Glutamate-cysteine ligase modifier subunit: mouse Gclm gene structure and regulation by agents that cause oxidative stress.
Glutamate-cysteine ligase is a heterodimer comprising a modifier (GCLM) and a catalytic (GCLC) subunit. In mouse Hepa-1c1c7 hepatoma cell cultures, we found that tert-butylhydroquinone (tBHQ; 50 microM) induces the GCLM and GCLC mRNAs approximately 10- and approximately 2-fold, respectively, and that these increases primarily reflect de novo transcription. We determined that the mouse Gclm gene has seven exons, spanning 22.3 kb; all exons, intron-exon junctions, and 4.7 kb of 5'-flanking region were sequenced. By RNase protection analysis, we identified two major and several minor transcription start-site clusters over a 300-bp region. The Gclm 5'-flanking region is GC-rich and lacks a canonical TATA box. Transient and stable transfection studies, using luciferase reporter constructs containing incremental Gclm 5'-flanking deletions (4.7-0.5 kb), showed high basal activity but only modest ( approximately 2-fold) inducibility by tBHQ. The only candidate motif for oxidative stress regulation (in the 4.7-kb region we sequenced) is a putative inverted electrophile response element (EPRE) 9 bp upstream from the 5'-most transcription start-site. Site-directed mutagenesis of this -9 EPRE demonstrated minimal (30-40%) decreases in tBHQ induction and no effect on basal activity-suggesting that this EPRE might be necessary but not sufficient. The nuclear erythroid factor-2 (NEF2)-related factor-2 (NRF2) is known to transactivate via EPRE motifs. In the presence of co-transfected NRF cDNA expression vector, however, no increase in Gclm promoter activity was observed. Thus, the endogenous Gclm gene shows robust transcriptional activation by tBHQ in the intact Hepa-1 cell, but reporter constructs containing up to 4.7 kb of promoter (having only the one EPRE at -9) demonstrate a disappointing response, indicating that the major tBHQ-responsive regulatory element of the mouse Gclm gene must exist either further 5'- or 3'-ward of the 4.7-kb region studied.
Solis
Willy A
WA
Department of Environmental Health, Center for Environmental Genetics, University of Cincinnati Medical Center, Cincinnati, OH 45267-0056, USA.
Dalton
Timothy P
TP
Dieter
Matthew Z
MZ
Freshwater
Sarah
S
Harrer
Judy M
JM
He
Lei
L
Shertzer
Howard G
HG
Nebert
Daniel W
DW
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001483:Base Sequence; D002104:Cadmium / Q000494:pharmacology; D004247:DNA / Q000032:analysis; D005786:Gene Expression Regulation / Q000187:drug effects*; D016678:Genome; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D005978:Glutathione / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006873:Hydroquinones / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic / Q000187:drug effects; D021122:Protein Subunits; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured; D024483:Vitamin K 3 / Q000494:pharmacology
D000975:Antioxidants; D006873:Hydroquinones; D021122:Protein Subunits; D012333:RNA, Messenger; D002104:Cadmium; D024483:Vitamin K 3; D004247:DNA; D006861:Hydrogen Peroxide; C018855:2-tert-butylhydroquinone; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione