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J. Biol. Chem., Vol. 282, Issue 48, 35024-35034, November 30, 2007

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Pax-6 and c-Maf Functionally Interact with the -Cell-specific DNA Element G1 in Vivo to Promote Glucagon Gene Expression^*

From the Diabetes Unit, Division of Endocrinology, Diabetes and Nutrition, University Hospital, University of Geneva Medical School, 1211 Geneva 14, Switzerland

Received for publication, April 2, 2007 , and in revised form, August 29, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Specific expression of the glucagon gene in the rat pancreas requires the presence of the G1 element localized at -100/-49 base pairs on the promoter. Although it is known that multiple transcription factors such as Pax-6, Cdx-2/3, c-Maf, Maf-B, and Brain-4 can activate the glucagon gene promoter through G1, their relative importance in vivo is unknown. We first studied the expression of Maf-B, c-Maf, and Cdx-2/3 in the developing and adult mouse pancreas. Although Maf-B was detectable in a progressively increasing number of -cells throughout development and in adulthood, c-Maf and Cdx-2/3 were expressed at low and very low levels, respectively. However, c-Maf but not Cdx-2/3 was detectable in adult islets by Western blot analyses. We then demonstrated the in vivo interactions of Pax-6, Cdx-2/3, Maf-B, and c-Maf but not Brain-4 with the glucagon gene promoter in glucagon-producing cells. Although Pax-6, Cdx-2/3, Maf-B, and c-Maf were all able to bind G1 by themselves, we showed that Pax-6 could interact with Maf-B, c-Maf, and Cdx-2/3 and activate transcription of the glucagon gene promoter. Overexpression of dominant negative forms of Cdx-2/3 and Mafs in -cell lines indicated that Cdx-2/3 and the Maf proteins interact on an overlapping site within G1 and that this binding site is critical in the activation of the glucagon gene promoter. Finally, we show that specific inhibition of Pax-6 and c-Maf but not Cdx-2/3 or Maf-B led to decreases in endogenous glucagon gene expression and that c-Maf binds the glucagon gene promoter in mouse islets. We conclude that Pax-6 and c-Maf interact with G1 to activate basal expression of the glucagon gene.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The glucagon gene is expressed in the -cells of the pancreatic islets, the L cells of the intestine, and specific areas of the brain (1). Although multiple transcription factors have been proposed to regulate glucagon gene transcription, the molecular mechanisms by which glucagon gene expression is restricted to -cells are still not known. Pancreas-specific expression of the glucagon gene is conferred by the islet-specific enhancer elements G2, G3, and G4 (2-5), whereas the proximal promoter element G1 is critical for -cell-specific expression (2-4, 6, 7). We and others have proposed Pax-6, Cdx-2/3, large Mafs, Brain-4, and Foxa-2 as potential factors implicated in glucagon gene expression through the transactivation of the G1 element in glucagon-producing cells by transfection studies (8-11). However, the relevance and relative importance of these factors in vivo is unknown. Pax-6 has previously been reported to be expressed in the endocrine pancreas and during pancreas development as early as embryonic day 10.5 and in the adult (12, 13). Pax-6 is critical for -cell development and activates glucagon gene promoter constructs through the enhancer element G3 and the -cell-specific element G1 (4). Furthermore, Pax-6 is essential for the transactivation of glucagon gene promoter constructs in the small and large intestine (14). Nevertheless, Pax-6 is expressed in all pancreatic endocrine cells and thus cannot explain the specificity of glucagon gene expression. Pax-6 has been shown to interact with Cdx-2/3 and Maf-A, resulting in a synergistic transactivation of the glucagon gene (9, 15). We and others have shown that Cdx-2/3 is able to form a heterocomplex with Pax-6 on the G1 element using InR1G9 glucagon-producing cell nuclear extracts and strongly activates gene transcription by transfection assays (15). Nevertheless, the expression of Cdx-2/3 in -cells is not established. Although Cdx-2/3 has been reported to be present in -cells of the adult mouse pancreas by immunocytochemistry (16), it is absent from the adult rat and human -cells as well as from glucagonomas (10, 17).

Similarly, the expression of the large Mafs in the -cells is controversial. Although Maf-A is specifically expressed in pancreatic β-cells (18, 19), the expression of Maf-B and c-Maf in insulin- and glucagon-producing cells has generated opposing results. It has, indeed, been reported by Artner et al. (20) that Maf-B is expressed in mouse - and β-cells during development but only in -cells in the adult islets, whereas c-Maf is not expressed at any stage. Others have found that c-Maf but not Maf-B is expressed in -cells (19). Functionally, Maf-B- or c-Maf-deficient mice show an important reduction in both insulin- and glucagon-positive cells throughout development, indicating a significant role of Maf-B and c-Maf in differentiation, replication, and/or survival of endocrine precursor cells (21, 22).

Brain-4 and Foxa-1/Foxa-2 have also been shown to activate glucagon gene expression and to be expressed in the -cells. Brain-4 is specifically expressed in -cells throughout development and in the adult and has been proposed as a candidate for -cell specific expression of the glucagon gene (10, 23); however, mutant mice lacking Brain-4 have no developmental and functional defect of the -cells, raising doubts regarding the relevance of Brain-4 in glucagon gene expression (23).

Foxa-1 and Foxa-2 have been proposed to regulate glucagon gene expression by binding to G1 and G2 of the rat glucagon promoter (11). Nevertheless, the binding site of Foxa-1 and -2 on G1 is overlapping with Pax-6, and overexpression of these factors in -cells results in an inhibition of Pax-6-induced expression of the glucagon gene, suggesting an inhibitory role for these factors on G1-mediated transactivation. The aim of this study was to better define and characterize the transcription factors interacting with G1 involved in the -cell-specific expression of the glucagon gene.

We show here by ChIP analyses that Pax-6, Cdx-2/3, Maf-B, and c-Maf interact with the glucagon gene promoter in InR1G9 cells, whereas only Pax-6, Maf-B and c-Maf do in -TC1 cells, suggesting that neither Cdx-2/3 nor Brain-4 are necessary for glucagon gene expression. Furthermore, whereas both Maf-B and c-Maf are expressed in the islets, Cdx-2/3 mRNA is expressed at very low levels in the embryonic mouse pancreas, and the protein is not detected by either immunocytochemistry or Western blot analyses. Functionally, Pax-6 and c-Maf are the most potent transactivators of the glucagon gene promoter compared with Pax-6/Cdx-2/3 or Pax-6/Maf-B. We show by specifically silencing the expression of c-Maf, Maf-B, and Cdx-2/3 by siRNA³ that c-Maf seems to be the critical partner of Pax-6 to activate endogenous glucagon gene expression. Finally, we demonstrate that c-Maf interacts with the glucagon gene promoter in adult mouse islets, indicating involvement of c-Maf on glucagon gene transcription in vivo.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—The glucagon-producing hamster InR1G9 (24) and mice -TC1 cells (25) as well as the non-islet Syrian baby hamster kidney (BHK-21) cell lines were grown in RPMI 1640 (R-6504; Sigma) supplemented with 5% fetal bovine serum and 5% newborn calf serum, 2 mM glutamine, 100 units/ml of penicillin, and 100 µg/ml of streptomycin.

RNA Preparation and RT-PCR Analysis—Total RNA was isolated from mouse embryonic and adult pancreas, mouse intestine, -TC1 and InR1G9 cell lines using TRIzol Reagent (Invitrogen) according to the manufacturer's specifications. First strand cDNA synthesis, from 1 µg of total RNA, was performed with random hexamer primers and Superscript II Reverse Transcriptase (Invitrogen) as recommended by the supplier. cDNA obtained from mouse pancreas and cell lines were analyzed by semi-quantitative PCR using Goldstar (Eurogenetech, Brussels, Belgium) DNA polymerase as recommended by the provider. PCR amplifications were analyzed on ethidium bromide-stained 3% agarose gels. Real time PCRs were performed for glucagon and TBP (TATA box-binding protein) gene expression in glucagon-producing cell lines using QuantiTect SYBR Green kit (Qiagen, Basel, CH) and Light-Cycler (Roche Applied Science). For quantification, a standard curve was systematically generated with four different amounts of purified target cDNA. Each assay was performed in duplicate, and validation of the real time PCR runs was assessed by evaluation of the melting temperature of the products and by the slope and error obtained with the standard curve. The analyses were performed using the Light-Cycler software (Roche Applied Science). The results were corrected by TBP mRNA levels.

Mouse Islet Purification—Mouse pancreatic islets were isolated as described previously (26, 27).

Western Blot Analyses—Nuclear extracts were isolated as described (28, 29) from InR1G9 or BHK cells. Ten to thirty µg of each protein extract were resolved on a 10% SDS-polyacrylamide gel and transferred electrophoretically to polyvinylidene difluoride membranes. Immunoblotting was performed with polyclonal antibodies to rabbit Pax-6 diluted 1/1000 (generous gift from S. Saule, CNRS/UMR 146, Institut Curie Section de Recherche, France), Cdx-2/3 diluted 1/500 (kind gift from M. German, University of California), Maf-B (BL658; Bethyl, Montgomery, TX), or c-Maf (BL662; Bethyl) diluted 1/1000 and goat anti-rabbit IgG-conjugated with horseradish peroxidase diluted 1/2000 (sc2030; Santa Cruz, CA). The signal was detected with Super Signal West Pico Trial Kit (Pierce). Protein loading was normalized by immunodetection of rabbit TFIIE- diluted 1/500 (C17, Santa Cruz). At least three independent experiments were performed with nuclear cell extracts.

c-Maf detection was performed with or without c-Maf blocking peptide (BP300-613; Bethyl). 5 µg of c-Maf blocking peptide was preincubated for 2 h at room temperature with 1 µg of specific c-Maf antibody (BL662).

Coimmunoprecipitation and Western Blot—-TC1 cells were trypsinized, washed, and resuspended in a buffer containing 10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2.5 mM dithiothreitol, 1.2 mM phenylmethylsulfonyl fluoride. Subsequent to a 15-min incubation on ice, the cells were lysed by the addition of 10% Nonidet P-40, and intact nuclei were sedimented by centrifugation. Buffer C containing 20 mM Hepes, pH 7.9, 25% glycerol, 250 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1.2 mM phenylmethylsulfonyl fluoride and complemented with a mixture of protease inhibitors (Complete mini; Roche Applied Science) was added to the pellet and incubated for 1 h. Nuclear proteins were purified from membrane debris by centrifugation. Approximately 800 µg of nuclear extracts were incubated overnight at 4 °C with a mouse anti-Pax-6 (MAB1260, R & D Systems, Abingdon, UK) coupled to sheep -mouse IgG magnetic Dynabeads® (Invitrogen). The beads were then washed extensively with Buffer C and resuspended in 50 µl of Laemmli buffer. 10-50 µg of total nuclear extracts (input) and 20 µl of the 50 µl of immunoprecipitate were separated on SDS-PAGE and transferred on to polyvinylidene difluoride membrane (Amersham Biosciences). The membranes were incubated with either -rabbit Pax-6 (S. Saule) or anti-rabbit Maf-B (BL 658) and c-Maf (BL 662) and subsequently with sheep -rabbit IgG coupled to horseradish peroxidase. Signals were detected using a Super Signal West Pico trial kit (Pierce).

Immunohistochemistry—Cryosections of adult or embryonic mouse pancreas fixed with 4% paraformaldehyde were treated with 0.1% Triton X-100 and immunolabeled using polyclonal goat glucagon antibody (N-17; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) combined with polyclonal rabbit Maf-B (Santa Cruz Biotechnology, LabForce, Nunningen, Switzerland, P-20) and Cdx-2/3 (M. German) antibodies. glucagon, Maf-B and Cdx-2/3 immunostaining were performed with confocal microscope (Zeiss LSM 510 confocal microscope; Carl Zeiss AG, Göttingen, Germany). The percentage of colocalization of glucagon and Maf-B were calculated with the Metamorph software version 6.2r4 (Universal Imaging, Puchheim, Germany). The percentages were determined on at least 100 (e12.5, e14.5, and e17.5) to 500 glucagon-positive cells (adult) in four to five distinct pancreas sections corresponding to at least three different animals.

Transient Transfection Assays—For all applications, BHK-21 cells were transfected by the calcium phosphate precipitation technique (30) as previously described (15). InR1G9 and -TC1 cell lines were transfected by the DEAE-dextran method (30, 31) for glucagon promoter studies. cDNAs encoding the dominant negative forms of Maf-A (DN-MafA) and Cdx-2/3 (DN-Cdx2/3) were transfected in InR1G9 and -TC1 cells using the Transfectin Reagent (Bio-Rad) according to the manufacturer's instructions.

Promoter Analysis—glucagon promoter transactivation was studied after transfection of -292 to +50 base pair sequence of the 5'-flanking region of the rat glucagon promoter (-292Glu-CAT) in BHK-21, InR1G9, and -TC1 cell lines. glucagon promoter activities were analyzed by chloramphenicol acetyl transferase (CAT) activity as previously described (32). The cells were cotransfected with either a plasmid containing the human placental alkaline phosphatase gene (pSV2A-PAP) or the luciferase gene (RSV-Luc; Promega, Wallisellen, Switzerland), driven by the simian virus 40 early promoter, to monitor transfection efficiency. Quantification of acetylated and nonacetylated forms was done with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Placental alkaline phosphatase and luciferase activities were measured by spectrophotometry and luminometry (Labsystem), respectively. A minimum of three independent transfections were performed, each of them carried out in duplicate.

Electrophoretic Mobility Shift Assays (EMSAs)—EMSAs were performed using the -100 to -49 sequence of the 5'-flanking region of rat glucagon promoter corresponding to the G1 element (5'-CAAAACCCCATTATTTACAGAT GAGAAATTTATATTGTCAGCGTAATATCT-3') and nuclear extracts from BHK-21 cells overexpressing either mouse Pax-6 (p46) (subcloned in the laboratory), hamster Cdx-2/3 (kindly provided by Dr. M. German (University of California, San Francisco, CA)), mouse Maf-B, or mouse c-Maf full-length proteins (generous gift by Dr. R. Stein (Vanderbilt University Medical Center, Nashville, TE)), respectively. EMSA reactions contained 20 mmol/liter Hepes, pH 7.9, 5 mmol/liter MgCl₂, 0.5 mmol/liter EDTA, 50 mmol/liter KCl, 1 mmol/liter dithiothreitol, 6.25% glycerol, 1 µg of bovine serum albumin, 2 µg of salmon sperm DNA, and 2 µg of poly(dIdc-dIdC) as described previously (5) with nuclear protein extracts (15-25 µg of proteins) and 50 000 cpm of 5'-end ³²P-labeled oligonucleotide probe. Anti-Pax-6 (serum 13) and anti-Cdx-2/3 antibodies were generously provided by Drs. S. Saule (CNRS/UMR 146, France) and M. German (University of California, San Francisco), respectively. Anti-Maf-B (BL658) and anti-c-Maf (BL662) antibodies were purchased at Bethyl (Bethyl Laboratories Inc., Montgomery, TX).

Chromatin Immunoprecipitation Assay (ChIP)—ChIP assays were performed according to Orlando et al. (33). Briefly, formaldehyde-cross-linked chromatin extracts were prepared from InR1G9 and -TC1 cells and fragmented by enzymatic digestion (Enzymatic shearing kit; Active Motif Europe, Belgium). 50 µg (InR1G9 and -TC1 cells) or 20 µg (adult mouse islets) of chromatin extract were first precleared with protein A-Sepharose beads (CL-4B; Pharmacia Biotech AB, Uppsala, Sweden) for 1 h. After centrifugation, the supernatants were incubated overnight at 4 °C with 8 µg of anti-Pax-6 (serum 13), anti-Maf-B (BL658, Bethyl), anti-c-Maf (BL662, Bethyl), anti-Cdx-2/3 (M. German, University of California, San Francisco), anti-Brain-4 (J. F. Habener, Harvard University, Boston, MA), and -acetyl-histone H4 antibodies (06-866; Upstate, Lake Placid, NY), as well as rabbit IgG (sc-2027; Santa Cruz). The immunoprecipitated DNA-protein complexes were bound to protein A-Sepharose beads after 3 h of incubation at 4 °C and washed in a low salt buffer, high salt buffer, LiCl buffer, and Tris-EDTA buffer in succession as described by Duong et al. (34). Proteins were eliminated using proteinase K (200 µg; Applichem) in the presence of 10% SDS by overnight incubation at 37 °C. After phenol extraction, the DNA was precipitated, suspended in water, and used as a template for PCR. The set of PCR primers used for analysis of binding on the glucagon gene proximal promoter (corresponding to G4-G1 box) in InR1G9 cells were 5'-GACTAGGCTCATTTGACGTC-3' and 5'-ATGGAAAGGGCAGTTTGGAG-3'; the two set of primers used for -TC1 cells and mouse adult islets were 5'-CAAAGCGAGTGGGTGAGTG-3' and 5'-GCCACGCAGATATTACGCTG-3' and equally 5'-CAGCGTAAAAAGCAGATGAGC-3' and 5'-AGGCTGTTTAGCCTTGCAGAT-3'. PCR products were verified on ethidium bromide-stained 3% agarose gels and analyzed by real time PCR using a Light-Cycler (Roche Applied Science).

RNA Interference—Two different specific sequences of siRNA were designed by Qiagen for Cdx-2/3 and Stealth siRNA by Invitrogen for Maf-B, c-Maf, and Pax-6 against hamster mRNA sequences. Appropriate scrambles were obtained at the same time (percentage of GC content identical to Pax-6, Maf-B, c-Maf, and Cdx-2/3 siRNA). InR1G9 cells grown in 6-well plates were transfected with 100 nM of siRNA using 5 µl of Lipofectamine 2000 (Invitrogen) as recommended by the supplier. The transfections were performed twice sequentially. Total RNA and nuclear extract were isolated 48 h after transfection as described above under "Experimental Procedures."

Data Analysis—The data are presented as the means ± S.E. and analyzed by Student's t test. A p value of less than 0.05 was considered to be statistically significant.

View larger version (7K): [in this window] [in a new window]   FIGURE 1. Organization of the rat glucagon promoter. Shown is a schematic representation of the transcription factor-binding sites located between -100 and -49 of the 5'-flanking sequence of the rat glucagon gene corresponding to the G1 element. The locations of Pax-6, Brain-4, Cdx-2/3, and large Mafs core sequence elements are framed.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We first examined the expression of Cdx-2/3, Maf-B, and c-Maf at different stages of pancreatic development (Fig. 2). As shown in Fig. 2A, Maf-B and c-Maf mRNAs can be detected from at least embryonic day 12.5 (e12.5) until the adult stage. At all stages, Maf-B mRNA levels are much higher compared with those of c-Maf. The Maf-B/c-Maf mRNA ratio increases during development to reach 10 in the adult. Large Mafs mRNAs are also present in the hamster and mouse glucagon-producing cell lines InR1G9 and -TC1, respectively. By contrast, Cdx-2/3 mRNA was hardly detected from total mouse pancreas during development and in the adult islets, although Southern blots of the RT-PCR samples using a specific Cdx-2/3 cDNA probe revealed the presence of Cdx-2/3 mRNA at all the different developmental stages (data not shown). However, we found substantial Cdx-2/3 mRNA levels in InR1G9 cells but not in -TC1 cells. We then performed immunohistochemistry on mouse pancreas sections prepared at e12.5, e14.5, and e17.5 and in the adult (Fig. 2B). We could not detect Cdx-2/3 at any stage of development, nor in the adult, whereas it was present in the adult mouse intestine, used as a positive control. Cdx-2/3 was not detected either by Western blot of adult islet nuclear extracts, whereas it was present in InR1G9 cells (data not shown). We also assessed for the presence of the transcription factor Maf-B in glucagon-positive cells (Fig. 2B). Maf-B is present as soon as e12.5 and can be detected until the adult stage. At e12.5, only 10% of the glucagon-positive cells expressed Maf-B. This percentage increased progressively until the adult stage to 88% (Fig. 2B). To detect c-Maf, we also used two antibodies (M-153 and BL662); the BL662 antibody was raised against mouse c-Maf and reported to be specific, whereas M-153 was not specific because it could also detect both Maf-B and Maf-A proteins (data not shown and Ref. 19). However, we could not detect c-Maf in pancreas sections using the specific BL662 antibody as reported by others (18, 20). To further evaluate the presence of c-Maf and Maf-B in -cells, we performed Western blots of -cell lines and adult mouse islet protein extracts; both Mafs were detected in -TC1 cells and adult islets, whereas only Maf-B was found in InR1G9 cells (Fig. 2, C and D). We hypothesize that the BL662 antibody raised against mouse c-Maf is not able to recognize hamster c-Maf by Western blots.

Overall, our results indicate that Cdx-2/3 is not required and may be dispensable for glucagon gene expression because it is absent from -TC1 cells and not or very weakly expressed in mouse pancreas during development and in the adult, although we cannot exclude formally a role of Cdx-2/3 at some stage of development in glucagon gene expression. By contrast, Maf-B and probably c-Maf are expressed throughout development and in the adult -cells; however, because a significant percentage of glucagon-producing cells do not express the large Mafs at least during development, these factors may not be absolutely required for glucagon gene expression, although it is possible that low amounts of large Mafs precluding detection by immunocytochemistry are present in all -cells.

To investigate the involvement of Pax-6, Cdx-2/3, Maf-B, and c-Maf on glucagon gene expression, we first performed ChIP assays on -pancreatic cell lines to determine the in vivo binding of these transcription factors to the glucagon promoter (Fig. 3). We also investigated the binding of Brain-4, an -cell-specific transcription factor present in both InR1G9 and -TC1 cells that has been proposed to bind G1 (10). In the InR1G9 hamster cell line, Pax-6, Maf-B, c-Maf, and Cdx-2/3 were found to strongly interact with the glucagon promoter but not Brain-4 (Fig. 3A). Similar results were obtained with -TC1 cells (Fig. 3B), although no interaction of Cdx-2/3 was observed in agreement with the fact that it is not expressed in these cells. These data indicate that Pax-6, Maf-B, and c-Maf interact with G1 as well as Cdx-2/3 in InR1G9 cells but not Brain-4.

To further characterize the respective transcriptional effects of Pax-6, Cdx-2/3, and large Mafs on the glucagon gene promoter, we performed transient transfections with the rat glucagon gene promoter (-292/+50), linked to the CAT gene with expression plasmids containing full-length cDNAs of these transcription factors in the BHK-21 cell line (Fig. 4). We first quantified the respective levels of Maf-B, c-Maf, and Cdx-2/3 in BHK nuclear extracts after transfection; after correction for TFIIE-, levels of Maf-B and c-Maf were equal but 4-fold higher than levels of Cdx-2/3 (data not shown). Pax-6, Cdx-2/3, Maf-B, and c-Maf can all activate glucagon gene promoter activity by 12.4-, 6.6-, 7.7-, and 19.6-fold, respectively. Because both Cdx-2/3 and Maf-A have been proposed to interact with Pax-6, we performed transfection studies with Pax-6, Cdx-2/3, and the large Mafs in combination. When Pax-6 and Cdx-2/3, Pax-6 and Maf-B, or Pax-6 and c-Maf cDNAs were cotransfected, we observed a synergistic activation on glucagon gene promoter activity reaching 38.3-, 83.9-, and 141.1-fold, respectively (Fig. 4). No additional activity was obtained with the combination of all these factors, nor did we observe any additive or synergistic activity between Cdx-2/3 and Maf-B or c-Maf. In addition, cotransfection of the coactivator p300 cDNA with Pax-6/Cdx-2/3 or Pax-6/large Maf did not increase further glucagon gene promoter transactivation (Fig. 4). Taken together, these results indicate that c-Maf is a stronger activator of the glucagon gene promoter compared with Maf-B or Cdx-2/3 and induces the strongest activation when combined with Pax-6. However, taking into account that levels of Cdx-2/3 are 4-fold lower than those of Maf-B and c-Maf, it is possible that the complexes Pax-6/c-Maf and Pax-6/Cdx-2/3 have similar transcriptional capacities.

View larger version (33K): [in this window] [in a new window]   FIGURE 2. Expression of the Maf-B, c-Maf, and Cdx-2/3 genes in mouse pancreas. A, real time quantitative RT-PCR from different developmental stages of mouse pancreas and glucagon-producing cell lines. The analyses were performed using the Light-Cycler software (Roche Applied Science). The results were corrected by TBP mRNA levels and are presented as the means ± S.E. for at least three independent experiments. B, immunohistochemistry of Maf-B and Cdx-2/3 in mouse pancreas development. Immunostaining were performed on cryosections of developing and adult mouse pancreas. The images were obtained after coincubation with anti-glucagon (in red) and anti-Cdx-2/3 or anti-Maf-B (in green) antibodies as described under "Experimental Procedures." Immunohistochemistry on mouse intestine was performed as a positive control of Cdx-2/3. The table represents the percentage of cells colocalizing glucagon and Maf-B at e12.5, e14.5, and e17.5 and in adult mouse pancreas as quantified with the confocal microscope with magnification x 63, zoom x 4 on more than 100 glucagon-positive cells from e12.5 to e17.5 and more than 500 cells in the adult stage. C and D, Western blot analyses of nuclear extracts from -pancreatic cell lines (-TC1 and InR1G9) and mouse adult islets. C, analyses with anti-Maf-B (BL658), and D, with anti-c-Maf (BL662). c-Maf detection was performed with or without 5 µg of c-Maf blocking peptide to assess the specificity of band clusters. NS indicates nonspecific band, and the asterisk indicates specific band corresponding to c-Maf proteins. The specificity of the antibodies was verified with overexpressed Maf-A, Maf-B, and c-Maf in BHK-21 cells.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. In vivo interaction of Pax-6, Cdx-2/3, large Mafs, and Brain-4 on the glucagon gene promoter. Histograms represent relative binding of Pax-6, Cdx-2/3, large Mafs, and Brain-4 proteins on the glucagon gene promoter in InR1G9 (A) and -TC1 cells (B). After cross-linking between chromatin and the interacting proteins, specific immunoprecipitations with anti-Pax-6, anti-Cdx-2/3, anti-Maf-B, anti-c-Maf, and anti-Brain-4 were performed as indicated under "Experimental Procedures." An -Histone H4 immunoprecipitation was also performed as a positive control. Binding was analyzed by real time PCR with a Light-Cycler (Roche Applied Science). Binding intensity data are expressed relative to IgG immunoprecipitation (nonspecific binding) and are presented as the means ± S.E. for at least three independent experiments. The asterisk indicates statistical significance with p < 0.05 value using a Student's t test.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Effects of Pax-6, Cdx-2/3, Maf-B, c-Maf, and p300 on the glucagon gene promoter activity. BHK-21 cells were cotransfected with 10 µg of -292GluCAT and 500 ng of Pax-6, Cdx-2/3, Maf-B, c-Maf, and p300 expression vectors in 100-mm culture dishes. Cell extracts were prepared 48 h after transfection, and CAT and placental alkaline phosphatase enzyme activities were measured as described under "Experimental Procedures." The data are expressed relative to the empty CAT reporter gene and are presented as the means ± S.E. for four different transfection experiments. The asterisk indicates statistical significance with p < 0.05 value using a Student's t test.

View larger version (54K): [in this window] [in a new window]   FIGURE 5. Cdx-2/3, Maf-B and c-Maf bind alone and in combination with Pax-6 on G1 element. EMSAs were performed with 5' end-labeled G1 oligonucleotide in the presence of 15 µg of Pax-6 and/or Cdx-2/3, Maf-B, c-Maf containing nuclear extracts from BHK-21 cells after overexpression. The arrows indicate specific single and combination complexes on the G1 probe. A, Cdx-2/3 binds alone and in combination with Pax-6 on G1. B, large Mafs bind alone and in combination with Pax-6 on G1. C, EMSA competition experiments analyzing the relative affinity of Pax-6, Cdx-2/3, Maf-B, and c-Maf as single complexes on the G1 element. D, EMSAs competition experiments analyzing the affinity of Pax-6/Cdx-2/3, Pax-6/Maf-B, and Pax-6/c-Maf complexes on G1. Relative binding affinities were estimated by the IC50 index (see in tables). The indices were measured by competition with increasing amounts of nonlabeled G1 ranging from 12.5- to 100-fold excess. E, Maf-B and c-Maf directly interact with Pax-6. Coimmunoprecipitation was performed on 500-µg nuclear extracts using an anti-mouse Pax-6 serum in the presence of sheep anti-mouse IgG magnetic Dynabeads (+). The experiments were also performed in the absence of Pax-6 sera (-). The precipitates were subjected to Western blotting for Pax-6, Maf-B, and c-Maf. Input for Pax-6, Maf-B, and c-Maf were 20 µg. IP and ID represent immunoprecipitated and immunodepleted extracts, respectively.

View larger version (12K): [in this window] [in a new window]   FIGURE 6. Effects of dominant negative forms of Cdx-2/3 and large Mafs on glucagon gene promoter activity in BHK cells. BHK-21 cells were cotransfected with 10 µg of -292GluCAT and 500 ng of Pax-6, Cdx-2/3, Maf-B, c-Maf, and dominant negative forms of Cdx-2/3 (DN-Cdx2/3) and Maf-A (DN-MafA) expression vectors. Histograms represent normalized CAT activity of -292GluCAT construct versus poCAT. The data are presented as the means ± S.E. for three different transfection experiments. *, p < 0.05 relative to CAT activity of Pax-6/Cdx-2/3; #, p < 0.05 relative to CAT activity of Pax-6/Maf-B; $, p < 0.05 relative to CAT activity of Pax-6/c-Maf condition using Student's t test.

View larger version (14K): [in this window] [in a new window]   FIGURE 7. Effects of dominant negative forms of Cdx-2/3 and large Mafs on glucagon gene promoter activity in glucagon-producing cells. InR1G9 (A) and -TC1 (B) cell lines were cotransfected with 10 µg of -292GluCAT and 500 ng of DN-Cdx2/3 or DN-MafA expression vectors. The data are expressed relative to the empty CAT reporter gene. C, InR1G9 cells were transfected with 10 µg of DN-Cdx2/3 and DN-MafA expression vectors. glucagon mRNA levels are expressed relative to the empty vector and corrected for TBP mRNA. All of the data are presented as the means ± S.E. for three different transfection experiments. The asterisk indicates statistical significance with p < 0.05 value using a Student's t test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

G1 is a large 50-base pair control element with three potential binding sites, a distal site to which Pax-6 and Brain-4 have been reported to bind, and two proximal sites for Cdx-2/3 and the large Mafs. The binding sites for Pax-6 and Brain-4 are overlapping (10, 15). Pax-6 is detected in glucagon-producing cells of the mouse embryonic pancreas at day 10.5 and in all pancreatic endocrine cells at later stages in development (12, 13, 35). Pax-6 interacts with G1 and transactivates the rat glucagon gene promoter both alone and synergistically with Cdx-2/3 and the large Mafs (9, 15). Brain-4 is also expressed in the developing and adult -cells from day 9.5 and is able to activate glucagon gene promoter constructs through G1 (10, 36). Furthermore, Brain-4 is specifically expressed in -cells and not in other islet cell types. However, by contrast to the dramatic effects observed in Pax-6 mutant mice where -cells are lacking, the absence of Brain-4 in homozygous mutant mice does not result in any abnormality in -cell development or function (23), suggesting that Pax-6 is a more likely candidate than Brain-4 in the activation of the -cell-specific element G1. In agreement with this hypothesis, we find by ChIP analyses that Pax-6 but not Brain-4 interacts in vivo with G1, whereas both factors are present in InR1G9 cells. Furthermore, the functional relevance of Pax-6 in glucagon gene expression is supported by the significant decrease in endogenous glucagon mRNA levels in the presence of siRNA specifically silencing Pax-6 gene expression. We thus conclude that Pax-6 binds the distal binding site of G1 and is functionally critical for glucagon gene expression.

View larger version (21K): [in this window] [in a new window]   FIGURE 8. Effects of specific inactivation of the Pax-6, Cdx-2/3, Maf-B, and c-Maf genes by siRNA on glucagon gene expression. InR1G9 cell line was transfected with 100 nM of specific siRNA or combinations of siRNA. A, effects of the respective specific siRNA on Pax-6, Cdx-2/3, Maf-B, and c-Maf gene expression by real time RT-PCR. mRNA levels are corrected by TBP mRNA values. B, Western blot analyses of Pax-6, Cdx-2/3, and Maf-B from transfected InR1G9 cells with scramble or specific siRNA. Anti-TFIIE- serves as a control for the specificity of siRNA effects. c-Maf protein quantification is not shown because the anti-c-Maf antibody does not detect hamster c-Maf. C, quantitative analyses of glucagon mRNA level after 48 h of siRNA transfection. The data are expressed relative to TBP mRNA values and are presented as the means ± S.E. for three different experiments. The asterisk indicates statistical significance with p < 0.05 value using a Student's t test.

The large Maf subfamily has multiple members including Maf-A, Maf-B, c-Maf, and Nrl. Although Maf-A has been clearly shown to be specifically expressed in β cells (18, 19), controversial data exist on the respective distribution of Maf-B and c-Maf in - and β-cells. It was first suggested that Maf-B was expressed more in - than β-cells, and c-Maf was present at low levels (18). Maf-B and c-Maf were then shown to be expressed in pancreatic -cell lines and c-Maf to be specific for -cells in the mouse (19). More recent data indicate that Maf-B is expressed as early as e10.5 and in both mouse glucagon- and insulin-producing cells although not uniformly and becomes restricted to -cells in the adult and a few β-cells (20, 39). It is to be noted that in the porcine pancreas, Maf-B is only expressed in β cells in the adult. c-Maf by contrast is present in both cell types during development and in the adult in the mouse (39) and porcine pancreas (40), although it has been proposed that c-Maf is not expressed in pancreatic endocrine cells (20). We find that Maf-B is expressed at much higher levels than c-Maf during mouse pancreatic development and in the adult. The same quantitative observation is found in glucagon-producing cell lines. We could not assess the presence and cell type distribution of c-Maf by immunocytochemistry because the different anti-c-Maf antibodies available were either nonspecific or did not generate any signal. By contrast, by Western blot analyses the anti-c-Maf BL662 antibody clearly demonstrated the presence of c-Maf in a reproductible pattern of bands in -TC1 cells and mouse adult islets confirming previous data (39). Both Maf-B and c-Maf are able to bind G1 in vitro and in vivo as indicated by EMSA and ChIP analyses; they bind with the same affinity both alone and in complexes with Pax-6. Indeed both Maf-B and c-Maf can directly interact with Pax-6 in the presence or absence of G1 as shown by EMSA and immunoprecipitation studies. Such an interaction between Pax-6 and the large Mafs was previously suggested for Maf-A (9). The transcriptional capacity of c-Maf was, however, significantly stronger compared with Maf-B in the presence or absence of Pax-6, suggesting that the large Mafs have clear functional differences. Similar observations have been previously reported for the A-crystallin gene promoter (41). Furthermore, silencing of c-Maf but not Maf-B in InR1G9 cells specifically affected endogenous glucagon mRNA levels, indicating that these factors may not be redundant at least in glucagon cell lines. We cannot exclude, however, the possibility that our results are somewhat biased by the fact that a 60% inhibition of the relatively abundant Maf-B might not be sufficient to affect glucagon gene expression, whereas the same decrease in c-Maf that is expressed at low levels results in clear effects; in the latter experiments, however, Maf-B was unable to compensate for the decrease of c-Maf. We propose that by analogy to the crystallin genes in the lens, Pax-6 might open the chromatin structure, activate c-Maf gene expression, and, along with c-Maf in turn, activate glucagon gene expression (41). These data then raise several questions, and more importantly the role of Maf-B, which is much more abundant in the mouse -cells than c-Maf and which is able to bind the glucagon gene promoter in vivo at e18.5 (21). Is Maf-B only implicated in endocrine cell development? Or alternatively in the regulated and not basal glucagon gene expression? Investigations of Maf-B- or c-Maf-deficient mice clearly showed a key role of large Mafs in the production of mature - and β-cells (21, 22). Furthermore, Maf-B is able to form homodimers through its leucine repeat structure and heterodimers with c-Maf, small Mafs, and Fos (42), suggesting a potential role in the regulated expression of the glucagon gene.

View larger version (29K): [in this window] [in a new window]   FIGURE 9. In vivo interactions of c-Maf on the glucagon gene promoter in adult mouse islets. Analyses of in vivo binding of c-Maf on the glucagon gene promoter in mouse adult islets by ChIP experiments. After pancreatic islets isolation, chromatin was sheared and extracted as described under "Experimental Procedures." Next, specific immunoprecipitations with an anti-histone H4 (used as a positive control) and anti-c-Maf were performed as indicated under "Experimental Procedures." A, c-Maf binding was analyzed with semi-quantitative PCR in ethidium bromide-stained 3% agarose gels. B, binding was also analyzed by real time PCR with a Light-Cycler (Roche Applied Science). To confirm c-Maf binding, a second set of glucagon gene promoter primers were used and gave identical results (data not shown). Binding intensity data are expressed relative to IgG immunoprecipitation (nonspecific binding) and are presented as the means ± S.E. for at least three independent experiments. The asterisk indicates statistical significance with p < 0.05 value using a Student's t test.

To verify whether the Maf- and Cdx-2/3-binding sites are overlapping, we analyzed the effects of dominant negative forms of the large Mafs or Cdx-2/3 on glucagon gene transcription in both glucagon- and non-glucagon-producing cells. Overexpression of DN-Cdx2/3 and DN-MafA markedly decreased glucagon promoter activity and glucagon gene expression in InR1G9 cells. We observed similar results in -TC1 cells, which do not express Cdx-2/3, thus suggesting that the large Mafs- and Cdx-2/3-binding sites are overlapping and that there is competition between the large Mafs and Cdx-2/3 to bind G1. These data also illustrate the critical importance of the distal (Pax-6-binding site) and proximal elements of G1 for glucagon gene expression.

Although c-Maf is expressed in -cells, it is also found in β-cells as well as Pax-6. We previously proposed the hypothesis that glucagon gene expression results from a default pathway (1). The absence of the β-cell-specific transcription factors, such as Pdx-1, Pax-4, and Nkx-6.1 through the action of Arx and other factors during development would allow for the glucagon gene to be expressed and for the differentiation of the -cell in the presence of islet-specific factors. The absence of the β-cell-specific factors Pax-4 or Pdx-1 in knock-out mice results in an increase in -cells and a large number of cells coexpressing glucagon and insulin (44, 45). We have also shown that ectopic expression of Pax-4 or Pdx-1 in glucagon-producing cell lines inhibit basal transcription of the glucagon gene promoter by 60-90%. Both Pax-4 and Pdx-1 are able to bind to the Pax-6-binding site with higher or similar affinity compared with Pax-6. In addition expression of Pdx-1 in glucagon-producing cells inhibits endogenous glucagon gene expression (46, 47). Finally, overexpression of a dominant negative form of Pdx-1 in insulin-producing cells leads to coexpression of insulin and glucagon in the same cell, suggesting an inhibitory role for Pdx-1 on glucagon gene expression (48). More recently, Nkx-6.1 has also been shown to play an inhibitory role on glucagon gene expression (49) through competition with Pax-6 (50). We thus conclude that -cell-specific expression of the glucagon gene is conferred by two potential mechanisms, the interaction of Pax-6 and c-Maf on G1 and the absence of β-cell specific transcription factors such as Pax-4, Pdx-1, and Nkx-6.1.

	FOOTNOTES

 
^* This work was supported by Swiss National Science Foundation Grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Present address: Dept. of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva 14, Switzerland.

¹ To whom correspondence should be addressed. Tel.: 41-22-372-42-37; Fax: 41-22-372-93-26; E-mail: Yvan.Gosmain{at}hcuge.ch.

³ The abbreviations used are: siRNA, small interfering RNA; BHK, baby hamster kidney; EMSA, electrophoretic mobility shift assay; ChIP, Chromatin Immunoprecipitation; CAT, chloramphenicol acetyltransferase; RT, reverse transcription; TBP, TATA box-binding protein; en, embryonic day n.

	ACKNOWLEDGMENTS

 
We thank Dr. S. Saule (CNRS/UMR 146, Institut Curie Section de Recherche, France) for generously providing Pax-6 antibody and Dr. T. Jin (Division of Cell and Molecular Biology, Toronto General Research Institute, University of Toronto, Toronto, Canada) and Dr. R. Stein (Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN) for providing the dominant negative forms of Cdx-2/3 and Maf-A, respectively. We also thank I. Dunand-Sauthier (Department of Pathology and Immunology, University of Geneva Medical School, CMU, Geneva, Switzerland) for helpful advice in coimmunoprecipitation assays.

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