Regulation of single-cell genome organization into TADs and chromatin nanodomains - PubMed (original) (raw)
Regulation of single-cell genome organization into TADs and chromatin nanodomains
Quentin Szabo et al. Nat Genet. 2020 Nov.
Abstract
The genome folds into a hierarchy of three-dimensional structures within the nucleus. At the sub-megabase scale, chromosomes form topologically associating domains (TADs)1-4. However, how TADs fold in single cells is elusive. Here, we reveal TAD features inaccessible to cell population analysis by using super-resolution microscopy. TAD structures and physical insulation associated with their borders are variable between individual cells, yet chromatin intermingling is enriched within TADs compared to adjacent TADs in most cells. The spatial segregation of TADs is further exacerbated during cell differentiation. Favored interactions within TADs are regulated by cohesin and CTCF through distinct mechanisms: cohesin generates chromatin contacts and intermingling while CTCF prevents inter-TAD contacts. Furthermore, TADs are subdivided into discrete nanodomains, which persist in cells depleted of CTCF or cohesin, whereas disruption of nucleosome contacts alters their structural organization. Altogether, these results provide a physical basis for the folding of individual chromosomes at the nanoscale.
Conflict of interest statement
Competing interest statement
The authors declare no competing interests.
Figures
Extended Data Fig. 1. Oligopaint probe design.
Hi-C maps from ESCs and NPCs along with the positions of FISH probes. Gray ticks indicate TAD borders defined from visual inspection.
Extended Data Fig. 2. Oligopaint probe coverage.
Oligopaint coverage for each probe. X axes represent the labeled regions (from their start to their end coordinates), each dot represents the number of oligos in a 10 kb bin.
Extended Data Fig. 3. Spatial segregation of TADs in ESCs.
a, 3D-SIM images of a control (Ctl) probe (5d2) simultaneously labeled by Alexa-488 and ATTO-565 fluorophores (85 alleles were analyzed). Maximum projections, scale bar = 500 nm. b, Distances between the centroids of the two segmented channels. n = 85 alleles. c, Pearson’s correlation coefficient (PCC) between probe intensities. Boxplots represent median, interquartile ranges, and Tukey-style whiskers. n = 48 and 50 for the pair 51a-51b and 51b-52, respectively; ***, P = 1.14e-5; two-sided Wilcoxon rank sum test. d, Mean (± SD) population fraction with OF < 0.1. n = 7 and 8 probe pairs within and between TADs, respectively; ***, P = 3.11e-4; two-sided Wilcoxon rank sum test. e, OFs and 3D distances between centroids. Boxplots represent median, interquartile ranges and Tukey-style whiskers. A mean of 65 alleles was analyzed per probe pair. f, Mean (± SD) PCC between probe intensities measured from each probe pair. n = 7 and 8 probe pairs within and between TADs, respectively; ***, P = 6.22e-4; two-sided Wilcoxon rank sum test. g, Representation of the crossing contact fraction, defined as the Hi-C contacts measured between the labeled regions (A) divided by the sum of contacts measured in regions A, B and C. h, scHi-C maps of a locus labeled by FISH probes (color-coded). Yellow lines indicate TADs detected from cell-population Hi-C. i, Crossing contact fraction from individual cells for each probe pair. Boxplots represent median, interquartile ranges and Tukey-style whiskers. n = 165 cells. j, Crossing contact fraction from individual cells for all probe pairs used in FISH. Boxplots represent median, interquartile ranges and Tukey-style whiskers. n = 1155 and 1320 for probe pairs within and between TADs, respectively; ***, P = 3.84e-158; two-sided Wilcoxon rank sum test.
Extended Data Fig. 4. Spatial segregation of TADs in NPCs.
a, Pax6 immunostaining (in all FISH experiments performed in NPCs, each analyzed nucleus was positive for Pax6 staining). Conventional wide field (WF) microscopy, scale bar: 10 μm. b, Mean (± SD) population fraction with OF < 0.1. n = 10 and 5 probe pairs within and between TADs, respectively; ***, P = 6.66e-4; two-sided Wilcoxon rank sum test. c, OFs and 3D distances between centroids. Boxplots represent median, interquartile ranges and Tukey-style whiskers. A mean of 36 alleles was analyzed per probe pair. d, Mean (± SD) OF fold change (NPC/ESC, probe pairs that do not change their TAD borders between ESCs and NPCs). n = 7 and 5 probe pairs within and between TADs, respectively; two-sided t-test. e, Mean probe volume as a function of genomic size. A mean of 70 and 47 alleles was analyzed per probe in ESCs and NPCs, respectively. f, Mean (± SD) of coefficient of variations (CV) for OFs and 3D distances (probe pairs that do not change their TAD borders between ESCs and NPCs). n = 7 and 5 probe pairs within and between TADs, respectively. g, Crossing contact fraction measured for the probe pair shown in Fig. 2e. h, Hi-C maps in ESCs, NPCs, and ncxNPCs along with probe pair location. Arrowhead indicates the expressed Zfp608 gene. i, 3D-SIM images of the probes shown in h (62, 34 and 48 alleles were analyzed in ESCs, NPCs and ncxNPCs, respectively). Maximum projections, scale bar: 500 nm. j, OFs and 3D distances between the centroids of the probes shown in h. Boxplots represent median, interquartile ranges and Tukey-style whiskers. n = 62, 34 and 48 for ESCs, NPCs, and ncxNPCs, respectively. k, Crossing contact fraction measured for the probe pair shown in i. Despite the appearance of a border within the locus labeled by the probe pair 11-12 in differentiated cells (panel h and Extended Data Fig. 1), and considering the absence of changes in crossing contact fraction neither in intermingling (j), we considered this probe pair as “Within TAD” in NPCs.
Extended Data Fig. 5. Spatial segregation of TADs in CTCF- and RAD21-depleted cells.
a, Western Blot of CTCF and Vinculin (loading control) in CTCF-AID, CTCF-AID + auxin (2 days), and wild-type ESCs. 4 reproducible western blots were performed from different biological replicates. b, OFs and 3D distances between centroids measured for each probe pair located within or between TADs as a function of the genomic distance separating their centers. Graph represents medians and interquartile ranges. A mean of 57 and 51 alleles was analyzed per probe pair in CTCF-AID and CTCF-AID + auxin cells, respectively. c, Mean OF measured from all probe pairs within TADs divided by the mean OF from all probe pairs between TADs. d, Western Blot of RAD21 and Vinculin (loading control) in wild-type ESCs, RAD21-AID and RAD21-AID + auxin (6 hours). 3 reproducible western blots were performed from different biological replicates. e, OFs and 3D distances between centroids measured for each probe pair located within or between TADs as a function of the genomic distance separating their centers. Graph represents medians and interquartile ranges. A mean of 75 and 47 alleles was analyzed per probe pair in RAD21-AID and RAD21-AID + auxin cells, respectively. f, Mean OF measured from all probe pairs within TADs divided by the mean OF from all probe pairs between TADs.
Extended Data Fig. 6. TAD and CND structures revealed by super-resolution imaging.
a, Hi-C map from ESCs along with probe position. b. Individual TADs labeled by the probe shown in a imaged with conventional WF microscopy (top) or with 3D-SIM (bottom). 23 and 33 alleles were analyzed with conventional WF and 3D-SIM microscopy, respectively. White lines represent the boundaries of probe segmentations (2D projections). Maximum projections, scale bar = 500 nm. c, Volumes, sphericities and principal axis lengths of individual TADs. Boxplots represent median, interquartile ranges and Tukey-style whiskers. n = 23 and 33 for conventional WF microscopy and 3D-SIM, respectively; ***, P < 0.001; two-sided Wilcoxon rank sum tests. d, Sphericity as a function of principal axis length for individual TADs. A mean of 73 TADs was analyzed per probe. e, 3D-SIM image of TAD #22 (top, maximum projection, scale bar = 500 nm), CND identification using 3D watershed segmentation (middle) or using local fluorescence intensity maxima (bottom) and number of CNDs per TAD. 51 alleles were analyzed. f, Stimulated emission depletion (STED) images of TAD #22 (single _z_-slice, scale bar = 500 nm) and extrapolated diameters of individual TADs and of CNDs within them. Bins represent 50 nm, n = 21 and 96 for TADs and CNDs, respectively. 21 alleles were analyzed. g, DAPI staining in ESC and watershed segmentation showing CNDs. 3 nuclei were analyzed. Single _z_-slice, scale bar = 5 μm. h, 3D-SIM image of DAPI staining in Drosophila male embryonic cell (single _z_-slice, scale bar = 5 μm or 500 nm in the magnification) and of an X-linked 110 kb Drosophila TAD (maximum projection, scale bar = 500 nm). 20 and 52 nuclei were analyzed with DAPI staining and FISH, respectively. i, Extrapolated diameters of the Drosophila TAD, of CNDs within it, and of CNDs measured with DAPI staining. Bins represent 50 nm, n = 52, 54 and 2250, respectively.
Extended Data Fig. 7. Structural organization of TADs and CNDs in CTCF- and RAD21-depleted cells.
a, TAD volumes, TAD sphericities, CND volumes, and number of CNDs per TAD (mean ± SD). Boxplots represent median, interquartile ranges, and Tukey-style whiskers. A mean of 84 and 64 alleles was analyzed per probe in CTCF-AID and CTCF-AID + auxin cells, respectively; ***, P < 0.001; **, P < 0.01; *, P < 0.05; two-sided Wilcoxon rank sum tests. b, 3D-SIM images of TAD #62 (59 and 41 alleles were analyzed in CTCF-AID and CTCF-AID + auxin, respectively). White lines represent the boundaries of probe segmentations (2D projections). Maximum projections, scale bar = 500 nm. c, TAD volumes, TAD sphericities, CND volumes, and number of CNDs per TAD (mean ± SD). Boxplots represent median, interquartile ranges and Tukey-style whiskers. A mean of 176 and 116 alleles was analyzed per probe in in RAD21-AID and RAD21-AID + auxin cells, respectively; ***, P < 0.001; **, P < 0.01; two-sided Wilcoxon rank sum tests. d, 3D-SIM images of TAD #112 showing alleles segmented as one (top) or two (bottom) objects. Lines represent the boundaries of probe segmentations (2D projections). Maximum projections, scale bar = 500 nm. e, Cell cycle profiling using DAPI staining (with examples of nucleus segmentation, scale bar = 10 μm). As nucleus size and DAPI intensity reflect cell cycle stage, cutoff values for nucleus area and DAPI integrated intensity were applied to define G1 ESCs. 27% of the population was defined as G1, consistently with flow cytometry measurements. 164 nuclei were analyzed. f, TAD volumes, TAD sphericities, CND volumes, and number of CNDs per TAD (mean ± SD). Boxplots represent median, interquartile ranges and Tukey-style whiskers. n = 48, 77 and 48 for ESC-G1, RAD21-AID + auxin and RAD21-AID + auxin single chromatids, respectively; ***, P < 0.001; two-sided Wilcoxon rank sum tests.
Extended Data Fig. 8. Structural organization of TADs and CNDs relates to chromatin state and histone acetylation.
a, Hierarchical clustering of the 26 labeled loci (Extended Data Figs. 1 and 9) using ATAC-seq, RNA-seq and ChIP-seq of the indicated histone post-translational modifications in ESCs. b, Mean (± SD) density (genomic size/mean volume) of the probes in ESCs. A mean of 70 alleles was analyzed per probe; **, P = 0.0019; two-sided Wilcoxon rank sum test. c, Mean (± SD) number of CNDs per probe divided by their genomic size (in Mb) in ESCs. A mean of 70 alleles was analyzed per probe; ***, P = 6.05e-4; two-sided Wilcoxon rank sum test. d, Hierarchical clustering of the 26 labeled loci in NPCs. e, Mean (±SD) density of the probes in NPCs. A mean of 47 alleles was analyzed per probe; **, P = 0.0013; two-sided Wilcoxon rank sum test. f, Mean (± SD) number of CNDs per probe divided by their genomic size (in Mb) in NPCs. A mean of 47 alleles was analyzed per probe; **, P = 0.001; two-sided Wilcoxon rank sum test. g, Western Blot of H4 (loading control) and H4-acetyl (H4ac). Two different protein concentrations were loaded. 3 reproducible western blots were performed from different biological replicates. h, Mean OF measured from probe pairs within TADs (102a-102b and 121a-121b) divided by the mean OF from probe pairs between TADs (101-102a, 101-103 and 111-112). A mean of 76 and 69 alleles was analyzed in ESCs and ESCs + TSA, respectively. i, Mean OF fold change (ESC + TSA/ESC) for each probe pair within TADs (n = 2; 102a-102b and 121a-121b) or between TADs (n = 3; 101-102a, 101-103 and 111-112). j, TAD volumes and sphericities. Boxplots represent median, interquartile ranges and Tukey-style whiskers. A mean of 93 and 95 alleles was analyzed in ESCs and ESCs + TSA, respectively; ***, P < 0.001; *, P < 0.05; two-sided Wilcoxon rank sum tests.
Extended Data Fig. 9. Oligopaint probe information.
Oligopaint IDs, coordinates, sizes, numbers of oligos, density of oligos, primer codes (see Supplementary Table 1), and clusters (1 for active or 0 for repressed; ESC/NPC; see Extended Data Fig. 8a, d).
Fig. 1. TADs form heterogeneous structures in single cells, favoring intra-TAD over inter-TAD chromatin intermingling.
a, Left: Hi-C maps from ESCs along with probe locations, either within a TAD (top) or between two adjacent TADs (bottom). Right: representative 3D-SIM images of the probes shown in a and averaged images generated from all segmented probe pairs (48 and 50 alleles were analyzed for the pair 51a-51b and 51b-52, respectively). Maximum projections, scale bars: 500 nm. b, 3D-views from different individual segmented probe pairs shown in a. c, Overlap fractions (OFs) and 3D distances between centroids of the probes shown in a. Boxplots represent median, interquartile ranges and Tukey-style whiskers. n = 48 and 50 for the pair 51a-51b and 51b-52, respectively; ***, P < 0.001; two-sided Wilcoxon rank sum tests. d, Experimental design to label individual or adjacent TADs. e, OFs and 3D distances between centroids measured for each probe pair as a function of the genomic distance separating their centers. Graph represents medians and interquartile ranges. A mean of 65 alleles was analyzed per probe pair. f, OFs and 3D distances between centroids as a function of crossing contact fraction measured in Hi-C along with the goodness of fit (R2) of a linear regression and the Pearson’s correlation coefficient ρ.
Fig. 2. The spatial segregation of TADs increases during cell differentiation.
a, NPCs were generated from in vitro differentiation of ESCs. b, OFs and 3D distances between centroids measured for each probe pair in NPCs as a function of the genomic distance separating their centers. Graph represents medians and interquartile ranges. A mean of 36 alleles was analyzed per probe pair. c, OFs and 3D distances between centroids as a function of crossing contact fraction measured in Hi-C along with the goodness of fit (R2) of a linear regression and the Pearson’s correlation coefficient ρ. d, Mean OF measured from all probe pairs within TADs divided by the mean OF from all probe pairs between TADs (using probe pairs that do not change their TAD borders between ESCs and NPCs). e, Hi-C maps from ESCs, NPCs, and ncxNPCs along with probe pair location. Arrowhead indicates the location of the expressed Zfp42 gene. f, Representative 3D-SIM images of the probes shown in e (50, 26 and 18 alleles were analyzed in ESCs, NPCs, ncxNPCs, respectively). Maximum projections, scale bar: 500 nm. g, OFs and 3D distances between the centroids of the probes shown in e. Boxplots represent median, interquartile ranges and Tukey-style whiskers. n = 50, 26 and 18 for ESCs, NPCs, and ncxNPCs, respectively; ***, P < 0.001; **, P < 0.01; *, P < 0.05; two-sided Wilcoxon rank sum tests.
Fig. 3. Cohesin generates chromatin intermingling while CTCF prevents inter-TAD contacts.
a, Hi-C maps from ESCs along with probe locations, either between two adjacent TADs (probe pair 101-102a) or within a TADs (probe pair 102a-102b). b, Representative 3D-SIM images of the probes shown in a in the indicated cell types (probe pairs 101-102a/102a-102b; 52/58, 47/57, 163/130 and 54/61 alleles were analyzed in CTCF-AID, CTCF-AID + auxin, RAD21-AID and RAD21-AID + auxin, respectively). Maximum projections, scale bar: 500 nm. c, OFs and 3D distances between the centroids of the probes shown in a. Boxplots represent median, interquartile ranges and Tukey-style whiskers. n (probe pairs 101-102a/102a-102b) = 52/58, 47/57, 163/130 and 54/61 for CTCF-AID, CTCF-AID + auxin, RAD21-AID and RAD21-AID + auxin, respectively; ***, P < 0.001; **, P < 0.01; *, P < 0.05; two-sided Wilcoxon rank sum tests. d, OFs and 3D distances between centroids measured for each probe pair in CTCF-AID + auxin cells as a function of the genomic distance separating their centers. Graph represents medians and interquartile ranges. A mean of 51 alleles was analyzed per probe pair. e, Mean (± standard deviation “SD”) OF fold change (CTCF-AID + auxin / CTCF-AID) for probe pairs within TADs (n = 7) or between TADs (n = 8); **, P = 0.0019; * P = 0.016; two-sided _t_-test. f, OFs and 3D distances between centroids measured for each probe pair in RAD21-AID + auxin cells as a function of the genomic distance separating their centers. Graph represents medians and interquartile ranges. A mean of 47 alleles was analyzed per probe pair. g, Mean (± SD) OF fold change (RAD21-AID + auxin / RAD21-AID) for probe pairs within TADs (n = 7) or between TADs (n = 8); **, P = 0.0012; two-sided _t_-test.
Fig. 4. TADs are subdivided into discrete chromatin nanodomains (CNDs) that depend on histone acetylation.
a, Hi-C map from ESCs along with probe location (TAD #22) and ChIP-seq tracks. b, Top: representative 3D-SIM images of the TAD shown in a (51 alleles were analyzed). White lines represent the boundaries probe segmentations (2D projections). Maximum projections, scale bar = 500 nm. Bottom: 3D views of the segmented TADs shown above (gray mesh) and watershed segmented CNDs (colored objects). c, DAPI staining in ESC (3 nuclei were analyzed). Single _z_-slice, scale bars = 5 μm and 500 nm in the magnification. d, Extrapolated diameters (diameter of a circle with the same area than the segmented object) of TADs (ranging from 215 to 990 kb), of CNDs within them, and of CNDs measured with DAPI staining. Bins represent 50 nm, n = 804, 1,413 and 3,068, respectively. e, Representative 3D-SIM images of TAD #62 in ESC and ESC + TSA (94 and 74 alleles were analyzed in ESCs and ESCs + TSA, respectively). White lines represent the boundaries of probe segmentations (2D projections). Maximum projections, scale bar = 500 nm. f, CND volumes. Boxplots represent median, interquartile ranges and Tukey-style whiskers. A mean of 296 and 417 CNDs was analyzed per probe in ESCs and ESCs + TSA, respectively; ***, P < 0.001; **, P < 0.01; *, P < 0.05; two-sided Wilcoxon rank sum tests. g, Mean (± SD) number of CNDs per TAD. A mean of 93 and 95 alleles was analyzed per probe in ESCs and ESCs + TSA, respectively; ***, P < 0.001; **, P < 0.01; two-sided Wilcoxon rank sum tests. h, Model of 3D TAD folding. TADs, which form variable structures and are subdivided into smaller CNDs, favor chromatin intermingling in most cells. Their spatial segregation is further exacerbated in differentiated NPCs. Upon CTCF depletion, the cohesin complex can extrude chromatin, through TAD borders, inducing ectopic contacts between adjacent TADs and abolishing preferential intra-TAD interactions. Upon RAD21 depletion, preferential intra-TAD interactions are lost due to the absence of intermingling generated by the cohesin complex. Upon TSA-mediated histone hyper-acetylation, TADs remain spatially segregated while the structural organization of CNDs is disrupted.
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