3D structures of individual mammalian genomes studied by single-cell Hi-C

a, Comparison of 3D images of CENP-A in haploid mouse ES cell nuclei, expressing mEos3.2-tagged CENP-A and tandem infrared fluorescent protein (iRFP)-tagged histone H2B, with their corresponding white-light images. b, Comparison of three single-cell Hi-C contact maps (above the diagonal; contacts coloured red, yellow and blue) with the population Hi-C map (below the diagonal). c, An analysis of the accuracy and precision of the 100-kb structure calculation procedure for cell 1. The graphs show how the global (dis)similarity of structures is affected by: the total number of contacts (left); the number of inter-chromosomal contacts (middle); and the number of random noise contacts (right). Mean r.m.s.d. values for all pairs of conformations ± s.e.m. are shown for: the precision within ensembles arising from ten re-calculations using the same contacts (red); the variation across ensembles that arises from different random resampling (blue); and, as a measure of accuracy, the similarity to the best ensemble of structures (yellow). d, An example of a structure calculation carried out using either a single dataset, or after randomly merging 50% of the data from two different cells. Strongly violated experimental restraints (>4 particle radii apart) are shown in red. The plot (right) shows the probability of any two particles connected by an experimental restraint being violated to different degrees. e, Left, the structure of chromosome 1 from cell 6, where part of the chromosome lies at the opposite side of the genome structure, with no intermediate chromosome folding, illustrating the presence of a chromosomal break or recombination event. Right, the contact map shows that there are no contacts from the disconnected region to any other part of chromosome 1, but clear contacts to chromosomes 3 and 7. f, An example of an attempted calculation of the haploid genome structure for a cell containing a duplicated chromosome 2 shows many violations of the experimental restraints for that chromosome and a much more compacted structure (here compared with chromosomes 1 and 3). The structures are coloured according to position in the chromosome sequence from red to purple (centromere to telomere).

a, Structure of the entire haploid mouse ES cell genome from cells 2 to 8. The structural ensemble is represented by five superimposed conformations from repeat calculations, and is shown in three different orientations (after rotation through 90° relative to each other), with the chromosomes coloured according to their position in the chromosome sequence from red to purple (centromere to telomere). b, Correspondence between the distribution of Hi-C contacts (both cis and trans), violations of the distance restraints in the 3D structures, and DNA replication timing¹³ for a representative chromosome (chromosome 12). c, Left, log-scale plots of contact probability (P_cont) against sequence separation (S). The slopes for a power law relationship (P_cont ∝ S^α) in which α is either −1.0 or −1.5 are also indicated. Data are shown for the combined single-cell Hi-C contact data, for all of the non-sequential particles that are close to each other in the structures (<2 particle radii apart), and for the population Hi-C data. Right, the distribution in the number of intra-chromosomal (cis) or inter-chromosomal (trans) contacts between 100-kb regions in the single-cell Hi-C data are shown for both the A and B compartments. d, Correlation of gene expression levels (left), and hierarchically clustered heat maps showing the pairwise enrichment of ChIP–seq peak overlaps between haploid and diploid ES cells (centre), and Nanog ChIP–seq peak overlaps between haploid and diploid ES cells used in this study, as well as that previously published from diploid ES cells (right)⁴¹.

a, Violin plot showing the proportion of each chromosome that intermingles with other chromosomes. b, Pairwise comparison of the chromosome structure in different cells by r.m.s.d. analysis. Four models of chromosome 9 from a selection of different cells are shown, coloured according to the chromosome sequence (from red to purple, centromere to telomere), together with a table showing the r.m.s.d. values between the chromosomal 3D coordinates for each cell (bottom). c, Further cross-sections through the structures of haploid genomes from cells 3–8 through the structures of haploid genomes (see Fig. 2e), coloured according to: whether the sequence is in the A or B compartment (top); whether the sequence is part of a cLAD or contains highly expressed genes (coloured yellow and blue, respectively) (centre); and the identity of the chromosomes (bottom). In each case, an ensemble of five superimposed conformations arising from repeat calculations starting from different randomly generated sets of coordinates is shown. d, An analysis of the genome depth of various chromatin class categories, determined by k-means clustering of 100-kb segments according to the presence of histone H3 ChIP–seq data¹⁵. The active class is associated with H3K4me3, Polycomb with H3K27me3, the inactive class with H3K9me3, and null denotes the remainder. Left, the probability distribution for each of the categories at different normalized nucleus depths. Right, the divergence of the probability distribution for each category from the whole-genome average. Data are shown for the genome structures of all cells. e, An analysis of the genome depth for regions with differing levels of gene expression, as measured by nuclear RNA-seq. Here, RNA-seq signal peaks were ranked and split into five classes. As in d, the probability distribution for each class with regard to genome depth is shown (left), together with the divergence of each distribution from the genome as a whole (right). f, Further comparisons of the structure of chromosome 3 from different cells, coloured according to whether the sequence is part of the cLAD domains (yellow), with the positions of highly expressed genes indicated by the blue rings (larger circles indicate higher expression).

a, Calculation of 3D spatial clustering compared to a random hypothesis in which the same data were circularly permuted around the sequence, and repeating the calculations, using the same structure. Two examples, with strong (Klf4/H3K4me1) and weaker (Nanog/H3K27me3) spatial co-localization compared to random, are shown. b, The enrichment in spatial density (after removal of any clustering expected from their being located in the same chromosome sequence) of histone H3 with various post-translational modifications and selected pluripotency factors as determined by ChIP–seq data. The enrichment is calculated over all cells as the Kullback–Liebler divergence of the normalized spatial density distribution from a random, circularly permuted, expectation (see Supplementary Methods for more details), and the data are presented in hierarchical cluster order, grouping the most similar datasets together. c, Box and whisker plots showing enhancer, promoter and repetitive sequence content (bottom), and the enrichment in spatial density of different types of enhancer, promoter and repetitive sequence (top), after the data have been divided into ten groups based on increasing distance from the nearest inter-chromosomal interface. Whiskers represent the tenth and ninetieth percentiles, boxes represent the range from the twenty-fifth to the seventy-fifth percentile, and outliers are shown as dots. Mean and median values are shown by black crosses and bars, respectively. The R values are the Pearson’s correlation coefficient on the underlying, unranked data. d, Plots of the level of gene expression as measured by the nuclear RNA-seq signal within 1-Mb regions against distance from the nearest inter-chromosomal interface (left) and the outer surface of the A compartment (right). e, Examples of inter-chromosomal interfaces from two different cells in which the chromosomes are coloured increasingly bright red for higher enrichment in the density of gene expression, compared to what would be expected for a given sequence separation. The remainder of the two chromosomes is coloured grey, and the positions of promoters are indicated by blue circles. The same views are shown with the two different chromosomes coloured yellow and blue (top), or with their regions in the A and B compartments coloured blue and red (bottom).

a, A contact map showing the population Hi-C data for chromosome 12 with TADs identified using the directionality index⁵ as blue squares. On the left and at the bottom, data tracks are shown identifying the A and B compartments (in blue and red, respectively), and highly expressed genes (in magenta). b, Further comparisons (see Fig. 4b) showing the structures (and their variability) of two B compartment TADs either side of a highly expressed gene(s) in a short region of A compartment (top), or at a boundary between the A and B compartments (bottom). Ensembles of five superimposed conformations, from repeat calculations using the same experimental data, are shown with pairs of TADs highlighted and coloured according to whether they are in the A or B compartments (blue and red, respectively), with white indicating a transitional segment (between A and B). TAD boundaries are marked by asterisks. c, Scatter plots of the mean radius of gyration for 1-Mb regions of genome structure compared to the average number of single-cell Hi-C contacts, within the same region, considering a 1-Mb sliding analysis window. Data are shown for all genome structures and split according to cis (left) and trans (right) contacts. d, Structure of chromosome 12, with the A compartment coloured blue and positions of CTCF/cohesin loops identified previously⁷ indicated by dotted red lines. The pie chart shows the numbers of loops between sequences in the A and B compartments.

Bar charts of the mean values of TADs identified using the directionality index⁵ for all the different chromosomes. The data are mean values over all structure conformations, scaled according to TAD size, and presented as quantile values for the chromosome. The fiftieth percentile value corresponds to the central grey line. Values below and above this are coloured blue and red respectively. TADs that contain both regions of early replication timing (above the ninetieth percentile) and moderate restraint violation (see Extended Data Fig. 2b) are excluded from the calculation. The errors in the are the percentiles ± the s.e.m. Values for multiple cells are presented in hierarchical cluster order, grouping the most similar cells together.

A genome-wide analysis illustrating whether CTCF/cohesin loops⁷ could be formed in the different single cells, in each chromosome. A black square indicates that the two boundaries in the loop could interact, a white square indicates that the two relevant particles are too far apart in the structure. The loop boundary separation, in particles, is shown along the x axis. The bar chart across the top shows the probability, for each loop, of random particles (pairs with the same sequence separation) forming the same number of contacts, or better. The probability of choosing a set of loop boundary points, which interact more frequently than we observed is 0.00072 (see Supplementary Methods).

a, Structures of cells 2–8 illustrating the interactions identified between the Nanog gene and other regions of the genome by population 4C (ref. 34). Chromosome 6 is coloured blue, with the position of the Nanog gene highlighted in yellow, and the remainder of the chromosomes are coloured grey. Interacting positions in the genome are indicated by red circles. b, Heat map showing the number of times a particular interaction is detected between two of the 4C Nanog gene-interacting points³⁵. c, Heat map showing the number of times a particular interaction is detected between two of the 4C Pou5f1 gene-interacting points³⁶. In b and c, the interaction points are presented in hierarchical order, grouping the regions that show the most interactions together. d, 2D single-molecule tracking using photo-activated light microscopy in live mouse ES cells shows clustering of CHD4 and MBD3. In both cases, a heat map of a single cell is shown in which the pixels have been colour-coded according to the density of molecules detected in that region.