Novel recombinant sensors to study histone ubiquitin signaling

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PROJECT SUMMARY Monoubiquitination (Ub) of histones is an important post-translational modification (PTM) that regulates multiple DNA-related processes including DNA replication, transcription, and repair. Aberrant regulation of histone Ub is strongly linked to the pathogenesis of diverse diseases, including cancer, aging, and inflammation. However, the ability to map the distribution and dynamics of histone Ub signals has been challenged by a lack of high-quality tools, most notably detection reagents. EpiCypher performed a screen of commercial histone Ub antibodies using a panel of monoubiquitinated recombinant designer nucleosomes (Ub-dNucs) and found that nearly every commercial histone ubiquitin antibody is not specific for its intended target. Thus, next-generation detection reagents are greatly needed to advance our understanding of histone Ub signaling, opening the door to new avenues of high value drug and biomarker discovery. In this Direct to Phase II application, EpiCypher is developing UbSensors™, a novel class of detection reagents that leverage chromatin reader domains to specifically detect Ub on nucleosomes to enable analysis of histone Ub signaling dynamics in vivo. Recombinant UbSensors are designed using a structure-guided approach to combine binding domains that interact with a nucleosomal surface (Anchor) and ubiquitin (UBD) with a rationally designed Linker, providing high selectivity towards site-specific nucleosomal Ub targets. In Phase I equivalent studies, we collaborated with Drs. Robert Cohen and Tingting Yao (Colorado State University), who developed UbSensors to target H2AK119ub1, H2BK120ub1, H2AK15ub1, or any histone Ub moiety (pan-Ub). We validated these reagents in a biochemical binding assay using Ub-dNucs, demonstrating that the UbSensors are highly specific for their intended Ub-Nuc targets and vastly outperform current best-in- class antibodies. In addition, we successfully applied our UbSensors to interrogate the distribution of histone Ub genome-wide using CUT&RUN (Cleavage Under Targets & Release Using Nuclease), a breakthrough genomic mapping technology that generates high quality data with low cell input and sequencing requirements. In Phase II, we will expand upon our structure-guided designs and develop additional UbSensors, and apply these reagents to characterize Ub-Nuc signaling dynamics using cutting-edge genomic mapping assays. In Aim 1, we will develop additional UbSensors to target new Ub-Nuc modifications as well as explore various epitope tags to improve performance in downstream assays. In Aim 2, we will further develop the application of UbSensors for CUT&RUN by developing a DNA-barcoded Ub-dNuc spike-in panel and performing rigorous validation with a range of cell types, sample processing methods, and inputs. In Aim 3, we will prepare for commercial launch at the conclusion of Phase II by scaling UbSensor manufacturing and developing a lot release strategy, as well as performing external validation and demonstrating the utility of UbSensors for high-value genomics research by generating the first reliable maps of nucleosomal Ub signals in DNA double strand break repair.