Abstract
During mitosis, condensin activity is thought to interfere with interphase chromatin structures. To investigate genome folding principles in the absence of chromatin loop extrusion, we codepleted condensin I and condensin II, which triggered mitotic chromosome compartmentalization in ways similar to that in interphase. However, two distinct euchromatic compartments, indistinguishable in interphase, emerged upon condensin loss with different interaction preferences and dependencies on H3K27ac. Constitutive heterochromatin gradually self-aggregated and cocompartmentalized with facultative heterochromatin, contrasting with their separation during interphase. Notably, some cis-regulatory element contacts became apparent even in the absence of CTCF/cohesin-mediated structures. Heterochromatin protein 1 (HP1) proteins, which are thought to partition constitutive heterochromatin, were absent from mitotic chromosomes, suggesting, surprisingly, that constitutive heterochromatin can self-aggregate without HP1. Indeed, in cells traversing from M to G1 phase in the combined absence of HP1α, HP1β and HP1γ, constitutive heterochromatin compartments are normally re-established. In sum, condensin-deficient mitotic chromosomes illuminate forces of genome compartmentalization not identified in interphase cells.
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Data availability
The Hi-C, Cut&Tag and ChIP-seq data generated in this study are deposited at the Gene Expression Omnibus (GEO) database with the accession GSE228402. External ChIP-seq data from previous studies are listed as follows: H3K27ac (GSE61349)39, H3K4me1 (GSM946535), H3K4me3 (GSM946533), H3K36me3 (GSM946529), H3K9me3 (GSM946542), H3K27me3 (GSM946531)40 and PolII (GSE168251)3. External Hi-C data of CTCF depleted and complete G1E-ER4 cells are available at GSE168251 (ref. 3). External Hi-C data of prometaphase G1E-ER4 parental cell line is available at GSE129997 (ref. 2). Source data are provided with this paper.
Code availability
Codes used in this study are available at GitHub (https://github.com/karazakarak/zhang_lab_pipeline). Codes are also available at Zenodo41.
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Acknowledgements
We thank L. Mirny at MIT; M. Vermunt at Goethe University, Frankfurt; X. Shen at Tsinghua University; and members of the Zhang and Blobel Labs for helpful discussions. We are indebted to M. Li, Z. Huang and D. Cheng from the Flow-cytometry Core at the Shenzhen Bay Laboratory for technical support on cell sorting. We thank S. Huang and M. Yu from the Bioimaging Core at the Shenzhen Bay Laboratory for imaging support. This work was supported by the National Science Foundation of China (grant 32100422 to H.Z.) and the National Institutes of Health (NIH; grants U01DK127405 and R01DK058044 to G.A.B.).
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Contributions
G.A.B. and H. Zhang conceived the study and designed experiments. H. Zhao and Y.L. created the inducible acute degradation cell line used in this study. H. Zhao performed sample preparation, FACS sorting, in situ Hi-C, ChIP-seq and microscopic experiment with help from E.L., L.S., D.J., B.W., M.W., L.Z., F.S., J.C.L. and S.C.M. C.A.K., B.M.G. and R.C.H. contributed to sequencing of in situ Hi-C materials. Data analysis was performed by H. Zhang with help from F.L. H. Zhang and G.A.B. wrote the paper with inputs from all authors.
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Nature Genetics thanks Anton Goloborodko, Wenbo Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Characterization of the G1E-ER4:SMC2-AID-mCherry cell line.
a, Left panel: representative image showing the condensation of mitotic chromosomes and correct nuclear localization of SMC2-mAID-mCherry fusion protein. Scale bar: 5 μm. Right panel: representative image showing the correct separation of chromatids during ana/telophase. Scale bar: 5 μm. Three independent experiments replicates were performed. b, Left panel: flow cytometry plots showing the rapid degradation of SMC2-mAID-mCherry fusion protein upon auxin treatment. Right panel: flow cytometry plot showing the shift of cell cycle distribution upon long-term auxin treatment. Two independent experiments were performed. c, Growth curve of G1E-ER4:SMC2-mAID-mCherry cells with or without auxin treatment. Error bar denotes SEM (n = 3). P values were calculated using a two-sided Student’s t test. d, Representative image showing the morphology of mitotic chromosomes after auxin treatment for indicated durations (indicated by yellow arrows). Cells were stained with anti-pMPM2 antibody to indicate mitotic population. Scale bars are indicated in the image. Three independent experiments were performed. e, Bar graph showing the percentage of mitotic cells with extensive chromatid entanglements after auxin treatment for indicated durations. Error bar denotes SEM (n = 3). P values were calculated using a two-sided Student’s t test.
Extended Data Fig. 2 Purification of condensin-deficient mitotic cells.
a, Flow cytometry plot showing the gating strategy to purify mitotic cells at different time points after addition of auxin. Cells were sorted by pMPM2, DAPI and mCherry signals. More than two independent experiments were performed. b, Bar graph showing the high stratum-adjusted correlation coefficient for each chromosome (n = 19) between biological replicates for each condition. c, KR balanced Hi-C contact matrices (chr2 vs. chr3) showing the gradual increase of trans-chromosome interactions over time upon condensin loss. d, Dot plot showing the percentage of tran-chromosome interactions for independent biological replicates at each tested time point. n = 2 for SMC2(+), 0.5 h, 1 h and 4 h auxin treated samples. n = 4 for 8 h auxin treated samples.
Extended Data Fig. 3 Progressively strengthened compartments in mitotic cells upon condensin loss.
a, Saddle plots of independent biological replicates of asynchronous and condensin deficient mitotic cells. b, Dot plot showing progressive gain of compartmental strength of each individual chromosome (n = 20). P values were calculated using a two-sided paired Wilcoxon signed-rank test. c, Raw Hi-C contact matrices showing the results of in-silicon simulation of various levels of G1 contamination in mitotic control cells. Note that even 20% of G1 contamination, failed to show as strong compartmentalization as mitotic cells without condensin.
Extended Data Fig. 4 Initial clustering of mitotic specific compartments based on EV1 values.
a, Scatter plot showing the EV1 values of 25 kb genomic bins in asynchronous control cells (x-axis) against condensin-deficient (4 h) mitotic cells (y-axis). Bins belonging to groups I, II and III were marked by red, blue and gray colors, respectively. b–h, Scatter plot showing the enrichment of indicated histone modification intensity or RNA polymerase binding intensity for 25 kb genomic bins (group I and II) in asynchronous control cells (x-axis) against condensin-deficient (4 h) mitotic cells (y-axis). i, Heatmap showing the clustering result to group the genome into different mitotic specific compartments. Note that bins from group I can be further categorized into mA1 and mB4 compartments and bins from group II can be categorized into mA2 and mB1 compartments.
Extended Data Fig. 5 Characterization of repressive compartments in the condensin-deficient mitotic cells.
a, Pie chart showing the fraction of different mitotic compartments in the genome. b, Bar graph showing the percentage of genomic bins with inverted EV1 values during mitosis in each type of compartment. P values were calculated using a two-sided Fisher’s exact test. c, Schematic illustration showing how to generate the chromosome-___location dependent attraction–repulsion curve. d, Pseudo-data showing analytical results of c. e, Chromosome-___location dependent attraction–repulsion curve between mB1 and mB4 compartments for condensin-deficient (4 h) mitotic and asynchronous cells.
Extended Data Fig. 6 Characterization of active compartments in the condensin-deficient mitotic cells.
a, KR balanced Hi-C contact matrices showing the prominent interactions among mA1 compartment (chr1:152–155.5 Mb) in the condensin-replete mitotic samples (from this study and a prior study by Zhang et al.2) as well as condensin-deficient (4 h) mitotic samples and asynchronous samples. b, Box plots showing the enrichment of indicated chromatin associating features (histone modification and transcription) for mA1 (n = 5,299 genomic bins), mA2 (n = 11,531 genomic bins) compartments and others (n = 77,997 genomic bins). For all 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. c, Box plots showing the asynchronous EV1 values for mA1 (n = 5,299 genomic bins) and mA2 (n = 11,531 genomic bins) compartments for both biological replicates. For all box plots, central lines denote medians; box limits denote 25th–75th percentile; whiskers denote 5th–95th percentile. d, Box plots showing the EV1 values for mA1 (n = 5,299 genomic bins) and mA2 (n = 11,531 genomic bins) compartments in the condensin-deficient (4 h) mitotic cells for both biological replicates. For all 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.
Extended Data Fig. 7 Comparison of the ‘extrusion-free’ mitotic and interphase chromatin.
a, Schematic illustration showing homozygous insertion of mAID-mCherry tag at the C terminus of endogenous SMC3. b, Flow-cytometry showing the rapid degradation of SMC3 upon auxin treatment. Two independent experiments were performed. c, Western blot showing the degradation of SMC3 upon auxin treatment. One experiment was performed. d, KR balanced Hi-C contact matrices (chr6:92–96 Mb) showing the loss of TADs boundaries (purple arrows) upon SMC3 depletion. Contact maps of both biological replicates and replicate-merged samples are shown. Bin size: 10 kb. TAD boundaries are indicated by purple arrows. e, Composite contact map of TAD boundary showing reduced insulation after SMC3 depletion. f, KR balanced Hi-C contact matrices (chr1:127.5-133.5 Mb) showing elimination of chromatin loops upon SMC3 depletion. Contact maps of both biological replicates and replicate-merged samples are shown. Bin size: 10 kb. Chromatin loops are indicated by purple arrows. g, APA plots showing disappearance of structural loops upon SMC3 loss. h, Scatter plot showing the EV1 values of 25 kb genomic bins in SMC3 (+) (x-axis) against SMC3 (−) G1 phase cells (y-axis). Bins were color coded by their compartment assignment. i, KR balanced Hi-C contact matrices (chr1:181–183.5 Mb and chr2:118–122 Mb) showing mild increase of mA1 homotypic interactions after SMC3 loss. Bin size: 25 kb. Browser tracks of EV1 values are shown. j, Dot plot showing the strength of mA1 homotypic interactions in indicated samples. Each dot represents an individual chromosome (n = 18). P values were calculated using a two-sided paired Wilcoxon signed-rank test. k, Dot plot showing the strength of mA2 homotypic interactions in indicated samples. Each dot represents an individual chromosome (n = 18). P values were calculated using a two-sided paired Wilcoxon signed-rank test. l, KR-balanced Hi-C contact matrices (chr2:166.5–170 Mb) showing the lack of mB1-to-mB4 interactions in interphase cells without SMC3. mB1 and mB4 compartments are highlighted by purple and blue bars, respectively. Browser tracks of EV1 values are shown. Bin size: 25 kb.
Extended Data Fig. 8 Epigenetic landscape of mitotic chromosomes in the absence of condensin.
a, KR balanced Hi-C contact matrices (chr1:152–152.5 Mb and chr1:152.8–153.8 Mb and chr1:154.6–155.6 Mb) showing mitotic compartments. Bin size: 25 kb. mA1, mA2 and mB1 compartments are labeled by red, yellow and purple bars, respectively. b, Representative tracks corresponding to the genomic regions in a, showing the ChIP–seq profiles of RNA PolII, H3K27ac, H3K36me3 and H3K27me3 in asynchronous as well as condensin-deficient (4 h) mitotic cells. c, Density plots showing the EV1 values as well as ChIP–seq signal strength of indicated marks in mA1 and mA2 compartments in the asynchronous and condensin-deficient (4 h) mitotic cells. d, Box plots showing the log2 fold change of indicated marks within mA1 (n = 2,096 compartments) or mA2 (n = 4,137 compartments) between condensin-deficient mitotic and asynchronous samples. For all 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.
Extended Data Fig. 9 Characterization of the G1E-ER4:mCherry-mAID-HP1α;GFP-FKBPF36V-HP1β cell line.
a, Schematic illustration showing the homozygous insertion of mCherry-mAID tag and GFP-FKBPF36V tag to the N terminus of endogenous Cbx5 and Cbx1 locus, respectively. b, Western blot showing the degradation of HP1α and HP1β upon 5-Ph-IAA or/and dTag13 treatment. One experiment was performed. Loading control was processed on a different gel. c, Schematic illustration showing the strategy of nocodazole-based arrest/release in conjunction with 5-Ph-IAA or/and dTag13 treatment. Early and late-G1 phase cells with four distinct HP1 protein configurations were collected. d, Flow cytometry plot showing the sorting strategy of early-G1 and late-G1 phase cells with distinct HP1 protein configurations. Two biological replicates were performed. e, Representative confocal images showing the successful depletion of HP1α or HP1β or both in the sorted G1 phase cells. Scale bar: 10 μm. Two biological replicates were performed. f, Flow cytometry plot showing the mitotic progression of cells under distinct configurations of HP1 proteins after nocodazole release. Two biological replicates were performed.
Extended Data Fig. 10 HP1α and HP1β are dispensable for post-mitotic genome refolding.
a, Bar graph showing the high stratum-adjusted correlation coefficient for each chromosome (n = 19) between biological replicates for each condition. b, KR balanced Hi-C contact matrices (chr1:3–160 Mb) showing chromatin the compartmentalization in early-G1 and late-G1 cells. Samples with four distinct HP1 protein configurations are shown. Bin size: 100 kb. Browser tracks of the corresponding EV1 values are shown for each contact map. c, Saddle plots showing the progressive compartmentalization of chromatin from early-G1 to late G1. Samples with four distinct HP1 protein configurations are shown. Compartmental strength for samples is labeled for each plot (purple number). d, P(s) curves for early-G1 and late-G1 phase samples under distinct HP1 protein configurations. e, APA plots for structural loop signals in early-G1 and late-G1 phase samples under distinct HP1 protein configurations. f, APA plots for CRE loop signals in early-G1 and late-G1 phase samples under distinct HP1 protein configurations.
Supplementary information
Supplementary Information
Supplementary Notes 1–5 and Supplementary Figs. 1–11.
Supplementary Table 1
Information on Hi-C statistics, ___domain calls, mitotic compartments and oligos used in this study.
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Zhao, H., Lin, Y., Lin, E. et al. Genome folding principles uncovered in condensin-depleted mitotic chromosomes. Nat Genet 56, 1213–1224 (2024). https://doi.org/10.1038/s41588-024-01759-x
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DOI: https://doi.org/10.1038/s41588-024-01759-x
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