The characterization and purification of a human transcription factor modulating the glutathione peroxidase gene in response to oxygen tension.
An oxygen responsive transcription factor regulating human glutathione peroxidase gene (GPx) through two oxygen responsive elements (ORE I 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.
Merante
Frank
F
Tm Bioscience Corporation, Toronto, Ontario, Canada.
Altamentova
Svetlana M
SM
Mickle
Donald A G
DA
Weisel
Richard D
RD
Thatcher
Bradley J
BJ
Martin
Brian M
BM
Marshall
John G
JG
Tumiati
Laura C
LC
Cowan
Douglas B
DB
Li
Ren-Ke
RK
D000906:Antibodies / Q000276:immunology; D034961:Antigens, Nuclear; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D005979:Glutathione Peroxidase / Q000235:genetics*; D006801:Humans; D000072200:Ku Autoantigen; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000737:chemistry / Q000276:immunology; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D020218:Response Elements; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D004247:DNA; D005979:Glutathione Peroxidase; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D010100:Oxygen