IDENTIFICATION OF A PUTATIVE ANTIOXIDANT RESPONSE ELEMENT IN THE 5'-FLANKING REGION OF THE HUMAN GAMMA-GLUTAMYLCYSTEINE SYNTHETASE HEAVY SUBUNIT GENE

We have cloned the human gamma-glutamylcysteine synthetase heavy subun it gene (GCS(h)) from a P1 library and isolated a 5.5kb fragment (P1-G CS5') from the 5'-end of the P1 clone. P1-GCS5' has been sequenced fro m -1460 to +547. Multiple transcription start sites were identified by primer extension and S1 nuclease protection. Two start sites were ide ntified by primer extension analysis within 23 bp (+1 and +10) of a co nsensus TATAAAA box; all sequences were numbered relative to the 5'-mo st of these two sites. Two additional major start sites were identifie d at -106 and +398. This latter site was the most prominent of all the initiation sites. In addition to a TATA box, the promoter contains a CCAAT box at -125 and GC boxes up- and down-stream of the TATAAAA. In addition, the first few hundred base pairs of the sequence are highly GC-rich(similar to 75%). This sequence also contains several Sp-l bind ing sites, a consensus AP-1 site and several AP-l-like binding sites, as well as putative AP-2 sites. A consensus metal responsive element ( MRE) was identified at position +198. Sequence analysis also identifie d a putative core (5'-TGACnnnGCA-3') antioxidant response element (ARE ) at -862 to -853. As is typical of other AREs, a second AP-l-like seq uence is located adjacent to the core sequence. These results suggest that GCS(h) gene expression in response to oxidative challenge may be regulated through an antioxidant response element similar to those rec ently detected in the promoter region of several Phase II enzymes. (Th e sequence of the 5'-flanking region of the human GCS heavy subunit ge ne has been assigned accession number L39773.) (C) 1995 Academic Press , Inc.