Glutamate–cysteine ligase modifier subunit: mouse Gclm gene structure and regulation by agents that cause oxidative stress

Abstract

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; 50μM) induces the GCLM and GCLC mRNAs ∼10- and ∼2-fold, respectively, and that these increases primarily reflect de novo transcription. We determined that the mouse Gclm gene has seven exons, spanning 22.3kb; all exons, intron–exon junctions, and 4.7kb 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 (4.7–0.5kb), showed high basal activity but only modest (∼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) 9bp 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 4.7kb 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.