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Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe

Abstract

Eukaryotic genomes are folded into three-dimensional structures, such as self-associating topological domains, the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF) required for long-range interactions1,2,3,4,5,6,7. How local chromatin interactions govern higher-order folding of chromatin fibres and the function of cohesin in this process remain poorly understood. Here we perform genome-wide chromatin conformation capture (Hi-C) analysis8 to explore the high-resolution organization of the Schizosaccharomyces pombe genome, which despite its small size exhibits fundamental features found in other eukaryotes9. Our analyses of wild-type and mutant strains reveal key elements of chromosome architecture and genome organization. On chromosome arms, small regions of chromatin locally interact to form ‘globules’. This feature requires a function of cohesin distinct from its role in sister chromatid cohesion. Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structures and global chromosome territories. By contrast, heterochromatin, which loads cohesin at specific sites including pericentromeric and subtelomeric domains9,10,11, is dispensable for globule formation but nevertheless affects genome organization. We show that heterochromatin mediates chromatin fibre compaction at centromeres and promotes prominent inter-arm interactions within centromere-proximal regions, providing structural constraints crucial for proper genome organization. Loss of heterochromatin relaxes constraints on chromosomes, causing an increase in intra- and inter-chromosomal interactions. Together, our analyses uncover fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions.

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Figure 1: High-resolution contact probability map reveals the higher-order organization of the S. pombe genome.
Figure 2: Cohesin is required for globule formation.
Figure 3: Globules are a feature of chromosome architecture in G1 cells.
Figure 4: Loss of heterochromatin affects genome organization.

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Gene Expression Omnibus

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Microarray and sequencing data are available at the NCBI Gene Expression Omnibus (GEO) repository under the accession number GSE56849.

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Acknowledgements

We thank Y. Watanabe and M. Yanagida for strains, M. Zofall for contributions, and A. Kelly for comments. This study used the Helix Systems and the Biowulf Linux cluster at the National Institutes of Health. This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute and by a grant from NHGRI (HG003143) to J.D. The work of G.F. and L.M is supported by NCI Physical Sciences-Oncology Center at MIT (U54CA143874).

Author information

Authors and Affiliations

Authors

Contributions

T.M. and S.I.S.G. designed the experiments. T.M. did most experiments including Hi-C with guidance from J.-M.B. and J.D.; G.F., S.M. and L.M. performed the Hi-C computational analyses; P.F. contributed to data analyses; N.T. and H.D.F. provided imaging analyses. All authors contributed to data interpretation. T.M., J.B. and S.I.S.G. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Shiv I. S. Grewal.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 S. pombe chromosomes are partitioned into complex domains.

The log2(observed/expected) contact probability maps for wild-type S. pombe chromosomes. The colour code indicates more (brown) or less (blue) interaction than expected depending on genomic distance (ranging from −2 to +2). These maps show an increasing extent of centromeric avoidance for regions along the arms of chromosomes (black outlines). Boxes indicate strong cross-like patterns of centromere proximal arm-arm interactions. The left arm of chromosome 1 is segregated into compartments (green arrow). The mating type locus on chromosome 2 (black arrow) separates two adjacent domains. Subtelomere 2R (tel2R) is partitioned into strongly interacting domains (red arrow).

Extended Data Figure 2 Contact probability as a function of genomic distance for different chromosomal arms.

The decay of intra-arm contact probability as a function of genomic distance, plotted for each chromosome arm. All chromosome arms behave similarly in terms of their scaling. Pc(s) decreases more slowly at short distances (grey shaded area). The black and grey dashed lines represent the slope for fractal globules (−1) and polymers in a melt (−3/2), respectively.

Extended Data Figure 3 Contact probability as a function of genomic distance in wild-type and rad21-K1.

Global decay of intra-arm contact probability as a function of genomic distance plotted for rad21-K1 (blue) and wild type (grey). Average inter-chromosomal interactions (flat lines) are markedly increased (twofold) in rad21-K1 compared to wild type. Short-range contact probability decays rapidly in rad21-K1, and no longer decreases more rapidly after 100 kb, probably reflecting the loss of globules in rad21-K1. The black and grey dashed lines represent the slope for fractal globules (−1) and polymers in a melt (−3/2), respectively.

Extended Data Figure 4 Insulation at globule boundaries.

The degree to which a locus displays decreased contact frequency between, or is insulated from, regions separated by that locus can be directly quantified from the corrected Hi-C contact map. Here we use Rj(s), the relative frequency of contacts occurring over a bin j at a distance s. Negative values of relative contact frequency, Rj(s), are indicative of insulation at a given locus. Rj(s) at a given distance s is calculated from a region within a rectangular band of a Hi-C contact map rotated by 45°. a, Diagram illustrating the concept of the insulation plot. At the location of the cohesin binding peak, interactions between two adjacent globules are less frequent (blue stripe). Within the globule domain, contact probability is high (red stripe). b, Relative contact probability around a cohesin peak as a function of insulation distance averaged over all cohesin peaks. Average insulation is examined by calculating the relative contact probability around cohesin peaks. Relative contact probability around the cohesin peak is depleted up to 50–100 kb, indicative of insulation at peaks of local cohesin enrichment at these scales. c, Relative contact probability averaged from 20–50 kb around positions of each cohesin peak (positions obtained in wild type were assayed in rad21-K1). d, Mean number of cohesin peaks as a function of distance from boundaries. Psc3 peaks are highly enriched at the boundary in wild type. e, The negative correlation between cohesin and relative contact frequency Rj(s) in wild type indicates that not only is insulation observed at peaks of cohesin enrichment, but that the inverse relationship between the local enrichment of cohesin (Psc3) and the relative contact frequency holds genome-wide for data binned to 10 kb. This indicates that it is not just the presence or absence of a cohesin peak, but the local amount of cohesin protein in the chromatin fibre that may be important for boundary formation, as well as the strength of a given boundary. The negative correlation holds up to 100 kb in wild type. In rad21-K1, however, there is no appreciable correlation with Psc3 at any distance. This indicates that there is no clear relationship between the distribution of cohesin and local chromatin organization in this mutant.

Extended Data Figure 5 Genomic rearrangements and transcriptional dysregulation in rad21-K1.

a, Microarray comparative genomic hybridization profile of rad21-K1. Genomic DNA isolated from rad21-K1 and wild type was labelled with cy5-dCTP and cy3-dCTP, respectively. The log2(cy5/cy3 signal ratio) was plotted to detect copy number differences between the two strains. Several copy number gains were identified in rad21-K1. All changes were flanked by highly homologous sequences. SPAC212.08c and SPAC212.12 share a 372-bp DNA stretch that shows 97% sequence similarity. SPAC27D7.09c and SPAC27D7.11c share a 560-bp DNA stretch that shows 88% sequence similarity. Pericentromeric heterochromatin contains a specific class of repeat elements, referred to as dg/dh repeats. b, Relative expression values (mutants/wild type) were plotted to detect read-through transcripts in the indicated strains. All pairs of convergent genes were aligned at the 3′ end of the second gene in the pair. Note that rad21-K1 cells show increased levels of read-through transcripts that were further enhanced in a pht1Δ rad21-K1 double mutant lacking the histone variant H2A.Z known to prevent their accumulation35.

Extended Data Figure 6 Hi-C analysis of G1-arrested cells.

a, Cohesin (Psc3) localization was examined by immunofluorescence in asynchronous wild-type cells. Cell cycle stage was determined by tubulin staining (TAT1). Psc3 was detected in the nucleus in both G2 and G1/S phase cells (top). Psc3–GFP localization was examined in G1 arrested cells (cdc10-v50). Predominant nuclear staining and Psc3–GFP dots were detected in both asynchronous cells and G1-arrested cells (bottom). Scale bars, 5 μm. b, All-by-all interaction heatmap for G1 cells. The inter-chromosomal cross-like pattern is more prominent in G1 cells than in asynchronous cells. c, 4C-like inter-chromosomal profiles for centromeres and telomeres. d, Global decay of intra-arm contact probability as a function of genomic distance in G1 cells (green) compared with wild type (grey); flat lines indicate average inter-chromosomal contact probability. Slower decay of contact probability over short distances, followed by a more rapid decrease after 100 kb, was observed in G1-arrested cells. The black and grey dashed lines represent the slope for fractal globules (−1) and polymers in a melt (−3/2), respectively. e, FACS analysis of cell populations used for Hi-C (left). Global decay of intra-arm contact probability as a function of genomic distance in G1-arrested rad21-K1 (orange) compared to G1 (green) cells is shown at right. f, Hi-C heatmaps of a segment of chromosome 2 for indicated samples overlaid with lines corresponding to cohesin peaks from the 10-kb binned cohesin (Psc3) profile. The Hi-C directional preference profile is shown below. Note the globules are not visible in G1-arrested rad21-K1 (cdc10-v50 rad21-K1). g, Insulation plot around cohesin peak sites (detected in G1-arrested cells) for G1 and G1-arrested rad21-K1.

Extended Data Figure 7 Globule boundaries in wild-type, rad21-K1, G1-arrested and clr4Δ.

a, Insulation effect for all cohesin (Psc3) peaks. Relative contact probability was averaged over 20–50 kb at each Psc3 peak region (−50 kb to +50 kb region) and sorted by Psc3 peak height. Insulation effect at Psc3 peaks in wild type, G1 and clr4Δ holds genome-wide, and not only at a small subset of peaks. b, The correlation between Psc3 and relative contact probability Rj(s) profile at a given distance s. The negative correlations hold up to 100 kb in wild type, G1-arrested cells and clr4Δ. Psc3 enrichment and the relative contact probability profile in rad21-K1 do not show an appreciable correlation at any distance. c, The fraction of overlapping boundaries (±1 bin) for each Hi-C data set is shown. Boundaries in clr4Δ and G1-arrested cells show high overlap with wild-type boundaries. The top 100 strongest boundaries from each data set were examined for comparison.

Extended Data Figure 8 Cohesin localization in wild-type and clr4Δ strains as determined by ChIP-chip.

a, Cohesin subunit Psc3 tagged with GFP (Psc3–GFP) is distributed broadly throughout the genome. Note that Psc3 localization in clr4Δ was specifically affected in heterochromatic regions, but not in chromosome arm regions. b, Psc3–GFP localization across pericentromere, subtelomere and mating type heterochromatic regions. Heterochromatic regions are highlighted. c, Psc3–GFP localization on chromosome arm regions (left). Green bars represent open reading frames according to the 2007 S. pombe genome assembly. Cohesin enrichment sites are highlighted. Genome-wide profile of Psc3–GFP chromatin immunoprecipitation enrichment at convergent genes in wild type and clr4Δ (right). All pairs of convergent genes were aligned at the 3′ end of the second gene (green box).

Extended Data Figure 9 Box plots showing contact probabilities for centromere–centromere/telomere–telomere inter-chromosomal interactions in wild type, clr4Δ and rad21-K1.

Box plots, overlaid with values for individual bin-pairs, showing contact probabilities for centromere–centromere/telomere–telomere inter-chromosomal interactions in wild type, clr4Δ and rad21-K1. Whiskers span from minimum to maximum of each set of points, boxes show twenty-fifth, median and seventy-fifth percentiles. Centromere–centromere/telomere–telomere inter-chromosomal interactions are less frequent in clr4Δ as compared to wild type.

Extended Data Figure 10 Effects of heterochromatin and cohesin on centromere proximal arm interactions, and a model showing their distinct effects on S. pombe genome organization.

a, Centromere proximal regions considered for intra-chromosomal and inter-chromosomal arm interaction Pc(s) are shown on a heatmap (left). Diagram showing intra- and inter-arm interactions within a chromosome, and inter-arm interactions between chromosomes in centromere proximal regions (right). Standard genomic distances were used to consider intra-arm contact probabilities. To consider inter-arm contacts within or between chromosomes, genomic distance was defined as the combined distance of two loci to the centromere, for regions at similar (<50 kb) distance from their respective centromeres. b, Pc(s) plotted for inter-arm interactions within a chromosome and between different chromosomes in wild-type cells. Inter-chromosomal inter-arm (red) Pc(s) falls below intra-chromosomal inter-arm (blue) and intra-arm (black), but have a similar rate of decay with distance. Note that inter-arm Pc(s) starts at a larger genomic distance, since centromere proximal bins were removed at the stage of bin-level filtering due to their low coverage. c, Pairwise comparisons of inter-arm Pc(s) of rad21-K1 and clr4Δ with wild type. In clr4Δ, both inter-arm Pc(s) are shifted lower, most notably for inter-chromosomal inter-arm contact probability. In rad21-K1, inter-arm scaling near centromere is similar to wild type. d, Model showing distinct roles of heterochromatin and cohesin-dependent globules in overall chromosome organization. In wild-type cells, non-random organization consistent with a degree of chromosome territoriality was evident. These levels of organization may underlie genomic integrity, both independently and collectively, for example, by effectively preventing interaction between repetitive elements. The peripheral positioning of centromere and telomere clusters promotes a Rabl configuration. Heterochromatin reinforces this configuration by compacting centromere and telomere proximal regions, promoting strong interactions and aligning arms to facilitate proper genome architecture. A newly identified layer of globules bounded by high amounts of arm cohesin organizes chromosome arms. The formation of globules depends on arm cohesin. Unlike in wild type, globules on arms are disrupted in rad21-K1, whereas constraints at pericentromeric regions are maintained. In clr4Δ, pericentromeric regions are decompacted, but globules are not disrupted along arms.

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Mizuguchi, T., Fudenberg, G., Mehta, S. et al. Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe. Nature 516, 432–435 (2014). https://doi.org/10.1038/nature13833

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