PDF(Pubmed)
Abstract：
Blue light illumination can be detected by Light-Oxygen-Voltage (LOV) photosensing proteins and translated into a range of biochemical responses, facilitating the generation of novel optogenetic tools to control cellular function. Here we develop new variants of our previously described VP-EL222 light-dependent transcription factor and apply them to study the phosphate-responsive signaling (PHO) pathway in the budding yeast Saccharomyces cerevisiae, exemplifying the utilities of these new tools. Focusing first on the VP-EL222 protein itself, we quantified the tunability of gene expression as a function of light intensity and duration, and demonstrated that this system can tolerate the addition of substantially larger effector domains without impacting function. We further demonstrated the utility of several EL222-driven transcriptional controllers in both plasmid and genomic settings, using the PHO5 and PHO84 promoters in their native chromosomal contexts as examples. These studies highlight the utility of light-controlled gene activation using EL222 tethered to either artificial transcription domains or yeast activator proteins (Pho4). Similarly, we demonstrate the ability to optogenetically repress gene expression with EL222 fused to the yeast Ume6 protein. We finally investigated the effects of moving EL222 recruitment sites to different locations within the PHO5 and PHO84 promoters, as well as determining how this artificial light-controlled regulation could be integrated with the native controls dependent on inorganic phosphate (Pi) availability. Taken together, our work expands the applicability of these versatile optogenetic tools in the types of functionality they can deliver and biological questions that can be probed.
摘要：
蓝光照明可以通过光氧电压（LOV）光敏蛋白检测，并转化为一系列生化反应，促进新型光遗传学工具的产生，以控制细胞功能。在这里，我们开发了我们先前描述的VP-EL222光依赖性转录因子的新变体，并将它们用于研究发芽酵母酿酒酵母中的磷酸盐响应信号（PHO）途径，举例说明了这些新工具的实用程序。首先关注VP-EL222蛋白本身，我们将基因表达的可调性量化为光照强度和持续时间的函数，并证明该系统可以容忍添加实质上更大的效应子结构域而不影响功能。我们进一步证明了几种EL222驱动的转录控制器在质粒和基因组环境中的实用性。使用PHO5和PHO84启动子在其天然染色体环境中作为例子。这些研究强调了使用连接到人工转录域或酵母激活蛋白（Pho4）的EL222进行光控基因激活的实用性。同样，我们证明了与酵母Ume6蛋白融合的EL222光遗传学抑制基因表达的能力。我们最终研究了将EL222招募位点移动到PHO5和PHO84启动子内不同位置的影响，以及确定如何将这种人工光控调节与取决于无机磷酸盐（Pi）可用性的天然控制相结合。一起来看,我们的工作扩展了这些多功能光遗传学工具在它们可以提供的功能类型和可以探索的生物学问题方面的适用性。
