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

项目总结 伏隔核(NAC)是大脑的一个关键区域，它编码对自然和药物的反应 增强剂。众所周知，所有滥用药物都会增加NAC中的多巴胺能张力，使其成为中枢 在协调药物反应方面的结构。滥用药物主要通过突触靶点改变细胞内 信号级联导致转录因子的激活或抑制，进而诱导或 抑制特定基因的表达。这些脑转录因子的药物诱导功能是 对神经元内的表观遗传格局敏感，并对其做出贡献，并与持久相关 神经功能和药物相关行为的改变。因此，了解药物诱导的作用 对大脑这一区域转录因子的研究可能会扩大我们对滥用药物如何协调的理解 毒品相关行为的持久变化。这一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，我提出了以合成生物学为中心的创新 对转录因子的功能进行重新编程的方法，以便在清醒和健康儿童的大脑中传递病毒 表现为小鼠，以解决确切的，具有破坏性的转录因子功能，这些功能与刺激剂和 阿片成瘾。我建议通过应用新的合成转录因子结构来实现这一点。 结合脑转录组图谱和药物自我给药程序。这个项目的重点是 ZFP189，一种新的药物激活的转录因子，我在我早期的毕业生中仔细描述了它的特征 工作。首先，在NAC，我将通过病毒传递合成的ZFP 189转录因子，它具有人工合成的 能够在体内对靶基因施加不同形式的转录控制的功能部分。这就做 执行RNA测序方法，以揭示ZFP 189的完整基因调节功能是如何 在兴奋剂与阿片类药物使用方面存在差异。其次，我将利用这些相同的合成材料 ZFP189转录因子在静脉药物强化中的因果作用研究 药物自我管理程序。通过创新合成转录因子的应用，可以 这些技术被编程为在整个基因组中施加新的基因调控形式，可以组合在一起 利用现有的RNA测序和复杂的行为程序作为工具 对转录因子功能的询问与滥用特定药物截然不同，并具有高度特异性 转录操作，以揭示驱动某些肥皂水的因果基因调控事件。总而言之，这 这项工作将提供新的方法来识别特定SUD核心的转录因子功能和 产生精制的候选基因，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.