Identification of cis-acting DNA elements of the human gamma-glutamylcysteine synthetase heavy subunit gene

Transcriptional activity of the 5'-flanking sequence of the human gamma-glutamylcysteine synthetase heavy subunit (gamma-GCS(h)) gene was investigated in COS7 cells transfected with hGH reporter constructs having successively deleted 5'-flanking sequence of the gamma-GCS(h) gene. Transcriptional activity was determined by the amounts of hGH secreted from She reporter constructs, Deletion of the sequence from -1,413 to -664 or -315 base pairs (bp) increased transcriptional activity from 100 to 138 or 136%. Further deletion from -315 to -241 bp, which contained all AP1 site, decreased transcriptional activity to 87%. Mutations introduced into the AP1 decreased transcriptional activity from 136 to 105%. These findings suggested that the AP1 increased transcriptional activity. When the sequence from -241 to -192 bp was deleted, transcriptional activity was restored from 87 to 128%. When this sequence was linked to the thymidine kinase promoter, it also decreased transcriptional activity by 38%. Deletion from -192 to -149, -116, or -108 bp did not significantly alter transcriptional activity. Further deletion of the GC-rich sequences from -108 to -70 and -28 bp dramatically decreased transcriptional activity from 135 to 87 and 34%, respectively. These findings indicate that multiple DNA elements, especially those in the proximal GC-rich sequences, are involved in the regulation of transcriptional activity of the gamma-GCS(h) gene. (C) 1997 Academic Press.