MEASURING NONCOVALENT INTERACTIONS INVOLVED IN THE RECOGNITION OF HISTONE POST-TRANSLATIONAL MODIFICATIONS AND ENGINEERING READER PROTEIN SCAFFOLDS INTO IMPROVED TOOLS FOR EPIGENETICS RESEARCH

Precise and temporal regulation of DNA-templated processes is maintained by the hierarchical systems of epigenetics. As a core structural unit of the epigenome, chromatin architecture is in part governed by the histone code – the covalent modifications to histone proteins that direct biological functions through the physiochemical properties of the posttranslational modifications (PTMs) themselves. As required features for maintaining the homeostasis of the phenotypic genome, dysregulation of these marks is equally devastating to eukaryotic biology, and aberrant levels of PTMs are associated with the development of disease. Consequentially, detailed investigations are needed for understanding the chemical basis by which PTMs influence dynamic epigenetic states. These insights contextualize the implications of the histone code among the other levels of epigenetic regulatory elements and inform the design of therapeutics that specifically target the macromolecular complexes that associate with these marks. The work herein describes our contributions to both mechanistically characterizing the discrete noncovalent interactions necessary for selective recognition of the important histone PTM trimethyllysine (Kme3), and engineering the protein scaffolds that natively bind these marks, called reader proteins, into beneficial detection reagents for epigenetics research. Towards our first aim, two classes of reader proteins for Kme3 were extensively studied to measure the contributions of individual residues within their conserved site for recognition called an aromatic cage. Using biophysical and chemical biology techniques, we evaluated differences in the noncovalent driving forces implicated in binding Kme3. In pursuit of this goal, we discovered several readers that do not selectively recognize this mark on the premise of its positive charge and characterizing the change in mechanism between binding Kme3 and a neutral analog is impactful for the development of chemical probes and inhibitors. Secondly, we capitalized on the inherent selectivity of reader proteins and through a rational design approach engineered a native Kme3 reader protein into a modular reagent that provides enhanced detection of Kme3 in standard chromatin assays. Together, our work furthers our understanding of the balance of noncovalent interactions that contribute to the dynamic regulation of epigenetic states by Kme3 and has provided additional tools for detecting these marks in chromatin research.Doctor of Philosoph