Genome-wide Light-inducible Tuning of Transcriptional Network Dynamic

描述（由申请人提供）：我们建议开发和应用一个范式转换的技术平台，该平台使用一系列光诱导转录效应物（LITEs）来协调体外单个细胞和完整生物体中多个基因的时间调控。我们提议开发的技术将非常广泛地适用，并有可能从根本上改变不同生物医学领域的发现规模和速度。这项新技术的应用将使高通量发现任何内源基因的上游转录调控元件，以及在本地基因组中基因表达的临时精确调节成为可能。在不同的组织和细胞类型中观察到精确的基因表达时间和空间模式，并通过涉及多个基因的复杂转录调控回路进行协调和维持。由于缺乏集成的控制和读出技术，能够同时对多个基因进行扰动和“快速”调整，我们将转录网络动力学与生理和发育因果联系起来的能力仍处于初级阶段。LITEs平台将使用非侵入性的光介导诱导策略，在数小时的时间尺度上修改和调节基因表达，从而使新一代的交互式基因研究成为可能，目前传统技术无法实现。我们的提案包括两个主要部分：1)光敏设计锌指（ZF）转录调节剂（LITEs-ZF）的新技术开发。基因组学、合成生物学和蛋白质工程方法将用于开发一套针对哺乳动物细胞原生基因组中特定基因的新型光诱导转录调节因子。由于ZF DNA结合域可以针对任何DNA序列进行工程设计，因此ZF- lite适用于各种不同生物体和细胞类型的广泛生物学研究。2)该技术在高通量体外和体内中枢神经系统转录网络动力学研究中的应用。体外应用将旨在鉴定对皮质脊髓运动神经元（CSMN）分化至关重要的上游基因表达效应物，CSMN是一种临床相关的神经元群体，在肌萎缩侧索硬化症（ALS）中退化，并在脊髓损伤中受损。在体内的应用将集中在通过模仿成人大脑中CSMN特异性发育线索的特定时间序列来指导CSMN的再生。鉴于这项技术的广泛适用性，这项提议的工作将产生深远的影响，并将从根本上改变生命科学和医学所有领域中研究基因相互作用的现有实验方法。

DESCRIPTION (provided by applicant): We propose to develop and apply a paradigm-shifting technological platform that uses a series of Light- Inducible Transcriptional Effectors (LITEs) to orchestrate the temporal regulation of multiple genes in both individual cells in vitro and in the intact organism. The technology we propose to develop will be very broadly applicable and has the potential to radically transform the scale and rate of discovery across different biomedical fields. Application of this novel technology will enable high throughput discovery of the upstream transcriptional regulatory elements of any endogenous gene, as well as temporally precise modulation of gene expression in the native genome. Precise temporal and spatial patterns of gene expression are observed in different tissue and cell types, and are orchestrated and maintained by complex transcriptionally regulated circuits involving multiple genes. Due to the lack of integrated control and readout technologies that enable simultaneous perturbation and "fast" tuning of multiple genes, our ability to causally link transcriptional network dynamics with physiology and development remains at the infancy stage. The LITEs platform will enable modification and regulation of gene expression on the time-scale of hours using a non-invasive, light-mediated inductive strategy, thereby enabling a new generation of interactive genetic studies currently inaccessible with conventional techniques. Our proposal consists of two main components: 1) Novel technology development of light sensitive designer zinc finger (ZF) transcriptional modulators (LITEs-ZF). Genomic, synthetic biology, and protein engineering approaches will be used to develop a suite of novel light-inducible transcriptional regulators targeted at specific genes in the native genome of mammalian cells. Since ZF DNA binding domains can be engineered to target any DNA sequence, ZF-LITEs are applicable to a broad range of biological research studies in a variety of different organisms and cells types. 2) Application of the technology toward high-throughput in vitro and in vivo interrogation of transcriptional network dynamics in the central nervous system. In vitro application will be aimed at identification of upstream effectors of gene expression critical to the differentiation of corticospinal motor neurons (CSMN), a clinically relevant neuronal population that degenerates in amyotrophic lateral sclerosis (ALS) and is injured in spinal cord injury. In vivo application will be focused on directing the regeneration of CSMN by mimicking specific temporal sequences of CSMN-specific developmental cues within the adult brain. Given the broad applicability of this technology, the impact of this proposed work will be far reaching and will radically transform existing experimental approaches for studying gene interactions in all fields of life science and medicine.