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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) ChIP analysis to determine the binding of nucleolin to the VEGF promoter region containing the polypurine/polypyrimidine tract in A498 renal carcinoma cells. Recruitment of RNA Polymerase II (Pol II) (lanes 3), Sp1 (lanes 4) and nucleolin (lanes 5) to the VEGF proximal promoter was assessed using primers specific to the VEGF promoter (–248 to +48). One percent of total input DNA was used as a loading control (lane 1) and isotype-matched IgG was used as an internal control for the immunoprecipitation (lane 2). (B) PCR amplification of immunoprecipitated DNAs using primers specific to the 5′ upstream promoter region (–1079 to –874) of the VEGF gene as a negative control. (C) PCR amplification of immunoprecipitated DNAs using primers specific to the proximal promoter region (−273 to +71) of the HIF−1α gene as a positive control. Data shown are representative of at least two experiments.
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Figure 5: (A) ChIP analysis to determine the binding of nucleolin to the VEGF promoter region containing the polypurine/polypyrimidine tract in A498 renal carcinoma cells. Recruitment of RNA Polymerase II (Pol II) (lanes 3), Sp1 (lanes 4) and nucleolin (lanes 5) to the VEGF proximal promoter was assessed using primers specific to the VEGF promoter (–248 to +48). One percent of total input DNA was used as a loading control (lane 1) and isotype-matched IgG was used as an internal control for the immunoprecipitation (lane 2). (B) PCR amplification of immunoprecipitated DNAs using primers specific to the 5′ upstream promoter region (–1079 to –874) of the VEGF gene as a negative control. (C) PCR amplification of immunoprecipitated DNAs using primers specific to the proximal promoter region (−273 to +71) of the HIF−1α gene as a positive control. Data shown are representative of at least two experiments.

Mentions: Next, we carried out a ChIP assay to determine whether nucleolin is associated with the proximal promoter region of the VEGF gene in A498 renal carcinoma cells. As shown in Figure 5A, we observed the PCR amplification product of the proximal promoter region (–242 to +48) after immunoprecipitation of the cross-linked chromatin with the anti-nucleolin antibody (lane 5) in addition to the anti-RNA polymerase II (lane 3) and anti-Sp1 antibody (lane 4), while immunoprecipitates with purified mouse IgG as a negative control resulted in no PCR amplified products (lane 2). These results support the idea that nucleolin as well as RNA polymerase II bind to the VEGF promoter spanning the nucleotides from −242 to +48, suggesting the presence of specific G-quadruplex structures within the proximal promoter region of this gene in vivo. To further determine the specificity of nucleolin binding to the G-quadruplex-forming region, we also amplified the 5′ upstream promoter region (–1079 to −874) of the VEGF gene, not containing any putative G-quadruplex-forming regions, and no PCR products indicative of the upstream region of the VEGF promoter were found (Figure 5B). As a positive control, we also amplified the proximal promoter region (–273 to +71) of the HIF-1α gene, which contains a putative G-quadruplex-forming region. As shown in Figure 5C, PCR products indicative of this region were observed in the input sample (lane 1), RNA polymerase, Sp1 and nucleolin-specific immunoprecipitations.Figure 5.


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) ChIP analysis to determine the binding of nucleolin to the VEGF promoter region containing the polypurine/polypyrimidine tract in A498 renal carcinoma cells. Recruitment of RNA Polymerase II (Pol II) (lanes 3), Sp1 (lanes 4) and nucleolin (lanes 5) to the VEGF proximal promoter was assessed using primers specific to the VEGF promoter (–248 to +48). One percent of total input DNA was used as a loading control (lane 1) and isotype-matched IgG was used as an internal control for the immunoprecipitation (lane 2). (B) PCR amplification of immunoprecipitated DNAs using primers specific to the 5′ upstream promoter region (–1079 to –874) of the VEGF gene as a negative control. (C) PCR amplification of immunoprecipitated DNAs using primers specific to the proximal promoter region (−273 to +71) of the HIF−1α gene as a positive control. Data shown are representative of at least two experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045601&req=5

Figure 5: (A) ChIP analysis to determine the binding of nucleolin to the VEGF promoter region containing the polypurine/polypyrimidine tract in A498 renal carcinoma cells. Recruitment of RNA Polymerase II (Pol II) (lanes 3), Sp1 (lanes 4) and nucleolin (lanes 5) to the VEGF proximal promoter was assessed using primers specific to the VEGF promoter (–248 to +48). One percent of total input DNA was used as a loading control (lane 1) and isotype-matched IgG was used as an internal control for the immunoprecipitation (lane 2). (B) PCR amplification of immunoprecipitated DNAs using primers specific to the 5′ upstream promoter region (–1079 to –874) of the VEGF gene as a negative control. (C) PCR amplification of immunoprecipitated DNAs using primers specific to the proximal promoter region (−273 to +71) of the HIF−1α gene as a positive control. Data shown are representative of at least two experiments.
Mentions: Next, we carried out a ChIP assay to determine whether nucleolin is associated with the proximal promoter region of the VEGF gene in A498 renal carcinoma cells. As shown in Figure 5A, we observed the PCR amplification product of the proximal promoter region (–242 to +48) after immunoprecipitation of the cross-linked chromatin with the anti-nucleolin antibody (lane 5) in addition to the anti-RNA polymerase II (lane 3) and anti-Sp1 antibody (lane 4), while immunoprecipitates with purified mouse IgG as a negative control resulted in no PCR amplified products (lane 2). These results support the idea that nucleolin as well as RNA polymerase II bind to the VEGF promoter spanning the nucleotides from −242 to +48, suggesting the presence of specific G-quadruplex structures within the proximal promoter region of this gene in vivo. To further determine the specificity of nucleolin binding to the G-quadruplex-forming region, we also amplified the 5′ upstream promoter region (–1079 to −874) of the VEGF gene, not containing any putative G-quadruplex-forming regions, and no PCR products indicative of the upstream region of the VEGF promoter were found (Figure 5B). As a positive control, we also amplified the proximal promoter region (–273 to +71) of the HIF-1α gene, which contains a putative G-quadruplex-forming region. As shown in Figure 5C, PCR products indicative of this region were observed in the input sample (lane 1), RNA polymerase, Sp1 and nucleolin-specific immunoprecipitations.Figure 5.

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
Related in: MedlinePlus