Functional characterization of transcription regulators that interact with the electrophile response element.
The electrophile response element (EpRE), also referred to as the antioxidant responsive element (ARE), is found in the 5'-regulatory region of a number of genes encoding phase II, drug-metabolizing enzymes. Gene knockout studies have demonstrated the primary regulatory role that an Nrf2:Maf dimer plays by binding to nucleotides within the EpRE consensus sequence. Current models of transcription regulation have also shown the involvement of higher-order transcriptional coactivators, proteins that nucleate around DNA sequence-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-binding 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 expression is regulated by a synthetic TK promoter containing a linked EpRE. To identify the involvement of specific, primary and higher-order transcriptional regulators in the EpRE-mediated regulation of the GFP reporter gene, we microinjected antibodies directed against specific transcription factors into the HepG2/GFP cells and determined their effect upon tBHQ-induced expression of the GFP gene. The results demonstrate that microinjected antibodies directed against Nrf2, MafK, CBP and p300 could each, individually, significantly inhibit tBHQ-induced GFP expression. This directly demonstrates the role that the tertiary regulators, CBP or p300, play in mediating EpRE activation of phase II genes, and also implicates the involvement of secondary, p160 family coactivators. Moreover, we found that the same anti-MafK antibody that blocked induction of the EpRE-regulated GFP gene completely ablated the gel-shift complex that we hypothesize contains an Nrf2:Maf dimer, ARE-binding protein-1, and CBP or p300.
Zhu
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McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706, USA.
Fahl
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D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D015870:Gene Expression / Q000187:drug effects; D005809:Genes, Regulator; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D051267:NF-E2-Related Factor 2; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051257:Proto-Oncogene Proteins c-maf; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D006873:Hydroquinones; D008164:Luminescent Proteins; C495607:MAF protein, human; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D011518:Proto-Oncogene Proteins; D051257:Proto-Oncogene Proteins c-maf; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; C018855:2-tert-butylhydroquinone