Regulation of Human B19 Parvovirus Promoter Expression by hGABP (E4TF1) Transcription Factor
1998; Elsevier BV; Volume: 273; Issue: 14 Linguagem: Inglês
10.1074/jbc.273.14.8287
ISSN1083-351X
AutoresIsabelle Vassias, Uriel Hazan, Yanne Michel, Chika Sawa, Hiroshi Handa, Laurent Gouya, Frédéric Morinet,
Tópico(s)Viral-associated cancers and disorders
ResumoThe genetic expression of human B19 parvovirus is only dependent on one promoter in vivo and in vitro. This is the P6 promoter, which is located on the left side of the genome and is a single-stranded DNA molecule. This led us to investigate the regulation of the P6 promoter and the possible resulting variability of the nucleotide sequence. After analysis of the promoter region of 17 B19 strains, only 1.5% variability was found. More exciting was the finding of mutations that were clustered around the TATA box and defined a highly conserved region (nucleotides 113–210) in the proximal part of the P6 promoter. HeLa and UT7/Epo cell extracts were found to protect this region, which contained a core motif for Ets family proteins, with YY1 and Sp1 binding sites on either side. Gel mobility shift assays performed with nuclear proteins from HeLa and UT7/Epo cells identified DNA-binding proteins specific for these sites. By supershift analysis, we demonstrated the binding of the hGABP (also named E4TF1) protein to the Ets binding site and the fixation of Sp1 and YY1 proteins on their respective motifs. in Drosophila SL2 cells, hGABPα and -β stimulated P6 promoter activity, and hGABPα/hGABPβ and Sp1 exerted synergistic stimulation of this activity, an effect diminished by YY1. The genetic expression of human B19 parvovirus is only dependent on one promoter in vivo and in vitro. This is the P6 promoter, which is located on the left side of the genome and is a single-stranded DNA molecule. This led us to investigate the regulation of the P6 promoter and the possible resulting variability of the nucleotide sequence. After analysis of the promoter region of 17 B19 strains, only 1.5% variability was found. More exciting was the finding of mutations that were clustered around the TATA box and defined a highly conserved region (nucleotides 113–210) in the proximal part of the P6 promoter. HeLa and UT7/Epo cell extracts were found to protect this region, which contained a core motif for Ets family proteins, with YY1 and Sp1 binding sites on either side. Gel mobility shift assays performed with nuclear proteins from HeLa and UT7/Epo cells identified DNA-binding proteins specific for these sites. By supershift analysis, we demonstrated the binding of the hGABP (also named E4TF1) protein to the Ets binding site and the fixation of Sp1 and YY1 proteins on their respective motifs. in Drosophila SL2 cells, hGABPα and -β stimulated P6 promoter activity, and hGABPα/hGABPβ and Sp1 exerted synergistic stimulation of this activity, an effect diminished by YY1. B19 parvovirus is the only member of the Parvoviridae family that is pathogenic for humans (1Berns K.I. Bergoin M. Lederman M. Muzyczka N. Siegl G. Tal J. Tattersall P. Arch. Virol. Suppl. 1994; 10: 166-178Google Scholar). It has been associated with a wide range of clinical symptoms and is responsible for erythema infectiosum in children and arthropathy in adults. B19 infections can be particularly severe, leading to hydrops fetalis during pregnancy, transient aplastic crisis in patients with underlying hemolytic diseases, or chronic bone marrow infection in immunocompromised patients (2Brown K.E. Young N.S. Liu J.M. Crit. Rev. Oncol. Hematol. 1994; 16: 1-31Crossref PubMed Scopus (122) Google Scholar). In vivo and in vitro, the infection of human bone marrow cells leads to the depletion of the immature erythroid progenitor cells,i.e. burst-forming unit erythroid and cluster-forming unit erythroid (3Mortimer P.P. Humphries R.K. Moore J.G. Purcell R.H. Young N.S. Nature. 1983; 302: 426-429Crossref PubMed Scopus (232) Google Scholar, 4Takahashi T. Ozawa K. Takahashi K. Asano S. Takaku F. Blood. 1990; 75: 603-610Crossref PubMed Google Scholar). In the latter cells, replication occurs and results in cell cytotoxicity (5Ozawa K. Kurtzman G. Young N. Science. 1986; 233: 883-886Crossref PubMed Scopus (257) Google Scholar). However, despite such remarkable erythroid tropism, which is still unexplained, B19 infection can also impair megakaryocytopoiesis (6Pallier C. Greco A. Le Junter J. Saib A. Vassias I. Morinet F. J. Virol. 1997; 71: 9482-9489Crossref PubMed Google Scholar, 7Srivastava A. Bruno E. Briddell R. Cooper R. Srivastava C. van Besien K. Hoffman R. Blood. 1990; 76: 1997-2004Crossref PubMed Google Scholar). Whereas virus replication is responsible for the disruption of erythropoiesis, only viral transcription occurs in megakaryocytes. In these cells, the accumulation of the nonstructural protein NS1 seems to be responsible for cell lysis (8Leruez M. Pallier C. Vassias I. Elouet J.F. Romeo P. Morinet F. J. Gen. Virol. 1994; 75: 1475-1478Crossref PubMed Scopus (25) Google Scholar).B19 virus, like other parvoviruses, is a nonenveloped icosahedral virus with a single-stranded DNA linear genome composed of 5596 nucleotides that encode one nonstructural protein (NS1), two structural proteins (VP1 and VP2), and several small polypeptides of unknown function (9Luo W. Astell C.R. Virology. 1993; 195: 448-455Crossref PubMed Scopus (59) Google Scholar, 10Ozawa K. Ayub J. Hao Y.S. Kurtzman G. Shimada T. Young N. J. Virol. 1987; 61: 2395-2406Crossref PubMed Google Scholar, 11Cotmore S.F. McKie V.C. Anderson L.J. Astell C.R. Tattersall P. J. Virol. 1986; 60: 548-557Crossref PubMed Google Scholar). Both ends of the genome are composed of identical inverted repeat sequences of 383 nucleotides (12Deiss V. Tratschin J.D. Weitz M. Siegl G. Virology. 1990; 175: 247-254Crossref PubMed Scopus (99) Google Scholar). The distal 365 nucleotides are imperfect palindromes that can form a hairpin structure. The transcription map of the B19 parvovirus has been determined in infected human bone marrow cells (9Luo W. Astell C.R. Virology. 1993; 195: 448-455Crossref PubMed Scopus (59) Google Scholar, 10Ozawa K. Ayub J. Hao Y.S. Kurtzman G. Shimada T. Young N. J. Virol. 1987; 61: 2395-2406Crossref PubMed Google Scholar). Its only known promoter, named P6 and located in the 5′-terminal region, directs the synthesis of up to nine viral transcripts (13Blundell M.C. Beard C. Astell C.R. Virology. 1987; 157: 534-538Crossref PubMed Scopus (74) Google Scholar, 14Doerig C. Hirt B. Antonietti J.P. Beard P. J. Virol. 1990; 64: 387-396Crossref PubMed Google Scholar). Although the mRNAs encoding for the capsid proteins and the small polypeptides are spliced, the NS1 mRNA is not (10Ozawa K. Ayub J. Hao Y.S. Kurtzman G. Shimada T. Young N. J. Virol. 1987; 61: 2395-2406Crossref PubMed Google Scholar).The regulation of the P6 promoter by viral or cellular proteins has not been extensively studied. In erythroid-permissive cells, this regulation might be preponderant. Thus, a recombinant adeno-associated virus, a defective parvovirus in which the P5 promoter has been substituted for the B19 P6 promoter, is able to replicate specifically and autonomously in erythroid cells (15Wang X.S. Yoder M.C. Zhou S.Z. Srivastava A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12416-12420Crossref PubMed Scopus (33) Google Scholar). However, isolated in front of a reporter gene, the P6 promoter exhibits strong activity in many cell lines, as demonstrated after transfection (14Doerig C. Hirt B. Antonietti J.P. Beard P. J. Virol. 1990; 64: 387-396Crossref PubMed Google Scholar, 16Liu J.M. Green S.W. Hao Y.S. McDonagh K.T. Young N.S. Shimada T. Virology. 1991; 185: 39-47Crossref PubMed Scopus (12) Google Scholar, 17Liu J.M. Fujii H. Green S.W. Komatsu N. Young N.S. Shimada T. Virology. 1991; 182: 361-364Crossref PubMed Scopus (45) Google Scholar). Like other parvoviruses, the nonstructural protein NS1 can up-regulate the P6 promoter (14Doerig C. Hirt B. Antonietti J.P. Beard P. J. Virol. 1990; 64: 387-396Crossref PubMed Google Scholar, 18Leruez-ville M. Vassias I. Pallier C. Cecille A. Hazan U. Morinet F. J. Gen. Virol. 1997; 78: 215-219Crossref PubMed Scopus (11) Google Scholar, 19Moffatt S. Tanaka N. Tada K. Nose M. Nakamura M. Muraoka O. Hirano T. Sugamura K. J. Virol. 1996; 70: 8485-8491Crossref PubMed Google Scholar, 20Sol N. Morinet F. Alizon M. Hazan U. J. Gen. Virol. 1993; 74: 2011-2014Crossref PubMed Scopus (34) Google Scholar), but the exact mechanism of this up-regulation is not yet clear. The result of a recent study argues in favor of an indirect effect involving Sp1 and cAMP-response element binding proteins, as already demonstrated for other parvoviruses. 1Gareus, R., Gigler, A., Hemauer, A., Leruez-Ville, M., Morinet, F., Wolf, H., and Modrow, S. (1998) J. Virol. 72, 609–616. 1Gareus, R., Gigler, A., Hemauer, A., Leruez-Ville, M., Morinet, F., Wolf, H., and Modrow, S. (1998) J. Virol. 72, 609–616. The Sp1 transcription factor has been implicated in the regulation of the P6 promoter (22Blundell M.C. Astell C.R. J. Virol. 1989; 63: 4814-4823Crossref PubMed Google Scholar). Indeed, two GC box motifs located upstream of the TATA box have been implicated in the in vitro up-regulation of promoter transcription. The YY1 transcription factor also binds the P6 promoter to three different motifs (23Momoeda M. Kawase M. Jane S.M. Miyamura K. Young N.S. Kajigaya S. J. Virol. 1994; 68: 7159-7168Crossref PubMed Google Scholar), which results in a positive P6 promoter regulation.In this investigation, we first studied the genetic diversity of the B19 P6 promoter. A highly conserved region was characterized after sequencing 17 B19 strains. Within this region, a large sequence protected by erythroid or nonerythroid nuclear proteins was observed using in vitro footprinting analysis. For the first time, as far as we know, we demonstrated the presence of an Ets binding site (EBS) 2The abbreviations used are: EBS, Ets binding site; EMSA, electrophoretic mobility shift assays; TK, thymidine kinase; bp, base pair(s); PCR, polymerase chain reaction; nts, nucleotides. 2The abbreviations used are: EBS, Ets binding site; EMSA, electrophoretic mobility shift assays; TK, thymidine kinase; bp, base pair(s); PCR, polymerase chain reaction; nts, nucleotides. in the conserved protected region using electrophoretic mobility shift assays (EMSA). By supershift analysis, we characterized the binding of hGABP proteins, an Ets-related transcription factor so far not found to be involved in regulating a parvoviral promoter. In addition to the YY1 transcription factor described above, we demonstrated the fixation of the Sp1 factor to a GC box placed just downstream of the EBS. We then defined a 3-fold sequence composed of the YY1, Ets, and Sp1 binding sites. By transfection analysis of a Drosophila cell line, we studied the effect of the B19 P6 promoter regulation by YY1, hGABP, and Sp1 factors. We showed that Sp1 and hGABP activated transcription synergistically throughout this 3-fold sequence. This synergy was abolished by YY1. Of greater interest was the fact that we observed the same results with the P6 native promoter.DISCUSSIONContrary to other parvoviruses whose genetic expression is controlled by two functional promoters located on the left and the middle of the genome, only one promoter has been described for the human parvovirus B19. We therefore attempted to analyze its variability by PCR amplification and sequencing of the promoter region of 17 B19 virus strains. Our data show an average variation of 1.5%, in agreement with previous results.1 The mutations were clustered around the TATA box, and a highly conserved region was observed in the area proximal of the P6 promoter. This region was located between nucleotides 113 and 210. When it was deleted, there was a 90% loss in transcriptional activity.1 Therefore we decided to examine the cellular factors that interact with this region. By DNA footprinting assays on both strands, a part of the conserved region was seen to be protected by nuclear extracts from the nonerythroid HeLa cell line or the erythroid UT7/Epo cell line. Previous footprinting experiments showed that this region interacts with the proteins of HeLa and CEM cells (16Liu J.M. Green S.W. Hao Y.S. McDonagh K.T. Young N.S. Shimada T. Virology. 1991; 185: 39-47Crossref PubMed Scopus (12) Google Scholar, 22Blundell M.C. Astell C.R. J. Virol. 1989; 63: 4814-4823Crossref PubMed Google Scholar). Here, we noticed that an 8-nucleotide EBS site CCGGAAGT (−208/−201) is located between the binding sites YY1 (−220/−212) and Sp1 (−200/−195). An EBS was described earlier in the P4 promoter of the minute virus of mice and was also located immediately upstream from the GC box that binds the Sp1 transcription factor (37Fuks F. Deleu L. Dinsart C. Rommelaere J. Faisst S. J. Virol. 1996; 70: 1331-1339Crossref PubMed Google Scholar). Using EMSA with synthetic oligonucleotidic probes and competition assays with the corresponding probes, we confirmed the binding of YY1 previously observed by Momoedaet al. (23Momoeda M. Kawase M. Jane S.M. Miyamura K. Young N.S. Kajigaya S. J. Virol. 1994; 68: 7159-7168Crossref PubMed Google Scholar). Binding was also detected at this site for Sp1, contrarily to the findings of Liu et al. (16Liu J.M. Green S.W. Hao Y.S. McDonagh K.T. Young N.S. Shimada T. Virology. 1991; 185: 39-47Crossref PubMed Scopus (12) Google Scholar). The results were similar whether the extract used was from HeLa or UT7/Epo cells. Supershift analysis allowed us to establish that the Ets motif at nucleotides −208/−201 in the conserved B19 promoter region is recognized by hGAPBα, a ubiquitously expressed Ets protein. In addition, antibodies confirmed that HeLa and UT7/Epo binding complexes contain proteins that are immunologically related to both hGABPα and hGABPβ. The complexes produced by the hGABP proteins were specific, as evaluated by probe competition. hGABP (also named E4TF1) is indeed composed of three distinct polypeptides: hGABPα (60 kDa), hGABPβ1 (53 kDa), and hGABPβ2 (47 kDa), all of which are required for high affinity DNA binding (α subunit) and transcriptional activation (α and β subunits) (38Sawada J. Goto M. Sawa C. Watanabe H. Handa H. EMBO J. 1994; 13: 1396-1402Crossref PubMed Scopus (46) Google Scholar, 39Watanabe H. Sawada J. Yano K. Yamaguchi K. Goto M. Handa H. Mol. Cell. Biol. 1993; 13: 1385-1391Crossref PubMed Scopus (90) Google Scholar). hGABP binds to a purine-rich cis-regulatory element required for the VP16-mediated activation of herpes simplex virus immediate early gene and regulates adenovirus E4 gene transcription (39Watanabe H. Sawada J. Yano K. Yamaguchi K. Goto M. Handa H. Mol. Cell. Biol. 1993; 13: 1385-1391Crossref PubMed Scopus (90) Google Scholar, 40Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (320) Google Scholar). We therefore investigated the possible involvement of hGABP in the regulation of the B19 promoter. hGABP was shown to activate a 3-fold sequence comprising YY1-GABP-Sp1 binding sites with the TK minimal promoter in Drosophila SL2 cells; this activation was also found with the P6 promoter. It is noteworthy that our GABP binding site was immediately adjacent to the SP1 site. Ets-related transcription factors such as hGABP are often found in large complexes with other transcription factors (41Wotton D. Ghysdael J. Wang S. Speck N.A. Owen M.J. Mol. Cell. Biol. 1994; 14: 840-850Crossref PubMed Scopus (198) Google Scholar, 42Pongubala J.M. Nagulapalli S. Klemsz M.J. McKercher S.R. Maki R.A. Atchison M.L. Mol. Cell. Biol. 1992; 12: 368-378Crossref PubMed Scopus (311) Google Scholar, 43Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (809) Google Scholar, 44Rosmarin A.G. Caprio D.G. Kirsch D.G. Handa H. Simkevich C.P. J. Biol. Chem. 1995; 270: 23627-23633Crossref PubMed Scopus (88) Google Scholar, 45Sadasivan E. Cedeno M.M. Rothenberg S.P. J. Biol. Chem. 1994; 269: 4725-4735Abstract Full Text PDF PubMed Google Scholar, 46Fitzsimmons D. Hodsdon W. Wheat W. Maira S.-M. Wasylyk B. Hagman J. Genes Dev. 1996; 10: 2198-2211Crossref PubMed Scopus (204) Google Scholar, 47Dittmer J. Pise-Masison C.A. Clemens K.E. Choi K.-S. Brady J.N. J. Biol. Chem. 1997; 272: 4953-4958Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). For example, Ets-1 and Sp1 interact to activate synergistically the human T-cell lymphotrophic virus long terminal repeat (29Gegonne A. Bosselut R. Bailly R.A. Ghysdael J. EMBO J. 1993; 12: 1169-1178Crossref PubMed Scopus (152) Google Scholar). In addition, Sp1 activity is known to be modulated by factors that recognize the DNA elements flanking or overlapping a GC box (48Fischer K.D. Haese A. Nowock J. J. Biol. Chem. 1993; 268: 23915-23923Abstract Full Text PDF PubMed Google Scholar, 49Perkins N.D. Edwards N.L. Duckett C.S. Agranoff A.B. Schmid R.M. Nabel G.J. EMBO J. 1993; 12: 3551-3558Crossref PubMed Scopus (399) Google Scholar). In the present work, Sp1 transactivated the 3-fold sequence and the P6 promoter and displayed synergistic activation with hGABP. Similarly, by co-transfection experiments using also Drosophila SL2 cells, the P4 promoter of minute virus of mice was found to be transactivated synergistically by Ets-1, the prototype member of the Ets family of transcription factors, and the Sp1 factor that binds to a GC box flanking the EBS motif (37Fuks F. Deleu L. Dinsart C. Rommelaere J. Faisst S. J. Virol. 1996; 70: 1331-1339Crossref PubMed Google Scholar). In our study, the mutations of the GABP and Sp1 sites suggest that the combined synergistic effect of the corresponding transcription factors seems to incriminate DNA binding but also protein interactions. Whatever the precise mechanism under investigation, this cooperation was partially inhibited by YY1 protein. In adeno-associated virus, YY1 was found to act as a repressor of transcription from the adeno-associated virus P5 promoter, which is relieved by EIA proteins (21Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Abstract Full Text PDF PubMed Scopus (809) Google Scholar). For B19 parvovirus, the positive effect of YY1 on transcription was described by Momoeda et al. in HeLa cells but was very weak, i.e. 1.3–1.9-fold above basal transcription (23Momoeda M. Kawase M. Jane S.M. Miyamura K. Young N.S. Kajigaya S. J. Virol. 1994; 68: 7159-7168Crossref PubMed Google Scholar). We did not find that YY1 had any effect on the 3-fold sequence or the P6 promoter in SL2 cells. This difference may be due to the type of cells transfected.The present study demonstrated, for the first time as far as we know, that the specific DNA-binding proteins for the CCGGAAGT motif of the human B19 parvovirus promoter is very likely to be hGABP, as indicated by the following results. (i) The DNA protein complex detected in the gel shift assay was abolished by the competition assay, (ii) antibodies against GABPα and -β subunits supershifted this complex, and (iii) the combination of in vitro translated hGABPα and -β proteins produced a complex with essentially the same mobility as that produced by the HeLa or UT7/Epo cell extract. Lastly, our results clearly demonstrated that in nonerythroid cells, hGABP proteins, ubiquitously expressed Ets protein, stimulate the expression of the human B19 parvovirus promoter. The precise mechanism of the synergy exerted by hGABP and Sp1, which is diminished by YY1, is currently under investigation. B19 parvovirus is the only member of the Parvoviridae family that is pathogenic for humans (1Berns K.I. Bergoin M. Lederman M. Muzyczka N. Siegl G. Tal J. Tattersall P. Arch. Virol. Suppl. 1994; 10: 166-178Google Scholar). It has been associated with a wide range of clinical symptoms and is responsible for erythema infectiosum in children and arthropathy in adults. B19 infections can be particularly severe, leading to hydrops fetalis during pregnancy, transient aplastic crisis in patients with underlying hemolytic diseases, or chronic bone marrow infection in immunocompromised patients (2Brown K.E. Young N.S. Liu J.M. Crit. Rev. Oncol. Hematol. 1994; 16: 1-31Crossref PubMed Scopus (122) Google Scholar). In vivo and in vitro, the infection of human bone marrow cells leads to the depletion of the immature erythroid progenitor cells,i.e. burst-forming unit erythroid and cluster-forming unit erythroid (3Mortimer P.P. Humphries R.K. Moore J.G. Purcell R.H. Young N.S. Nature. 1983; 302: 426-429Crossref PubMed Scopus (232) Google Scholar, 4Takahashi T. Ozawa K. Takahashi K. Asano S. Takaku F. Blood. 1990; 75: 603-610Crossref PubMed Google Scholar). In the latter cells, replication occurs and results in cell cytotoxicity (5Ozawa K. Kurtzman G. Young N. Science. 1986; 233: 883-886Crossref PubMed Scopus (257) Google Scholar). However, despite such remarkable erythroid tropism, which is still unexplained, B19 infection can also impair megakaryocytopoiesis (6Pallier C. Greco A. Le Junter J. Saib A. Vassias I. Morinet F. J. Virol. 1997; 71: 9482-9489Crossref PubMed Google Scholar, 7Srivastava A. Bruno E. Briddell R. Cooper R. Srivastava C. van Besien K. Hoffman R. Blood. 1990; 76: 1997-2004Crossref PubMed Google Scholar). Whereas virus replication is responsible for the disruption of erythropoiesis, only viral transcription occurs in megakaryocytes. In these cells, the accumulation of the nonstructural protein NS1 seems to be responsible for cell lysis (8Leruez M. Pallier C. Vassias I. Elouet J.F. Romeo P. Morinet F. J. Gen. Virol. 1994; 75: 1475-1478Crossref PubMed Scopus (25) Google Scholar). B19 virus, like other parvoviruses, is a nonenveloped icosahedral virus with a single-stranded DNA linear genome composed of 5596 nucleotides that encode one nonstructural protein (NS1), two structural proteins (VP1 and VP2), and several small polypeptides of unknown function (9Luo W. Astell C.R. Virology. 1993; 195: 448-455Crossref PubMed Scopus (59) Google Scholar, 10Ozawa K. Ayub J. Hao Y.S. Kurtzman G. Shimada T. Young N. J. Virol. 1987; 61: 2395-2406Crossref PubMed Google Scholar, 11Cotmore S.F. McKie V.C. Anderson L.J. Astell C.R. Tattersall P. J. Virol. 1986; 60: 548-557Crossref PubMed Google Scholar). Both ends of the genome are composed of identical inverted repeat sequences of 383 nucleotides (12Deiss V. Tratschin J.D. Weitz M. Siegl G. Virology. 1990; 175: 247-254Crossref PubMed Scopus (99) Google Scholar). The distal 365 nucleotides are imperfect palindromes that can form a hairpin structure. The transcription map of the B19 parvovirus has been determined in infected human bone marrow cells (9Luo W. Astell C.R. Virology. 1993; 195: 448-455Crossref PubMed Scopus (59) Google Scholar, 10Ozawa K. Ayub J. Hao Y.S. Kurtzman G. Shimada T. Young N. J. Virol. 1987; 61: 2395-2406Crossref PubMed Google Scholar). Its only known promoter, named P6 and located in the 5′-terminal region, directs the synthesis of up to nine viral transcripts (13Blundell M.C. Beard C. Astell C.R. Virology. 1987; 157: 534-538Crossref PubMed Scopus (74) Google Scholar, 14Doerig C. Hirt B. Antonietti J.P. Beard P. J. Virol. 1990; 64: 387-396Crossref PubMed Google Scholar). Although the mRNAs encoding for the capsid proteins and the small polypeptides are spliced, the NS1 mRNA is not (10Ozawa K. Ayub J. Hao Y.S. Kurtzman G. Shimada T. Young N. J. Virol. 1987; 61: 2395-2406Crossref PubMed Google Scholar). The regulation of the P6 promoter by viral or cellular proteins has not been extensively studied. In erythroid-permissive cells, this regulation might be preponderant. Thus, a recombinant adeno-associated virus, a defective parvovirus in which the P5 promoter has been substituted for the B19 P6 promoter, is able to replicate specifically and autonomously in erythroid cells (15Wang X.S. Yoder M.C. Zhou S.Z. Srivastava A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12416-12420Crossref PubMed Scopus (33) Google Scholar). However, isolated in front of a reporter gene, the P6 promoter exhibits strong activity in many cell lines, as demonstrated after transfection (14Doerig C. Hirt B. Antonietti J.P. Beard P. J. Virol. 1990; 64: 387-396Crossref PubMed Google Scholar, 16Liu J.M. Green S.W. Hao Y.S. McDonagh K.T. Young N.S. Shimada T. Virology. 1991; 185: 39-47Crossref PubMed Scopus (12) Google Scholar, 17Liu J.M. Fujii H. Green S.W. Komatsu N. Young N.S. Shimada T. Virology. 1991; 182: 361-364Crossref PubMed Scopus (45) Google Scholar). Like other parvoviruses, the nonstructural protein NS1 can up-regulate the P6 promoter (14Doerig C. Hirt B. Antonietti J.P. Beard P. J. Virol. 1990; 64: 387-396Crossref PubMed Google Scholar, 18Leruez-ville M. Vassias I. Pallier C. Cecille A. Hazan U. Morinet F. J. Gen. Virol. 1997; 78: 215-219Crossref PubMed Scopus (11) Google Scholar, 19Moffatt S. Tanaka N. Tada K. Nose M. Nakamura M. Muraoka O. Hirano T. Sugamura K. J. Virol. 1996; 70: 8485-8491Crossref PubMed Google Scholar, 20Sol N. Morinet F. Alizon M. Hazan U. J. Gen. Virol. 1993; 74: 2011-2014Crossref PubMed Scopus (34) Google Scholar), but the exact mechanism of this up-regulation is not yet clear. The result of a recent study argues in favor of an indirect effect involving Sp1 and cAMP-response element binding proteins, as already demonstrated for other parvoviruses. 1Gareus, R., Gigler, A., Hemauer, A., Leruez-Ville, M., Morinet, F., Wolf, H., and Modrow, S. (1998) J. Virol. 72, 609–616. 1Gareus, R., Gigler, A., Hemauer, A., Leruez-Ville, M., Morinet, F., Wolf, H., and Modrow, S. (1998) J. Virol. 72, 609–616. The Sp1 transcription factor has been implicated in the regulation of the P6 promoter (22Blundell M.C. Astell C.R. J. Virol. 1989; 63: 4814-4823Crossref PubMed Google Scholar). Indeed, two GC box motifs located upstream of the TATA box have been implicated in the in vitro up-regulation of promoter transcription. The YY1 transcription factor also binds the P6 promoter to three different motifs (23Momoeda M. Kawase M. Jane S.M. Miyamura K. Young N.S. Kajigaya S. J. Virol. 1994; 68: 7159-7168Crossref PubMed Google Scholar), which results in a positive P6 promoter regulation. In this investigation, we first studied the genetic diversity of the B19 P6 promoter. A highly conserved region was characterized after sequencing 17 B19 strains. Within this region, a large sequence protected by erythroid or nonerythroid nuclear proteins was observed using in vitro footprinting analysis. For the first time, as far as we know, we demonstrated the presence of an Ets binding site (EBS) 2The abbreviations used are: EBS, Ets binding site; EMSA, electrophoretic mobility shift assays; TK, thymidine kinase; bp, base pair(s); PCR, polymerase chain reaction; nts, nucleotides. 2The abbreviations used are: EBS, Ets binding site; EMSA, electrophoretic mobility shift assays; TK, thymidine kinase; bp, base pair(s); PCR, polymerase chain reaction; nts, nucleotides. in the conserved protected region using electrophoretic mobility shift assays (EMSA). By supershift analysis, we characterized the binding of hGABP proteins, an Ets-related transcription factor so far not found to be involved in regulating a parvoviral promoter. In addition to the YY1 transcription factor described above, we demonstrated the fixation of the Sp1 factor to a GC box placed just downstream of the EBS. We then defined a 3-fold sequence composed of the YY1, Ets, and Sp1 binding sites. By transfection analysis of a Drosophila cell line, we studied the effect of the B19 P6 promoter regulation by YY1, hGABP, and Sp1 factors. We showed that Sp1 and hGABP activated transcription synergistically throughout this 3-fold sequence. This synergy was abolished by YY1. Of greater interest was the fact that we observed the same results with the P6 native promoter. DISCUSSIONContrary to other parvoviruses whose genetic expression is controlled by two functional promoters located on the left and the middle of the genome, only one promoter has been described for the human parvovirus B19. We therefore attempted to analyze its variability by PCR amplification and sequencing of the promoter region of 17 B19 virus strains. Our data show an average variation of 1.5%, in agreement with previous results.1 The mutations were clustered around the TATA box, and a highly conserved region was observed in the area proximal of the P6 promoter. This region was located between nucleotides 113 and 210. When it was deleted, there was a 90% loss in transcriptional activity.1 Therefore we decided to examine the cellular factors that interact with this region. By DNA footprinting assays on both strands, a part of the conserved region was seen to be protected by nuclear extracts from the nonerythroid HeLa cell line or the erythroid UT7/Epo cell line. Previous footprinting experiments showed that this region interacts with the proteins of HeLa and CEM cells (16Liu J.M. Green S.W. Hao Y.S. McDonagh K.T. Young N.S. Shimada T. Virology. 1991; 185: 39-47Crossref PubMed Scopus (12) Google Scholar, 22Blundell M.C. Astell C.R. J. Virol. 1989; 63: 4814-4823Crossref PubMed Google Scholar). Here, we noticed that an 8-nucleotide EBS site CCGGAAGT (−208/−201) is located between the binding sites YY1 (−220/−212) and Sp1 (−200/−195). An EBS was described earlier in the P4 promoter of the minute virus of mice and was also located immediately upstream from the GC box that binds the Sp1 transcription factor (37Fuks F. Deleu L. Dinsart C. Rommelaere J. Faisst S. J. Virol. 1996; 70: 1331-1339Crossref PubMed Google Scholar). Using EMSA with synthetic oligonucleotidic probes and competition assays with the corresponding probes, we confirmed the binding of YY1 previously observed by Momoedaet al. (23Momoeda M. Kawase M. Jane S.M. Miyamura K. Young N.S. Kajigaya S. J. Virol. 1994; 68: 7159-7168Crossref PubMed Google Scholar). Binding was also detected at this site for Sp1, contrarily to the findings of Liu et al. (16Liu J.M. Green S.W. Hao Y.S. McDonagh K.T. Young N.S. Shimada T. Virology. 1991; 185: 39-47Crossref PubMed Scopus (12) Google Scholar). The results were similar whether the extract used was from HeLa or UT7/Epo cells. Supershift analysis allowed us to establish that the Ets motif at nucleotides −208/−201 in the conserved B19 promoter region is recognized by hGAPBα, a ubiquitously expressed Ets protein. In addition, antibodies confirmed that HeLa and UT7/Epo binding complexes contain proteins that are immunologically related to both hGABPα and hGABPβ. The complexes produced by the hGABP proteins were specific, as evaluated by probe competition. hGABP (also named E4TF1) is indeed composed of three distinct polypeptides: hGABPα (60 kDa), hGABPβ1 (53 kDa), and hGABPβ2 (47 kDa), all of which are required for high affinity DNA binding (α subunit) and transcriptional activation (α and β subunits) (38Sawada J. Goto M. Sawa C. Watanabe H. Handa H. EMBO J. 1994; 13: 1396-1402Crossref PubMed Scopus (46) Google Scholar, 39Watanabe H. Sawada J. Yano K. Yamaguchi K. Goto M. Handa H. Mol. Cell. Biol. 1993; 13: 1385-1391Crossref PubMed Scopus (90) Google Scholar). hGABP binds to a purine-rich cis-regulatory element required for the VP16-mediated activation of herpes simplex virus immediate early gene and regulates adenovirus E4 gene transcription (39Watanabe H. Sawada J. Yano K. Yamaguchi K. Goto M. Handa H. Mol. Cell. Biol. 1993; 13: 1385-1391Crossref PubMed Scopus (90) Google Scholar, 40Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (320) Google Scholar). We therefore investigated the possible involvement of hGABP in the regulation of the B19 promoter. hGABP was shown to activate a 3-fold sequence comprising YY1-GABP-Sp1 binding sites with the TK minimal promoter in Drosophila SL2 cells; this activation was also found with the P6 promoter. It is noteworthy that our GABP binding site was immediately adjacent to the SP1 site. Ets-related transcription factors such as hGABP are often found in large complexes with other transcription factors (41Wotton D. Ghysdael J. Wang S. Speck N.A. Owen M.J. Mol. Cell. Biol. 1994; 14: 840-850Crossref PubMed Scopus (198) Google Scholar, 42Pongubala J.M. Nagulapalli S. Klemsz M.J. McKercher S.R. Maki R.A. Atchison M.L. Mol. Cell. Biol. 1992; 12: 368-378Crossref PubMed Scopus (311) Google Scholar, 43Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (809) Google Scholar, 44Rosmarin A.G. Caprio D.G. Kirsch D.G. Handa H. Simkevich C.P. J. Biol. Chem. 1995; 270: 23627-23633Crossref PubMed Scopus (88) Google Scholar, 45Sadasivan E. Cedeno M.M. Rothenberg S.P. J. Biol. Chem. 1994; 269: 4725-4735Abstract Full Text PDF PubMed Google Scholar, 46Fitzsimmons D. Hodsdon W. Wheat W. Maira S.-M. Wasylyk B. Hagman J. Genes Dev. 1996; 10: 2198-2211Crossref PubMed Scopus (204) Google Scholar, 47Dittmer J. Pise-Masison C.A. Clemens K.E. Choi K.-S. Brady J.N. J. Biol. Chem. 1997; 272: 4953-4958Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). For example, Ets-1 and Sp1 interact to activate synergistically the human T-cell lymphotrophic virus long terminal repeat (29Gegonne A. Bosselut R. Bailly R.A. Ghysdael J. EMBO J. 1993; 12: 1169-1178Crossref PubMed Scopus (152) Google Scholar). In addition, Sp1 activity is known to be modulated by factors that recognize the DNA elements flanking or overlapping a GC box (48Fischer K.D. Haese A. Nowock J. J. Biol. Chem. 1993; 268: 23915-23923Abstract Full Text PDF PubMed Google Scholar, 49Perkins N.D. Edwards N.L. Duckett C.S. Agranoff A.B. Schmid R.M. Nabel G.J. EMBO J. 1993; 12: 3551-3558Crossref PubMed Scopus (399) Google Scholar). In the present work, Sp1 transactivated the 3-fold sequence and the P6 promoter and displayed synergistic activation with hGABP. Similarly, by co-transfection experiments using also Drosophila SL2 cells, the P4 promoter of minute virus of mice was found to be transactivated synergistically by Ets-1, the prototype member of the Ets family of transcription factors, and the Sp1 factor that binds to a GC box flanking the EBS motif (37Fuks F. Deleu L. Dinsart C. Rommelaere J. Faisst S. J. Virol. 1996; 70: 1331-1339Crossref PubMed Google Scholar). In our study, the mutations of the GABP and Sp1 sites suggest that the combined synergistic effect of the corresponding transcription factors seems to incriminate DNA binding but also protein interactions. Whatever the precise mechanism under investigation, this cooperation was partially inhibited by YY1 protein. In adeno-associated virus, YY1 was found to act as a repressor of transcription from the adeno-associated virus P5 promoter, which is relieved by EIA proteins (21Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Abstract Full Text PDF PubMed Scopus (809) Google Scholar). For B19 parvovirus, the positive effect of YY1 on transcription was described by Momoeda et al. in HeLa cells but was very weak, i.e. 1.3–1.9-fold above basal transcription (23Momoeda M. Kawase M. Jane S.M. Miyamura K. Young N.S. Kajigaya S. J. Virol. 1994; 68: 7159-7168Crossref PubMed Google Scholar). We did not find that YY1 had any effect on the 3-fold sequence or the P6 promoter in SL2 cells. This difference may be due to the type of cells transfected.The present study demonstrated, for the first time as far as we know, that the specific DNA-binding proteins for the CCGGAAGT motif of the human B19 parvovirus promoter is very likely to be hGABP, as indicated by the following results. (i) The DNA protein complex detected in the gel shift assay was abolished by the competition assay, (ii) antibodies against GABPα and -β subunits supershifted this complex, and (iii) the combination of in vitro translated hGABPα and -β proteins produced a complex with essentially the same mobility as that produced by the HeLa or UT7/Epo cell extract. Lastly, our results clearly demonstrated that in nonerythroid cells, hGABP proteins, ubiquitously expressed Ets protein, stimulate the expression of the human B19 parvovirus promoter. The precise mechanism of the synergy exerted by hGABP and Sp1, which is diminished by YY1, is currently under investigation. Contrary to other parvoviruses whose genetic expression is controlled by two functional promoters located on the left and the middle of the genome, only one promoter has been described for the human parvovirus B19. We therefore attempted to analyze its variability by PCR amplification and sequencing of the promoter region of 17 B19 virus strains. Our data show an average variation of 1.5%, in agreement with previous results.1 The mutations were clustered around the TATA box, and a highly conserved region was observed in the area proximal of the P6 promoter. This region was located between nucleotides 113 and 210. When it was deleted, there was a 90% loss in transcriptional activity.1 Therefore we decided to examine the cellular factors that interact with this region. By DNA footprinting assays on both strands, a part of the conserved region was seen to be protected by nuclear extracts from the nonerythroid HeLa cell line or the erythroid UT7/Epo cell line. Previous footprinting experiments showed that this region interacts with the proteins of HeLa and CEM cells (16Liu J.M. Green S.W. Hao Y.S. McDonagh K.T. Young N.S. Shimada T. Virology. 1991; 185: 39-47Crossref PubMed Scopus (12) Google Scholar, 22Blundell M.C. Astell C.R. J. Virol. 1989; 63: 4814-4823Crossref PubMed Google Scholar). Here, we noticed that an 8-nucleotide EBS site CCGGAAGT (−208/−201) is located between the binding sites YY1 (−220/−212) and Sp1 (−200/−195). An EBS was described earlier in the P4 promoter of the minute virus of mice and was also located immediately upstream from the GC box that binds the Sp1 transcription factor (37Fuks F. Deleu L. Dinsart C. Rommelaere J. Faisst S. J. Virol. 1996; 70: 1331-1339Crossref PubMed Google Scholar). Using EMSA with synthetic oligonucleotidic probes and competition assays with the corresponding probes, we confirmed the binding of YY1 previously observed by Momoedaet al. (23Momoeda M. Kawase M. Jane S.M. Miyamura K. Young N.S. Kajigaya S. J. Virol. 1994; 68: 7159-7168Crossref PubMed Google Scholar). Binding was also detected at this site for Sp1, contrarily to the findings of Liu et al. (16Liu J.M. Green S.W. Hao Y.S. McDonagh K.T. Young N.S. Shimada T. Virology. 1991; 185: 39-47Crossref PubMed Scopus (12) Google Scholar). The results were similar whether the extract used was from HeLa or UT7/Epo cells. Supershift analysis allowed us to establish that the Ets motif at nucleotides −208/−201 in the conserved B19 promoter region is recognized by hGAPBα, a ubiquitously expressed Ets protein. In addition, antibodies confirmed that HeLa and UT7/Epo binding complexes contain proteins that are immunologically related to both hGABPα and hGABPβ. The complexes produced by the hGABP proteins were specific, as evaluated by probe competition. hGABP (also named E4TF1) is indeed composed of three distinct polypeptides: hGABPα (60 kDa), hGABPβ1 (53 kDa), and hGABPβ2 (47 kDa), all of which are required for high affinity DNA binding (α subunit) and transcriptional activation (α and β subunits) (38Sawada J. Goto M. Sawa C. Watanabe H. Handa H. EMBO J. 1994; 13: 1396-1402Crossref PubMed Scopus (46) Google Scholar, 39Watanabe H. Sawada J. Yano K. Yamaguchi K. Goto M. Handa H. Mol. Cell. Biol. 1993; 13: 1385-1391Crossref PubMed Scopus (90) Google Scholar). hGABP binds to a purine-rich cis-regulatory element required for the VP16-mediated activation of herpes simplex virus immediate early gene and regulates adenovirus E4 gene transcription (39Watanabe H. Sawada J. Yano K. Yamaguchi K. Goto M. Handa H. Mol. Cell. Biol. 1993; 13: 1385-1391Crossref PubMed Scopus (90) Google Scholar, 40Thompson C.C. Brown T.A. McKnight S.L. Science. 1991; 253: 762-768Crossref PubMed Scopus (320) Google Scholar). We therefore investigated the possible involvement of hGABP in the regulation of the B19 promoter. hGABP was shown to activate a 3-fold sequence comprising YY1-GABP-Sp1 binding sites with the TK minimal promoter in Drosophila SL2 cells; this activation was also found with the P6 promoter. It is noteworthy that our GABP binding site was immediately adjacent to the SP1 site. Ets-related transcription factors such as hGABP are often found in large complexes with other transcription factors (41Wotton D. Ghysdael J. Wang S. Speck N.A. Owen M.J. Mol. Cell. Biol. 1994; 14: 840-850Crossref PubMed Scopus (198) Google Scholar, 42Pongubala J.M. Nagulapalli S. Klemsz M.J. McKercher S.R. Maki R.A. Atchison M.L. Mol. Cell. Biol. 1992; 12: 368-378Crossref PubMed Scopus (311) Google Scholar, 43Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (809) Google Scholar, 44Rosmarin A.G. Caprio D.G. Kirsch D.G. Handa H. Simkevich C.P. J. Biol. Chem. 1995; 270: 23627-23633Crossref PubMed Scopus (88) Google Scholar, 45Sadasivan E. Cedeno M.M. Rothenberg S.P. J. Biol. Chem. 1994; 269: 4725-4735Abstract Full Text PDF PubMed Google Scholar, 46Fitzsimmons D. Hodsdon W. Wheat W. Maira S.-M. Wasylyk B. Hagman J. Genes Dev. 1996; 10: 2198-2211Crossref PubMed Scopus (204) Google Scholar, 47Dittmer J. Pise-Masison C.A. Clemens K.E. Choi K.-S. Brady J.N. J. Biol. Chem. 1997; 272: 4953-4958Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). For example, Ets-1 and Sp1 interact to activate synergistically the human T-cell lymphotrophic virus long terminal repeat (29Gegonne A. Bosselut R. Bailly R.A. Ghysdael J. EMBO J. 1993; 12: 1169-1178Crossref PubMed Scopus (152) Google Scholar). In addition, Sp1 activity is known to be modulated by factors that recognize the DNA elements flanking or overlapping a GC box (48Fischer K.D. Haese A. Nowock J. J. Biol. Chem. 1993; 268: 23915-23923Abstract Full Text PDF PubMed Google Scholar, 49Perkins N.D. Edwards N.L. Duckett C.S. Agranoff A.B. Schmid R.M. Nabel G.J. EMBO J. 1993; 12: 3551-3558Crossref PubMed Scopus (399) Google Scholar). In the present work, Sp1 transactivated the 3-fold sequence and the P6 promoter and displayed synergistic activation with hGABP. Similarly, by co-transfection experiments using also Drosophila SL2 cells, the P4 promoter of minute virus of mice was found to be transactivated synergistically by Ets-1, the prototype member of the Ets family of transcription factors, and the Sp1 factor that binds to a GC box flanking the EBS motif (37Fuks F. Deleu L. Dinsart C. Rommelaere J. Faisst S. J. Virol. 1996; 70: 1331-1339Crossref PubMed Google Scholar). In our study, the mutations of the GABP and Sp1 sites suggest that the combined synergistic effect of the corresponding transcription factors seems to incriminate DNA binding but also protein interactions. Whatever the precise mechanism under investigation, this cooperation was partially inhibited by YY1 protein. In adeno-associated virus, YY1 was found to act as a repressor of transcription from the adeno-associated virus P5 promoter, which is relieved by EIA proteins (21Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Abstract Full Text PDF PubMed Scopus (809) Google Scholar). For B19 parvovirus, the positive effect of YY1 on transcription was described by Momoeda et al. in HeLa cells but was very weak, i.e. 1.3–1.9-fold above basal transcription (23Momoeda M. Kawase M. Jane S.M. Miyamura K. Young N.S. Kajigaya S. J. Virol. 1994; 68: 7159-7168Crossref PubMed Google Scholar). We did not find that YY1 had any effect on the 3-fold sequence or the P6 promoter in SL2 cells. This difference may be due to the type of cells transfected. The present study demonstrated, for the first time as far as we know, that the specific DNA-binding proteins for the CCGGAAGT motif of the human B19 parvovirus promoter is very likely to be hGABP, as indicated by the following results. (i) The DNA protein complex detected in the gel shift assay was abolished by the competition assay, (ii) antibodies against GABPα and -β subunits supershifted this complex, and (iii) the combination of in vitro translated hGABPα and -β proteins produced a complex with essentially the same mobility as that produced by the HeLa or UT7/Epo cell extract. Lastly, our results clearly demonstrated that in nonerythroid cells, hGABP proteins, ubiquitously expressed Ets protein, stimulate the expression of the human B19 parvovirus promoter. The precise mechanism of the synergy exerted by hGABP and Sp1, which is diminished by YY1, is currently under investigation. We would like to thank C. Rahuel (INSERM U91, INTS, Paris, France) for help with footprinting assays. We are also grateful to J. Ghysdael (Institut Curie, Orsay, France) for the generous gift of pPac plasmids and to T. Shenk (Princeton University, NJ) for the generous gift of YY1 cDNA. Thanks are due to F. Moreau-Gachelin (Institut Curie, Paris, France), T. Brown (Pfizer), and S. T. Smale (UCLA school of Medicine, Los Angeles, CA) who, respectively, contributed kind gifts of antibodies against PU-1 and Spi-B, GABPα and GABPβ, and Ets-1 and Elf-1 and to F. Thierry (Institut Pasteur, Paris, France) for the gift of pTK-50 plasmid. Lastly, we are indebted to the Laboratoire Photographique d'Hématologie for photographic work and to M. C. Daudon for typing the manuscript.
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