Maf Transcriptionally Activates the Mouse p53Promoter and Causes a p53-dependent Cell Death
2000; Elsevier BV; Volume: 275; Issue: 24 Linguagem: Inglês
10.1074/jbc.m000921200
ISSN1083-351X
AutoresTracy K. Hale, Colleen J. Myers, Rupa Maitra, T Kolzau, Makoto Nishizawa, Antony W. Braithwaite,
Tópico(s)RNA Research and Splicing
ResumoAn increase in the level of the tumor suppressor protein p53 can induce cell cycle arrest or cell death. Although mechanisms for regulating the life span of p53 have been described, there is growing evidence that transcriptional regulation of thep53 gene contributes significantly to controlling p53 protein levels and therefore the fate of a cell. However, the signal transduction pathways that lead to transcriptional activation of thep53 gene are poorly understood. The oncoprotein v-Maf and its cellular counterparts belong to the large combinatorially complex basic leucine zipper family of transcription factors, which include the AP1 family. To date few cellular targets of c-Maf have been identified. It is demonstrated here that v-Maf can bind as a homodimer to a variant Maf recognition element located between −66 and −54 upstream in the mouse p53 promoter. V-Maf and its cellular counterparts are shown to activate p53 expression through this site. The ability of v-Maf to activate p53 expression is modulated by AP1 family members. In addition, overexpression of v-Maf in primary cells leads to a p53-dependent cell death. Thus, Maf and members of the AP1 family are able to regulate p53expression through this site in the p53 promoter. An increase in the level of the tumor suppressor protein p53 can induce cell cycle arrest or cell death. Although mechanisms for regulating the life span of p53 have been described, there is growing evidence that transcriptional regulation of thep53 gene contributes significantly to controlling p53 protein levels and therefore the fate of a cell. However, the signal transduction pathways that lead to transcriptional activation of thep53 gene are poorly understood. The oncoprotein v-Maf and its cellular counterparts belong to the large combinatorially complex basic leucine zipper family of transcription factors, which include the AP1 family. To date few cellular targets of c-Maf have been identified. It is demonstrated here that v-Maf can bind as a homodimer to a variant Maf recognition element located between −66 and −54 upstream in the mouse p53 promoter. V-Maf and its cellular counterparts are shown to activate p53 expression through this site. The ability of v-Maf to activate p53 expression is modulated by AP1 family members. In addition, overexpression of v-Maf in primary cells leads to a p53-dependent cell death. Thus, Maf and members of the AP1 family are able to regulate p53expression through this site in the p53 promoter. basic leucine zipper Maf recognition element 12-O-tetradecanoate-13-acetate responsive element cAMP responsive element p53 factor 1 chloramphenicol acetyltransferase polypeptide chain elongation factor 1α cytomegalovirus in vitro translation electrophoretic mobility shift assay dithiothreitol rat embryo fibroblasts interleukin-4 T helper 2 The v-maf oncogene was identified as the transforming gene of the avian retrovirus AS42 and was first isolated from a spontaneous musculoaponeurotic fibrosarcoma of chicken (1.Nishizawa M. Kataoka K. Goto N. Fujiwara K. Kawai S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7711-7715Crossref PubMed Scopus (220) Google Scholar). Inoculation of this virus into newborn chickens induced tumors that were pathologically indistinguishable from the original tumor and when introduced into chicken embryo fibroblasts led to cellular transformation (1.Nishizawa M. Kataoka K. Goto N. Fujiwara K. Kawai S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7711-7715Crossref PubMed Scopus (220) Google Scholar, 2.Kawai S. Goto N. Kataoka K. Saegusa T. Shinno-Kohno H. Nishizawa M. Virology. 1992; 188: 778-784Crossref PubMed Scopus (37) Google Scholar). In human carcinogenesis, c-MAF was shown to be overexpressed in 25% of multiple myelomas tested due to the translocation of c-MAF to the IgH locus (3.Chesi M. Bergsagel P.L. Shonukan O.O. Martelli M.L. Brents L.A. Chen T. Schrock E. Ried T. Kuehl W.M. Blood. 1998; 91: 4457-4463Crossref PubMed Google Scholar). A cellular homologue of v-maf, c-MAF type I, was identified in chickens. Comparison of the amino acid sequences revealed that the only differences between chicken c-MAF and v-Maf are the substitution at position 257 of a methionine residue for valine and the presence of the viral Gag sequence at the amino terminus (4.Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar). Comparison of the mouse, rat, and chicken c-Maf sequences shows them to be very highly conserved at the primary amino acid level. c-Maf was found to be but one member of an extended multigene family, members of which all share homology in a basic leucine zipper (b-Zip)1 motif, and hence form a distinct subgroup of the b-Zip family of transcription factors (5.Kerppola T.K. Curran T. Oncogene. 1994; 9: 675-684PubMed Google Scholar). Members of the Maf family are themselves divided into two subgroups as follows: the large Maf proteins c-Maf (4.Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar), MafB (6.Sakai M. Imaki J. Yoshida K. Ogata A. Matsushima-Hibiya Y. Kuboki Y. Nishizawa M. Nishi S. Oncogene. 1997; 14: 745-750Crossref PubMed Scopus (83) Google Scholar), and neural retina-specific leucine zipper (7.Swaroop A. Xu J. Pawar H. Jackson A. Skolnick C. Agarwal N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 266-270Crossref PubMed Scopus (265) Google Scholar), and the small Maf proteins MafG (8.Kataoka K. Igarashi K. Itoh K. Fujiwara K.T. Noda M. Yamamoto M. Nishizawa M. Mol. Cell. Biol. 1995; 15: 2180-2190Crossref PubMed Scopus (199) Google Scholar), MafF, and MafK (9.Fujiwara K.T. Kataoka K. Nishizawa M. Oncogene. 1993; 8: 2371-2380PubMed Google Scholar). Consistent with being a member of the b-Zip family of transcription factors, c-Maf contains a typical acidic type transactivation domain in the amino terminus, and a carboxyl terminus consisting of a basic DNA binding domain and an adjacent leucine-rich region. This domain shares 20–30% homology with those of other b-Zip proteins (4.Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar). The leucine motif is somewhat atypical for b-Zip proteins as position 5 in the heptad repeat of hydrophobic residues is occupied by a tyrosine rather than a leucine residue. Maf is able to form homodimers through the leucine zipper motif, bind DNA, and transactivate promoter/reporter constructs in a sequence-specific manner (4.Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar, 10.Kataoka K. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 700-712Crossref PubMed Google Scholar). Interestingly, the ability to form homodimers appears to be critical for v-Maf to transform primary chicken embryo fibroblasts (4.Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar). There are two known DNA consensus sequences bound by Maf homodimers, a 13-base pair 12-O-tetradecanoate-13-acetate-responsive element (TRE) type Maf recognition element (MARE) and a 14-base pair cAMP-responsive element (CRE) type MARE (10.Kataoka K. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 700-712Crossref PubMed Google Scholar). The former MARE contains an AP1 (TGCTGACTCAGCA)-binding site, and the latter harbors a CRE (TGCTGACGTCAGCA) bound by members of CREB/ATF family of transcription factors (10.Kataoka K. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 700-712Crossref PubMed Google Scholar). The various dimers formed among Maf family members and other b-Zip proteins have been shown to bind to MAREs and MARE-like sequences with varying affinities and with varying transactivation potentials (10.Kataoka K. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 700-712Crossref PubMed Google Scholar, 11.Kataoka K. Noda M. Nishizawa M. Oncogene. 1996; 12: 53-62PubMed Google Scholar), creating a combinatorially complex and sensitive network of b-Zip transcription factors. c-MAF is widely expressed to modest levels in adult and embryonic tissues and highly expressed in some developing skeletal tissues (6.Sakai M. Imaki J. Yoshida K. Ogata A. Matsushima-Hibiya Y. Kuboki Y. Nishizawa M. Nishi S. Oncogene. 1997; 14: 745-750Crossref PubMed Scopus (83) Google Scholar). Currently, there are few cellular genes known to be transcriptionally regulated by Maf. One is the interleukin-4 (IL-4) gene (12.Ho I.-C. Hodge M.R. Rooney J.W. Glimcher L.H. Cell. 1996; 85: 973-983Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). IL-4 is known to affect T helper cell subset ratios. The expression of c-Maf in the immune system has been shown to be specific to the T helper 2 (Th2) subset of T helper cells. Maf binds to a MARE-like element in the IL-4 promoter, resulting in increased expression and secretion of IL-4 by Th2 cells, causing the preferential differentiation of naive T cells into Th2 cells (12.Ho I.-C. Hodge M.R. Rooney J.W. Glimcher L.H. Cell. 1996; 85: 973-983Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). Another known target gene for c-Maf is the L7 gene, which is expressed in all adult cerebellar Purkinje cells as well as in cells in certain functional domains of the developing nervous system. Mouse c-MAF was shown to activate transcription from two sites within the L7promoter in mouse cerebellar cells (13.Kurschner C. Morgan J.I. Mol. Cell. Biol. 1995; 15: 246-254Crossref PubMed Scopus (69) Google Scholar). In addition, induction of lens differentiation in the chicken was shown to be triggered by a lens-specific Maf that regulates multiple genes expressed in the lens (14.Ogino H. Yasuda K. Science. 1998; 280: 115-118Crossref PubMed Scopus (233) Google Scholar). A data base search of sequence segments differing from the MARE by fewer than 5 base pairs revealed several other possible target genes for Maf, including the placental-type glutathioneS-transferase gene and the proto-oncogenes pim-1and c-erbB, although none of these has been confirmed (10.Kataoka K. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 700-712Crossref PubMed Google Scholar). Another possible gene target for Maf, which was also identified by the data base search, is the p53 tumor suppressor gene (10.Kataoka K. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 700-712Crossref PubMed Google Scholar). Between −66 and −54 (all numbering is relative to the start site of transcription) in the mouse p53 promoter is a sequence that differs from the TRE-type MARE consensus sequence by only 2 base pairs. This region is completely conserved between the mouse and humanp53 promoters (Fig. 1, see Refs. 15.Bienz-Tadmor B. Zakut-Houri R. Libresco S. Givol D. Oren M. EMBO J. 1985; 4: 3209-3213Crossref PubMed Scopus (103) Google Scholar and 16.Tuck S.P. Crawford L. Mol. Cell. Biol. 1989; 9: 2163-2172Crossref PubMed Scopus (105) Google Scholar). The AP1 site within the p53 MARE (which differs by 1 base pair to the AP1 consensus sequence) has been designated the p53 factor 1 (PF1) site (17.Ginsberg D. Oren M. Yaniv M. Piette J. Oncogene. 1990; 5: 1285-1290PubMed Google Scholar). This site is important in regulating p53 promoter activity (18.Kirch H.-C. Flaswinkel S. Rumpf H. Brockmann D. Esche H. Oncogene. 1999; 18: 2728-2738Crossref PubMed Scopus (117) Google Scholar, 19.Schreiber M. Kolbus A. Piu F. Szabowski A. Mohle-Steinlein U. Tian J. Karin M. Angel P. Wagner E.F. Genes Dev. 1999; 13: 607-619Crossref PubMed Scopus (467) Google Scholar) and has been shown to bind several activities, some of which contain the AP1 components, c-Fos and c-Jun (18.Kirch H.-C. Flaswinkel S. Rumpf H. Brockmann D. Esche H. Oncogene. 1999; 18: 2728-2738Crossref PubMed Scopus (117) Google Scholar, 19.Schreiber M. Kolbus A. Piu F. Szabowski A. Mohle-Steinlein U. Tian J. Karin M. Angel P. Wagner E.F. Genes Dev. 1999; 13: 607-619Crossref PubMed Scopus (467) Google Scholar). These observations led us to test the possibility that Maf could transcriptionally regulate the p53 gene. We demonstrate that v-Maf binds to the MARE-like site in the mouse p53 promoter and transactivates the p53 gene in a sequence-specific manner. Results show that overexpression of v-Maf causes cells to die in a p53-dependent manner. Furthermore, the ability of v-Maf to activate p53 expression is modulated by members of the AP1 family. Therefore, we conclude that a cellular target of the Maf transcription factor is the p53 gene and suggest that Maf has the potential to act as a “tumor suppressor” since it is able to induce cell death through its control of p53expression. The plasmid pCAT3M contains the bacterial chloramphenicol acetyltransferase (CAT) gene but no eukaryotic promoter sequences upstream of this gene (20.Laimins L.A. Gruss P. Pozatti R. Khoury G. J. Virol. 1984; 49: 183-189Crossref PubMed Google Scholar). pAACAT contains −224 to +116 of the mouse p53 promoter blunt-end cloned in front of the CAT gene in pCAT3M (15.Bienz-Tadmor B. Zakut-Houri R. Libresco S. Givol D. Oren M. EMBO J. 1985; 4: 3209-3213Crossref PubMed Scopus (103) Google Scholar). The expression plasmids v-mafPT/pEFBssHII,ND5/pEFBssHII,CD3/pEFBssHII,CD4/pEFBssHII,R22E/pEFBssHII,L2PL4P/pEFBssHII,MAFB/pEFBssHII, c-MAF type I/pEFBssHII, and c-MAF type II/pEFBssHII were used to express the various Maf proteins and v-Maf mutants under the control of the human polypeptide chain elongation factor 1α (EF) promoter. These constructs are based on the expression vector pEFBssHII and are described in detail in Kataoka et al. (11.Kataoka K. Noda M. Nishizawa M. Oncogene. 1996; 12: 53-62PubMed Google Scholar). pEF/luc expresses the firefly luciferase gene under control of the EF promoter and was created by cloning the firefly luciferase gene into the BssHII site of pEFBssHII. In the cytomegalovirus (CMV)-based plasmids, transcription is controlled by the immediate early enhancer-promoter of human CMV. pCMVE1b58 kDa expresses the adenovirus 2 E1b 58-kDa protein (21.Ridgway P. Soussi T. Braithwaite A.W. J. Virol. 1994; 68: 7178-7187Crossref PubMed Google Scholar). pCMVjun expresses rat c-Jun, and pCMVfosexpresses c-Fos (22.Cohen D.R. Ferreira P.C.P. Gentz R. Franza Jr., B.R. Curran T. Genes Dev. 1989; 3: 173-184Crossref PubMed Scopus (223) Google Scholar). The construction of pCMVmfra2, which expresses mouse FRA-2, is described in McHenry et al. (23.McHenry J.Z. Leon A. Matthaei K.I. Cohen D.R. Oncogene. 1998; 17: 1131-1140Crossref PubMed Scopus (21) Google Scholar). pCMVneo was used for selecting cells in the colony formation assay as it contains the Tn5 gene that encodes resistance to the neomycin family of antibiotics (24.Southern P.J. Berg P. J. Mol. Appl. Genet. 1982; 1: 327-341PubMed Google Scholar). pBR322 was used as the control plasmid in these assays. pCMVE1a (25.Morris G.F. Mathews M.B. J. Virol. 1991; 65: 6397-6406Crossref PubMed Google Scholar) contains a genomic fragment of adenovirus early region 1 that encodes all the E1a proteins. pCMV280-390 encodes only the carboxyl-terminal region of p53 from amino acids 280 to 390 (26.Reed M. Wang Y. Mayr G. Anderson M.E. Schwedes J.F. Tegtmeyer P. Gene Exp. 1993; 3: 95-107PubMed Google Scholar). In order to create the reporter plasmid, pAAmut, that contains 4-base pair substitutions in the p53 MARE-like site present within pAACAT, the technique of inverse polymerase chain reaction was used (27.Imai Y. Matsushima Y. Sugimura T. Terada M. Nucleic Acids Res. 1991; 19: 2785Crossref PubMed Scopus (314) Google Scholar). The following primer pairs were synthesized: 5′-TCCTC AAGTC CCGCC TCCAT TTC-3′ and 5′-GTCCT GATTC TCCCT GAGAT GTTGC-3′ (substitutions are underlined). Polymerase chain reaction was performed with 1 cycle at 96 °C for 3 min, followed by 25 cycles at 96 °C for 1 min, 67 °C for 15 s and 72 °C for 5 min, and then 1 cycle of 72 °C for 9 min in 50 μl of reaction mixture containing 20 mm Tris-HCl, pH 8.8, 10 mm KCl, 10 mm (NH4)2SO4, 2–10 mm MgSO4, 0.1% Triton X-100, 100 mm each of dNTPs, 10 ng of the template pAACAT, 40 pmol each of the primers, and 0.5 units of Vent DNA polymerase (New England Biolabs). The amplified linear DNA was agarose gel-purified, T4polynucleotide kinase-treated, and then a portion was self-ligated in 15 mm Tris-HCl, pH 7.8, 5 mm MgCl2, 5 mm DTT, 0.25 mm ATP, 30 mm KCl, 1 mm hexamine cobalt chloride, and 8 units of T4DNA ligase at 14 °C for 16 h and then used to transform competent Escherichia coli DH5 α cells. The substitutions within pAAmut were confirmed by sequencing. pRAM-GEM is a subclone of the v-maf gene based on the pGEM-4 vector, which was used to in vitro translate (IVT) v-Maf PT (4.Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar). The structure and construction of the deletion and point mutants of the v-maf PT gene used are based on pRAM-GEM and have been described previously (4.Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar). The plasmids pSP6jun and pSP6fos (kindly provided by Dr. Donna Cohen, JCSMR, Canberra, Australia) were used to IVT rat c-Jun and c-Fos proteins, respectively. All plasmids were transcribed and translated in vitro using the TNT-coupled Wheat Germ Extract kit (Promega). The sequence of Oligomer M, which contains the mouse p53 MARE and flanking p53 promoter sequences, is shown in Fig.2 a. Also shown in Fig.2 a is the sequence of Oligomer Mmut that contains a mutant MARE. The sequence of Oligomer 11, which contains a MARE known to bind both the v-Maf PT and c-Jun homodimers and the c-Fos/c-Jun heterodimer (10.Kataoka K. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 700-712Crossref PubMed Google Scholar), is 5′-GGGAG CTCGG AATTG ATGAC TCATC ATTAC TC-3′. Binding reactions were performed in a volume of 15 μl containing 6 μl of IVT product, 0.1–0.5 μg of poly(dI·dC)·poly(dI·dC), 20 mm HEPES-KOH, pH 7.9, 1 mm EDTA, 20 mm KCl, 4 mm MgCl2, 5 mm DTT. Reactions were allowed to proceed for 15 min at room temperature. 1 × 104 cpm of target oligomer, end-labeled with [32P]dCTP, was added to the binding reaction and incubated at room temperature for a further 15 min. Following this, 1.5 μl of 10× DNA loading dye was added, and the binding reaction was immediately loaded onto a pre-electrophoresed 5% polyacrylamide gel. After electrophoresis, gels were fixed in 10% acetic acid for 10 min, dried for 30 min at 80 °C, and exposed to Kodak X-Omat AR film at −70 °C, usually for 18 h. The mouse NIH3T3 and human HeLa cell lines were maintained at 37 °C, 10% CO2 in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. Rat embryo fibroblasts (REFs) were prepared from 15- to 17-day-old Wistar rat embryos as described previously (28.Bellett A.J.D. Younghusband H.B. J. Cell. Physiol. 1979; 101: 33-48Crossref PubMed Scopus (37) Google Scholar) and routinely cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. REFs were used up to passage six and then replaced with new cells. For all cell types, 2.5 × 105 cells were seeded into 35-mm dishes and transfected with FuGENE 6 (Roche Molecular Biochemicals) after 18 h. For each dish transfected the total amount of DNA was kept at 4 μg (except for the transfection performed in Fig. 9 c, where the total amount of DNA was 4.5 μg). The amounts of reporter and expression plasmids used are indicated in the figure legends, and sonicated salmon sperm DNA was used as carrier DNA to keep the total amount of DNA constant. The ratio of DNA (μg) to the volume of FuGENE 6 Reagent (μl) used was kept at 2:3 for each transfection. Sixty hours after transfection cells were washed twice in ice-cold phosphate-buffered saline and harvested by scraping into 1 ml of ice-cold phosphate-buffered saline, centrifuged, then resuspended in 100 μl of 0.25 m Tris-HCl, pH 7.5. Extracts of transfected cells were then prepared by three rounds of freezing and thawing followed by centrifugation for 15 min at 12,000 × g and 4 °C to remove cellular debris. The supernatant was then heated to 65 °C for 10 min to inactivate a CAT inhibitor (29.Sleigh M.J. Anal. Biochem. 1986; 156: 251-256Crossref PubMed Scopus (297) Google Scholar). Cell lysates were then normalized for protein content using the BCA Protein Assay Reagent Kit (Pierce), and CAT activities were determined essentially as described in Sleigh (29.Sleigh M.J. Anal. Biochem. 1986; 156: 251-256Crossref PubMed Scopus (297) Google Scholar). Extracts of transfected cells were prepared as described above (see “CAT Assay”). Western immunoblot analysis was performed according to a standard procedure (30.Harlow E. Lane D. Immunoblotting: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 471-510Google Scholar). Briefly, protein fractionation was performed by 10% SDS-polyacrylamide gel electrophoresis using 11 μg of cell extract per lane. Extracts were diluted in loading buffer containing 40% glycerol, 4% SDS, 50 mm Tris-HCl, pH 6.8, 80 μm DTT, 0.08% bromphenol blue, and heated to 100 °C for 5 min before loading. Gels were transferred to polyvinylidene difluoride membrane (Millipore) in transfer buffer (192 mm glycine, 25 mm Tris, 0.05% SDS, 20% methanol). The filters were incubated for 1 h at room temperature in 1% non-fat dried milk and TBS buffer (20 mm Tris-HCl, pH 7.9, 0.5 m NaCl). After washing in TTBS buffer (20 mm Tris-HCl, pH 7.9, 0.5 mNaCl, 0.35% Tween), the filters were incubated for 1 h in TTBS buffer containing a 1:50 dilution of the commercial monoclonal p53 antibody DO-7 (Dako). After three washes, blots were incubated with anti-mouse antibodies conjugated with alkaline phosphatase for 1 h in TTBS. After further washes, detection of bound antibody was performed with the Immun-star Chemiluminescent Protein Detection System (Bio-Rad). To investigate the survival and growth of cells, 1.5 × 105 REFs were cotransfected, with the appropriate plasmid DNA, using FuGENE 6 Reagent (as described above). pCMVneo was included as one of the transfected plasmids at an amount of 0.2 μg to selectively permit survival of the transfected cells in the presence of the neomycin analogue G418 (24.Southern P.J. Berg P. J. Mol. Appl. Genet. 1982; 1: 327-341PubMed Google Scholar). Forty eight hours post-transfection REFs were maintained in medium containing 200 μg/ml G418 (Life Technologies, Inc.) for 21 days. Cells were then fixed and stained with 0.1% crystal violet, 20% ethanol. The extent of cell survival was reflected by the number of G418-resistant colonies present. To determine whether v-Maf binds to the MARE-like site within the mouse p53 promoter, EMSAs were performed where an end-labeled synthetic double-stranded oligonucleotide, Oligomer M (Fig.2 a), was mixed with IVT v-Maf PT. Oligomer M spans 43 base pairs of the mouse p53 promoter from −81 to −39, and this region encompasses the MARE-like site situated between −66 and −54. Whereas v-Maf PT is a v-Maf protein that lacks the first 18 amino acids of the amino terminus, however, it has been shown to be as active as full-length v-Maf and c-MAF in DNA binding, transactivation and cell transformation abilities (4.Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar, 10.Kataoka K. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 700-712Crossref PubMed Google Scholar). Results (Fig. 2 b) showed that IVT v-Maf PT bound to Oligomer M. Furthermore, four nucleotide substitutions within the MARE-like site of Oligomer M (Oligomer Mmut, Fig. 2 a) abolished the ability of v-Maf PT to bind to this oligomer (Fig. 2 b). Therefore, we conclude that v-Maf binds to the MARE-like site located from −66 to −54 in the mouse p53 promoter. To determine whether the binding of v-Maf to the p53 promoter results in transcriptional activation, the plasmid, v-mafPT/pEFBssHII, expressing v-Maf PT under control of the human EF promoter (31.Kataoka K. Fujiwara K.T. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 7581-7591Crossref PubMed Scopus (197) Google Scholar) was cotransfected into NIH3T3 cells with the reporter construct, pAACAT. pAACAT contains the region of the mouse p53 promoter from −224 to +116 cloned in front of the reporter CAT gene. This 340-base pair region of thep53 promoter, contained within pAACAT, has been shown to be sufficient to drive maximal expression of the mouse p53promoter (15.Bienz-Tadmor B. Zakut-Houri R. Libresco S. Givol D. Oren M. EMBO J. 1985; 4: 3209-3213Crossref PubMed Scopus (103) Google Scholar, 32.Hale T.K. Braithwaite A.W. Nucleic Acids Res. 1995; 23: 663-669Crossref PubMed Scopus (34) Google Scholar). Results of an experiment (Fig.3 a) in which pAACAT activity was measured after titration of v-mafPT/pEFBssHII showed that v-Maf PT transactivated the p53 promoter in a dose-dependent manner. At the ratio of 3:1 (v-mafPT/pEFBssHII:pAACAT), over six independent experiments, v-Maf PT increased p53 promoter activity between 3- and 10-fold (data not shown), although typically activation was between 3- and 4-fold as shown in Fig. 3 a. In contrast, expression of v-Maf PT had little effect on pCAT3M, a “promoter-less” CAT construct into which the 340-base pairp53 promoter fragment was cloned to create pAACAT. Similar experiments were then performed in REFs and HeLa cells (Figs.3 b and 3 c). At the ratio of 3:1 (v-mafPT/pEFBssHII:pAACAT), v-Maf PT activatedp53 expression 17-fold in REFs (Fig. 3 b), whereas in HeLa cells expression of v-Maf PT increased p53 promoter activity by approximately 2.5-fold (Fig. 3 c). Similar results have been obtained in repeat experiments. Therefore, these experiments demonstrate that v-Maf is able to activatep53 expression in at least three different cellular environments, although the level of activation varies. To test if v-Maf requires the MARE-like site located in the mousep53 promoter for transactivation, 4-base pair substitutions were introduced into the MARE-like site within the p53promoter/reporter construct pAACAT. The 4-base pair substitutions are the same as those that abolished the binding of v-Maf PT to the MARE-like site within Oligomer M (Fig. 2 b). This mutant construct called pAAmut was then tested for its response to v-Maf PT when cotransfected into NIH3T3 cells with v-mafPT/pEFBssHII. In this experiment v-Maf PT transactivated pAACAT about 3-fold, whereas it had no effect on pAAmut (Fig. 4). This activation of thep53 promoter is specific to v-Maf as expression of luciferase or the adenovirus 2 E1b 58-kDa protein has no effect on pAACAT or pAAmut activity (Fig. 4). Consistent with the promoter/reporter experiments, overexpression of v-Maf results in an increase in p53 protein levels (Fig.5). When NIH3T3 cells were transfected with the v-Maf PT expression construct, v-mafPT/pEFBssHII, Western immunoblot analysis demonstrated that 30 h post-transfection, endogenous p53 protein was detectable in cells overexpressing v-Maf PT. In comparison, no p53 protein was detectable in mock-transfected NIH3T3 cells or in cells that had been transfected with a construct expressing the v-Maf PT deletion mutant ND5. This mutant lacks the amino-terminal activation domain (its structure is shown in Fig.6 a) and as demonstrated below cannot transcriptionally activate the p53 promoter (Fig. 6 c).Figure 6Functional domain analysis of v-Maf regulation of p53 expression. a, schematic representation of v-Maf mutant proteins (4.Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar). PT is a nearly full-length version of the v-Maf protein. The amino acid substitutions in the L2PL4P and R22E mutants are shown. H indicates a tract of eight histidine residues, and G indicates three clusters of 8–10 glycine residues. Numbering indicates amino acid position. b, DNA binding abilities of the v-Maf PT mutants. Deletion or substitution mutants of v-Maf PT, as well as v-Maf PT, were translated in vitro and then assayed for their ability to bind to Oligomer M in an EMSA. Arrowsindicate IVT protein-Oligomer M complexes. Lane P refers to the reaction containing only Oligomer M; lane L represents wheat germ extract added to the reaction in place of IVT v-Maf PT.c, NIH3T3 cells were cotransfected with the mousep53 promoter/reporter construct pAACAT (1 μg) and either 3 μg of carrier DNA (mock), v-mafPT/pEFBssHII, or the mutant v-Maf PT expression constructs CD3/ pEFBssHII, CD4/pEFBssHII,ND5/pEFBssHII,R22E/pEFBssHII, orL2PL4P/pEFBssHII. Sixty hours post-transfection cells were harvested and assayed for CAT activity. The promoter-less CAT construct pCAT3M (1 μg) was also cotransfected with 3 μg of either carrier DNA (mock) or v-mafPT/pEFBssHII as a control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) These data show that v-Maf transcriptionally activates the mousep53 promoter and that this transactivation requires the MARE-like site located between −66 to −54 in the mouse p53promoter. As a result of this transactivation, overexpression of v-Maf results in an increase in endogenous p53 protein levels. To determine which functional domains of v-Maf are required for transcriptional activation ofp53 expression, a series of mutants based on v-Maf PT (their structures are summarized in Fig. 6 a) were tested for their ability to bind to the p53 MARE and transcriptionally activate p53 expression. These v-Maf PT mutants were translated in vitro, and EMSAs were performed to test their ability to bind to Oligomer M. Fig.6 b shows that although v-Maf PT binds to its site within Oligomer M as demonstrated above (Fig. 2 b), the v-Maf PT mutants CD3, CD4, and L2PL4P, which have previously been shown not to form homodimers (4.Kataoka K. Nishizawa M. Kawai S. J. Virol. 1993; 67: 2133-2141Crossref PubMed Google Scholar), failed to bind t
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