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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Oct 25;91(22):10465–10469. doi: 10.1073/pnas.91.22.10465

Transcriptional regulation of the human insulin gene is dependent on the homeodomain protein STF1/IPF1 acting through the CT boxes.

H V Petersen 1, P Serup 1, J Leonard 1, B K Michelsen 1, O D Madsen 1
PMCID: PMC45041  PMID: 7937976

Abstract

Insulin gene transcription is a unique feature of the pancreatic beta cells and is increased in response to glucose. The recent cloning of insulin promoter factor 1 (IPF1) and somatostatin transcription factor 1 (STF1) unexpectedly revealed that these are mouse and rat homologues of the same protein mediating transactivation through binding of CT box-like elements in rat insulin 1 and somatostatin promoter/enhancer regions, respectively. By using oligonucleotides representing each of the three CT boxes of the human insulin (HI) gene enhancer and nuclear extracts from the mouse islet tumor cell lines beta TC3 and alpha TC1, we have identified a beta-cell-specific binding activity as reported for IPF1, which has maximal affinity toward the CT2 box. However, in pluripotent, HI-transfected rat islet tumor cells, NHI-6F, this binding activity is present prior to induction of (human) insulin gene transcription. Its migration is identical to that of in vitro translated STF1 in electrophoretic mobility-shift assays; it is specifically recognized by anti-STF1 antibodies and has an apparent molecular mass of 46 kDa. Mutation of the CT2 box decreases transcriptional activity of a HI reporter plasmid by approximately 65% in beta TC3 cells and blocks the glucose response in isolated newborn rat islet cells. Furthermore, cotransfection with STF1 cDNA into the glucagon-producing alpha TC1 cells increases the activity of the HI enhancer 4- to 5-fold, suggesting that STF1/IPF1 can confer on alpha TC1 cells the ability to transcribe the HI gene. We conclude that STF1/IPF1 is a necessary but not sufficient key regulator of insulin gene activity, possibly also involved in glucose-regulated transcription.

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Selected References

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  1. 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]
  2. 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]
  3. Blume N., Petersen J. S., Andersen L. C., Kofod H., Dyrberg T., Michelsen B. K., Serup P., Madsen O. D. Immature transformed rat islet beta-cells differentially express C-peptides derived from the genes coding for insulin I and II as well as a transfected human insulin gene. Mol Endocrinol. 1992 Feb;6(2):299–307. doi: 10.1210/mend.6.2.1569972. [DOI] [PubMed] [Google Scholar]
  4. Boam D. S., Clark A. R., Docherty K. Positive and negative regulation of the human insulin gene by multiple trans-acting factors. J Biol Chem. 1990 May 15;265(14):8285–8296. [PubMed] [Google Scholar]
  5. Boam D. S., Docherty K. A tissue-specific nuclear factor binds to multiple sites in the human insulin-gene enhancer. Biochem J. 1989 Nov 15;264(1):233–239. doi: 10.1042/bj2640233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brunstedt J. Rapid isolation of functionally intact pancreatic islets from mice and rats by percollTM gradient centrifucation. Diabete Metab. 1980 Jun;6(2):87–89. [PubMed] [Google Scholar]
  7. Bucchini D., Ripoche M. A., Stinnakre M. G., Desbois P., Lorès P., Monthioux E., Absil J., Lepesant J. A., Pictet R., Jami J. Pancreatic expression of human insulin gene in transgenic mice. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2511–2515. doi: 10.1073/pnas.83.8.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clark A. R., Docherty K. Cell-specific gene expression in the islets of Langerhans: E boxes and TAAT boxes. Biochem Soc Trans. 1993 Feb;21(1):154–159. doi: 10.1042/bst0210154. [DOI] [PubMed] [Google Scholar]
  9. Clark A. R., Petersen H. V., Read M. L., Scott V., Michelsen B. K., Docherty K. Human insulin gene enhancer-binding proteins in pancreatic alpha and beta cell lines. FEBS Lett. 1993 Aug 23;329(1-2):139–143. doi: 10.1016/0014-5793(93)80210-l. [DOI] [PubMed] [Google Scholar]
  10. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Efrat S., Surana M., Fleischer N. Glucose induces insulin gene transcription in a murine pancreatic beta-cell line. J Biol Chem. 1991 Jun 15;266(17):11141–11143. [PubMed] [Google Scholar]
  13. Efrat S., Teitelman G., Anwar M., Ruggiero D., Hanahan D. Glucagon gene regulatory region directs oncoprotein expression to neurons and pancreatic alpha cells. Neuron. 1988 Sep;1(7):605–613. doi: 10.1016/0896-6273(88)90110-9. [DOI] [PubMed] [Google Scholar]
  14. Fromont-Racine M., Bucchini D., Madsen O., Desbois P., Linde S., Nielsen J. H., Saulnier C., Ripoche M. A., Jami J., Pictet R. Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice. Mol Endocrinol. 1990 May;4(5):669–677. doi: 10.1210/mend-4-5-669. [DOI] [PubMed] [Google Scholar]
  15. German M. S., Moss L. G., Rutter W. J. Regulation of insulin gene expression by glucose and calcium in transfected primary islet cultures. J Biol Chem. 1990 Dec 25;265(36):22063–22066. [PubMed] [Google Scholar]
  16. German M. S., Wang J., Chadwick R. B., Rutter W. J. Synergistic activation of the insulin gene by a LIM-homeo domain protein and a basic helix-loop-helix protein: building a functional insulin minienhancer complex. Genes Dev. 1992 Nov;6(11):2165–2176. doi: 10.1101/gad.6.11.2165. [DOI] [PubMed] [Google Scholar]
  17. Goodison S., Kenna S., Ashcroft S. J. Control of insulin gene expression by glucose. Biochem J. 1992 Jul 15;285(Pt 2):563–568. doi: 10.1042/bj2850563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Karlsson O., Thor S., Norberg T., Ohlsson H., Edlund T. Insulin gene enhancer binding protein Isl-1 is a member of a novel class of proteins containing both a homeo- and a Cys-His domain. Nature. 1990 Apr 26;344(6269):879–882. doi: 10.1038/344879a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Madsen O. D., Andersen L. C., Michelsen B., Owerbach D., Larsson L. I., Lernmark A., Steiner D. F. Tissue-specific expression of transfected human insulin genes in pluripotent clonal rat insulinoma lines induced during passage in vivo. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6652–6656. doi: 10.1073/pnas.85.18.6652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Madsen O. D., Karlsen C., Nielsen E., Lund K., Kofod H., Welinder B., Rehfeld J. F., Larsson L. I., Steiner D. F., Holst J. J. The dissociation of tumor-induced weight loss from hypoglycemia in a transplantable pluripotent rat islet tumor results in the segregation of stable alpha- and beta-cell tumor phenotypes. Endocrinology. 1993 Nov;133(5):2022–2030. doi: 10.1210/endo.133.5.8404649. [DOI] [PubMed] [Google Scholar]
  22. Madsen O. D., Larsson L. I., Rehfeld J. F., Schwartz T. W., Lernmark A., Labrecque A. D., Steiner D. F. Cloned cell lines from a transplantable islet cell tumor are heterogeneous and express cholecystokinin in addition to islet hormones. J Cell Biol. 1986 Nov;103(5):2025–2034. doi: 10.1083/jcb.103.5.2025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Madsen O. D., Nielsen J. H., Michelsen B., Westermark P., Betsholtz C., Nishi M., Steiner D. F. Islet amyloid polypeptide and insulin expression are controlled differently in primary and transformed islet cells. Mol Endocrinol. 1991 Jan;5(1):143–148. doi: 10.1210/mend-5-1-143. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Murre C., McCaw P. S., Vaessin H., Caudy M., Jan L. Y., Jan Y. N., Cabrera C. V., Buskin J. N., Hauschka S. D., Lassar A. B. Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell. 1989 Aug 11;58(3):537–544. doi: 10.1016/0092-8674(89)90434-0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Ohlsson H., Karlsson O., Edlund T. A beta-cell-specific protein binds to the two major regulatory sequences of the insulin gene enhancer. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4228–4231. doi: 10.1073/pnas.85.12.4228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ohlsson H., Thor S., Edlund T. Novel insulin promoter- and enhancer-binding proteins that discriminate between pancreatic alpha- and beta-cells. Mol Endocrinol. 1991 Jul;5(7):897–904. doi: 10.1210/mend-5-7-897. [DOI] [PubMed] [Google Scholar]
  29. Olson L. K., Redmon J. B., Towle H. C., Robertson R. P. Chronic exposure of HIT cells to high glucose concentrations paradoxically decreases insulin gene transcription and alters binding of insulin gene regulatory protein. J Clin Invest. 1993 Jul;92(1):514–519. doi: 10.1172/JCI116596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Read M. L., Clark A. R., Docherty K. The helix-loop-helix transcription factor USF (upstream stimulating factor) binds to a regulatory sequence of the human insulin gene enhancer. Biochem J. 1993 Oct 1;295(Pt 1):233–237. doi: 10.1042/bj2950233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Scott V., Clark A. R., Hutton J. C., Docherty K. Two proteins act as the IUF1 insulin gene enhancer binding factor. FEBS Lett. 1991 Sep 23;290(1-2):27–30. doi: 10.1016/0014-5793(91)81217-v. [DOI] [PubMed] [Google Scholar]
  32. Selden R. F., Skośkiewicz M. J., Howie K. B., Russell P. S., Goodman H. M. Regulation of human insulin gene expression in transgenic mice. 1986 May 29-Jun 4Nature. 321(6069):525–528. doi: 10.1038/321525a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Teitelman G., Alpert S., Polak J. M., Martinez A., Hanahan D. Precursor cells of mouse endocrine pancreas coexpress insulin, glucagon and the neuronal proteins tyrosine hydroxylase and neuropeptide Y, but not pancreatic polypeptide. Development. 1993 Aug;118(4):1031–1039. doi: 10.1242/dev.118.4.1031. [DOI] [PubMed] [Google Scholar]
  35. Walker M. D., Edlund T., Boulet A. M., Rutter W. J. Cell-specific expression controlled by the 5'-flanking region of insulin and chymotrypsin genes. Nature. 1983 Dec 8;306(5943):557–561. doi: 10.1038/306557a0. [DOI] [PubMed] [Google Scholar]

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