Identification of a putative antioxidant response element in the 5'-flanking region of the human gamma-glutamylcysteine synthetase heavy subunit gene

Abstract

We have cloned the human gamma-glutamylcysteine synthetase heavy subunit gene (GCSh) from a P1 library and isolated a 5.5kb fragment (P1-GCS5') from the 5'-end of the P1 clone. P1-GCS5' has been sequenced from -1460 to +547. Multiple transcription start sites were identified by primer extension and S1 nuclease protection. Two start sites were identified by primer extension analysis within 23 bp (+1 and +10) of a consensus TATAAAA box; all sequences were numbered relative to the 5'-most of these two sites. Two additional major start sites were identified 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 (approximately 75%). This sequence also contains several Sp-1 binding sites, a consensus AP-1 site and several AP-1-like binding sites, as well as putative AP-2 sites. A consensus metal responsive element (MRE) was identified at position +198. Sequence analysis also identified a putative core (5'-TGACnnnGCA-3') antioxidant response element (ARE) at -862 to -853. As is typical of other AREs, a second AP-1-like sequence is located adjacent to the core sequence. These results suggest that GCSh gene expression in response to oxidative challenge may be regulated through an antioxidant response element similar to those recently detected in the promoter region of several Phase II enzymes.