Differential regulation of gene expression in vivo by triple helix-forming oligonucleotides as detected by a reporter enzyme.
In an attempt to assay the capability of various oligonucleotides to inhibit gene transcription in vivo through triplex formation, we developed a cellular system employing transfection of a reporter plasmid and putative triplex-forming oligonucleotides targeted to Sp1-binding sites contained within the SV40 early promoter. Using this approach, we demonstrated that the activity of the reporter enzyme, alkaline phosphatase, was highly dependent on the sequence of the oligonucleotides: oligonucleotides utilizing G:GC triplets, but not C:GC triplets, promoted a dose-dependent decrease in reporter enzyme activity. Evidence of physical interaction between Sp1-binding sites within the SV40 promoter and sequence-specific G-rich oligonucleotides has been demonstrated, suggesting triple-helix formation as the most probable explanation for the inhibitory effect on alkaline phosphatase activity observed for these oligonucleotides. Surprisingly, Southern analysis of isolated nuclear DNA indicates that the differences in alkaline phosphatase activity associated with transfection of the different oligonucleotides appear to correlate with internalized plasmid DNA copy number rather than inhibition of transcription. It is intriguing to postulate the existence of a nuclease that is able to recognize and cleave triple-helical DNA structures. This hypothesis implies the existence of a novel mechanism of gene regulation specific for triplex structures and, presumably, independent of transcription.
Hobbs
C A
CA
Department of Molecular Biology, Sterling Winthrop Pharmaceuticals Research Division, Collegeville, Pennsylvania 19426.
Yoon
K
K
D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D016466:CHO Cells; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006224:Cricetinae; D004247:DNA / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D016329:Sp1 Transcription Factor; C051802:triplex DNA; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D000469:Alkaline Phosphatase