Transcriptional Repression by the Human Homeobox Protein EVX1 in Transfected Mammalian Cells
1995; Elsevier BV; Volume: 270; Issue: 46 Linguagem: Inglês
10.1074/jbc.270.46.27695
ISSN1083-351X
AutoresPaola Briata, Rinke Van De Werken, Irma Airoldi, Cristina Ilengo, Erica Di Blas, Edoardo Boncinelli, Giorgio Corte,
Tópico(s)Developmental Biology and Gene Regulation
ResumoThe human homeobox protein EVX1 (EVX1) is thought to play an important role during embryogenesis. In this study, the effect of EVX1 on gene transcription has been investigated in transfected mammalian cells. EVX1 expression represses transcription of a reporter gene directed by either cell-specific or viral promoter/enhancer sequences in a variety of mammalian cell lines and in a concentration-dependent manner. Transcriptional repression is independent of the presence of DNA-binding sites for EVX1 in all the promoters we tested. Furthermore, repression by EVX1 is evident also using a TATA-less minimal promoter in the reporter construct. A carboxyl-terminal proline/alanine-rich region of EVX1 seems to be responsible for the transcriptional repression activity, as suggested by transfection of EVX1 mutants. We speculate that the repressor function of EVX1 contributes to its proposed role in embryogenesis. The human homeobox protein EVX1 (EVX1) is thought to play an important role during embryogenesis. In this study, the effect of EVX1 on gene transcription has been investigated in transfected mammalian cells. EVX1 expression represses transcription of a reporter gene directed by either cell-specific or viral promoter/enhancer sequences in a variety of mammalian cell lines and in a concentration-dependent manner. Transcriptional repression is independent of the presence of DNA-binding sites for EVX1 in all the promoters we tested. Furthermore, repression by EVX1 is evident also using a TATA-less minimal promoter in the reporter construct. A carboxyl-terminal proline/alanine-rich region of EVX1 seems to be responsible for the transcriptional repression activity, as suggested by transfection of EVX1 mutants. We speculate that the repressor function of EVX1 contributes to its proposed role in embryogenesis. INTRODUCTIONHomeobox genes code for transcription factors containing a trihelical DNA-binding motif, termed homeodomain (HD) 1The abbreviations used are: HDhomeodomainTRE12-O-tetradecanoylphorbol 13-acetate response elementEVX1human homeobox protein EVX1CATchloramphenicol acetyltransferaseDEAE-dextrandiethylaminoethyl-dextranPCRpolymerase chain reactionRT-PCRreverse transcriptase-PCREveeven-skipped productmAbmonoclonal antibodybpbase pair(s). ((1Levine M. Hoey T. Cell. 1988; 55: 537-540Google Scholar, 2Hayashi S. Scott M.P. Cell. 1990; 63: 883-894Google Scholar, 3McGinnis W. Krumlauf R. Cell. 1992; 68: 283-302Google Scholar), and references cited therein). The HD is highly conserved with respect to structure and function throughout evolution and is a common component of numerous proteins that regulate transcription during development (for reviews, see (4Scott M.P. Tammkun J.W. Hartzell G.W. Biochim. Biophys. Acta. 1989; 989: 25-48Google Scholar, 5Laughon A. Biochemistry. 1991; 30: 11357-11367Google Scholar, 6Gehring W.J. Gene (Amst.). 1993; 135: 215-221Google Scholar)). The functions of HD proteins range from activities in directing pattern formation to more restricted roles in regulating specific cell fate(3McGinnis W. Krumlauf R. Cell. 1992; 68: 283-302Google Scholar, 7Rosenfeld M.G. Genes & Dev. 1991; 5: 897-907Google Scholar). The products of vertebrate homeobox-containing genes have been shown to bind DNA in vitro(8Odenwald W.F. Garbern J. Arnheiter H. Tournier-Lasserve E. Lazzarini R.A. Genes & Dev. 1989; 3: 158-172Google Scholar, 9Corsetti M.T. Briata P. Sanseverino L. Daga A. Airoldi I. Simeone A. Palmisano G. Angelini C. Boncinelli E. Corte G. Nucleic Acids Res. 1992; 20: 4465-4472Google Scholar) and to regulate gene transcription through specific target sequences in cell culture(10Zappavigna V. Renucci A. Izpisua-Belmonte J.C. Urier G. Peschle C. Duboule D. EMBO J. 1991; 10: 4177-4187Google Scholar, 11Jones F.S. Prediger E.A. Bittner D.A. De Robertis E.M. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2086-2090Google Scholar). It is unlikely that HD-containing proteins exert their different selective functions in vivo only through protein-DNA interactions, since they share similar DNA-binding specificities in vitro(4Scott M.P. Tammkun J.W. Hartzell G.W. Biochim. Biophys. Acta. 1989; 989: 25-48Google Scholar, 5Laughon A. Biochemistry. 1991; 30: 11357-11367Google Scholar). Thus, it is presumed that although a single HD has the potential to bind to many DNA-target sites it may be specifically recruited into a functional complex at only a subset of those sites by selective protein-protein interactions(12Stern, S., Tanaka, M., Herr, W., Nature, 345, 783–785.Google Scholar, 13Pomerantz J.L. Kristie T.M. Sharp P.A. Genes & Dev. 1992; 6: 2047-2057Google Scholar, 14Zappavigna V. Sartori D. Mavilio F. Genes & Dev. 1994; 8: 732-744Google Scholar, 15Johnson F.B. Parker E. Krasnow M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 739-743Google Scholar). Furthermore, in some cases the HD has been demonstrated to be dispensable for activity in vivo(16Ananthan J. Baler R. Morrissey D. Zuo J. Lan Y. Weir M. Voellmy R. Mol. Cell. Biol. 1993; 13: 1599-1609Google Scholar, 17Fitzpatrick V.D. Percival-Smith A. Ingles C.J. Krause H.M. Nature. 1992; 356: 610-612Google Scholar).Recently, the human homeobox genes EVX1 and EVX2 have been isolated and sequenced(18Faiella A. D'Esposito M. Rambaldi M. Acampora D. Balsofiore S. Stornaiuolo A. Mallamaci A. Migliaccio E. Gulisano M. Simeone A. Boncinelli E. Nucleic Acids Res. 1992; 23: 6541-6545Google Scholar, 19D'Esposito M. Morelli F. Acampora D. Migliaccio E. Simeone A. Boncinelli E. Genomics. 1991; 10: 43-50Google Scholar). These genes encode proteins containing a HD closely related to that encoded by the Drosophila even-skipped, which belongs to the pair-rule class of segmentation genes and is required for the proper development of the metameric body plan of the fruit fly(20Macdonald P.M. Ingham P. Struhl G. Cell. 1986; 47: 721-734Google Scholar, 21Frasch M. Hoey T. Rushlow C. Doyle H Levine M. EMBO J. 1987; 6: 749-759Google Scholar, 22Jäckle H. Sauer F. Curr. Opin. Cell Biol. 1993; 5: 505-512Google Scholar). Also the murine Evx 1 and Evx 2 genes have been cloned, and Evx 1 expression pattern during mouse embryogenesis has been studied(23Bastian H. Gruss P. EMBO J. 1990; 9: 1839-1852Google Scholar). During early embryogenesis, Evx 1 is expressed in a biphasic manner. From day 7 to 9 of development its expression emerges at the posterior end of the embryo within the primitive ectoderm and later in the mesoderm and neuroectoderm. From day 10 to 12.5, Evx 1 transcripts are restricted to specific cells within the neural tube and hindbrain, while no expression is detectable in a variety of adult tissues. Spyropoulos et al.(24Spyropoulos D.D Capecchi M.R. Genes & Dev. 1994; 8: 1949-1961Google Scholar) have recently shown that the targeted disruption of the Evx 1 gene in mice causes early postimplantation lethality of the conceptus.Several authors have reported that the even-skipped gene product acts as a transcriptional repressor both in in vitro assays (25Biggin M.D. Tjian R. Cell. 1989; 58: 433-440Google Scholar, 26Johnson F.B. Krasnow M.A. Genes & Dev. 1992; 6: 2177-2189Google Scholar, 27TenHarmsel A. Austin R.J. Savenelli N. Biggin M.D. Mol. Cell. Biol. 1993; 13: 2742-2752Google Scholar) and in cell transfection experiments(28Han K. Levine M.S. Manley J. Cell. 1989; 56: 573-583Google Scholar, 29Han K. Manley J.L. Genes & Dev. 1993; 7: 491-503Google Scholar). On the contrary, Jones et al.(30Jones F.S. Chalepakis G. Gruss P. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2091-2095Google Scholar) showed that the mouse Evx 1 protein induces the expression of a reporter gene driven by the chicken Tenascin-C promoter. The same authors restricted the sequences that contributed to the activation to a segment containing a TRE known to bind transcription factors belonging to the AP-1 family. Currently, the role of the human EVX1 and EVX2 proteins in gene transcription is unknown.The aim of our study was to investigate the transcriptional activity of EVX1 in transfected mammalian cells. Our data indicate that EVX1 expression strongly reduces, in a concentration-dependent manner, the basal and activated transcription of the reporter gene CAT directed by a variety of cell-specific and viral promoters in several different mammalian cell lines. EVX1 transcriptional repressor function is evident using both TATA-containing and TATA-less promoters. We also show that the repressor function of EVX1 is contained within a C-terminal region rich in alanine and proline residues and is independent of the presence of DNA-binding sites for EVX1.EXPERIMENTAL PROCEDURESExpression and Purification of EVX1 Recombinant ProteinEVX1 protein was produced in the baculovirus system according to the method previously described in detail (9Corsetti M.T. Briata P. Sanseverino L. Daga A. Airoldi I. Simeone A. Palmisano G. Angelini C. Boncinelli E. Corte G. Nucleic Acids Res. 1992; 20: 4465-4472Google Scholar) with minor modifications. Briefly, pVL941/EVX1 was obtained by inserting the filled in Sau3AI-Sau3AI (1453 bp) EVX1 cDNA fragment (18Faiella A. D'Esposito M. Rambaldi M. Acampora D. Balsofiore S. Stornaiuolo A. Mallamaci A. Migliaccio E. Gulisano M. Simeone A. Boncinelli E. Nucleic Acids Res. 1992; 23: 6541-6545Google Scholar) in the filled in BamHI cloning site of pVL941 (Pharmingen, San Diego, CA). Recombinant baculovirus particles (Baculogold/EVX1) were obtained by transfecting Sf9 cells with the wild type Baculogold™ (Pharmingen) viral DNA together with pVL941/EVX1 and purified by limiting dilution and dot hybridization. For the production of EVX1 protein, 500 × 106 Sf9 cells were infected with five plaque-forming units/cell of the recombinant virus, and cells were recovered and lysed as described previously(9Corsetti M.T. Briata P. Sanseverino L. Daga A. Airoldi I. Simeone A. Palmisano G. Angelini C. Boncinelli E. Corte G. Nucleic Acids Res. 1992; 20: 4465-4472Google Scholar). Both the cytoplasmic and the nuclear lysates were analyzed by SDS-polyacrylamide gel electrophoresis and stained with silver. A band that was absent in lysates from cells infected with a control virus was present in the profile of the nuclear lysates, accounting for approximately 2% of the total proteins (not shown). EVX1 protein was purified by ion exchange chromatography using Q and S Sepharose Fast Flow columns (Pharmacia Biotech, Inc.). After elution, the fractions containing the recombinant protein were renatured by extensive dialysis against buffer D (20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM 1,4-dithiothreitol) at 4°C. The recombinant protein was stored in buffer D supplemented with 20% glycerol at -80°C.General handling techniques for baculovirus expression system were performed essentially as described in (31Summers M.D. Smith G.E. A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures. Texas Agricultural Experiment Station, 1987Google Scholar).Cells, Antibodies, and Western BlottingHeLa human cervix carcinoma cells, F9 teratocarcinoma cells, and InR1-G9 hamster glucagonoma cells (32Takaki R. Ono J. Nakamura M. Yokogawa Y. Kumae S. Hiroaka T. Yamaguchi K. Uchida S. In Vitro Cell. & Dev. Biol. 1986; 22: 120-126Google Scholar) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. β-TC1 mouse insulinoma cells (33Efrat S. Linde S. Kofod M. Spector D. Delamoy M. Grant S. Hanahan D. Baekkeskov S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9037-9041Google Scholar) were cultured in Dulbecco's modified Eagle's medium supplemented with 12.5% horse serum, 2.5% fetal calf serum, glutamine, penicillin, and streptomycin as above. NIH-3T3, mouse fibroblast cells, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum, glutamine, penicillin, and streptomycin as detailed above.Mouse monoclonal antibodies to EVX1 were produced and characterized according to methods previously described(34Corte G. Moretta L. Damiani G. Mingari M.C. Bargellesi A. Eur. J. Immunol. 1981; 11: 162-170Google Scholar, 35Gefter M.L. Margulies D.H. Scharff M.D. Somatic Cell Genet. 1977; 3: 231-237Google Scholar). Determination of Ig classes was carried out by standard procedures utilizing the Mouse Typer Sub-Isotyping kit (Bio-Rad).For immunoblot analysis, nuclear lysates of infected Sf9 cells were electrophoresed in 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes according to standard procedures. The filters were incubated with the supernatant of hybridoma cells, followed by an alkaline phosphatase-conjugated goat anti-mouse antibody from Sigma. Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate were used as the substrates (Promega, Madison, WI) with a positive reaction resulting in purple color.Immunofluorescence and ImmunohistochemistryFor immunofluorescence, cytospins were prepared and fixed in acetone at -20°C for 5 min. Cells were incubated for 2-16 h at room temperature with supernatant of hybridoma cells, followed by extensive washes and by incubation with rhodaminated goat anti-mouse IgG + IgM (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature.Immunohistochemistry was performed according to the streptavidin-biotin complex technique using a commercially available kit system (Dako, Glostrup, Denmark). Nuclei were counterstained for 5 min with Mayer's hematoxylin solution (Sigma).Plasmidsp[-1.1]GLU-CAT and p[-0.35]GLU-CAT were previously described and characterized (36Drucker D.J. Philippe J. Jepeal L. Habener J.F. J. Biol. Chem. 1987; 262: 15659-15665Google Scholar, 37Knepel W. Chafitz J. Habener J.F. Mol. Cell. Biol. 1990; 10: 6799-6804Google Scholar, 38Gherzi R. Fehmann H.-C. Volz A. Ponassi M. Göke B. Exp. Cell Res. 1995; 218: 460-468Google Scholar) and were a gift from Drs. R. Gherzi and B. Göke (Marburg, Germany), pCAT™-Promoter was purchased from Promega, pRSV-CAT (39Gorman C.M. Merlino G.T. Willingham M.C. Pastan I. Howard B.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6777-6781Google Scholar) was obtained from Dr. M. Beato (Marburg, Germany), and p7B2-I-CAT was described previously (40Waldbieser G.C. Aimi J. Dixon J. Endocrinology. 1991; 128: 3228-3236Google Scholar) and obtained from Dr. B. Göke (Marburg, Germany). pInr-CAT was constructed by cloning synthetic oligonucleotides containing the appropriate sequence (41Smale S.T. Baltimore D. Cell. 1989; 57: 103-113Google Scholar) into the PstI and XbaI sites of pCAT™-Basic vector purchased from Promega. The pCMV-EVX1 expression construct was generated by cloning the Klenow-filled in Sau3AI-Sau3AI fragment containing the complete open reading frame of the EVX1 cDNA (18Faiella A. D'Esposito M. Rambaldi M. Acampora D. Balsofiore S. Stornaiuolo A. Mallamaci A. Migliaccio E. Gulisano M. Simeone A. Boncinelli E. Nucleic Acids Res. 1992; 23: 6541-6545Google Scholar) into the Klenow-filled in HindIII site of the pRC-CMV expression vector purchased from Invitrogen Corp. (San Diego, CA). The pCMV-HOXD4 expression construct was generated by cloning the ApaI-ApaI fragment containing the complete open reading frame of the HOXD4 cDNA (42Mavilio F. Simeone A. Giampaolo A. Faiella A. Zappavigna V. Acampora D. Poiana G. Russo G. Peschle C. Boncinelli E. Nature. 1986; 324: 664-668Google Scholar) into the ApaI site of the pRC-CMV expression vector. pCMV-ΔEVX1 and pCMV-ɛEVX1 expression constructs were generated by cloning PCR-amplified fragments containing the 5′ 750 bp and the 3′ 675 bp of the open reading frame of EVX1 cDNA, respectively, in the pRC-CMV expression vector. The chimeric construct pCMV-HOXC6/EVX1 was created by ligating the 5′ 612-bp fragment of HOXC6 (indicated as pCMV-HOXC6N when cloned into pRC-CMV and utilized as the control for transfection experiments; (9Corsetti M.T. Briata P. Sanseverino L. Daga A. Airoldi I. Simeone A. Palmisano G. Angelini C. Boncinelli E. Corte G. Nucleic Acids Res. 1992; 20: 4465-4472Google Scholar) and references cited therein) with the 3′ 459-bp fragment of EVX1 and cloning the construct into the pRC-CMV vector. For PCR reactions, ULTma™ DNA polymerase from Perkin-Elmer was used. Oligonucleotides were obtained from TIB MolBiol, Genova, Italy.General handling of plasmid DNA was performed according to previously described techniques(43Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar).DNA Transfections and CAT AssaysInR1-G9 and β-TC1 cells transient transfection was performed essentially as described by Fehmann and Habener (44Fehmann H.-C. Habener J.F. Endocrinology. 1982; 130: 159-166Google Scholar) with the modification reported in (38Gherzi R. Fehmann H.-C. Volz A. Ponassi M. Göke B. Exp. Cell Res. 1995; 218: 460-468Google Scholar). HeLa and F9 cells were transiently transfected by the calcium phosphate technique, performed exactly as reported in (45Ausubel F.A. Brent R. Kingstone R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing and Wiley-Interscience, New York, NY1990Google Scholar). Transient transfection of NIH-3T3 by the electroporation technique was performed using a Gene Pulser™ apparatus (Bio-Rad) as follows: 4 × 106 NIH-3T3 cells in complete medium were electroporated by applying a voltage of 0.25 kV and a capacitance of 960 microfarads. In each transfection experiment the cell number of each well was controlled by both protein concentration assays and cell counting, and variation never exceeded 10%. pRSV-CAT was used in each transfection as an internal control of transfection efficiency. Cell treatment with signal-transducing agents started 30 h after transfection and was carried out for 16 h in medium without serum. Thereafter, cells were harvested, extracts were prepared, and CAT activity was measured as detailed by Sambrook et al.(43Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), according to the modifications described in (46Pothier F. Ouellet M. Julien J-P. Guerin S.L. DNA Cell Biol. 1992; 11: 83-90Google Scholar). Experiments were performed in duplicate and repeated a minimum of three times.RNase Protection and RT-PCR AnalysisTotal RNA was prepared from InR1-G9 cells transfected with p[-1.1]GLU-CAT and either pRC-CMV or pCMV-EVX1 by the guanidine/cesium chloride method(45Ausubel F.A. Brent R. Kingstone R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing and Wiley-Interscience, New York, NY1990Google Scholar). RNase protection analysis was performed using the RPA II kit (Ambion, Austin, TX) following the manufacturer's instructions. As a probe, an antisense strand RNA provided by Ambion (CAT-Direct) was used. RT-PCR was performed exactly as reported in (47Gherzi R. Ponassi M. Gaggero B. Zardi L. FEBS Lett. 1995; 369: 335-339Google Scholar).RESULTSExpression of the EVX1 Protein and Production of Monoclonal AntibodiesEVX1 was expressed in the baculovirus system and purified to near homogeneity (Fig. 1A), as described under "Experimental Procedures." When analyzed by SDS-polyacrylamide gel electrophoresis, the recombinant protein showed an apparent molecular mass higher (62 kDa) than predicted by its amino acid sequence (42.5 kDa). Mouse monoclonal antibodies were prepared against EVX1 as described under "Experimental Procedures." The antibody used in this study (mAb B6-41) is an IgG1 that strongly reacts in Western blot with EVX1 in total nuclear lysates of Sf9 cells infected with the recombinant virus Baculogold/EVX1. No immunoreactivity to HOXB7, HOXC6, HOXD4, and HOXA10 in the nuclear lysates of Sf9 cells infected with the corresponding recombinant viruses was detected (Fig. 1A and data not shown). Furthermore, nuclei of Sf9 cells infected with the Baculogold/EVX1 recombinant virus showed a bright staining in immunofluorescence when reacted with mAb B6-41, while nuclei of Sf9 cells either noninfected or infected with Baculogold/HOXB7, HOXC6, HOXD4, or HOXA10, were negative (data not shown).In order to determine whether mAb B6-41 is able to recognize the fully processed EVX1 expressed in human cells, HeLa cells were transiently transfected with the expression construct pCMV-EVX1 (see "Experimental Procedures"). Both in immunofluorescence and immunohistochemistry assays nuclei of approximately 20% of HeLa cells showed a strong staining when reacted with mAb B6-41, while cells transiently transfected with pRC-CMV were negative (Fig. 1, B and C).EVX1 Represses Basal and Activated Transcription from Several PromotersTo investigate the transcriptional properties of the human EVX1 in vivo we transiently cotransfected glucagonoma cells InR1-G9 with an EVX1 expression vector (pCMV-EVX1) together with two plasmids (p[-1.1]GLU-CAT and p[-0.35]GLU-CAT) in which the transcription of the reporter CAT is directed by regions of the rat proglucagon gene promoter(36Drucker D.J. Philippe J. Jepeal L. Habener J.F. J. Biol. Chem. 1987; 262: 15659-15665Google Scholar, 37Knepel W. Chafitz J. Habener J.F. Mol. Cell. Biol. 1990; 10: 6799-6804Google Scholar, 38Gherzi R. Fehmann H.-C. Volz A. Ponassi M. Göke B. Exp. Cell Res. 1995; 218: 460-468Google Scholar). p[-1.1]GLU-CAT contains 1.1 kilobases of the promoter sequence(38Gherzi R. Fehmann H.-C. Volz A. Ponassi M. Göke B. Exp. Cell Res. 1995; 218: 460-468Google Scholar), which includes, between positions -503 and -487, 2EMBL accession number to the sequence: Z35161. the element ANATTANNNNNTAATNG (which is very similar to the consensus TNATTANNNNNTAATNG found to be bound with high affinity by the human recombinant EVX1). 3Antonio Daga and G. Corte, submitted for publication. On the contrary, p[-0.35]GLU-CAT does not contain this consensus sequence. Homeobox proteins have been reported to play important roles in cell-specific gene transcription in pancreatic islet cells(48Karlsson O. Thor S. Norberg T. Ohlsson H. Edlund T. Nature. 1990; 344: 879-882Google Scholar, 49Miller C.P. McGehee Jr., E. Habener J.F. EMBO J. 1994; 13: 1145-1156Google Scholar, 50Leonard J. Peers B. Johnson T. Ferreri K. Lee S. Montminy M.R. Mol. Endocrinol. 1993; 7: 1275-1283Google Scholar). Thus, it was not surprising to find a putative EVX1 DNA-binding site in the proglucagon gene promoter. As previously reported, p[-1.1]GLU-CAT and p[-0.35]GLU-CAT direct high levels of CAT transcription in InR1-G9 cells, p[-0.35]GLU-CAT being less effective than p[-1.1]GLU-CAT(38Gherzi R. Fehmann H.-C. Volz A. Ponassi M. Göke B. Exp. Cell Res. 1995; 218: 460-468Google Scholar). As shown in Fig. 2A and inFig. 4, the basal transcriptional activity of either p[-1.1]GLU-CAT or p[-0.35]GLU-CAT was approximately 90% reduced when each of the reporter plasmids was cotransfected with the expression plasmid pCMV-EVX1 in InR1-G9 cells in comparison with either the pRC-CMV alone or the expression plasmid containing the HOXD4 cDNA (pCMV-HOXD4; see "Experimental Procedures"). These observations, together with results presented below (seeFig. 6), suggest that EVX1 represses transcription directed by a promoter either containing or not containing its high affinity DNA target sites.Figure 2:EVX1 represses basal and activated transcriptional activity of the rat proglucagon gene promoter in transiently transfected InR1-G9 cells. A, cells were transiently transfected in suspension using the DEAE-dextran technique (see "Experimental Procedures") with 10 μg of the reporter plasmid p[-1.1]GLU-CAT and 10 μg of the effector plasmids indicated. 30 h later cells were changed to serum-free medium with or without 20 μM forskolin. After a further 16 h, cells were harvested, and the CAT activity present in aliquots of cell extracts was measured, as detailed under "Experimental Procedures." The results are the average (± S.E.) of five independent experiments (performed in duplicate). In the lower part of the panel, a representative autoradiogram of one experiment (performed in duplicate) is shown. B, cells were transfected with 10 μg of the reporter plasmid p[-1.1]GLU-CAT and 10 μg of the effector plasmids indicated. 30 h later cells were changed to serum-free medium with or without 160 nM phorbol 12-myristate 13-acetate. After a further 16 h, cells were harvested, and the CAT activity present in aliquots of cell extracts was measured, as detailed under "Experimental Procedures." In the lower part of the panel, a representative autoradiogram of one experiment (performed in duplicate) is shown.View Large Image Figure ViewerDownload (PPT)Figure 4:EVX1 represses the transcriptional activity of several cell-specific and viral promoter/enhancer sequences in several different transiently transfected cells. Indicated cells were transiently transfected (detailed under "Experimental Procedures") with 10 μg of the reporter plasmids indicated and 10 μg of the pCMV-EVX1 expression plasmid. 46 h later, cells were harvested, and the CAT activity present in aliquots of cell extracts was measured as detailed under "Experimental Procedures." Results represent the average (± S.E.) of three independent experiments performed in duplicate.View Large Image Figure ViewerDownload (PPT)Figure 6:EVX1 represses the transcriptional activity of a minimal TATA-less promoter in transiently transfected InR1-G9 and NIH-3T3 cells. Cells were transiently transfected in suspension using the DEAE-dextran technique with 10 μg of the reporter construct and 10 μg of effector plasmids, as indicated. 46 h after the transfection, cells were harvested, and the CAT activity present in aliquots of cell lysates was assayed, as detailed under "Experimental Procedures." Results represent the average (± S.E.) of three independent experiments (performed in duplicate). In the lower part of the figure, a representative autoradiogram of one experiment (performed in duplicate) for each cell line is shown.View Large Image Figure ViewerDownload (PPT)Next we investigated whether transcriptional repression is exerted by EVX1 at any concentration of the transfected expression vector. As shown in Fig. 3, repression by EVX1 is a concentration-dependent phenomenon in a range of transfected pCMV-EVX1 vector from 0.1 to 8 μg.Figure 3:EVX1 functions as a transcriptional repressor in a concentration-dependent manner. Subconfluent InR1-G9 cells were transfected in suspension using the DEAE-dextran technique with 10 μg of the reporter plasmid p[-1.1]GLU-CAT and different amounts (as indicated) of pCMV-EVX1. 46 h after transfection, cells were harvested, and aliquots of lysates were assayed for CAT activity as detailed under "Experimental Procedures." The results are the average (± S.E.) of three independent experiments performed in duplicate.View Large Image Figure ViewerDownload (PPT)It has been reported that the mouse Evx 1 activates the chicken Tenascin-C promoter in transfected cells by interacting with a TRE(30Jones F.S. Chalepakis G. Gruss P. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2091-2095Google Scholar). The rat proglucagon gene promoter contains binding sites for transcription factors belonging to the AP1 and CREB families and is regulated by protein kinase A and protein kinase C activators in pancreatic cell lines(36Drucker D.J. Philippe J. Jepeal L. Habener J.F. J. Biol. Chem. 1987; 262: 15659-15665Google Scholar, 37Knepel W. Chafitz J. Habener J.F. Mol. Cell. Biol. 1990; 10: 6799-6804Google Scholar, 38Gherzi R. Fehmann H.-C. Volz A. Ponassi M. Göke B. Exp. Cell Res. 1995; 218: 460-468Google Scholar). The transcriptional activation of p[-1.1]GLU-CAT by forskolin (Fig. 2A) and phorbol 12-myristate 13-acetate (Fig. 2B) was approximately 9O% reduced by cotransfection of pCMV-EVX1 in comparison with pRC-CMV alone. Similar results were obtained using β-TC1 insulinoma cells (not shown).To investigate whether the transcriptional repression is restricted to the specific promoter we used in the above described experiments, other reporter plasmids containing mammalian and viral promoter/enhancer regions were tested in different mammalian cell lines. The cotransfection of pCMV-EVX1 with pRSV-CAT, pCAT-promoter, and p7B2-I-CAT, containing 524 bp of the 3′ LTR of the Rous sarcoma virus (39Gorman C.M. Merlino G.T. Willingham M.C. Pastan I. Howard B.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6777-6781Google Scholar), 202 bp of the SV40 promoter(51Rosenthal N. Berger S.L. Kimmel A.R. Methods in Enzymology. 152. Academic Press, Inc., San Diego1987: 704-720Google Scholar), and 1.5 kilobases of the 5′-flanking region plus 1.5 kilobases of the first intron of the human 7B2 gene (which encodes a neuroendocrine molecular chaperone expressed in insulinoma and glucagonoma cells)(52Gherzi R. Fehmann H.-C. Eissele R. Göke B. Exp. Cell Res. 1994; 213: 20-27Google Scholar), respectively, decreased the transcriptional activity of the reporter by 50-90% in different cell lines (Fig. 4, and data not shown). These results indicate that the transfected EVX1 exerts its transcriptional repressor activity in cells either expressing or not expressing the endogenous EVXI and that this effect is not promoter-specific.The observed repression of CAT activity by EVX1 corresponds to a decrease in CAT mRNA, as measured either by RNase protection analysis or by RT-PCR experiments (Fig. 5
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