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.

Chromosome structure in mammals is thought to regulate transcription by modulating three-dimensional interactions between enhancers and promoters, notably through CTCF-mediated loops and topologically associating domains (TADs)1–4. However, how chromosome interactions are actually translated into transcriptional outputs remains unclear. Here, to address this question, we use an assay to position an enhancer at large numbers of densely spaced chromosomal locations relative to a fixed promoter, and measure promoter output and interactions within a genomic region with minimal regulatory and structural complexity. A quantitative analysis of hundreds of cell lines reveals that the transcriptional effect of an enhancer depends on its contact probabilities with the promoter through a nonlinear relationship. Mathematical modelling suggests that nonlinearity might arise from transient enhancer–promoter interactions being translated into slower promoter bursting dynamics in individual cells, therefore uncoupling the temporal dynamics of interactions from those of transcription. This uncovers a potential mechanism of how distal enhancers act from large genomic distances, and of how topologically associating domain boundaries block distal enhancers. Finally, we show that enhancer strength also determines absolute transcription levels as well as the sensitivity of a promoter to CTCF-mediated transcriptional insulation. Our measurements establish general principles for the context-dependent role of chromosome structure in long-range transcriptional regulation.

In mammals, interactions between sequences within topologically associating domains enable control of gene expression across large genomic distances. Yet it is unknown how frequently such contacts occur, how long they last and how they depend on the dynamics of chromosome folding and loop extrusion activity of cohesin. By imaging chromosomal locations at high spatial and temporal resolution in living cells, we show that interactions within topologically associating domains are transient and occur frequently during the course of a cell cycle. Interactions become more frequent and longer in the presence of convergent CTCF sites, resulting in suppression of variability in chromosome folding across time. Supported by physical models of chromosome dynamics, our data suggest that CTCF-anchored loops last around 10 min. Our results show that long-range transcriptional regulation might rely on transient physical proximity, and that cohesin and CTCF stabilize highly dynamic chromosome structures, facilitating selected subsets of chromosomal interactions.

Chromosome structure in mammals is thought to regulate transcription by modulating the three-dimensional interactions between enhancers and promoters, notably through CTCF-mediated interactions and topologically associating domains (TADs)1–4. However, how chromosome interactions are actually translated into transcriptional outputs remains unclear. To address this question we use a novel assay to position an enhancer at a large number of densely spaced chromosomal locations relative to a fixed promoter, and measure promoter output and interactions within a genomic region with minimal regulatory and structural complexity. Quantitative analysis of hundreds of cell lines reveal that the transcriptional effect of an enhancer depends on its contact probabilities with the promoter through a non-linear relationship. Mathematical modeling and validation against experimental data further provide evidence that nonlinearity arises from transient enhancer-promoter interactions being memorized into longer-lived promoter states in individual cells, thus uncoupling the temporal dynamics of interactions from those of transcription. This uncovers a potential mechanism for how enhancers control transcription across large genomic distances despite rarely meeting their target promoters, and for how TAD boundaries can block distal enhancers. We finally show that enhancer strength additionally determines not only absolute transcription levels, but also the sensitivity of a promoter to CTCF-mediated functional insulation. Our unbiased, systematic and quantitative measurements establish general principles for the context-dependent role of chromosome structure in long-range transcriptional regulation.

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