The caudal-related Homeodomain Protein Cdx-2/3 Regulates Glucagon Gene Expression in Islet Cells

Abstract

Glucagon gene transcription in the endocrine pancreas is regulated by at least four cis-acting DNA control elements. We showed previously that G1 is critical for alpha cell-specific expression. G1 contains three AT-rich sequences important for promoter function, which represent candidate binding sites for homeodomain transcription factors. Performing reverse transcription-polymerase chain reaction amplifications with degenerate oligonucleotide primers homologous to the Antennapedia homeobox, cDNA clones corresponding to the caudal-related gene cdx-2/3 were predominantly obtained from glucagon-producing cells and primary non-beta cells. From RNase protection and polymerase chain reaction analyses, cdx-2/3 turned out to be the only caudal-related gene that is expressed at significant levels in cells of the endocrine pancreas. Cdx-2/3 binds with high affinity to an AT-rich motif of G1, which matches the consensus binding site of caudal-related proteins. In the glucagon-producing hamster cell line InR1G9, Cdx-2/3 is a subunit of complex B3 formed on G1. Alternative splicing generates two cdx-2/3 transcripts in islet cells, coding for a full-length protein and an amino-terminally truncated isoform. Although both isoforms bind G1 with similar affinity, only the full-length Cdx-2/3 A protein activates glucagon gene transcription in non-glucagon-producing cells, transcriptional activation being dose-dependent. We therefore conclude that the caudal-related gene cdx-2/3 is implicated in the transcriptional control of glucagon gene expression in the alpha cells of the islets of Langerhans. Glucagon gene transcription in the endocrine pancreas is regulated by at least four cis-acting DNA control elements. We showed previously that G1 is critical for alpha cell-specific expression. G1 contains three AT-rich sequences important for promoter function, which represent candidate binding sites for homeodomain transcription factors. Performing reverse transcription-polymerase chain reaction amplifications with degenerate oligonucleotide primers homologous to the Antennapedia homeobox, cDNA clones corresponding to the caudal-related gene cdx-2/3 were predominantly obtained from glucagon-producing cells and primary non-beta cells. From RNase protection and polymerase chain reaction analyses, cdx-2/3 turned out to be the only caudal-related gene that is expressed at significant levels in cells of the endocrine pancreas. Cdx-2/3 binds with high affinity to an AT-rich motif of G1, which matches the consensus binding site of caudal-related proteins. In the glucagon-producing hamster cell line InR1G9, Cdx-2/3 is a subunit of complex B3 formed on G1. Alternative splicing generates two cdx-2/3 transcripts in islet cells, coding for a full-length protein and an amino-terminally truncated isoform. Although both isoforms bind G1 with similar affinity, only the full-length Cdx-2/3 A protein activates glucagon gene transcription in non-glucagon-producing cells, transcriptional activation being dose-dependent. We therefore conclude that the caudal-related gene cdx-2/3 is implicated in the transcriptional control of glucagon gene expression in the alpha cells of the islets of Langerhans. INTRODUCTIONGlucagon and the glucagon-like peptides are synthesized as a common precursor, preproglucagon, encoded by the glucagon gene and are involved in the control of glucose homeostasis. Expression of the glucagon gene is highly restricted to the alpha cells of the endocrine pancreas, the L cells of the intestine, and certain areas of the brain (1Drucker D.J. Asa S. J. Biol. Chem. 1988; 263: 13475-13478Google Scholar, 2Mojsov S. Heinrich G. Wilson I.B. Ravazzola M. Orci L. Habener J.F. J. Biol. Chem. 1986; 261: 11880-11889Google Scholar, 3Novak U. Wilks A. Buelln G. McEwen S. Eur. J. Biochem. 1987; 164: 553-558Google Scholar). The factors controlling glucagon gene expression are poorly understood. In addition, they may differ depending on the tissue examined; studies in transgenic mice expressing the simian virus (SV) 1The abbreviations used are: SVsimian virusbpbase pair(s)PCRpolymerase chain reactionntnucleotide(s)BHKbaby hamster kidneyEMSAelectrophoretic mobility shift assayCATchloramphenicol acetyltransferaseRSVRous sarcoma virus. large T antigen under the control of 1300 bp of the rat glucagon gene 5′-flanking sequences demonstrate expression in the pancreas and the brain, whereas additional upstream sequences are necessary for expression in the gut (4Efrat S. Teitelman G. Anwar M. Ruggiero D. Hanahan D. Neuron. 1988; 1: 605-613Google Scholar, 5Lee Y.C. Asa S.L. Drucker D.J. J. Biol. Chem. 1992; 267: 10705-10708Google Scholar).Tissue-specific expression of the glucagon gene in the pancreas is conferred in two steps by the islet-specific enhancer elements G2, G3, and G4 (6Cordier-Bussat M. Morel C. Philippe J. Mol. Cell. Biol. 1995; 15: 3904-3916Google Scholar, 7Philippe J. Drucker D. Knepel W. Jepal L. Misulovin Z. Habener J.F. Mol. Cell. Biol. 1988; 8: 4877-4888Google Scholar, 8Philippe J. Morel C. Cordier-Bussat M. J. Biol. Chem. 1995; 270: 3039-3045Google Scholar) and the alpha cell-specific proximal promoter element G1 (7Philippe J. Drucker D. Knepel W. Jepal L. Misulovin Z. Habener J.F. Mol. Cell. Biol. 1988; 8: 4877-4888Google Scholar, 9Morel C. Cordier-Bussat M. Philippe J. J. Biol. Chem. 1995; 270: 3046-3055Google Scholar, 10Philippe J. Endocr. Rev. 1994; 2: 21-27Google Scholar). G1 contains three AT-rich sequences that are candidate binding sites for homeodomain transcription factors. Two of these sequences represent nearly identical 7-bp direct repeats and are important for transcriptional activity. At least four protein complexes were found to interact with G1, and the integrity of the AT-rich 7-bp direct repeats was shown to be critical for their binding (9Morel C. Cordier-Bussat M. Philippe J. J. Biol. Chem. 1995; 270: 3046-3055Google Scholar).In a first attempt to identify the proteins implicated in the control of glucagon gene expression, we designed degenerate oligonucleotide primers based on highly conserved sequences in the first and third helix of the Antennapedia class of homeodomain transcription factors. Recent studies indicate that homeobox genes are involved in the control of pancreatic hormone genes, such as insulin and somatostatin. Several homeodomain proteins have been isolated from islet cells by cDNA cloning or PCR amplification of reverse transcribed RNA (11Miller C. McGehee R. Habener J. EMBO J. 1994; 13: 1145-1156Google Scholar, 12Rudnick A. Ling T.Y. Odagiri H. Rutter W.J. German M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12203-12207Google Scholar) and proposed to be implicated in insulin and somatostatin gene regulation (13Emens L.A. Landers D.W. Moss L.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7300-7304Google Scholar, 14Karlsson O. Thor S. Norberg T. Ohlsson H. Edlund T. Nature. 1990; 344: 879-882Google Scholar, 15German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes & Dev. 1992; 6: 2165-2176Google Scholar, 16Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Google Scholar).In this study, we show that the caudal-related gene cdx-3, which has been proposed to regulate insulin gene transcription through its binding to the FLAT element (15German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes & Dev. 1992; 6: 2165-2176Google Scholar) is expressed in glucagon-producing cells. Since cdx-3 represents the hamster homologue of the mouse cdx-2 gene (15German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes & Dev. 1992; 6: 2165-2176Google Scholar, 17James R. Erler T. Kazenwadel J. J. Biol. Chem. 1994; 269: 15229-15237Google Scholar, 18Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Google Scholar), this gene will be referred to here as cdx-2/3. In cell lines of the endocrine pancreas, alternative splicing generates two cdx-2/3 mRNAs, encoding a full-length protein (termed Cdx-2/3 A) and an amino-terminally truncated isoform (Cdx-2/3 B). Cdx-2/3 binds the proximal AT-rich direct repeat motif of the glucagon promoter element G1, which matches the consensus binding site of caudal-related genes (18Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Google Scholar, 19Margalit Y. Yarus S. Shapira E. Gruenbaum Y. Fainsod A. Nucleic Acids Res. 1993; 21: 4915-4922Google Scholar) and is contained in complex B3 binding to G1. Interestingly, the full-length Cdx-2/3 A, but not the truncated Cdx-2/3 B isoform activates glucagon gene expression in non-glucagon-producing cells. Different transactivating properties of the two isoforms exhibiting the same affinity for G1 suggests that differential expression of cdx-2/3 A and cdx-2/3 B may regulate transcriptional activation. We conclude that the intestine-specific gene cdx-2/3 is the only caudal-related gene expressed in the endocrine pancreas and that it may be involved in the control of pancreatic glucagon gene expression.DISCUSSIONDifferentiation of multicellular organisms is based on a precise temporal and spatial pattern of cell specific gene expression. In vertebrates, as in all other metazoa analyzed today, homeotic genes are major control genes determining cell fate and function (42Gehring W. Affolter J.M. Bürglin T. Annu. Rev. Biochem. 1994; 63: 487-526Google Scholar). Islet cells express a variety of homeobox genes, and recent data indicate that Idx-1/STF1/IPF1, a homeodomain protein of the Antennapedia class, plays a major role in the cell-specific expression of the insulin gene, its regulation by glucose, and the differentiation of the pancreas (16Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Google Scholar, 43Leonard J. Peers B. Johnson T. Ferreri K. Lee S. Montminy M.R. Mol. Endocrinol. 1993; 7: 1275-1283Google Scholar, 54MacFarlane W.M. Read M.L. Gilligan M. Bujalska I. Docherty K. Biochem. J. 1994; 303: 625-631Google Scholar, 55Petersen H.V. Serup P. Leonard J. Michelsen B.K. Madsen O.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10465-10469Google Scholar, 56Serup P. Petersen H.V. Pedersen E.E. Edlund H. Leonard J. Petersen J.S. Larsson L.-I. Madsen O.D. Biochem. J. 1995; 310: 997-1003Google Scholar, 57Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 371: 606-609Google Scholar).To better understand the molecular mechanisms involved in the islet cell-specific expression of the glucagon gene, we searched for homeotic cDNA sequences in glucagon-producing cells by reverse transcription-PCR. cdx-2/3 appears from our PCR analysis to be the predominant homeobox gene from the Antennapedia class in glucagon-producing cells, although it is also present in the intestine and in pancreatic beta cells (15German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes & Dev. 1992; 6: 2165-2176Google Scholar, 17James R. Erler T. Kazenwadel J. J. Biol. Chem. 1994; 269: 15229-15237Google Scholar, 18Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Google Scholar). Quantification of cdx-2/3 by RNase protection experiments, Western blots, and EMSAs on RNA and nuclear extracts from glucagon-, insulin-, and somatostatin-producing cell lines show roughly equivalent cdx-2/3 transcript and protein levels in all cell types. In addition, the amount of PCR-amplified full-length cdx-2/3 cDNA products from primary beta and non-beta cells does not indicate major differences. The reason for the preferential amplification of Idx-1/STF1/IPF1 sequences in primary beta cells is then likely due to the relatively higher Idx-1/STF1/IPF1 mRNA levels compared with cdx-2/3. The relative distribution of Antennapedia class homeobox sequences identified in our study is slightly different from previous PCR analyses in islet cell lines and primary islets (11Miller C. McGehee R. Habener J. EMBO J. 1994; 13: 1145-1156Google Scholar, 12Rudnick A. Ling T.Y. Odagiri H. Rutter W.J. German M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12203-12207Google Scholar); some homeobox genes reported to be expressed in primary islets could not be detected among the cDNA clones sequenced (Table I). Likely explanations for these differences are statistical variance, primer choice, and the source of RNA analyzed.Cdx-2/3 is a member of the family of homeodomain proteins related to the Drosophila melanogaster gene caudal (cad) (58Mlodzik M. Fjose A. Gehring W.J. EMBO J. 1985; 4: 2961-2969Google Scholar). caudal-related genes have been found in Bombyx mori (59Xu X. Xu P.-X. Suzuki Y. Development. 1994; 120: 277-285Google Scholar), Caenorhabditis elegans (60Burglin T.R. Finney M. Coulson A. Ruvkun G. Nature. 1989; 341: 239-243Google Scholar), zebrafish, Xenopus laevis, and chicken, as well as in the mammals mouse, rat, hamster, and human (50Stein S. Fritsch R. Lemaire L. Kessel M. Mech. Dev. 1996; 55: 91-108Google Scholar, 61Bonner C.A. Loftus S.K. Wasmuth J.J. Genomics. 1995; 28: 206-211Google Scholar). In vertebrates, expression onset of most caudal-related genes is observed at the state of gastrulation exhibiting a gradient along the anteroposterior axis. With the exception of Chox-cad2 (62Serrano J. Scavo L. Roth J. de la Rosa E.J. de Pablo F. Biochem. Biophys. Res. Commun. 1993; 190: 270-276Google Scholar) and Xcad-3 (63Northrop J.L. Kimelman D. Dev. Biol. 1994; 161: 490-503Google Scholar), transcripts of caudal-related genes are detected predominantly in endodermally derived tissues, playing a major role in the development of the intestine (17James R. Erler T. Kazenwadel J. J. Biol. Chem. 1994; 269: 15229-15237Google Scholar, 23Gamer L.W. Wright C.V.E. Mech. Dev. 1993; 43: 71-81Google Scholar, 64Frumkin A. Pillemer G. Haffner R. Tarcic N. Gruenbaum Y. Fainsod A. Development. 1994; 120: 253-263Google Scholar, 65Suh E. Traber P.G. Mol. Cell. Biol. 1996; 16: 619-625Google Scholar). For cdxA, early expression pattern is reported to cover several organs of endodermal origin like liver, pancreas, lung, and the epithelial lining of the intestine, whereas later expression is restricted to the intestine. The presence of cdx-2/3 in cells of the endocrine pancreas (15German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes & Dev. 1992; 6: 2165-2176Google Scholar, T. Drucker D.J. Mol. Cell. Biol. 1996; 16: Scholar) may indicate a role of genes not only in the development of endodermally derived but also in transcriptional control of islet cells. In to the intestine, cdx-2/3 is the only caudal-related gene expressed at significant levels in the islets of Langerhans. Interestingly, alternative splicing of the cdx-2/3 mRNA to an amino-terminally truncated isoform protein with at an upstream to be (15German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes & Dev. 1992; 6: 2165-2176Google Scholar). all caudal-related genes a upstream of the (17James R. Erler T. Kazenwadel J. J. Biol. Chem. 1994; 269: 15229-15237Google Scholar, 23Gamer L.W. Wright C.V.E. Mech. Dev. 1993; 43: 71-81Google Scholar, Y. Kazenwadel J. R. J. Biol. Chem. 1993; Scholar). With the exception of cdx-2/3 these upstream only the consensus for vertebrates, which may the of the first The for cdx-2/3 B is to the consensus in the of the of Cdx-2/3 B may not be it is of Cdx-2/3 A in in indicate that of the of cdx-2/3 may be the regulation of glucagon gene expression. Cdx-2/3 binds the upstream control element G1 at the proximal AT-rich repeat which to the consensus binding site for caudal-related genes Y. Yarus S. Shapira E. Gruenbaum Y. Fainsod A. Nucleic Acids Res. 1993; 21: 4915-4922Google Scholar). is also although with affinity, of binding the repeat motif G1. in to the proximal the element contains a at in the consensus as well as in the binding sites is a for T. used for EMSAs both repeat Cdx-2/3 A is observed with nuclear extracts high of Cdx-2/3 A and of the repeat elements is data indicate that this is only in the presence of large of Cdx-2/3 A. With cdx-2/3 extracts the of complex is that of the Cdx-2/3 A is not with G1, an in with the affinity of the AT-rich motif for Cdx-2/3 A. with nuclear only the complex B3 is by and a Cdx-2/3 A only be detected or that may the of higher molecular with extracts from glucagon-producing cells, of is binding sites in Drosophila J. Nature. 1989; 341: Scholar), the rat insulin gene (15German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes & Dev. 1992; 6: 2165-2176Google Scholar), and the mouse gene (18Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Google Scholar) represent direct or and for the was Although most homeodomain proteins bind to sequences with it is well that also on repeats such as G1 J. Mol. Cell. Biol. 1993; 13: Scholar, C. J. Cell. 1988; Scholar, G. Finney M. Cell. Scholar, C.A. Mol. Cell. Biol. 1993; 13: Scholar). in to the on the gene the Cdx-2/3 A B3 binding to G1 is by the state not In a recent and Drucker T. Drucker D.J. Mol. Cell. Biol. 1996; 16: Scholar) have observed binding of Cdx-2/3 A as a to the G1 the used for in their study only Cdx-2/3 binding site and not of Cdx-2/3 A to the glucagon promoter to a activation of transcription in non-glucagon-producing cells, and of the Cdx-2/3 A binding site to a in transcriptional a for Cdx-2/3 A in glucagon gene expression (9Morel C. Cordier-Bussat M. Philippe J. J. Biol. Chem. 1995; 270: 3046-3055Google Scholar). The presence of an amino-terminally truncated Cdx-2/3 isoform in glucagon-producing cells that binds to G1 but does not transcription additional for gene The of Cdx-2/3 B with the transcriptional of Cdx-2/3 A in and cells suggests a role of Cdx-2/3 B as a of glucagon gene expression. could be due to of both isoforms for binding to the glucagon promoter or to the activation properties of Cdx-2/3 A. will be to the alternative splicing to both Cdx-2/3 isoforms is regulated development or in to and to in glucagon gene is a large DNA control element that likely with transcription Cdx-2/3 A may be of present in complex We previously identified at least three additional protein and (9Morel C. Cordier-Bussat M. Philippe J. J. Biol. Chem. 1995; 270: 3046-3055Google Scholar), that also be important for glucagon gene expression. the homeobox gene was shown to interact with the most proximal AT-rich motif to of the glucagon promoter element G1 M. Drucker D.J. J. Biol. Chem. 1995; 270: Scholar). in with bp of the 5′-flanking of the rat glucagon gene promoter to the gene, was to and with Cdx-2/3 A on transcription of the glucagon gene could be P. I. and J. Cdx-2/3 A is to interact with which by does not to a major role in glucagon gene A has been implicated in the control of cell and differentiation in the intestine E. Traber P.G. Mol. Cell. Biol. 1996; 16: 619-625Google Scholar) as well as in the regulation of intestine-specific gene at least for the and genes (18Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Google Scholar, F. J. K. Eur. J. Biochem. 1996; Scholar). Cdx-2/3 A is also to bind and the rat insulin gene promoter at the (15German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes & Dev. 1992; 6: 2165-2176Google Scholar), although other homeodomain factors such as Idx-1/STF1/IPF1 are likely of insulin gene transcription (11Miller C. McGehee R. Habener J. EMBO J. 1994; 13: 1145-1156Google Scholar, 16Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Google Scholar, 54MacFarlane W.M. Read M.L. Gilligan M. Bujalska I. Docherty K. Biochem. J. 1994; 303: 625-631Google Scholar, 55Petersen H.V. Serup P. Leonard J. Michelsen B.K. Madsen O.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10465-10469Google Scholar, 56Serup P. Petersen H.V. Pedersen E.E. Edlund H. Leonard J. Petersen J.S. Larsson L.-I. Madsen O.D. Biochem. J. 1995; 310: 997-1003Google Scholar). data and a recent T. Drucker D.J. Mol. Cell. Biol. 1996; 16: Scholar) indicate that Cdx-2/3 A is involved in glucagon gene expression both in the endocrine pancreas and in cell lines expressing In both cell Cdx-2/3 A was shown to the glucagon gene promoter T. Drucker D.J. Mol. Cell. Biol. 1996; 16: Scholar). are with the endodermal origin of both and indicate that some of the mechanisms of glucagon gene regulation in the intestine and endocrine pancreas be The presence of two Cdx-2/3 isoforms with different properties not only in but also in cells as well as in the intestine to control gene expression in these INTRODUCTIONGlucagon and the glucagon-like peptides are synthesized as a common precursor, preproglucagon, encoded by the glucagon gene and are involved in the control of glucose homeostasis. Expression of the glucagon gene is highly restricted to the alpha cells of the endocrine pancreas, the L cells of the intestine, and certain areas of the brain (1Drucker D.J. Asa S. J. Biol. Chem. 1988; 263: 13475-13478Google Scholar, 2Mojsov S. Heinrich G. Wilson I.B. Ravazzola M. Orci L. Habener J.F. J. Biol. Chem. 1986; 261: 11880-11889Google Scholar, 3Novak U. Wilks A. Buelln G. McEwen S. Eur. J. Biochem. 1987; 164: 553-558Google Scholar). The factors controlling glucagon gene expression are poorly understood. In addition, they may differ depending on the tissue examined; studies in transgenic mice expressing the simian virus (SV) 1The abbreviations used are: SVsimian virusbpbase pair(s)PCRpolymerase chain reactionntnucleotide(s)BHKbaby hamster kidneyEMSAelectrophoretic mobility shift assayCATchloramphenicol acetyltransferaseRSVRous sarcoma virus. large T antigen under the control of 1300 bp of the rat glucagon gene 5′-flanking sequences demonstrate expression in the pancreas and the brain, whereas additional upstream sequences are necessary for expression in the gut (4Efrat S. Teitelman G. Anwar M. Ruggiero D. Hanahan D. Neuron. 1988; 1: 605-613Google Scholar, 5Lee Y.C. Asa S.L. Drucker D.J. J. Biol. Chem. 1992; 267: 10705-10708Google Scholar).Tissue-specific expression of the glucagon gene in the pancreas is conferred in two steps by the islet-specific enhancer elements G2, G3, and G4 (6Cordier-Bussat M. Morel C. Philippe J. Mol. Cell. Biol. 1995; 15: 3904-3916Google Scholar, 7Philippe J. Drucker D. Knepel W. Jepal L. Misulovin Z. Habener J.F. Mol. Cell. Biol. 1988; 8: 4877-4888Google Scholar, 8Philippe J. Morel C. Cordier-Bussat M. J. Biol. Chem. 1995; 270: 3039-3045Google Scholar) and the alpha cell-specific proximal promoter element G1 (7Philippe J. Drucker D. Knepel W. Jepal L. Misulovin Z. Habener J.F. Mol. Cell. Biol. 1988; 8: 4877-4888Google Scholar, 9Morel C. Cordier-Bussat M. Philippe J. J. Biol. Chem. 1995; 270: 3046-3055Google Scholar, 10Philippe J. Endocr. Rev. 1994; 2: 21-27Google Scholar). G1 contains three AT-rich sequences that are candidate binding sites for homeodomain transcription factors. Two of these sequences represent nearly identical 7-bp direct repeats and are important for transcriptional activity. At least four protein complexes were found to interact with G1, and the integrity of the AT-rich 7-bp direct repeats was shown to be critical for their binding (9Morel C. Cordier-Bussat M. Philippe J. J. Biol. Chem. 1995; 270: 3046-3055Google Scholar).In a first attempt to identify the proteins implicated in the control of glucagon gene expression, we designed degenerate oligonucleotide primers based on highly conserved sequences in the first and third helix of the Antennapedia class of homeodomain transcription factors. Recent studies indicate that homeobox genes are involved in the control of pancreatic hormone genes, such as insulin and somatostatin. Several homeodomain proteins have been isolated from islet cells by cDNA cloning or PCR amplification of reverse transcribed RNA (11Miller C. McGehee R. Habener J. EMBO J. 1994; 13: 1145-1156Google Scholar, 12Rudnick A. Ling T.Y. Odagiri H. Rutter W.J. German M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12203-12207Google Scholar) and proposed to be implicated in insulin and somatostatin gene regulation (13Emens L.A. Landers D.W. Moss L.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7300-7304Google Scholar, 14Karlsson O. Thor S. Norberg T. Ohlsson H. Edlund T. Nature. 1990; 344: 879-882Google Scholar, 15German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes & Dev. 1992; 6: 2165-2176Google Scholar, 16Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Google Scholar).In this study, we show that the caudal-related gene cdx-3, which has been proposed to regulate insulin gene transcription through its binding to the FLAT element (15German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes & Dev. 1992; 6: 2165-2176Google Scholar) is expressed in glucagon-producing cells. Since cdx-3 represents the hamster homologue of the mouse cdx-2 gene (15German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes & Dev. 1992; 6: 2165-2176Google Scholar, 17James R. Erler T. Kazenwadel J. J. Biol. Chem. 1994; 269: 15229-15237Google Scholar, 18Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Google Scholar), this gene will be referred to here as cdx-2/3. In cell lines of the endocrine pancreas, alternative splicing generates two cdx-2/3 mRNAs, encoding a full-length protein (termed Cdx-2/3 A) and an amino-terminally truncated isoform (Cdx-2/3 B). Cdx-2/3 binds the proximal AT-rich direct repeat motif of the glucagon promoter element G1, which matches the consensus binding site of caudal-related genes (18Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Google Scholar, 19Margalit Y. Yarus S. Shapira E. Gruenbaum Y. Fainsod A. Nucleic Acids Res. 1993; 21: 4915-4922Google Scholar) and is contained in complex B3 binding to G1. Interestingly, the full-length Cdx-2/3 A, but not the truncated Cdx-2/3 B isoform activates glucagon gene expression in non-glucagon-producing cells. Different transactivating properties of the two isoforms exhibiting the same affinity for G1 suggests that differential expression of cdx-2/3 A and cdx-2/3 B may regulate transcriptional activation. We conclude that the intestine-specific gene cdx-2/3 is the only caudal-related gene expressed in the endocrine pancreas and that it may be involved in the control of pancreatic glucagon gene expression.