Quantitative mapping of dynamic epigenetic states in rare and stimulated immune cells

PROJECT SUMMARY Histone post-translational modifications (PTMs) play important roles in modulating chromatin structure and gene expression, and dysregulation of these marks is associated with immune and inflammatory diseases. Recent work by Dr. Steven Josefowicz and EpiCypher highlights the dynamic regulation of histone phosphorylation (i.e. phospho-PTMs) at induced genes in stimulated primary macrophages, revealing new roles for these underappreciated marks. Indeed, our studies strongly suggest that the study of stimulation-responsive chromatin dynamics in diverse primary immune cell types will unlock novel regulatory mechanisms directly related to immune and inflammatory diseases. Phospho-PTMs and other stimulation-responsive marks are transient in nature, and must be studied in primary cells (vs. proliferative cell lines). However, analysis of PTMs (phospho-PTMs and other types) in primary immune cells has been limited by existing chromatin mapping assays (i.e. chromatin immunoprecipitation [ChIP-seq]), which display poor signal-to-noise (S/N), are extremely low-throughput and expensive, and lack the sensitivity to study rare cell populations. Even CUT&RUN (Cleavage Under Targets & Release Using Nuclease), a new immunotethering approach that vastly outperforms ChIP-seq, still lacks the sensitivity to map histone PTMs in primary cells at low inputs (≤10K cells). To meet these needs, EpiCypher is developing EpiPrime-seqTM, an ultra-sensitive genomics platform for immunology research. A key innovation of our approach is the development of a novel CUT&RUN-based protocol to profile PTMs, including stimulation-responsive PTMs, from primary immune cells. This approach includes development of a novel "direct-to-PCR" approach that drastically improves assay sensitivity to enable low primary cell inputs (≤10K cells). We will leverage EpiCypher’s designer nucleosome (dNuc) technology to develop controls carrying diverse phospho-PTMs, enabling antibody validation and in-assay technical monitoring and normalization. For Phase I feasibility, we developed a set of phosphorylated dNucs and used them to identify highly specific phospho-PTM antibodies. These tools were used to develop EpiPrime-seq, mapping phospho- PTM at induced genes following stimulation in both abundant and rare, sorted primary immune cells. In Phase II, we will extend EpiPrime-seq development for improved sensitivity and throughput. We will develop an expanded set of phosphorylated dNuc spike-ins (PhosphoStat panel) and validate phospho-PTM antibodies. We will develop robust EpiPrime-seq protocols for ≤10K primary cells, using our novel direct-to-PCR approach to map stimulation-responsive phospho- and methyl-lysine PTMs in diverse primary immune cells. Following these studies, we will prepare for commercial release by establishing lot-release strategies and performing validation studies for EpiPrime-seq kits and PhosphoStat spike-ins. Finally, we will leverage EpiCypher’s recent advances in high-throughput CUT&RUN assays to develop automated EpiPrime-seq protocols, which we will leverage to launch assay services and create novel stimulation-responsive epigenomic atlases.