Bay Daniyah H, Busch Annika, Lisdat Fred, Iida Keisuke, Ikebukuro Kazunori, Nagasawa Kazuo, Karube Isao, Yoshida Wataru
School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakuramachi, Hachioji, Tokyo, 192-0982, Japan.
Biology Department, Umm Al-Qura University, Makkah, Kingdom of Saudi Arabia.
BMC Mol Biol. 2017 Jun 27;18(1):17. doi: 10.1186/s12867-017-0094-z.
G-quadruplex is a DNA secondary structure that has been shown to play an important role in biological systems. In a previous study, we identified 1998 G-quadruplex-forming sequences using a mouse CpG islands DNA microarray with a fluorescent-labeled G-quadruplex ligand. Among these putative G-quadruplex-forming sequences, G-quadruplex formation was verified for 10 randomly selected sequences by CD spectroscopy and DMS footprinting analysis. In this study, the biological function of the 10 G-quadruplex-forming sequences in the transcriptional regulation has been analyzed using a reporter assay.
When G-quadruplex-forming sequences from the Dele and Cdc6 genes have been cloned in reporter vectors carrying a minimal promoter and the luciferase gene, luciferase expression is activated. This has also been detected in experiments applying a promoterless reporter vector. Mutational analysis reveals that guanine bases, which form the G-tetrads, are important in the activation. In addition, the activation has been found to decrease by the telomestatin derivative L1H1-7OTD which can bind to the G-quadruplex DNA. When Dele and Cdc6 CpG islands, containing the G-quadruplex-forming sequence, have been cloned in the promoterless reporter vector, the luciferase expression is activated. Mutational analysis reveals that the expression level is decreased by mutation on Dele G-quadruplex; however, increased by mutation on Cdc6 G-quadruplex.
Dele and Cdc6 G-quadruplex formation is significant in the transcriptional regulation. Dele and Cdc6 G-quadruplex DNA alone possess enhancer and promotor function. When studied in more complex CpG islands Dele G-quadruplex also demonstrates promotor activity, whereas Cdc6 G-quadruplex may possess a dual function of transcriptional regulation.
G-四链体是一种DNA二级结构，已被证明在生物系统中发挥重要作用。在先前的一项研究中，我们使用带有荧光标记的G-四链体配体的小鼠CpG岛DNA微阵列鉴定了1998个形成G-四链体的序列。在这些推定的形成G-四链体的序列中，通过圆二色光谱和二甲基亚砜足迹分析对10个随机选择的序列进行了G-四链体形成验证。在本研究中，使用报告基因测定法分析了这10个形成G-四链体的序列在转录调控中的生物学功能。
当将来自Dele和Cdc6基因的形成G-四链体的序列克隆到携带最小启动子和荧光素酶基因的报告载体中时，荧光素酶表达被激活。在应用无启动子报告载体的实验中也检测到了这一点。突变分析表明，形成G-四联体的鸟嘌呤碱基在激活中很重要。此外，发现端粒酶抑制剂衍生物L1H1-7OTD可与G-四链体DNA结合，从而降低激活作用。当将含有形成G-四链体序列的Dele和Cdc6 CpG岛克隆到无启动子报告载体中时，荧光素酶表达被激活。突变分析表明，Dele G-四链体上的突变会降低表达水平；然而，Cdc6 G-四链体上的突变会增加表达水平。
Dele和Cdc6 G-四链体的形成在转录调控中具有重要意义。单独的Dele和Cdc6 G-四链体DNA具有增强子和启动子功能。在更复杂的CpG岛中研究时，Dele G-四链体也表现出启动子活性，而Cdc6 G-四链体可能具有转录调控的双重功能。
