Characterization of the G-quadruplexes in the duplex nuclease hypersensitive element of the PDGF-A promoter and modulation of PDGF-A promoter activity by TMPyP4.
The proximal 5'-flanking region of the human platelet-derived growth factor A (PDGF-A) promoter contains one nuclease hypersensitive element (NHE) that is critical for PDGF-A gene transcription. On the basis of circular dichroism (CD) and electrophoretic mobility shift assay (EMSA), we have shown that the guanine-rich (G-rich) strand of the DNA in this region can form stable intramolecular parallel G-quadruplexes under physiological conditions. A Taq polymerase stop assay has shown that the G-rich strand of the NHE can form two major G-quadruplex structures, which are in dynamic equilibrium and differentially stabilized by three G-quadruplex-interactive drugs. One major parallel G-quadruplex structure of the G-rich strand DNA of NHE was identified by CD and dimethyl sulfate (DMS) footprinting. Surprisingly, CD spectroscopy shows a stable parallel G-quadruplex structure formed within the duplex DNA of the NHE at temperatures up to 100 degrees C. This structure has been characterized by DMS footprinting in the double-stranded DNA of the NHE. In transfection experiments, 10 microM TMPyP4 reduced the activity of the basal promoter of PDGF-A approximately 40%, relative to the control. On the basis of these results, we have established that ligand-mediated stabilization of G-quadruplex structures within the PDGF-A NHE can silence PDGF-A expression.
Qin
Yong
Y
College of Pharmacy, 1703 E. Mabel, University of Arizona, Tucson, Arizona 85721, USA.
Rezler
Evonne M
EM
Gokhale
Vijay
V
Sun
Daekyu
D
Hurley
Laurence H
LH
D045744:Cell Line, Tumor; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D018983:DNA Footprinting; D054856:G-Quadruplexes / Q000187:drug effects; D006147:Guanine / Q000737:chemistry; D006801:Humans; D008024:Ligands; D009154:Mutation; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D011166:Porphyrins / Q000737:chemistry / Q000494:pharmacology*; D011188:Potassium / Q000737:chemistry; D011401:Promoter Regions, Genetic; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*
D008024:Ligands; D010982:Platelet-Derived Growth Factor; D011166:Porphyrins; C075278:platelet-derived growth factor A; C021096:tetra(4-N-methylpyridyl)porphine; D006147:Guanine; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases; D011188:Potassium