Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Jul;17(7):3987–3996. doi: 10.1128/mcb.17.7.3987

Functional characterization of the transactivation properties of the PDX-1 homeodomain protein.

M Peshavaria 1, E Henderson 1, A Sharma 1, C V Wright 1, R Stein 1
PMCID: PMC232251  PMID: 9199333

Abstract

Pancreas formation is prevented in mice carrying a null mutation in the PDX-1 homeoprotein, demonstrating a key role for this factor in development. PDX-1 can also bind to and activate transcription from cis-acting regulatory sequences in the insulin and somatostatin genes, which are expressed in pancreatic islet beta and delta cells, respectively. In this study, we compared the functional properties of PDX-1 with those of the closely related Xenopus homeoprotein XIHbox8. Analysis of chimeras between PDX-1, XIHbox8, and the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that their transactivation domain was contained within the N-terminal region (amino acids 1 to 79). Detailed mutagenesis of this region indicated that transactivation is mediated by three highly conserved sequences, spanning amino acids 13 to 22 (subdomain A), 32 to 38 (subdomain B), and 60 to 73 (subdomain C). These sequences were also required by PDX-1 to synergistically activate insulin enhancer-mediated transcription with another key insulin gene activator, the E2A-encoded basic helix-loop-helix E2-5 and E47 proteins. These results indicated that N-terminal sequences conserved between the mammalian PDX-1 and Xenopus XIHbox8 proteins are important in transcriptional activation. Stable expression of the PDX-1 deltaABC mutant in the insulin- and PDX-1-expressing betaTC3 cell line resulted in a threefold reduction in the rate of endogenous insulin gene transcription. Strikingly, the level of the endogenous PDX-1 protein was reduced to very low levels in these cells. These results suggest that PDX-1 is not absolutely essential for insulin gene expression in betaTC3 cells. We discuss the possible significance of these findings for insulin gene transcription in islet beta cells.

Full Text

The Full Text of this article is available as a PDF (838.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahlgren U., Jonsson J., Edlund H. The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. Development. 1996 May;122(5):1409–1416. doi: 10.1242/dev.122.5.1409. [DOI] [PubMed] [Google Scholar]
  2. Alpert S., Hanahan D., Teitelman G. Hybrid insulin genes reveal a developmental lineage for pancreatic endocrine cells and imply a relationship with neurons. Cell. 1988 Apr 22;53(2):295–308. doi: 10.1016/0092-8674(88)90391-1. [DOI] [PubMed] [Google Scholar]
  3. Andersson S., Davis D. L., Dahlbäck H., Jörnvall H., Russell D. W. Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem. 1989 May 15;264(14):8222–8229. [PubMed] [Google Scholar]
  4. Aronheim A., Ohlsson H., Park C. W., Edlund T., Walker M. D. Distribution and characterization of helix-loop-helix enhancer-binding proteins from pancreatic beta cells and lymphocytes. Nucleic Acids Res. 1991 Jul 25;19(14):3893–3899. doi: 10.1093/nar/19.14.3893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Aronheim A., Shiran R., Rosen A., Walker M. D. The E2A gene product contains two separable and functionally distinct transcription activation domains. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8063–8067. doi: 10.1073/pnas.90.17.8063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bretherton-Watt D., Gore N., Boam D. S. Insulin upstream factor 1 and a novel ubiquitous factor bind to the human islet amyloid polypeptide/amylin gene promoter. Biochem J. 1996 Jan 15;313(Pt 2):495–502. doi: 10.1042/bj3130495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chan S. K., Jaffe L., Capovilla M., Botas J., Mann R. S. The DNA binding specificity of Ultrabithorax is modulated by cooperative interactions with extradenticle, another homeoprotein. Cell. 1994 Aug 26;78(4):603–615. doi: 10.1016/0092-8674(94)90525-8. [DOI] [PubMed] [Google Scholar]
  8. Cordle S. R., Henderson E., Masuoka H., Weil P. A., Stein R. Pancreatic beta-cell-type-specific transcription of the insulin gene is mediated by basic helix-loop-helix DNA-binding proteins. Mol Cell Biol. 1991 Mar;11(3):1734–1738. doi: 10.1128/mcb.11.3.1734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Crowe D. T., Tsai M. J. Mutagenesis of the rat insulin II 5'-flanking region defines sequences important for expression in HIT cells. Mol Cell Biol. 1989 Apr;9(4):1784–1789. doi: 10.1128/mcb.9.4.1784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. De Krijger R. R., Aanstoot H. J., Kranenburg G., Reinhard M., Visser W. J., Bruining G. J. The midgestational human fetal pancreas contains cells coexpressing islet hormones. Dev Biol. 1992 Oct;153(2):368–375. doi: 10.1016/0012-1606(92)90121-v. [DOI] [PubMed] [Google Scholar]
  11. Eckner R., Yao T. P., Oldread E., Livingston D. M. Interaction and functional collaboration of p300/CBP and bHLH proteins in muscle and B-cell differentiation. Genes Dev. 1996 Oct 1;10(19):2478–2490. doi: 10.1101/gad.10.19.2478. [DOI] [PubMed] [Google Scholar]
  12. Efrat S., Linde S., Kofod H., Spector D., Delannoy M., Grant S., Hanahan D., Baekkeskov S. Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9037–9041. doi: 10.1073/pnas.85.23.9037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gamer L. W., Wright C. V. Autonomous endodermal determination in Xenopus: regulation of expression of the pancreatic gene XlHbox 8. Dev Biol. 1995 Sep;171(1):240–251. doi: 10.1006/dbio.1995.1275. [DOI] [PubMed] [Google Scholar]
  14. German M. S., Blanar M. A., Nelson C., Moss L. G., Rutter W. J. Two related helix-loop-helix proteins participate in separate cell-specific complexes that bind the insulin enhancer. Mol Endocrinol. 1991 Feb;5(2):292–299. doi: 10.1210/mend-5-2-292. [DOI] [PubMed] [Google Scholar]
  15. German M. S., Moss L. G., Wang J., Rutter W. J. The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical beta-cell nuclear complexes. Mol Cell Biol. 1992 Apr;12(4):1777–1788. doi: 10.1128/mcb.12.4.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. German M., Ashcroft S., Docherty K., Edlund H., Edlund T., Goodison S., Imura H., Kennedy G., Madsen O., Melloul D. The insulin gene promoter. A simplified nomenclature. Diabetes. 1995 Aug;44(8):1002–1004. doi: 10.2337/diab.44.8.1002. [DOI] [PubMed] [Google Scholar]
  17. Gittes G. K., Rutter W. J. Onset of cell-specific gene expression in the developing mouse pancreas. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):1128–1132. doi: 10.1073/pnas.89.3.1128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Guz Y., Montminy M. R., Stein R., Leonard J., Gamer L. W., Wright C. V., Teitelman G. Expression of murine STF-1, a putative insulin gene transcription factor, in beta cells of pancreas, duodenal epithelium and pancreatic exocrine and endocrine progenitors during ontogeny. Development. 1995 Jan;121(1):11–18. doi: 10.1242/dev.121.1.11. [DOI] [PubMed] [Google Scholar]
  19. Henthorn P., Kiledjian M., Kadesch T. Two distinct transcription factors that bind the immunoglobulin enhancer microE5/kappa 2 motif. Science. 1990 Jan 26;247(4941):467–470. doi: 10.1126/science.2105528. [DOI] [PubMed] [Google Scholar]
  20. Henthorn P., McCarrick-Walmsley R., Kadesch T. Sequence of the cDNA encoding ITF-1, a positive-acting transcription factor. Nucleic Acids Res. 1990 Feb 11;18(3):677–677. doi: 10.1093/nar/18.3.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jonsson J., Carlsson L., Edlund T., Edlund H. Insulin-promoter-factor 1 is required for pancreas development in mice. Nature. 1994 Oct 13;371(6498):606–609. doi: 10.1038/371606a0. [DOI] [PubMed] [Google Scholar]
  22. Kamei Y., Xu L., Heinzel T., Torchia J., Kurokawa R., Gloss B., Lin S. C., Heyman R. A., Rose D. W., Glass C. K. A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell. 1996 May 3;85(3):403–414. doi: 10.1016/s0092-8674(00)81118-6. [DOI] [PubMed] [Google Scholar]
  23. Karlsson O., Edlund T., Moss J. B., Rutter W. J., Walker M. D. A mutational analysis of the insulin gene transcription control region: expression in beta cells is dependent on two related sequences within the enhancer. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8819–8823. doi: 10.1073/pnas.84.24.8819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee J. E., Hollenberg S. M., Snider L., Turner D. L., Lipnick N., Weintraub H. Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. Science. 1995 May 12;268(5212):836–844. doi: 10.1126/science.7754368. [DOI] [PubMed] [Google Scholar]
  25. Leonard J., Peers B., Johnson T., Ferreri K., Lee S., Montminy M. R. Characterization of somatostatin transactivating factor-1, a novel homeobox factor that stimulates somatostatin expression in pancreatic islet cells. Mol Endocrinol. 1993 Oct;7(10):1275–1283. doi: 10.1210/mend.7.10.7505393. [DOI] [PubMed] [Google Scholar]
  26. Lillie J. W., Green M. R. Transcription activation by the adenovirus E1a protein. Nature. 1989 Mar 2;338(6210):39–44. doi: 10.1038/338039a0. [DOI] [PubMed] [Google Scholar]
  27. Lukinius A., Ericsson J. L., Grimelius L., Korsgren O. Ultrastructural studies of the ontogeny of fetal human and porcine endocrine pancreas, with special reference to colocalization of the four major islet hormones. Dev Biol. 1992 Oct;153(2):376–385. doi: 10.1016/0012-1606(92)90122-w. [DOI] [PubMed] [Google Scholar]
  28. MacFarlane W. M., Read M. L., Gilligan M., Bujalska I., Docherty K. Glucose modulates the binding activity of the beta-cell transcription factor IUF1 in a phosphorylation-dependent manner. Biochem J. 1994 Oct 15;303(Pt 2):625–631. doi: 10.1042/bj3030625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Massari M. E., Jennings P. A., Murre C. The AD1 transactivation domain of E2A contains a highly conserved helix which is required for its activity in both Saccharomyces cerevisiae and mammalian cells. Mol Cell Biol. 1996 Jan;16(1):121–129. doi: 10.1128/mcb.16.1.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Melloul D., Ben-Neriah Y., Cerasi E. Glucose modulates the binding of an islet-specific factor to a conserved sequence within the rat I and the human insulin promoters. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3865–3869. doi: 10.1073/pnas.90.9.3865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Miller C. P., McGehee R. E., Jr, Habener J. F. IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene. EMBO J. 1994 Mar 1;13(5):1145–1156. doi: 10.1002/j.1460-2075.1994.tb06363.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Naya F. J., Stellrecht C. M., Tsai M. J. Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor. Genes Dev. 1995 Apr 15;9(8):1009–1019. doi: 10.1101/gad.9.8.1009. [DOI] [PubMed] [Google Scholar]
  33. Nordeen S. K., Green P. P., 3rd, Fowlkes D. M. A rapid, sensitive, and inexpensive assay for chloramphenicol acetyltransferase. DNA. 1987 Apr;6(2):173–178. doi: 10.1089/dna.1987.6.173. [DOI] [PubMed] [Google Scholar]
  34. Offield M. F., Jetton T. L., Labosky P. A., Ray M., Stein R. W., Magnuson M. A., Hogan B. L., Wright C. V. PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development. 1996 Mar;122(3):983–995. doi: 10.1242/dev.122.3.983. [DOI] [PubMed] [Google Scholar]
  35. Ohlsson H., Karlsson K., Edlund T. IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J. 1993 Nov;12(11):4251–4259. doi: 10.1002/j.1460-2075.1993.tb06109.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Olson L. K., Sharma A., Peshavaria M., Wright C. V., Towle H. C., Rodertson R. P., Stein R. Reduction of insulin gene transcription in HIT-T15 beta cells chronically exposed to a supraphysiologic glucose concentration is associated with loss of STF-1 transcription factor expression. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9127–9131. doi: 10.1073/pnas.92.20.9127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Peers B., Leonard J., Sharma S., Teitelman G., Montminy M. R. Insulin expression in pancreatic islet cells relies on cooperative interactions between the helix loop helix factor E47 and the homeobox factor STF-1. Mol Endocrinol. 1994 Dec;8(12):1798–1806. doi: 10.1210/mend.8.12.7708065. [DOI] [PubMed] [Google Scholar]
  38. Peers B., Sharma S., Johnson T., Kamps M., Montminy M. The pancreatic islet factor STF-1 binds cooperatively with Pbx to a regulatory element in the somatostatin promoter: importance of the FPWMK motif and of the homeodomain. Mol Cell Biol. 1995 Dec;15(12):7091–7097. doi: 10.1128/mcb.15.12.7091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Peshavaria M., Gamer L., Henderson E., Teitelman G., Wright C. V., Stein R. XIHbox 8, an endoderm-specific Xenopus homeodomain protein, is closely related to a mammalian insulin gene transcription factor. Mol Endocrinol. 1994 Jun;8(6):806–816. doi: 10.1210/mend.8.6.7935494. [DOI] [PubMed] [Google Scholar]
  40. Petersen H. V., Serup P., Leonard J., Michelsen B. K., Madsen O. D. Transcriptional regulation of the human insulin gene is dependent on the homeodomain protein STF1/IPF1 acting through the CT boxes. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10465–10469. doi: 10.1073/pnas.91.22.10465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Peyton M., Moss L. G., Tsai M. J. Two distinct class A helix-loop-helix transcription factors, E2A and BETA1, form separate DNA binding complexes on the insulin gene E box. J Biol Chem. 1994 Oct 14;269(41):25936–25941. [PubMed] [Google Scholar]
  42. Pictet R. L., Rall L. B., Phelps P., Rutter W. J. The neural crest and the origin of the insulin-producing and other gastrointestinal hormone-producing cells. Science. 1976 Jan 16;191(4223):191–192. doi: 10.1126/science.1108195. [DOI] [PubMed] [Google Scholar]
  43. Quong M. W., Massari M. E., Zwart R., Murre C. A new transcriptional-activation motif restricted to a class of helix-loop-helix proteins is functionally conserved in both yeast and mammalian cells. Mol Cell Biol. 1993 Feb;13(2):792–800. doi: 10.1128/mcb.13.2.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Robinson G. L., Cordle S. R., Henderson E., Weil P. A., Teitelman G., Stein R. Isolation and characterization of a novel transcription factor that binds to and activates insulin control element-mediated expression. Mol Cell Biol. 1994 Oct;14(10):6704–6714. doi: 10.1128/mcb.14.10.6704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Robinson G. L., Peshavaria M., Henderson E., Shieh S. Y., Tsai M. J., Teitelman G., Stein R. Expression of the trans-active factors that stimulate insulin control element-mediated activity appear to precede insulin gene transcription. J Biol Chem. 1994 Jan 28;269(4):2452–2460. [PubMed] [Google Scholar]
  46. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  47. Schreiber E., Matthias P., Müller M. M., Schaffner W. Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. Nucleic Acids Res. 1989 Aug 11;17(15):6419–6419. doi: 10.1093/nar/17.15.6419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Serup P., Jensen J., Andersen F. G., Jørgensen M. C., Blume N., Holst J. J., Madsen O. D. Induction of insulin and islet amyloid polypeptide production in pancreatic islet glucagonoma cells by insulin promoter factor 1. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):9015–9020. doi: 10.1073/pnas.93.17.9015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Serup P., Petersen H. V., Pedersen E. E., Edlund H., Leonard J., Petersen J. S., Larsson L. I., Madsen O. D. The homeodomain protein IPF-1/STF-1 is expressed in a subset of islet cells and promotes rat insulin 1 gene expression dependent on an intact E1 helix-loop-helix factor binding site. Biochem J. 1995 Sep 15;310(Pt 3):997–1003. doi: 10.1042/bj3100997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sharma A., Stein R. Glucose-induced transcription of the insulin gene is mediated by factors required for beta-cell-type-specific expression. Mol Cell Biol. 1994 Feb;14(2):871–879. doi: 10.1128/mcb.14.2.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shieh S. Y., Tsai M. J. Cell-specific and ubiquitous factors are responsible for the enhancer activity of the rat insulin II gene. J Biol Chem. 1991 Sep 5;266(25):16708–16714. [PubMed] [Google Scholar]
  52. Stein R. W., Corrigan M., Yaciuk P., Whelan J., Moran E. Analysis of E1A-mediated growth regulation functions: binding of the 300-kilodalton cellular product correlates with E1A enhancer repression function and DNA synthesis-inducing activity. J Virol. 1990 Sep;64(9):4421–4427. doi: 10.1128/jvi.64.9.4421-4427.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Steiner D. F., Chan S. J., Welsh J. M., Kwok S. C. Structure and evolution of the insulin gene. Annu Rev Genet. 1985;19:463–484. doi: 10.1146/annurev.ge.19.120185.002335. [DOI] [PubMed] [Google Scholar]
  54. Sun X. H., Baltimore D. An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but not in E12 heterodimers. Cell. 1991 Jan 25;64(2):459–470. doi: 10.1016/0092-8674(91)90653-g. [DOI] [PubMed] [Google Scholar]
  55. Teitelman G. Insulin cells of pancreas extend neurites but do not arise from the neuroectoderm. Dev Biol. 1990 Dec;142(2):368–379. doi: 10.1016/0012-1606(90)90357-o. [DOI] [PubMed] [Google Scholar]
  56. Waeber G., Thompson N., Nicod P., Bonny C. Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor. Mol Endocrinol. 1996 Nov;10(11):1327–1334. doi: 10.1210/mend.10.11.8923459. [DOI] [PubMed] [Google Scholar]
  57. Watada H., Kajimoto Y., Umayahara Y., Matsuoka T., Kaneto H., Fujitani Y., Kamada T., Kawamori R., Yamasaki Y. The human glucokinase gene beta-cell-type promoter: an essential role of insulin promoter factor 1/PDX-1 in its activation in HIT-T15 cells. Diabetes. 1996 Nov;45(11):1478–1488. doi: 10.2337/diab.45.11.1478. [DOI] [PubMed] [Google Scholar]
  58. Wentworth B. M., Schaefer I. M., Villa-Komaroff L., Chirgwin J. M. Characterization of the two nonallelic genes encoding mouse preproinsulin. J Mol Evol. 1986;23(4):305–312. doi: 10.1007/BF02100639. [DOI] [PubMed] [Google Scholar]
  59. Wessells N. K., Evans J. Ultrastructural studies of early morphogenesis and cytodifferentiation in the embryonic mammalian pancreas. Dev Biol. 1968 Apr;17(4):413–446. doi: 10.1016/0012-1606(68)90073-0. [DOI] [PubMed] [Google Scholar]
  60. Whelan J., Cordle S. R., Henderson E., Weil P. A., Stein R. Identification of a pancreatic beta-cell insulin gene transcription factor that binds to and appears to activate cell-type-specific expression: its possible relationship to other cellular factors that bind to a common insulin gene sequence. Mol Cell Biol. 1990 Apr;10(4):1564–1572. doi: 10.1128/mcb.10.4.1564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Whelan J., Poon D., Weil P. A., Stein R. Pancreatic beta-cell-type-specific expression of the rat insulin II gene is controlled by positive and negative cellular transcriptional elements. Mol Cell Biol. 1989 Aug;9(8):3253–3259. doi: 10.1128/mcb.9.8.3253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. van Dijk M. A., Murre C. extradenticle raises the DNA binding specificity of homeotic selector gene products. Cell. 1994 Aug 26;78(4):617–624. doi: 10.1016/0092-8674(94)90526-6. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES