Functional characterization of transcription regulators that interact withthe electrophile response element

The electrophile response element (EpRE), also referred to as the antioxida nt responsive element (ARE), is found in the 5'-regulatory region of a numb er of genes encoding phase II, drug-metabolizing enzymes. Gene knockout stu dies have demonstrated the primary regulatory role that an Nrf2:Maf dimer p lays by binding to nucleotides within the EpRE consensus sequence. Current models of transcription regulation have also shown the involvement of highe r-order transcriptional coactivators, proteins that nucleate around DNA seq uence-specific transcription factors, enhancing transcription of the target gene by interacting with components of the basal transcriptional apparatus and by enabling chromatin remodeling. Here, we hypothesized that multiple transcriptional regulators, including: (i) a primary Nrf2-Maf heterodimer, (ii) a proposed secondary, EpRE-specific, p160 family coactivator, ARE-bind ing protein-1, and (iii) a tertiary coactivator, CBP/p300, nucleate to form a complex at the EpRE that regulates transcription of the dependent gene. To test this hypothesis, we constructed a HepG2 cell line which contains a stably integrated green fluorescent protein (GFP) gene; its inducible expre ssion is regulated by a synthetic TK promoter containing a linked EpRE. To identify the involvement of specific, primary and higher-order transcriptio nal regulators in the EpRE-mediated regulation of the GFP reporter gene, we microinjected antibodies directed against specific transcription factors i nto the HepG2/GFP cells and determined their effect upon tBHQ-induced expre ssion of the GFP gene. The results demonstrate that microinjected antibodie s directed against Nrf2, MafK, CBP and p300 could each, individually, signi ficantly inhibit tBHQ-induced GFP expression. This directly demonstrates th e role that the tertiary regulators, CBP or p300, play in mediating EpRE ac tivation of phase Il genes, and also implicates the involvement of secondar y, p160 family coactivators. Moreover, we found that the same anti-MafK ant ibody that blocked induction of the EpRE-regulated GFP gene completely abla ted the gel-shift complex that we hypothesize contains an Nrf2:Maf dimer, A RE-binding protein-1, and CBP or p300. (C) 2001 Academic Press.