The tert-butylhydroquinone-mediated activation of the human thioredoxin gene reveals a novel promoter structure
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Thioredoxin is a redox-active protein that plays multiple roles in regulating cell growth, cell signalling and apoptosis. Here we have demonstrated that a complex mechanism involving multiple regulatory elements is involved in the tert-butylhydroquinone mediated activation of the thioredoxin gene. Luciferase assays, utilising various wild-type and mutated thioredoxin promoter fragments, revealed roles for the oxidative stress responsive element, antioxidant responsive element, three Sp1 binding sites and the TATA box in the activation of the thioredoxin gene by tert-butylhydroquinone. The oxidative stress responsive element required the presence of the antioxidant responsive element to elicit its response, whilst the independent removal of three Sp1 binding sites and the TATA box also decreased activation of the thioredoxin gene, with mutation of the TATA box having the greatest effect. Real-time RT-PCR analysis also revealed varying roles for two transcription start sites in the activation of the thioredoxin gene by tert-butylhydroquinone. Transcription was initiated from both transcription start sites, however different response rates and fold-inductions were observed. Together these results suggest that the thioredoxin gene is controlled by a novel arrangement of two overlapping core promoter regions, one containing a TATA box and the other TATA-less. Altering the intracellular levels of thioredoxin in a breast cancer cell line also influenced the induction of thioredoxin transcription in response to tert-butylhydroquinone. Stable transfections with a redox-inactive thioredoxin mutant produced 3.6 times higher induction levels of thioredoxin transcription compared to control cells, indicating an intrinsic form of control of promoter activity by the thioredoxin system itself.