DNA triplex formation selectively inhibits granulocyte-macrophage colony-stimulating factor gene expression in human T cells.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hemopoietic growth factor that is expressed in activated T cells, fibroblasts, macrophages, and endothelial cells. Although GM-CSF does not appear to be essential for normal hemopoiesis, overexpression of GM-CSF has been implicated in the pathogenesis of some diseases such as myeloid leukemia and chronic inflammation. An NF-kappaB/Rel binding site within the GM-CSF promoter, termed the kappaB element appears to be important for controlling expression in reporter gene assays in response to a number of stimuli in T cells. We investigated oligonucleotide-directed triple helix formation across this regulatory sequence as a potential tool to inhibit GM-CSF gene transcription. A 15-base oligonucleotide, GM3, was targeted to a purine-rich region in the GM-CSF proximal promoter, which overlaps the kappaB element. Gel mobility shift assays and DNase I footprinting demonstrated that GM3 formed a sequence-specific collinear triplex with its double-stranded DNA target. Triplex formation by GM3 blocked recombinant and nuclear NF-kappaB proteins binding to the GM-CSF element. GM3 also caused selective inhibition of the human T-cell lymphotrophic virus-1 Tax transactivator-induced luciferase activity from a reporter construct driven by the GM-CSF promoter in Jurkat T cells. Finally, GM3 greatly reduced the concentration of endogenous GM-CSF mRNA induced by different stimuli in Jurkat T cells but did not affect interleukin 3 mRNA levels in the same cells. We conclude that the kappaB element in the GM-CSF promoter plays a central role in the transcriptional activation of the endogenous GM-CSF gene. Colinear triplex formation acts as a selective transcriptional repressor of the GM-CSF gene and may have potential therapeutic application in cases of undesirable overexpression of this protein.
Kochetkova
M
M
Division of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide 5000, South Australia, Australia.
Shannon
M F
MF
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D003850:Deoxyribonuclease I; D016356:Gene Products, tax / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics; D015368:Human T-lymphotropic virus 1 / Q000235:genetics*; D006801:Humans; D007951:Leukemia, Myeloid; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D013601:T-Lymphocytes; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D016356:Gene Products, tax; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004247:DNA; D008156:Luciferases; D003850:Deoxyribonuclease I; D013755:Tetradecanoylphorbol Acetate