Single-cell Hi-C reveals cell-to-cell variability in chromosome structure

Abstract

Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual chromosomes maintain domain organization at the megabase scale, but show variable cell-to-cell chromosome structures at larger scales. Despite this structural stochasticity, localization of active gene domains to boundaries of chromosome territories is a hallmark of chromosomal conformation. Single-cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organization underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns.

Figure 1. Single cell and ensemble Hi-C

a, Single cell Hi-C method. b, Single cell Hi-C heatmap (cell-5), coverage for 10 Mb bins. c, Contact enrichment versus genomic distance, from ensemble Hi-C, pool of 60 single cells and 10 individual cells, scaled to normalise sequencing depths. d, Normalising by the trends in c, intra-chromosomal contact enrichments for 1 Mb square bins, comparing ensemble and pooled single cell Hi-C (Spearman correlation = 0.56). e, Intra-chromosomal contact enrichment maps of ensemble and pooled single cell Hi-C, for chromosome 10 (top) and chromosome 2 (bottom), using variable bin sizes.

Figure 2. Conserved intra-domain, but not inter-domain structure in single cells

a, Individual intra-chromosomal contacts of 50 Mb region of chromosome 2 up to 3 Mb distance (blue dots), domains (grey). b, Ratios between intra-domain and inter-domain contact enrichments over genomic distance. Control is combined trend of 10 single cells calculated by repeatedly shifting the domains randomly. c, Distribution of intra-domain contact enrichments per domain from 9 cells (where Bgl II was used) and reshuffled datasets (black bars, standard errors). d, Maps of inter-domain contacts intensities for chromosome 2 from individual cells and reshuffled controls using variable bin sizes. e, Distribution of percentage of loci with high insulation scores in single vs. reshuffled cells. f, For all pairs of single cells, the correlations between inter-domain contact numbers of all pairs of domains within the same chromosome were computed. Shown are the distributions of these correlations in the real and reshuffled cells.

Figure 3. Structural modeling of X chromosomes

a, Distribution of longest diameter of X chromosome paint DNA FISH signals in 62 male Th1 cells (real), 200 structural models calculated for each single cell (cell-1 to -9), 200 structures from combined dataset (cell-1 and -2; comb) and 200 structures from 20 randomised cell-1 datasets (random; 10 calculations per dataset). Whiskers denote minimum and maximum. b, Average coordinate root-mean-square deviation (RMSD) values in microns comparing 200 low-resolution structural models for each cell and between cells. c, Four surface-rendered models of the X chromosome from cell-1, which are most representative of the data based on hierarchical clustering of pair-wise RMSD values (Supplementary Information). Scale bar, 1 μm. d, Structural ensembles of the four most representative fine-scale models for cell-1 and cell-6, with four large regions coloured. Scale bar, 1 μm. e, Mean structural density rank for 500 kb regions (black) from 6 × 200 fine-scale models from cell-1 to -6. Standard deviation (blue/pink). Abundance of intra-chromosomal restraints (grey, right axis). DNA FISH probes (P1-P5) are indicated. f, DNA FISH on Th1 cells. X chromosome paint (green) and specific locus signals (red). g, Distribution of DNA FISH distance measurements between signal centres for probes P1 - P5 and edge of the X chromosome territory in Th1 cells (n = 114, 113, 105, 115, 108 for P1-P5). Whiskers denote 10 and 90 percentiles. h, Enrichment of cis-, trans-contacts and Lamin-B1 associated domains at various depths on the chromosome models relative to null hypothesis of random positions.

Figure 4. Active domains localise to territory interfaces

a, Distribution of trans-chromosomal contact enrichments of each domain averaged across real and reshuffled cells. Reshuffling maintains the number of cis and trans contacts within each cell and chromosome. b, Intra-domain contact enrichment over genomic distance for high vs. low trans-chromosomal contacting domains selected independently in each cell, with 95% confidence intervals. c, Same sets as in b but plotting the enrichment of inter-domain contacts. d, Distribution of H3K4me3 peak density in domains (number of peaks divided by size), color-coded according to density. e, Domains plotted according to number of trans- and cis-chromosomal (excluding intra-domain) contacts, color coded for H3K4me3 density as in d.

Figure 5. Chromosomal interfaces

a, All trans-chromosomal contacts formed by chromosome 2 in real cells (blue) and reshuffled (red). b, Schematic diagram of a chromosomal interface between linearly adjacent domains, their borders marked in black on two chromosomes, A and B. We considered each of the two contacting fragments of every trans-chromosomal contact and classified every nearby trans-chromosomal contact as domain-domain, domain-chromosome and chromosome-chromosome, the latter being used as background for normalisation (Supplementary Information). Contact under consideration (red), nearby contacts (blue). Fold enrichments shown for each group type (error bars, standard deviation). c, Trans-chromosomal contacts are highly significantly enriched between active domains (H3K4me3 enriched) or between inactive domains, but not mixed interaction (chi-square test; p = 5.8e-18; even after taking account of the generally higher connectivity of active domains). d, Bar graph depicting mouse autosomes ordered by size with number of interacting chromosomes per single cell (black circles depict the distribution over individual cells). Mean number of interacting chromosomes changes modestly (30%) with chromosome size, suggesting a highly organized territory structure with surface that is not scaling with chromosome length.