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

Biochem Pharmacol. 2002 May 1;63(9):1739-54. doi: 10.1016/s0006-2952(02)00897-3.


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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 5' Flanking Region / genetics
  • Animals
  • Antioxidants / pharmacology*
  • Base Sequence
  • Cadmium / pharmacology
  • DNA / analysis
  • Gene Expression Regulation / drug effects*
  • Genome
  • Glutamate-Cysteine Ligase / genetics*
  • Glutathione / metabolism
  • Hydrogen Peroxide / pharmacology
  • Hydroquinones / pharmacology*
  • Mice
  • Molecular Sequence Data
  • Oxidative Stress
  • Promoter Regions, Genetic / drug effects
  • Protein Subunits
  • RNA, Messenger / drug effects
  • RNA, Messenger / metabolism
  • Sequence Homology, Nucleic Acid
  • TATA Box
  • Transcription Initiation Site
  • Transcription, Genetic / drug effects
  • Tumor Cells, Cultured
  • Vitamin K 3 / pharmacology


  • Antioxidants
  • Hydroquinones
  • Protein Subunits
  • RNA, Messenger
  • Cadmium
  • Vitamin K 3
  • DNA
  • Hydrogen Peroxide
  • 2-tert-butylhydroquinone
  • Glutamate-Cysteine Ligase
  • Glutathione