Research Article

A pathway for mitotic chromosome formation

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Science  09 Feb 2018:
Vol. 359, Issue 6376, eaao6135
DOI: 10.1126/science.aao6135
  • A pathway for mitotic chromosome formation.

    In prophase, condensins mediate the loss of interphase chromosome conformation, and loop arrays are formed. In prometaphase, the combined action of condensin I (blue spheres in the bottom diagram) and II (red spheres) results in helically arranged nested loop arrays.

  • Fig. 1 Chromosome morphogenesis during synchronous mitosis.

    (A) Representative DAPI images of nuclei and chromosomes in CDK1as DT40 cells taken at the indicated time points (in minutes) after release from 1NM-PP1–induced G2 arrest show mitotic chromosome formation. NEBD, nuclear envelope breakdown. (B) Hi-C interaction maps of chromosome 7 (binned at 100 kb) from cells collected at the indicated time points in prophase and prometaphase show large-scale changes in contact frequencies as cells progress through mitosis. (C) The average interaction maps centered around G2 TAD (topologically associating domain) boundaries. TAD boundaries disappear. (D) Compartmentalization saddle plots: average distance-normalized interaction frequencies between cis-pairs of 100-kb bins arranged by their G2 eigenvector value (EV1). Compartments disappear.

  • Fig. 2 Prophase chromosomes fold as axially compressed loop arrays.

    (A) Genome-wide curves of contact frequency P(s) versus genomic distance s, normalized to unity at s = 100 kb. The curves are derived from prophase Hi-C data at the indicated time points after release from G2 arrest. The dotted line indicates P(s) = s−0.5 observed for mitotic chromosomes (8). log, asynchronous culture. (B) Overview of the coarse-grained model of prophase chromosomes. The chromosome is compacted into a series of consecutive loops and compressed into a cylindrical shape. The loop bases form a scaffold at the chromosomal axis, each loop occupies a cylindrical sector of height h and angular size Embedded Image, oriented at angle Embedded Image. Δz, loop separation. The coarse-grained model predicts the P(s) curve to have three distinct regions: intraloop (I), intralayer (II), and interlayer (III) regions. (C) Best-fitting P(s) predictions by the coarse-grained model for late prophase (t = 7.5 min) under two different assumptions on loop orientations: (top) uncorrelated and (bottom) correlated orientations of consecutive loops. Uncorrelated angular loop orientations lead to a plateau in P(s) in the intralayer, whereas correlated angles lead to the experimentally observed P(s) = s−0.5 (right panels). (D) Polymer models of prophase chromosomes. A chromosome is modeled by a polymer (dark gray circles), arranged into an array of consecutive loops (loop bases indicated in orange) and compacted into a cylinder with a specified volume density (bottom right). (E) Goodness of fit for simulated versus experimental P(s). Polymer simulations were performed for a range of loop densities and loop lengths, and P(s) was calculated for each simulation. The heat map shows the quality of a match between the predicted and experimental P(s) curves at late prophase (t = 7.5 min). (F) P(s) derived from late prophase Hi-C experiments (green line) and the best-fitting polymer models (grayscale lines). Average loop size and linear density of loops along the chromosome axis are listed. (G) Top and side view of the best-fitting polymer model of late prophase chromosomes. Loop bases are shown in red and several loops rendered in different colors. (H) Average loop size and linear density of the three best-fitting models of prophase chromosomes at different time points.

  • Fig. 3 Helical organization of prometaphase chromosomes.

    (A) Genome-wide curves of contact frequency P(s) versus genomic distance (separation, s), normalized to unity at s = 100 kb. The curves are derived from Hi-C data obtained from prometaphase cells (t = 10 to 60 min after release from G2 arrest). The dashed line indicates P(s) = s−0.5. Arrows indicate positions of a local peak in P(s) representing the second diagonal band observed in Hi-C interaction maps. (B) Coarse-grained model of prometaphase chromosomes with staircase loop arrangement. (Left, top) The staircase loop arrangement implies that loops rotate in genomic order around a central scaffold (supplementary materials). (Left, bottom) Angles of adjacent loops are correlated and steadily increasing, reflecting a helical arrangement of loops. (Right) This helical arrangement can be observed as gyres by DNA staining, and a helical scaffold can be observed in cells expressing GFP-tagged condensins. (C) Best-fitting P(s) predictions by the staircase coarse-grained model for late prometaphase t = 30 min (left panel) and t = 60 min (right panel) after release from G2 arrest (Hi-C data: colored lines; model; gray lines). (D) Polymer model of prometaphase chromosomes. Chromosomes are modeled as arrays of consecutive nested loops with a helical scaffold (outer loops in red; inner loops in blue; also indicated diagrammatically at bottom right). (E) Goodness of fit for simulated versus experimental P(s). Polymer simulations were performed with varying the helix height (nanometers), the size of a helical turn (megabases), and the sizes of the inner and outer loops. P(s) was calculated for each simulation. The heat maps show the quality of the best match between the predicted and experimental P(s) at prometaphase (t = 30 min), when two out of four parameters were fixed to the specified values. (F) P(s) derived from prometaphase Hi-C experiments (colored lines) and the best-fitting polymer models (gray lines). (Left) t = 30 min; (right) t = 60 min after release from G2 arrest. The average size of outer and inner loops, length of a helix turn, and helical pitch are indicated. (G) Parameters of the helical scaffolds from the best-fitting polymer models. x axis: ratio of the radius of the helical scaffold to that of the whole chromatid; y axis: ratio of the pitch to the helix radius. The dashed lines show the corresponding values (0.46 and 2.5122) for the optimal space-filling helix (84). Classical solenoid configurations are predicted to be in sector III, whereas the spiraling staircase configurations are in I and II. On the right, three examples of models of type I, II, and III are shown with loops bases in red and several individual loops rendered in different colors. Also shown is a schematic of a prometaphase chromosome with the helical winding of loops indicated by an arrow around the loop array. (H) Parameters of the best three models of prometaphase chromosomes at different time points.

  • Fig. 4 Defects in chromosome morphogenesis in condensin-depleted cells.

    (A to C). Hi-C interaction frequency maps (binned at 100 kb) for chromosome 7 at the indicated time points (top right in each heat map) after release from G2 arrest. The first plot below each Hi-C interaction map displays the compartment signal (eigenvector 1). The bottom graph shows the insulation score (TADs; binned at 50 kb). (A) SMC2-mAID cells were treated with auxin for 3 hours before release from G2 arrest to deplete SMC2. SMC2+: Hi-C interaction map for G2-arrested cells before auxin treatment. SMC2: Hi-C interaction map for G2-arrested cells after 3 hours of auxin treatment. (B) Hi-C data for CAP-H2–mAID cells treated for 3 hours with auxin before release from G2 arrest to deplete CAP-H2. (C) Hi-C data for CAP-H–mAID cells treated for 3 hours with auxin before release from G2 arrest to deplete CAP-H.

  • Fig. 5 Distinct roles for condensin I and II in mitotic chromosome formation.

    (A) Genome-wide curves of contact frequency P(s) versus genomic distance s, normalized to unity at s = 100 kb. The curves are P(s) derived from Hi-C data obtained from CAP-H2–depleted (left) and CAP-H–depleted (right) cells, at t = 7 to 60 min after release from G2 arrest. Dashed line indicates P(s) = s−0.5. (B) Overlayed P(s) curves of WT, CAP-H–, and CAP-H2–depleted chromosomes show independent contributions of two condensin complexes to short- and long-distance contacts. (C) Polymer models of CAP-H2–depleted (top) and CAP-H–depleted (bottom) chromosomes. (Top) Depletion of CAP-H2 is modeled via removal of outer loops and relaxation of the helix. (Bottom) Depletion of CAP-H is modeled via removal of the inner loops while preserving the helical arrangement of the scaffold. Condensin II loop anchors are shown in red; condensin I loop anchors are shown in blue. (D) P(s) derived from late prometaphase CAP-H2–depletion Hi-C experiments (red line) and the three best-fitting polymer models (grayscale lines). The average loop size and linear density of loops along the chromosome axis are indicated. (E) Average loop size and linear DNA density of the three best-fitting models of CAP-H2–depleted chromosomes at different time points. (F) P(s) derived from late prometaphase CAP-H–depletion Hi-C experiments (red line) and the best-fitting polymer models with and without nested inner loops (grayscale lines). The average size of the outer and inner loops, the length of a helix turn in megabases, and the helical pitch are indicated. (G) Best-fitting P(s) predictions by the staircase coarse-grained model for late prometaphase CAP-H–depletion Hi-C experiments at t = 30 min after release of G2 arrest (gray lines). Red lines denote experimental P(s). (Top) Loop size is 200 kb; (bottom) loop size is 400 kb.

  • Fig. 6 A mitotic chromosome morphogenesis pathway.

    In prophase, condensin II compacts chromosomes into arrays of consecutive loops and sister chromatids split along their length. The scaffold of condensin II–mediated loop bases is indicated in red. Upon nuclear envelope breakdown and entry into prometaphase, condensin II–mediated loops become increasingly large as they split into smaller ~80-kb loops by condensin I. Chromosomes are shown as arrays of loops. Top, cross section; bottom, side view. (Only inner loops can be observed microscopically. For clarity, loops are indicated as separate entities pointing in one direction, though in reality loops are unstructured and can mix.) The nested arrangements of centrally located condensin II–mediated loop bases and more peripherally located condensin I–mediated loop bases are indicated in red and blue, respectively. During prometaphase, the central scaffold acquires a helical arrangement with loops rotating around the scaffold as steps in a spiral staircase (the helical path of the loops is indicated by arrows). As prometaphase progresses, outer loops grow, the number of loops per turn increases, and chromosomes shorten to form the mature mitotic chromosome.

Supplementary Materials

  • A pathway for mitotic chromosome formation

    Johan H. Gibcus, Kumiko Samejima, Anton Goloborodko, Itaru Samejima, Natalia Naumova, Johannes Nuebler, Masato T. Kanemaki, Linfeng Xie, James R. Paulson, William C. Earnshaw, Leonid A. Mirny, Job Dekker

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S27
    • Captions for Tables S1 to S4
    • Caption for Movie S1
    • References
    Table S1
    Sample and FACS summary. All samples with their cell type used for Hi-C libraries (name_galGal5) are listed by the names used throughout all figures. For each cell collection (time), the main cell cycle phase, treatments and drugs used are listed. FACS analysis was used to determine AID-GFP presence (and level of protein degradation). A subset of cells was sorted (sorted) for GFP positivity or negativity respectively, as indicated.
    Table S2
    Hi-C dataset overview. The names used corresponds to those used in figures and Table S1. Each harvested cell sample was submitted to Hi-C as part of a set (Set#) and the sequencing results and mapping statistics for galGal5 (gg5) are listed. Valid pair: reads mapped for both ends; NRVP (non-redundant valid pairs): valid pairs with duplicate reads removed (see Supplementary Methods); %Dangling: percentage of dangling ends (inward reads [→|←] derived from false biotin incorporation); %cis: percentage of intra-chromosomal interactions; %redundant: percentage of exact (PCR) duplicate reads (used to determine NRVP).
    Table S3
    DNA constructs used for generation of cell lines. Mutant: name used throughout the manuscript; background: cell line used to generate modified cell line; construct: vector used to generate mutants; FBOX: the specific FBOX protein used to degrade the AID-fused mutant; endogenous: the modification to the endogenous locus using homologous recombination (HR) or CRISPR; alleles: the number and location of altered alleles in DT40 mutants; selection: antibiotic resistance used for mutant selection; publication: previous work using these mutants.
    Table S4
    Short- and long-distance parts of the P(s).Equalization of the contribution of the short- and longdistance parts of the P(s) was calculated using the r.m.s. P(s) difference in each of these regions separately and reporting the mean of the two.

    Images, Video, and Other Media

    Movie S1
    The emergence of nested loop architecture in loop extrusion lattice simulations of slow- and fastexchanging condensin populations. The video shows the simulated dynamics of loops extruded by condensins I and II on a 2Mb chromatin segment during two hours of simulated time. The positions of loops formed by fast-exchanging condensins I are shown as blue semi-transparent arcs and those formed by slow-exchanging condensin II are shown in red. The detailed description of simulations is available in Supplemental Materials and Methods.

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