Cloning and analysis of the rat glutamate-cysteine ligase modifier subunitpromoter

Glutamate-cysteine ligase (GCL), the rate-limiting enzyme in glutathione (G SH) synthesis, is made up of two subunits, a catalytic (GCLC) and a modifie r (GCLM) subunit, which are differentially regulated. Increased GCLM expres sion occurs under certain oxidative stress conditions. To facilitate studie s of GCLM transcriptional regulation, we have cloned and characterized a 1. 86-kb 5 ' -flanking region of the rat GCLM (GenBank Accession No. AF311745) . A TATA-like element and one transcriptional start sites are located at 36 4 and 93 nucleotides upstream of the translational start site, respectively . The promoter contains consensus binding sites for many transcription fact ors including activator protein 1 (AP-1), transcription factor 11 (TCF11), heat shock transcription factor (HSF), and nuclear factor kappa B (NF kappa B). The rat GCLM promoter was able to drive efficiently luciferase expressi on in H4IIE cells. Sequential deletion analysis revealed DNA regions, -649 to -154 and -1251 to -649, are involved in positive and negative gene regul ation, respectively. Candidate transcription factors were identified by DNa se I footprinting. (C) 2001 Academic Press.