Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Biomedical Synthetic Biology Research Center, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Dongchuan Road 500, Shanghai, 200241, China.
Wuhu Hospital, Health Science Center, East China Normal University, Middle Jiuhua Road 263, Wuhu City, China.
Nat Commun. 2024 Jun 8;15(1):4894. doi: 10.1038/s41467-024-49254-5.
Synthetic biology applications require finely tuned gene expression, often mediated by synthetic transcription factors (sTFs) compatible with the human genome and transcriptional regulation mechanisms. While various DNA-binding and activation domains have been developed for different applications, advanced artificially controllable sTFs with improved regulatory capabilities are required for increasingly sophisticated applications. Here, in mammalian cells and mice, we validate the transactivator function and homo-/heterodimerization activity of the plant-derived phytochrome chaperone proteins, FHY1 and FHL. Our results demonstrate that FHY1/FHL form a photosensing transcriptional regulation complex (PTRC) through interaction with the phytochrome, ΔPhyA, that can toggle between active and inactive states through exposure to red or far-red light, respectively. Exploiting this capability, we develop a light-switchable platform that allows for orthogonal, modular, and tunable control of gene transcription, and incorporate it into a PTRC-controlled CRISPRa system (PTRC) to modulate endogenous gene expression. We then integrate the PTRC with small molecule- or blue light-inducible regulatory modules to construct a variety of highly tunable systems that allow rapid and reversible control of transcriptional regulation in vitro and in vivo. Validation and deployment of these plant-derived phytochrome chaperone proteins in a PTRC platform have produced a versatile, powerful tool for advanced research and biomedical engineering applications.
合成生物学应用需要精细调节基因表达，通常通过与人类基因组和转录调控机制兼容的合成转录因子（sTF）来介导。虽然已经开发了各种 DNA 结合和激活结构域用于不同的应用，但对于越来越复杂的应用，需要具有改进的调控能力的先进的人工可控 sTF。在这里，我们在哺乳动物细胞和小鼠中验证了植物来源的光敏色素伴侣蛋白 FHY1 和 FHL 的转录激活功能和同/异二聚化活性。我们的结果表明，FHY1/FHL 通过与光敏色素ΔPhyA 的相互作用形成一个光感应转录调控复合物（PTRC），通过分别暴露于红光或远红光，该复合物可以在活性和非活性状态之间切换。利用这一特性，我们开发了一种光控可切换平台，允许对基因转录进行正交、模块化和可调谐的控制，并将其整合到一个由 PTRC 控制的 CRISPRa 系统（PTRC）中，以调节内源性基因表达。然后，我们将 PTRC 与小分子或蓝光诱导的调节模块集成在一起，构建了各种高度可调的系统，允许在体外和体内快速和可逆地控制转录调控。在 PTRC 平台中验证和部署这些植物来源的光敏色素伴侣蛋白已经产生了一种通用、强大的工具，用于高级研究和生物医学工程应用。
