Comprehensive analysis of proteins which interact with the antioxidant responsive element: correlation of ARE-BP-1 with the chemoprotective induction response.
Transcriptional activation of the mouse glutathione S-transferase Ya gene by chemoprotective molecules is mediated through the interaction of trans-acting factors with an antioxidant responsive element (ARE) in the promoter region of this gene. In a step toward identifying those factors which bind productively to the GST Ya ARE, all of the discernible, specific ARE-binding proteins (ARE-BP) in nuclear extracts from HepG2 cells were systematically characterized. By gel-mobility-shift analysis, seven specific ARE-BPs, termed ARE-BP-1 through 7 in order of increasing mobility, were observed that did not vary in concentration or migration between induced and uninduced cell extracts. The molecular weights of the individual ARE-BP subunits were determined by a two-dimensional electrophoresis protocol. Ferguson gel analysis of native protein size indicated that several of the ARE-BP-DNA complexes are composed of multiple protein subunits. Wild-type AREs and GST Ya ARE fragments and mutant sequences were evaluated for their ability to mediate induction in a reporter gene system in HepG2 cells. This same panel of sites was tested in an in vitro binding assay for the ability to compete for the ARE-BPs. A binding profile for each ARE-BP was compiled. Correlation between the ARE-BP binding profiles and induction results indicated that: (i) the ARE-BP-1 and ARE-BP-2 complexes formed only with AREs that supported induction, and (ii) the ARE-BP-4 complex formed with all inducible AREs, but it also bound to ARE mutants that failed to support induction. Based on the studies, an early composite regulatory element model for ARE-mediated expression is presented. ARE-BP-1 is proposed to be the mediator of the ARE's unique induction response to chemoprotective agents.
Wasserman
W W
WW
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706, USA.
Fahl
W E
WE
D000975:Antioxidants / Q000494:pharmacology*; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D015972:Gene Expression Regulation, Neoplastic; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D008970:Molecular Weight; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D000975:Antioxidants; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D006873:Hydroquinones; D016328:NF-kappa B; D009687:Nuclear Proteins; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D004247:DNA; C018855:2-tert-butylhydroquinone; D005982:Glutathione Transferase