Skip to main content
SpringerLink
Log in

The characterization and purification of a human transcription factor modulating the glutathione peroxidase gene in response to oxygen tension

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

An oxygen responsive transcription factor regulating human glutathione peroxidase gene (GPx) through two oxygen responsive elements (ORE1 and ORE2) has been purified and characterized by sequence-specific DNA affinity chromatography. The DNA binding activity, termed Oxygen Responsive Element Binding Protein (OREBP), was partially represented by a 77 kD polypeptide (p70) possessing a blocked N-terminus. The p70 subunit co-eluted with an 86 kD subunit (p80) from affinity columns. N-terminal sequencing analysis of the 86 kD component revealed that this protein represented the larger member of the Ku antigen complex. The identity of the purified 77 kD subunit was determined by Western blot analysis using an antibody directed against the p70 protein. In addition to binding the GPx-ORE, the OREBP was itself regulated by oxygen tension. It was found that the abundance of the ORE binding activity was decreased in cells maintained at low oxygen tension (40 mm Hg). Anti-Ku-antibodies specifically supershifted the OREBP-ORE DNA complex. These observations further add to the numerous nuclear roles of the Ku-transcription factor.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Merante F, Mickle DAG, Weisel RD, Li R-K, Tumiati LC, Rao V, Williams WG, Robinson BH: Myocardial aerobic metabolism is impaired in a cell culture model of cyanotic heart disease. Am J Physiol 275: 673–681, 1998

    Google Scholar 

  2. Teoh KH, Mickle DAG, Li R-K, Tumiati LC, Coles JC, Williams WG: Effect of oxygen tension and cardiovascular operations on the myocardial antioxidant enzyme activities in patients with tetralogy of Fallot and aorta-coronary bypass. J Thorac Cardiovasc Surg 104: 159–164, 1992

    Google Scholar 

  3. Cowan DB, Weisel RD, Williams WG, Mickle DAG: The regulation of glutathione peroxidase gene expression by oxygen tension in cultured human cardiomyocytes. J Mol Cell Cardiol 24: 423–433, 1992

    Google Scholar 

  4. Cowan DB, Weisel RD, Williams WG, Mickle DAG: Identification of oxygen responsive elements in the 5′-flanking region of the human glutathione peroxidase gene. J Biol Chem 268: 26904–26910, 1993

    Google Scholar 

  5. Del Nido PJ, Mickle DAG, Wilson GJ, Bensen LN, Weisel RD, Coles JG, Trustler GA, Williams WG: Inadequate myocardial protection with cold cardioplegic arrest during repair of tetralogy of Fallot. J Thorac Cardiovasc Surg 95: 223–229, 1988

    Google Scholar 

  6. Wang GL, Semenza GL: Purification and characterization of hypoxiainducible factor 1. J Biol Chem 270: 1230–1237, 1995

    Google Scholar 

  7. Wang GL, Jiang B-H, Rue EA, Semenza GL: Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 92: 5510–5514, 1995

    Google Scholar 

  8. Semenza GL, Roth PH, Fang H-M, Wang GL: Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem 269: 23757–23763, 1994

    Google Scholar 

  9. Griffith AJ, Craft J, Evans J, Mimori T, Hardin JA: Nucleotide sequence and genomic structure analyses of the p70 subunit of the human Ku autoantigen: Evidence for a family of genes encoding Ku (p70)-related polypeptides. Mol Biol Rep 16: 91–97, 1992

    Google Scholar 

  10. Reeves WH, Sthoeger ZM: Molecular cloning of cDNA encoding the p70 (Ku) Lupus autoantigen. J Biol Chem 264: 5047–5052, 1989

    Google Scholar 

  11. Yaneva M, Wen J, Ayala A, Cook R: cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen. J Biol Chem 264: 13407–13411, 1989

    Google Scholar 

  12. Hahn C-G, Covault J: Isolation of transcriptionally active nuclei from striated muscle using percoll density gradients. Anal Biochem 190: 193–197, 1990

    Google Scholar 

  13. Li R-K, Weisel RD, Williams WG, Mickle DAG: Method of culturing cardiomyocytes from human pediatric ventricular myocardium. J Tiss Cult Meth 14: 93–100, 1992

    Google Scholar 

  14. Andrews NC, Faller DV: A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res 19: 2499–2500, 1991

    Google Scholar 

  15. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976

    Google Scholar 

  16. Schmitz GG, Walter T, Seibl R, Kessler C: Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal Biochem 192: 222–231, 1991

    Google Scholar 

  17. Holtke HJ, Sagner G, Kessler C, Schmitz G: Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: A fast and simple protocol and its applications. BioTechniques 12: 104–113, 1992

    Google Scholar 

  18. Zhang X-Y, Asiedu CK, Supakar PC, Ehrilich M: Increasing the activity of affinity-purified DNA-binding proteins by adding high concentrations of nonspecific proteins. Anal Biochem 201: 336–374, 1991

    Google Scholar 

  19. Tomáška L, Nosek J: Several polymers enhance the sensitivity of the southwestern assay. Anal Biochem 227: 387–389, 1995

    Google Scholar 

  20. Klug J: Ku autoantigen is a potential major cause of nonspecific bands in electrophoretic mobility shift assays. BioTechniques 22: 212–216, 1997

    Google Scholar 

  21. Demczuk S, Donovan M, Franklin G, Ohlsson R: Order of probe and nuclear protein extract addition can determine specificity of protein—DNA complexes in tested mobility shift assays. Nucleic Acids Res 19: 677–678, 1991

    Google Scholar 

  22. Bliss MB, Lane DP: Ku selectively transfers between DNA molecules with homologous ends. J Biol Chem 272: 5765–5773, 1997

    Google Scholar 

  23. Tuteja N, Tuteja R, Ochem A, Taneja P, Huang NW, Simoncsits A, Susic S, Rahman K, Marusic L, Chen J, Zhang J, Wang S, Pongor S, Falaschi A: Human DNA helicase II: A novel DNA unwinding enzyme identified as the Ku autoantigen. EMBO J 13: 4991–5001, 1994

    Google Scholar 

  24. Avissar N, Eisenmann C, Breen JG, Horowitz S, Miller RK, Cohen HJ: Human placenta makes extracellular glutathione peroxidase and secretes it into maternal circulation. Am J Physiol 267: E68–76, 1994

    Google Scholar 

  25. Genersch E, Eckerskorn C, Lottspeich F, Herzog C, Kuhn K, E. Pöschl E: Purification of the sequence-specific transcription factor CTCBF, involved in the control of human collagen IV genes: Subunits with homology to Ku antigen. EMBO J 14: 791–800, 1995

    Google Scholar 

  26. Mimori T, Hardin JA: Mechanism of interaction between Ku protein and DNA. J Biol Chem 261: 10375–10379, 1986

    Google Scholar 

  27. Ochem AE, Skopac D, Costa M, Rabilloud T, Vuillard L, Simoncsits A, Giacca M, Falaschi A: Functional properties of the separate subunits of human DNA Helicase II/Ku autoantigen. J Biol Chem 272: 29919–29926, 1997

    Google Scholar 

  28. Wang J, Satoh M, Pierani A, Schmitt J, Chou C-H, Stunnenberg HG, Roeder RG, Reeves WH: Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers. J Cell Sci 107: 3223–3233, 1994

    Google Scholar 

  29. Tuteja R, Tuteja N: Ku autoantigen: A multifunctional DNA-binding protein. Crit Rev Biochem Mol Biol 35: 1–33, 2000

    Google Scholar 

  30. Dynan WS, Yoo S: Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids. Nucleic Acids Res 26: 1551–1559, 1998

    Google Scholar 

  31. Gravel S, Larrivee M, Labrecque P, Wellinger RJ: Yeast Ku as a regulator of chromosomal DNA end structure. Science 280: 741–744, 1998

    Google Scholar 

  32. Kunth MW, Gunderson SI, Thompson NE, Strasheim LA, Burgess RR: Purification and characterization of proximal sequence element-binding protein 1, a transcription activating protein related to Ku and TREF that binds the proximal sequence element of the human U1 promoter. J Biol Chem 265: 17911–17920, 1990

    Google Scholar 

  33. Giffin W, Torrance H, Rodda DJ, Prefontaine GG, Pope L, Hache RJG: Sequence-specific DNA binding by Ku autoantigen and its effects on transcription. Nature 380: 265–268, 1996

    Google Scholar 

  34. Falzon M, Fewell JW, Kuff EL: EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single-to double-strand transitions in DNA. J Biol Chem 268: 10546–10552, 1993

    Google Scholar 

  35. May G, Sutton C, Gould H: Purification and characterization of Ku-2, an octamer-binding protein related to the autoantigen Ku. J Biol Chem 266: 3052–3059, 1991

    Google Scholar 

  36. Stuiver MH, Coenjaerts FEJ, van der Vliet PC: The autoantigen Ku is indistinguishable from NFIV, a protein forming multimeric protein —DNA complexes. J Exp Med 172: 1049–1054, 1990

    Google Scholar 

  37. Rodríguez JC, Ortiz JA, Hegardt FG, Haro D: The hepatocyte nuclear factor 4 (HNF-4) represses the mitochondrial HMG-CoA synthase gene. Biochem Biophys Res Commun 242: 692–696, 1998

    Google Scholar 

  38. Yoshida E, Aratani S, Itou H, Miyagishi M, Takiguchi M, Osumu T, Murakami K, Fukamizu A: Functional asociation between CBP and HNF4 in trans-activation. Biochem Biophys Res Commun 241: 664–669, 1997

    Google Scholar 

  39. Chung U, Igarashi T, Nishishita T, Iwanari H, Iwamatsu A, Suwa A, Mimori T, Hata K, Ebisu S, Ogata E, Fujita T, Okazaki T: The interaction between Ku antigen and REF1 protein mediates negative gene regulation by extracellular calcium. J Biol Chem 271: 8593–8598, 1996

    Google Scholar 

  40. Satoh M, Ajmani AK, Stojanov L, Langdon JJ, Ogasawara T, Wang J, Dooley MA, Richards HB, Winfield JB, Carter TH, Reeves WH: Autoantibodies that stabilize the molecular interaction of Ku antigen with DNA-dependent protein kinase catalytic subunit. Clin Exp Immunol 105: 460–467, 1996

    Google Scholar 

  41. Giffin W, Kwast-Welfeld J, Rodda DJ, Prefontaine GG, Traykova-Andronova M, Zhang Y, Weigel NL, Lefebvre YA, Hache RJG: Sequence-specific DNA binding and transcription factor phosphorylation by Ku autoantigen/DNA-dependent protein kinase. J Biol Chem 272: 5647–5658, 1997

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Tm Bioscience Corporation, Toronto, Ontario, Canada

    Frank Merante

  2. Centre for Cardiovascular Research, Department of Clinical Biochemistry and the Division of Cardiovascular Surgery, The Toronto Hospital, University of Toronto, Toronto, Ontario, Canada

    Svetlana M. Altamentova, Donald A.G. Mickle, Richard D. Weisel, Laura C. Tumiati, Douglas B. Cowan & Ren-Ke Li

  3. Bethesda, National Institutes of Medical Health (NIH), MD, USA

    Bradley J. Thatcher, Brian M. Martin & John G. Marshall

Authors
  1. Frank Merante
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Svetlana M. Altamentova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Donald A.G. Mickle
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Richard D. Weisel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bradley J. Thatcher
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Brian M. Martin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. John G. Marshall
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Laura C. Tumiati
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Douglas B. Cowan
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ren-Ke Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Donald A.G. Mickle.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Merante, F., Altamentova, S.M., Mickle, D.A. et al. The characterization and purification of a human transcription factor modulating the glutathione peroxidase gene in response to oxygen tension. Mol Cell Biochem 229, 73–83 (2002). https://doi.org/10.1023/A:1017921110363

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1017921110363

Search

Navigation