Fig 1: Global chromatin states are highly variable and dynamic in single cells.a, The intensities of IF markers show heterogeneities in single cells. Images are from the same z section. Scalebars, 10 µm. b, Heatmap of cell clusters with distinct IF profiles. Bimodally expressed Nanog, Esrrb and Zfp4241 are distributed over several IF clusters. n = 326 cells in the center field of views from two biological replicates. c, Schematic of colony tracing experiments. Intensity of markers with fast dynamics are expected to be heterogeneous within a colony. d, Representative maximum intensity z-projected images for one 48-hour colony, showing heterogeneities in mRNA (left) and IF markers (right). Scalebars, 20 µm. e, Mean Pearson correlation between cells within colonies decays slowly for mRNA and chromatin states, and quickly for chromosome proximities. Control measures correlation between colonies for both 24- and 48-hour datasets. f, Standard deviation of individual IF marker intensities in 48-hour colonies compared to those between colonies. H3K27me3 and mH2A1 have less variance in cells within a colony, which can be seen in d. Mean values from 20 bootstrap trials are shown with error bars corresponding to standard errors (e, f). n = 117 unlabeled cells within colonies in 48-hour dataset. n=53 cells in 24-hour dataset.
Fig 2: Additional analysis for colony level cell state heterogeneity.a, mRNA and IF images in a colony in the 48 hour clonal tracing experiment. H3K27me3 and mH2A1 overall intensities are similar in WT cells (GFP/Neo negative) in the colony. b, Standard deviation of normalized mRNA levels within colonies (red) and between colonies (grey). Error bars are standard errors for 20 bootstrap trials. Tbx3 and Nanog are more homogeneous within colonies, consistent with previous findings of the long-lived transcriptional states of these genes across several generations by single cell live imaging experiments41,49. n = 117 unlabeled cells within colonies from a 48-hour dataset. c, Histogram of cell-to-cell correlations of chromosome to chromosome proximity maps for cells within colonies (red) and between colonies (grey). Cells with similar chromosome structures (red dots with high correlation values) are likely to be sister cells. Y-axis represents Pearson correlation coefficient, computed by 20 × 20 chromosome proximity matrices from pairs of cells. p values were from a two-sided Wilcoxon’s rank sum test with pairs of cells of 180, 1,198, 966 and 5,820 (from left to right). d, Correlation of chromosome proximities between cells in colonies in the 48 hour clonal tracing experiment. Strong correlations are seen between putative sister cells suggesting that gross chromosome proximities are preserved for 1 generation. Color bars represent Pearson correlation coefficient computed in c. e, Chromosome images for unlabeled cells from a 24-hour colony shows similarities between two sets of neighboring cells (maximum z projection). Chromosome organizations in single cells are highly correlated between pairs of cells that were physically close, possibly sister cells, and are mostly uncorrelated with other cells in the colonies. 6 chromosomes are shown for visual clarity. r represents Pearson correlation coefficient computed in c.
Fig 3: Comparison between population level and single cell level chromosome organization in association with chromatin markers.a, Clustering of the ensemble-averaged IF spatial proximity profile of individual loci. n = 2,460 loci (n = 805, 278, 877, 500 loci in each cluster, respectively). b, In individual cells, loci associated with each cluster are mapped onto their spatial location. Note that cluster definitions for DNA loci were obtained from population-averaged data, and those cluster-assigned loci distribution may not necessarily reflect IF marker localization in single cells. c, Boxplot of IF marks for the loci in each of the clusters. Cluster 1 is enriched in repressive markers such as H3K9me3, mH2A1, DAPI. Cluster 2 is enriched in interactions with Fibrillarin. Cluster 3 is enriched in active marks such as RNAPII Ser5-P, H3K27ac and SF3a66 (nuclear speckle marker). Cluster 4 is enriched in Lamin B1. For the boxplots in c, d, h, i, the center line in the boxes marks median, the upper and lower limits of the boxes mark the interquartile range, the whiskers extend to the farthest data points within 1.5 times the interquartile range, and the gray points mark outliers. d, The probability of loci of certain cluster pairs within 1 µm search radius in individual cells. Cluster definitions follow those in a-c. Randomized data were generated by scrambling the cluster identities of individual loci in cells while keeping the total number of loci within each cluster the same within that cell. The probability for observed and randomized data for each cell are shown as boxplots. e, The probability that pairs of loci with cluster assignments are found within a given search radius, as a function of search radius. Error bars represent standard error over 20 bootstrap trials. f, Mapping of the A/B compartment definitions23 onto the tSNE plot based on the ensemble-averaged loci-IF mark spatial proximity map. Note that regions that are not assigned to one of the compartments were excluded from the analysis. (n = 1,188 and 960 loci in A and B compartment). g, Reconstructions of individual cells with loci assigned as A or B compartment mapped onto their spatial location. Observed compared to randomized data for 2 cells shown in b. h, Boxplot of the IF marks for the loci assigned to A or B compartments. i, The probability that loci in A/B compartments are within 1 µm search radius in individual cells, similar to d. j, The probability that pairs of loci with A/B assignments are found within a given search radius, as a function of search radius for spatial proximity, similar to e. n = 446 cells from two biological replicates in a-j.
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