Rapid and robust assay for measurement of in vivo activity of chromatin-interacting proteins

PROJECT SUMMARY The activity of chromatin-associating proteins (CAPs) is fundamental to regulation of gene expression. Elucidating CAP activity is central to understanding the regulatory mechanisms that drive cell biology, both in the context of health and disease, and to guide development of novel therapeutics. However, this work is not straightforward, as CAP activity is regulated by multiple factors including post-translational modifications and subcellular localization. Thus, assays to study CAP activity must specifically detect CAPs in their active form (i.e., bound to chromatin, directly or in complex). Genomic mapping assays can provide detailed information on CAP engagement and localization; however, these assays are inaccessible to a majority of researchers and unsuitable for large-scale, high-throughput studies due to a high barrier of entry associated with cost, complexity, equipment, and data analysis. Other existing assays to study CAPs capture total cellular abundance (e.g., EMSAs, ELISAs, IHC) or transcript levels (e.g., qPCR, RNA-seq), and do not specifically quantify CAPs actually associated with chromatin in vivo. Thus, there is a field need for a high-throughput, low-cost assay that is accessible to all researchers, and can inform CAP activity via quantification of in vivo engagement on chromatin. Market availability of such an assay would be transformative for biomedical research by providing convenient access to study the role and response of CAP in gene regulatory programs, including analysis of drug mechanism of action and immune stimulus responses, as well as for high-throughput drug screening. Here, EpiCypher will develop QuantiCAPTM, a breakthrough assay that will enable the study of in vivo activity of CAPs. The innovation of this proposal is the development of a targeted approach to directly quantify the total amount of CAP-bound chromatin in cells. Our approach leverages an in situ immunotargeting strategy to excise CAP-bound chromatin combined with highly sensitive detection by a fluorescent DNA stain. Overall, this platform will provide the first quantitative, low-cost, and scalable approach to leverage analysis of CAPs for biomedical research. In this Direct to Phase II research program, we are highlighting development of the platform to study transcription factors (TFs) and chromatin reader proteins, though our platform will be broadly applicable for the study of any CAP. In Phase I, we developed an automated QuantiCAP workflow, demonstrating feasibility for the assay to quantify changes in a CAP following drug treatment, and identified assay conditions to further improve sensitivity. In Phase II, we will extend QuantiCAP assay development to cover five (5) high value targets using both manual and automated workflows, including genomics validation. We will validate our assays across diverse primary immune cells and stimuli, then determine cell input thresholds, a robust normalization strategy, and quantitative assay parameters. Finally, we will prepare for marketing and commercialization by developing a QuantiCAP beta kit and applying the automated assay to perform high-impact drug development studies.