Cloning and characterization of the human glutathione synthetase 5'-flanking region.
GSH synthesis occurs through a two-step enzymatic reaction driven by GCL (glutamate-cysteine ligase; made up of catalytic and modifying subunits) and GSS (glutathione synthetase). In humans, oxidative stress regulates GCL expression in an antioxidant response element-dependent manner via Nrf2 [NFE (nuclear factor erythroid)-related factor 2]. In the rat, GSS and GCL are regulated co-ordinately by oxidative stress, and induction of GSS further increases GSH synthetic capacity. Transcriptional regulation of the human GSS has not been examined. To address this, we have cloned and characterized a 2.2 kb 5'-flanking region of the human GSS. The transcriptional start site is located 80 nt upstream of the translation start site. The human GSS promoter efficiently drove luciferase expression in Chang cells. Overexpression of either Nrf1 or Nrf2 induced the GSS promoter activity by 130 and 168% respectively. Two regions homologous to the NFE2 motif are demonstrated to be important for basal expression of human GSS, as mutation of these sites reduced the promoter activity by 66%. Nrf1, Nrf2 and c-Jun binding to these NFE2 sites under basal conditions was demonstrated using chromatin immunoprecipitation assays. In summary, two NFE2 sites in the human GSS promoter play important roles in the basal expression of GSS and, similar to the GCL subunits, the human GSS gene expression is also regulated by Nrf2.
Lee
Taunia D
TD
Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, Department of Medicine, Keck School of Medicine USC, 2011 Zonal Ave., Los Angeles, CA 90033, USA.
Yang
Heping
H
Whang
Janet
J
Lu
Shelly C
SC
D024506:5' Flanking Region / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D005981:Glutathione Synthase / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051267:NF-E2-Related Factor 2 / Q000235:genetics; D051266:Nuclear Respiratory Factor 1 / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D024363:Transcription Initiation Site
D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; C495633:NRF1 protein, human; D051266:Nuclear Respiratory Factor 1; D005981:Glutathione Synthase