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CTCF–CTCF loops and intra-TAD interactions show differential dependence on cohesin ring integrity

Nature Cell Biology volume 24, pages 1516–1527 (2022)Cite this article

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Abstract

The ring-like cohesin complex mediates sister-chromatid cohesion by encircling pairs of sister chromatids. Cohesin also extrudes loops along chromatids. Whether the two activities involve similar mechanisms of DNA engagement is not known. We implemented an experimental approach based on isolated nuclei carrying engineered cleavable RAD21 proteins to precisely control cohesin ring integrity so that its role in chromatin looping could be studied under defined experimental conditions. This approach allowed us to identify cohesin complexes with distinct biochemical, and possibly structural, properties that mediate different sets of chromatin loops. When RAD21 is cleaved and the cohesin ring is opened, cohesin complexes at CTCF sites are released from DNA and loops at these elements are lost. In contrast, cohesin-dependent loops within chromatin domains that are not anchored at pairs of CTCF sites are more resistant to RAD21 cleavage. The results show that the cohesin complex mediates loops in different ways depending on the genomic context and suggests that it undergoes structural changes as it dynamically extrudes and encounters CTCF sites.

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Fig. 1: RAD21 degradation in G1 cells eliminates CTCF–CTCF loops and TADs but enhances compartmentalization.
Fig. 2: RAD21 cleavage reduces the CTCF–CTCF loop interactions.
Fig. 3: RAD21 cleavage in NBS1 dissociates cohesin, eliminates CTCF–CTCF loops and reduces intra-TAD interactions.
Fig. 4: Compartmentalization remains after nucleus expansion and contraction.
Fig. 5: Intra-TAD interactions remain after nuclear expansion and contraction.

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Data availability

Deep sequencing data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under the accession code GSE182500. All Hi-C and ChIP–seq libraries are listed in Supplementary Tables 2 and 3. The protein structure in Fig. 2 was drawn from 6WG3 from the Protein Data Bank. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

Both cWorld scripts and cooltools (0.3.0) used in this study are publicly available in GitHub: https://github.com/dekkerlab/cworld-dekker and https://github.com/mirnylab/cooltools. No other customized codes were developed for this study.

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Acknowledgements

We thank all of the members of the Dekker laboratory for their helpful discussion and R. McCord for advice on the nuclear expansion assay. We thank the Deep Sequencing Core and the Flow Cytometry Core (supported by grant no. S10 OD028576) at UMass Chan Medical School. We thank C. Navarro for help with editing the manuscript. We acknowledge support from the National Institutes of Health Common Fund 4D Nucleome Program (grant nos DK107980 and HG011536) and the National Human Genome Research Institute (grant no. HG003143). J.D. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Systems Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Yu Liu & Job Dekker

  2. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    Job Dekker

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  1. Yu Liu
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Contributions

Y.L. and J.D. conceived and designed the project. Y.L. performed all the experiments and analysed all of the data. Y.L. and J.D. wrote the manuscript.

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Correspondence to Job Dekker.

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Extended data

Extended Data Fig. 1 Cell-cycle profiles of the HCT-RAD21-mAC cells in different conditions for Hi-C experiments.

(a) Schematic of HCT-RAD21-mAC cells (a gift from Dr. Kanemaki’s lab)47. After treatment with 500 µM IAA, RAD21-mAC was degraded. (b) Western blot analysis of RAD21 degradation in HCT116-RAD21-mAC cells after 500 µM IAA treatments for various times as indicated. GAPDH was used as loading control. Numbers indicate the relative level of intact RAD21. (c) FACS analysis of non-synchronous HCT116-mAC cells treated with IAA as shown. DNA was stained using fxCycle-far red and RAD21 was tagged with mClover. Upper panels are 2D scatter plots indicating DNA content and RAD21 levels before and after IAA treatment. Lower panels, histograms indicating cell cycle stage distributions for cultures with and without IAA treatment. The same staining method was used for (d) and (e). (d) FACS analysis of G1 synchronized HCT116-mAC cells treated with IAA as shown. Upper panels: plots of DNA contents vs RAD21 levels treated with IAA as shown. Lower panels: cell cycle stage distributions treated with IAA as shown. (e) FACS analysis of non-synchronous HCT116-mAC cells treated with IAA as shown. G1 cells were sorted from these non-synchronous cells for Hi-C analysis. Upper panels are 2D scatter plots DNA content and RAD21 levels treated with IAA as shown. Lower panels, histogram graphics indicate cell cycle profiles treated with IAA as shown. Red dashed boxes indicate the G1 population that were sorted and collected for Hi-C analysis. (f) Hi-C interaction maps for non-synchronous (NS), synchronous (Syn), and G1 cells treated with IAA as shown. Data for the 29-34 Mb region of chromosome 14 are shown. (g) Insulation profiles for the same region as in f. The blue, grey and red lines represent 0, 2 and 6 hour IAA treatment, respectively. The lower panels indicate compartment Eigenvector value E1 across the same region. (h) Aggregate Hi-C data at TAD boundaries that were identified in each condition without IAA treatments. The numbers at the sides of the cross indicate the boundary strength.

Source data

Extended Data Fig. 2 Three replicates of Hi-C analysis of G1 cells without and with IAA treatment.

(a) Hi-C interaction maps for three independent Hi-C experiments using G1-sorted cells treated with IAA as shown. Data for the 29-34 Mb regions of chromosome 14 is shown. (b) Insulation profiles for the same region as in (a). The blue, grey and red lines represent 0, 2 and 6 hour IAA treatments, respectively. Blue arrow shows weakened insulation at boundaries (c). Aggregate Hi-C data at TAD boundaries that were identified in each replicate without IAA treatments. The numbers at the sides of the cross indicate the strength of boundary-anchored stripes using the mean values of interaction frequency within the white dashed boxes. (d). Aggregated Hi-C data at a set of 3169 loops identified in HCT116-RAD21-mAC cells with intact RAD21 identified by23. Plots at the bottom show average Hi-C signals along the dotted blue lines representing signals from the bottom-left corner to the top-right corner of the loop-aggregated heatmaps shown in upper panels. (e). P(s) plots (upper panels) and derivative from P(s) plots (lower panels) for Hi-C data as indicated. The arrows on the derivative plots indicate cohesin loops. (f). Hi-C interaction maps for three independent Hi-C experiments using G1-sorted cells treated with IAA as shown. Data for the 18–107.3 Mb regions of chromosome 14 is shown. (g). E1 across the same region as in (f). Bottom panels, E1 for the 77.4-89 Mb region is shown and the colour assignments for the lines are the same as for the other panels. (h). Saddle plots for three independent Hi-C experiments using G1-sorted cells treated with IAA as shown. Saddle plots for each cell condition were calculated using the E1 calculated from the Hi-C data obtained with G1 cells grown without IAA treatments. The numbers indicate compartment strength.

Extended Data Fig. 3 A replicate Hi-C analysis of nuclei with RAD21 cleaved in NB buffer.

(a) The TEV motif insertion site is conserved between human and mouse. Amino acid sequences flanking TEV motif insertion sites in mouse Scc1 and human RAD21 are shown. (b) TEV proteases at different concentrations cleave RAD21 from HAP1-RAD21TEV purified nuclei. Left panels, RAD21TEV and cleaved fragments were detected using an antibody recognizing the N terminus of RAD21TEV. Right panels, RAD21TEV and cleaved fragments were detected using an antibody recognizing C-terminus of RAD21TEV. LMNA was a loading control. (c). Hi-C maps for HAP1-RAD21TEV nuclei treated with TEV as shown. Data for the 18-107.3 Mb region of chromosome 14 is shown. Bottom panel, E1 across the same region as in (c). (d). Saddle plots for HAP1-RAD21TEV nuclei treated with TEV as shown. Numbers indicate compartment strength. (e) Interaction strength of compartments. Bars represent strength of compartment interactions for each sample as described in Fig1h. (f). Hi-C maps for HAP1-RAD21TEV nuclei treated with TEV as shown. Data for the 29-34 Mb region of chromosome 14 is shown. (g) Insulation profiles for the same region as in f. Blue and red lines represent without and with TEV protease treatment, respectively. Lower panels indicate E1 across the same region. (h) Aggregate Hi-C data at TAD boundaries identified in the sample in NB buffer without TEV treatment. Numbers at the sides of the cross indicate boundary strength. (i) P(s) plots (upper panels), and derivatives of P(s) plots (lower panels) for Hi-C data from nuclei treated with TEV as shown. Blue arrows indicate the signature of cohesin loops in each condition. (j) Aggregated Hi-C data at loops as in Fig. 2j. Right panel: average Hi-C signals along the blue dashed line shown in the left Hi-C panel. (k) Aggregated Hi-C data at chromatin loops of three different loop sizes, 100–500 kb, 500kb-1Mb, and >1 Mb. Right panels: average Hi-C signals along the blue dashed line shown in the left Hi-C map in panel Fig. 2j. See source data for numerical data and unprocessed blots.

Source data

Extended Data Fig. 4 Cleaving RAD21 in NBS1 dissociates and releases cohesin components.

Western blot analysis of nuclear retention of cohesin in RAD21TEV nuclei treated with TEV in the specified buffer as shown. Left panels indicated as nuclei show cohesin components in nuclei detected using antibodies as described in the right panels of a-c. Right panels show supernatant of released cohesin components. Cohesin components from supernatants were separated and detected using the same antibodies as used for the Western blots shown on the right panels of a-c. For all western blot analyses, LMNA was used as the loading controls. (a) A biological replicate of cohesin subunits retained in nuclei or released to the supernatant treated with TEV in specified buffer as shown. The antibodies used here are the same as Fig. 3a. (b) Western blot analysis of PDS5A, PDS5B and NIPBL retained in nuclei or released to the supernatant treated with TEV in specified buffer as shown. (c) and (d) Western analysis of MAU2 (SCC4) retained in nuclei (c) and released to the supernatant (d) treated with TEV in the specified buffer as shown. Cleaving RAD21 in NBS1 has little effects on compartmentalization and CTCF binding (e) Hi-C interaction maps for HAP1-RAD21TEV nuclei treated with TEV as shown. Data for the 18-107.3 Mb region of chromosome 14 is shown. Bottom, eigenvector E1 profiles across the same region. (f) Saddle plots for HAP1-RAD21TEV nuclei treated with TEV as shown. The numbers indicate compartment strength. (g) Interaction strength of compartments. The bars represent the strength of compartment interactions for each sample as described in Fig1h. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 5 A replicate of ChIP analysis of the nuclei with cleaved RAD21 in NBS1.

(a) Profiles of ChIP signals of CTCF and RAD21 at CTCF binding sites, from nuclei treated with TEV in the specified buffers as shown. Data shown are for biological replicate 2, an independent replicate is shown in Fig. 3h. 25,879 CTCF binding sites were identified from CTCF ChIP data of nuclei without TEV treatment in NB. Upper panel, average CTCF and RAD21 ChIP–seq signals for each condition for the set of 25,879 CTCF binding sites. Two different RAD21 antibodies were used, nRAD21 recognizes the N-terminal domain of RAD21, as used in Fig. 3h; cRAD21 recognizes C-terminal domain of RAD21. Lower panel, heatmap of CTCF and RAD21 ChIP–seq signals of each condition at each of the 25,879 CTCF binding sites. (b) Profiles of ChIP signals of CTCF and RAD21 at active transcription start sites (TSS) treated with TEV in the specified buffers as shown. Of 13,412 active TSS of HAP1 cells, 1,951 overlapped with CTCF binding sites while 8,777 did not overlap with CTCF binding sites (2 kb away from CTCF binding sites). Both average ChIP signals (upper panels) and heatmap of ChIP signals of CTCF and RAD21 for these two groups of TSS sites are shown. (c) Biological replicate of the experiment shown in panel b. Of 13412 active TSS sites, 1,588 were overlapped with CTCF binding sites while 9,423 did not overlap with CTCF binding sites. Dark blue and orange lines indicate TSSs that overlapped or did not overlap with CTCF binding sites, respectively. Both average ChIP signals (upper panels) and heatmap of ChIP signals of CTCF and RAD21 on these two groups of TSS sites are shown. Right panel includes a RAD21 ChIP data using the RAD21 antibody that recognizes C-terminal of RAD21. Dark blue and orange lines indicate TSS that overlapped or did not overlap with CTCF binding sites, respectively.

Extended Data Fig. 6 Cleaving RAD21 in NBS2 dissociates and releases cohesin components.

(a) Western blot analysis of salt effects on nuclear retention of cohesin complex subunits in HAP-1RAD21TEV nuclei treated with TEV as shown. Western blot analysis was performed as indicated in Extended Data Fig. 4ab except that 200 mM NaCl was used, instead of 132 mM. (b) Quantification of levels of SMC1/3, N-terminal cleaved RAD21 and C-terminal cleaved RAD21. The levels of indicated proteins or fragments were normalized to LMNA as loading controls first, then the ratio was normalized to the same protein or fragments in NB without TEV treatment. For SMC proteins, the average relative percentage of SMC1 and SMC3 levels, from panel (a) and Fig. 3a, is presented. See source data for numerical data and unprocessed blots.

Source data

Extended Data Fig. 7 Two biological replicates of Hi-C analysis of nuclei with RAD21 cleaved in NBS2 buffer (with Supplementary Fig. 2).

(a) Hi-C maps for HAP1-RAD21TEV nuclei treated with TEV in specified buffer sas shown. Data for the 18-107.3 Mb region of chromosome 14 is shown. Bottom, E1 across the 18-107.3 Mb region of chromosome 14. (b) Saddle plots for HAP1-RAD21TEV nuclei treated with TEV in specified buffers as shown. Numbers indicate compartment strength. (c) Interaction strength of compartments. Bars represent strength of compartment interactions for each sample as described in Fig1h. (d) Hi-C maps for HAP1-RAD21TEV nuclei treated with TEV in specified buffers as shown. Data for the 29-34 Mb region of chromosome 14 is shown. Middle panels indicate insulation profiles for the 29-34 Mb regions of chromosome 14. Blue and red lines represent without and with TEV protease treatment, respectively, as in panel a. Lower panels indicate E1 across the same region. (e) Aggregate Hi-C data at TAD boundaries identified in the sample in NB buffer without TEV treatment. Numbers at the sides of the cross indicate strength of boundary-anchored stripes using the mean values of interaction frequency within the white dashed boxes. (f) P(s) plots (left panels), and derivatives of P(s) plots (right panels) for Hi-C data from nuclei treated with TEV as shown. Blue arrows indicate the signature of cohesin loops in each condition. Red arrows indicate changes of contact frequency at 2 Mb. (g) Aggregated Hi-C data at loops as in Fig. 2j. Lower panels: average Hi-C signals along the blue dashed line shown in the upper left Hi-C panel. (h) Aggregated Hi-C data at chromatin loops of three different loop sizes as indicated. Lower panels: average Hi-C signals along the blue dashed line shown in the left Hi-C map in Fig. 2j. See source data for numerical data.

Source data

Extended Data Fig. 8 Two biological replicates of Hi-C analysis of G1-sorted nuclei with RAD21 cleaved in NBS1 (with Supplementary Fig. 3).

(a) Hi-C maps for G1-sorted HAP1-RAD21TEV nuclei treated with TEV in specified buffers as shown. Data for the 18-107.3 Mb region of chromosome 14 is shown. Bottom, E1 cross the same region. (b) Saddle plots for G1-sorted HAP1-RAD21TEV nuclei treated with TEV in specified buffer as shown. Numbers indicate compartment strength. (c) Interaction strength of compartments. Bars represent strength of compartment interactions for each sample as described in Fig. 1h. (d) Hi-C interaction maps for G1-sorted HAP1-RAD21TEV nuclei treated with TEV in specified buffers as shown. Data for the 29-34 Mb region of chromosome 14 is shown. Middle panels indicate insulation profiles for the 29-34 Mb regions of chromosome 14. Blue and red lines represent without and with TEV protease treatment, respectively. The lower panels indicate compartment E1 across the region. (e) Aggregate Hi-C data at TAD boundaries identified in each condition as shown. Numbers at the sides of the cross indicate strength of boundary-anchored stripes using the mean values of interaction frequency within the white dashed boxes. (f) P(s) plots (left panels), and derivatives of P(s) plots (right panels) for Hi-C data from nuclei treated with TEV as shown. Blue arrows indicate the signature of cohesin loops in each condition. Red arrows indicate changes of contact frequency at 2 Mb. (g) Aggregated Hi-C data at loops as in Fig. 2j. Lower panel: average Hi-C signals along the blue dashed line shown in the left Hi-C panel. (h) Aggregated Hi-C data at chromatin loops of three different loop sizes as shown. Lower panels: average Hi-C signals along the blue dashed line shown in the left Hi-C map in Fig. 2j. (i) Quantification of loop strength and intra-TAD interaction strength obtained with G1-sorted HAP1-RAD21TEV nuclei treated with TEV in specified buffer as shown (two biological replicates). (j) Quantification of loop strength and intra-TAD interaction strength in each condition as shown. Loop strength and intra-TAD interaction strength were normalized as in Fig. 3i. See source data for numerical data.

Source data

Extended Data Fig. 9 Two replicates of Hi-C analysis of the nuclei with RAD21 cleaved in HBSS buffer.

(a) Western blot analysis of RAD21 and cohesin components treated with TEV in specified buffer as shown. (b) Hi-C interaction maps for HAP1-RAD21TEV nuclei without and with TEV protease treatment in HBSS buffer, respectively. Data for the 18-107.3 Mb region of chromosome 14 is shown. Bottom, eigenvector E1 cross the same region. (c) Saddle plots for HAP1-RAD21TEV nuclei treated with TEV in specified buffer as shown. The numbers indicate compartment strength. (d) Interaction strength of compartments. The bars represent the strength of compartment interactions for each sample as described in Fig. 1h. (e) Hi-C interaction maps for HAP1-RAD21TEV nuclei treated with TEV in HBSS buffer as shown. Data for the 29-34 Mb region of chromosome 14 is shown. Middle panels indicate insulation profiles for the same region. The blue and red lines represent without and with TEV protease treatments, respectively, as in panel b. The lower panels indicate compartment Eigenvector value E1 across the same region. (f) Aggregate Hi-C data at TAD boundaries identified in the sample without TEV treatment in each replicate. The numbers at the sides of the cross indicate the boundary strength. (g) P(s) plots (upper panels), and the derivatives of P(s) plots (lower panels) for Hi-C data from nuclei treated with TEV as shown. The blue arrows indicate the signature of cohesin loops in each condition. (h) Aggregated Hi-C data at loops as in Fig. 2j. Lower panel: average Hi-C signals along the blue dashed line shown in the left Hi-C panel. See source data for numerical data and unprocessed blots.

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Extended Data Fig. 10 Responses of stripes to RAD21 cleavage in different salt buffers.

(a) The examples of 3’- and 5’-stripes treated with TEV as shown. The left and right columns of the Hi-C interaction maps showed the 72-75 Mb region of chromosome 14 and the 76-79 Mb region of chromosome 15 at 10 kb resolution, respectively. On each Hi-C interaction map, the boxes with blue dashed lines highlighted the stripes. The upper and lower Hi-C interaction maps of each column are treated with TEV as shown. (b) Aggregate Hi-C data binned at 10 kb resolution at both 3’- and 5’-stripes identified in the sample in NB buffer without TEV treatment. The first two rows indicated the first replicate including Hi-C data without and with TEV treatment in NB, NBS1 and NBS2 as indicated. The third and fourth rows are the second replicate across all the conditions as shown. (c) The relative strength of 3’- and 5’-stripes for all the samples in (b). The median of six replicates without TEV protease treatment in NB buffer was used for normalization. (d) Comparison of stripe strength to the strength of intra-TAD and CTCF–CTCF loop interactions in all samples. The strength of intra-TAD and CTCF–CTCF loop interactions are averages of all replicates in each condition as indicated and were calculated as in Fig. 3i. See source data for numerical data.

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Supplementary information

Supplementary Information

Supplementary Materials and Methods, Supplementary Figs. 1–12 and unprocessed western blots for the supplementary figures.

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Supplementary Table 1

Supplementary Tables 1–3.

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Liu, Y., Dekker, J. CTCF–CTCF loops and intra-TAD interactions show differential dependence on cohesin ring integrity. Nat Cell Biol 24, 1516–1527 (2022). https://doi.org/10.1038/s41556-022-00992-y

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