Designing synthetic transcription factors to study drug-specific transcription and behaviors

项目摘要 脑桥核（NAc）是编码对天然物质和药物反应的关键脑区 咖啡机。所有的药物滥用是众所周知的增加多巴胺能紧张的NAc，使其成为一个中央大脑 协调药物反应的结构。滥用药物主要通过突触靶点改变细胞内 信号级联，其导致转录因子的激活或抑制，转录因子又诱导或 抑制特定基因的表达。这些药物诱导的脑转录因子的功能是 对神经元内的表观遗传景观敏感并做出贡献，并与持久的 神经元功能和药物相关行为的改变。因此，了解药物诱导功能 在这个大脑区域的转录因子可能会扩大我们对滥用药物如何协调的理解 药物相关行为的持久变化。这些知识对下一代的发展至关重要 特定物质使用障碍（SUD）药物。在这里，我提出以合成生物学为中心的创新 重新编程转录因子功能的方法，用于在清醒的大脑中进行病毒递送， 行为小鼠，以解决确切的，破坏性的转录因子功能，这是不同的刺激与 阿片类药物成瘾我建议通过应用新的合成转录因子构建体来实现这一点 结合脑转录组分析和药物自我给药程序。该项目的重点是 ZFP 189，一种新的药物激活转录因子，我在我早期的毕业论文中仔细描述了它的特征。 工作首先，在NAc中，我将通过病毒传递合成的ZFP 189转录因子， 能够对所有体内靶基因施加不同形式的转录控制的功能部分。我会 进行RNA测序方法，以揭示ZFP 189的完整基因调控功能是如何 在兴奋剂和阿片类药物使用的背景下，其次，我将利用这些相同的合成 ZFP189转录因子，以研究其对静脉注射药物强化的因果作用 药物自我给药程序。通过创新合成转录因子的应用， 编程以在整个基因组中发挥新的基因调控形式，这些技术可以结合起来 利用现有的RNA测序和复杂的行为程序， 询问转录因子的功能与特定的滥用药物不同， 转录操作，以揭示驱动某些SUD的因果基因调控事件。在一起，这 这项工作将提供新的方法来确定特定SUD核心的转录因子功能， 产生精炼的候选基因作为未来SUD药物的靶点。

PROJECT SUMMARY The nucleus accumbens (NAc) is a key brain region which encodes responses to both natural and drug reinforcers. All drugs of abuse are known to increase the dopaminergic tone in the NAc, making it a central brain structure in coordinating drug response. Drugs of abuse act largely through synaptic targets to alter intracellular signaling cascades which leads to the activation or inhibition of transcription factors, which in turn induce or repress the expression of specific genes. These drug-induced functions of brain transcription factors are sensitive to, and contribute to, the epigenetic landscape within a neuron and have been associated with lasting alterations in neuronal function and drug-related behaviors. Therefore, understanding the drug-induced function of transcription factors in this brain area may expand our understanding of how drugs of abuse can coordinate lasting changes in drug-related behaviors. This knowledge is critical for the development of the next generation of specific substance use disorder (SUD) medications. Here, I propose innovation centered on synthetic biology approaches to re-program the function of transcription factors, for viral delivery in the brain of awake and behaving mice, to resolve the exact, damaging transcription factor functions that are distinct to stimulant versus opioid addiction. I propose to accomplish this by applying novel synthetic transcription factor constructs combined with brain transcriptome profiling and drug self-administration procedures. This project focuses on ZFP189, a novel drug-activated transcription factor that I have carefully characterized in my earlier graduate work. First, in the NAc, I will virally deliver synthetic ZFP189 transcription factors, which possess artificial functional moieties capable of exerting distinct forms of transcriptional control at all in vivo target genes. I will perform RNA sequencing approaches to uncover how the complete gene-regulatory functions of a ZFP189 are differentially engaged in the context of stimulant versus opioid use. Second, I will utilize these same synthetic ZFP189 transcription factors to investigate their causal contributions to drug reinforcement using intravenous drug self-administration procedures. By innovating the application of synthetic transcription factors that can be programmed to exert novel forms of gene regulation across the genome, these techniques can be combined with existing RNA sequencing and complex behavioral procedures to serve as tools for both molecular interrogation into the transcription factor functions distinct to specific drugs of abuse and as highly specific transcription manipulations to uncover the causal gene regulatory events that drive certain SUDs. Together, this work will provide new approaches to identify the transcription factor functions at the core of specific SUDs and yield refined gene candidates as targets for future SUD medications.