title: Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents.
abstract: The proximal promoter region of the human vascular endothelial growth factor (VEGF) gene contains a polypurine/polypyrimidine tract that serves as a multiple binding site for Sp1 and Egr-1 transcription factors. This tract contains a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif for the formation of an intramolecular G-quadruplex. In this study, we observed the progressive unwinding of the oligomer duplex DNA containing this region into single-stranded forms in the presence of KCl and the G-quadruplex-interactive agents TMPyP4 and telomestatin, suggesting the dynamic nature of this tract under conditions which favor the formation of the G-quadruplex structures. Subsequent footprinting studies with DNase I and S1 nucleases using a supercoiled plasmid DNA containing the human VEGF promoter region also revealed a long protected region, including the guanine-rich sequences, in the presence of KCl and telomestatin. Significantly, a striking hypersensitivity to both nucleases was observed at the 3'-side residue of the predicted G-quadruplex-forming region in the presence of KCl and telomestatin, indicating altered conformation of the human VEGF proximal promoter region surrounding the guanine-rich sequence. In contrast, when specific point mutations were introduced into specific guanine residues within the G-quadruplex-forming region (Sp1 binding sites) to abolish G-quadruplex-forming ability, the reactivity of both nucleases toward the mutated human VEGF proximal promoter region was almost identical, even in the presence of telomestatin with KCl. This comparison of wild-type and mutant sequences strongly suggests that the formation of highly organized secondary structures such as G-quadruplexes within the G-rich region of the human VEGF promoter region is responsible for observed changes in the reactivity of both nucleases within the polypurine/polypyrimidine tract of the human VEGF gene. The formation of the G-quadruplex structures from this G-rich sequence in the human VEGF promoter is further confirmed by the CD experiments. Collectively, our results provide strong evidence that specific G-quadruplex structures can naturally be formed by the G-rich sequence within the polypurine/polypyrimidine tract of the human VEGF promoter region, raising the possibility that the transcriptional control of the VEGF gene can be modulated by G-quadruplex-interactive agents.
authors.affiliation: College of Pharmacy, University of Arizona, Tucson, AZ 85721, USA. sun@pharmacy.arizona.edu
authors.forename: Daekyu
authors.initials: D
authors.lastname: Sun
authors.forename: Kexiao
authors.initials: K
authors.lastname: Guo
authors.forename: Jadrian J
authors.initials: JJ
authors.lastname: Rusche
authors.forename: Laurence H
authors.initials: LH
authors.lastname: Hurley
mesh_terms: D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000187:drug effects; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D054856:G-Quadruplexes; D006147:Guanine / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010080:Oxazoles / Q000494:pharmacology; D011089:Polydeoxyribonucleotides / Q000737:chemistry; D011166:Porphyrins / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011687:Purines / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
chemical_list: D010080:Oxazoles; D011089:Polydeoxyribonucleotides; D011166:Porphyrins; D011687:Purines; D011743:Pyrimidines; D042461:Vascular Endothelial Growth Factor A; C431888:telomestatin; C021096:tetra(4-N-methylpyridyl)porphine; D006147:Guanine; D011189:Potassium Chloride; D004247:DNA; D003850:Deoxyribonuclease I; D015719:Single-Strand Specific DNA and RNA Endonucleases