Abstract
Chromosome folding is modulated as cells progress through the cell cycle. During mitosis, condensins fold chromosomes into helical loop arrays. In interphase, the cohesin complex generates loops and topologically associating domains (TADs), while a separate process of compartmentalization drives segregation of active and inactive chromatin. We used synchronized cell cultures to determine how the mitotic chromosome conformation transforms into the interphase state. Using high-throughput chromosome conformation capture (Hi-C) analysis, chromatin binding assays and immunofluorescence, we show that, by telophase, condensin-mediated loops are lost and a transient folding intermediate is formed that is devoid of most loops. By cytokinesis, cohesin-mediated CTCF–CTCF loops and the positions of TADs emerge. Compartment boundaries are also established early, but long-range compartmentalization is a slow process and proceeds for hours after cells enter G1. Our results reveal the kinetics and order of events by which the interphase chromosome state is formed and identify telophase as a critical transition between condensin- and cohesin-driven chromosome folding.
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Data availability
Sequencing data generated in this study have been deposited in the Gene Expression Omnibus (GEO) repository under accession number GSE133462. The dataset titled R1 refers to replicate 1, which is used in Figs. 1–5 and Extended Data Figs. 1–5. The dataset titled R2 refers to replicate 2, which is used in Fig. 2 and Extended Data Figs. 1 and 6. The dataset titled R3 refers to replicate 3, which is used in Fig. 2 and Extended Data Figs. 1 and 7. Data from this publication can also be accessed in the 4DN Data Portal at https://data.4dnucleome.org/abramo_et_al_2019. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are available online for Figs. 2, 3, 6 and 7 and Extended Data Figs. 1, 3, 4 and 8.
Code availability
Code for Hi-C analyses are available at the following links: distiller-nf (https://github.com/mirnylab/distiller-nf), pairtools (https://github.com/mirnylab/pairtools), cooltools (https://github.com/mirnylab/cooltools) and cworld (https://github.com/dekkerlab/cworld-dekker).
Change history
19 November 2019
In the HTML version of this article originally published, the links for the unprocessed western blots of ‘Source Data Fig. 7’ and the statistical data for ‘Source Data Extended Data Fig. 1’ were incorrectly swapped. This has now been corrected.
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Acknowledgements
We thank C. Baer from the UMass SCOPE Imaging Core for advice on imaging and help with the classification pipeline on CellProfiler. We thank members of the Dekker and Mirny laboratories for discussions. We acknowledge support from the National Institutes of Health Common Fund 4D Nucleome Program (DK107980) and the National Human Genome Research Institute (HG003143). J.D. is an investigator of the Howard Hughes Medical Institute.
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Contributions
K.A. and J.D. conceived and designed the project. K.A. and A.-L.V. performed time-course experiments. K.A. performed Hi-C, imaging and chromatin-association experiments. A.N.F. generated the HeLa S3 NCAPH–dTomato cells and performed imaging experiments. K.A., S.V.V. and H.O. analysed data. K.A. and J.D. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Cell cycle analysis of mitotic exit time courses.
a, FACS analysis of non-synchronous and prometaphase-arrested cultures and of cultures at different time points after release from prometaphase-arrest in time course replicate 1. Percentages in the upper right corner represent the number of cells with a G1 DNA content. b-d, Quantification of the fraction of cells in G1 at each time point from time course replicate 1 normalized to t = 8 hours (b), time course replicate 2 normalized to t = 8 hours (c), and time course replicate 3 normalized to a G1 maximum assumed to be 80% (d). Three independent experiments were performed with similar results. Source Data are provided in Source data Extended Data Fig. 1.
Extended Data Fig. 2 Compartment analysis for time course replicate 1.
a, Principal component 1 (PC1) along Chromosome 14 for Hi-C data obtained from cells at different time points after release from prometaphase. Principal component analysis was performed on Hi-C data binned at 200 kb resolution. PC1 detects A and B compartments starting at t = 3 hours. Lower left corner represents Pearson correlation value of each track compared to non-synchronous PC1. b, Principal component 3 (PC3) along Chromosome 14 for Hi-C data obtained from cells at different time points after release from prometaphase. Principal component analysis was performed on Hi-C data binned at 200 kb resolution. PC3 detects some A and B compartments starting at t = 2.75 hours, but at later time points, PC1 captures compartments. Lower left corner represents Pearson correlation value of each track compared to non-synchronous PC1. Three independent experiments were performed with similar results.
Extended Data Fig. 3 TAD and compartment domain boundaries form with similar kinetics.
a, Aggregate Hi-C data binned at 40 kb resolution at domain boundaries (top = all 724 boundaries, middle = 657 TAD boundaries, bottom = 67 compartment boundaries) at different time points after release from prometaphase. b, Average insulation profile across averaged domain boundaries shown in panel a (left = TAD boundaries, right = compartment boundaries) for different time points. c, Normalized strength for domain boundaries as a function of time after release from prometaphase. The strength for each of these features was set at 1 for the 8 hour time point. TAD boundaries and compartment boundaries form with similar kinetics. Three independent replicate experiments yielded similar results. Source Data are provided in Source Data Extended Data Fig. 3.
Extended Data Fig. 4 Compartment analysis for chromosome 14.
a, Saddle plots of Hi-C data for chromosome 14 binned at 200 kb resolution for different time points and split into genomic distance bands, as shown in gray in the first row. b, Normalized compartmentalization strength of different genomic distances as a function of time and split by interaction type (A-A, B-B, A-B). c, Normalized compartmentalization strength of interaction types as a function of time and split by genomic distance. Three independent experiments were performed with similar results. Source Data are provided in Source data Extended Data Fig. 4.
Extended Data Fig. 5 Kinetics of loop formation for loops of different size.
Loops were grouped according to size: a, loops less than or equal to 125 kb, b, loops greater than 125 kb and less than or equal to 200 kb, c, loops greater than 200 kb and less than or equal to 325 kb, d, loops greater than 325 kb. For each panel, top row: log2(observed/expected) Hi-C data for experimental time course, middle row: log2(observed/expected) Hi-C data for simulated time course, bottom row: the difference between experimental and simulated Hi-C data. Kinetics of loop formation is similar for all loop sizes. Three independent experiments were performed with similar results.
Extended Data Fig. 6 Analysis of time course replicate 2.
a, Aggregated Hi-C data binned at 20 kb resolution at chromatin loops at different time points. Top row: Experimental Hi-C data. Middle row: Simulated Hi-C data. Bottom row: The difference between experimental and simulated Hi-C data. Loops are more prominent in experimental Hi-C data than in the simulated data between 3 and 4.5 hours. This analysis included loops larger than 200 kb to avoid the strong signal at the diagonal of the interaction matrix. Simulations were performed with experimental data from this time course (mixing Hi-C data for t = 0 and t = 8 hours). b, Aggregate Hi-C data binned at 40 kb resolution at TAD boundaries for different time points. Top row: Experimental Hi-C data. Middle row: Simulated Hi-C data. Bottom row: The difference between experimental and simulated Hi-C data. Insulation strength is stronger in experimental Hi-C data than in simulated Hi-C data at t = 3.5 and t = 4.5 hours. c, Saddle plots of Hi-C data binned at 200 kb resolution for different time points. Top row: Experimental Hi-C data. Middle row: Simulated Hi-C data. Bottom row: The difference between experimental and simulated Hi-C data. Compartmentalization is weaker in experimental Hi-C than in simulated Hi-C data as illustrated by the fact that A-B interactions are less depleted in the experimental data (upper right and lower left corner of saddle plots). d, Derivative from P(s) plots. Black lines represent the derivative of P(s) for experimental Hi-C data and the dashed green lines represent the derivative of P(s) for the simulated Hi-C datasets for corresponding time points. Three independent experiments were performed with similar results.
Extended Data Fig. 7 Analysis of time course replicate 3.
a, Aggregated Hi-C data binned at 20 kb resolution at chromatin loops at different time points. Top row: Experimental Hi-C data. Middle row: Simulated Hi-C data. Bottom row: The difference between experimental and simulated Hi-C data. Loops are more prominent in experimental Hi-C data than in the simulated data between 3.25 and 4 hours. This analysis included loops larger than 200 kb to avoid the strong signal at the diagonal of the interaction matrix. Simulations were performed with experimental data from this time course (mixing Hi-C data for t = 0 and t = 6 hours). b, Aggregate Hi-C data binned at 40 kb resolution at TAD boundaries for different time points. Top row: Experimental Hi-C data. Middle row: Simulated Hi-C data. Bottom row: The difference between experimental and simulated Hi-C data. Insulation strength is stronger in experimental Hi-C data than in simulated Hi-C data at t = 3.25 and t = 4 hours. c, Saddle plots of Hi-C data binned at 200 kb resolution for different time points. Top row: Experimental Hi-C data. Middle row: Simulated Hi-C data. Bottom row: The difference between experimental and simulated Hi-C data. Compartmentalization is weaker in experimental Hi-C than in simulated Hi-C data as illustrated by the fact that A-B interactions are less depleted in the experimental data (upper right and lower left corner of saddle plots). d, Derivative from P(s) plots. Black lines represent the derivative of P(s) for experimental Hi-C data and the dashed green lines represent the derivative of P(s) for the simulated Hi-C datasets for corresponding time points. Three independent experiments were performed with similar results.
Extended Data Fig. 8 Chromatin association dynamics of CTCF, condensin, and cohesion.
a, Classification of cell cycle stages based on DAPI staining and tubulin organization. Scale bar = 5µm. b, Localization of Lamin A/C, NCAPH, and CTCF during different cell cycle stages shown in panel a. Scale bar = 5µm. c, Quantification of CTCF and NCAPH colocalization with chromatin in single cells at different cell cycle stages. Left plot represents data from all cells with color indicating cell cycle stage. Right plots represent the data separated into each individual cell cycle stage. d, Top: Western blot analysis of chromatin-associated proteins purified from HeLaS3-NCAPH-dTomato cells at different time points after release from prometaphase. Bottom: Quantification of the western blot shown above. NCAPH and Rad21 were analysed on the same gel. The samples for Histone H3 analysis were run on another gel. Four independent experiments were performed with similar results. Source Data for microscopy are provided in Source data Fig. 7. Unprocessed blots are provided in Source data Extended Data Fig. 8.
Supplementary information
Supplementary Tables
Supplementary Tables 1–4: gRNA sequences for genome editing, PCR primers for NCAPH homology arms, gBlock Gene Fragment and Hi-C Library Statistics.
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Abramo, K., Valton, AL., Venev, S.V. et al. A chromosome folding intermediate at the condensin-to-cohesin transition during telophase. Nat Cell Biol 21, 1393–1402 (2019). https://doi.org/10.1038/s41556-019-0406-2
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DOI: https://doi.org/10.1038/s41556-019-0406-2
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