Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Oct;15(10):5453–5460. doi: 10.1128/mcb.15.10.5453

Overproduction of a truncated hepatocyte nuclear factor 3 protein inhibits expression of liver-specific genes in hepatoma cells.

V Vallet 1, B Antoine 1, P Chafey 1, A Vandewalle 1, A Kahn 1
PMCID: PMC230795  PMID: 7565696

Abstract

Transcription of hepatocyte-specific genes requires the interaction of their regulatory regions with several nuclear factors. Among them is the hepatocyte nuclear factor 3 (HNF3) family, composed of the HNF3 alpha, HNF3 beta, and HNF3 gamma proteins, which are expressed in the liver and have very similar fork head DNA binding domains. The regulatory regions of numerous hepatocyte-specific genes contain HNF3 binding sites. We examined the role of HNF3 proteins in the liver-specific phenotype by turning off the HNF3 activity in well-differentiated mhAT3F hepatoma cells. Cells were stably transfected with a vector allowing the synthesis of an HNF3 beta fragment consisting of the fork head DNA binding domain without the transactivating amino- and carboxy-terminal domains. The truncated protein was located in the nuclei of cultured hepatoma cells and competed with endogenous HNF3 proteins for binding to cognate DNA sites. Overproduction of this truncated protein, lacking any transactivating activity, induced a dramatic decrease in the expression of liver-specific genes, including those for albumin, transthyretin, transferrin, phosphoenolpyruvate carboxykinase, and aldolase B, whereas the expression of the L-type pyruvate kinase gene, containing no HNF3 binding sites, was unaltered. Neither were the concentrations of various liver-specific transcription factors (HNF3, HNF1, HNF4, and C/EBP alpha) affected. In partial revertants, with a lower ratio of truncated to full-length endogenous HNF3 proteins, previously extinguished genes were re-expressed. Thus, the transactivating domains of HNF3 proteins are needed for the proper expression of a set of liver-specific genes but not for expression of the genes encoding transcription factors found in differentiated hepatocytes.

Full Text

The Full Text of this article is available as a PDF (636.2 KB).

Selected References

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

  1. Ang S. L., Rossant J. HNF-3 beta is essential for node and notochord formation in mouse development. Cell. 1994 Aug 26;78(4):561–574. doi: 10.1016/0092-8674(94)90522-3. [DOI] [PubMed] [Google Scholar]
  2. Antoine B., Levrat F., Vallet V., Berbar T., Cartier N., Dubois N., Briand P., Kahn A. Gene expression in hepatocyte-like lines established by targeted carcinogenesis in transgenic mice. Exp Cell Res. 1992 May;200(1):175–185. doi: 10.1016/s0014-4827(05)80086-2. [DOI] [PubMed] [Google Scholar]
  3. Augé-Gouillou C., Petropoulos I., Zakin M. M. Liver-enriched HNF-3 alpha and ubiquitous factors interact with the human transferrin gene enhancer. FEBS Lett. 1993 May 24;323(1-2):4–10. doi: 10.1016/0014-5793(93)81436-4. [DOI] [PubMed] [Google Scholar]
  4. Bassel-Duby R., Hernandez M. D., Yang Q., Rochelle J. M., Seldin M. F., Williams R. S. Myocyte nuclear factor, a novel winged-helix transcription factor under both developmental and neural regulation in striated myocytes. Mol Cell Biol. 1994 Jul;14(7):4596–4605. doi: 10.1128/mcb.14.7.4596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baumhueter S., Mendel D. B., Conley P. B., Kuo C. J., Turk C., Graves M. K., Edwards C. A., Courtois G., Crabtree G. R. HNF-1 shares three sequence motifs with the POU domain proteins and is identical to LF-B1 and APF. Genes Dev. 1990 Mar;4(3):372–379. doi: 10.1101/gad.4.3.372. [DOI] [PubMed] [Google Scholar]
  6. Blumenfeld M., Maury M., Chouard T., Yaniv M., Condamine H. Hepatic nuclear factor 1 (HNF1) shows a wider distribution than products of its known target genes in developing mouse. Development. 1991 Oct;113(2):589–599. doi: 10.1242/dev.113.2.589. [DOI] [PubMed] [Google Scholar]
  7. Clark K. L., Halay E. D., Lai E., Burley S. K. Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5. Nature. 1993 Jul 29;364(6436):412–420. doi: 10.1038/364412a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Costa R. H., Grayson D. R. Site-directed mutagenesis of hepatocyte nuclear factor (HNF) binding sites in the mouse transthyretin (TTR) promoter reveal synergistic interactions with its enhancer region. Nucleic Acids Res. 1991 Aug 11;19(15):4139–4145. doi: 10.1093/nar/19.15.4139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Descombes P., Chojkier M., Lichtsteiner S., Falvey E., Schibler U. LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein. Genes Dev. 1990 Sep;4(9):1541–1551. doi: 10.1101/gad.4.9.1541. [DOI] [PubMed] [Google Scholar]
  11. Dubois N., Bennoun M., Allemand I., Molina T., Grimber G., Daudet-Monsac M., Abelanet R., Briand P. Time-course development of differentiated hepatocarcinoma and lung metastasis in transgenic mice. J Hepatol. 1991 Sep;13(2):227–239. doi: 10.1016/0168-8278(91)90819-w. [DOI] [PubMed] [Google Scholar]
  12. Friedman A. D., McKnight S. L. Identification of two polypeptide segments of CCAAT/enhancer-binding protein required for transcriptional activation of the serum albumin gene. Genes Dev. 1990 Aug;4(8):1416–1426. doi: 10.1101/gad.4.8.1416. [DOI] [PubMed] [Google Scholar]
  13. Gregori C., Kahn A., Pichard A. L. Activity of the rat liver-specific aldolase B promoter is restrained by HNF3. Nucleic Acids Res. 1994 Apr 11;22(7):1242–1246. doi: 10.1093/nar/22.7.1242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gregori C., Kahn A., Pichard A. L. Competition between transcription factors HNF1 and HNF3, and alternative cell-specific activation by DBP and C/EBP contribute to the regulation of the liver-specific aldolase B promoter. Nucleic Acids Res. 1993 Feb 25;21(4):897–903. doi: 10.1093/nar/21.4.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grossniklaus U., Pearson R. K., Gehring W. J. The Drosophila sloppy paired locus encodes two proteins involved in segmentation that show homology to mammalian transcription factors. Genes Dev. 1992 Jun;6(6):1030–1051. doi: 10.1101/gad.6.6.1030. [DOI] [PubMed] [Google Scholar]
  16. Hatini V., Tao W., Lai E. Expression of winged helix genes, BF-1 and BF-2, define adjacent domains within the developing forebrain and retina. J Neurobiol. 1994 Oct;25(10):1293–1309. doi: 10.1002/neu.480251010. [DOI] [PubMed] [Google Scholar]
  17. Hoppe-Seyler F., Butz K., Rittmüller C., von Knebel Doeberitz M. A rapid microscale procedure for the simultaneous preparation of cytoplasmic RNA, nuclear DNA binding proteins and enzymatically active luciferase extracts. Nucleic Acids Res. 1991 Sep 25;19(18):5080–5080. doi: 10.1093/nar/19.18.5080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ingraham H. A., Flynn S. E., Voss J. W., Albert V. R., Kapiloff M. S., Wilson L., Rosenfeld M. G. The POU-specific domain of Pit-1 is essential for sequence-specific, high affinity DNA binding and DNA-dependent Pit-1-Pit-1 interactions. Cell. 1990 Jun 15;61(6):1021–1033. doi: 10.1016/0092-8674(90)90067-o. [DOI] [PubMed] [Google Scholar]
  19. Ip Y. T., Poon D., Stone D., Granner D. K., Chalkley R. Interaction of a liver-specific factor with an enhancer 4.8 kilobases upstream of the phosphoenolpyruvate carboxykinase gene. Mol Cell Biol. 1990 Jul;10(7):3770–3781. doi: 10.1128/mcb.10.7.3770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jackson D. A., Rowader K. E., Stevens K., Jiang C., Milos P., Zaret K. S. Modulation of liver-specific transcription by interactions between hepatocyte nuclear factor 3 and nuclear factor 1 binding DNA in close apposition. Mol Cell Biol. 1993 Apr;13(4):2401–2410. doi: 10.1128/mcb.13.4.2401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Lai E., Prezioso V. R., Smith E., Litvin O., Costa R. H., Darnell J. E., Jr HNF-3A, a hepatocyte-enriched transcription factor of novel structure is regulated transcriptionally. Genes Dev. 1990 Aug;4(8):1427–1436. doi: 10.1101/gad.4.8.1427. [DOI] [PubMed] [Google Scholar]
  23. Lai E., Prezioso V. R., Tao W. F., Chen W. S., Darnell J. E., Jr Hepatocyte nuclear factor 3 alpha belongs to a gene family in mammals that is homologous to the Drosophila homeotic gene fork head. Genes Dev. 1991 Mar;5(3):416–427. doi: 10.1101/gad.5.3.416. [DOI] [PubMed] [Google Scholar]
  24. Landschulz W. H., Johnson P. F., Adashi E. Y., Graves B. J., McKnight S. L. Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev. 1988 Jul;2(7):786–800. doi: 10.1101/gad.2.7.786. [DOI] [PubMed] [Google Scholar]
  25. Levrat F., Vallet V., Berbar T., Miquerol L., Kahn A., Antoine B. Influence of the content in transcription factors on the phenotype of mouse hepatocyte-like cell lines (mhAT). Exp Cell Res. 1993 Dec;209(2):307–316. doi: 10.1006/excr.1993.1315. [DOI] [PubMed] [Google Scholar]
  26. Lichtsteiner S., Schibler U. A glycosylated liver-specific transcription factor stimulates transcription of the albumin gene. Cell. 1989 Jun 30;57(7):1179–1187. doi: 10.1016/0092-8674(89)90055-x. [DOI] [PubMed] [Google Scholar]
  27. Liu J. K., DiPersio C. M., Zaret K. S. Extracellular signals that regulate liver transcription factors during hepatic differentiation in vitro. Mol Cell Biol. 1991 Feb;11(2):773–784. doi: 10.1128/mcb.11.2.773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Margolskee R. F., Kavathas P., Berg P. Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells. Mol Cell Biol. 1988 Jul;8(7):2837–2847. doi: 10.1128/mcb.8.7.2837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McPherson C. E., Shim E. Y., Friedman D. S., Zaret K. S. An active tissue-specific enhancer and bound transcription factors existing in a precisely positioned nucleosomal array. Cell. 1993 Oct 22;75(2):387–398. doi: 10.1016/0092-8674(93)80079-t. [DOI] [PubMed] [Google Scholar]
  30. Mendel D. B., Hansen L. P., Graves M. K., Conley P. B., Crabtree G. R. HNF-1 alpha and HNF-1 beta (vHNF-1) share dimerization and homeo domains, but not activation domains, and form heterodimers in vitro. Genes Dev. 1991 Jun;5(6):1042–1056. doi: 10.1101/gad.5.6.1042. [DOI] [PubMed] [Google Scholar]
  31. Miura N., Wanaka A., Tohyama M., Tanaka K. MFH-1, a new member of the fork head domain family, is expressed in developing mesenchyme. FEBS Lett. 1993 Jul 12;326(1-3):171–176. doi: 10.1016/0014-5793(93)81785-x. [DOI] [PubMed] [Google Scholar]
  32. Monaghan A. P., Kaestner K. H., Grau E., Schütz G. Postimplantation expression patterns indicate a role for the mouse forkhead/HNF-3 alpha, beta and gamma genes in determination of the definitive endoderm, chordamesoderm and neuroectoderm. Development. 1993 Nov;119(3):567–578. doi: 10.1242/dev.119.3.567. [DOI] [PubMed] [Google Scholar]
  33. Mueller C. R., Maire P., Schibler U. DBP, a liver-enriched transcriptional activator, is expressed late in ontogeny and its tissue specificity is determined posttranscriptionally. Cell. 1990 Apr 20;61(2):279–291. doi: 10.1016/0092-8674(90)90808-r. [DOI] [PubMed] [Google Scholar]
  34. Murphy D. B., Wiese S., Burfeind P., Schmundt D., Mattei M. G., Schulz-Schaeffer W., Thies U. Human brain factor 1, a new member of the fork head gene family. Genomics. 1994 Jun;21(3):551–557. doi: 10.1006/geno.1994.1313. [DOI] [PubMed] [Google Scholar]
  35. Pani L., Overdier D. G., Porcella A., Qian X., Lai E., Costa R. H. Hepatocyte nuclear factor 3 beta contains two transcriptional activation domains, one of which is novel and conserved with the Drosophila fork head protein. Mol Cell Biol. 1992 Sep;12(9):3723–3732. doi: 10.1128/mcb.12.9.3723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pani L., Quian X. B., Clevidence D., Costa R. H. The restricted promoter activity of the liver transcription factor hepatocyte nuclear factor 3 beta involves a cell-specific factor and positive autoactivation. Mol Cell Biol. 1992 Feb;12(2):552–562. doi: 10.1128/mcb.12.2.552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Raymondjean M., Cereghini S., Yaniv M. Several distinct "CCAAT" box binding proteins coexist in eukaryotic cells. Proc Natl Acad Sci U S A. 1988 Feb;85(3):757–761. doi: 10.1073/pnas.85.3.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Raymondjean M., Pichard A. L., Gregori C., Ginot F., Kahn A. Interplay of an original combination of factors: C/EBP, NFY, HNF3, and HNF1 in the rat aldolase B gene promoter. Nucleic Acids Res. 1991 Nov 25;19(22):6145–6153. doi: 10.1093/nar/19.22.6145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rey-Campos J., Chouard T., Yaniv M., Cereghini S. vHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1. EMBO J. 1991 Jun;10(6):1445–1457. doi: 10.1002/j.1460-2075.1991.tb07665.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ruiz i Altaba A., Cox C., Jessell T. M., Klar A. Ectopic neural expression of a floor plate marker in frog embryos injected with the midline transcription factor Pintallavis. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8268–8272. doi: 10.1073/pnas.90.17.8268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sasaki H., Hogan B. L. Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development. 1993 May;118(1):47–59. doi: 10.1242/dev.118.1.47. [DOI] [PubMed] [Google Scholar]
  42. Sasaki H., Hogan B. L. HNF-3 beta as a regulator of floor plate development. Cell. 1994 Jan 14;76(1):103–115. doi: 10.1016/0092-8674(94)90176-7. [DOI] [PubMed] [Google Scholar]
  43. Sladek F. M., Zhong W. M., Lai E., Darnell J. E., Jr Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily. Genes Dev. 1990 Dec;4(12B):2353–2365. doi: 10.1101/gad.4.12b.2353. [DOI] [PubMed] [Google Scholar]
  44. Strähle U., Blader P., Henrique D., Ingham P. W. Axial, a zebrafish gene expressed along the developing body axis, shows altered expression in cyclops mutant embryos. Genes Dev. 1993 Jul;7(7B):1436–1446. doi: 10.1101/gad.7.7b.1436. [DOI] [PubMed] [Google Scholar]
  45. Tao W., Lai E. Telencephalon-restricted expression of BF-1, a new member of the HNF-3/fork head gene family, in the developing rat brain. Neuron. 1992 May;8(5):957–966. doi: 10.1016/0896-6273(92)90210-5. [DOI] [PubMed] [Google Scholar]
  46. Theill L. E., Castrillo J. L., Wu D., Karin M. Dissection of functional domains of the pituitary-specific transcription factor GHF-1. Nature. 1989 Dec 21;342(6252):945–948. doi: 10.1038/342945a0. [DOI] [PubMed] [Google Scholar]
  47. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Vallet V., Bens M., Antoine B., Levrat F., Miquerol L., Kahn A., Vandewalle A. Transcription factors and aldolase B gene expression in microdissected renal proximal tubules and derived cell lines. Exp Cell Res. 1995 Feb;216(2):363–370. doi: 10.1006/excr.1995.1046. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Weigel D., Jürgens G., Küttner F., Seifert E., Jäckle H. The homeotic gene fork head encodes a nuclear protein and is expressed in the terminal regions of the Drosophila embryo. Cell. 1989 May 19;57(4):645–658. doi: 10.1016/0092-8674(89)90133-5. [DOI] [PubMed] [Google Scholar]
  51. Weinstein D. C., Ruiz i Altaba A., Chen W. S., Hoodless P., Prezioso V. R., Jessell T. M., Darnell J. E., Jr The winged-helix transcription factor HNF-3 beta is required for notochord development in the mouse embryo. Cell. 1994 Aug 26;78(4):575–588. doi: 10.1016/0092-8674(94)90523-1. [DOI] [PubMed] [Google Scholar]

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

RESOURCES