Abstract. The integral roles of histone post-translational modifications (PTMs), including lysine methylation and acylation, in chromatin remodeling is well established, but new roles of histone PTMs in diseases, including many types of cancer, are continually discovered. This rapidly evolving field necessitates the development of new molecular approaches to probe the biological impacts of these PTMs. Herein we focus on the reader proteins for lysine methylation (Kme) and crotonylation (Kcr), as reader proteins are responsible for sensing the PTM and triggering the biological outcome, and both Kme and Kcr have biomedical relevance. Aims 1 and 2 make use of global analysis strategies to determine distinct molecular recognition features across Kme and Kcr reader proteins, respectively, that will advance the ability to develop selective inhibitors and molecular probes for these two classes of proteins. Aims 3 and 4 will exploit the inherent selectivity of reader proteins to develop engineered Kme and Kcr reader proteins (eReaders and eWriters) for sensing, labeling, and introduction of dual PTMs in a site selective manner. This combined effort will advance the field by providing new insight and new tools to study the biological pathways that depend on these PTMs and their role in disease.