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Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast

Abstract

The eukaryotic genome consists of DNA molecules far longer than the cells that contain them. They reach their greatest compaction during chromosome condensation in mitosis. This process is aided by condensin, a structural maintenance of chromosomes (SMC) family member1,2. The spatial organization of mitotic chromosomes and how condensin shapes chromatin architecture are not yet fully understood. Here we use chromosome conformation capture (Hi-C)3,4 to study mitotic chromosome condensation in the fission yeast Schizosaccharomyces pombe5,6,7. This showed that the interphase landscape characterized by small chromatin domains is replaced by fewer but larger domains in mitosis. Condensin achieves this by setting up longer-range, intrachromosomal DNA interactions, which compact and individualize chromosomes. At the same time, local chromatin contacts are constrained by condensin, with profound implications for local chromatin function during mitosis. Our results highlight condensin as a major determinant that changes the chromatin landscape as cells prepare their genomes for cell division.

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Figure 1: Hi-C identifies genome-wide contact changes during chromosome condensation.
Figure 2: Mitotic conformational changes depend on the condensin complex.
Figure 3: Condensin replaces local contacts with longer-range interactions in mitosis.
Figure 4: Condensin-dependent chromatin domain expansion in mitosis.
Figure 5: Condensin confines local chromatin motility.

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Acknowledgements

We would like to thank J. Abella and M. Way for their help with high-speed microscopy, A. Stewart for bioinformatic support, C. Haering (EMBL, Heidelberg) for Slp1 shutoff and chromosomal loci–tagged strains and H. Masukata (Osaka University) for the Skp1–Tir1 strain and AID plasmids, and P. Bates, E. Wershof, B. Khatri, Y. Murayama, T. Toda and our laboratory members for discussions and critical reading of the manuscript. This work was supported by the European Research Council and the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001198), the UK Medical Research Council (FC001198) and the Wellcome Trust (FC001198). Y.K. was supported by the Japanese Society for the Promotion of Science (JSPS Overseas Research Fellowships).

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Authors and Affiliations

Authors

Contributions

Y.K. and F.U. conceived the study, Y.K. performed the experiments, Y.K., A.R. and D.J.B. analyzed the data, and Y.K. and F.U. wrote the manuscript with input from A.R.

Corresponding author

Correspondence to Frank Uhlmann.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Confirmation of mitotic arrest and condensin depletion.

(a) Chromosome morphology in mitotically arrested wild-type cells (WT; left) and following condensin depletion (cut14SO; right). DNA was stained with DAPI and visualized together with microtubules (GFP-Atb2) to confirm mitotic arrest. Dotted squares indicate the areas of the magnified images on the right. Scale bar, 1 μm. (b) Depletion of the condensin subunits Cut14 or Cnd3 in the presence of thiamine to repress gene transcription and auxin (NAA) to promote protein degradation via an auxin-inducible degron (aid)16, was confirmed by western blotting. Tat1 (α-tubulin) served as a loading control. (c) Schematic of the positions on chromosome I, where LacO repeats (green) and TetO repeats (red) were inserted and visualized by LacI-GFP and TetR-tdTomato repressor fusion proteins, respectively13. (d) Examples of images to visualize LacO and TetO localization in mitotically arrested wild-type cells and following condensin depletion. Nuclear DNA was counterstained with DAPI. Dotted squares indicate areas corresponding to the individual color images on the right. Scale bar, 1 μm. (e) Box plots of the LacO–TetO distance distributions (n ≥ 155 cells for each condition). The box shows the 25th, 50th and 75th percentiles of distance between two dots. Whiskers indicate 1.58 times the interquantile distance divided by the square roots of total cell number. Distances more than 1.5 times the 75th percentile or less than 1.5 times the 25th percentile are shown as outliers. (f) Chromosome morphology in interphase, mitosis and mitosis following chemical inhibition of Ark1/Aurora B kinase (+1NM-PP1). A Cnd3-GFP fusion visualizes condensin, DNA was stained with DAPI. Magnified individual color images of the highlighted squares are shown at the bottom. Scale bar, 1 μm.

Supplementary Figure 2 Quality control of the Hi-C data sets.

(a) Distribution of read counts within each 2-kb bin. The blue dotted line demarcates bins containing less than 500 reads that were discounted from the analysis. The red dashed line indicates the median read count. Distributions of one replicate of wild-type cells in interphase (WT Interphase Replicate 1) and in mitosis (WT Mitosis Replicate 1) are shown. (b) log2 directionality plots, with the identified boundary positions indicated (red lines), along the chromosome I left arm of the same samples shown in a. (c) Clustering of Euclidean distances between the interaction directionality plots of all the samples included in this study. (d) Smoothed mean normalized log2 directionalities at the identified boundaries. Gray areas show the 95% confidential interval. (e) Average normalized log2 insulation scores around boundaries determined by log2 directionality are shown as a function of insulation distance in interphase (left) and mitosis (right).

Supplementary Figure 3 Schematic Hi-C map, illustrating chromosomal interactions.

(a) The different types of chromosomal interactions studied in our Hi-C analyses are illustrated. Interactions within a chromosome arm (Intra-arm) are shown in red; those between the two arms of one chromosome (Inter-arm) are shown in dark blue. Interactions between different chromosomes (Inter-chr) are represented in light blue. Inter-centromeric and inter-telomeric interactions (Inter-cen and Inter-tel) are highlighted in yellow and green, respectively. (b) Schematic Hi-C map, highlighting the areas corresponding to each of the interactions shown in a, identified by the use of the same color. Note that rDNA repeats are found at the ends of chromosome III. Because of their repetitive nature, we could not analyze chromosome interactions close to the ends of chromosome III.

Supplementary Figure 4 The effect of cnd3SO and mitotic Ark1 inhibition on contact probability changes between interphase and mitosis.

(a,b) Hi-C difference maps from experiments comparing interphase to mitosis following depletion of the condensin subunit Cnd3 (cnd3SO) or mitotic Ark1 inhibition (+1NM-PP1). (c) Distribution of normalized contact probabilities between chromosomes (Inter-chr), within chromosome arms (Intra-arm) or between the two arms of the same chromosome (Inter-arm). (d) Median contact probabilities in interphase and mitosis as a function of genomic distance along the chromosome II right arm are shown, corresponding to the above comparisons.

Supplementary Figure 5 Moving intra-arm median interacting distances along chromosome II.

As in Figure 3g, the moving intra-arm median interacting distances along chromosome II (solid lines) are shown together with shaded areas representing the 25th and 75th percentiles in each indicated condition. In contrast to the iteratively normalized matrices used in Figure 3g, interacting distances were calculated from HiCNorm-normalized matrices (a) or from raw count matrices (b).

Supplementary Figure 6 Chromatin domain boundaries following Cnd3 depletion or mitotic Ark1 inhibition.

(a,b) Hi-C contact probability maps along a section of chromosome I are shown under the indicated conditions. Domain boundaries are indicated (black triangles). (c) Effect of Cnd3 depletion or mitotic Ark1 inhibition on domain size distribution.

Supplementary Figure 7 Numbers and overlap of boundary positions in interphase and mitosis.

(a) Numbers and overlap of boundary positions in interphase and mitosis. Boundary positions were determined using either log2 directionality or the TopDom algorithm. (b) Correlation between boundaries determined by log2 directionality and by the TopDom algorithm in each of the indicated conditions.

Supplementary Figure 8 Boundaries overlap with condensin peaks.

(ac) Correlation between condensin peaks in mitosis and boundary positions in mitosis (a), in interphase (b) and in mitosis following depletion of condensin (c).

Supplementary Figure 9 Chromatin motility in interphase and mitosis.

(a) Schematic of the locations where LacO (green) or TetO (red) repeats were inserted in chromosomes I and II, respectively. (b) A typical image of a cell harboring LacO and TetO repeats bound by LacI-GFP and TetR-tdTomato, respectively. (c) Mean square displacement (MSD) of the TetO locus in wild-type interphase and mitosis. Mean ± s.e.m. is shown (n = 22 interphase, n = 26 mitosis).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Tables 2 and 3. (PDF 1974 kb)

Life Sciences Reporting Summary (PDF 159 kb)

Supplementary Table 1

Condensin ChIP peaks along fission yeast chromosomes I–III. (XLSX 80 kb)

Supplementary Table 4

Hi-C library sequencing metrics. (XLSX 47 kb)

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Kakui, Y., Rabinowitz, A., Barry, D. et al. Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast. Nat Genet 49, 1553–1557 (2017). https://doi.org/10.1038/ng.3938

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