Triplex formation by the human Ha-ras promoter inhibits Sp1 binding and in vitro transcription.
The central role of the ras oncogenes in the pathogenesis of a wide variety of human malignancies is well established. Toward developing specific transcriptional inhibitors of the human Ha-ras oncogene, we have designed oligonucleotides to target a region of the Ha-ras promoter (-8 to -28) which contains two of the three Sp1 binding sites essential for transcriptional activity. Gel mobility analysis and DNase I footprinting demonstrate that an oligonucleotide (HR21ap) forms a sequence-specific triple helix with its target site in an antiparallel orientation with respect to the purine-rich duplex strand through predominantly G*G:C triplets. Within the Ha-ras promoter, HR21ap binds exclusively to the proximal target Sp1 sites over a similar nontarget distal sequence which, like the target, contains a consensus Sp1 site. Protein binding assays demonstrate that triplex formation by HR21ap inhibits Sp1 binding to the Ha-ras promoter. Moreover, oligonucleotide-directed triplex formation arrests Ha-ras promoter-dependent transcription in vitro. The results presented here suggest that triplex formation by the Ha-ras promoter targeted oligonucleotide may provide a means to specifically inhibit transcription of this oncogene in vivo.
Mayfield
C
C
Bolden Laboratory, Department of Medicine, University of Alabama at Birmingham 35294-3300.
Ebbinghaus
S
S
Gee
J
J
Jones
D
D
Rodu
B
B
Squibb
M
M
Miller
D
D
D001483:Base Sequence; D004247:DNA; D011905:Genes, ras; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic
D016329:Sp1 Transcription Factor; D004247:DNA