Eukaryotic genetic material is stored as chromatin, wherein DNA is wound around repeating octameric units of histone proteins. This nucleosomal subunit represents the most basic level of DNA compaction. Chromatin remodeling complexes are multi-subunit nucleosome translocases that can physically reposition nucleosomal subunits for the purpose of major biological processes such as DNA replication. The RSC complex is the most abundant remodelling complex of budding yeast, with high evolutionary conservation in animals and plants. Current research of RSC’s function and mechanics is limited. In this work, we map interactions between the catalytic subunit of RSC, Sth1, and the nucleosome of living yeast using a genetically encoded unnatural amino acid photo-crosslinker. The crosslinker, p-benzoylphenylalanine (pBPA), is site-specifically encoded into histone proteins and covalently traps histone-remodeler interactions upon UV exposure. Remodeler binding is then identified using short peptide fusion tags to identify the Sth1 protein. This allows for the illumination of detailed interaction maps of the chromatin remodeler complex, which provides new insights into the biological relevance of previously determined structural data. We verify the interactions by western blot analysis and clearly visualize crosslinked products between a myc-tagged Sth1 protein with histones H2A and H3. We show that an acetylation event at H3K14 controls recruitment and binding of Sth1 to histone H3. Histone H3 that lack this essential post-translational modification are unable to sequester Sth1 to the nucleosome, shedding new biological relevance on the RSC-nucleosomal mechanistic/functional relationship. Additionally, we believe the bromodomain of Sth1 to be the recognition module for the acetylation signal.

Exploring interactions between chromatin and essential chromatin remodeling complex <i>in vivo</i>

2019 Fall National Meeting

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Exploring interactions between chromatin and essential chromatin remodeling complex in vivo