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Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes

Nature Genetics volume 52, pages 840–848 (2020)Cite this article

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Abstract

The human genome can be segmented into topologically associating domains (TADs), which have been proposed to spatially sequester genes and regulatory elements through chromatin looping. Interactions between TADs have also been suggested, presumably because of variable boundary positions across individual cells. However, the nature, extent and consequence of these dynamic boundaries remain unclear. Here, we combine high-resolution imaging with Oligopaint technology to quantify the interaction frequencies across both weak and strong boundaries. We find that chromatin intermingling across population-defined boundaries is widespread but that the extent of permissibility is locus-specific. Cohesin depletion, which abolishes domain formation at the population level, does not induce ectopic interactions but instead reduces interactions across all boundaries tested. In contrast, WAPL or CTCF depletion increases inter-domain contacts in a cohesin-dependent manner. Reduced chromatin intermingling due to cohesin loss affects the topology and transcriptional bursting frequencies of genes near boundaries. We propose that cohesin occasionally bypasses boundaries to promote incorporation of boundary-proximal genes into neighboring domains.

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Fig. 1: Boundary permissibility is a widespread feature of the human genome.
Fig. 2: Variable interactions across boundaries occur independently from intra-domain compaction.
Fig. 3: Cohesin promotes interactions within and across domain boundaries.
Fig. 4: WAPL and CTCF restrict cohesin-dependent interactions across domain boundaries.
Fig. 5: Cohesin alters the topological context of boundary-proximal genes.
Fig. 6: Cohesin alters the transcriptional bursting frequency of boundary-proximal genes.

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Data availability

All data that support the findings of this study are included in manuscript or are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We would like to thank G. Blobel, L. Rosin, M. Lakadamyali, and members of the Joyce laboratory for helpful discussions and critical reading of the manuscript. In addition, we thank M. Kanemaki for the HCT-116-RAD21-AID cell line and S. Rao and E. Lieberman-Aiden for sharing critical primary datasets. We also thank A. Stout at the Penn Cell and Developmental Biology Microscopy core and C. Eberling from Bruker for assistance with super-resolution imaging and analysis. This work was supported by a Charles E. Kaufman grant from The Pittsburgh Foundation (KA2017-91787) to E.F.J. and NIH grants R35GM128903 to E.F.J. and T32GM008216 to J.M.L.

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  1. Department of Genetics, Penn Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Jennifer M. Luppino, Daniel S. Park, Son C. Nguyen, Zhuxuan Xu, Rebecca Yunker & Eric F. Joyce

  2. Department of Cell and Developmental Biology, Penn Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Yemin Lan

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Contributions

J.M.L. and E.F.J. designed the study. J.M.L., D.S.P., S.C.N. and E.F.J. analyzed the results. J.M.L. and E.F.J. wrote the manuscript. J.M.L. and D.S.P. performed all of the experiments, except the RNA FISH experiments, which were performed by S.C.N. Y.L. performed the Hi-C data analysis. Z.X. and R.Y. generated all of the Oligopaint probes used in this study. All authors discussed the results and commented on the manuscript.

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Correspondence to Eric F. Joyce.

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Extended data

Extended Data Fig. 1 Genomic landscapes at Oligopaint target regions.

Genomic profiles of loci imaged by FISH. Hi-C contact matrices visualized by Juicebox (v1.9.0)56. Data from HCT-116-RAD21-AID8 cells. Solid and dashed lines indicate domains and subdomains, respectively. The gene density, eigenvectors, and insulation score (computed by the TopDom) are noted below. Insulation score computed prior to (solid) and following 6 hours of auxin treatment (dashed). Published ChIP–seq tracks8 depict protein binding and histone modifications in the HCT-116-RAD21-AID cell line prior to and following 6 hours of auxin treatment (-/+ Auxin). Genomic tracks visualized using Integrative Genomics Viewer57.

Extended Data Fig. 2 Additional information related to Fig. 1.

a, HCT-116-RAD21-AID cells were synchronized at the G1/S transition. Immunofluorescence for CENPF (green) to indicate cells in G2 and PCNA (gray) to mark cells in S phase. DNA (Hoescht stain) is shown in gray in first column. Dashed lines represent nuclear edges. Scale bar equals 5 μm. b, Cumulative frequency distribution of spatial overlap between neighboring domains on chr12:11.6Mb-13.6 Mb (n = 716 chromosomes) and chr22:33.2Mb-36.8 Mb (n = 1410 in asynchronous HCT-116 cells. Overlap normalized to the volume of the upstream domain. n > 716 chromosomes. c, Hi-C contact matrix and Oligopaint designs corresponding to (c-e). d, Representative FISH images of each subdomain and the downstream D2. Scale bar equals 1 μm. Corresponding 3D segmentation of FISH signals below each image. e, Cumulative distribution plot of spatial overlap between the subdomains [S1 (n = 1932); S2 (n = 2283); S3 (n = 1977)] and D2, normalized to the volume of D2. *** P < 0.001, two-tailed Mann-Whitney test. f, Frequency of contact between each subdomain and D2 from data in e. Contact defined as > 500 nm3 overlap. **** P < 0.0001, two-tailed Fisher’s exact test. g, Scatterplot of spatial overlap volume across the strong and weak boundaries on the same allele at the chr22:33.2-36.8 Mb locus. n = 1060 chromosomes. See Fig. 2e for corresponding Oligopaint design.

Extended Data Fig. 3 Boundary permissibility is a widespread feature of the human genome (additional loci).

a, Distribution of spatial overlap between neighboring domains (D1 & D2) at chr1:36.5-40.3 Mb. Overlap normalized to the volume of the upstream domain. n = 2294 chromosomes. b, Distribution of spatial overlap between neighboring domains (D2 & D3) at chr1:36.5-40.3 Mb. Overlap normalized to the volume of the upstream domain. n = 2553 chromosomes. c, Distribution of spatial overlap between neighboring domains (D3 & D4) at chr1:36.5-40.3 Mb. Overlap normalized to the volume of the upstream domain. n = 2502 chromosomes. d, Distribution of spatial overlap between neighboring domains (D4 & D5) at chr1:36.5-40.3 Mb. Overlap normalized to the volume of the upstream domain. n = 9443 chromosomes. e, Distribution of spatial overlap between neighboring domains (D1 & D2) at chr2:217.45-223 Mb. Overlap normalized to the volume of the upstream domain. n = 1850 chromosomes. f, Distribution of spatial overlap between neighboring domains (D2 & D3) at chr2:217.45-223 Mb. Overlap normalized to the volume of the upstream domain. n = 1903 chromosomes. g, Distribution of spatial overlap between neighboring domains (D2 & D3) at chr3:44.2-47.55 Mb. Overlap normalized to the volume of the upstream domain. n = 1657 chromosomes. h, Distribution of spatial overlap between neighboring domains (D2 & D3) at chr12:11.6-13.6 Mb. Overlap normalized to the volume of the upstream domain. n = 1606 chromosomes. i, Distribution of spatial overlap between neighboring domains (S1 & S2) at chr12:11.6-13.6 Mb. Overlap normalized to the volume of the upstream domain. n = 1479 chromosomes. j, Distribution of spatial overlap between neighboring domains (S2 & S3) at chr12:11.6-13.6 Mb. Overlap normalized to the volume of the upstream domain. n = 1912 chromosomes. k, Distribution of spatial overlap between neighboring domains (D1 & D2) at chr19:17.35-18.6 Mb. Overlap normalized to the volume of the upstream domain. n = 1406 chromosomes. l, Distribution of spatial overlap between neighboring domains (S1 & S2) at chr22:33.2-36.8 Mb. Overlap normalized to the volume of the upstream domain. n = 1634 chromosomes. m, Distribution of spatial overlap between neighboring domains (S2 & S3) at chr22:33.2-36.8 Mb. Overlap normalized to the volume of the upstream domain. n = 1640 chromosomes. n, Distribution of spatial overlap between neighboring domains (S4 & S5) at chr22:33.2-36.8 Mb. Overlap normalized to the volume of the upstream domain. n = 1494 chromosomes.

Extended Data Fig. 4 Additional information related to Fig. 2.

a, Localization density per domain quantified from 3D-STORM images. Chr12.D1 (median = 0.0002707, n = 91); Chr12.D2 (median = 0.0002076, n = 91); Chr22.D1 (median = 0.0002376, n = 95); Chr22.D2 (median = 0.0002245, n = 95). b, Scatterplot depicting the relationship between domain volumes on the same chromosome by 3D-STORM on chr12:11.6Mb-13.6 Mb. n = 91 chromosomes. c, Scatterplot depicting the relationship between domain volumes between homologs by 3D-STORM on chr12:11.6Mb-13.6 Mb. n = 82 chromosomes. d, Scatterplot depicting the relationship between domain volumes on the same chromosome by 3D-STORM on chr22:33.2Mb-36.8 Mb. n = 95 chromosomes. e, Scatterplot depicting the relationship between domain volumes between homologs by 3D-STORM on chr22:33.2Mb-36.8 Mb. n = 86 chromosomes. f, Scatterplot of overlap volume (x-axis) versus domain density (y-axis) by 3D-STORM for the chr22:33.2Mb-36.8 Mb locus. n = 95 chromosomes.

Extended Data Fig. 5 Confirmation and quantification of RAD21 degradation.

a, Immunofluorescence for RAD21 (cyan). DNA (Hoescht stain) is shown in gray. Dashed lines represent nuclear edges. Scale bar equals 5 μm. b, Western blot to RAD21 protein in the chromatin-bound fraction of HCT-116-RAD21-AID cells with no auxin treatment (-) or following 6 hours of auxin treatment (+). Histone H3 as loading control. Protein was labeled using fluorescent secondary antibodies. c, Fluorescence quantification of RAD21 and H3 isolated from the chromatin-bound fraction of protein corresponding to blot in b using Image Lab v5.2.1. Protein intensity normalized to total protein per well (via stain-free technology) and presented as fraction of protein observed in untreated (- auxin) conditions; we observe a 96% reduction in chromatin-bound RAD21 following auxin treatment.

Source data

Extended Data Fig. 6 Cohesin promotes interactions across domain boundaries (additional loci).

Hi-C contact matrix and corresponding cumulative distribution of spatial overlap between the neighboring domains prior to or after auxin treatment. Overlap normalized to the volume of the upstream domain. a, Map of chr1:36.5-40.3 Mb for b-e. b, D1 & D2 (-aux n = 2294, +aux n = 2175). c, D2 & D3 (-aux n = 2553, +aux n = 2353). d, D3 & D4 (-aux n = 2502, +aux n = 2488), e. D4 & D5 (-aux n = 9443, +aux n = 8695). f, Map of chr2:217.45-223 Mb for g-h. g, D1 & D2 (-aux n = 1850, +aux n = 1665). h, D2 & D3 (-aux n = 1903, +aux n = 1717). i, Map of chr3:44.2-47.55 Mb for j-k. j, D1 & D2 (-aux n = 1643, +aux n = 2186). k, D2 & D3 (-aux n = 1657, +aux n = 2181). l, Map of chr12:11.6-13.6 Mb for m-o. m, D2 & D3 (-aux n = 1606, +aux n = 1570). n, S1 & S2 (-aux n = 1479, +aux n = 1220). o, S2 & S3 (-aux n = 731, +aux n = 678). p, Map of chr19:17.35-18.6 Mb for q. q, D1 & D2 (-aux n = 1406, +aux n = 1419). r, Map of chr22:33.2-36.8 Mb for s-u. s, S1 & S2 (-aux n = 1634, +aux n = 1703). t, S2 & S3 (-aux n = 1640, +aux n = 1702). u, S4 & S5 (-aux n = 1494, +aux n = 1718). P < 0.001, two-tailed Mann-Whitney test for all domain pairs.

Extended Data Fig. 7 Additional information related to Fig. 3.

a, Minimum distances between localizations contained within each domain on chr12:11.6-13.6 Mb as measured from 3D-STORM data (prior to auxin: median = 0.008292 μm, n = 91; after auxin: median = 0.01234 μm, n = 76). P < 0.001, two-tailed Mann-Whitney test. b, Minimum distances between localizations contained within each domain on chr22:33.2Mb-36.8 Mb as measured from 3D-STORM data (prior to auxin: median = 0.009470 μm, n = 95; after auxin: median = 0.01563 μm, n = 105). P < 0.001, two-tailed Mann-Whitney test. c, Violin plots of domain volumes as measured from 3D-STORM data prior to [Chr12.D1 (median = 0.2629 μm3, n = 91); Chr12.D2 (median = 0.1830 μm3, n = 91); Chr22.D1 (median = 0.2749 μm3, n = 95); Chr22.D2 (median = 0.2320 μm3, n = 95)] or after auxin treatment [Chr12.D1 (median = 0.1703 μm3, n = 91); Chr12.D2 (median = 0.1671 μm3, n = 76); Chr22.D1 (median = 0.2284 μm3, n = 105); Chr22.D2 (median = 0.2213μm3, n = 105)]. Chr12.D1 P = 0.008; Chr12.D2 P = 0.368; Chr22.D1 P = 0.076, Chr22.D2 P = 0.907; two-tailed Mann-Whitney test. d, Violin plots of domain densities as measured from 3D-STORM data prior to [Chr12.D1 (median = 0.0002707, n = 91); Chr12.D2 (median = 0.0002076, n = 91); Chr22.D1 (median = 0.0002376, n = 95); Chr22.D2 (median = 0.0002245, n = 95)] or after auxin treatment [Chr12.D1 (median = 0.0003080, n = 76); Chr12.D2 (median = 0.0002385, n = 76); Chr22.D1 (median = 0.0002651, n = 105); Chr22.D2 (median = 0.0002353, n = 105)]. Chr12.D1 P = 0.247; Chr12.D2 P = 0.014; Chr22.D1 P = 0.025, Chr22.D2 P = 0.602; two-tailed Mann-Whitney test.

Extended Data Fig. 8 Additional information related to Fig. 4.

a, Western blot of NIPBL, WAPL, and CTCF protein of HCT-116 cells following RNAi. H3 as loading control. b, Representative FISH images of neighboring domains on chr12:11.6Mb-13.6 Mb in RNAi control, NIPBL-, WAPL-, or CTCF-depleted cells. Dashed lines represent nuclear edges. Scale bar equals 5 μm (left) or 1 μm (zoomed images, below). c, Spatial overlap between neighboring domains on chr12:11.6Mb-13.6 Mb in control (n = 643), NIPBL- (n = 636), WAPL- (n = 819) or both NIPBL and WAPL- (n = 922) depleted cells. ***P < 0.001, two-tailed Mann-Whitney test. d, Spatial overlap between neighboring domains on chr22:33.2Mb-36.8 Mb in control (n = 1722), NIPBL- (n = 1440), WAPL- (n = 1769) or both NIPBL and WAPL- (n = 1762) depleted cells. Overlap normalized to the volume of the upstream domain. ***P < 0.001, two-tailed Mann-Whitney test. e, Western blot to RAD21 and CTCF in whole cell lysate of HCT-116 cells following RNAi to CTCF and/or 6 hours auxin to degrade RAD21. Alpha tubulin as loading control. Spatial overlap between neighboring domains across six loci (f-k). f, D4 and D5 on chr1:36.5-40.3 Mb in control (n = 2362) or CTCF- (n = 1611) depleted cells. ***P < 0.001. two-tailed Mann-Whitney test. g, D1 and D2 on chr2:217.45-223 Mb in control (n = 1484) or CTCF- (n = 1466) depleted cells. P = 0.604 (ns). two-tailed Mann-Whitney test. h, D2 and D3 on chr2:217.45-223 Mb in control or CTCF-depleted cells prior to (control n = 1145; CTCF n = 1133) or after auxin treatment (control n = 1039; CTCF n = 877). P < 0.001 (***), P = 0.008 (**). two-tailed Mann-Whitney test. i, D1 and D2 on chr3:44.2-47.55 Mb in control or CTCF-depleted cells prior to (control n = 1272; CTCF n = 1215) or after auxin treatment (control n = 1254; CTCF n = 1090). P < 0.001 (***) or P = 0.996. two-tailed Mann-Whitney test. j, D1 and D2 on chr12:11.6Mb-13.6 Mb in control or CTCF-depleted cells prior to (control n = 1236; CTCF n = 1250) or after auxin treatment (control n = 1174; CTCF n = 1231). P < 0.001 (***) or P = 0.314. two-tailed Mann-Whitney test. k, D1 and D2 on chr22:33.2-36.8 Mb in control or CTCF-depleted cells prior to (control n = 1258; CTCF n = 1370) or after auxin treatment (control n = 1170; CTCF n = 1157). P < 0.001 (***) or P = 0.89, two-tailed Mann-Whitney test.

Source data

Extended Data Fig. 9 Additional information related to Fig. 5.

a, Hi-C contact matrix chr12:11.6-13.6 Mb and corresponding Oligopaint design for (b-c). Blue represents the boundary proximal gene, CREBL2. b, Cartoon representations for three possible interactions between boundary-proximal genes and their neighboring domains: domain maintenance, switching, and exclusion (top). Representative images of three-color FISH to the chr12:11.6-13.6 Mb locus illustrating the three domain configurations (middle); scale bar equals 1 μm. Corresponding 3D segmentation of FISH signals below each image. c, Frequencies of domain configurations at chr12:11.6-13.6 Mb prior to (n = 1074) or after auxin treatment (n = 979). P < 0.0001 (***) or P = 0.0046 (**), two-sided Fisher’s exact test.

Extended Data Fig 10 Additional information related to Fig. 6.

a, Distribution of domain sizes that harbor either a significantly differentially expressed (median = 183734, n = 4196) or non-differentially expressed genes (median = 194447, n = 8026) in the HCT-116 cell line following auxin treatment. P = 0.102, two-tailed Mann-Whitney test. b, Scatterplot of log2(fold change)] of HCT-116 differentially expressed genes (DEGs) versus the distance between their TSS and the center of the nearest domain boundary. c, Distance to nearest super enhancer defined by H3K27ac signal58 in HCT-116 cells for DEGs with >30% fold change in expression following auxin treatment. Genes were categorized by their proximity to a domain boundary (< 5 kb or > 5 kb away) and whether they were up or down regulated following auxin treatment. d, Gene ontology enrichment analysis for HCT-116 differentially expressed genes (> 30% fold change, n = 68) within 5 kb of a domain boundary. e, Gene ontology enrichment analysis for HCT-116 differentially expressed genes (> 30% fold change, n = 1593) not within 5 kb of a domain boundary. f, Scatterplot of [fold change] of differentially expressed genes (DEGs) associated with CdLS versus the distance between their TSS and the center of the nearest domain boundary in GM12878 cells. n = 1569, P = 0.0017, Spearman correlation. g, Fraction of genes that are up or down regulated in CdLS at binned distances from the nearest domain boundary. h, Fold change in burst volume by RNA FISH in HCT-116 cells following auxin treatment. Each dot represents the fold change in average burst volume per biological replicate; horizonal line indicates mean.

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Source Data Extended Data Fig. 5

Unprocessed full Western Blots corresponding to Extended Data Fig. 5.

Source Data Extended Data Fig. 8

Unprocessed full Western Blots corresponding to Extended Data Fig. 8.

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Luppino, J.M., Park, D.S., Nguyen, S.C. et al. Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes. Nat Genet 52, 840–848 (2020). https://doi.org/10.1038/s41588-020-0647-9

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