A novel platform for quantification of acute neuronal transcriptional responses

项目总结 急性转录反应是重要的神经生物学过程的基础，从发育到 学习和记忆，并被假设为物质使用障碍背后的驱动力。 然而，目前测量转录急剧变化的方法缺乏对能量的敏感性和吞吐量。 对神经元刺激的即时转录反应的详细的机械研究。广泛使用的信使核糖核酸 序列分析通过量化稳定的、多腺化的RNA的整体水平来测量转录，这些RNA掩盖了早期 转录反应。Mna-seq也不能捕获非蛋白质编码的rna，如增强子rna。 (ERNAs)，它们与增强子活性相关，并在神经元的增强子功能中发挥重要作用。 测量新合成的“新生RNA”的分析方法能够测量转录的早期变化， 包括eRNA。然而，目前用于测量新生RNA合成的分析需要复杂的工作流程和 表现出较差的分析灵敏度，限制了它们在神经科学研究中的使用。与其测量新生的RNA， 直接测量RNA聚合酶为研究转录提供了一种强有力的方法 这些限制。RNA聚合酶II(RNAPII)是一种转录编码mRNAs和 非编码RNA，包括eRNAs，并包含差异表达的羧基末端结构域(CTD) 磷酸化以促进转录起始和延伸。定位RNAPII和RNAPII的基因组位置 它的磷酸盐为直接测量转录动力学提供了一个独特的机会，但目前的分析 要映射RNAPII(例如，CHIP-SEQ)缺乏实现这一点的敏感性。 在这里，EpiCypher正在开发Cutana-RNAPII，一种超灵敏的RNAPII图谱分析，直接 量化神经科学研究和药物对细胞外刺激的全球转录动力学 发展。我们提案的创新之处在于开发了修改后的Cut&Tag工作流，其目标是 不同的RNAPII CTD磷酸化位点来测量转录起始和延伸的早期变化。 这项第一阶段可行性研究的目标是证明Cutana-RNAPII分析方法对测量的敏感性 神经元的早期转录反应。在目标1中，我们将开发Cutana-RNAPII工作流程和 将我们的检测与当前的金标准新生RNA检测(PRO-SEQ检测)进行比较 细胞对激素处理的反应的转录动力学。在《目标2》中，我们将与佐伊·麦克埃利戈特博士合作 证明Cutana-RNAPII在神经科学中测量早期转录动力学的能力 阿片类药物暴露模型。在第一阶段取得成功后，在第二阶段我们将开发强大的Cutana-RNAPII Beta试剂盒和自动分析服务，使用低样本投入针对脑组织进行优化，包括 单细胞。这项工作将是部署Cutana-RNAPII来研究高度异质性大脑样本的关键 用于突破性的神经科学和物质使用研究。这个低成本的化验平台将彻底改变 可以在复杂的组织中研究转录动力学，用于临床研究和药物开发研究。

PROJECT SUMMARY Acute transcriptional responses underlie essential neurobiological processes from development to learning and memory, and have been hypothesized to be a driving force behind substance use disorders. However, current assays to measure acute changes in transcription lack the sensitivity and throughput to power detailed, mechanistic studies of the immediate transcriptional response to neuronal stimuli. Widely used mRNA- seq assays measure transcription by quantifying bulk levels of stable, polyadenylated RNA, which mask early transcriptional responses. mRNA-seq also fails to capture non-protein coding RNAs, such as enhancer RNAs (eRNAs), which are correlated with enhancer activity and play essential roles in enhancer function in neurons. Assays that measure newly synthesized ‘nascent RNAs’ are capable of measuring early changes in transcription, including eRNAs. However, current assays to measure nascent RNA synthesis require complex workflows and exhibit poor assay sensitivity, limiting their use for neuroscience research. Rather than measuring nascent RNAs, direct measurement of RNA polymerase provides a powerful approach to study transcription that overcomes these limitations. RNA polymerase II (RNAPII) is the polymerase that transcribes protein coding mRNAs and non-coding RNAs, including eRNAs, and contains a carboxyl-terminal domain (CTD) that is differentially phosphorylated to promote transcription initiation and elongation. Mapping the genomic position of RNAPII and its phosphoforms provides a unique opportunity to directly measure transcriptional dynamics, but current assays to map RNAPII (e.g. ChIP-seq) lack the sensitivity to accomplish this. Here, EpiCypher is developing CUTANA-RNAPII, an ultra-sensitive RNAPII mapping assay that directly quantifies global transcriptional dynamics in response to extracellular stimuli for neuroscience research and drug development. The innovation of our proposal is the development of a modified CUT&Tag workflow that targets various RNAPII CTD phosphorylation sites to measure early changes in transcription initiation and elongation. The goal of this Phase I feasibility study is to demonstrate the sensitivity of CUTANA-RNAPII assays to measure early transcriptional responses in neurons. In Aim 1, we will develop the CUTANA-RNAPII workflow and benchmark our assay against the current gold-standard nascent RNA assay (PRO-seq assay) to measure transcriptional dynamics in cells in response to hormone treatment. In Aim 2, we will work with Dr. Zoe McElligott to demonstrate the ability of CUTANA-RNAPII to measure early transcriptional dynamics in a neuroscience model of opioid exposure. Following success in Phase I, in Phase II we will develop robust CUTANA-RNAPII beta kits and automated assay services that are optimized for brain tissue using low sample inputs, including single cells. This work will be key for deploying CUTANA-RNAPII to study highly heterogeneous brain samples for breakthrough neuroscience and substance use research. This low-cost assay platform will revolutionize how transcriptional dynamics can be studied in complex tissues for clinical research and drug development research.