Abstract
Mammalian genomes are folded through the distinct actions of structural maintenance of chromosome (SMC) complexes, which include the chromatin loop-extruding cohesin (extrusive cohesin), the sister chromatid cohesive cohesin and the mitotic chromosome-associated condensins1,2,3. Although these complexes function at different stages of the cell cycle, they exist together on chromatin during the G2-to-M phase transition, when the genome structure undergoes substantial reorganization1,2. Yet, how the different SMC complexes affect each other and how their interactions orchestrate the dynamic folding of the three-dimensional genome remain unclear. Here we engineered all possible cohesin and condensin configurations on mitotic chromosomes to delineate the concerted, mutually influential action of SMC complexes. We show that condensin disrupts the binding of extrusive cohesin at CCCTC-binding factor (CTCF) sites, thereby promoting the disassembly of interphase topologically associating domains (TADs) and loops during mitotic progression. Conversely, extrusive cohesin impedes condensin-mediated mitotic chromosome spiralization. Condensin reduces peaks of cohesive cohesin, whereas cohesive cohesin antagonizes condensin-mediated longitudinal shortening of mitotic chromosomes. The presence of both extrusive and cohesive cohesin synergizes these effects and inhibits mitotic chromosome condensation. Extrusive cohesin positions cohesive cohesin at CTCF-binding sites. However, cohesive cohesin by itself cannot be arrested by CTCF molecules and is insufficient to establish TADs or loops. Moreover, it lacks loop-extrusion capacity, which indicates that cohesive cohesin has nonoverlapping functions with extrusive cohesin. Finally, cohesive cohesin restricts chromatin loop expansion mediated by extrusive cohesin. Collectively, our data describe a three-way interaction among major SMC complexes that dynamically modulates chromatin architecture during cell cycle progression.
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Data availability
The Hi-C and ChIP–seq data generated in this study have been deposited into the Gene Expression Omnibus (GEO) database with accession number GSE269952. The following ChIP–seq data from previous studies were used: H3K27ac (GSE61349)55, H3K4me1 (GSM946535), H3K4me3 (GSM946533), H3K36me3 (GSM946529), H3K9me3 (GSM946542), H3K27me3 (GSM946531)56 and CTCF (GSE98671)57. External Hi-C data of condensin I or condensin II deleted mitotic cells are available from the GEO (GSE228402)38. External ChIP–seq data of mitotically retained CTCF are available from the GEO (GSE129997)24. Source data are provided with this paper.
Code availability
The following codes used in this study are available from Zenodo: sequencing analysis pipelines (https://doi.org/10.5281/zenodo.10968270)58 and polymer simulation (https://doi.org/10.5281/zenodo.14545472)59.
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Acknowledgements
We thank M. Vermunt, H. Long, J. Lam and members of the Zhang Laboratory for discussions; M. Li, Z. Huang and D. Cheng from the Flow cytometry Core of Shenzhen Bay Laboratory for technical support on cell sorting; S. Huang and M. Yu from the Bioimaging Core of Shenzhen Bay Laboratory for imaging support; and staff from the Shenzhen Bay Laboratory High Performance Computing and Informatics Core for computational support. This work was supported by the National Natural Science Foundation of China (32100422 and 82471197 to H. Zhang; 22203055 to K.H.; and 22403068 to S.Q.); the Shenzhen Medical Research Fund (D2401002 to H. Zhang); and the Major Program of Shenzhen Bay Laboratory (S201101004 to H. Zhang; and S241101001 to K.H.). We thank J.-M. Peters’ group for the schematic of Sororin protein as illustrated in Supplementary Fig. 4a.
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Contributions
H. Zhang conceived the study and designed experiments. H. Zhang and K.H. supervised the study. H. Zhao and L.S. created the inducible acute degradation cell line used in this study. H. Zhao and L.S. performed sample preparation, FACS sorting, in situ Hi-C, ChIP–seq and microscopy experiments with help from E.L., B.W., M.W., F.S., Y.L., L.Z. and Y.G. S.Q. and F. Lyu performed polymer modelling. Data analyses and interpretation were performed by H. Zhang and G.A.B., with help from F. Liu, S.X., S.Q. and F. Lyu. H. Zhang wrote the manuscript with input from all authors.
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Extended data figures and tables
Extended Data Fig. 1 Characterization of the WapldTag/Smc2mAID cell line.
a, Schematic showing the edited Wapl locus and genotyping strategy. b, Genotyping results of positive clones targeting the Wapl locus. One experiment was performed. c, Schematic showing the edited Smc2 locus and genotyping strategy. d, Genotyping results of positive clones targeting the Smc2 locus. One experiment was performed. e, Flow cytometry plots showing progressive loss of WAPL or/and SMC2 fluorescence signals upon dTag13 or/and 5-Ph-IAA treatment. Two independent clones were tested. The parental cell line was shown as negative control. f, Western blots showing degradation of WAPL or/and SMC2 upon dTag13 or/and 5-Ph-IAA treatment. One experiment was performed. g, Immunofluorescence image showing mitotic retention of RAD21 upon WAPL depletion. Scale bar: 5 μm. Three independent experiments were performed for each clone. h, Quantification of the percentage of mitotic RAD21 retention upon WAPL loss. Data are presented as mean values +/- SEM (n = 3 independent experiments for both conditions). P values were calculated using two-sided student’s t-test. i, Quantification of the SMC2-mAID-mCherry fluoresence signal on mitotic chromosomes with or without WAPL. Data are presented as mean values +/- SEM (n = 3 independent experiments for both conditions). j, Metaphase spread analysis showing “closed-arm” mitotic cells in the absence of WAPL, indicating mitotic retention of cohesin. Images are zoomed-in views from a single nucleus. Scale bar: 5 μm. Three independent experiments were performed. P values were calculated using two-sided student’s t-test. k, Representative image showing the presence of lagging chromosomes in ana/telophase upon WAPL depletion. Scale bar: 2 μm. Two independent clones were shown. l, Quantification of lagging chromosomes in WAPL-replete and depleted mitotic cells. Data are presented as mean values +/- SEM (n = 3 independent experiments for both conditions). P values were calculated using two-sided student’s t-test. m, Line graph showing cell growth defects in WAPL depleted, SMC2 depleted or WAPL and SMC2 co-depleted cells. Data are presented as mean values +/- SEM (n = 3 independent experiments for all conditions). P values were calculated using two-sided student’s t-test.
Extended Data Fig. 2 Characterization of mitotic cohesin peaks.
a, Experimental design, showing the strategy of prometaphase arrest in combination with 5-Ph-IAA or/and dTag13 treatment. b, Flow cytometry plot showing the gating strategy to purify WAPL-depleted and WAPL and SMC2 co-depleted mitotic cells. More than three independent experiments were performed. c, Representative images showing FACS purified WAPL-depleted or WAPL and SMC2 co-depleted mitotic cells. Two independent experiments were performed. Scale bar: 10 μm. d, Venn diagram showing the intersection results of RAD21 peaks in the WAPL deficient G1-phase samples and WAPL and SMC2 co-depleted mitotic samples. e, Density heatmap plots showing the ChIP-seq signals of RAD21 peaks that were lost, maintained or gained during mitosis. f, Meta region plots showing the corresponding mitotic CTCF ChIP-seq signals for RAD21 peaks that were lost, maintained or gained. Note that compared to the lost ones, the maintained RAD21 peaks were co-localized by stronger mitotic CTCF binding. CTCF ChIP-seq data was adapted from our previous study24. g, Dot plots showing the average RAD21 ChIP-seq signals for peaks that were lost, maintained or gained during mitosis. Central line denotes mean values (n = 4 and 5 biological replicates for G1-phase and mitotic samples respectively). P values were calculated using two-sided student’s t-test. h, De-novo motif enrichment analysis for the mitotic specific RAD21 peaks. i, Box plot showing that mitotic specific RAD21 peaks (n = 3,642) were significantly larger compared to the interphase Rad21 peaks (n = 29,756). For both box plots, central lines denote medians; box limits denote 25th–75th percentile; whiskers denote 5th–95th percentile. P values were calculated using a two-sided Wilcoxon signed-rank test. j, Meta region plots showing that mitotic specific RAD21 peaks were heavily decorated with active histone marks. k, Pie charts showing that the mitotic specific RAD21 peaks were significantly more likely to co-localize with CREs. P values were calculated using two-sided Fisher’s exact test.
Extended Data Fig. 3 Condensin relocates mitotically retained RAD21 peaks.
a, Venn diagram showing the intersection results of RAD21 peaks identified in WAPL-deficient mitotic cells with or without condensin. b, Density heatmap plots of RAD21 ChIP-seq signals in WAPL-deficient mitotic cells with or without condensin. Peaks that were lost or maintained upon the introduction of condensin were shown. c, Upper panel: dot plots showing the significant reduction of mitotic RAD21 ChIP-seq signals in the presence of condensin. Rad21 peaks co-localized to CTCF, CRE or both were separately shown. Central line denotes mean values (n = 5 biological replicates for both conditions). P values were computed using two-sided student’s t-test. Lower panel: similar to the upper panel showing that the mitotic CTCF ChIP-seq signals were not significantly perturbed by condensin. Central line denotes mean values (n = 4 biological replicates for both conditions). P values were computed using two-sided student’s t-test. d, Browser tracks of independent biological replicates showing the reduced RAD21 peaks (co-localized to CTCF, indicated by Red arrows) in response to condensin loading during mitosis. Tracks of interphase samples were shown as control. Tracks of mitotic and interphase CTCF ChIP-seq as well as interphase H3K27ac ChIP-seq results were also shown. e, Similar to (d), browser tracks of independent biological replicates showing the reduced RAD21 peaks (co-localized to CREs, indicated by orange arrows) in response to condensin loading during mitosis.
Extended Data Fig. 4 Characterization of the WapldTagCdca5mAID and WapldTagSmc2mAIDCdca5mAID cell lines.
a, Schematic showing the edited Cdca5 (encoding Sororin), Wapl and Smc2 loci and the respective genotyping strategy. b, Genotyping results of the WapldTagCdca5mAID clones. One experiment was performed. c, Fluorescence images showing loss of WAPL and Sororin fluorescence signals upon 4 h of dTag13 and 5-Ph-IAA treatment in the WapldTagCdca5mAID cells. Two independent clones were tested. Scale Bar: 10 μm. d, Schematic showing the strategy to determine G2/M arrest length. e, Metaphase spread analysis showing a more extensive dissociation of sister-chromatids in the WapldTagCdca5mAID cells after 16 h compared to 6 h of RO-3306 mediated G2 arrest. Scale Bar: 10 μm. Four independent experiments were performed. f, Quantification of (d & e), showing progressive dissociation of sister-chromatids as G2-arrest duration were extended. Longer G2-arrest allowed more complete removal of cohesive-cohesin by WAPL. Data are presented as mean values +/- SEM (n = 4 independent experiments for all time points). P values were calculated by two-sided student’s t-test. g, Genotyping results of WapldTagSmc2mAIDCdca5mAID clones. One experiment was performed. h, Fluorescence images showing loss of WAPL, Sororin and SMC2 fluorescence signals upon 4 h of dTag13 and 5-Ph-IAA treatment in the WapldTagSmc2mAIDCdca5mAID cells. Three independent clones were tested. Scale Bar: 10 μm.
Extended Data Fig. 5 Condensin facilitates the disassembly of structural loops during mitotic progression.
a, Schematic showing the RO-3306 arrest/release strategy to obtain condensin replete or deficient cells from G2 phase to mitosis. b, Flow chart showing the gating strategy to obtain pMPM2-negative, -low, -mid and -high populations from condensin-replete control samples. Two independent experiments were performed. c, Flow chart showing the gating strategy to obtain pMPM2-negative, -low, -mid and -high populations from condensin-deficient samples. Two independent experiments were performed. d, Representative images of DAPI signals from cells sorted in (b) and (c). Scale bar: 10 μm. Two independent experiments were performed. e, Upper panel: KR-balanced Hi-C contact maps showing the dynamic change of compartments when cells enter mitosis with or without condensin. Bin size: 100 kb. Lower panel: Saddle plots showing the preservation of compartments in condensin deficient cells during mitotic entry. f, KR-balanced Hi-C contact maps showing the dynamic change of structural loops (green arrow) in cells transiting from G2-phase to mitosis with or without condensin. Bin size: 10 kb. g, Bar graph showing the number of structural loops identified by HICCUPS in each population. h, APA plots showing the delayed disappearance of structural loops during mitotic entry without condensin. i, Bar graph showing the quantification of APA signals of structural loops during mitotic entry with or without condensin. Data are presented as mean values (n = 2 biological replicates for both conditions).
Extended Data Fig. 6 G2/M short-arrest and release protocol confirms that cohesive-cohesin alone does not form focalized peaks without extrusive-cohesin.
a, Schematic showing the G2/M short-arrest and release protocol to obtain condensin-deficient mitotic chromosomes loaded with cohesive-cohesin alone. b, Metaphase spread analysis using the G2/M short-arrest and release protocol showing extensive sister-chromatid cohesion in the WAPL replete mitotic cells with Sororin(9A) mutant. In comparison, cells without Sororin(9A) mutant lost the cohesion between sister chromatids. Two independent experiments were performed. Scale bar: 10 μm. c, Quantification of (b), showing the percentage of “closed arm” during mitosis for indicated conditions. Data are presented as mean values (n = 2 independent experiments for both conditions). d, Venn-diagrams showing very few RAD21 peak calls in condensin-deficient mitotic cells with cohesive- but not extrusive-cohesin (G2/M short-arrest and release protocol). e, Venn-diagrams showing very few Sororin(9A) peak calls in condensin-deficient mitotic cells with cohesive- but not extrusive-cohesin (G2/M short-arrest and release protocol). There are 13 peaks that do not fall into the three listed categories. f, CPM normalized browser tracks showing loss of flag-tagged Sororin(9A) (cohesive-cohesin) peaks in the condensin-deficient mitotic chromosomes without extrusive-cohesin. Samples obtained through original or short-arrest and released protocols were shown. Tracks for independent biological replicates were shown. g, CPM normalized density heatmap plots of the flag-tagged Sororin(9A), showing that cohesive-cohesin peaks were almost completely lost in the absence of extrusive-cohesin. Samples generated by both original or short-arrest and release protocols were shown. h, Bar graphs showing the dramatic loss of flag-tagged Sororin(9A) ChIP-seq signals in the absence of extrusive-cohesin. Peaks colocalized to CTCF, CRE or both were shown separately. Data are presented as mean values +/- SEM (n = 5, 2 and 3 biological replicates for extrusive-cohesin replete, extrusive-cohesin deficient (long-arrest/release) and extrusive-cohesin deficient (short-arrest/release) samples respectively). P values were calculated using two-sided student’s t-test.
Extended Data Fig. 7 Influence of cohesive- or/and extrusive-cohesin on mitotic chromosome architecture.
a, P(s) curve of the control mitotic cells. The three regimes were indicated by dotted lines. The secondary diagonal between regime (ii) and (iii) were indicated by arrow. b, Upper panel: P(s) curves of independent clones showing the influence of extrusive-cohesin (“heavy load”) on mitotic chromosome architecture. Lower panel: Zoom in P(s) curves showing the influence of heavily or lightly loaded extrusive-cohesin on mitotic chromosome architecture. c, P(s) curves of independent clones showing the influence of cohesive-cohesin on mitotic chromosome architecture. d, P(s) curves of independent clones showing the influence of cohesive-cohesin (G2/M short-arrest and release protocol) on mitotic chromosome architecture. e, KR-balanced Hi-C contact matrices for independent clones showing the log2 fold change of contact probabilities when cohesive-cohesin was loaded (G2/M short-arrest and release protocol). f, P(s) curves of independent clones showing the combined influence of cohesive-cohesin and extrusive-cohesin on mitotic chromosome architecture. g, P(s) curve showing gain of short-range contact frequencies in the condensin-deficient mitotic chromosomes by extrusive-cohesin. Note that light-loaded extrusive-cohesin displayed similar but milder effects. Curves for independent clone were shown. h, P(s) curve showing the influence of cohesive-cohesin alone on the condensin-deficient mitotic chromosomes. i, P(s) curves showing the effect of condensin II depletion on mitotic chromosome architecture. Data adapted from our previous study38. j, P(s) curves showing the effect of condensin I depletion on mitotic chromosome architecture. Data adapted from our previous study38.
Extended Data Fig. 8 Extrusive- but not cohesive-cohesin influences the compartmentalization pattern of the condensin-deficient mitotic chromosomes.
a, KR-balanced Hi-C contact matrices of condensin-deficient mitotic chromosomes, showing the checkerboard compartmentalization patterns in condensin-deficient mitotic chromosomes with indicated configurations of cohesin. Bin size: 100 kb. b, Saddle plots of condensin-deficient mitotic chromosomes. c, KR-balanced Hi-C contact matrices showing loss of mA1 homotypic interactions (red arrows) in the presence of extrusive-cohesin (“heavy” or “light load”) but not cohesive-cohesin. Bin size: 24 kb. EV1 tracks were shown in parallel. d, KR-balanced Hi-C contact matrices showing the increased homotypic interactions among mB4 mitotic compartments in the presence of extrusive-cohesin (“heavy” or “light load”) but not cohesive-cohesin. Bin size: 100 kb. EV1 tracks were shown in parallel. mB4 compartments were marked by green bars. e, Aggregation-repulsion plots showing the impacts of extrusive-cohesin (“heavy-” or “light-load”) or/and cohesive-cohesin on mitotic chromosome compartmentalization patterns. Note that extrusive- but not cohesive-cohesin disrupted euchromatin (mA1, mA2) self-aggregation. f, Scatter plots showing the EV1 values of 25 kb genomic bins in the condensin-deficient mitotic chromosomes without any cohesin (x-axis) against condensin-deficient mitotic chromosomes with cohesive-cohesin, extrusive-cohesin (“heavy” or “light load”) or both (y-axes). Bins were color coded based on their compartment assignment. Green and red arrows indicate the reduction of EV1 values of bins within mB4 and mA1 compartments respectively.
Extended Data Fig. 9 Extrusive- but not cohesive-cohesin facilitates chromatin individualization.
a, KR-balanced Hi-C contact matrices showing the trans-chromosomal interactions among chromosomes 1 to 6 in mitotic cells with eight distinct SMC protein complex configurations. Maps of independent clones were shown. Scale bars were normalized to the respective size of each library. b, Bar graph showing the percentage of trans contacts in mitotic cells with eight distinct SMC protein complex configurations. Data are presented as mean values +/- SEM (n = 2 and 3 biological replicates for condensin replete and deficient conditions respectively). P values were calculated using two-sided student’s t-test.
Extended Data Fig. 10 Cohesive-cohesin displays relatively low mobility on chromatin without extrusive-cohesin.
a, Schematic showing the segmentation of domains into 100kb-wide stripes. b, Line plots showing the gain of short-range interactions (purple arrows) within TADs in the presence of cohesive-cohesin. TADs of different size-range were separately shown. Note that random position control did not show such gain of interactions. c, Line plots showing the gain of short-range interactions within pre-defined TAD regions (purple arrows) induced by cohesive-cohesin in the absence of extrusive-cohesin. d, KR-balanced Hi-C contact matrices of two independent loci showing that cohesive-cohesin from the short-arrest/release protocol induced contacts within TADs without extrusive-cohesin. Bin size: 25 kb. e, Line plots showing the gain of short-range interactions within pre-defined TAD regions (purple arrows) induced by cohesive-cohesin in the absence of extrusive-cohesin from the short arrest/release protocol.
Extended Data Fig. 11 Structural loops are attenuated in G2 phase compared to late-G1 phase.
a, Flow cytometry plot showing the gating strategy to sort late-G1 and G2 phase cells from asynchronous population expressing the mCherry-MD cell cycle phase reporter. Two independent experiments were performed. b, Representative images showing the DAPI signal of sorted late-G1 or G2-phase cells. Scale Bar: 5 μm. Note that G2-phase cells displayed significantly larger nuclei size. P values were calculated using a two-sided Wilcoxon signed-rank test. n > 200 nuclei from two independent experiments. c, KR-balanced Hi-C contact maps showing the weakening of structural loops (indicated by green arrows) in G2-phase cells compared to late-G1. Bin size: 25 kb. Note that small structural loops were not affected (blue arrow). d, APA plots showing the dramatic attenuation of large structural loops in G2-phase compared to G1. e, Bar plot showing the APA signals corresponding to (d). Data are presented as mean values (n = 2 biological replicates for both conditions).
Extended Data Fig. 12 Polymer simulation unveils extrusive- and cohesive-cohesin interaction pattern.
a, Schematic illustration showing the three modes (I, II and III) of interaction between extrusive- and cohesive-cohesin. b, Simulated tracks showing the distribution of CTCF, extrusive-cohesin and cohesive-cohesin for each mode of interaction. c, Cis- (upper panel) and trans- (lower panel) sister-chromatid contact maps showing the simulation of distinct modes of interaction between extrusive- and cohesive-cohesin. Cohesive-cohesin are randomly positioned (3/Mb) before extrusive-cohesin loading. In this simulation, the cohesive-cohesin resistance coefficient α is set to be 0. Example weakened and unaffected loops were marked by green and blue arrows respectively. d, Scatter plot showing that larger loops were more susceptible to cohesive-cohesin in our simulation (cohesive-cohesin density = 3/Mb and resistance coefficient α = 0). Results of mode II and mode III were plotted separately. The trend lines were generated using the geom_smooth function of ggplot2 in R software with shaded area representing 95% confidence interval of the corresponding fitted lines. e, Heatmaps showing the impacts of various cohesive-cohesin density and resistance coefficient on loop formation. Relative strength of all loops and two subsets of loops with indicated size ranges were shown.
Extended Data Fig. 13 The tripartite pattern of interactions among extrusive-cohesin, cohesive-cohesin and condensin.
a, Schematic illustration showing two potential mechanisms (relocation model and eviction model) through which condensin disrupts extrusive-cohesin positioning. b, Schematic illustration showing the influence of extrusive-cohesin or cohesive-cohesin on mitotic chromosome structure. c, Schematic illustration showing the two potential models (passive model and reliance model) through which condensin disrupts cohesive-cohesin focal peaks. In the second model, condensin bypasses cohesive-cohesin. d, Schematic showing that cohesive-cohesin cannot be arrested at CTCF binding sites. e, Schematic showing the classic loop extrusion process without cohesive-cohesin. f, Schematic showing that extrusive-cohesin pushes cohesive-cohesin along one sister-chromatid without affecting the other chromatid. Cohesive-cohesin is positioned by extrusive-cohesin at CTCF binding sites.
Supplementary information
Supplementary Information
This file contains Supplementary Notes 1–14, Supplementary Figs. 1–14 and Supplementary References.
Supplementary Table 1
List of domains identified by Arrowhead and insulation score profiles.
Supplementary Table 2
List of loops identified by HICCUPS.
Supplementary Table 3
Hi-C statistics.
Supplementary Table 4
CRE contact list.
Supplementary Table 5
Structural loop anchor list.
Supplementary Table 6
Oligonucleotides used for gene editing and genotyping.
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Zhao, H., Shu, L., Qin, S. et al. Extensive mutual influences of SMC complexes shape 3D genome folding. Nature 640, 543–553 (2025). https://doi.org/10.1038/s41586-025-08638-3
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DOI: https://doi.org/10.1038/s41586-025-08638-3
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