Abstract
Chromosomes in proliferating metazoan cells undergo marked structural metamorphoses every cell cycle, alternating between highly condensed mitotic structures that facilitate chromosome segregation, and decondensed interphase structures that accommodate transcription, gene silencing and DNA replication. Here we use single-cell Hi-C (high-resolution chromosome conformation capture) analysis to study chromosome conformations in thousands of individual cells, and discover a continuum of cis-interaction profiles that finely position individual cells along the cell cycle. We show that chromosomal compartments, topological-associated domains (TADs), contact insulation and long-range loops, all defined by bulk Hi-C maps, are governed by distinct cell-cycle dynamics. In particular, DNA replication correlates with a build-up of compartments and a reduction in TAD insulation, while loops are generally stable from G1 to S and G2 phase. Whole-genome three-dimensional structural models reveal a radial architecture of chromosomal compartments with distinct epigenomic signatures. Our single-cell data therefore allow re-interpretation of chromosome conformation maps through the prism of the cell cycle.
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Acknowledgements
The authors thank M. Leeb, A. Wutz and J. Gribnau for haploid and diploid ES cells, and the Babraham Flow Cytometry and Sequencing Facilities for assistance. This work was supported by grants from the National Institutes of Health 4D Nucleome 1U01HL129971-01, European Research Council 340152 and 309706, and the Flight Attendant Medical Research Institute.
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T.N., Y.L., P.F. and A.T. conceived the project and designed the experiments. T.N. devised new single-cell Hi-C protocol. T.N. and W.L. carried out experiments. Y.L., C.V., C.D., Y.B., N.M.C., S.W. and A.T. analysed data. T.N., Y.L., C.V., C.D., P.F. and A.T. wrote the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Technical quality controls.
Data for all the diploid cells analysed (as specified in i). a, Testing library saturation. Showing the fraction of segment chains supported by a single read in each batch, batch colours match their sorting criteria, see i for details. b, Number of unique molecules captured per cell against the number of sequenced reads. c, Number of reads per unique molecule against number of sequenced reads. d, Observed (red) and expected (by binomial reshuffling of the observed contacts) number of cells each trans-chromosomal contact appears in. e, Correlation of the fraction of trans-chromosomal contacts and close cis-chromosomal contacts (<1 kb, non-digested) with the fraction of contacts in different distance bins. f, Stratification of contacts by the orientation of contacting fragments against contact distance. g, Distribution of the logarithmic decay bin with the most contacts per cell, dashed line at 15.5 mark quality control threshold below which cells are discarded. h, Quality control metrics of single cells by batch ID shown in the same colour as i. Vertical lines mark experimental batches. Shown from top to bottom: total number of contacts (coverage); fraction of inter-chromosomal contacts (%trans); fraction of very close (<1 kb) intra-chromosomal contacts (%no dig); early-replicating coverage enrichment (repli-score); fraction of fragment ends (fends) covered more than once (%dup fend). Horizontal dashed lines mark thresholds used to filter good cells. i, Details on the experimental batches, showing the number of cells in each batch, number of cells passed quality control, mean number of reads per cell (MRPC, in million reads), FACS sorting criteria and the medium used to grow the cells. Batches are coloured by FACS sorting criteria: ‘2n DNA’ and ‘2n < DNA ≤ 4n’ by Hoechst staining (H), ‘G1’, ‘early-S’, ‘mid-S’ and ‘late-S/G2’ by Hoechst and geminin (H + G) staining. j, Coverage enrichment per cell (column) and chromosome (rows), in which the expected coverage is calculated from the frequency in the pooled cells and the total number of contacts in each cell. We discard cells that have any aberrantly covered chromosome (at least twofold enrichment or depletion, shown on the right ‘bad’ panel). Left panel shows all cells ordered by batch ID, with lower panel coloured by FACS sorting criteria (as in h and i).
Extended Data Figure 2 Trans-chromosomal contacts.
a, Examples of inter-chromosomal contact maps for several pairs of chromosomes. Showing contact maps of single cells (blue, each point is a contact) and the pooled contact map on the same chromosomes, using 500-kb square bins. b, Distribution of the number of contacts between selected pairs of chromosomes. Showing the number of contacts per square megabase; red dashed line marks the cutoff for interacting chromosomes. c, Fraction of cells in which each pair of chromosomes was interacting (the pair of chromosomes had normalized interaction above the cutoff shown in b).
Extended Data Figure 3 Pooled contact map and bulk data.
a, Normalized chromosomal contact maps of the pooled diploid 2i single cells (showing chromosomes 10 and 11). b, A chromosome idiogram showing the division of domains to the A and B compartments by k-means clustering of domains trans-chromosomal contact profiles. c, A chromosome idiogram showing the inferred A-score of each domain. d, Comparing A and B domains by their lengths. e, Comparing A and B domains by their mean time of replication (ToR); percentage of H3K4me3 peaks; mean H3K27me3; mean laminB1 values; mean RNA-seq. f, Genome-wide comparison of insulation scores across reference bulk Hi-C dataset (from ref. 23) and different pools of single cells. Insulation is calculated every 10 kb using a scale of 300 kb; Pearson’s correlation coefficient is shown.
Extended Data Figure 4 Mitotic cells and dynamics of coverage by replication time.
a, Contact decay profile of our pool of diploid 2i singles (pool), a reference mouse ES cell bulk Hi-C dataset (Bulk mES, from ref. 23), human K562 mitotic population Hi-C (K562 M, from ref. 11) and our pooled 8 most mitotic-like cells (top M). b, Genome-wide contact maps of 8 putative mitotic diploid cells (cells with the highest fraction of contacts at 2–12 Mb distances). c, Correlation matrix of domain coverage across cells. Ordered by the mean correlation to the top 50 earliest replicating domains minus the mean correlation with the latest 50 domains. Time of replication of each domain is shown at the bottom. d, Comparing the fraction of contacts associated with the latest- and earliest-replicating fends (ToR less than −0.5 and more than 0.5, respectively). Cells are coloured as in Fig. 1e. e, Projection of diploid 2i cells onto a two-dimensional plane using their contact decay profile (>1 kb) by a nonlinear dimensionality reduction algorithm (see Supplementary Methods). Cells are coloured by their repli-score, which was not used to position them on the plane.
Extended Data Figure 5 Clustering cells by contact decay and phasing over the cell cycle.
a, k-means (k = 12) clusters of single-cell contact decay profiles. b, Chromosomal contact maps (chromosome 6) of pooled cells per cluster defined in a and the mean decay trend of each cluster (red) compared to the rest of clusters (grey). c, Mean far contact distances and repli-score against the fraction of short-range contacts per clusters in a (red, cells in cluster; grey, all cells), ordering clusters by their mean percentage short-range.
Extended Data Figure 6 Phasing cells over the cell cycle and quality controls.
a, Mean contact distance among the long-range (4.5–225 Mb) contacts against the fraction of short-range (23 kb–2 Mb) contacts. Dashed red lines mark the cutoffs used to divide cells into the main three groups (G1, early-S and late-S–G2). b, Similar to a but showing repli-score against the fraction of short-range contacts. c, Batch of origin of each phased cell using all diploid 2i and serum cells, coloured by the batch FACS sorting criteria (see Extended Data Fig. 1h for batch information). d, Similar to c but for haploid 2i and serum cells (see Extended Data Fig. 8b for batch information). e, Testing the stability of our phasing, we compare the positions of cells by phasing using only half the chromosomes (first half: 2, 3, 5, 6, 9, 10, 12, 15, 17, 19; second half: X, 1, 4, 7, 8, 11, 13, 14, 16, 18). Position of only 38 cells (<2%) differ in more than 10% of the number of cells (outliers marked in red, 10% margin in dashed grey). Phasing groups marked in black lines. f, Chromosomal comparison of the contact decay metrics used to phase the cells in each group: mean far contact distance for the G1 cells (left) and percentage short-range for G1, S and G2 cells (right). Showing smoothed (n = 51) trends per chromosome on top, chromosomes coloured by length (light grey, chromosome 19; black, chromosome 1). Data for specific chromosomes: chromosome 1 (middle) and chromosome 11 (bottom) as red dots. g, Hoechst and geminin FACS indices of cells in batches for cells in batches 27–35 (Ext Data Fig. 1i) against the cells’ inferred phasing position. h, Genome-wide contact maps of representative cells along the inferred G1 phase, their position marked at the bottom of Fig. 2g.
Extended Data Figure 7 Insulation and loops by cell cycle phase.
a, Insulation profiles of the main three phased groups (G1, early-S and late-S–G2) over 6 Mb regions in chromosomes 1–5. b, Comparing border insulation of phased groups. Dashed lines show the mean insulation value per group. c, Showing mean insulation per cell on mouse ES cell TAD borders taken from ref. 5 (using the centre point of borders smaller than 80 kb) compared to the mean cell insulation over our inferred borders. d, Insulation profile over a 6 Mb region in chromosome 1 (same as a), showing insulation of pooled cells (black), pooled mitotic cells (orange) and a shuffled pool of mitotic cells (red). e, Genome-wide distribution of insulation values for pooled-, pooled-mitotic- and shuffled-pooled-mitotic cells, same colours as in d. f, Mean border insulation per cell against the fraction of short-range (<2 Mb) contacts. g, A/B compartment score on trans-chromosomal contacts, single-cell mean values in dots coloured by FACS sorting, mean trend (black) and mean trend of cis-chromosomal A/B compartment in dashed black (same trend as in Fig. 3b). h, Normalized contact maps of the regions in a, circling the loops detected in distances 200 kb to 1 Mb (marked in dashed black line). i, Comparing loop foci enrichment (log2) calculated per phased group, showing the Pearson’s correlation coefficient.
Extended Data Figure 8 Haploid cells technical quality controls.
a, Quality control metrics of single cells. Vertical lines separate experimental batches shown at bottom in the same colour as b. Shown from top to bottom: total number of contacts (coverage); fraction of inter-chromosomal contacts (%trans); fraction of very close (<1 kb) intra-chromosomal contacts (%no dig); early-replicating coverage enrichment (repli-score); fraction of fragment ends (fends) covered more than once (%dup fend). Horizontal dashed lines mark thresholds used to filter good cells. b, Details on the experimental batches, showing the number of cells in each batch, the number of cells that passed quality control, mean number of reads per cell (in million reads), the medium used to grow the cells and FACS sorting criteria: G1 (H) and G1/S (H) by Hoechst staining. c, Coverage enrichment per cell (column) and chromosome (rows), with expected coverage calculated from the pooled cells. We discard cells that have any aberrantly covered chromosome (at least twofold enrichment or depletion, shown on the left ‘bad’ panel). The right panel shows all cells. d, Similar to Fig. 2d–g but produced from the haploid cells, outliers coloured in black. e, Similar to Fig. 3a, b but produced from the haploid cells, outliers coloured in black. f, Similar to Fig. 3e but produced from the haploid cells. g, Similar to Fig. 3g but produced from the haploid cells, outliers coloured in black.
Extended Data Figure 9 Haploid cells clustering, diploid cells pseudo-compartment analysis.
a, Comparing trans A-score of a reference bulk Hi-C dataset (from ref. 23) and different pools of single cells, using the set of domains inferred from the pool of diploid 2i cells (A domains in red and B in black). Showing on top of each panel the correlation of the domains’ A-score in each dataset. b, Per-domain distributions of A-association score across haploid cells in each of the three main cell cycle inferred phases (from left to right: G1, early S, late S to G2). Domains were clustered (k-means, k = 30) by their distribution in all groups. Inter- and intra-cluster ordering by mean A-association score at late-S–G2 phase. c, Similar to Fig. 4g but for diploid 2i cells. d, Contingency table of haploid and diploid inferred pseudo-compartments, considering all overlapping intervals between the haploid and diploid sets of domains. e, Long-range (>2 Mb) cis-chromosomal contact enrichments between TAD groups according to pseudo-compartments for haploid (top) and 2i diploid (bottom) cells. Showing the expected contact frequency by contacts in the shuffled pooled contact maps on the right. f, Long-range (>2 Mb) cis-chromosomal (top) and trans-chromosomal (bottom) 2i diploid contact enrichments between TAD groups according to pseudo-compartments. Showing the expected contact frequency by the marginal pseudo-compartments coverage on the right. g, TADs are sorted by their mean A-association score in late S–G2 phase (x axis, left, strongly B; right, strongly A), and their mean RNA, H3K4me1, laminB1 and H3K4me3 levels are shown (y axis).
Extended Data Figure 10 Whole-genome structure modelling, quality control and clustering of peri-centromeric regions.
a, The distribution of the fraction of unsupported contacts in the pre-filtered contact set in all 190 haploid cells in 2i inferred as being in G1. b, The distribution of the fraction of violated constraints in the 190 haploid G1 cells. c, The fraction of violated constraints in the 3D models of 190 haploid G1 cells at 500 kb versus 100 kb resolutions (Pearson’s r = 0.96). Black dots represent cells that have at least 10,000 contacts and no more than 0 violations at 1 Mb, 0.5% violations at 500 kb and 0.1% violations at 100 kb. Cells with no violations at either 500 kb or 100 kb resolution are not shown. Red dots, cells with low quality models that were filtered out. d, Mean root mean squared deviation (r.m.s.d.) values between all models of the same cell with at least 10,000 (black) and fewer than 10,000 (red) filtered contacts, for cells at 1 Mb (dots), 500 kb (open diamonds) and 100 kb (triangles) resolutions. e, Model reproducibility across cells. The r.m.s.d. distribution between all models for the same cell (red) compared to the models for different cells (blue), for the 126 cells with the highest quality models. The images show aligned structures of 106 × 1 Mb models (top), 80 × 500 kb models (centre) and 5 × 100 kb models (bottom) for NXT-1091 (38th of 126 ordered G1 cells) with a mean r.m.s.d. at the peak of the red curves. f, Cross-validation test. Red curve: average trans-chromosomal 3D distance distribution of the five most strongly contacting chromosome pairs, measured at 200 randomly chosen loci, using all supported contacts. Blue curve: same as the red curve, except after removal of the trans-contacts between one of the five chromosome pairs. Green curve: trans-chromosomal distance distribution control from models of all cells. Cyan curve: trans-distance distribution of loci involved in unsupported contacts. b0 is the bond length parameter. g, Nucleus and chromosome shape properties in the 126 cells with high-quality models: the nuclear radius (in arbitrary units), the longest-to-shortest (a/c) semi-axis ratio of the ellipsoid fitted to the whole nucleus model, and the longest-to-shortest (a/c) and middle-to-shortest (b/c) semi-axis ratios of the ellipsoids fitted to each chromosome of the 3D models. For the nucleus size and a/c ratio, values in each model are shown, for the chromosome fitted ellipsoid semi-axis ratios the model-averaged value is shown for each cell. h, The 7 cell groups in the inferred G1 time order for the 126 cells with the highest-quality models. i, Distances of the top third shortest distances between nucleolar organizer region (NOR) chromosomes or non-NOR chromosomes in 7 cell groups in h. Distances are normalized by the nucleus diameter.
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Nagano, T., Lubling, Y., Várnai, C. et al. Cell-cycle dynamics of chromosomal organization at single-cell resolution. Nature 547, 61–67 (2017). https://doi.org/10.1038/nature23001
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DOI: https://doi.org/10.1038/nature23001
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