College of Pharmacy, University of Arizona, Tucson, AZ 85721, USA.
Nucleic Acids Res. 2011 Mar;39(4):1256-65. doi: 10.1093/nar/gkq926. Epub 2010 Oct 18.
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.
人血管内皮生长因子（VEGF）基因的多嘌呤/多嘧啶（pPu/pPy）区被认为在结构上是动态的，并有可能采用非 B 型 DNA 结构。在本研究中，我们使用二甲硫酸盐（DMS）足迹技术和核仁素作为专门识别 G-四链体结构的结构探针，进一步提供了该区域内存在 G-四链体结构的证据，无论是在体外还是在体内。我们观察到，与侧翼区域的其他鸟嘌呤残基相比，该区域内的鸟嘌呤残基对 DMS 的整体反应性在体外和体内足迹实验中均显著降低。我们还证明，核仁素已知能与 G-四链体结构结合，能够特异性地结合到该区域富含 G 的序列在负超螺旋 DNA 中。我们的染色质免疫沉淀分析进一步揭示了核仁素在体内与 VEGF 基因启动子区域的结合。总之，我们的结果与我们的假设一致，即在生理条件下，类似于在单链 DNA 模板中形成的条件下，超螺旋双链 DNA 和染色质中的 DNA 可以形成二级 DNA 结构，如 G-四链体。
