Asymmetric nucleosome substrates to advance the study of the histone code

PROJECT SUMMARY Chromatin function is regulated by histone post-translational modifications (PTMs) on nucleosomes, which act in concert to recruit chromatin-associated proteins (CAPs) and modulate gene expression. This integrative signaling mechanism is termed the “histone code”. Recent work by EpiCypher and others has shown that histone PTM (a)symmetry (i.e., symmetric vs. asymmetric conformation) is an important factor in controlling CAP interactions, providing a basis for a previously unappreciated layer of complexity underlying chromatin regulation. Mass spectrometry data shows that nearly half of all nucleosomes are asymmetrically modified, including those carrying the canonical bivalent PTM signature (co-occurring active H3K4me3 and repressive H3K27me3) that denotes poised promoters. However, there are currently no available substrates or detection reagents to study PTM (a)symmetry in vitro or in vivo. Thus, new tools are needed to understand how PTM (a)symmetry regulates chromatin-CAP interactions, which will enable researchers to fully leverage an understanding of the histone code to develop next-generation biomarkers and epigenetic-targeted therapies. EpiCypher is developing a first-in-class toolbox to study how PTM (a)symmetry regulates nucleosome signaling. The technical innovation of this project is the development of a novel manufacturing method to efficiently generate asymmetric designer nucleosomes (a-dNucs) at commercial scale. The conceptual innovation of this project is the application of a-dNucs to drive advances in chromatin biology, including: 1) uncovering mechanisms of CAP interactions; and 2) developing genomic mapping assays using a novel recombinant detection reagent. To date, commercial dNuc manufacture has been limited to symmetric dNucs, as existing methods for generation of a-dNucs are highly inefficient. In Phase I equivalent studies, we developed a novel strategy that bridges asymmetric histone tail dimers for highly efficient a-dNuc manufacturing. We then applied these a-dNucs to characterize CAP interactions, demonstrating the role of PTM (a)symmetry in the histone code. In Phase II, we will generate additional a-dNucs and apply them as substrates for biochemical assays and for development of first-to-market epigenomic assays that map asymmetrically modified bivalent promoters in vivo. First, we will generate an expanded set of a-dNucs at commercial scale, including disease- relevant bivalent marks (Aim 1). Next, we will demonstrate the application of a-dNucs to perform mechanistic dissection of CAP interactions (Aim 2). Finally, we will leverage a-dNucs to develop a novel detection reagent that binds the highly studied bivalent promoter signature, then utilize this detection reagent to develop next- generation genomic mapping assays using EpiCypher’s well-established CUT&RUN platform (Aim 3). These tools will be first-to-market products to study how nucleosome (a)symmetry impacts chromatin regulation, and their availability has significant impact potential, driving advances for important clinical applications including development, aging, cancer and drug discovery.