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.
Triple-helix formation interferes with the transcription and hinged DNA structure of the interferon-inducible 6-16 gene promoter
Eur J Biochem
1994-03-01 / vol 220 / pages 493-503
Humans; Molecular Sequence Data; Protein Binding; Base Sequence; Kinetics; Transfection; *Nucleic Acid Conformation; Hela Cells; Cloning, Molecular; *Promoter Regions (Genetics); Chloramphenicol O-Acetyltransferase/biosynthesis/metabolism; DNA/*chemistry/drug effects/genetics; Exodeoxyribonucleases; Gene Expression Regulation/*drug effects; Interferons/*pharmacology; Oligodeoxyribonucleotides/chemical synthesis/chemistry/*pharmacology; Plasmids; Sulfuric Acid Esters; Transcription, Genetic/*drug effects