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 - PubMed (original) (raw)

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

Daekyu Sun et al. Nucleic Acids Res. 2005.

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.

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Figures

Figure 1

Polypurine/polypyrimidine sequence located upstream (−89 to −43) of the promoter region of the VEGF gene. Runs of guanines (GR-I through GR-V) are boxed. Binding sites of the transcriptional factors Egr-1 and Sp1 are underlined.

Figure 2

(A) H-bonding pattern in a G-tetrad, (B) schematic diagram of the G-tetrad and (C) cartoon of an 18mer parallel G-quadruplex representing that found in the c-MYC promoter region.

Figure 3

The effect of increasing TMPyP4 and telomestatin concentrations on the conversion of 59mer duplex DNA (59WT) to alternative secondary structures. (A) Nucleotide sequence of 59WT. (B) Structures of TMPyP4 and telomestatin. (C) Autoradiogram of DNA breathing assay. Duplex DNA was titrated with TMPyP4 or telomestatin in 100 mM KCl/TE buffer at 37 or 42°C. Two bands corresponding to Watson–Crick duplex (DS) and single-stranded DNA (S) were identified, along with an isomeric form. (D) Graphical representation of data described in (C).

Figure 4

In vitro footprinting of the VEGF promoter region with DNase I. (A) Autoradiograms showing DNase I cleavage sites on the top strand of a supercoiled pGL3-VEGF plasmid. The plasmid DNA was incubated in the absence of salt (lane 1), or in the presence of 100 mM KCl without (lane 2) and with (lane 3) 1 µM telomestatin at 37°C for 1 h before digesting with DNase I. DNase I cleavage sites were mapped using linear amplification by PCR with 32P-labeled gene-specific plasmid DNA pretreated with DNase I. Arrows A and B indicate the hypersensitive cleavage sites to nucleases. (B) Densitometric scanning of the autoradiogram in (A). The bars indicate the guanine repeats involved in the formation of the G-quadruplex structures. Arrows A and B indicate the hypersensitive cleavage sites to nucleases. (C) Autoradiograms showing DNase I cleavage sites on the bottom strand of a supercoiled pGL3-VEGF plasmid. The designation of lanes 1–3 was as in (A) above. DNase I cleavage sites were mapped using linear amplification by PCR with 32P-labeled gene-specific plasmid DNA pretreated with DNase I. The vertical bar next to the gel indicates the polypyrimidine tract.

Figure 5

In vitro footprinting of the VEGF promoter region with S1 nuclease. (A) Autoradiograms showing S1 nuclease cleavage sites on the top strand of a supercoiled pGL3-VEGF plasmid. Arrow A indicates the hypersensitive cleavage sites to S1 nuclease. (B) Densitometric scanning of the autoradiogram in (A). The plasmid DNA was incubated in the absence of salt (lane 1) or in the presence of 100 mM KCl without (lane 2) and with (lane 3) 1 µM telomestatin at 37°C for 1 h before digesting with S1 nuclease. S1 nuclease cleavage sites were mapped using linear amplification by PCR with 32P-labeled gene-specific primers on plasmid DNA pretreated with S1 nuclease. Arrow A indicates the hypersensitive cleavage sites to S1 nuclease. (C) Autoradiograms showing S1 cleavage sites on the bottom strand of a supercoiled pGL3-VEGF plasmid. The designation of lanes 1–3 was as in (A) above. The vertical bar next to the gel indicates the polypyrimidine tract and the arrows indicate the S1 nuclease hypersensitivity sites.

Figure 6

DNA polymerase stop assay to determine the ability of the VEGF promoter to form G-quadruplex structures. (A and B) The wild-type (WT) and mutant (MT) template sequences (shown below the gels) with increasing concentrations of K+ (0–100 mM). Arrows indicate the positions of the full-length product (F) of DNA synthesis, the G-quadruplex pause site (S), and the free primer (P). Lanes A, G, T and C represent dideoxy sequencing reactions with the same template as a size marker for the precise arrest sites.

Figure 7

In vitro footprinting of the mutant VEGF promoter region with DNase I and S1 nuclease. Autoradiograms showing DNase I (lanes 1–3) and S1 (lanes 4–6) cleavage sites on the top strand of a supercoiled pGL3-VEGFM17 plasmid. This plasmid was incubated in the absence of salt (lanes 1 and 4) or in the presence of 100 mM KCl without (lanes 2 and 5) and with (lanes 3 and 6) 1 µM telomestatin at 37°C for 1 h before digesting with nucleases. Nuclease cleavage sites were mapped using linear amplification by PCR with 32P-labeled gene-specific primers on mutant plasmid DNA pretreated with S1 nuclease or DNase I.

Figure 8

CD spectra of the VEGF-Pu20T, d(T5G3CG3C2G5CG3T5), in Tris–HCl buffer (20 mM, pH 7.6) in the presence of increasing concentrations of KCl (0, 10, 50 and 100 mM). Each spectrum corresponds to four averaged scans taken at 25°C and is baseline corrected for signal contributions due to the buffer.

Figure 9

Summary of the results from both DNase I and S1 nuclease footprinting (Figures 4 and 5). The arrow heads and filled circles indicate the hypersensitive sites to S1 nuclease and DNase I, respectively.

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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 - PubMed (original) (raw)