Genome-wide Light-inducible Tuning of Transcriptional Network Dynamic

描述（由申请人提供）：我们提出开发和应用一种范式转换技术平台，其使用一系列光诱导转录效应子（LITE）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具有非常广泛的适用性，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。这种新技术的应用将使得能够高通量地发现任何内源基因的上游转录调控元件，以及天然基因组中基因表达的时间精确调节。 在不同的组织和细胞类型中观察到基因表达的精确的时间和空间模式，并且通过涉及多个基因的复杂转录调控回路来协调和维持。由于缺乏集成的控制和读出技术，使多个基因的同时扰动和“快速”调整，我们的能力，因果关系的转录网络动态与生理和发展仍然处于起步阶段。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对基因表达进行修饰和调控，从而实现目前无法使用常规技术进行的新一代交互式遗传研究。我们的计划包括两个主要部分：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天然基因组中特定基因的新型光诱导转录调控因子。由于ZF DNA结合结构域可以被改造为靶向任何DNA序列，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)该技术在中枢神经系统转录网络动力学的体外和体内高通量询问中的应用。体外应用的目的是鉴定对皮质脊髓运动神经元（CSMN）分化至关重要的基因表达的上游效应子，CSMN是一种临床相关的神经元群体，在肌萎缩性侧索硬化症（ALS）中退化，并在脊髓损伤中受损。体内应用将集中在通过模仿成年大脑内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，这项拟议工作的影响将是深远的，并将从根本上改变现有的实验方法，研究基因相互作用的所有领域的生命科学和医学。 公共卫生相关性：理解驱动细胞发育和修复的转录网络在再生医学领域具有改变景观的影响。在这里，我们开发了一种非侵入性的光学技术，使基因组范围内的基因表达动态调整对皮质脊髓运动神经元的再生。

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. PUBLIC HEALTH RELEVANCE: Understanding the transcriptional networks that drive cellular development and repair has landscape- shifting impacts in the field of regenerative medicine. Here we develop a non-invasive, optical technology to enable genome-wide tuning of gene expression dynamics toward regeneration of corticospinal motor neurons.