Glucagon Gene Transcription Activation Mediated by Synergistic Interactions of pax-6 and cdx-2 with the p300 Co-activator

In the endocrine pancreas, α-cell-specific expression of the glucagon gene is mediated by DNA-binding proteins that interact with the proximal G1 promoter element, which contains several AT-rich domains. The homeodomain transcription factors brain-4, pax-6, and cdx-2 have been shown to bind to these sites and to transactivate glucagon gene expression. In the present study, we investigated the interaction of cdx-2 and pax-6 with p300, a co-activator coupled to the basal transcription machinery. In transient transfection-expression experiments, we found that the transactivating effects of cdx-2 and pax-6 on the glucagon gene were greatly enhanced by the additional expression of p300. This enhancement was due to direct protein-protein interactions of both pax-6 and cdx-2 with the N-terminal C/H1 domain of p300. pax-6 and cdx-2 also directly interacted with one another at the protein level. pax-6, bound to its DNA recognition site in the glucagon G1 promoter element, tethered cdx-2 to the molecular complex of pax-6 and p300. Further, we found that the presence of cdx-2 enhanced the interaction of pax-6 with p300, thus establishing a molecular complex of transcription factors implicated in tissue-specific glucagon gene expression with the basal transcriptional machinery. In the endocrine pancreas, α-cell-specific expression of the glucagon gene is mediated by DNA-binding proteins that interact with the proximal G1 promoter element, which contains several AT-rich domains. The homeodomain transcription factors brain-4, pax-6, and cdx-2 have been shown to bind to these sites and to transactivate glucagon gene expression. In the present study, we investigated the interaction of cdx-2 and pax-6 with p300, a co-activator coupled to the basal transcription machinery. In transient transfection-expression experiments, we found that the transactivating effects of cdx-2 and pax-6 on the glucagon gene were greatly enhanced by the additional expression of p300. This enhancement was due to direct protein-protein interactions of both pax-6 and cdx-2 with the N-terminal C/H1 domain of p300. pax-6 and cdx-2 also directly interacted with one another at the protein level. pax-6, bound to its DNA recognition site in the glucagon G1 promoter element, tethered cdx-2 to the molecular complex of pax-6 and p300. Further, we found that the presence of cdx-2 enhanced the interaction of pax-6 with p300, thus establishing a molecular complex of transcription factors implicated in tissue-specific glucagon gene expression with the basal transcriptional machinery. cyclic AMP response element-binding protein base pair(s) CAAT element-binding protein β C/EBP homologous protein CREB-binding protein electrophoretic mobility shift assay EDTA low salt buffer glutathione S-transferase NaCl-EDTA-Nonidet P-40, p300, protein homologous to CBP Rous sarcoma virus polyacrylamide gel electrophoresis Dulbecco's modified Eagle's medium The glucagon gene is expressed in the pancreatic endocrine α-cells of the pancreatic islets, the L cells of the intestine, and specific areas of the brain (1Habener J.F. Fehmann H.C. Goeke B. The Insulinotropic Gut Hormone Glucagon-like Peptide-I. Karger, Basel, Switzerland1996Google Scholar). The cell-specific expression of the proglucagon gene is best studied in the pancreatic α-cells, in which expression is conferred by the proximal G1 promoter element (1Habener J.F. Fehmann H.C. Goeke B. The Insulinotropic Gut Hormone Glucagon-like Peptide-I. Karger, Basel, Switzerland1996Google Scholar). The G1 element contains two AT-rich motifs that are binding sites for homeodomain transcription factors. Recently, the transcription factors cdx-2, pax-6, and brain-4 have been shown to bind the AT-rich elements and transactivate glucagon gene expression (2Laser B. Meda P. Constant I. Philippe J. J. Biol. Chem. 1996; 271: 28984-28994Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 3Jin T. Trinh D.K.Y. Wang F. Drucker D.J. Mol. Endocrinol. 1997; 11: 203-209Crossref PubMed Scopus (38) Google Scholar, 4Hussain M.A. Lee J. Miller C.P. Habener J.F. Mol. Cell. Biol. 1997; 17: 7186-7194Crossref PubMed Google Scholar). Nuclear proteins of glucagon-producing cell lines form three main protein complexes with the G1 promoter element (5Philippe J. Drucker D.J. Knepel W. Jepeal L. Misulovin Z. Habener J.F. Mol. Cell. Biol. 1988; 8: 4877-4888Crossref PubMed Scopus (125) Google Scholar). One of these protein complexes contains the POU domain transcription factor brain-4 (4Hussain M.A. Lee J. Miller C.P. Habener J.F. Mol. Cell. Biol. 1997; 17: 7186-7194Crossref PubMed Google Scholar). Of the two other complexes, the lower molecular weight complex has been reported to contain the paired-domain factor pax-6 as monomer, whereas the higher molecular weight form contains pax-6 in a heterodimer with the caudal-related factor cdx-2 (6Andersen F. Heller R. Petersen H. Jensen J. Madsen O. Serup P. FEBS Lett. 1999; 445: 306-310Crossref PubMed Scopus (32) Google Scholar, 7Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Of these transcription factors, brain-4 is expressed specifically in pancreatic α-cells (4Hussain M.A. Lee J. Miller C.P. Habener J.F. Mol. Cell. Biol. 1997; 17: 7186-7194Crossref PubMed Google Scholar), and pax-6 is expressed in all pancreatic endocrine cells implicated in islet cell development, predominantly the pancreatic α-cells (8Sander M. Neubüser A. Kalamaras J. Ee H.C. Martin G.R. German M.S. Genes Dev. 1997; 11: 1662-1673Crossref PubMed Scopus (475) Google Scholar, 9St.-Onge L. Sosa-Pineda B. Nature. 1997; 387: 406-409Crossref PubMed Scopus (671) Google Scholar). pax-6 also binds to the G3 element of the proglucagon gene enhancer at a site that confers insulin inhibition of proglucagon gene expression (8Sander M. Neubüser A. Kalamaras J. Ee H.C. Martin G.R. German M.S. Genes Dev. 1997; 11: 1662-1673Crossref PubMed Scopus (475) Google Scholar). cdx-2 is found in both pancreatic α- and β-cell lines (3Jin T. Trinh D.K.Y. Wang F. Drucker D.J. Mol. Endocrinol. 1997; 11: 203-209Crossref PubMed Scopus (38) Google Scholar), as well as in intestinal epithelial cells (3Jin T. Trinh D.K.Y. Wang F. Drucker D.J. Mol. Endocrinol. 1997; 11: 203-209Crossref PubMed Scopus (38) Google Scholar, 4Hussain M.A. Lee J. Miller C.P. Habener J.F. Mol. Cell. Biol. 1997; 17: 7186-7194Crossref PubMed Google Scholar, 10Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (386) Google Scholar). Recently, pax-6 and cdx-2 have been shown to directly bind to each other and to synergistically transactivate the proglucagon gene via interaction with the G1 element of the proglucagon gene promoter (6Andersen F. Heller R. Petersen H. Jensen J. Madsen O. Serup P. FEBS Lett. 1999; 445: 306-310Crossref PubMed Scopus (32) Google Scholar,7Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). cAMP response element-binding protein (CREB)1-binding protein (CBP) and a closely related homologue p300 are known to integrate a number of cell-signaling pathways and to serve as co-activators of various transcription factors, including helix-loop-helix and homeodomain proteins (11Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.E. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (937) Google Scholar, 12Arany Z. Sellers W. Livingston D. Eckner R. Cell. 1994; 77: 799-800Abstract Full Text PDF PubMed Scopus (380) Google Scholar, 13Lundblad J.R. Kwok R.P. Laurence M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (534) Google Scholar, 14Xu L. Lavinsky R.M. Dasen J.S. Flynn S.E. McInerney E.M. Mullen T.-M. Heinzel T. Szeto D. Korzus E. Kurokawal R. Aggarwal A.K. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1998; 395: 301-306Crossref PubMed Scopus (251) Google Scholar). Both CBP and p300 serve as adapter proteins linking DNA binding transcription factors with the basal transcriptional machinery. Furthermore, CBP and p300 are proposed to both activate histone acetyltransferase and displace nucleosomes, as well as recruit RNA polymerase II to the transcription complex (15Ogryzko V.V. Schiltz L. Russanova V. Howard B.H. Nakatni Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2445) Google Scholar). In an attempt to understand the mechanisms underlying the tissue-specific transcription of the proglucagon gene, we have examined the mediation of the transactivating properties of pax-6 and cdx-2. We report here that the transactivating effects of both pax-6 and cdx-2 are mediated through their interaction with the transcriptional co-activator p300. Furthermore, our results suggest that the binding of pax-6 to p300 is weak and that the presence of cdx-2 enhances the physical interaction of pax-6 with p300. Whereas the transcriptional activity of pax-6 is dependent on its binding to its DNA recognition site within the G1 promoter element, binding of cdx-2 to the promoter is not absolutely required since pax-6 tethers cdx-2 to form a complex with cdx-2 and p300. Baby hamster kidney (BHK)-21 cells were obtained from the American cells were a from S. of cells were at in in Dulbecco's modified Eagle's medium of with and of and were with to the of the for were in One of the was of expression were as in the was for of for activity obtained was to the activity obtained of the in the were on three of these are were all by a promoter with of expression and a which are by a Rous sarcoma virus and and various of p300 were by L. M. Rosenfeld of M. of P. Traber of and M. R. Goodman and H. proglucagon gene from and to to a have been (4Hussain M.A. Lee J. Miller C.P. Habener J.F. Mol. Cell. Biol. 1997; 17: 7186-7194Crossref PubMed Google Scholar, T. Drucker D.J. Mol. Endocrinol. 1995; PubMed Google Scholar). in the G1 element were by polymerase and are to of the for the electrophoretic mobility shift assay The were by and which a of to have been Habener J.F. S. A. 1994; PubMed Scopus Google for from to with the from to with in the AT-rich site of the G1 element of the glucagon gene promoter sites are from to with in the AT-rich site of the G1 element of the glucagon gene promoter sites are from to with the from to with in the AT-rich site of the G1 element of the glucagon gene promoter sites are from to with in the AT-rich site of the G1 element of the glucagon gene promoter sites are in a cells were in in and with of expression as cells were with were in of with of for were with and proteins were to the E. P. W. 17: PubMed Scopus Google Scholar). was as in T. A. D. H. Cell. Full Text PDF PubMed Scopus Google of were to with EDTA low salt buffer P-40, T. A. D. H. Cell. Full Text PDF PubMed Scopus Google and to a of The was with of from the was with a specific to cdx-2, pax-6, p300 at on a of a and an additional for the was and the was in The proteins in the were by and by In with binding of with p300 cells were with and for a of to protein-protein interaction in of was as of the cells to of the the proteins were to a The of p300, pax-6, and cdx-2 was with the with as the proteins were in and on proteins were on in the assay to of proteins were with an in and to the a an with was of proteins were bound to of in a of of buffer for at were three in of and with of protein cdx-2, p300, for at with at the proteins by the were on and by the of pax-6 interaction with p300 through cdx-2, cdx-2 protein was to the assay in and were by the by R. J.A. in Scholar). and have been D. Habener J.F. Genes Dev. PubMed Scopus Google Scholar). of p300 were a by H. Nuclear proteins from cell lines and cells with were as E. P. W. 17: PubMed Scopus Google Scholar). The in to glucagon promoter G1 were with with and a were by of DNA of with protein in a binding buffer for at experiments, proteins were with specific in the binding buffer at for to the DNA of the been to the to The was by electrophoresis on a polyacrylamide the gel was and to for cdx-2, and p300 were by P. Traber and M. pax-6 were from the of which a of the not and Habener J.F. S. A. 1994; PubMed Scopus Google Scholar). mobility shift of with proximal element G1 of the glucagon promoter Of complex which is found at various and is complexes and is by a heterodimer of pax-6 and cdx-2 F. Heller R. Petersen H. Jensen J. Madsen O. Serup P. FEBS Lett. 1999; 445: 306-310Crossref PubMed Scopus (32) Google B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). contains the POU domain transcription factor brain-4 (4Hussain M.A. Lee J. Miller C.P. Habener J.F. Mol. Cell. Biol. 1997; 17: 7186-7194Crossref PubMed Google Scholar), and complex contains pax-6 (6Andersen F. Heller R. Petersen H. Jensen J. Madsen O. Serup P. FEBS Lett. 1999; 445: 306-310Crossref PubMed Scopus (32) Google Scholar, 7Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). to contain all three an of with the present Of not the of complex A. This that complex is of a as has been reported by (6Andersen F. Heller R. Petersen H. Jensen J. Madsen O. Serup P. FEBS Lett. 1999; 445: 306-310Crossref PubMed Scopus (32) Google Scholar, 7Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In with from cells pax-6 and cdx-2, a heterodimer of pax-6 and cdx-2 form complex both AT-rich binding sites are interaction of pax-6 and cdx-2 not the proximal AT-rich the binding site for cdx-2 (3Jin T. Trinh D.K.Y. Wang F. Drucker D.J. Mol. Endocrinol. 1997; 11: 203-209Crossref PubMed Scopus (38) Google Scholar, F. Heller R. Petersen H. Jensen J. Madsen O. Serup P. FEBS Lett. 1999; 445: 306-310Crossref PubMed Scopus (32) Google Scholar, 7Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), is is by pax-6 the binding sites of pax-6, cdx-2, and the interaction of pax-6 and cdx-2, transient with expression of pax-6 and cdx-2 with of the glucagon G1 element were Both pax-6 and cdx-2 transactivate the glucagon the binding and AT-rich sites of the promoter are the activity of pax-6 and cdx-2 is pax-6 and cdx-2 in a on the of the proglucagon gene the DNA binding site for cdx-2 proximal AT-rich is a transactivating of pax-6 and cdx-2 is are in with reported (6Andersen F. Heller R. Petersen H. Jensen J. Madsen O. Serup P. FEBS Lett. 1999; 445: 306-310Crossref PubMed Scopus (32) Google Scholar, 7Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In in a of pax-6 DNA recognition site AT-rich of pax-6 a not of the through cdx-2 We that pax-6 cdx-2 to a transcriptional a co-activator we studied the transactivating properties of pax-6 and cdx-2 in with p300. of p300 expression through pax-6 and cdx-2 and in the in The of pax-6 and cdx-2 with the of p300 was also present the AT-rich site site of was This that the cdx-2 DNA recognition site is not for cdx-2 to in the transcription of the glucagon In of the pax-6 binding site not pax-6 to the transcription by cdx-2 with p300. that the pax-6 DNA recognition site in the presence of is required for pax-6 to in the of the glucagon Furthermore, of the cdx-2 site to a of the of pax-6 through interaction with p300 This for and interaction with p300, an of the DNA within the promoter is This the of of proteins and DNA in transcriptional we to the through by of the which specifically binds the domain of p300 and CBP and transcriptional (11Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.E. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (937) Google Scholar, 12Arany Z. Sellers W. Livingston D. Eckner R. Cell. 1994; 77: 799-800Abstract Full Text PDF PubMed Scopus (380) Google Scholar, 13Lundblad J.R. Kwok R.P. Laurence M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (534) Google Scholar). protein which is a of transcriptional through was as a J.R. Kwok R.P. Laurence M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (534) Google Scholar). shown in of not of the basal expression of a glucagon in Furthermore, of not the in the by cdx-2, pax-6, and cdx-2 and pax-6 of the glucagon results suggest that an co-activator homologue of is in the transcription of the glucagon gene in and that by pax-6 and cdx-2 the co-activator We that cdx-2 binds to (6Andersen F. Heller R. Petersen H. Jensen J. Madsen O. Serup P. FEBS Lett. 1999; 445: 306-310Crossref PubMed Scopus (32) Google Scholar, 7Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). we cdx-2 and pax-6 bind to the CBP homologue p300. interacted with in p300 in a Furthermore, also interacted with p300. Furthermore, we that both in p300 and pax-6 both in p300 and cdx-2 interacted with in that interactions of pax-6 and cdx-2 with p300 not the of the transient we that cdx-2 the interaction of pax-6 and the basal transcription through interaction with a co-activator cdx-2 the physical interaction of pax-6 and p300, we of cdx-2 to the shown in of of cdx-2 to in enhanced the of p300 protein by In of pax-6 to of p300 through not directly the that cdx-2 the protein-protein interaction of pax-6 with p300. In of proteins in the glucagon-producing cell cells were in with the in an for the experiments, we the known interaction of with p300 pax-6 and cdx-2 with co-activator at we with of shown in with p300 and for pax-6 cdx-2 also an interaction of the with with cdx-2 pax-6 and of p300 that both homeodomain transcription factors interact with p300 in We to the site of interaction pax-6 and cdx-2 with p300. with of p300 protein that both pax-6 and cdx-2 interact with the domain of the p300 protein This that both homeodomain proteins pax-6 and cdx-2 interact with co-activator p300 in the transcription of the proglucagon of pax-6 and cdx-2 is by of p300 in the of Furthermore, our the that pax-6 and cdx-2 interact synergistically on the proximal G1 promoter element of the glucagon gene (6Andersen F. Heller R. Petersen H. Jensen J. Madsen O. Serup P. FEBS Lett. 1999; 445: 306-310Crossref PubMed Scopus (32) Google Scholar, 7Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (82) Google and the by that transcriptional is mediated by the CBP homologue p300. The proximal promoter element G1 to of the proglucagon gene is for cell-specific expression of the proglucagon gene in pancreatic α-cells (5Philippe J. Drucker D.J. Knepel W. Jepeal L. Misulovin Z. Habener J.F. Mol. Cell. Biol. 1988; 8: 4877-4888Crossref PubMed Scopus (125) Google Scholar). Nuclear form pancreatic lines contain brain-4, pax-6, and cdx-2, all which bind to the G1 element of the proglucagon gene (3Jin T. Trinh D.K.Y. Wang F. Drucker D.J. Mol. Endocrinol. 1997; 11: 203-209Crossref PubMed Scopus (38) Google Scholar, 4Hussain M.A. Lee J. Miller C.P. Habener J.F. Mol. Cell. Biol. 1997; 17: 7186-7194Crossref PubMed Google Scholar, F. Heller R. Petersen H. Jensen J. Madsen O. Serup P. FEBS Lett. 1999; 445: 306-310Crossref PubMed Scopus (32) Google Scholar, M. Neubüser A. Kalamaras J. Ee H.C. Martin G.R. German M.S. Genes Dev. 1997; 11: 1662-1673Crossref PubMed Scopus (475) Google Scholar). complexes are and the G1 element F. Heller R. Petersen H. Jensen J. Madsen O. Serup P. FEBS Lett. 1999; 445: 306-310Crossref PubMed Scopus (32) Google and 7Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). molecular weight complex is by a heterodimer of pax-6 and cdx-2. The of complex in is dependent on the presence of both AT-rich sites present in the G1 promoter element The complex contains the homeodomain protein brain-4 4Hussain M.A. Lee J. Miller C.P. Habener J.F. Mol. Cell. Biol. 1997; 17: 7186-7194Crossref PubMed Google Scholar), and complex contains pax-6 (6Andersen F. Heller R. Petersen H. Jensen J. Madsen O. Serup P. FEBS Lett. 1999; 445: 306-310Crossref PubMed Scopus (32) Google Scholar, 7Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The of the of complexes and are in with (6Andersen F. Heller R. Petersen H. Jensen J. Madsen O. Serup P. FEBS Lett. 1999; 445: 306-310Crossref PubMed Scopus (32) Google Scholar, 7Ritz-Laser B. Estreicher A. Klages N. Saule S. Philippe J. J. Biol. Chem. 1999; 274: 4124-4132Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The protein complex for binding by brain-4 since of cells brain-4 form a which is in complex not In a of brain-4 and pax-6 cdx-2 with the G1 element to form complex in not a protein in to brain-4 to in the of complex (4Hussain M.A. Lee J. Miller C.P. Habener J.F. Mol. Cell. Biol. 1997; 17: 7186-7194Crossref PubMed Google Scholar). are required to the of additional that present in all and of p300 not in in is shown to a co-activator present in that transcription factors for the interaction with co-activator D. Habener J.F. Genes Dev. PubMed Scopus Google Scholar). is that of p300 to enhanced transactivating effects as shown in the present the of pax-6 and cdx-2 with p300 on the transcription of the proglucagon gene at of pax-6 and cdx-2 the that CBP in The results of the present the that pax-6 and cdx-2 each interaction with the co-activator p300 in that both and p300. the of p300 through was in cdx-2 to the the of p300 the not absolutely with the results of the transient that the interaction of pax-6 and p300 is weak and enhanced by the presence of cdx-2 In the interaction of pax-6 with p300 by cdx-2 not absolutely binding of cdx-2 to the proximal AT-rich site of the proglucagon gene The transient the in the of a cdx-2 DNA binding site and cdx-2 in the transcriptional of the proglucagon with the that pax-6 binds cdx-2 in and in is that pax-6 tethers cdx-2 to the complex with p300. the other in the presence of cdx-2, of pax-6 a not to transcription its recognition site is in of pax-6 is not that the DNA binding site for pax-6 is for pax-6 to in the transcription of the glucagon gene through interaction with the G1 is that of the AT-rich site binding the of pax-6 in the with p300 in the of cdx-2, an interaction of pax-6 with p300 a DNA which is present the AT-rich site is a pax-6 binding site within the AT-rich site of which to a of pax-6 the not suggest that pax-6 binds to the AT-rich The from that both pax-6 and cdx-2 interact with the of p300. from the of transient that the interaction site with p300 absolutely since pax-6 and cdx-2 not to with the binding to p300, synergistically at the level. Furthermore, the in that cdx-2 binding of pax-6 to p300, by the interaction of these proteins One for our that cdx-2 a pax-6 and p300, a direct whereas in the of cdx-2, pax-6 has the of directly with p300. the and physical interaction of pax-6 with p300 in the presence of cdx-2 due to a in of the proteins with each other and thus their to with other of homeodomain transcription factors has been L. Aggarwal A.K. Nature. 1999; PubMed Scopus Google Scholar). all the that pax-6, cdx-2, and p300 interact with each other in the of the interaction of these proteins an proximal G1 element of the proglucagon gene This is by the which that both AT-rich sites of the G1 element for the of a both pax-6 and cdx-2 is to at that the in the promoter also the of the transcription factors, and the basal transcription to an that of the results by these not for by the presence of DNA binding sites for transcription factors also by in the of the transcriptional pax-6 has been shown to bind not to the G1 element also to the G3 enhancer element (8Sander M. Neubüser A. Kalamaras J. Ee H.C. Martin G.R. German M.S. Genes Dev. 1997; 11: 1662-1673Crossref PubMed Scopus (475) Google Scholar). pax-6 bound to the G3 element also with cdx-2 p300 with the present cdx-2 has been reported to the of intestinal epithelial cells E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (386) Google Scholar). In other the inhibition of through the cell is reported to mediated by the of cdx-2 in pancreatic α-cells not the expression of the glucagon gene also inhibition of is on the pancreatic in cdx-2 pax-6, on the other has been implicated in pancreatic islet cell and (8Sander M. Neubüser A. Kalamaras J. Ee H.C. Martin G.R. German M.S. Genes Dev. 1997; 11: 1662-1673Crossref PubMed Scopus (475) Google Scholar, 9St.-Onge L. Sosa-Pineda B. Nature. 1997; 387: 406-409Crossref PubMed Scopus (671) Google Scholar). pax-6 and have pancreatic endocrine cell with glucagon-producing cells (8Sander M. Neubüser A. Kalamaras J. Ee H.C. Martin G.R. German M.S. Genes Dev. 1997; 11: 1662-1673Crossref PubMed Scopus (475) Google Scholar, 9St.-Onge L. Sosa-Pineda B. Nature. 1997; 387: 406-409Crossref PubMed Scopus (671) Google Scholar). The present with the that the transcription factors implicated in islet cell are also in gene expression of cells (8Sander M. Neubüser A. Kalamaras J. Ee H.C. Martin G.R. German M.S. Genes Dev. 1997; 11: 1662-1673Crossref PubMed Scopus (475) Google Scholar, 9St.-Onge L. Sosa-Pineda B. Nature. 1997; 387: 406-409Crossref PubMed Scopus (671) Google Scholar, B. M. P. Nature. 1997; PubMed Scopus Google Scholar). The in of our with to glucagon gene expression to by the present the of the interaction of the transcription factors pax-6 and cdx-2 with the α-cell-specific G1 element of the glucagon gene promoter and with the co-activator p300, that expression of pax-6 cdx-2 is to pancreatic Of the transcription factors known to expressed in pancreatic α-cells, brain-4 to to the glucagon-producing cells (4Hussain M.A. Lee J. Miller C.P. Habener J.F. Mol. Cell. Biol. 1997; 17: 7186-7194Crossref PubMed Google Scholar). is that the interaction of cell-specific with transcription factors to an expression of a in we have found that in of brain-4 in the of other transcription factors required of pancreatic endocrine cell to expression of the glucagon A. and J. F. in In the present that pax-6 and cdx-2 both interact with p300 at the protein-protein level. Further, the interaction of pax-6 with p300 is enhanced by the presence of cdx-2. cdx-2 in the of the proglucagon gene in of its DNA binding site in the G1 promoter element, is that pax-6 tethers cdx-2 to the complex by pax-6, cdx-2, and the transcriptional co-activator p300. Both pax-6 and cdx-2 proglucagon gene expression in pancreatic α-cells through interaction with the cell-specific G1 pax-6, cdx-2, and p300 form a complex in the α-cell-specific expression of the proglucagon as that additional protein complexes also bind to the G1 element of the proglucagon gene which confers α-cell-specific expression of the glucagon The of these additional transcription factors to a of the complex of the tissue-specific expression of the glucagon We L. D. J. S. R. H. M. M. and P. Traber for their of for the We the of the of and M. for and and for in the of the