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.

Targeting the c-Kit Promoter G-quadruplexes with 6-Substituted Indenoisoquinolines

“…7,8 Their unique structural features and possible biological functions make them attractive targets for drug design. 9,10 Genomic locations that are known to possess quadruplex structures include the immunoglobulin switch region and the promoters of numerous proto-oncogenes, such as c-MYC, 11 VEGF, 12 BCL-2, 13 and K-RAS. 14 Two quadruplex-forming motifs have been identified in the c-Kit promoter (c-Kit 1 15,16 and c-Kit 2 17,18 ).…”

Targeting G-Quadruplex DNA with Small Molecules

“…3. The equilibrium between duplex and G-quadruplex/i-motif structures can be influenced by DNA binding proteins, [7] small-molecule binding, [10][11][12] negative supercoiling, [12][13][14][15][16] changes in pH and temperature, [29] and molecular crowding. [30] plex-specific antibodies.…”

“…[6] The thermodynamic and kinetic barriers of duplex dissociation, a prerequisite for singlestranded DNA folding, might be off-set in part or in whole by single-stranded DNA binding proteins, [7] chaperones, [8,9] smallmolecule binding, [10][11][12] and/or negative supercoiling. [12][13][14][15][16] Polypurine-polypyrimidine tracts and other repetitive sequences can form nonduplex and/or higher-order chromatin structures possibly related to a wide variety of biological activities. One family of structures originating from guanosine-rich tracts is referred to as G-quadruplex (or G-tetraplex).…”

“…[22] But much faster rates of folding have been observed when G-quadruplex DNA was imbedded in a molecular matrix at temperatures slightly below the T m (k f ~1.0 s -1 ). [43] Molecular crowding, [44] synthetic and endogenous chaperones, [8][9][10][11][12] and dehydrating conditions inside the nucleus might also accelerate the rates of G-quadruplex formation in vivo. [22] Due to the self-complementary nature of duplex DNA, approximately 370'000 Crich motifs (C 3+ N 1-7 ) 4+ (where C = cytosine and N = any base) are present in the human genome.…”

Constrained bisantrene derivatives as G-quadruplex binders

“…G-quadruplex DNA structures play important roles in the indirect inhibition of telomerase in cancerous cells, [1][2][3][4] in the regulation of the oncogenetic expression [5][6][7][8][9] and in viral functions. [10][11][12] The action of a small molecule directed towards these targets could result in contrasting cancerous cells immortalization or modifying the expression of the related genes by stabilizing this peculiar DNA arrangement.…”