The packaging of genomic DNA into nucleosomes is a ubiquitous feature of eukaryotic genomes. Conventionally, nucleosomes have been viewed as DNA storage units that obstruct the accessibility of DNA to the transcription machinery, thereby suppressing gene expression. However, the nucleosome’s unique topology and its hierarchical organization into chromatin suggest a multifaceted regulatory repertoire encoded by its physical traits (shape, dimension, thermodynamics, etc.). This concept is fortified by recent studies, including work from our lab, that employ quantitative biophysical tools to dissect the dynamic interaction between the nucleosome and its associated factors. These findings establish nucleosomes as major activity hubs for chromatin regulators. The experimental platforms that we set up—capable of directly following transient and heterogeneous molecular events—pave the way for deriving mechanistic models of how the nucleosome harnesses its biophysical properties to direct genomic activities, and how disease-associated mutations in chromatin and its regulators perturb genome function.