Transcription of the human c-Src promoter is dependent on Sp1, a novel pyrimidine binding factor SPy, and can be inhibited by triplex-forming oligonucleotides.
The tyrosine kinase pp60(c-src) has been implicated in the regulation of numerous normal physiological processes as well the development of several human cancers. However, the mechanisms regulating its expression have not been addressed. In the present study, we report the presence of two Sp1/Sp3 binding sites and three polypurine:polypyrimidine (Pu:Py) tracts in the c-Src promoter that are essential for controlling expression. We demonstrate that Sp1, but not Sp3, is capable of activating the c-Src promoter and that Sp3 is also capable of inhibiting Sp1-mediated transactivation. The presence of multiple Pu:Py tracts conferred S1 sensitivity on plasmids in vitro, suggesting they are capable of adopting non B-DNA conformations. These tracts specifically bind a nuclear factor we named SPy (Src pyrimidine binding factor), which demonstrates both novel double- and single-stranded binding specificities. Mutations eliminating SPy binding compromised Src transcriptional activity, especially in concert with additional mutations affecting Sp1 binding, suggesting the two factors may cooperate in regulating c-Src expression. Finally, we demonstrate that triplex-forming oligonucleotides designed to target both Sp1 and SPy binding sites can down-regulate c-Src expression in vitro, suggesting a potential therapeutic approach to controlling c-Src expression in diseases where aberrant expression or activity has been documented.
Ritchie
S
S
Saskatoon Cancer Center Research Unit, Saskatchewan Cancer Agency. Division of Oncology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 4H4, Canada.
Boyd
F M
FM
Wong
J
J
Bonham
K
K
D000230:Adenocarcinoma; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003110:Colonic Neoplasms; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D016391:Genes, src; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D016392:Proto-Oncogene Proteins pp60(c-src)