Up-regulation of the human gamma-glutamylcysteine synthetase regulatory subunit gene involves binding of Nrf-2 to an electrophile responsive element.
The rate-limiting step in the de novo synthesis of the cellular protectant glutathione is catalyzed by gamma-glutamylcysteine synthetase (GCS; also known as glutamine-L-cysteine ligase, GLCL), a heterodimer consisting of catalytic (GCS(h)) and regulatory (GCS(l)) subunits. Regulation of expression of the human gamma-glutamylcysteine synthetase regulatory subunit gene in response to beta-NF is mediated by an Electrophile Responsive Element (EpRE) [Moinova, H., and Mulcahy, R. T. (1998) J. Biol. Chem. 273, 14683-14689]. Oligonucleotide probes corresponding to wild-type and mutant EpRE sequences were used in gel-shift and super-shift analyses to identify proteins binding. Four protein:DNA complexes (a-d) with distinct mobilities were detected when the wild-type EpRE probe was incubated with nuclear extracts from control or beta-NF-treated HepG2 cells. Following beta-NF treatment, there was an increase in the intensity of a single band, band b. This band was eliminated in gel shifts employing mutant EpRE probes which abolish beta-NF inducibility, demonstrating a correlation between band b and transactivation. Super-shift analysis identified JunD, Nrf1, and Nrf2 in the EpRE-binding complexes. Antibodies to Nrf2 completely super-shifted the band b protein:DNA complex. These studies demonstrate that Nrf2 proteins recognize and bind the GCS(l) EpRE sequence to affect transactivation of the gene.
Moinova
H R
HR
Department of Human Oncology, University of Wisconsin Medical School, 600 Highland Avenue, K4/316 CSC, Madison, Wisconsin, 53792, USA.
Mulcahy
R T
RT
D001483:Base Sequence; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D051268:GA-Binding Protein Transcription Factor; D015971:Gene Expression Regulation, Enzymologic; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms; D009154:Mutation; D012045:Regulatory Sequences, Nucleic Acid; D020218:Response Elements; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D019324:beta-Naphthoflavone / Q000494:pharmacology
D004268:DNA-Binding Proteins; D051268:GA-Binding Protein Transcription Factor; D014157:Transcription Factors; D019324:beta-Naphthoflavone; D004247:DNA; D005721:Glutamate-Cysteine Ligase