Abstract

Aims/hypothesis. The transcription factor Pdx1 is re-quired for the development and differentiation of allpancreatic cells. Beta-cell specific inactivation ofPdx1 in developing or adult mice leads to an increasein glucagon-expressing cells, suggesting that absenceof Pdx1could favour glucagon gene expression by adefault mechanism.Method. We investigated the inhibitory role of Pdx1on glucagon gene expression in vitro. The glucagono-ma cell line InR1G9 was transduced with a Pdx1-encoding lentiviral vector and insulin and glucagonmRNA levels were analysed by northern blot and real-time PCR. To understand the mechanism by whichPdx1 inhibits glucagon gene expression, we studied itseffect on glucagon promoter activity in non-islet cellsusing transient transfections and gel-shift analysis.Results. In glucagonoma cells transduced with a Pdx1-encoding lentiviral vector, insulin gene expression wasinduced while glucagon mRNA levels were reduced by

50 to 60%. In the heterologous cell line BHK-21, Pdx1inhibited by 60 to 80% the activation of the α-cell spe-cific element G1 conferred by Pax-6 and/or Cdx-2/3.Although Pdx1 could bind three AT-rich motifs withinG1, two of which are binding sites for Pax-6 and Cdx-2/3, the affinity of Pdx1 for G1 was much loweras compared to Pax-6. In addition, Pdx1 inhibited Pax-6 mediated activation through G3, to which Pdx1 wasunable to bind. Moreover, a mutation impairing DNAbinding of Pdx1 had no effect on its inhibition on Cdx-2/3. Since Pdx1 interacts directly with Pax-6 andCdx-2/3 forming heterodimers, we suggest that Pdx1inhibits glucagon gene transcription through protein toprotein interactions with Pax-6 and Cdx-2/3.Conclusion/interpretation. Cell-specific expression ofthe glucagon gene can only occur when Pdx1 expres-sion extinguishes from the early α cell precursor.[Diabetologia (2003) 46:810–821]

Keywords Endocrine pancreas, Pdx1, Pax-6, Cdx-2/3,glucagon, transcriptional regulation, α-cell.

Received: 21 January 2003 / Revised: 14 March 2003Published online: 3 June 2003© Springer-Verlag 2003

Corresponding author: B. Ritz-Laser PhD, Diabetes Unit, University Hospital Geneva, 24, rue Micheli-du-Crest, 1211Geneva 14, SwitzerlandE-mail: Beate.Laser@medecine.unige.chAbbreviations: E, embryonic day; EMSA, electrophoretic mo-bility shift assay; GST, glutathione S-transferase; PD, paireddomain; HD, homeodomain; PDHD, paired-linker-homeodo-main.

Diabetologia (2003) 46:810–821DOI 10.1007/s00125-003-1115-7

Ectopic expression of the beta-cell specific transcription factor Pdx1 inhibits glucagon gene transcriptionB. Ritz-Laser1, B. R. Gauthier1, A. Estreicher1, A. Mamin1, T. Brun1, F. Ris2, P. Salmon3, P. A. Halban2,D. Trono3, J. Philippe1

1 Diabetes Unit, University Hospital Geneva, Geneva, Switzerland2 Jeantet Research Laboratories, Geneva, Switzerland3 Department of Microbiology, Centre Médical Universitaire, Geneva, Switzerland

num. Determination of the pancreatic fate of the dor-sal anlage requires signals from the notochord repress-ing endodermal Sonic hedgehog expression, whereasthe ventral anlage forms independently from the noto-chord [1, 2]. In the mouse, the first two transcriptionfactors expressed in the prospective pancreatic endo-derm are HB9 and Pdx1, which are detected at the 8 to10 somite stage, respectively (embryonic day (E)8–8.5) [3, 4, 5]; both factors are transiently expressedin the entire pancreatic anlage and are crucial for itsdevelopment. Mice deficient in HB9 selectively lackthe dorsal pancreas due to a defect in specification ofthe pancreatic epithelium, whereas the ventral pancre-as develops and exhibits more subtle defects in beta-cell differentiation and islet organization [3, 4]. Inacti-

The mammalian pancreas arises as two evaginations,the dorsal and ventral pancreatic anlage, from the em-bryonic endoderm in the region of the future duode-

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vation of Pdx1 results in pancreatic agenesis through afailure in proliferation of the pancreatic epithelium aswell as in the branching and differentiation of the pan-creatic buds [5, 6]. The widespread expression of HB9and Pdx1 in the embryonic pancreas declines atE9.5–10.5 (HB9) and E10.5 (Pdx1) and they are laterrestricted to beta cells (HB9) or beta and δ cells(Pdx1). Similarly, the homeobox gene Isl-1 is tran-siently expressed in the entire dorsal bud and later re-stricted to pancreatic endocrine cells; mice deficient inIsl-1 lack the dorsal exocrine pancreas and have nodifferentiated islet cells [7, 8, 9]. Another transcriptionfactor expressed very early (E9) during developmentin discrete cells of the pancreatic epithelium is Pax-6,a member of the paired-homeobox family. Pax-6 iscrucial for the differentiation of glucagon-producing α cells inasmuch as Pax-6 homozygous mutant micehave no or few α cells [10, 11]. In contrast, inacti-vation of Pax-4, another paired homeobox gene, re-sults in the selective lack of beta and δ cells; micelacking both Pax-6 and Pax-4 have no endocrine cells[12].

Several lines of evidence suggest that Pdx1 plays akey role not only in islet cell differentiation but also inmaintaining the differentiated state and control of is-let-hormone gene expression. Pdx1 is a major transac-tivator of the insulin and somatostatin genes throughits synergistic interaction with E47/Beta2 and Pax6 orPbx/Prep1, respectively [13, 14, 15, 16, 17, 18]. Beta-cell specific inactivation of Pdx1 in developing miceresulted in a decrease in beta-cells with a concomitant2.5-fold increase in Glu+ cells and to coexpression ofinsulin and glucagon in 22% of cells suggesting thatα-cell differentiation and glucagon gene expressionare favored by the absence of Pdx1 in vivo [19]. Fur-thermore, transient inhibition of Pdx1 in pancreaticbeta cells of adult mice by antisense RNA expressionusing a Tet-On system led to reduction of Pdx1 depen-dent beta-cell specific gene expression and to a strik-ing increase in the number of glucagon gene express-ing cells that were homogeneously distributed withinthe islet [20]. Similarly, we could recently show in a Tet-On system in insulinoma cells that functional inactivation of Pdx1 resulted in differentiation of insulin-producing beta cells into glucagon-producingα-cells [21].

In this work, we aimed to complement the studiesof Pdx1 function in beta cells by analysing the effectof ectopic Pdx1 expression in α cells. Since α-cellspecificity is mainly characterized by glucagon geneexpression, we studied Pdx1 effects on glucagon genetranscription in glucagonoma (InR1G9) cells byLentiviral transduction and on glucagon promoter ac-tivity in non-islet cells. We show a new potentialfunction of Pdx1 as mediator of cell-specific expres-sion of the glucagon gene through inhibition of tran-scription.

Materials and methods

Cell culture and DNA transfection. The glucagon-producinghamster InRIG9 [22] and mouse αTC1 [23], the insulin-pro-ducing hamster HIT-T15 [24], the non-islet Syrian baby ham-ster kidney (BHK-21) and human embryonic kidney (HEK)cell lines were grown in RPMI 1640 (Seromed; Basel, Switzer-land) supplemented with 5% heat-inactivated fetal calf serumand 5% heat-inactivated newborn calf serum, 2 mmol/l gluta-mine, 100 units/ml of penicillin, and 100 µg/ml of streptomy-cin. BHK-21 and HEK cells were transfected by the calciumphosphate precipitation technique [25] using 10–15 µg of totalplasmid DNA per 10-cm petri dish. One µg of pSV2A PAP, aplasmid containing the human placental alkaline phosphatasegene, driven by the simian virus 40 (SV40) early promoter wasadded to monitor transfection efficiency [26]. Transfection ofInR1G9 cells was done using the DEAE-dextran method as described previously [27]. cDNAs for the hamster Cdx-2/3 andPdx1 (M.S. German, University of California, San Francisco,Calif., USA), rat Isl-1 (D. Drucker, University of Toronto, Toronto, Canada), mouse Meis2 (N. Copeland, National Can-cer Institute, Fredrick, USA), and quail Pax-6 (S. Saule, Insti-tut Curie, Orsay, France), were cloned in the expression vectorpSG5 (Stratagene). In experiments using variable quantities ofexpression vectors coding for Pdx1, Pax-6, or Cdx-2/3, totalamount of DNA was kept constant by adding appropriateamounts of the empty vector pSG5. Reporter plasmids consist-ed of the CAT reporter gene driven by different fragments ofthe rat glucagon gene promoter (-292GluCAT, -138GluCAT,G3–138GluCAT, G3–31GluCAT [28] or the rat insulin I genepromoter (-410InsCAT, comprising 410 bp of the 5’flankingsequence and 49 bp of exon 1 and intron 1 of the rat insulin Igene) [29]. Data are presented as fold stimulation of the CATactivity obtained with the reporter plasmid alone and are themeans +/- SEM of at least three experiments.

Transduction of InR1G9 cells with a lentiviral vector encodinghPDX1. A Human PDX1 cDNA (hPDX1), kindly provided byV. Schwitzgebel (University Hospital Geneva, Geneva, Swit-zerland), was subcloned into pBluescript (Stratagene) and theninserted into an optimized HIV-based vector (pWPT, kindlyprovided by M. Wiznerowicz, Faculty of Medecine, Geneva,Switzerland), generating the pWPT-PDX transfer plasmid.Map and sequence are available at http://www.medecine.un-ige.ch/~salmon/pWPT-PDX.html. Stocks of pWPT-PDX lenti-viral vectors were produced using transient cotransfection of293T cells with pWPT-PDX, R8.91 (second generation HIV-packaging plasmid [30], and pMDG (VSV envelope-expres-sion plasmid) as described previously [31]. Vectors were con-centrated by ultracentrifugation and resuspended in serum-freemedium (CellGro SCGM, CellGenix, Freiburg, Germany) asdescribed previously [32]. Vector stocks were stored at −70°Cand titres were determined by enzymatic assay of HIV reversetranscriptase. For hPDX1 transduction, 3×104 INR1G9 cellswere transduced in 50 µl of RPMI 1640 10% FCS with2.5×104 pPWT-PDX transducing units (as determined by RTassay). After 24 h, cells were washed twice with RPMI 10%FCS and cultured for further 72 h before testing for Pdx1 ex-pression by immunocytochemistry using anti-Pdx1 antibodieskindly provided by C. V. E. Wright (Vanderbilt Medical Cen-ter, Nashville, Tenn., USA). RNA was isolated for northernblotting using Trizol (Invitrogen) and whole-cell protein ex-tracts were prepared for EMSA. Northern blots were sequen-tially hybridized with random labelled complementary DNA(cDNA) probes for rat glucagon and proinsulin I and an 18S ribosomal (rRNA) oligonucleotide probe (5′-GCCGTCCCTC-TTAATCATGGCCTCAGTTCC). Hybridization signals of the

812 B. Ritz-Laser et al.: Ectopic expression of the beta-cell specific transcription factor Pdx1

pWPT-PDX transduced InR1G9 cells were quantified using aPhosphoImager (Molecular Dynamics) and expressed as gluca-gon mRNA/18S rRNA ratio relative to the control cells.

Quantitative RT-PCR. Reverse transcription was done by using2 µg of total RNA isolated from HIT-T15 and from non-infect-ed or lentivirus-infected InR1G9 cells, random hexanucleotideprimers and Superscript II reverse transcriptase (Invitrogen).One tenth of the resulting cDNA was used for real-time PCRin the LightCycler instrument (Roche) using the QuantiTectSYBR Green PCR kit (Qiagen) as recommended by the pro-vider. The following primers were used: insulin sense 5′-tcttctacacacccaagacc, antisense 5′-gttccacaatgccacgcttc; gluca-gon sense 5′-gatcattcccagcttcccag, antisense 5′-ctggtaaaggtcc-cttcagc; 28S rRNA sense 5′-tagccaaatgcctcgtcatc, antisense 5′-acctctcatgtctcttcacc.

Chloramphenicol aetyltransferase (CAT) and protein assays.Cell extracts were prepared 48 h after transfection and analy-sed for CAT and alkaline phosphatase activities as describedpreviously [28]. Quantification of acetylated and non-acetyla-ted forms was done with a PhosphorImager (Molecular Dy-namics). A minimum of three independent transfections wasdone; each of them carried out in duplicate.

Electrophoretic Mobility Shift Assays (EMSAs). Nuclear ex-tracts from InR1G9, HIT-T15 or BHK-21 cells transfectedwith Pax-6 or Pdx1 expression plasmids or the empty vectorpSG5 were prepared according to [33]. EMSAs were carriedout as described previously [34] adding first 8 to 28 µg (8 µg instandard reactions) of nuclear extracts and subsequently la-belled oligonucleotides containing the rat glucagon gene G1(G1–56, G1–52, G1–54, G1–50) or G3 (5′-GCTGAAGTA-GTTTTTCACGCCTGACTGAGATTGAAGGGTGTATTTC)sequence [35, 36], or the A3/A4 element of the rat insulin Igene (InsA3/A4, 5′-GATCTTGTTAATAATCTAATTACCCT-AGAACAATTATTAGATTAATGG) [37]. In competition ex-periments, labelled and cold oligonucleotides were addedsimulaneously and when specific antibodies were used, theywere added after half of the incubation time. Antisera weregenerously provided by H. Edlund (anti-Pdx1; University ofUmea, Umea, Sweden), S. Saule (anti-Pax-6 homeodomain,serum no. 13; Institut Curie, Orsay, France), D. Melloul (antifull-length Pdx1; Hebrew University Hadassah Medical Cen-ter, Jerusalem, Israel), and M.S. German (anti-Cdx-2/3; Uni-versity of California, San Francisco, Calif., USA).

Mutagenesis of Pdx1. In vitro mutagenesis was done by usingthe QuikChange Site-Directed Mutagenesis Kit (Stratagene,The Netherlands) according to the manufacturer’s protocol us-ing the following primers for the I192Q mutation of Pdx1: for-ward 5′-gagacacatcaagcagtggttccaaaacc and reverse 5′-ggttttg-gaaccactgcttgatgtgtctc.

GST fusion proteins and GST-precipitation. Hamster Pax-6GST fusion proteins have been described [36]. GST fusionproteins were expressed in E. coli and purified according to themanufacturer recommendations (Pharmacia). L-[35S]methio-nine-labelled Pdx1 was generated in vitro using the TNT wheatgerm extract system (Promega) and GST-precipitations weredone as described previously [36].

Data analysis. Data are presented as means ± SE and statisticalsignificance was tested by analysis of variance and Student’s t test where applicable. The threshold for statistical signifi-cance was a p value of less than 0.05 (* and ** indicate statis-tical significance with p<0.05 and p<0.01, respectively).

Results

Lentiviral transduction of Pdx1 in InR1G9 cells inhibitstranscription of the endogenous glucagon gene. To as-sess the effect of ectopic Pdx1 expression in glucagon-producing cells, we transduced the hamster glucagono-ma cell line InR1G9 with a lentiviral vector coding forhPdx1. After 4 days, expression of Pdx1 in two inde-pendent transductions of InR1G9 cells was confirmedby immunocytochemistry and EMSA in 90% of thecells (data not shown). Northern blot and real-time PCRanalysis showed that Pdx1 repressed endogenous gluca-gon mRNA levels by 50% and induced the endogenousinsulin gene in the glucagonoma cell line (Fig. 1).

Pdx1 inhibits transcriptional activation of the gluca-gon gene promoter by Pax-6 and Cdx-2/3. To investi-gate the mechanisms by which Pdx1 decreases gluca-gon mRNA levels, we analysed its effect on transacti-vation of glucagon gene reporter constructs in thenon-islet cell line BHK-21. The paired-homeodomainprotein Pax-6, which is essential for the developmentof α cells, acts as a major transactivator of the gluca-gon gene by its interaction with the α-cell-specificpromoter element G1 and the enhancer G3 [10, 11,36]. In addition, the homeodomain protein Cdx-2/3transactivates the glucagon gene promoter indepen-dently through G1 and acts synergistically with Pax-6[36, 38]. Since Pdx1 has been reported to interact withboth Pax-6 and Cdx-2/3 in vitro [15, 38], we investi-gated the effects of Pdx1 on Pax-6 and Cdx-2/3 induced activation of glucagon gene promoter con-structs. Overexpression of Pdx1 resulted in a dose-dependent inhibition, up to 90%, of Pax-6 mediatedtranscriptional stimulation whether G1 (-138GluCAT),G3 (G3–31GluCAT), or both G1 and G3 (G3–138Glu-CAT) were present in the reporter construct (Fig. 2).Pdx1 is thus able to inhibit Pax-6 mediated activationof glucagon gene expression through both the G1 andG3 elements in BHK-21 cells. In addition to its inhibi-tory action on Pax-6, Pdx1 reduced the Cdx-2/3 andPax6/Cdx-2/3-mediated activations of G1 (-138Glu-CAT) by 65% and 85%, respectively (Fig. 2C). Con-trol cotransfections with Isl-1 [39] and Meis2 [40] hadno effect on Pax-6 or Cdx-2/3 induced transcriptionalactivation (Fig. 2C).

When Pdx1 was transfected in BHK-21 cells in theabsence of Pax-6 and Cdx-2/3, CAT activity driven by-138Glu was stimulated (Fig. 2C, Fig. 3). This effectwas dose-dependent and quantitatively similar to theactivation observed with the rat insulin I promoterconstruct -410InsCAT (Fig. 3). However, the maximal4.6-fold stimulation of the glucagon gene promoter byPdx1 was weak when compared to that obtained withPax-6 or Pax6/Cdx-2/3.

Pdx1 binds to the G1 element. The G1 element of theglucagon gene promoter contains three AT-rich se-

B. Ritz-Laser et al.: Ectopic expression of the beta-cell specific transcription factor Pdx1 813

quences, two of which form a nearly perfect direct re-peat with binding sites for the homeodomain contain-ing proteins Pax-6 and Cdx-2/3 (Fig. 4A) [35, 36, 38,41]. Since Pdx1 is a member of the Antennapediafamily of homeodomain proteins [42] and able totransactivate -138Glu in the absence of Pax-6 andCdx-2/3, we tested whether Pdx1 could bind to theglucagon gene promoter. With glucagon-producingInR1G9 cell extracts, at least three protein complexesare formed on G1–56 comprising the direct repeat ele-ment within G1; Pax-6 as a monomer, Pax-6/Cdx-2/3as a heterodimer, and HNF3β [36, 43] (Fig. 4A,B,G1–56). Using nuclear extracts from the insulin-pro-ducing cell line HIT-T15, which contains Pdx1, an ad-ditional complex was detected. This band co-migratedwith a complex formed in BHK-21 cells overexpress-ing Pdx1 and was recognized by anti-Pdx1 antibodies.

Fig. 1A, B. Pdx1 induces insulin mRNA and reduces endoge-nous glucagon mRNA levels in glucagonoma cells. Northernblot (A) and Real-time RT-PCR (B) analysis of glucagon-pro-ducing InR1G9 cells (contr. A, contr. B), from two experi-ments of unselected InR1G9 cellpools transduced with a lenti-viral vector coding for hPdx1 (Pdx1 A, Pdx1 B,), and Hit-T15cells. The proportion of infected cells was roughly 90% (im-munocytochemistry), but Pdx1 infected cellpool A showed aslightly stronger Pdx1 protein complex in EMSA that cellpoolB. (A) Northern blot sequentially hybridized with probes forinsulin, glucagon and 18S rRNA. (B) Quantitative RT-PCRshowing that Pdx1 reduces glucagon mRNA levels to roughly36% and 62% of wild-type levels (cellpool A and B, respec-tively). Whereas insulin mRNA was undetectable in noninfect-ed InR1G9 cells by Northern blot (A), quantitative RT-PCRprovided evidence of as little as 0.2% of insulin mRNA levelsfound in HIT-T15 cells. Pdx1 infection increased insulinmRNA levels by 21 and 27-fold reaching 4% and 6% (cellpoolA and B, respectively) of HIT-T15 insulin mRNA levels

814 B. Ritz-Laser et al.: Ectopic expression of the beta-cell specific transcription factor Pdx1

Pdx1 is thus able to interact with the G1 element. Tolocalize the binding site of Pdx1 within G1, we usedoligonucleotides G1–54, G1–52, and G1–50 repre-senting the three AT-rich elements. Pdx1 bound to allthese sites (Fig. 4B) and is thus capable of interactingwith three AT-rich motifs within the first 100 bp of theglucagon gene promoter.

Fig. 2A–C. Pdx1 interferes with transcriptional activation ofthe glucagon gene promoter by Pax-6 and Cdx-2/3. (A) Sche-matic representation of glucagon reporter gene constructs usedin this study. Characterized cis-acting control elements are des-ignated as G1 to G4. (B) Dose-dependent inhibition of thePax-6-mediated activation of the glucagon gene elements G1(-138GluCAT) and/or G3 by Pdx1 in BHK-21 cells. (C) To as-sess their effect on Pax-6 and/or Cdx-2/3 mediated activationof the glucagon gene promoter, BHK-21 cells were cotransfec-ted with 1 µg of Pdx1, Pax-4, Meis2, and/or Isl-1 expressionvectors, 250 ng of expression vectors containing the Pax-6and/or Cdx-2/3 cDNAs and 10 µg of -138GluCAT

Fig. 3. Dose-dependent activation of the glucagon gene ele-ment G1 by Pdx1. BHK-21 cells were cotransfected with 10 µgof the indicated reporter plasmids and increasing amounts ofPdx1 expression vectors

To assess the relative affinity of Pdx1 for G1 com-pared to the A3/A4 of the rat insulin I gene, a knownPdx1 binding site, we carried out EMSA competitionexperiments. A similar molar excess of cold G1–56and InsA3/A4 oligonucleotides competed for the Pdx1complex formed with InsA3/A4 indicating that Pdx1binds to both elements with roughly the same affinity(Fig. 4C). These data are in agreement with the equiv-alent transactivation of the glucagon and insulin genepromoters by Pdx1. When the individual glucagongene binding sites were used for EMSA, the mostproximal motif G1–50 displayed a slightly higher affinity for Pdx1 compared to G1–52 and G1–54 lo-cated within G1–56 but lower compared to InsA3/A4(Fig. 4D).

B. Ritz-Laser et al.: Ectopic expression of the beta-cell specific transcription factor Pdx1 815

Fig. 4A–D. Pdx1 binds to three sites within the glucagon geneG1 element. (A) Schematic diagram illustrating oligonucleo-tides for the glucagon gene G1 element and protein complexesformed on G1. The AT-rich elements are underlined. (B) EMSAusing nuclear extracts from InR1G9, Hit-T15, or BHK-21 cellsoverexpressing Pdx1. Pdx1 was able to bind the three AT-richelements contained within G1. Ab indicates the addition of anti-Pdx1 antibodies. A and C represent yet unidentified protein-DNA complexes.(C, D) Nuclear extracts of BHK-21 cells over-expressing Pdx1 were incubated with the InsA3/A4 or G1–50oligonucleotide and the indicated excess of cold competitor oli-gonucleotides. Bold arrowheads and asterisks indicate specificand non-specific complexes, respectively

816 B. Ritz-Laser et al.: Ectopic expression of the beta-cell specific transcription factor Pdx1

Fig. 5A–E. Pdx1 does not impair Pax-6 binding to the gluca-gon gene promoter. (A, B) Competition experiments analysingthe relative affinity of Pdx1 and Pax-6 for the G1 element. Pro-tein-DNA complexes formed with nuclear extracts from BHK-21 cells overexpressing Pdx1, Pax-6 or Cdx-2/3 and G1–56were competed for by the indicated molar excess of cold oligo-nucleotides. (C) Pax-6 containing extracts from BHK-21 cellswere mixed with increasing amounts of Pdx1 containing ex-tracts and incubated with the G3 oligonucleotide. No impair-ment of Pax-6 binding by Pdx1 was observed. Heterodimers ofthe widely expressed Prep1/Pbx proteins formed on G3 [44]are observed in all BHK-21 extracts. (D) Pdx1 formed a het-erodimer with Pax-6 on G1–56; this complex was displaced byadding anti-Pax6 (P6) or anti-Pdx1 (P1) antibodies. The identi-ty of two further slower migrating complexes containing Pdx1has yet to be determined. (E) When Pdx1 containing extractswere mixed with Cdx-2/3 containing extracts, Cdx-2/3 formeda heterodimer with Pdx1 on G1-56; this complex was dis-placed by adding anti-Cdx-2/3 antibodies. Note that when anti-Pdx1 antibodies were used, the Cdx-2/3-Pdx1 heterodimer wasdisplaced and a Cdx-2/3 homodimer with only slightly slowermigration than Cdx-2/3-Pdx1 was formed

Pdx1 forms heterodimers with Pax-6 and Cdx-2/3 onG1. To investigate the mechanism by which Pdx1 affects the Pax-6 and Cdx-2/3 mediated activation ofthe glucagon gene, we assessed whether Pdx1 couldaffect Pax-6 and Cdx-2/3 binding to DNA in vitro.When BHK-21 nuclear extracts containing Pax-6 weremixed with Pdx-containing extracts, Pdx1 formed aweaker complex on G1–56 as compared with thebinding reaction without Pax-6 (Fig. 5A). Further-more, competition with cold G1–56 showed a betteraffinity of Pax-6 compared with Pdx1 for G1. By con-trast with Pax-6, Cdx-2/3 and Pdx1 displayed similaraffinity for G1–56 (Fig. 5B).

Alternatively to a mechanism of direct competitionfor the binding site, Pdx1 could interact with Pax-6and/or Cdx-2/3 and either form a heterodimer or im-pair DNA binding by protein to protein interaction.We therefore analysed the effect of adding variousamounts of Pdx1 on Pax-6 binding to G3 and G1.Pdx1 overexpressed in BHK-21 cells did not interactwith G3, only heterodimers of the ubiquitous proteinsPbx and Prep1 [44] were observed (Fig. 5C). Pax-6formed a complex with G3 that was not affected bythe addition of increasing amounts of Pdx1-containingextracts (Fig. 5C). In contrast to G3, Pdx1 bound as amonomer and a homodimer to G1 (Fig. 5D). In addi-tion, a Pax6:Pdx1 heterodimer was formed that wasrecognized by both anti-Pdx1 and anti-Pax-6 antibod-ies, thus confirming the protein to protein interactionof both transcription factors (Fig. 5D). When Pdx1and Cdx-2/3 containing extracts were mixed, bothproteins bound as monomers and Pdx1 did not impairCdx-2/3 binding. In addition, both proteins formed aheterodimer that was recognized by anti-Cdx-2/3 andanti-Pdx1 antibodies (Fig. 5E). Note that when anti-Pdx1 antibodies were used, the Cdx-2/3-Pdx1 hetero-dimer was displaced by a Cdx-2/3 homodimer withonly slightly slower migration than Cdx-2/3-Pdx1. Wetherefore conclude that Pdx1 could exert its transcrip-tional inhibition on the glucagon gene promoterthrough protein to protein interaction with Pax-6 andCdx-2/3 that could change their conformation and in-terfere with their transcriptional properties. Further-more, inhibition of Pax-6 mediated activation couldalso in part be mediated by competition of binding tothe G1 but not the G3 element.

Pdx1 interacts with the homeodomain of Pax-6. To testfor direct physical interactions of Pdx1 and Pax-6, wedid GST precipitation assays using GST fusion pro-teins containing either the Pax-6 paired domain (PD),homeodomain (HD) or paired-linker-homeodomain(PDHD) and 35S-labelled Pdx1. Protein to protein in-teractions with Pdx1 were observed with fusion pro-teins comprising the Pax-6 HD (10% of the input),whereas precipitation of Pdx1 with the Pax-6 PD wasweak. However, adding the PD and linker domain tothe Pax-6 HD increased interaction to 17% (Fig. 6).

B. Ritz-Laser et al.: Ectopic expression of the beta-cell specific transcription factor Pdx1 817

These data show that Pdx1 interacts with Pax-6 in theabsence of DNA and that major contacts are mediatedby the Pax-6 homeodomain although the linker domainand/or PD might be required for full interactions.

Inhibition of Pax-6 and Cdx-2/3 mediated activationis independent of Pdx1 DNA binding. To further testour hypothesis that Pdx1 represses Pax-6 and Cdx-2/3mediated activation of the glucagon gene promoterthrough protein to protein interactions, we mutatedPdx1 in an amino acid (I192Q) of the DNA recogni-tion helix within the homeodomain that has been re-ported to abolish binding to the A3/A4 element of therat insulin I gene promoter [45]. This mutant Pdx1protein was produced at equivalent levels in fibroblastcells and in in vitro translation assays (Fig. 7A), andits binding activity to the G1 element was reduced bymore than 90% (Fig. 7B).

In cotransfection assays, a quantitatively similar re-pression of Cdx-2/3 was observed with the Pdx1 wtand mutant I192Q protein (Fig. 7C) confirming thatthe inhibition of Cdx-2/3 mediated transcriptional ac-tivation is independent of Pdx1 DNA binding andrather occurs through protein to protein interactions.In contrast, repression of Pax-6 mediated activationwas attenuated by the Pdx1 mutation (78% and 54%

Fig. 6A, B. Pdx1 interacts with the HD of Pax-6. (A) GST-pre-cipitation assay using 10 µg of GST alone, GST-Pax-6 paired(PD), homeo (HD) or paired-linker-homeodomain (PDHD, fu-sion proteins immobilized on sepharose beads and in vitro syn-thesized, 35S-labelled Pdx1. Lane input, 10% of the respectivein vitro translation reaction used for protein to protein interac-tion. (B) Quantification of protein interactions shown in (A) asthe precipitation rates relative to the respective input. Data arepresented as the mean ± SEM of at least three experiments

818 B. Ritz-Laser et al.: Ectopic expression of the beta-cell specific transcription factor Pdx1

Fig. 7A–D. Mutant Pdx1 protein impaired in DNA bindingstill repressed transcriptional activation of the glucagon genepromoter by Pax6 and Cdx-2/3. (A) Western blot analysisshowing similar synthesis of wild-type (wt) and mutant(I192Q) Pdx1 in HEK cells and in in vitro transcription/trans-lation (TNT). (B) EMSA analysis showing 90% reduced bind-ing activity of Pdx1 I192Q to G1 as compared to Pdx1 wt. (C)To assess the effect of mutant Pdx1 on Pax-6 and Cdx-2/3 me-diated activation of the glucagon gene promoter, BHK-21 cellswere cotransfected with 1 µg of wt Pdx or Pdx1 I192Q expres-sion vectors and 250 ng of expression vectors containing thePax-6 or Cdx-2/3 cDNAs as well as 10 µg of -138GluCAT. (D)GST-precipitation assay using 10 µg of GST alone or GST-Pax-6 paired-linker-homeodomain fusion protein immobilizedon sepharose beads and in vitro synthesized, 35S-labelled Pdx1wt or I192Q. Lanes input, 10% of the respective in vitro trans-lation reactions used for protein to protein interaction

by wt and I192Q Pdx1, respectively). We thereforetested by GST pulldown whether the I192Q mutationaffected interactions between Pdx1 and Pax-6. Proteinto protein interactions of both transcription factorswere reduced by approximately 65% indicating thatthe Pdx1 homeodomain is critical for protein interac-tions with Pax-6 (Fig. 7D). Since the reduced repres-sor activity of Pdx1 I192Q on Pax-6 correspondsquantitatively with the decreased protein to protein in-teraction of both factors, we suggest that Pdx1 couldexert its repressor activity primarily through protein toprotein interaction. Our data cannot, however, rule outa contribution of DNA binding competition to its in-hibitory effect on Pax-6.

Discussion

We show that Pdx1 is able to act as a repressor of theendogenous glucagon gene in InR1G9 glucagonoma

potentially neutralizing Cdx-2/3 function independentof DNA binding. In the same study, Pdx1 was able toinhibit the Cdx-2/3 mediated activation of the sucrose-isomaltase gene in heterologous cells. (ii) Alternative-ly, free Pdx1 might efficiently compete for commoncoactivators and impair their interaction with DNA-bound transcription factors thus blocking transcrip-tional activation. In this regard it is interesting thatoverexpression of Pdx1 in insulin-producing cells hasrecently been shown to repress insulin promoter activ-ity [50]. Similarly, an excess of Pdx1 impaired its syn-ergy with E47 on the insulin mini-enhancer in non-is-let cells [16]. One common cofactor linking specifictranscription factors to the basal transcription machin-ery, CBP/p300, is able to physically interact with Pax-6, Cdx-2/3, and Pdx1 [38, 51]. We therefore cotrans-fected BHK-21 cells with increasing amounts of CBPor p300; however, this complementation with tran-scriptional coactivators failed to compensate for thetranscriptional repression by Pdx1. Pax-6 contains anunusually long transactivation domain that has beenreported to directly interact with the TATA bindingprotein within TFIID (TBP) [52]. Pdx1 could thus im-pair Pax-6 interactions with TBP or alternatively, actby disturbing a multifactorial transcription complexby altering the ratio of its free components.

Pdx1 has been implicated in the tissue-specific acti-vation of different genes through cell type and pro-moter context dependent protein to protein interac-tions. In acinar cells, Pdx1 associates with Pbx1 andMeis2 on the B element of the elastase I gene [40] andendocrine-specific complexes consist of Pdx1/Pbx,Pdx1/Pax-6 or E47/Beta2/Pdx1 on the somatostatinand insulin gene promoter, respectively [13, 14, 15,16, 17]. These protein contacts allow for cooperativeDNA binding and synergistic transcriptional activa-tion. We show a new potential function of Pdx1 asmediator of cell-specific expression of the glucagongene through its inhibition of Pax-6 and Cdx-2/3 me-diated transcription.

Acknowledgements. We thank V. Schwitzgebel, D. Drucker, H.Edlund, M.S. German, S. Saule, C.V.E. Wright, D. Melloul, N.Copeland, and M.D. Walker for generously providing molecu-lar probes. This work was supported by the Swiss NationalFund (J. Philippe, P.A. Halban), the Institute for Human Genet-ics and Biochemistry (J. Philippe), the Berger Foundation (J.Philippe), Juvenile Diabetes Research Foundation International(P.A. Halban) and the Carlos and Elsie de Reuters Foundation(J. Philippe).

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