Kexiao Guo

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

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.

Identification and Characterization of Nucleolin as a c-myc G-quadruplex-binding Protein

myc is a proto-oncogene that plays an important role in the promotion of cellular growth and proliferation. Understanding the regulation of c-myc is important in cancer biology, as it is overexpressed in a wide variety of human cancers, including most gynecological, breast, and colon cancers. We previously demonstrated that a guanine-rich region upstream of the P1 promoter of c-myc that controls 85–90% of the transcriptional activation of this gene can form an intramolecular G-quadruplex (G4) that functions as a transcriptional repressor element. In this study, we used an affinity column to purify proteins that selectively bind to the human c-myc G-quadruplex. We found that nucleolin, a multifunctional phosphoprotein, binds in vitro to the c-myc G-quadruplex structure with high affinity and selectivity when compared with other known quadruplex structures. In addition, we demonstrate that upon binding, nucleolin facilitates the formation and increases the stability of the c-myc G-quadruplex structure. Furthermore, we provide evidence that nucleolin overexpression reduces the activity of a c-myc promoter in plasmid presumably by inducing and stabilizing the formation of the c-myc G-quadruplex. Finally, we show that nucleolin binds to the c-myc promoter in HeLa cells, which indicates that this interaction occurs in vivo. In summary, nucleolin may induce c-myc G4 formation in vivo.

Intramolecularly folded G-quadruplex and i-motif structures in the proximal promoter of the vascular endothelial growth factor gene

A polyguanine/polycytosine (polyG/polyC) tract in the proximal promoter of the vascular endothelial growth factor (VEGF) gene is essential for transcriptional activation. The guanine-rich (G-rich) and cytosine-rich (C-rich) strands on this tract are shown to form specific secondary structures, characterized as G-quadruplexes and i-motifs, respectively. Mutational analysis of the G-rich strand combined with dimethyl sulfate (DMS) footprinting, a polymerase stop assay, and circular dichroism (CD) spectroscopy revealed that the G-quadruplex containing a 1:4:1 double-chain reversal loop is the most thermodynamically stable conformation that this strand readily adopts. These studies provide strong evidence that the size of loop regions plays a critical role in determining the most favored folding pattern of a G-quadruplex. The secondary structure formed on the complementary C-rich strand was also determined by mutational analysis combined with Br2 footprinting and CD spectroscopy. Our results reveal that at a pH of 5.9 this strand is able to form an intramolecular i-motif structure that involves six C–C+ base pairs and a 2:3:2 loop configuration. Taken together, our results demonstrate that the G-quadruplex and i-motif structures are able to form on the G- and C-rich strands, respectively, of the polyG/polyC tract in the VEGF proximal promoter under conditions that favor the transition from B-DNA to non-B-DNA conformations.

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

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.

Heterogeneous Nuclear Ribonucleoprotein K and Nucleolin as Transcriptional Activators of the Vascular Endothelial Growth Factor Promoter through Interaction with Secondary DNA Structures

The human vascular endothelial growth factor (VEGF) promoter contains a polypurine/polypyrimidine (pPu/pPy) tract that is known to play a critical role in its transcriptional regulation. This pPu/pPy tract undergoes a conformational transition between B-DNA, single-stranded DNA, and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We studied the interaction of the cytosine-rich (C-rich) and guanine-rich (G-rich) strands of this tract with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of VEGF. Using chromatin immunoprecipitation (ChIP) assay for our in vivo studies and electrophoretic mobility shift assay (EMSA) for our in vitro studies, we demonstrated that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the VEGF promoter. The small interfering RNA (siRNA)-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within the pPu/pPy tract will provide new insight into mechanisms of transcriptional regulation of the VEGF gene.

Solution structure of the major G-quadruplex formed in the human VEGF promoter in K+: insights into loop interactions of the parallel G-quadruplexes

Vascular endothelial growth factor (VEGF) proximal promoter region contains a poly G/C-rich element that is essential for basal and inducible VEGF expression. The guanine-rich strand on this tract has been shown to form the DNA G-quadruplex structure, whose stabilization by small molecules can suppress VEGF expression. We report here the nuclear magnetic resonance structure of the major intramolecular G-quadruplex formed in this region in K+ solution using the 22mer VEGF promoter sequence with G-to-T mutations of two loop residues. Our results have unambiguously demonstrated that the major G-quadruplex formed in the VEGF promoter in K+ solution is a parallel-stranded structure with a 1:4:1 loop-size arrangement. A unique capping structure was shown to form in this 1:4:1 G-quadruplex. Parallel-stranded G-quadruplexes are commonly found in the human promoter sequences. The nuclear magnetic resonance structure of the major VEGF G-quadruplex shows that the 4-nt middle loop plays a central role for the specific capping structures and in stabilizing the most favored folding pattern. It is thus suggested that each parallel G-quadruplex likely adopts unique capping and loop structures by the specific middle loops and flanking segments, which together determine the overall structure and specific recognition sites of small molecules or proteins. LAY SUMMARY: The human VEGF is a key regulator of angiogenesis and plays an important role in tumor survival, growth and metastasis. VEGF overexpression is frequently found in a wide range of human tumors; the VEGF pathway has become an attractive target for cancer therapeutics. DNA G-quadruplexes have been shown to form in the proximal promoter region of VEGF and are amenable to small molecule drug targeting for VEGF suppression. The detailed molecular structure of the major VEGF promoter G-quadruplex reported here will provide an important basis for structure-based rational development of small molecule drugs targeting the VEGF G-quadruplex for gene suppression.

Abstract 5134: G-Quadruplex structure formation in the proximal promoter region of the human vascular endothelial growth factor gene

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The human vascular endothelial growth factor gene, VEGF, is a key regulator of angiogenesis. It plays an important role in tumor survival, growth and metastasis. It is over-expressed in many types of human cancers including glioma and renal cell carcinoma, ovarian and pancreatic cell carcinomas. The VEGF proximal promoter region contains a poly G/poly C rich tract that is essential for basal and inducible VEGF expression. The guanine-rich (G-rich) strand on this tract is shown to form a specific secondary structure, the G-quadruplex. Here we have elucidated the solution structure of the major intramolecular G-quadruplex formed on the G-rich strand of this region in K+ solution by proton Nuclear Magnetic Resonance (NMR) spectroscopy followed by the structural calculation of the G-quadruplex using Distance Geometry Simulated Annealing (DGSA) module of XPLOR-NIH software. It is inferred that this strand adopts a well-defined parallel-stranded G-quadruplex structure, which contains three G-tetrads stabilized by Hoogsteen hydrogen bonding, as well as three double-chain reversal loops containing 1:4:1 bases, a feature important for the formation of the most thermodynamically stable conformation. The size of the 4-base loop plays a critical role in determining the most favored folded pattern of a G-quadruplex. These 4-base loop residues interact with the residues of the 3’ flanking region of VEGF sequence; also, the 1-base double-chain reversal loop on two edges of the tetrad are very stable, which overall contribute in stabilizing the 1:4:1 conformer. We have found that the two cytosines in this central 4-base loop are involved in hydrogen bonding with the 3’ terminal end residues of VEGF, which is a salient feature as this interaction is sequence-specific and differs from that in other parallel-stranded structures, e.g. c-myc, significantly. These results are also supported by the DMS footprinting results by Guo et al. (2008) which show that the guanines involved in the tetrad formation in this major G-quadruplex loop isomer 1:4:1 are well-protected against DMS methylation. Therefore, the knowledge about the secondary structure of this non B-DNA conformation of the VEGF promoter region is essential for using the G- quadruplex as a target for anticancer drugs, which could be a novel approach to anti-angiogenesis drug discovery in cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5134. doi:10.1158/1538-7445.AM2011-5134

Abstract A203: Heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds to the vascular endothelial growth factor (VEGF) promoter and plays a role in its transcriptional control

The vascular endothelial growth factor (VEGF) promoter is capable of forming secondary DNA structures, called G‐quadruplex and i‐motif, in its proximal promoter region (−85 to −50 from the transcription initiation site) that can mask transcription factor binding sites thereby inhibiting transcription. To understand this dynamic region and its implications in transcriptional control we studied protein‐DNA interactions of this structurally versatile region, specifically those involving transcription factors. Our candidate transcription factor, heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a single‐stranded DNA binding protein able to binding to cytosine‐rich sequences such as the VEGF polypurine/polypyrimidine tract. We found that the cytosine‐rich 24‐bp oligomer has the potential of forming an i‐motif at pH 6.5 and formed a complex with hnRNP K as shown by CD spectra analysis and electrophoretic mobility shift assay (EMSA), respectively. The binding of hnRNP K to the VEGF promoter was confirmed through chromatin immunoprecipitation (ChIP) assay, and its effect on transcriptional control was demonstrated through RT/PCR of hnRNP K siRNA KO and overexpression assays. We also observed through EMSA/Footprint that the hnRNP K protein changes the oligo conformation from an i‐motif structure to a more unstructured linear conformation. In summary, this work is the first to show that hnRNP K serves a role in the transcriptional control of VEGF and that a bromine solution left to react to the protein‐complex can be used to footprint its binding motif. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A203.