Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Mar;11(3):1547–1552. doi: 10.1128/mcb.11.3.1547

Extinction of insulin gene expression in hybrids between beta cells and fibroblasts is accompanied by loss of the putative beta-cell-specific transcription factor IEF1.

D Leshkowitz 1, M D Walker 1
PMCID: PMC369442  PMID: 1996108

Abstract

Insulin-producing cells and fibroblasts were fused to produce hybrid lines. In hybrids derived from both hamster and rat insulinoma cells, no insulin mRNA could be detected in any of seven lines examined by Northern (RNA) analysis despite the presence in each line of the insulin genes of both parental cells. Hybrid cells were transfected with recombinant chloramphenicol acetyltransferase plasmids containing defined segments of the rat insulin I gene 5' flank. We observed no transcriptional activity of the intact insulin enhancer or of IEB2, a critical cis-acting element of the insulin enhancer. IEB2 has previously been shown to interact in vitro with IEF1, a DNA-binding activity observed selectively in insulin-producing cells. Hybrid cells showed no detectable IEF1 activity. Furthermore, the insulin enhancer was unable to reduce transcription directed by the Moloney sarcoma virus enhancer in a double-enhancer construct. Thus, extinction of insulin gene expression in the hybrids apparently does not operate through a direct action of repressors on the insulin enhancer; rather, extinction is accompanied by, and may be caused by, reduced DNA-binding activity of the putative transcriptional activator IEF1.

Full text

PDF
1547

Images in this article

1548Image
on p.1548
1549Image
on p.1549
1549Image
on p.1549
1550Image
on p.1550
1551Image
on p.1551

Selected References

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

  1. Bergman Y., Strich B., Sharir H., Ber R., Laskov R. Extinction of Ig genes expression in myeloma x fibroblast somatic cell hybrids is accompanied by repression of the oct-2 gene encoding a B-cell specific transcription factor. EMBO J. 1990 Mar;9(3):849–855. doi: 10.1002/j.1460-2075.1990.tb08182.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bernard H. U., Krämmer G., Röwekamp W. G. Construction of a fusion gene that confers resistance against hygromycin B to mammalian cells in culture. Exp Cell Res. 1985 May;158(1):237–243. doi: 10.1016/0014-4827(85)90446-x. [DOI] [PubMed] [Google Scholar]
  3. Besnard C., Lores P., Daegelen D., Jami J. Reversible extinction of insulin gene expression in insulinoma x fibroblast somatic cell hybrids. Exp Cell Res. 1989 Nov;185(1):101–108. doi: 10.1016/0014-4827(89)90040-2. [DOI] [PubMed] [Google Scholar]
  4. Boyd A. E., 3rd, Riser M. E., Bolton W. E., Siciliano M. J. Human insulinoma hybrids produce proinsulin-like material. Diabetes. 1982 Dec;31(12):1035–1040. doi: 10.2337/diacare.31.12.1035. [DOI] [PubMed] [Google Scholar]
  5. Cereghini S., Yaniv M., Cortese R. Hepatocyte dedifferentiation and extinction is accompanied by a block in the synthesis of mRNA coding for the transcription factor HNF1/LFB1. EMBO J. 1990 Jul;9(7):2257–2263. doi: 10.1002/j.1460-2075.1990.tb07396.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cordell B., Diamond D., Smith S., Pünter J., Schöne H. H., Goodman H. M. Disproportionate expression of the two nonallelic rat insulin genes in a pancreatic tumor is due to translational control. Cell. 1982 Dec;31(3 Pt 2):531–542. doi: 10.1016/0092-8674(82)90309-9. [DOI] [PubMed] [Google Scholar]
  7. Davidson R. L., Ephrussi B., Yamamoto K. Regulation of pigment synthesis in mammalian cells, as studied by somatic hybridization. Proc Natl Acad Sci U S A. 1966 Nov;56(5):1437–1440. doi: 10.1073/pnas.56.5.1437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Gazdar A. F., Chick W. L., Oie H. K., Sims H. L., King D. L., Weir G. C., Lauris V. Continuous, clonal, insulin- and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3519–3523. doi: 10.1073/pnas.77.6.3519. [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. Gough N. M. Rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells. Anal Biochem. 1988 Aug 15;173(1):93–95. doi: 10.1016/0003-2697(88)90164-9. [DOI] [PubMed] [Google Scholar]
  12. Ivarie R. D., Schacter B. S., O'Farrell P. H. The level of expression of the rat growth hormone gene in liver tumor cells is at least eight orders of magnitude less than that in anterior pituitary cells. Mol Cell Biol. 1983 Aug;3(8):1460–1467. doi: 10.1128/mcb.3.8.1460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Junker S., Pedersen S., Schreiber E., Matthias P. Extinction of an immunoglobulin kappa promoter in cell hybrids is mediated by the octamer motif and correlates with suppression of Oct-2 expression. Cell. 1990 May 4;61(3):467–474. doi: 10.1016/0092-8674(90)90528-m. [DOI] [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. Killary A. M., Fournier R. E. A genetic analysis of extinction: trans-dominant loci regulate expression of liver-specific traits in hepatoma hybrid cells. Cell. 1984 Sep;38(2):523–534. doi: 10.1016/0092-8674(84)90507-5. [DOI] [PubMed] [Google Scholar]
  17. Laimins L. A., Khoury G., Gorman C., Howard B., Gruss P. Host-specific activation of transcription by tandem repeats from simian virus 40 and Moloney murine sarcoma virus. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6453–6457. doi: 10.1073/pnas.79.21.6453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Laimins L., Holmgren-König M., Khoury G. Transcriptional "silencer" element in rat repetitive sequences associated with the rat insulin 1 gene locus. Proc Natl Acad Sci U S A. 1986 May;83(10):3151–3155. doi: 10.1073/pnas.83.10.3151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. McCormick A., Wu D., Castrillo J. L., Dana S., Strobl J., Thompson E. B., Karin M. Extinction of growth hormone expression in somatic cell hybrids involves repression of the specific trans-activator GHF-1. Cell. 1988 Oct 21;55(2):379–389. doi: 10.1016/0092-8674(88)90061-x. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. Renkawitz R. Transcriptional repression in eukaryotes. Trends Genet. 1990 Jun;6(6):192–197. doi: 10.1016/0168-9525(90)90176-7. [DOI] [PubMed] [Google Scholar]
  25. Santerre R. F., Cook R. A., Crisel R. M., Sharp J. D., Schmidt R. J., Williams D. C., Wilson C. P. Insulin synthesis in a clonal cell line of simian virus 40-transformed hamster pancreatic beta cells. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4339–4343. doi: 10.1073/pnas.78.7.4339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schneider J. A., Weiss M. C. Expression of differentiated functions in hepatoma cell hybrids. I. Tyrosine aminotransferase in hepatoma-fibroblast hybrids. Proc Natl Acad Sci U S A. 1971 Jan;68(1):127–131. doi: 10.1073/pnas.68.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  29. 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]
  30. Takeda J., Ishii S., Seino Y., Imamoto F., Imura H. Negative regulation of human insulin gene expression by the 5'-flanking region in non-pancreatic cells. FEBS Lett. 1989 Apr 10;247(1):41–45. doi: 10.1016/0014-5793(89)81236-0. [DOI] [PubMed] [Google Scholar]
  31. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tripputi P., Guérin S. L., Moore D. D. Two mechanisms for the extinction of gene expression in hybrid cells. Science. 1988 Sep 2;241(4870):1205–1207. doi: 10.1126/science.2842865. [DOI] [PubMed] [Google Scholar]
  33. Ullrich A., Shine J., Chirgwin J., Pictet R., Tischer E., Rutter W. J., Goodman H. M. Rat insulin genes: construction of plasmids containing the coding sequences. Science. 1977 Jun 17;196(4296):1313–1319. doi: 10.1126/science.325648. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. Yu H., Porton B., Shen L. Y., Eckhardt L. A. Role of the octamer motif in hybrid cell extinction of immunoglobulin gene expression: extinction is dominant in a two enhancer system. Cell. 1989 Aug 11;58(3):441–448. doi: 10.1016/0092-8674(89)90425-x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES