Heterogeneous nuclear ribonucleoprotein K and nucleolin as transcriptional activators of the vascular endothelial growth factor promoter through interaction with secondary DNA structures.
Heterogeneous nuclear ribonucleoprotein K and nucleolin as transcriptional activators of the vascular endothelial growth factor promoter through interaction with secondary DNA structures.
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DOI：
10.1021/bi101633b
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发表时间：
2011-05-10
期刊：
影响因子：
2.9
通讯作者：
Sun D
中科院分区：
文献类型：
--
作者：
Uribe DJ;Guo K;Shin YJ;Sun D
人血管内皮生长因子(VEGF)启动子含有一个多嘌呤/多嘧啶(PPU/pPy)链，已知在其转录调控中起关键作用。这个PPU/pPy链经历了B-DNA、单链DNA和非典型二级DNA结构(如G-四链和I-基序)之间的构象转换。我们在体内和体外研究了富含胞嘧啶(C)和富鸟嘌呤(G)的链分别与转录因子异相核糖核蛋白(HnRNP)K和核素的相互作用，以及它们在转录调控血管内皮生长因子(VEGF)中的潜在作用。用染色质免疫沉淀法(ChIP)进行体内研究，用凝胶迁移率改变分析(EMSA)进行体外研究，我们证明了核仁素和hnRNP K分别选择性地与VEGF启动子的PPU/pPy区的G-和C-富含序列结合。小干扰RNA(SiRNA)介导的核仁素或hnRNP K沉默导致基本的血管内皮生长因子基因表达下调，提示它们可能是血管内皮生长因子转录的激活剂。综上所述，能够识别和结合PPU/pPy区域内非典型DNA结构的转录因子的鉴定将为研究VEGF基因的转录调控机制提供新的视角。
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.
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