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 g ene by chemoprotective molecules is mediated through the interaction o f 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 discerni ble, specific ARE-binding proteins (ARE-BP) in nuclear extracts from H epG2 cells were systematically characterized, By gel-mobility-shift an alysis, 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 molecula r weights of the individual ARE-BP subunits were determined by a two-d imensional electrophoresis protocol, Ferguson gel analysis of native p rotein size indicated that several of the ARE-BP-DNA complexes are com posed of multiple protein subunits, Wild-type AREs and GST Ya ARE frag ments and mutant sequences were evaluated for their ability to mediate induction in a reporter gene system in HepG2 cells. This same panel o f sites was tested in an in vitro binding assay for the ability to com pete 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-S complexes formed only wi th 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 model for ARE-mediated expression is pres ented, ARE-BP-1 is proposed to be the mediator of the ARE's unique ind uction response to chemoprotective agents. (C) 1997 Academic Press.