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Evidence of the formation of G-quadruplex structures in the promoter region of the human vascular endothelial growth factor gene.

Sun D, Guo K, Shin YJ - Nucleic Acids Res. (2010)

Bottom Line: We observed that the overall reactivity of the guanine residues within this tract toward DMS was significantly reduced compared with other guanine residues of the flanking regions in both in vitro and in vivo footprinting experiments.Our chromatin immunoprecipitation analysis further revealed binding of nucleolin to the promoter region of the VEGF gene in vivo.Taken together, our results are in agreement with our hypothesis that secondary DNA structures, such as G-quadruplexes, can be formed in supercoiled duplex DNA and DNA in chromatin in vivo under physiological conditions similar to those formed in single-stranded DNA templates.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, University of Arizona, Tucson, AZ 85721, USA. sun@pharmacy.arizona.edu

ABSTRACT
The polypurine/polypyrimidine (pPu/pPy) tract of the human vascular endothelial growth factor (VEGF) gene is proposed to be structurally dynamic and to have potential to adopt non-B DNA structures. In the present study, we further provide evidence for the existence of the G-quadruplex structure within this tract both in vitro and in vivo using the dimethyl sulfate (DMS) footprinting technique and nucleolin as a structural probe specifically recognizing G-quadruplex structures. We observed that the overall reactivity of the guanine residues within this tract toward DMS was significantly reduced compared with other guanine residues of the flanking regions in both in vitro and in vivo footprinting experiments. We also demonstrated that nucleolin, which is known to bind to G-quadruplex structures, is able to bind specifically to the G-rich sequence of this region in negatively supercoiled DNA. Our chromatin immunoprecipitation analysis further revealed binding of nucleolin to the promoter region of the VEGF gene in vivo. Taken together, our results are in agreement with our hypothesis that secondary DNA structures, such as G-quadruplexes, can be formed in supercoiled duplex DNA and DNA in chromatin in vivo under physiological conditions similar to those formed in single-stranded DNA templates.

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(A) DMS footprinting of the VEGF G-rich single strand in the presence of 100 mM KCl. AG and TC lanes represent purine and pyrimidine- specific Maxam–Gilbert sequencing reactions, respectively. Lanes 1 and 2 correspond to the G-rich strand DMS treated and untreated, respectively. (B and C) Autoradiogram showing DMS modification sites on the G-rich strand of a supercoiled (B) or linearized (C) form of pGL3-VEGF plasmid. Lanes 1 and 2 represent respective cytosine and guanine-specific dideoxysequencing reactions using the same primer as for the extension reactions. Lanes 3 and 4 correspond to the pGL3-VEGF plasmids that are untreated and treated with DMS (0.25%), respectively. Each band reflects extension reaction products of a gene specific primer designed to anneal to the G-rich strand of the plasmid DNA to map the DMS modification sites on the same strand. The bracket ‘G4’ represents a proposed G-quadruplex-forming region and brackets ‘UF’ and ‘DF’ represent the up- and downstream flanking regions of the G-quadruplex-forming sequence. (D) Summary of the results from both DMS footprinting experiments. The DMS-protected guanine residues within the G-rich sequences of the VEGF promoter are indicated by open circles, and closed circles indicate the guanine residues methylated by DMS. Data shown are representative of at least two experiments.
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Figure 2: (A) DMS footprinting of the VEGF G-rich single strand in the presence of 100 mM KCl. AG and TC lanes represent purine and pyrimidine- specific Maxam–Gilbert sequencing reactions, respectively. Lanes 1 and 2 correspond to the G-rich strand DMS treated and untreated, respectively. (B and C) Autoradiogram showing DMS modification sites on the G-rich strand of a supercoiled (B) or linearized (C) form of pGL3-VEGF plasmid. Lanes 1 and 2 represent respective cytosine and guanine-specific dideoxysequencing reactions using the same primer as for the extension reactions. Lanes 3 and 4 correspond to the pGL3-VEGF plasmids that are untreated and treated with DMS (0.25%), respectively. Each band reflects extension reaction products of a gene specific primer designed to anneal to the G-rich strand of the plasmid DNA to map the DMS modification sites on the same strand. The bracket ‘G4’ represents a proposed G-quadruplex-forming region and brackets ‘UF’ and ‘DF’ represent the up- and downstream flanking regions of the G-quadruplex-forming sequence. (D) Summary of the results from both DMS footprinting experiments. The DMS-protected guanine residues within the G-rich sequences of the VEGF promoter are indicated by open circles, and closed circles indicate the guanine residues methylated by DMS. Data shown are representative of at least two experiments.

Mentions: DMS footprinting is useful for fine mapping the presence of G-quadruplex structures within the promoter region (20,21). The formation of G-quadruplex structures requires N7 of guanine, which is subsequently protected from N7 methylation by DMS (20,21). Consistent with our previous studies (20), DMS footprinting revealed that a unique G-quadruplex structure is formed in the G-rich single strand of the pPu/pPy tract of the VEGF promoter region. This tract has the capacity to form a unique intramolecular G-quadruplex structure in the presence of K+, which requires four guanine blocks (GR-I to GR-IV), consisting of 12 total guanines (Figure 1A). DMS cleavage of the VEGF G-rich single strand showed that three stacked G-tetrads formed by four G-stretches were protected from methylation by DMS (except one guanine residue indicated by an asterisk), whereas two guanine residues within the central loop showed hypersensitivity to DMS (Figure 2A, lane 1). We further examined G-quadruplex formation in the promoter DNA duplex of the VEGF gene (787 to +50) using in vitro DMS footprinting experiments with a supercoiled form of plasmid pGL3-VEGFP. As shown in Figure 2B (lane 4), under supercoiled conditions DMS shows dramatically reduced reactivity within the four 5′-end runs of guanines (bracket ‘G4′) on the G-rich strand of the pPu/pPy tract of the VEGF promoter region, where G-quadruplex structures are proposed to be present. In contrast, the flanking regions (Brackets ‘UF’ and ‘DF’) of the proposed G-quadruplex-forming sequence show enhanced reactivity to DMS (Figure 2B, lane 4), presumably because of the presence of locally unwound structures at the junctions between normal duplex regions and stable secondary structures. Interestingly, a moderate reactivity of DMS toward some of the guanine residues from GR-III was found, suggesting a loop region of the G-quadruplex structures as observed in studies using a single-stranded G-rich DNA (Figure 2A, lane 1). The reactivity of DMS toward the guanine residues within the complete G-rich region is normal in the linearized form of plasmid pGL3-VEGFP (Figure 2C, lane 4), showing that negative supercoiling is required to drive the local unwinding of the pPu/pPy tract, allowing a specific G-rich region to form a G-quadruplex. The results of DMS footprinting for the VEGF G-rich single strand and the G-rich region within a supercoiled plasmid pGL3-VEGFP in the presence of 100 mM KCl are summarized in Figure 2D.Figure 2.


Evidence of the formation of G-quadruplex structures in the promoter region of the human vascular endothelial growth factor gene.

Sun D, Guo K, Shin YJ - Nucleic Acids Res. (2010)

(A) DMS footprinting of the VEGF G-rich single strand in the presence of 100 mM KCl. AG and TC lanes represent purine and pyrimidine- specific Maxam–Gilbert sequencing reactions, respectively. Lanes 1 and 2 correspond to the G-rich strand DMS treated and untreated, respectively. (B and C) Autoradiogram showing DMS modification sites on the G-rich strand of a supercoiled (B) or linearized (C) form of pGL3-VEGF plasmid. Lanes 1 and 2 represent respective cytosine and guanine-specific dideoxysequencing reactions using the same primer as for the extension reactions. Lanes 3 and 4 correspond to the pGL3-VEGF plasmids that are untreated and treated with DMS (0.25%), respectively. Each band reflects extension reaction products of a gene specific primer designed to anneal to the G-rich strand of the plasmid DNA to map the DMS modification sites on the same strand. The bracket ‘G4’ represents a proposed G-quadruplex-forming region and brackets ‘UF’ and ‘DF’ represent the up- and downstream flanking regions of the G-quadruplex-forming sequence. (D) Summary of the results from both DMS footprinting experiments. The DMS-protected guanine residues within the G-rich sequences of the VEGF promoter are indicated by open circles, and closed circles indicate the guanine residues methylated by DMS. Data shown are representative of at least two experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: (A) DMS footprinting of the VEGF G-rich single strand in the presence of 100 mM KCl. AG and TC lanes represent purine and pyrimidine- specific Maxam–Gilbert sequencing reactions, respectively. Lanes 1 and 2 correspond to the G-rich strand DMS treated and untreated, respectively. (B and C) Autoradiogram showing DMS modification sites on the G-rich strand of a supercoiled (B) or linearized (C) form of pGL3-VEGF plasmid. Lanes 1 and 2 represent respective cytosine and guanine-specific dideoxysequencing reactions using the same primer as for the extension reactions. Lanes 3 and 4 correspond to the pGL3-VEGF plasmids that are untreated and treated with DMS (0.25%), respectively. Each band reflects extension reaction products of a gene specific primer designed to anneal to the G-rich strand of the plasmid DNA to map the DMS modification sites on the same strand. The bracket ‘G4’ represents a proposed G-quadruplex-forming region and brackets ‘UF’ and ‘DF’ represent the up- and downstream flanking regions of the G-quadruplex-forming sequence. (D) Summary of the results from both DMS footprinting experiments. The DMS-protected guanine residues within the G-rich sequences of the VEGF promoter are indicated by open circles, and closed circles indicate the guanine residues methylated by DMS. Data shown are representative of at least two experiments.
Mentions: DMS footprinting is useful for fine mapping the presence of G-quadruplex structures within the promoter region (20,21). The formation of G-quadruplex structures requires N7 of guanine, which is subsequently protected from N7 methylation by DMS (20,21). Consistent with our previous studies (20), DMS footprinting revealed that a unique G-quadruplex structure is formed in the G-rich single strand of the pPu/pPy tract of the VEGF promoter region. This tract has the capacity to form a unique intramolecular G-quadruplex structure in the presence of K+, which requires four guanine blocks (GR-I to GR-IV), consisting of 12 total guanines (Figure 1A). DMS cleavage of the VEGF G-rich single strand showed that three stacked G-tetrads formed by four G-stretches were protected from methylation by DMS (except one guanine residue indicated by an asterisk), whereas two guanine residues within the central loop showed hypersensitivity to DMS (Figure 2A, lane 1). We further examined G-quadruplex formation in the promoter DNA duplex of the VEGF gene (787 to +50) using in vitro DMS footprinting experiments with a supercoiled form of plasmid pGL3-VEGFP. As shown in Figure 2B (lane 4), under supercoiled conditions DMS shows dramatically reduced reactivity within the four 5′-end runs of guanines (bracket ‘G4′) on the G-rich strand of the pPu/pPy tract of the VEGF promoter region, where G-quadruplex structures are proposed to be present. In contrast, the flanking regions (Brackets ‘UF’ and ‘DF’) of the proposed G-quadruplex-forming sequence show enhanced reactivity to DMS (Figure 2B, lane 4), presumably because of the presence of locally unwound structures at the junctions between normal duplex regions and stable secondary structures. Interestingly, a moderate reactivity of DMS toward some of the guanine residues from GR-III was found, suggesting a loop region of the G-quadruplex structures as observed in studies using a single-stranded G-rich DNA (Figure 2A, lane 1). The reactivity of DMS toward the guanine residues within the complete G-rich region is normal in the linearized form of plasmid pGL3-VEGFP (Figure 2C, lane 4), showing that negative supercoiling is required to drive the local unwinding of the pPu/pPy tract, allowing a specific G-rich region to form a G-quadruplex. The results of DMS footprinting for the VEGF G-rich single strand and the G-rich region within a supercoiled plasmid pGL3-VEGFP in the presence of 100 mM KCl are summarized in Figure 2D.Figure 2.

Bottom Line: We observed that the overall reactivity of the guanine residues within this tract toward DMS was significantly reduced compared with other guanine residues of the flanking regions in both in vitro and in vivo footprinting experiments.Our chromatin immunoprecipitation analysis further revealed binding of nucleolin to the promoter region of the VEGF gene in vivo.Taken together, our results are in agreement with our hypothesis that secondary DNA structures, such as G-quadruplexes, can be formed in supercoiled duplex DNA and DNA in chromatin in vivo under physiological conditions similar to those formed in single-stranded DNA templates.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, University of Arizona, Tucson, AZ 85721, USA. sun@pharmacy.arizona.edu

ABSTRACT
The polypurine/polypyrimidine (pPu/pPy) tract of the human vascular endothelial growth factor (VEGF) gene is proposed to be structurally dynamic and to have potential to adopt non-B DNA structures. In the present study, we further provide evidence for the existence of the G-quadruplex structure within this tract both in vitro and in vivo using the dimethyl sulfate (DMS) footprinting technique and nucleolin as a structural probe specifically recognizing G-quadruplex structures. We observed that the overall reactivity of the guanine residues within this tract toward DMS was significantly reduced compared with other guanine residues of the flanking regions in both in vitro and in vivo footprinting experiments. We also demonstrated that nucleolin, which is known to bind to G-quadruplex structures, is able to bind specifically to the G-rich sequence of this region in negatively supercoiled DNA. Our chromatin immunoprecipitation analysis further revealed binding of nucleolin to the promoter region of the VEGF gene in vivo. Taken together, our results are in agreement with our hypothesis that secondary DNA structures, such as G-quadruplexes, can be formed in supercoiled duplex DNA and DNA in chromatin in vivo under physiological conditions similar to those formed in single-stranded DNA templates.

Show MeSH