Triple-helix formation interferes with the transcription and hinged DNA structure of the interferon-inducible 6-16 gene promoter.
The interferon responsive element (IRE) of the 6-16 gene lies within two 39-bp elements in tandem. A purine-rich oligodeoxynucleotide, oligo(dN), was found to be able to pair with the purine-rich strand of the IRE in an antiparallel orientation which led to triple-helix formation with Mg2+ being necessary for triplex stability. Footprinting analysis confirmed these results. The interaction between the IRE and the oligo(dN) was reversible and had a Kd equal to 20 nM. The two repeats of the 6-16 gene IRE can form a hinged DNA structure through pairing of their purine-rich regions; exonuclease III experiments support this model. The hybrid DNA structure leads to a parallel pairing of the purine strands of the 6-16 gene IRE and this conformation was shown to be destabilized by triplex formation. When co-transfected with a reporter gene whose promoter was under the control of the 6-16 gene IRE, the triple-helix-forming oligo(dN)s inhibit the interferon-induced stimulation of the reporter gene with complete inhibition being obtained with 1 microM oligo(dN) at the time of transfection. When added to the cell culture medium after transfection, the concentrations of oligo(dN) needed to obtain 50% inhibition of the interferon effect on gene transcription must be 50-100 times higher. Besides the existence of a peculiar structure for the 6-16 gene IRE, the possibility of interfering with gene expression by means of oligo(dN)s is demonstrated.
Roy
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Institut de Génétique Moléculaire, UMR 9942, C. N. R. S., Montpellier, France.
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D005090:Exodeoxyribonucleases; D005786:Gene Expression Regulation / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D007372:Interferons / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013463:Sulfuric Acid Esters; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D013463:Sulfuric Acid Esters; D004247:DNA; D007372:Interferons; D015500:Chloramphenicol O-Acetyltransferase; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; C007482:dimethyl sulfate