Endogenous GATA factors bind the core sequence of the tetO and influence gene regulation with the tetracycline system.
The tetracycline-regulated eukaryotic gene expression systems have been applied in numerous areas of bioscience. The systems utilize a tetracycline-responsive promoter (P(tet)) and synthetic transactivators (tTA or rtTA) that bind to the promoter in the presence or absence of doxycycline, regulating gene expression. Both the basal activity of the P(tet) and the magnitude of regulation by the system vary between cell types. In this investigation we have mapped the positions of endogenous transcription factor binding sites within the P(tet) and through deletion studies determined the portion of the promoter that contributes to basal activity. The tetracycline operator (tetO) repeats appear to be the source of basal activity and they were shown to harbor motifs for GATA transcription factors. The GATA motif is located within the central core of the tetO and so has the potential to compete with tTA and rtTA binding. The molecular interactions of endogenous and overexpressed GATA factors with the GATA motif in the tetO were demonstrated and effects on function of the tetracycline-regulated gene expression system investigated. GATA factors are widespread in embryonic tissues, are expressed within several adult cell types, and display altered expression in disease states. We suggest that endogenous GATA factor expression may influence the degree of gene regulation by the tetracycline system between different cell types. The findings of this study may have implications for the application of the tetracycline system in gene therapy.
Gould
David J
DJ
Bone & Joint Research Unit, Barts and The London, Queen Mary's School of Medicine and Dentistry, London University, Charterhouse Square, London EC1M 6BQ, UK. d.j.gould@qmul.ac.uk
Chernajovsky
Yuti
Y
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009875:Operator Regions, Genetic / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D014157:Transcription Factors; D008156:Luciferases; D013752:Tetracycline