ml, washed with STBS (25 mm Tris-HCl (pH 7.5), 137 mm NaCl, 5 mm KCl, 700 μm CaCl2, 500 μmMgCl2, 600 μmNa2PO4), and resuspended at a concentration of 6 × 106 in 600 μl of a mixture containing 500 ng of the pGL3-BASIC-derived constructs (with or without cotransfected DNAs) and 450 μg of DEAE-dextran (Amersham Biosciences)/ml in STBS. Cells were incubated for 1 h at 37 °C, washed twice with STBS and once with culture medium, and grown in 3 ml of supplemented medium for 40–44 h. Cells were then lysed and assayed for luciferase activity (Promega). Luciferase activities derived from BLV LTRs were normalized with respect to protein concentrations using the Detergent-compatible Protein Assay (Bio-Rad). Daudi cells were transfected by electroporation. Cells were harvested in exponential growth phase and resuspended in supplemented medium at a concentration of 5 × 106 per 400 μl. The 400 μl of cells were mixed with 8 μg of pGL3-BASIC-derived constructs and 50 ng of pRL-TK (Promega) and incubated for 15 min at room temperature, transferred to electroporation vials, and electroporated at 250 V with a capacitance of 960 microfarads (by using a Bio-Rad gene pulser). Transfected cells were collected, plated out immediately in 4 ml of preheated medium (a 1:1 mixture of fresh culture medium and supernatant of a 24-h culture), and grown for 48 h at 37 °C. All transfection mixtures contained the pRL-TK, in which a cDNA encoding Renilla luciferase is under the control of the herpes simplex virus thymidine kinase promoter region and is used as an internal control for transfection efficiency. At 48 h post-transfection, luciferase activities (firefly andRenilla) were measured in cell lysates, and firefly luciferase activities derived from the BLV promoters were normalized with respect to the Renilla luciferase activity by using the dual luciferase reporter assay system (Promega). HeLa cells were transfected using FuGENETM-6 (Roche Molecular Biochemicals) according to the manufacturer's protocol. Briefly, cells were seeded at a density of 2 × 105cells/well in 6-well plates. FuGENETM-6 was added directly to serum-free medium 5 min prior to addition to the DNA. Hundred microliters of this FuGENETM-6/serum-free medium mix was added to the DNA mixture in microcentrifuge tubes. The mixture was incubated for 15 min at room temperature and, finally, was added to each well of a 6-well plate. Transfected cells were grown in 2 ml of supplemented medium for 40–44 h. Cells were then lysed and assayed for luciferase activity (Promega). Luciferase activities derived from BLV or TK promoters were normalized with respect to protein concentrations using the Detergent-Compatible Protein Assay (Bio-Rad). Nuclear extracts were prepared by a rapid method described by Osborn et al. (48Osborn L. Kunkel S. Nabel G.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Google Scholar). All buffers contained the protease inhibitors antipain (10 μg/ml), aprotinin (2 μg/ml), chymostatin (10 μg/ml), leupeptin (1 μg/ml), and pepstatin (1 μg/ml). Protein concentrations were determined by the method of Bradford (49Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (201652) Google Scholar). The DNA sequences of the coding strand of the double-stranded oligonucleotides used for this study are listed in Fig. 3 A or in the figure legends. EMSAs were performed as described previously (47Van Lint C. Ghysdael J. Paras Jr., P. Burny A. Verdin E. J. Virol. 1994; 68: 2632-2648Crossref PubMed Google Scholar). Briefly, nuclear extract (4 μg of protein) was first incubated at room temperature for 10 min in the absence of probe and specific competitor DNA in a 16-μl reaction mixture containing 10 μg of DNase-free bovine serum albumin (Amersham Biosciences), 6 μg of poly(dI-dC) (Amersham Biosciences) as nonspecific competitor DNA, 1 mmdithiothreitol, 20 mm Tris-HCl (pH 7.5), 60 mmKCl, 1 mm MgCl2, 0.1 mm EDTA, and 10% (v/v) glycerol. Fifteen thousand cpm of probe (10–40 fmol) was then added to the mixture with or without a molar excess of an unlabeled specific DNA competitor, and the mixture was incubated for 20 min at room temperature. Samples were subjected to electrophoresis at room temperature on 6% polyacrylamide gels at 150 V for 2–3 h in 1× TGE buffer (25 mm Tris acetate (pH 8.3), 190 mmglycine, 1 mm EDTA). Gels were dried and autoradiographed for 24–48 h at −70 °C. For supershift assays, polyclonal antibodies against USF1 (sc-229), USF2 (sc-862), Max (sc-197), Mad-1 (sc-222), Mad-2 (sc-1720), Mad-3 (sc-933), Mad-4 (sc-771), c-Myc (sc-764), and Mnt (sc-769) (Santa Cruz Biochemicals, Santa Cruz, CA) were added at a final concentration of 2 μg/reaction to the binding reaction mixture at the end of the binding reaction for an additional 30 min incubation at room temperature before electrophoresis. In vitro translated Ebox-binding proteins Max, Mad1, and c-Myc were used in control EMSAs. These proteins were generated by using the TNT-coupled Reticulocyte Lysate System (Promega) with the pGEM expression plasmid encoding mouse Max (p22 long form), the pRC/CMV expression vector for human Mad1 (both kindly provided by Dr. S. Segal), and the pcDNA3 expression vector for ovine c-Myc, pcDNA3-cMyc. Eight μl of in vi" @default.
• W2036745364 created "2016-06-24" @default.
• W2036745364 creator A5015075050 @default.
• W2036745364 creator A5026780492 @default.
• W2036745364 creator A5034397098 @default.
• W2036745364 creator A5035706656 @default.
• W2036745364 creator A5040770301 @default.
• W2036745364 creator A5060863599 @default.
• W2036745364 creator A5077662799 @default.
• W2036745364 date "2002-03-01" @default.
• W2036745364 modified "2023-10-18" @default.
• W2036745364 title "Upstream Stimulatory Factors Binding to an E Box Motif in the R Region of the Bovine Leukemia Virus Long Terminal Repeat Stimulates Viral Gene Expression" @default.
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