Quantitative mapping of combinatorial histone modifications

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PROJECT SUMMARY Nucleosomes are the repeating building blocks of chromatin, composed of a histone octamer wrapped with DNA. Histone tails are decorated with a variety of post-translational modifications (PTMs), which together form a combinatorial molecular language (i.e. “the histone code”) that regulates gene expression and other physiological processes. Defects in this complex landscape are associated with vast human pathologies, most notably cancer, and histone PTMs are rapidly emerging diagnostic / prognostic indicators. For instance, H3K4me3 + H3K27me3 “bivalent” promoters are frequent targets of DNA hypermethylation in cancer, resulting in drastically altered expression of important developmental regulators. Reliable quantification of dual (i.e. combinatorial) PTMs may provide novel access to new biomarkers or drug targets with disease specificity, a major limitation of single PTM biomarkers to date. However, tools to study dual PTMs in vivo are lacking. Here, EpiCypher is developing EpiTandem™ Sensors, a first-in-class technology that uses combinations of chromatin reader domains as next-generation affinity reagents to directly detect dual PTMs. These breakout tools leverage the enhanced avidity of multivalent interactions for combinatorial PTMs, a key mechanism displayed by dual chromatin reader domains in vivo. A central innovation of this project is the application of EpiCypher’s recombinant designer nucleosomes (dNuc) technology to characterize EpiTandem Sensor binding specificity against singly- and combinatorially-modified nucleosome substrates. dNucs faithfully replicate the endogenous three-dimensional nucleosome structure, which is crucial to accurately define cooperative, multivalent chromatin interactions. We will apply our validated EpiTandem Sensors to CUT&RUN, an ultra- sensitive chromatin profiling method that generates high quality mapping data with significantly lower input and sequencing requirements vs. ChIP-seq. We will develop optimized CUT&RUN protocols for EpiTandem Sensors, demonstrating their utility to interrogate combinatorial PTMs genome-wide. In Phase I, we developed two EpiTandem Sensors and utilized dNucs (singly- and combinatorially-modified) to characterize and validate their exquisite binding specificity for dual PTMs (up to 90-fold vs. single PTMs). We then applied an EpiTandem Sensor and DNA-barcoded dNuc spike-ins to CUT&RUN, providing strong proof-of-concept for genomic mapping applications. In Phase II, we will develop a collection of five EpiTandem Sensors and complementary dNuc spike-ins (Aim 1), and rigorously validate them in CUT&RUN assays using a range of cell types, sample processing methods, and inputs, including drug treatment time course experiments to highlight their value in clinical research projects (Aim 2). In Aim 3 we will optimize commercial-scale manufacturing of the five Sensors and dNuc panels, assemble EpiTandem beta kits, and launch in-house CUT&RUN assay services for dual PTM mapping studies. We will provide beta kits to leading epigenetics laboratories for external testing, generating essential protocols and product literature in preparation for market release.