In a collection of ten studies, published jointly in Science and Science Advances, researchers highlight advances in understanding the so-called “4D nucleome” (4DN). The human genome is not simply a static blueprint but a dynamic, three-dimensional structure. The structure’s looping and folding, and changes in its organization over time, help determine when and where genes are expressed. This 4D nucleome has emerged as a critical layer of gene regulation, with disruptions increasingly linked to human disease. To better understand this dynamic system, the National Institutes of Health created the 4D Nucleome Program, which has since developed powerful datasets, technologies, and analytical tools to map genome architecture at single-cell resolution across healthy and diseased tissues. The studies highlighted in this collection apply these cutting-edge approaches across a wide range of biological systems, including the aging brain, Alzheimer's disease, heart disease, immune cell development, and other human tissues. Collectively, they reveal how the physical organization of DNA shapes cell identity, stores epigenetic information, and changes during disease. Together with new computational and imaging methods, these advances mark a step forward in spatial genomics, providing the broader scientific community with valuable resources to deepen our understanding of human development, aging, and disease. “Collectively, this package illustrates how far single-cell three-dimensional genomics has advanced since the 4DN program’s inception and how integrating chromatin architecture with gene expression and epigenetic data is reshaping our understanding of development, aging, and disease,” write Science Editors Di Jiang, Yevgenia Nusinovich, and Sara Ross. “The resources and methods introduced here are poised to accelerate discovery across the broader research community.”