The nucleus is the most prominent structure in eukaryotic cells. It houses the cell’s DNA and is the hub for DNA replication, transcription, and RNA processing. Despite its prominence and importance, our understanding of how the nucleus and its DNA are organized in space and time—and the implications of that organization for proper function—has lagged behind that of other cellular structures. Written and edited by experts in the field, this collection from Cold Spring Harbor Perspectives in Biology covers recent advances in our understanding of nuclear organization and function. The contributors discuss the 3D organization of chromatin, the various nuclear bodies and compartments that have been identified, and the roles of RNA and actin in shaping nuclear organization, as well as how these structures interact with each other and with peripheral features (e.g., the nuclear pore complex and inner nuclear membrane proteins) to carry out the work of the nucleus. Insights into DNA replication timing and RNA processing dynamics based on new technologies aimed at examining chromatin and other intranuclear structures at high resolution are also included. Multiple chapters are devoted to physiological and disease processes involving disruption of nuclear structure and function (e.g., viral infection). This volume is therefore essential reading for all cell and molecular biologists, as well as pathologists interested in the role of nuclear architecture in disease.