Molecular Cloning of the Human Platelet-Derived Growth Factor Receptor β (PDGFR-β) Promoter and Drug Targeting of the G-Quadruplex-Forming Region To Repress PDGFR-β Expression

摘要

To understand the mechanisms controlling platelet-derived growth factor receptor β (PDGFR-β)nexpression in malignancies, we have cloned and characterized the first functional promoter of the humannPDGFR-β gene, which has been confirmed by luciferase reporter gene assays. The transcription initiationnsites were mapped by primer extension. Promoter deletion experiments demonstrate that the proximal, highlynGC-rich region (positions -165 to -139) of the human PDGFR-β promoter is crucial for basal promoternactivity. This region is sensitive to S1 nuclease and likely to assume a non-B-form DNA secondary structurenwithin the supercoiled plasmid. The G-rich strand in this region contains a series of runs of three or morenguanines that can formmultiple differentG-quadruplex structures, which have been subsequently assessed byncircular dichroism. A Taq polymerase stop assay has shown that three different G-quadruplex-interactivendrugs can each selectively stabilize different G-quadruplex structures of the human PDGFR-β promoter.nHowever, in transfection experiments, only telomestatin significantly reduced the human PDGFR-β basalnpromoter activity relative to the control. Furthermore, the PDGFR-β mRNA level in Daoy cells wasnsignificantly decreased after treatment with 1 μM telomestatin for 24 h. Therefore, we propose that ligand-nmediated stabilization of specific G-quadruplex structures in the human PDGFR-β promoter can modulatenits transcription.