Abstract 1532 Using an Expanded Genetic Code to Enrich Spatial Clarity for Protein Chromosomal Occupancy

Our lab focuses on histone interactions with the yeast chromatin remodeler, RSC, which has a total of seventeen subunits, making it difficult to mechanistically study, in real time. In particular, the RSC complex is notoriously known for being difficult to probe using chromatin immunoprecipitation (ChIP) methods. Our group has previously employed a photoreactive unnatural amino acid, pBpa (p-benzoyl-l-phenylalanine), that allowed us to covalently trap RSC subunit contacts to histone proteins and analyze mechanistic details in the living nucleus. Expanding on that work, we are developing an alternative ChIP protocol that first establishes a target protein (i.e. RSC subunit) crosslink to a histone and then apply standard ChIP procedures to isolate the DNA fragments that make contacts to the pBpa-histone, rather than the protein that experiences weaker DNA contacts. The advantage of this approach is that it provides a way to study proteins that make strong contacts to the nucleosome but have more transient associations with DNA. ChIP works especially well when studying histone positioning and we envision the analysis of the bridged (crosslinked) protein by tethering it to the histone prior to DNA isolation and analysis via ChIP. This allows for a spatiotemporal understanding of proteins that interact with histones at specialized nucleosomes, such as promoter regions. Using strains that produce tagged versions of RSC proteins we confidently identify histone-RSC associations under true physiological conditions. Our work has focused on studying the Rsc4 subunit and understanding how it is distributed amongst specialized genomic regions. We apply qPCR analysis across four genomic loci to ensure that histones harboring the unnatural amino acids are distributed evenly and randomly across the genome and then using a double IP process we can quantify the positioning of Rsc4 along the genome. National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM124600.