Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1989 Aug;9(8):3253–3259. doi: 10.1128/mcb.9.8.3253

Pancreatic beta-cell-type-specific expression of the rat insulin II gene is controlled by positive and negative cellular transcriptional elements.

J Whelan 1, D Poon 1, P A Weil 1, R Stein 1
PMCID: PMC362369  PMID: 2552288

Abstract

The insulin gene is expressed almost exclusively in pancreatic beta-cells. The DNA sequences that control cell-specific expression are located upstream of the transcription initiation site. To identify the cis-acting transcriptional control regions within the rat insulin II gene that are responsible for this tissue-specific expression pattern, we constructed a series of 5'-flanking deletion mutants and analyzed their expression in vivo in transfected insulin-producing and -nonproducing cell lines. Pancreatic beta-cell-specific expression was shown to be controlled by enhancer sequences lying between nucleotides -342 and -91 relative to the transcription start site. The rat insulin II enhancer appears to be a chimera, composed of a number of distinct cis-acting DNA elements. Both positive and negative transcriptional regulatory elements appear to be responsible for this cell-type-specific expression. We have shown that expression from one element within the enhancer, which is found between nucleotides -100 and -91, is regulated by both positive- and negative-acting cellular transcription factors. Expression from chimeras containing only the enhancer element sequences from -100 to -91 were active only in insulin-producing cells, indicating that the positive-acting factor(s) required for this activity may be active only in beta-cells. In contrast to the enhancer region, the rat insulin II gene promoter did not appear to require cell-specific transcription factors. Promoter mutants with 5'-flanking sequences extending to nucleotides -90 and -73 were constitutively active in both insulin-producing and -nonproducing cells. These results suggest that rat insulin II gene transcription in pancreatic beta-cells is imparted by a combination of both negative- and positive-acting cellular factors interacting with the gene enhancer.

Full text

PDF
3254

Images in this article

3256Image
on p.3256
3256Image
on p.3256
3258Image
on p.3258

Selected References

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

  1. Borrelli E., Hen R., Chambon P. Adenovirus-2 E1A products repress enhancer-induced stimulation of transcription. Nature. 1984 Dec 13;312(5995):608–612. doi: 10.1038/312608a0. [DOI] [PubMed] [Google Scholar]
  2. Chan S. J., Episkopou V., Zeitlin S., Karathanasis S. K., MacKrell A., Steiner D. F., Efstratiadis A. Guinea pig preproinsulin gene: an evolutionary compromise? Proc Natl Acad Sci U S A. 1984 Aug;81(16):5046–5050. doi: 10.1073/pnas.81.16.5046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  4. Clark J. L., Steiner D. F. Insulin biosynthesis in the rat: demonstration of two proinsulins. Proc Natl Acad Sci U S A. 1969 Jan;62(1):278–285. doi: 10.1073/pnas.62.1.278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Edlund T., Walker M. D., Barr P. J., Rutter W. J. Cell-specific expression of the rat insulin gene: evidence for role of two distinct 5' flanking elements. Science. 1985 Nov 22;230(4728):912–916. doi: 10.1126/science.3904002. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Episkopou V., Murphy A. J., Efstratiadis A. Cell-specified expression of a selectable hybrid gene. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4657–4661. doi: 10.1073/pnas.81.15.4657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goodbourn S., Maniatis T. Overlapping positive and negative regulatory domains of the human beta-interferon gene. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1447–1451. doi: 10.1073/pnas.85.5.1447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hanahan D. Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature. 1985 May 9;315(6015):115–122. doi: 10.1038/315115a0. [DOI] [PubMed] [Google Scholar]
  12. Hen R., Borrelli E., Chambon P. Repression of the immunoglobulin heavy chain enhancer by the adenovirus-2 E1A products. Science. 1985 Dec 20;230(4732):1391–1394. doi: 10.1126/science.2999984. [DOI] [PubMed] [Google Scholar]
  13. Hwung Y. P., Crowe D. T., Wang L. H., Tsai S. Y., Tsai M. J. The COUP transcription factor binds to an upstream promoter element of the rat insulin II gene. Mol Cell Biol. 1988 May;8(5):2070–2077. doi: 10.1128/mcb.8.5.2070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Karlsson O., Walker M. D., Rutter W. J., Edlund T. Individual protein-binding domains of the insulin gene enhancer positively activate beta-cell-specific transcription. Mol Cell Biol. 1989 Feb;9(2):823–827. doi: 10.1128/mcb.9.2.823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lomedico P., Rosenthal N., Efstratidadis A., Gilbert W., Kolodner R., Tizard R. The structure and evolution of the two nonallelic rat preproinsulin genes. Cell. 1979 Oct;18(2):545–558. doi: 10.1016/0092-8674(79)90071-0. [DOI] [PubMed] [Google Scholar]
  17. Magnuson M. A., Quinn P. G., Granner D. K. Multihormonal regulation of phosphoenolpyruvate carboxykinase-chloramphenicol acetyltransferase fusion genes. Insulin's effects oppose those of cAMP and dexamethasone. J Biol Chem. 1987 Nov 5;262(31):14917–14920. [PubMed] [Google Scholar]
  18. Maniatis T., Goodbourn S., Fischer J. A. Regulation of inducible and tissue-specific gene expression. Science. 1987 Jun 5;236(4806):1237–1245. doi: 10.1126/science.3296191. [DOI] [PubMed] [Google Scholar]
  19. McKnight S. L., Gavis E. R., Kingsbury R., Axel R. Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region. Cell. 1981 Aug;25(2):385–398. doi: 10.1016/0092-8674(81)90057-x. [DOI] [PubMed] [Google Scholar]
  20. Moss L. G., Moss J. B., Rutter W. J. Systematic binding analysis of the insulin gene transcription control region: insulin and immunoglobulin enhancers utilize similar transactivators. Mol Cell Biol. 1988 Jun;8(6):2620–2627. doi: 10.1128/mcb.8.6.2620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nir U., Walker M. D., Rutter W. J. Regulation of rat insulin 1 gene expression: evidence for negative regulation in nonpancreatic cells. Proc Natl Acad Sci U S A. 1986 May;83(10):3180–3184. doi: 10.1073/pnas.83.10.3180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
  24. Shapiro D. J., Sharp P. A., Wahli W. W., Keller M. J. A high-efficiency HeLa cell nuclear transcription extract. DNA. 1988 Jan-Feb;7(1):47–55. doi: 10.1089/dna.1988.7.47. [DOI] [PubMed] [Google Scholar]
  25. Soares M. B., Schon E., Henderson A., Karathanasis S. K., Cate R., Zeitlin S., Chirgwin J., Efstratiadis A. RNA-mediated gene duplication: the rat preproinsulin I gene is a functional retroposon. Mol Cell Biol. 1985 Aug;5(8):2090–2103. doi: 10.1128/mcb.5.8.2090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stein R. W., Ziff E. B. Repression of insulin gene expression by adenovirus type 5 E1a proteins. Mol Cell Biol. 1987 Mar;7(3):1164–1170. doi: 10.1128/mcb.7.3.1164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Velcich A., Kern F. G., Basilico C., Ziff E. B. Adenovirus E1a proteins repress expression from polyomavirus early and late promoters. Mol Cell Biol. 1986 Nov;6(11):4019–4025. doi: 10.1128/mcb.6.11.4019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Velcich A., Ziff E. Adenovirus E1a proteins repress transcription from the SV40 early promoter. Cell. 1985 Mar;40(3):705–716. doi: 10.1016/0092-8674(85)90219-3. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Weil P. A., Luse D. S., Segall J., Roeder R. G. Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on purified RNA polymerase II and DNA. Cell. 1979 Oct;18(2):469–484. doi: 10.1016/0092-8674(79)90065-5. [DOI] [PubMed] [Google Scholar]
  31. Westin G., Gerster T., Müller M. M., Schaffner G., Schaffner W. OVEC, a versatile system to study transcription in mammalian cells and cell-free extracts. Nucleic Acids Res. 1987 Sep 11;15(17):6787–6798. doi: 10.1093/nar/15.17.6787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zinn K., DiMaio D., Maniatis T. Identification of two distinct regulatory regions adjacent to the human beta-interferon gene. Cell. 1983 Oct;34(3):865–879. doi: 10.1016/0092-8674(83)90544-5. [DOI] [PubMed] [Google Scholar]

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

RESOURCES