Cloning and analysis of the rat glutamate-cysteine ligase modifier subunit promoter.
Glutamate-cysteine ligase (GCL), the rate-limiting enzyme in glutathione (GSH) synthesis, is made up of two subunits, a catalytic (GCLC) and a modifier (GCLM) subunit, which are differentially regulated. Increased GCLM expression occurs under certain oxidative stress conditions. To facilitate studies 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 364 and 93 nucleotides upstream of the translational start site, respectively. The promoter contains consensus binding sites for many transcription factors including activator protein 1 (AP-1), transcription factor 11 (TCF11), heat shock transcription factor (HSF), and nuclear factor kappa B (NFkappaB). The rat GCLM promoter was able to drive efficiently luciferase expression in H4IIE cells. Sequential deletion analysis revealed DNA regions, -649 to -154 and -1251 to -649, are involved in positive and negative gene regulation, respectively. Candidate transcription factors were identified by DNase I footprinting.
Yang
H
H
Division of Gastroenterology and Liver Diseases, USC Liver Disease Research Center, University of Southern California, Los Angeles, CA 90033, USA.
Wang
J
J
Ou
X
X
Huang
Z Z
ZZ
Lu
S C
SC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D018983:DNA Footprinting; D003850:Deoxyribonuclease I; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D005978:Glutathione / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D021122:Protein Subunits; D051381:Rats; D016385:TATA Box; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D021122:Protein Subunits; D014157:Transcription Factors; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione