Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Nov 1;343(Pt 3):687–695.

Delineation of the insulin-responsive sequence in the rat cytosolic aspartate aminotransferase gene: binding sites for hepatocyte nuclear factor-3 and nuclear factor I.

F Beurton 1, U Bandyopadhyay 1, B Dieumegard 1, R Barouki 1, M Aggerbeck 1
PMCID: PMC1220603  PMID: 10527950

Abstract

Expression of the rat cytosolic aspartate aminotransferase gene is stimulated by glucocorticoids and repressed by insulin in the liver. The regulation by insulin and part of the glucocorticoid effect are mediated by a distal region in the promoter. A 142 bp fragment (-1844 to -1702) confers hormonal sensitivity to the heterologous thymidine kinase promoter in transient-transfection assays in H4IIEC3 hepatoma cells. Footprinting and gel-shift assays showed that several nuclear proteins bind to this region at conserved CCAAT-enhancer binding protein (C/EBP), activator protein (AP-1) and E-box sequences. Hepatocyte nuclear factor-3alpha (HNF-3)alpha and beta bind to sequences upstream of a glucocorticoid-responsive element (GRE) half-site as demonstrated by supershift experiments. Nuclear factor I (NFI)-like proteins bind downstream of the GRE half-site. These sites around the GRE motif overlap with five insulin responsive element (IRE) -like sequences (TG/ATTT). The effect of insulin was not prevented by any single mutation in the IRE-like sites. However, mutation of two IRE sites (namely IREc and d) prevented the insulin effect although only marginally affecting the glucocorticoid effect. The results suggest that the effect of insulin is due to a complex interplay of factors requiring the synergistic contribution of at least two sites and underline the contribution of HNF-3 and NFI-like proteins.

Full Text

The Full Text of this article is available as a PDF (249.5 KB).

Selected References

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

  1. Aggerbeck M., Garlatti M., Feilleux-Duché S., Veyssier C., Daheshia M., Hanoune J., Barouki R. Regulation of the cytosolic aspartate aminotransferase housekeeping gene promoter by glucocorticoids, cAMP, and insulin. Biochemistry. 1993 Sep 7;32(35):9065–9072. doi: 10.1021/bi00086a011. [DOI] [PubMed] [Google Scholar]
  2. Allander S. V., Durham S. K., Scheimann A. O., Wasserman R. M., Suwanichkul A., Powell D. R. Hepatic nuclear factor 3 and high mobility group I/Y proteins bind the insulin response element of the insulin-like growth factor-binding protein-1 promoter. Endocrinology. 1997 Oct;138(10):4291–4300. doi: 10.1210/endo.138.10.5268. [DOI] [PubMed] [Google Scholar]
  3. Barouki R., Pavé-Preux M., Bousquet-Lemercier B., Pol S., Bouguet J., Hanoune J. Regulation of cytosolic aspartate aminotransferase mRNAs in the Fao rat hepatoma cell line by dexamethasone, insulin and cyclic AMP. Eur J Biochem. 1989 Dec 8;186(1-2):79–85. doi: 10.1111/j.1432-1033.1989.tb15180.x. [DOI] [PubMed] [Google Scholar]
  4. Biggs W. H., 3rd, Meisenhelder J., Hunter T., Cavenee W. K., Arden K. C. Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7421–7426. doi: 10.1073/pnas.96.13.7421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Brunet A., Bonni A., Zigmond M. J., Lin M. Z., Juo P., Hu L. S., Anderson M. J., Arden K. C., Blenis J., Greenberg M. E. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell. 1999 Mar 19;96(6):857–868. doi: 10.1016/s0092-8674(00)80595-4. [DOI] [PubMed] [Google Scholar]
  7. Cooke D. W., Lane M. D. A sequence element in the GLUT4 gene that mediates repression by insulin. J Biol Chem. 1998 Mar 13;273(11):6210–6217. doi: 10.1074/jbc.273.11.6210. [DOI] [PubMed] [Google Scholar]
  8. Cooke D. W., Lane M. D. The transcription factor nuclear factor I mediates repression of the GLUT4 promoter by insulin. J Biol Chem. 1999 Apr 30;274(18):12917–12924. doi: 10.1074/jbc.274.18.12917. [DOI] [PubMed] [Google Scholar]
  9. Costa R. H., Grayson D. R., Darnell J. E., Jr Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and alpha 1-antitrypsin genes. Mol Cell Biol. 1989 Apr;9(4):1415–1425. doi: 10.1128/mcb.9.4.1415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deschatrette J., Weiss M. C. Characterization of differentiated and dedifferentiated clones from a rat hepatoma. Biochimie. 1974;56(11-12):1603–1611. doi: 10.1016/s0300-9084(75)80286-0. [DOI] [PubMed] [Google Scholar]
  11. Durham S. K., Suwanichkul A., Scheimann A. O., Yee D., Jackson J. G., Barr F. G., Powell D. R. FKHR binds the insulin response element in the insulin-like growth factor binding protein-1 promoter. Endocrinology. 1999 Jul;140(7):3140–3146. doi: 10.1210/endo.140.7.6856. [DOI] [PubMed] [Google Scholar]
  12. Faber S., O'Brien R. M., Imai E., Granner D. K., Chalkley R. Dynamic aspects of DNA/protein interactions in the transcriptional initiation complex and the hormone-responsive domains of the phosphoenolpyruvate carboxykinase promoter in vivo. J Biol Chem. 1993 Nov 25;268(33):24976–24985. [PubMed] [Google Scholar]
  13. Feilleux-Duché S., Garlatti M., Aggerbeck M., Poyard M., Bouguet J., Hanoune J., Barouki R. Cell-specific regulation of cytosolic aspartate aminotransferase by glucocorticoids in the rat kidney. Am J Physiol. 1993 Nov;265(5 Pt 1):C1298–C1305. doi: 10.1152/ajpcell.1993.265.5.C1298. [DOI] [PubMed] [Google Scholar]
  14. Feilleux-Duché S., Garlatti M., Burcelin R., Aggerbeck M., Bouguet J., Girard J., Hanoune J., Barouki R. Acinar zonation of the hormonal regulation of cytosolic aspartate aminotransferase in the liver. Am J Physiol. 1994 Apr;266(4 Pt 1):C911–C918. doi: 10.1152/ajpcell.1994.266.4.C911. [DOI] [PubMed] [Google Scholar]
  15. Ganss R., Weih F., Schütz G. The cyclic adenosine 3',5'-monophosphate- and the glucocorticoid-dependent enhancers are targets for insulin repression of tyrosine aminotransferase gene transcription. Mol Endocrinol. 1994 Jul;8(7):895–903. doi: 10.1210/mend.8.7.7984151. [DOI] [PubMed] [Google Scholar]
  16. Garlatti M., Daheshia M., Slater E., Bouguet J., Hanoune J., Beato M., Barouki R. A functional glucocorticoid-responsive unit composed of two overlapping inactive receptor-binding sites: evidence for formation of a receptor tetramer. Mol Cell Biol. 1994 Dec;14(12):8007–8017. doi: 10.1128/mcb.14.12.8007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gorski K., Carneiro M., Schibler U. Tissue-specific in vitro transcription from the mouse albumin promoter. Cell. 1986 Dec 5;47(5):767–776. doi: 10.1016/0092-8674(86)90519-2. [DOI] [PubMed] [Google Scholar]
  18. Guo S., Rena G., Cichy S., He X., Cohen P., Unterman T. Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence. J Biol Chem. 1999 Jun 11;274(24):17184–17192. doi: 10.1074/jbc.274.24.17184. [DOI] [PubMed] [Google Scholar]
  19. Hirai S. I., Ryseck R. P., Mechta F., Bravo R., Yaniv M. Characterization of junD: a new member of the jun proto-oncogene family. EMBO J. 1989 May;8(5):1433–1439. doi: 10.1002/j.1460-2075.1989.tb03525.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kuo C. J., Conley P. B., Chen L., Sladek F. M., Darnell J. E., Jr, Crabtree G. R. A transcriptional hierarchy involved in mammalian cell-type specification. Nature. 1992 Jan 30;355(6359):457–461. doi: 10.1038/355457a0. [DOI] [PubMed] [Google Scholar]
  21. Lewis E. D., Manley J. L. Control of adenovirus late promoter expression in two human cell lines. Mol Cell Biol. 1985 Sep;5(9):2433–2442. doi: 10.1128/mcb.5.9.2433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li W. W., Dammerman M. M., Smith J. D., Metzger S., Breslow J. L., Leff T. Common genetic variation in the promoter of the human apo CIII gene abolishes regulation by insulin and may contribute to hypertriglyceridemia. J Clin Invest. 1995 Dec;96(6):2601–2605. doi: 10.1172/JCI118324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lichtsteiner S., Wuarin J., Schibler U. The interplay of DNA-binding proteins on the promoter of the mouse albumin gene. Cell. 1987 Dec 24;51(6):963–973. doi: 10.1016/0092-8674(87)90583-6. [DOI] [PubMed] [Google Scholar]
  24. Massaad C., Coumoul X., Sabbah M., Garlatti M., Redeuilh G., Barouki R. Properties of overlapping EREs: synergistic activation of transcription and cooperative binding of ER. Biochemistry. 1998 Apr 28;37(17):6023–6032. doi: 10.1021/bi972445e. [DOI] [PubMed] [Google Scholar]
  25. Morel Y., Barouki R. Down-regulation of cytochrome P450 1A1 gene promoter by oxidative stress. Critical contribution of nuclear factor 1. J Biol Chem. 1998 Oct 9;273(41):26969–26976. doi: 10.1074/jbc.273.41.26969. [DOI] [PubMed] [Google Scholar]
  26. Nagata K., Guggenheimer R. A., Enomoto T., Lichy J. H., Hurwitz J. Adenovirus DNA replication in vitro: identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6438–6442. doi: 10.1073/pnas.79.21.6438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nakae J., Park B. C., Accili D. Insulin stimulates phosphorylation of the forkhead transcription factor FKHR on serine 253 through a Wortmannin-sensitive pathway. J Biol Chem. 1999 Jun 4;274(23):15982–15985. doi: 10.1074/jbc.274.23.15982. [DOI] [PubMed] [Google Scholar]
  28. Nishio Y., Isshiki H., Kishimoto T., Akira S. A nuclear factor for interleukin-6 expression (NF-IL6) and the glucocorticoid receptor synergistically activate transcription of the rat alpha 1-acid glycoprotein gene via direct protein-protein interaction. Mol Cell Biol. 1993 Mar;13(3):1854–1862. doi: 10.1128/mcb.13.3.1854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nitsch D., Boshart M., Schütz G. Activation of the tyrosine aminotransferase gene is dependent on synergy between liver-specific and hormone-responsive elements. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5479–5483. doi: 10.1073/pnas.90.12.5479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nolten L. A., van Schaik F. M., Steenbergh P. H., Sussenbach J. S. Expression of the insulin-like growth factor I gene is stimulated by the liver-enriched transcription factors C/EBP alpha and LAP. Mol Endocrinol. 1994 Dec;8(12):1636–1645. doi: 10.1210/mend.8.12.7708053. [DOI] [PubMed] [Google Scholar]
  31. O'Brien R. M., Granner D. K. Regulation of gene expression by insulin. Physiol Rev. 1996 Oct;76(4):1109–1161. doi: 10.1152/physrev.1996.76.4.1109. [DOI] [PubMed] [Google Scholar]
  32. O'Brien R. M., Lucas P. C., Forest C. D., Magnuson M. A., Granner D. K. Identification of a sequence in the PEPCK gene that mediates a negative effect of insulin on transcription. Science. 1990 Aug 3;249(4968):533–537. doi: 10.1126/science.2166335. [DOI] [PubMed] [Google Scholar]
  33. O'Brien R. M., Noisin E. L., Suwanichkul A., Yamasaki T., Lucas P. C., Wang J. C., Powell D. R., Granner D. K. Hepatic nuclear factor 3- and hormone-regulated expression of the phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein 1 genes. Mol Cell Biol. 1995 Mar;15(3):1747–1758. doi: 10.1128/mcb.15.3.1747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pavé-Preux M., Aggerbeck M., Veyssier C., Bousquet-Lemercier B., Hanoune J., Barouki R. Hormonal discrimination among transcription start sites of aspartate aminotransferase. J Biol Chem. 1990 Mar 15;265(8):4444–4448. [PubMed] [Google Scholar]
  35. Pavé-Preux M., Ferry N., Bouguet J., Hanoune J., Barouki R. Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase. J Biol Chem. 1988 Nov 25;263(33):17459–17466. [PubMed] [Google Scholar]
  36. Rena G., Guo S., Cichy S. C., Unterman T. G., Cohen P. Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B. J Biol Chem. 1999 Jun 11;274(24):17179–17183. doi: 10.1074/jbc.274.24.17179. [DOI] [PubMed] [Google Scholar]
  37. Robins D. M., Scheller A., Adler A. J. Specific steroid response from a nonspecific DNA element. J Steroid Biochem Mol Biol. 1994 Jun;49(4-6):251–255. doi: 10.1016/0960-0760(94)90265-8. [DOI] [PubMed] [Google Scholar]
  38. Roux J., Pictet R., Grange T. Hepatocyte nuclear factor 3 determines the amplitude of the glucocorticoid response of the rat tyrosine aminotransferase gene. DNA Cell Biol. 1995 May;14(5):385–396. doi: 10.1089/dna.1995.14.385. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Schüle R., Muller M., Otsuka-Murakami H., Renkawitz R. Cooperativity of the glucocorticoid receptor and the CACCC-box binding factor. Nature. 1988 Mar 3;332(6159):87–90. doi: 10.1038/332087a0. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Streeper R. S., Svitek C. A., Chapman S., Greenbaum L. E., Taub R., O'Brien R. M. A multicomponent insulin response sequence mediates a strong repression of mouse glucose-6-phosphatase gene transcription by insulin. J Biol Chem. 1997 May 2;272(18):11698–11701. doi: 10.1074/jbc.272.18.11698. [DOI] [PubMed] [Google Scholar]
  43. Suwanickul A., Morris S. L., Powell D. R. Identification of an insulin-responsive element in the promoter of the human gene for insulin-like growth factor binding protein-1. J Biol Chem. 1993 Aug 15;268(23):17063–17068. [PubMed] [Google Scholar]
  44. Suzuki T., Okuno H., Yoshida T., Endo T., Nishina H., Iba H. Difference in transcriptional regulatory function between c-Fos and Fra-2. Nucleic Acids Res. 1991 Oct 25;19(20):5537–5542. doi: 10.1093/nar/19.20.5537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tang E. D., Nuñez G., Barr F. G., Guan K. L. Negative regulation of the forkhead transcription factor FKHR by Akt. J Biol Chem. 1999 Jun 11;274(24):16741–16746. doi: 10.1074/jbc.274.24.16741. [DOI] [PubMed] [Google Scholar]
  46. Umesono K., Giguere V., Glass C. K., Rosenfeld M. G., Evans R. M. Retinoic acid and thyroid hormone induce gene expression through a common responsive element. Nature. 1988 Nov 17;336(6196):262–265. doi: 10.1038/336262a0. [DOI] [PubMed] [Google Scholar]
  47. Unterman T. G., Fareeduddin A., Harris M. A., Goswami R. G., Porcella A., Costa R. H., Lacson R. G. Hepatocyte nuclear factor-3 (HNF-3) binds to the insulin response sequence in the IGF binding protein-1 (IGFBP-1) promoter and enhances promoter function. Biochem Biophys Res Commun. 1994 Sep 30;203(3):1835–1841. doi: 10.1006/bbrc.1994.2401. [DOI] [PubMed] [Google Scholar]
  48. Vaulont S., Puzenat N., Levrat F., Cognet M., Kahn A., Raymondjean M. Proteins binding to the liver-specific pyruvate kinase gene promoter. A unique combination of known factors. J Mol Biol. 1989 Sep 20;209(2):205–219. doi: 10.1016/0022-2836(89)90273-8. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES