Department of Pharmaceutical and Pharmacological Sciences, University of Padua, 35131 Padua, Italy.
Department of Comparative Biomedicine and Food Science, University of Padua, 35020 Legnaro, Italy.
Int J Mol Sci. 2020 Dec 30;22(1):329. doi: 10.3390/ijms22010329.
The regulation of conformational arrangements of gene promoters is a physiological mechanism that has been associated with the fine control of gene expression. Indeed, it can drive the time and the location for the selective recruitment of proteins of the transcriptional machinery. Here, we address this issue at the proximal promoter where three G-quadruplex forming sites are present (kit1, kit2 and kit*). On this model, we focused on the interplay between G-quadruplex (G4) formation and SP1 recruitment. By site directed mutagenesis, we prepared a library of plasmids containing mutated sequences of the WT promoter that systematically exploited different G4 formation attitudes and SP1 binding properties. Our transfection data showed that the three different G4 sites of the promoter impact on SP1 binding and protein expression at different levels. Notably, kit2 and kit* structural features represent an on-off system for expression through the recruitment of transcription factors. The use of two G4 binders further helps to address kit2-kit* as a reliable target for pharmacological intervention.
基因启动子构象排列的调控是一种生理机制，与基因表达的精细调控有关。事实上，它可以驱动转录机制蛋白的选择性募集的时间和位置。在这里，我们在近端启动子上解决了这个问题，在近端启动子上存在三个 G-四链体形成位点（kit1、kit2 和 kit*）。在这个模型上，我们专注于 G-四链体（G4）形成和 SP1 募集之间的相互作用。通过定点诱变，我们制备了一个包含 WT 启动子突变序列的质粒文库，该文库系统地利用了不同的 G4 形成倾向和 SP1 结合特性。我们的转染数据表明， 启动子的三个不同的 G4 位点在不同水平上影响 SP1 结合和蛋白质表达。值得注意的是，kit2 和 kit* 的结构特征通过转录因子的募集代表了 表达的开/关系统。使用两种 G4 结合物进一步有助于将 kit2-kit* 确定为药物干预的可靠靶标。
