The folding of genomic DNA from the beads-on-a-string like structure of nucleosomes into higher order assemblies is critically linked to nuclear processes. We have calculated the first 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. This has allowed us to study genome folding down to a scale of <100 kb and to validate the structures. We show that the structures of individual topological-associated domains and loops vary very substantially from cell-to-cell. By contrast, A/B compartments, lamin-associated domains and active enhancers/promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. Through studying pluripotency factor- and NuRD-regulated genes, we illustrate how single cell genome structure determination provides a novel approach for investigating biological processes.

Recent developments have for the first time allowed the determination of three-dimensional structures of individual chromosomes and genomes in nuclei of single haploid mouse embryonic stem (ES) cells based on Hi-C chromosome conformation contact data. Although these first structures have a relatively low resolution, they provide the first experimental data that can be used to study chromosome and intact genome folding. Here we further analyze these structures and provide the first evidence that G1 phase chromosomes are knotted, consistent with the fact that plots of contact probability vs sequence separation show a power law dependence that is intermediate between that of a fractal globule and an equilibrium structure.

Fluorescence imaging and chromosome conformation capture assays such as Hi-C are key tools for studying genome organization. However, traditionally, they have been carried out independently, making integration of the two types of data difficult to perform. By trapping individual cell nuclei inside a well of a 384-well glass-bottom plate with an agarose pad, we have established a protocol that allows both fluorescence imaging and Hi-C processing to be carried out on the same single cell. The protocol identifies 30,000-100,000 chromosome contacts per single haploid genome in parallel with fluorescence images. Contacts can be used to calculate intact genome structures to better than 100-kb resolution, which can then be directly compared with the images. Preparation of 20 single-cell Hi-C libraries using this protocol takes 5 d of bench work by researchers experienced in molecular biology techniques. Image acquisition and analysis require basic understanding of fluorescence microscopy, and some bioinformatics knowledge is required to run the sequence-processing tools described here.