Controlled transcription of genes is critical for cell differentiation and development. Gene expression regulation therefore involves a multilayered control from nucleosome composition in histone variants and their post-translational modifications to higher-order folding of chromatin fibers and chromatin interactions in nuclear space. Recent technological advances have allowed gaining insight into these mechanisms, the interplay between local and higher-order chromatin organization, and the dynamic changes that occur during stress response and developmental transitions. In this review, we will discuss chromatin organization from the nucleosome to its three-dimensional structure in the nucleus, and consider how these different layers of organization are maintained during the cell cycle or rapidly reprogrammed during development.