Quantification of combinatorial epigenetic modifications using defined nucleosome standards

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PROJECT SUMMARY Post-translational modification of histone tails (histone PTMs) and DNA methylation (DNAme) on nucleosomes form a sophisticated molecular code that regulates gene transcription. Aberrant regulation of these chromatin modifications is associated with a vast array of human pathologies. While the majority of work in the field has focused on signatures of individual modifications, combinations of histone PTMs and/or DNAme can be more specific and informative than single marks alone. For instance, although healthy cells and cancerous cells both have H3K27me3 and DNAme distributed genome-wide, the co-localization of these two modifications occurs uniquely in cancer cells. However, existing tools to measure global levels of chromatin modifications are low-throughput, display low sensitivity, and are unable to measure combinatorial modifications (e.g. immunoblot). The development of assays that overcome these limitations and are compatible with multiple sample types (including cellular samples or plasma [for detection of circulating nucleosomes, i.e. liquid biopsy]) will make the study of chromatin modifications widely accessible for academic, clinical, and pharmaceutical research. Here, EpiCypher will develop QuantiNucTM assays, a breakthrough epigenetics platform to quantify single and combinatorial chromatin modifications directly on nucleosomes from cells or plasma samples. The innovation of this proposal includes the a) application of designer nucleosomes (dNucs) to systematically identify top-performing detection reagents and to serve as quantitative assay standards, b) development of recombinant EpiSensors for unbiased detection of DNA and DNAme, and c) development of a proprietary targeted sample processing method for high-throughput cell-based assays. Overall, this platform will provide a quantitative, low- cost, and scalable approach to leverage analysis of chromatin modifications (i.e. histone PTMs and/or DNAme) for chromatin research, drug development, and novel biomarker discovery. In Phase I, we developed a QuantiNuc assay targeting combinatorial H3K4me3+H3K27ac, PTMs that are co-enriched at actively expressed genes. We validated the specificity and performance of this QuantiNuc assay by establishing key analytical parameters and applying the assay to quantify levels of H3K4me3+H3K27ac nucleosomes from human plasma samples. In Phase II, we will develop new QuantiNuc assays to measure other high-value single and combinatorial chromatin modifications and further validate these assays for use with human plasma samples (i.e. liquid biopsy). In addition, we will develop a novel targeted sample processing method for cell-based QuantiNuc assays, which will streamline the process of cell lysis and chromatin fragmentation to deliver a high- throughput, low-cost approach for clinical research. Finally, we will prepare for commercial launch of QuantiNuc assays by assembling beta-kits and performing internal and external validation testing of both liquid biopsy and cell-based assays, which will be used to develop reliable assay protocols and product literature. Market availability of these assays will transform biomarker discovery and accelerate epigenetic drug development.