Abstract
Eukaryotic genomes are highly ordered through various mechanisms, including topologically associating domain (TAD) organization. We employed an in situ Hi-C approach to follow the 3D organization of the fission yeast genome during the cell cycle. We demonstrate that during mitosis, large domains of 300 kb–1 Mb are formed by condensin. This mitotic domain organization does not suddenly dissolve, but gradually diminishes until the next mitosis. By contrast, small domains of 30–40 kb that are formed by cohesin are relatively stable across the cell cycle. Condensin and cohesin mediate long- and short-range contacts, respectively, by bridging their binding sites, thereby forming the large and small domains. These domains are inversely regulated during the cell cycle but assemble independently. Our study describes the chromosomal oscillation between the formation and decay phases of the large and small domains, and we predict that the condensin-mediated domains serve as chromosomal compaction units.
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Acknowledgements
We would like to thank the Wistar Institute Genomics and Bioinformatics Facilities for high-throughput sequencing and genomic data analyses, the Wistar Imaging Facility for microscopic analysis, and the Yeast Genetic Resource Center (YGRC) for fission yeast strains. We also thank L. Showe and R. Locke for critically reading the manuscript and S. Shaffer for editorial assistance. This work was supported by the G. Harold & Leila Y. Mathers Charitable Foundation and by grants from the National Institutes of Health/National Institute of General Medical Sciences (R01GM124195 to K.N.) and the NIH Director's New Innovator Award Program (DP2-OD004348 to K.N.). Support for Shared Resources used in this study was provided by Cancer Center Support Grant (CCSG) P30CA010815 to the Wistar Institute.
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H.T. performed the in situ Hi-C experiments and bioinformatics analyses. K.K. performed the ChIP, FISH and RNA-seq experiments. O.I. performed the FISH experiments. K.N. conceived and designed the study. All authors contributed to the data analyses and the writing of the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Schematic representation of the in situ Hi-C procedure optimized for fission yeast cells.
(a) The in situ Hi-C experimental procedure was detailed in Online Methods .
(b) The bioinformatics processing of in situ Hi-C data was explained in Online Methods . Read numbers remaining after the filtering processes are summarized in Supplementary Table 1.
(c) Convergence of root-mean-square deviation (RMSD) of in situ Hi-C matrix biases during iterative correction by the ICE method. Total contact scores from respective lines (5, 10, or 20 kb) were compared to those obtained after 50 iterations. As an example, the WT1 in situ Hi-C data processed by the ICE method are shown.
Supplementary Figure 2 Validation of the fission yeast in situ Hi-C data.
(a) Pie charts showing ratios of low quality reads (MapQ < 30), reads derived from repeats, PCR bias, self-ligation and undigested products, and usable reads. Reads from in situ Hi-C experiment (WT1: asynchronous cells) and from the standard Hi-C data (Mizuguchi et al., Nature, 516, 432-435, 2014) were processed by the filters, as described in Supplementary Fig. 1b (Online Methods ). Source data were shown in Supplementary Table 1.
(b) Distribution of sum of distances between mapped genomic positions and nearest restriction sites. Total distances between positions of paired reads and their nearest restriction sites were considered.
(c) Correlations between the indicated biological replicas (left) and between the in situ Hi-C and standard Hi-C data (right). WT1 and WT2 indicate the in situ Hi-C data derived from 26°C and 36°C cultures, respectively. Correlations between contact maps were evaluated by the HiCRep program (Yang et al., Genome Res., gr.220640.117, 2017). Note that the HiCRep algorithm is designed to avoid genomic distance effects by determining Pearson correlation coefficients between contact scores in the same distance groups, subsequently estimating an overall correlation coefficient based on the coefficients from the respective distance groups. Correlation scores range from -1 (opposite) to 1 (identical).
(d) Bar charts showing %reads reflecting intra- and inter-chromosomal contacts in the in situ Hi-C data (left) and standard Hi-C (right). Intra-chromosomal contacts were subdivided into intra- and inter-arm contacts.
(e) Correlation between FISH data and contact scores derived from the in situ Hi-C data (WT1). Eighteen pairs of gene loci had previously been analyzed by FISH (Tanizawa et al., Nucleic Acids Res., 38, 8164-8177, 2010).
(f) The cell cycle was synchronized using the temperature-sensitive cdc25-22 mutation, and monitored by two indexes (binucleated anaphase cells and septation).
Supplementary Figure 3 Genome-wide contact maps during the cell cycle.
(a) Cells in the different cell-cycle stages were subjected to in situ Hi-C genomic analysis. Contact scores estimated from the in situ Hi-C data were plotted across the fission yeast genome during the cell cycle.
(b) Relations between contact scores and genomic distances. Contact scores between gene loci separated by same distances were used to calculate average scores, which were plotted against distance. Dotted lines show the fitting lines. Open circles indicate the specific points, where the two fitting lines cross, predicting that distance-dependent decay of genomic contacts change at those distances.
Supplementary Figure 4 Genome-wide architectural changes during the cell cycle.
(a) Maps showing difference of contact scores between two consecutive time points. Red and blue dots indicate that contact scores are enhanced and reduced, respectively, upon cell-cycle progression between the indicated time points.
(b) Maps representing log2 ratios of contact scores between consecutive time points.
Supplementary Figure 5 Centromeric and telomeric contacts during the cell cycle.
(a) Cell cycle-dependent alterations of centromeric and telomeric contacts. Inter-chromosomal contact scores (chromosomes I and II) were plotted during the cell cycle.
(b) Difference of inter-chromosomal contact scores between two consecutive time points was plotted. Red and blue dots indicate that contact scores are increased and decreased, respectively, upon cell-cycle progression between the indicated time points.
(c) The centromeric and telomeric regions indicated by the open boxes in panel b were magnified in the top and bottom panels, respectively. P-values were calculated as follows: One hundred combinations of centromeric DNA sections were randomly selected. Log2 ratios of contact scores between the two consecutive time points and between the biological replicas (120-minute point) were extracted for the 100 combinations and subjected to two-sided paired Student’s t-test. Random sampling was repeated 1,000 times, and a median of P-values was indicated. The same analysis was also performed for telomeric contacts.
(d, e) FISH visualization of centromeres and telomeres during the cell cycle. The cdc25-22 mutant was used for the cell-cycle synchronization. Representative images are shown above. Centromeres of chromosomes I, II, and III were co-visualized using the pRS140 plasmid (d), and left and right subtelomeric regions of chromosomes I and II were visualized using the cos212 cosmid (e) (Online Methods ). The number of FISH foci was counted in more than 100 cells at the indicated time points, and cells were classified into the respective groups (bottom).
Supplementary Figure 6 Large-domain organization during the cell cycle.
(a) Border strength scores were estimated for the 40-minute time point based on contact scores as described in Online Methods . Furthermore, the large domains were identified based on the presence of potential borders. Since there were several overlap** domains, the two domain organization patterns are shown (gray and dotted). The domain pattern #1 shows the clearer domains than the domain pattern #2 and was subjected to the following data analyses.
(b) Enlarged contact maps for the indicated regions in panel a during the cell cycles.
Supplementary Figure 7 Large and small domains modulated during the cell cycle.
(a) Difference of contact scores between two consecutive time points during the cell cycle was plotted for the indicated region.
(b) A map showing significantly up- (red) and down- (blue) regulated contacts between the 40- and 120-minute points. Significantly altered contacts were identified as described in Online Methods .
(c) Frequencies of significantly up- and down-regulated contacts (top right) between the 40- and 120-minute points were plotted against distance (left). Note that down-regulated contacts in green circle were positioned in the less than 75 kb range (small domains).
(d) The statistical analysis as described in Fig. 2b was applied to the biological replicas from the 60-minute point (G1).
(e) The 2.4 Mb area (open box) indicated in panel b was enlarged (left). The right panel shows the 40-minute contact map for the same region. Dotted lines indicate the large domains.
(f) The up-regulated contacts at the 40-minute point comparing to the 120-minute point were frequently positioned within the large domains. One thousand combinations of DNA sections were randomly selected from the up-regulated contacts. Combinations of DNA sections with same distances were randomly selected, i.e., distance-conserved random sampling from every combination. This sampling was repeated 10,000 times, and distributions of %overlap with the large domains were plotted. For P-value calculation, the same sampling was repeated 100 times, and distributions of %overlap scores were subjected to one-sided Student’s t-test.
(g) The 200 kb region (open box) indicated in panel e was enlarged (left). The right panel represents the 120-minute contact map for the same region. Dotted lines indicate the small domains.
(h) The down-regulated contacts between the 40- and 120-minute points were often located within the small domains. The same analysis as described in panel f was performed for the down-regulated contacts and for the small domains.
(i) Averages and log2 ratios of contact scores between the 40- and 120-minute points were plotted for combinations of DNA sections within the large domains. The significantly up- (red) and down- (blue) regulated contacts are highlighted.
(j) Frequencies of significantly up- and down-regulated contacts in panel i were plotted against distance. Note that significantly up-regulated contacts were detected in the 75– 800 kb range (large domains), whereas significantly down-regulated contacts were positioned in the less than 75 kb range (small domains).
(k) Averages and log2 ratios of contact scores between two consecutive time points. Significantly up- (red) and down- (blue) regulated contacts were determined between the indicated time points (FDR < 0.05). Solid and dotted circles indicate significantly up- or down-regulated contacts in the 75 – 800 kb and < 75 kb ranges, respectively.
Supplementary Figure 8 Effects of 36°C culture and the cut3-477 condensin mutation on the domain organizations.
(a) A map showing difference of contact scores between the WT2 and WT1. WT2 cells were cultured at 36°C for 2 hours, while WT1 cells were cultured at 26°C.
(b) The 1.9 Mb area (open box) indicated in panel a was enlarged. The scale on top indicates chromosome I position. The bottom panel shows the 40-minute contact map for the same area. Dotted lines indicate the large domains.
(c) The 200 kb region (yellow box) indicated in panel b was enlarged. The bottom panel represents the 120-minute contact map for the same region. Dotted lines indicate the small domains.
(d) Averages and log2 ratios of contact scores between the WT2 and WT1 (left) and between the biological replica (120-minute point; top right) were plotted. Combinations of DNA sections within the large domains (red) were separated from other combinations (gray) and were used in the analysis. One hundred combinations of genomic loci were randomly selected from the large domains. Log2 ratios of contact scores between the WT2 and WT1 and between the biological replicas were extracted for the selected 100 combinations and subjected to two-sided paired Student’s t-test. Random sampling was repeated 1,000 times and a median of P-values is represented.
(e) Combinations of DNA sections within the small domains (red) were separated from other combinations (gray) and were used in the analysis. Log2 ratios of contact scores between the WT2 and WT1 and between the biological replicas were subjected to the same analysis as described in panel d for the small domains.
(f) Correlations of the WT1 and WT2 in situ Hi-C matrices with those from the cut14-208 and cut3-477 condensin mutants. Correlations of contact scores (> 1 Mb) were evaluated by the HiCRep program (Yang et al., Genome Res., gr.220640.117, 2017). cut14-208 and cut3-477 cells were cultured at 36°C for 1 and 2 hours, respectively, when most cells were in interphase.
(g) A contact map in the cut3-477 mutant.
(h) Relations between contact scores and genomic distances in WT2 and cut3-477 cells. Dotted lines show the fitting lines against the slopes in the 75 – 800 kb and more than 800 kb ranges.
(i) Log2 ratios of contact scores between cut3-477 and WT2 cells were plotted against distance.
(j) A map showing difference of contact scores between cut3-477 and WT2 cells.
(k) The genomic region marked by the open box in panel j was enlarged (top). Enlarged contact map at the 40-minute time point is also shown as a reference (bottom). Dotted lines indicate the large domains.
(l) Averages and log2 ratios of contact scores between the cut3-477 mutant and WT2 (left) and between the biological replicas (120-minute point; right) were plotted. Log2 ratios of contact scores between the cut3-477 mutant and WT2 and between the biological replicas were subjected to the statistical analysis as described in panel d.
(m) The same analysis as in panel l was performed for the indicated combinations.
Supplementary Figure 9 Condensin and cohesin localization during the cell cycle.
(a) Condensin (Cut14-Pk, red) and cohesin (Rad21-Myc, green) were co-visualized by IF at the indicated time points. The cdc25-22 mutant was used for the cell-cycle synchronization.
(b) Representative images showing condensin and cohesin localization during the cell cycle.
(c) Quantification of IF images. When condensin- or cohesin-derived fluorescent signals are more intense in the nucleus than the cytoplasm, cells were counted as those carrying condensin- or cohesin-enriched nuclei. More than 100 cells from the respective time points were subjected to the same analysis, and the %population of cells carrying the condensin- or cohesin-enriched nuclei were plotted against time.
Supplementary Figure 10 Binding of condensin and cohesin to the fission yeast genome during the cell cycle.
(a) Schematic representation of the centromere I. Fission yeast centromeres consist of the dg, dh, and imr repeats (Wood et al., Nature, 415, 871-880, 2002), and sequences and positions of ChIP primers are shown beneath the diagram.
(b) ChIP primers were also designed against the right telomere of chromosome II.
(c) ChIP results showing enrichment of the condensin subunit Cut14-Pk at the indicated gene loci during the cell cycle. The cdc25-22 mutant was used for the cell-cycle synchronization. The leu1 gene serves as a negative control. Fission yeast condensin binds to centromeres, telomeres, Pol II-transcribed genes (Ace2 target genes), Pol III-transcribed genes (tRNA and 5S rRNA), and LTR retrotransposons (Kim et al., Nat. Genet., 48, 1242-1252, 2016). Ace2 is a mitotically expressed transcription factor, which controls the cell cycle-dependent expression of genes required for septin ring formation and cell separation (Alonso-Nuñez et al., Mol. Biol. Cell, 16, 2003-2017, 2005; Petit et al., J. Cell Sci., 118, 5731-5742, 2005).
(d) ChIP results showing enrichment of the cohesin subunit Rad21-Myc during the cell cycle. Cohesin is known to bind to centromeres, telomeres, convergent gene loci, Pol III-transcribed genes (tRNA and 5S rRNA), and LTR retrotransposons (Kim et al., Nat. Genet., 48, 1242-1252, 2016).
Supplementary Figure 11 Subtelomeric genes are repressed in condensin mutant.
(a) Expression ratios between the cut14-208 condensin mutant and wild type.
(b) Distributions of significantly up- and down-regulated genes across the fission yeast genome. The significantly affected genes were determined by edgeR at FDR < 0.05 (Online Methods ).
(c) Significance of localization of down-regulated genes at subtelomeric region. There were 46 significantly down-regulated genes. Total 46 loci were randomly selected from the entire genome, and the number of genes present at subtelomeric regions was counted. Distribution of gene numbers located at subtelomeric regions was built by 10,000 permutation and was plotted (gray color) with the observed number (n=21).
(d) Subtelomeric region of the right arm of chromosome II consisted of the significantly down-regulated genes.
(e) RT-PCR primers were designed for subtelomeric genes. Note that positions of those genes are shown in panel d.
(f) qRT-PCR data showing that the subtelomeric genes were down-regulated in the condensin mutant (top). The same primers were used to amplify the indicated genes using genomic DNA (bottom). Note that the genomic DNA was prepared from the same samples used for RNA purification.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–11, Supplementary Tables 1, 2 and 4, and Supplementary Note 1 (PDF 4348 kb)
Supplementary Table 3
List of significantly up- (orange) or down- (blue) regulated genes in the cut14-208 condensin mutant compared to the wild type. (XLSX 16 kb)
Supplementary Data Set 1
Data for Fig 1f (top) and for L2 of Fig 2e (XLSX 16 kb)
Supplementary Data Set 2
Data for Fig 1f (bottom) (XLSX 695 kb)
Supplementary Data Set 3
Data for Fig 2d (XLSX 11794 kb)
Supplementary Data Set 4
Data for L1 of Fig 2e (XLSX 14 kb)
Supplementary Data Set 5
Data for Fig 2f (XLSX 683 kb)
Supplementary Data Set 6
Data for Fig 3b (XLSX 23 kb)
Supplementary Data Set 7
Data for Fig 3c (XLSX 6660 kb)
Supplementary Data Set 8
Data for Fig 5d (XLSX 24 kb)
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Tanizawa, H., Kim, KD., Iwasaki, O. et al. Architectural alterations of the fission yeast genome during the cell cycle. Nat Struct Mol Biol 24, 965–976 (2017). https://doi.org/10.1038/nsmb.3482
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DOI: https://doi.org/10.1038/nsmb.3482
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