Thioredoxin-mediated redox control of the transcription factor Sp1 and regulation of the thioredoxin gene promoter.
In recent years, redox control has emerged as a fundamental mechanism of gene regulation through transcriptional control. Thioredoxin (Trx) is a dithiol-reducing enzyme known to be involved in the redox regulation of a number of transcription factors, and in this study, we have investigated the redox-dependent regulation of the DNA binding activity of Sp1 by thioredoxin. Electrophoretic mobility shift assays were used to show that both recombinant Sp1 produced in Escherichia coli and endogenous Sp1 expressed by MDA-MB-231 breast cancer cells is subject to redox regulation. We found that thioredoxin alone or in conjunction with the full thioredoxin system (comprising thioredoxin, thioredoxin reductase [TR], and alpha-nicotinamide adenine dinucleotide phosphate [NADPH]) can increase Sp1 DNA binding activity in vitro to an oligonucleotide containing the Sp1 consensus sequence. Furthermore, we have provided evidence that recombinant Sp1 can bind to Sp1 consensus sequences within a 330-base pair (bp) thioredoxin promoter fragment and that this interaction can also be enhanced by the presence of thioredoxin. Luciferase reporter assays using this same minimal thioredoxin promoter region demonstrated that both Sp1 and Sp3 can bind to the promoter and act to enhance transcription. When the three identified Sp1 consensus sequences within the reporter construct were deleted, there was a loss of basal promoter activity, showing that these closely positioned sites are important for regulation of thioredoxin gene expression.
Bloomfield
Kelly L
KL
School of Biomolecular and Biomedical Science, Griffith University, Nathan, Qld, 4111, Australia. K.Tonissen@griffith.edu.au
Osborne
Simone A
SA
Kennedy
Derek D
DD
Clarke
Frank M
FM
Tonissen
Kathryn F
KF
D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
D009841:Oligonucleotides; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D013879:Thioredoxins