A Genetically Encoded Approach for Breaking Chromatin Symmetry. Author Bradley Lukasak, Robert Thompson, Michelle Mitchener, Vanessa Feng, John Bagert, Tom Muir Publication Year 2022 Type Journal Article Abstract Nucleosomes frequently exist as asymmetric species in native chromatin contexts. Current methods for the traceless generation of these heterotypic chromatin substrates are inefficient and/or difficult to implement. Here, we report an application of the SpyCatcher/SpyTag system as a convenient route to assemble desymmetrized nucleoprotein complexes. This genetically encoded covalent tethering system serves as an internal chaperone, maintained through the assembly process, affording traceless asymmetric nucleosomes following proteolytic removal of the tethers. The strategy allows for generation of nucleosomes containing asymmetric modifications on single or multiple histones, thereby providing facile access to a range of substrates. Herein, we use such constructs to interrogate how nucleosome desymmetrization caused by the incorporation of cancer-associated histone mutations alters chromatin remodeling processes. We also establish that our system provides access to asymmetric dinucleosomes, which allowed us to query the geometric/symmetry constraints of the unmodified histone H3 tail in stimulating the activity of the histone lysine demethylase, KDM5B. By providing a streamlined approach to generate these sophisticated substrates, our method expands the chemical biology toolbox available for interrogating the consequences of asymmetry on chromatin structure and function. Journal ACS Cent Sci Volume 8 Issue 2 Pages 176-183 Date Published 2022 Feb 23 ISSN Number 2374-7943 DOI 10.1021/acscentsci.1c01332 Alternate Journal ACS Cent Sci PMCID PMC8875426 PMID 35233450 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML