Inhibition of in vitro transcription by a triplex-forming oligonucleotide targeted to human c-myc P2 promoter.
Triplex-forming oligonucleotides (TFOs) have been shown to bind in a sequence-specific manner to polypurine/polypyrimidine sequences in several human gene promoters, including the c-myc P1 promoter. TFOs have been shown to inhibit transcription in vitro and the expression of target genes in cell culture. The human c-myc protooncogene contains a 23 base pair purine-pyrimidine-rich motif (-62 to -40) within its predominant promoter, P2, that is a potential target for purine-purine-pyrimidine triplex formation. Using electrophoretic mobility shift analysis (EMSA) and competition experiments, we have demonstrated that a MAZ (myc-associated zinc finger protein) consensus sequence is capable of competing with the purine-pyrimidine motif for the binding of a HeLa nuclear protein. We have shown the formation of an intermolecular triplex using a 23-base purine-rich oligonucleotide antiparallel to the purine-rich target sequence. DNase I footprinting was performed to confirm the exact location of triplex formation. Triplex formation by this oligonucleotide prevents binding of a HeLa nuclear protein (presumably MAZ) to the target site. We have also shown that the P2-targeted TFO is a potent and specific inhibitor of c-myc transcription in vitro. These data demonstrate that this novel TFO inhibits transcription of the c-myc P2 promoter. We propose that the P2-targeted TFO has its effect by blocking the binding of the regulatory factor MAZ.
Kim
H G
HG
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham 35294-0001, USA.
Miller
D M
DM
D001483:Base Sequence; D001667:Binding, Competitive; D002467:Cell Nucleus; D016384:Consensus Sequence; D003850:Deoxyribonuclease I; D016259:Genes, myc; D006367:HeLa Cells / Q000648:ultrastructure; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000187:drug effects*; D016335:Zinc Fingers
D009841:Oligonucleotides; D003850:Deoxyribonuclease I