Histone post-translational modifications play pivotal roles in eukaryotic gene expression through regulation of chromatin structure and/or recruitment of regulatory proteins to chromatin. To date, most studies have focused on modifications in unstructured histone N-terminal tail domains. In contrast, histone globular domains surrounded by DNA have been largely overlooked, despite the richness of their multiple modifications. Application of nucleosomes with homogeneous modifications (e.g., acetylation, methylation and phosphorylation), generated using recently developed genetic and chemical engineering techniques, will provide powerful tools for identifying and characterizing globular domain-modifying enzymes and globular domain-interacting proteins that transcend the capabilities of existing methods. I will discuss several current works in my laboratory to reveal novel mechanisms of gene expression regulation through systematic identification and analysis of new proteins.