title: Identification of a variant antioxidant response element in the promoter of the human glutamate-cysteine ligase modifier subunit gene. Revision of the ARE consensus sequence.
abstract: Constitutive and inducible expression of the gene encoding the modulator subunit of human glutamate-cysteine ligase (GCLM) is regulated by either of two regions of the promoter; an antioxidant response element (ARE) at -302:-291 and a 44-bp fragment (-346:-303) upstream of the ARE. This second region includes a consensus AP-1 site previously considered responsible for the enhancer activity of the upstream fragment. Deletion of a 165-bp fragment (-348:-183) including the ARE and upstream 44-bp fragment totally ablated t-butyl hydroquinone (tBHQ) inducibility of a GCLM promoter/luciferase transgene. Mutation analyses confirmed that both the ARE and the -346:-303 fragment could support induction following tBHQ exposure but demonstrated that induction in the latter case did not involve the AP-1 site at -341:-335. A region sharing significant homology with the consensus ARE sequence except for a single nucleotide mismatch at -330 (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') was identified at the 5'-end of the 44-bp fragment immediately adjacent to the AP-1 site. A G in this position has been considered an invariant requirement of functional ARE sequences. Mutation of T(-330) to A (a substitution known to ablate ARE function) or C eliminated basal and inducible expression. Substitution of a G at -330 enhanced basal expression relative to the wild-type sequence, but induction following tBHQ exposure was comparable, indicating that either sequence (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') may function as an ARE, although the former sequence is less effective at directing basal expression. This possibility was confirmed by similar mutational analyses of the core sequence of hNQO1, a prototypic ARE. Electrophoretic mobility shift competition assays revealed that the 5'-TTACnnnGCA-3' sequence could compete with the hNQO1 ARE for protein binding but was less effective than a similar probe containing the 5'-TGACnnnGCA-3' motif. Probes including the T(-330)A or T(-330)C mutations were ineffective. These results reveal that the GCLM promoter includes two functional AREs, one having a variant sequence. The results indicate that the consensus ARE sequence should be revised to 5'-RTKAYnnnGCR-3'.
authors.affiliation: Department of Pharmacology, University of Wisconsin Medical School, 600 Highland Avenue, Madison, WI 53792.
authors.forename: Aileen M
authors.initials: AM
authors.lastname: Erickson
authors.forename: Zulimar
authors.initials: Z
authors.lastname: Nevarea
authors.forename: Jerry J
authors.initials: JJ
authors.lastname: Gipp
authors.forename: R Timothy
authors.initials: RT
authors.lastname: Mulcahy
mesh_terms: D000595:Amino Acid Sequence; D000975:Antioxidants / Q000494:pharmacology*; D016384:Consensus Sequence; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D021122:Protein Subunits; D020218:Response Elements; D018808:Transcription Factor AP-1 / Q000737:chemistry
chemical_list: D000975:Antioxidants; D006873:Hydroquinones; D021122:Protein Subunits; D018808:Transcription Factor AP-1; C018855:2-tert-butylhydroquinone; D005721:Glutamate-Cysteine Ligase