Interaction of Maf Transcription Factors with Pax-6 Results in Synergistic Activation of the Glucagon Promoter
2001; Elsevier BV; Volume: 276; Issue: 38 Linguagem: Inglês
10.1074/jbc.m104523200
ISSN1083-351X
AutoresNathalie Planque, Laurence Leconte, Frédéric M. Coquelle, Sofia Benkhelifa‐Ziyyat, Patrick Martin, Marie‐Paule Felder‐Schmittbuhl, Simon Saule,
Tópico(s)Diabetes Treatment and Management
ResumoIn the endocrine pancreas, α-cell-specific expression of the glucagon gene is mediated by DNA-binding proteins that interact with the G1 proximal promoter element. Among these proteins, the paired domain transcription factor Pax-6 has been shown to bind to G1 and to transactivate glucagon gene expression. Close to the Pax-6-binding site, we observed the presence of a binding site for a basic leucine zipper transcription factor of the Maf family. In the present study, we demonstrate the presence of Maf family members in the endocrine pancreas that bind to G1 and transactivate glucagon promoter expression. In transient transfection experiments, we found that the transactivating effect on the glucagon promoter was greatly enhanced by the simultaneous expression of Maf transcription factors and Pax-6. This enhancement on glucagon transactivation could be correlated with the ability of these proteins to interact together but does not require binding of Maf proteins to the G1 element. Furthermore, we found that Maf enhanced the Pax-6 DNA binding capacity. Our data indicate that Maf transcription factors may contribute to glucagon gene expression in the pancreas. In the endocrine pancreas, α-cell-specific expression of the glucagon gene is mediated by DNA-binding proteins that interact with the G1 proximal promoter element. Among these proteins, the paired domain transcription factor Pax-6 has been shown to bind to G1 and to transactivate glucagon gene expression. Close to the Pax-6-binding site, we observed the presence of a binding site for a basic leucine zipper transcription factor of the Maf family. In the present study, we demonstrate the presence of Maf family members in the endocrine pancreas that bind to G1 and transactivate glucagon promoter expression. In transient transfection experiments, we found that the transactivating effect on the glucagon promoter was greatly enhanced by the simultaneous expression of Maf transcription factors and Pax-6. This enhancement on glucagon transactivation could be correlated with the ability of these proteins to interact together but does not require binding of Maf proteins to the G1 element. Furthermore, we found that Maf enhanced the Pax-6 DNA binding capacity. Our data indicate that Maf transcription factors may contribute to glucagon gene expression in the pancreas. basic region leucine zipper glutathione S-transferase chloramphenicol acetyltransferase baby hamster kidney long term repeat phosphate-buffered saline 1,4-piperazinediethanesulfonic acid reverse transcriptase-polymerase chain reaction green fluorescent protein enhanced GFP Discosoma striata red protein Pax-6 is a member of the paired domain family of transcription factors and has a specialized homeodomain downstream from the DNA-binding paired domain and upstream from the C-terminal activation domain (1Martin P. Carriere C. Dozier C. Quatannens B. Mirabel M.A. Vandenbunder B. Stehelin D. Saule S. Oncogene. 1992; 7: 1721-1728PubMed Google Scholar, 2Carriere C. Plaza S. Caboche J. Dozier C. Bailly M. Martin P. Saule S. Cell Growth Differ. 1995; 6: 1531-1540PubMed Google Scholar). Pax-6 encodes five proteins through alternative splicing and internal initiations (3Carriere C. Plaza S. Martin P. Quatannens B. Bailly M. Stehelin D. Saule S. Mol. Cell. Biol. 1993; 13: 7257-7266Crossref PubMed Scopus (112) Google Scholar). Three proteins of 48, 46, and 43 kDa contain the paired domain, whereas two proteins of 33 and 32 kDa are devoid of this DNA binding domain. Pax-6 is known to be critical for eye development (4Callaerts P. Halder G. Gehring W.J. Annu. Rev. Neurosci. 1997; 20: 483-532Crossref PubMed Scopus (392) Google Scholar, 5Chow R.L. Altmann C.R. Lang A.R. Hemmati-Brivanlou A. Development. 1999; 126: 4213-4222Crossref PubMed Google Scholar), where it is required for lens differentiation and crystalline gene expression (6Duncan M.K. Kozmik Z. Cveklova K. Piatigorsky J. Cvekl A. J. Cell Sci. 2000; 113: 3173-3185Crossref PubMed Google Scholar, 7Cvekl A. Piatigorsky J. BioEssays. 1996; 18: 621-630Crossref PubMed Scopus (247) Google Scholar). Pax-6 is also required for the development of all pancreatic endocrine cells and of duodenal GIP-positive cells and gastrin- and somatostatin-producing cells in the stomach (8Larsson L.I. St-Onge L. Hougaard D.M. Sosa-Pineda B. Gruss P. Mech. Dev. 1998; 79: 153-1599Crossref PubMed Scopus (117) Google Scholar, 9St-Onge L. Sosa-Pineda B. Chowdhury K. Mansouri A. Gruss P. Nature. 1997; 387: 406-409Crossref PubMed Scopus (664) Google Scholar, 10Sander M. Neubuser A. Kalamaras J. Ee H.C. Martin G.R. German M.S. Genes Dev. 1997; 11: 1662-1673Crossref PubMed Scopus (461) Google Scholar). The α-cell-specific glucagon gene expression in pancreas is conferred by the proximal G1 promoter element (11Phillipe J. Drucker D.J. Knepel W. Jepeal L. Misulovin Z. Habener J.F. Mol. Cell. Biol. 1988; 11: 4877-4888Crossref Scopus (116) Google Scholar). The G1 element contains two AT-rich sequences that are recognized by the homeodomain containing Cdx-2/3, Pax-6, and brain-4 (12Laser B. Meda P. Constant I. Philippe J. J. Biol. Chem. 1996; 271: 28984-28994Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 13Hussain M.A. Lee J. Miller C.P. Habener J.F. Mol. Cell. Biol. 1997; 17: 7186-7194Crossref PubMed Google Scholar, 14Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Pax-6 and Cdx-2/3 have been shown to bind directly to each other and to transactivate synergistically the glucagon gene via their interaction with the G1 element (14Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This effect is even enhanced by interaction of these proteins with the p300 coactivator (15Hussain M.A. Habener J.F. J. Biol. Chem. 1999; 274: 28950-28957Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Fine-tuning of cell type-specific gene expression and lineage-specific cell differentiation seems to be achieved by cooperative and inhibitory interactions of transcription factors. To understand the mechanisms of transcriptional regulation in pancreas, it is important to clarify the regulatory cross-talk that occurs with other transcription factors on the G1 element. Examination of the G1 element nucleotide sequence revealed the presence of a potential binding site (16Kataoka K. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 700-712Crossref PubMed Google Scholar) for a basic region leucine zipper (bZip) domain transcription factor of the Maf family, located close to the Pax-6-binding element. Loss-of-function of the c-Maf bZip1 factor results in defective differentiation of lens fiber cells (17Kawauchi S. Takahashi S. Nakajima O. Ogino H. Morita M. Nishizawa M. Yasuda K. Yamamoto M. J. Biol. Chem. 1999; 274: 19254-19260Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 18Ring B.Z. Cordes S.P. Overbeek P.A. Barsh G.S. Development. 2000; 127: 307-317Crossref PubMed Google Scholar). Ectopic expression of L-Maf/MafA results in conversion of ectodermal cells into lens fibers cells, and this factor is able to activate the promoters of crystalline genes (19Ogino H. Yasuda K. Science. 1998; 280: 115-118Crossref PubMed Scopus (233) Google Scholar). L-Maf/MafA, which is expressed in both lens and retina, displays mitogenic capacity when overexpressed in avian neuroretina cells (20Benkhelifa S. Provot S. Lecoq O. Pouponnot C. Calothy G. Felder-Schmittbuhl M.P. Oncogene. 1998; 17: 247-254Crossref PubMed Scopus (53) Google Scholar). A variety of developmental roles and transcriptional targets has been proposed for Maf transcription factors. The v-maf oncogene is the earliest described member of the family (21Nishizawa M. Kataoka K. Goto N. Fujiwara K.T. Kawai S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7711-7715Crossref PubMed Scopus (220) Google Scholar). Large Maf subfamily members (c-Maf, L-Maf/MafA, MafB, and Nrl) contain an activation domain at the N terminus, whereas small Maf subfamily members (MafF, MafK, and MafG) lack a distinct activation domain (22Blank V. Andrews N.C. Trends Biochem. Sci. 1997; 22: 437-441Abstract Full Text PDF PubMed Scopus (220) Google Scholar). Maf transcription factors share structural similarity both within and outside the basic leucine zipper domain and bind common recognition elements, 12-O-tetradecanoylphorbol 13-acetate type Maf response element or cyclic AMP-response element type (16Kataoka K. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 700-712Crossref PubMed Google Scholar, 23Kerppola T.K. Curran T. Oncogene. 1994; 9: 3149-3158PubMed Google Scholar). Homo- and heterodimerization through leucine zipper domains is one of the most important mechanisms underlying transcriptional regulation by bZip factors. All the Maf family members can form heterodimers with other bZip factors like Fos and Jun, and these heterodimers are different in their DNA binding specificity from Maf homodimers or AP-1 complexes (16Kataoka K. Noda M. Nishizawa M. Mol. Cell. Biol. 1994; 14: 700-712Crossref PubMed Google Scholar, 24Kerppola T.K Curran T. Oncogene. 1994; 9: 675-684PubMed Google Scholar). BZip transcription factors are also able to interact with unrelated transcription factors like glucocorticoid receptors or Ets family members (25Jonat C. Rahmsdorf H.J. Park K.K. Cato A.C. Gebel S. Ponta H. Herrlich P. Cell. 1990; 62 (204): 1189Abstract Full Text PDF PubMed Scopus (1371) Google Scholar, 26Basuyaux J.P. Ferreira E. Stehelin D. Buttice G. J. Biol. Chem. 1997; 272: 26188-26195Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). In the case of c-Maf, interaction with the transcription factor c-Myb plays a role during myeloid cell differentiation (27Hedge S.P. Kumar A. Kurschner C. Shapiro L.H. Mol. Cell. Biol. 1998; 18: 2729-2737Crossref PubMed Scopus (81) Google Scholar), whereas MafB interaction with c-Ets-1 represses its transcriptional activity, resulting in the inhibition of erythroid cells differentiation (28Sieweke M.H. Tekotte H. Frampton J. Graf T. Cell. 1996; 85: 49-60Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Recently, Maf family members were shown to associate with a set of Hox proteins, resulting in the inhibition of Maf DNA binding, transactivation, and transforming activities (29Kataoka K. Yoshitomo-Nakagawa K. Shioda S. Nishizawa M. J. Biol. Chem. 2001; 276: 819-826Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Thus, in view of the involvement of both Pax-6 and Maf in common developmental processes (such as lens formation), and the presence of the binding sites for both factors in the G1 element, we tested whether they could interact with and influence each other. We show that Pax-6 and MafA/L-Maf interact through their transactivation domains. This interaction results in Pax-6-enhanced DNA binding capacity on the G1 element where MafA/L-Maf is indeed able to bind. Cotransfection of a MafA/L-Maf expression plasmid and the G1-containing glucagon promoter results in an increase in CAT activity relatively to the control vector, showing that MafA can activate the glucagon promoter. Cotransfection of MafA/L-Maf expression plasmid with Pax-6 expression vector results in a 5-fold increase in CAT activity over the effect of each factor alone, suggesting a synergistic effect in expression of glucagon promoter through the G1 element by Pax-6 and MafA. This synergistic effect requires a functional paired domain but not the homeodomain. In contrast, as shown with two DNA-binding mutants, the DNA binding capacity of Maf is dispensable. Surprisingly, when the transactivation domain of MafA/Maf-L is removed, this truncated factor still increases the Pax-6 effect on the glucagon promoter. Maf protein strongly enhances the binding of Pax-6 to the G1 element, even when truncated in the transactivation domain or without DNA-binding properties. Finally, we demonstrate the presence of MafA/L-Maf in pancreas and c-Maf in endocrine cells, suggesting that Maf family members may play a role in the pancreas function. pcDNA3-MafA expression vector was obtained by inserting an EcoRI fragment corresponding to MafA open reading frame into the EcoRI site of a modified pcDNA3 vector, in frame with HA1 epitope coding sequence. Generation of mutant MafA containing the L2PL4P mutation and of truncated MafA corresponding to the bZip domain was described previously (20Benkhelifa S. Provot S. Lecoq O. Pouponnot C. Calothy G. Felder-Schmittbuhl M.P. Oncogene. 1998; 17: 247-254Crossref PubMed Scopus (53) Google Scholar). For eukaryotic expression, EcoRI fragments corresponding to these open reading frames were inserted into the EcoRI site of modified pcDNA3 vector as for wild type MafA. For the synthesis of GST-MafA recombinant proteins, theEcoRI fragment corresponding to MafA open reading frame was subcloned into the EcoRI site of pGex-4T1 vector (Amersham Pharmacia Biotech). For the synthesis of GST-MafA(1–151) proteins anEagI deletion, eliminating the bZip domain, was performed in the previous construct. For the synthesis of GST-MafA (151) proteins, an EagI fragment corresponding to MafA bZip domain was subcloned into the NotI site of pGex-4T2 (Amersham Pharmacia Biotech). Reporter construct (QR1-Abox(X4)-TK) carrying a multimerized A box from the neuroretina-specific QR1 gene, upstream of the thymidine kinase promoter, was described previously (20Benkhelifa S. Provot S. Lecoq O. Pouponnot C. Calothy G. Felder-Schmittbuhl M.P. Oncogene. 1998; 17: 247-254Crossref PubMed Scopus (53) Google Scholar). The glucagon promoter construct −138M3 was mutated in the MafA-binding site. To generate this mutant we used the Chameleon double-stranded site-directed mutagenesis kit (Stratagene) using the −138 CAT as a template and oligonucleotide G1–51M3 (5′-CAA AAC CCC ATT ATT TAC AGA TGA GAA ATT TAT ATT GTC CTG GTA ATA TCT-3′). The 35S-radiolabeled proteins were translated in vitro using the TNT system (Promega). The GST chimerical proteins were extracted from bacteria following the Amersham Pharmacia Biotech instructions. The labeled proteins were preincubated with empty glutathione-Sepharose beads 30 min at 4 °C. The GST proteins on glutathione-Sepharose beads were preincubated with 40 μg of BSA. Bead volumes were kept constant by adding empty beads. The pull-down assays were performed in 25 mm Hepes, pH 7.5, 150 mm KCl, 12.5 mm MgCl2, 0.1% Nonidet P-40, 20% glycerol. Proteins were incubated for 1 h at 4 °C and then the beads were washed four times in 20 mmTris-HCl, pH 8, 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40. Baby hamster kidney (BHK)-21 cells were cultured in Dulbecco's modified Eagle's medium/F-12 containing 10% fetal calf serum, 1% vitamins, 100× minimum Eagle's medium, and 10 μg/ml conalbumin. Cells were seeded 24 h prior to transfection. BHK21 cells transfections were performed with polyethyleneimine (exgen 500, Euromedex, Souffelweyersheim) reagent according to the instructions of the manufacturer. 200 ng of expression plasmids were cotransfected except where mentioned in the figure legend. αTC cells transfections were performed by the calcium-phosphate method. The total amount of transfected DNA was kept constant by addition of empty expression vector DNA. A LRTRSV-LacZ vector or a pcDNA3-LacZ was cotransfected for normalization of CAT assays by controlling the β-galactosidase activity. CAT assays were performed as described previously (30Plaza S. Dozier C. Saule S. Cell Growth Differ. 1993; 4: 1041-1050PubMed Google Scholar). Levels of CAT activity were quantified after exposure of the thin layer chromatograms to a PhosphorImager screen (Molecular Dynamics). The DNA used as probes were G1–51, G1–51M10, and G1–51M3 and as a control the Mitf-binding site in the QNR-71 promoter. G1–51 (5′-CAA AAC CCC ATT ATT TAC AGA TGA GAA ATT TAT ATT GTC AGC GTA ATA TCT-3′) is a wild type glucagon promoter element described previously (14Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) to bind Pax-6. G1–51M10 (5′-CAA AAC CCC ATT CCCGCCCCA GGA GAA ATT TAT ATT GTC AGC GTA ATA TCT-3′) is mutated in the Pax-6-binding site (14Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). G1–51M3 (5′-CAA AAC CCC ATT ATT TAC AGA TGA GAA ATT TAT ATT GTC CTG GTA ATA TCT-3′) is mutated in the presumptive Maf-binding site. Underlines represent mutations introduced in the oligonucleotides. QNR-71 oligonucleotide is 5′-GCT TTA ATT CCA TCA CAT GAT GAG TCC TG-3′. The DNA probes were double-strand oligonucleotides [γ-32P]ATP-labeled with the polynucleotide kinase T4. Gel retardation assays were performed as described previously (30Plaza S. Dozier C. Saule S. Cell Growth Differ. 1993; 4: 1041-1050PubMed Google Scholar) with 100 ng of bacterially expressed proteins. To visualize Pax-6 and MafA proteins, green fluorescent protein (GFP, CLONTECH) and Discosoma striatared protein (DsRed,CLONTECH) were used as fusion partners of p46 and MafA, respectively, and the following plasmids were constructed. AnNheI-BglII EGFP fragment was ligated to theXbaI and BglII sites of pVNC3 (modified from pVM116, see Ref. 31Klempnauer K.-H. Arnold H. Biedenkapp H. Genes Dev. 1989; 3: 1582-1589Crossref PubMed Scopus (73) Google Scholar) to produce pVNC3 EGFP C1. ABamHI-ApaI MafA fragment was inserted into theBglII and ApaI sites of pVNC3 EGFP to make pVNC EGFP MafA. pVNC3 MiRed (32Planque N. Leconte L. Coquelle F.M. Martin P. Saule S.L. J. Biol. Chem. 2001; 276: 29330-29337Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) was digested with XhoI and Bsp120I to excise the Mitf open reading frame and to replace it by aXhoI-NotI p46 fragment to generate pVNC3 Pax6Red. As controls, we used EGFPMyc and Pax6EGFP (32Planque N. Leconte L. Coquelle F.M. Martin P. Saule S.L. J. Biol. Chem. 2001; 276: 29330-29337Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). pVNC3 Pax6Red and pVNC EGFP MafA were cotransfected in BHK21 cells. To determine the localization of the chimerical proteins into the nuclei, cells were fixed for 20 min at room temperature in 3% paraformaldehyde in PHEM buffer (60 mm Pipes, 25 mm Hepes, 10 mm EGTA, 1 mmmagnesium acetate, pH 6.9). Cells were washed three times in PBS and permeabilized for 25 min in 0.1% Triton X-100 in PBS. Chromosomes were stained with 4,6-diamidino-2-phenylindole (Sigma) for 5 min. After a rinse in PBS, coverslips were mounted in 50% PBS/glycerol containing anti-fading reagent 1.4 diazabicyclo-(2-2-2)octane (Sigma) at 100 mg/ml. Pictures of fixed cells were collected using a three-dimensional deconvolution imaging system, the detailed description and validation of which will be published elsewhere. 2J. B. Sibarita et al., submitted for publication. Briefly, it consisted of a Leica DM RXA microscope, equipped with a piezoelectric translator (PIFOC, PI, Germany) placed at the base of a 100× PlanApo N.A. 1.4 objective, and a 5-MHz Micromax 1300Y interline CCD camera (Roper Instruments, France). For the acquisition of Z-series, the camera was operated at full speed and controlled the piezo translator at the start of each CCD chip read out. Stacks containing fluorescence images were collected automatically at 0.2-μm Z-intervals (Metamorph software, Universal Imaging). Wavelength selection was achieved by switching to the corresponding motorized selective Leica filter block before each stack acquisition. Tests using 50 nm tetraspec beads (Molecular Probes) showed that the system did not generatex-y pixel shifts and that Z-plane shifts between colors, due to chromatic aberrations, were reproducible and could be corrected. Exposure times were adjusted to provide ∼3000 gray levels at sites of strong labeling. Automated batch deconvolution of each Z-series was computed using a measured point spread function and constrained iterative deconvolution with a custom made software package. The point spread function of the optical system was extracted from three-dimensional images of fluorescent beads of 0.1 μm in diameter (Molecular Probes) collected at each wavelength. The Z-series were pseudo-colored and overlaid, and maximal pixel intensity projections were calculated with the help of Metamorph software (Universal Imaging). Total RNA from pancreas from 8-day-old quail embryos were digested with RQI DNase. After phenol-chloroform extraction and ethanol precipitation, 2 μg of these RNA were annealed with 0.5 μl of oligonucleotide dT (Perkin-Elmer) (50 μm) for 10 min at 70 °C. The cDNA were obtained by adding 1 μl of 0.1m dithiothreitol, 2 μl of 5 mm dNTP mix (Eurogentec), 200 units of Moloney murine leukemia virus-RT (Life Technologies, Inc.), and 4 μl of 5× buffer in a final volume of 20 μl. The mixture was incubated for 1 h at 42 °C. For the PCR, a 2-μl aliquot was added to 1.6 μl of 25 mmMgCl2, 1 μl of 5 mm dNTP mix (Eurogentec), 0.5 units of Goldstar Taq (Eurogentec), 2 μl of 10× buffer, 1 μl of 20 μm oligonucleotide primers 5′-TTC CAC CCC TCT CAG CAC-3′ and 5′-CTC CCG AAC CGA CAT ACT-3′, in a final volume of 20 μl. Amplifications were carried out in a PTC-200 (MJ Research) as follows: 96 °C for 2 min; 30 cycles at 94 °C for 45 s, 50 °C for 45 s, and 72 °C for 45 s; 72 °C for 10 min. Reaction products were cloned into PCR2.1 (TA cloning Kit, Invitrogen). Individual clones were isolated and sequenced. Similar experiments were performed using αTC cells RNA and oligonucleotide primers Maf5′1, Maf5′2, and Maf3′2 as described (20Benkhelifa S. Provot S. Lecoq O. Pouponnot C. Calothy G. Felder-Schmittbuhl M.P. Oncogene. 1998; 17: 247-254Crossref PubMed Scopus (53) Google Scholar). αTC cells cultured on 16-mm microscope coverslips were fixed for 1 h with 2% paraformaldehyde in PBS and then treated with anti v-Maf serum (a gift of Dr. M. Castellazzi). Anti-c-Maf-reactive proteins were detected with CY3-labeled goat anti-rabbit immunoglobulins secondary reagent (Jackson ImmunoResearch). To test the hypothesis that Pax-6 physically interacts with MafA, we prepared glutathione-Sepharose beads coupled with a GST-paired fusion protein containing the paired domain from the p46 (GST-Prd46(3–131)) and GST-Hom (224), containing the p46 homeodomain, and the GST-p46(1–416), containing the full-length Pax-6 product. We also used GST fusion proteins containing either the N-terminal (residues 1–151) or the C-terminal domains (residues 151–286) of MafA or the full-length protein. Glutathione-Sepharose beads coupled to GST served as a negative control. In vitroradiolabeled MafA/L-Maf or Pax-6 proteins were loaded onto these beads and washed, and bound proteins were eluted by boiling and analyzed in SDS-PAGE. The percentage of bound radioactivity was calculated using PhosphorImager. A similar amount of radiolabeled MafA was recovered from GST-Prd46 (0.4%) and from GST-Hom (0.2%). In contrast, a much higher amount of radiolabeled MafA protein (8.6%) was recovered from the GST-p46, suggesting that the transactivation domain of Pax-6 is involved in the protein-protein interaction (Fig.1 A). As shown in Fig. 1 B, both the N terminus of MafA (amino acids 1–151, 1%, lane 2) and the C-terminal (amino acids 151–286, 0.8%, lane 3) domains were independently able to bind the radiolabeled p46. However, it is important to note that a greater amount of GST-N-terminal protein has been used in the experiment (Fig. 1 C, compare lanes 1 with2 and 3) suggesting in fact a lower efficiency of this part of the MafA protein in the interaction with the p46. The full size Maf bound with a greater efficiency (19%) than the two separated parts of the protein. The GST alone did not interact with the radiolabeled MafA (Fig. 1 B, lane 1). As a positive control, we incubated the full size GST-MafA(1–286) with the radiolabeled MafA (Fig. 1 D). The amount of recovered protein (10%, lane 1) was similar to the amount p46 recovery (Fig. 1 B, lane 4), suggesting a strong interaction between the two proteins. As a negative control, we incubated the full size GST-MafA(1–286) with the nonrelevant radiolabeled GFP protein. No interaction could be observed between these two proteins (Fig. 1 E). As for Engrailed (33Plaza S. Langlois M.C. Turque N. LeCornet S. Bailly M. Begue A. Quatannens B. Dozier C. Saule S. Cell Growth Differ. 1997; 8: 1115-1125PubMed Google Scholar) or Mitf (32Planque N. Leconte L. Coquelle F.M. Martin P. Saule S.L. J. Biol. Chem. 2001; 276: 29330-29337Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), two p46-interacting proteins, the paired-less p30 proteins did not significantly bind to either part of MafA (Fig.1 B). Nrl, a large Maf family member, acts synergistically with the paired-like homeodomain transcription factor Crx to activate the rhodopsin gene (34Chen S. Wang Q.L. Nie Z. Sun H. Lennon G. Copeland N.G. Gilbert D.J. Jenkins N.A. Zack D.J. Neuron. 1997; 19: 1017-1030Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 35Chau K.Y. Chen S. Zack D.J. Ono S.J. J. Biol. Chem. 2000; 275: 37264-37270Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). A potential Maf-binding site (TCAGCG) is present close to the Pax-6-binding site in the G1 element of the glucagon promoter. Therefore, we tested if the observed Pax-6/Maf interaction was able to regulate expression of Pax-6-dependent glucagon promoter. BHK21 cell lysates were collected 2 days after transfection, and the levels of CAT and β-galactosidase activities present in the lysates were determined. Cotransfection of the G1 containing −138 glucagon promoter with the vector expressing the MafA/L-Maf protein resulted in an increase of CAT activity relative to that of the control vector (Fig.2). Similar increase of CAT activity was observed with the −138 glucagon promoter in response to Pax-6 protein expression. Cotransfection of the −138 glucagon promoter with the vectors expressing MafA and Pax-6 proteins resulted in a 5-fold increase of CAT activity relative to the level of expression obtained with each transcription factor alone. The synergistic activation obtained was specific of this promoter because no synergistic effect was observed on the CMV- or RSVLTR-lacZ cotransfected in the experiment. The G3–138, containing an additional Pax-6-binding site (14Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), also exhibited this synergistic response, except that the CAT activity was increased (Fig. 2 B) and the promoter −350 behaved similarly (Fig. 2 C). In contrast, the G3 element linked to the −31 sequence, showed a marginal activation by both Pax-6 and MafA/L-Maf, suggesting that the G1 element is indeed essential in the Maf response of the glucagon promoter and synergy with Pax-6. Another Pax family member (Pax-2) has been described to activate the glucagon promoter through the G1 element (36Ritz-Laser B. Estreicher A. Gauthier B. Philippe J. J. Biol. Chem. 2000; 275: 32708-32715Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). We tested the effect of the simultaneous expression of Pax-2 and MafA on the G3–138 glucagon promoter. The D2 form of Pax-2 (a splicing variant in the transactivation domain) was a better transactivator than Pax-6, whereas the C form (36Ritz-Laser B. Estreicher A. Gauthier B. Philippe J. J. Biol. Chem. 2000; 275: 32708-32715Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) exhibited only a marginal effect on glucagon expression (Fig. 3 A). On these two Pax-2 isoforms, MafA/L-Maf had a rather additional than synergistic effect (Fig. 3 A). In contrast to Pax-6, Pax-2 contains only a partial homeodomain (37Dahl E. Koseki H. Balling R. BioEssays. 1997; 19: 755-765Crossref PubMed Scopus (311) Google Scholar). Therefore, we asked whether the homeodomain of Pax-6 was required for the synergistic transactivation of the glucagon promoter observed with MafA. We used a Pax-6 mutant, which is devoid of homeodomain, (Pax-6Δ170–270, see Ref. 2Carriere C. Plaza S. Caboche J. Dozier C. Bailly M. Martin P. Saule S. Cell Growth Differ. 1995; 6: 1531-1540PubMed Google Scholar). This Pax-6 mutant transactivated the G3–138 glucagon promoter to an extent similar to that of the wild type, suggesting that the homeodomain is dispensable for G3–138 glucagon promoter activation (Fig.3 B). Cotransfection of the MafA/L-Maf revealed that the Pax-6Δ170–270 mutant increased glucagon promoter transactivation with MafA. We next asked whether a functional paired domain was required. For that purpose, we used the p48 isoform of Pax-6, which recognizes a distinct DNA-binding sequence due to the insertion of 14 amino acid residues in the paired domain (38Epstein J.A. Glaser T. Cai J. Jepeal L. Walton D.S. Maas R.L. Genes Dev. 1994; 8: 2022-2034Crossref PubMed Scopus (317) Google Scholar). p48 is unable to transactivate the glucagon promoter (Fig. 3 B). Coexpression of p48 and MafA proteins did not modify the level of CAT activity when compared with MafA alone (Fig. 3 B), indicating that the binding of Pax-6 to DNA through the paired domain is required in order to increase the MafA/L-Maf effect on this promoter. These results prompted us to study the effect of MafA/L-Maf truncated proteins devoid of transactivation domain in the cooperative transcriptional activation with Pax-6. Fig. 3 C shows that this mutant MafA(173–286) was no longer able to activate the glucagon promoter but still increased the effect of Pax-6 on the G3–138 promoter, suggesting that binding of MafA to the DNA was sufficient to
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