A novel platform for quantification of acute neuronal transcriptional responses

PROJECT SUMMARYAcute 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 CUTandTag 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.