Molecular cloning of the human platelet-derived growth factor receptor beta (PDGFR-beta) promoter and drug targeting of the G-quadruplex-forming region to repress PDGFR-beta expression.
To understand the mechanisms controlling platelet-derived growth factor receptor beta (PDGFR-beta) expression in malignancies, we have cloned and characterized the first functional promoter of the human PDGFR-beta gene, which has been confirmed by luciferase reporter gene assays. The transcription initiation sites were mapped by primer extension. Promoter deletion experiments demonstrate that the proximal, highly GC-rich region (positions -165 to -139) of the human PDGFR-beta promoter is crucial for basal promoter activity. This region is sensitive to S1 nuclease and likely to assume a non-B-form DNA secondary structure within the supercoiled plasmid. The G-rich strand in this region contains a series of runs of three or more guanines that can form multiple different G-quadruplex structures, which have been subsequently assessed by circular dichroism. A Taq polymerase stop assay has shown that three different G-quadruplex-interactive drugs can each selectively stabilize different G-quadruplex structures of the human PDGFR-beta promoter. However, in transfection experiments, only telomestatin significantly reduced the human PDGFR-beta basal promoter activity relative to the control. Furthermore, the PDGFR-beta mRNA level in Daoy cells was significantly decreased after treatment with 1 muM telomestatin for 24 h. Therefore, we propose that ligand-mediated stabilization of specific G-quadruplex structures in the human PDGFR-beta promoter can modulate its transcription.
Qin
Yong
Y
College of Pharmacy, 1703 East Mabel, University of Arizona, Tucson, Arizona 85721, USA.
Fortin
Jessica S
JS
Tye
Denise
D
Gleason-Guzman
Mary
M
Brooks
Tracy A
TA
Hurley
Laurence H
LH
D001483:Base Sequence; D002478:Cells, Cultured; D002942:Circular Dichroism; D003001:Cloning, Molecular; D054856:G-Quadruplexes / Q000187:drug effects; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000235:genetics* / Q000378:metabolism
D008024:Ligands; D012333:RNA, Messenger; D020797:Receptor, Platelet-Derived Growth Factor beta