ABSTRACT One of the epigenomic changes frequently observed during eukaryotic differentiation is the proteolysis of the histone H3 N-terminal tail (H3NT) within chromatin. Although H3NT proteolysis was first described over 60 years ago and is a common feature of diverse eukaryotic developmental pathways, the functional significance of this programmed epigenetic event remains largely undetermined. Progress in the field has been significantly hindered by the lack of methods to identify the specific loci targeted for H3NT proteolysis. To overcome this barrier we pioneered the first genome-wide method to discover and investigate H3NT-cleaved (H3cl) loci, called ChIPac-Seq. The goal of this proposal is to optimize the technical and computational aspects of ChIPac- Seq, which would support future efforts to adapt the ChIPac-Seq approach for high-throughput applications. To this end we will leverage ChIPac-Seq to discover the cell type-specific loci selectively targeted for H3NT proteolysis in differentiated osteoblasts, myocytes and adipocytes derived from the same multipotent progenitor cell in vitro. The expected low concordance of H3cl loci between cell types provides the unique opportunity to optimize the ChIPac-Seq computational analysis to identify bona fide H3cl loci with the highest degree of rigor and to simplify and streamline the pipeline for use by the broad scientific community. Furthermore, the direct functional effects of H3NT proteolysis in generating specific epigenetic changes at the H3cl loci in terms of histone modifications, chromatin structure and transcription factor binding will be examined by leveraging the wealth of publically available NGS datasets. The anticipated outcomes of this study will yield transformative insights into the sites and mechanistic functions of H3NT proteolysis as a novel epigenetic regulator and, importantly, produce an optimized ChIPac-Seq pipeline for use by the general scientific community.