在……中使用单组分融合蛋白对磷酸盐响应基因进行光遗传学控制
Optogenetic control of phosphate-responsive genes using single component fusion proteins in .
作者信息
Cleere Matthew M, Gardner Kevin H
机构信息
Structural Biology Initiative, CUNY Advanced Science Research Center, New York, NY 10031.
Biology Ph.D. Program, Graduate Center, City University of New York, New York, NY 10016.
出版信息
bioRxiv. 2024 Oct 29:2024.08.02.605841. doi: 10.1101/2024.08.02.605841.
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 () pathway in the budding yeast , 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 and 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 and promoters, as well as determining how this artificial light-controlled regulation could be integrated with the native controls dependent on inorganic phosphate (P) availability. Taken together, our work expands the applicability of these versatile optogenetic tools in the types of functionalities they can deliver and biological questions that can be probed.
蓝光照射可被光-氧-电压(LOV)光敏蛋白检测到,并转化为一系列生化反应,有助于生成控制细胞功能的新型光遗传学工具。在此,我们开发了先前描述的VP-EL222光依赖性转录因子的新变体,并将其应用于研究出芽酵母中的磷酸盐响应信号通路,例证了这些新工具的实用性。首先聚焦于VP-EL222蛋白本身,我们量化了基因表达的可调性作为光强度和持续时间的函数,并证明该系统能够耐受添加大得多的效应结构域而不影响功能。我们还以天然染色体背景下的和启动子为例,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。这些研究突出了使用与人工转录结构域或酵母激活蛋白(Pho4)相连的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能够光遗传学抑制基因表达。我们最终研究了将EL222募集位点移至和启动子内不同位置的影响,以及确定这种人工光控调节如何与依赖无机磷酸盐(P)可用性的天然控制整合。综上所述,我们的工作扩展了这些多功能光遗传学工具在其可提供的功能类型和可探究的生物学问题方面的适用性。