Abstract
Structural maintenance of chromosomes (SMC) protein complexes are key determinants of chromosome conformation. Using Hi-C and polymer modelling, we study how cohesin and condensin, two deeply conserved SMC complexes, organize chromosomes in the budding yeast Saccharomyces cerevisiae. The canonical role of cohesin is to co-align sister chromatids, while condensin generally compacts mitotic chromosomes. We find strikingly different roles for the two complexes in budding yeast mitosis. First, cohesin is responsible for compacting mitotic chromosome arms, independently of sister chromatid cohesion. Polymer simulations demonstrate that this role can be fully accounted for through cis-looping of chromatin. Second, condensin is generally dispensable for compaction along chromosome arms. Instead, it plays a targeted role compacting the rDNA proximal regions and promoting resolution of peri-centromeric regions. Our results argue that the conserved mechanism of SMC complexes is to form chromatin loops and that distinct SMC-dependent looping activities are selectively deployed to appropriately compact chromosomes.
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Acknowledgements
We thank B. Lajoie and J. Gibcus for aid in processing the Hi-C data sets. We thank L. Aragon, K. Nasmyth and J. Diffley for yeast strains. This work was funded by the Biotechnology and Biological Sciences Research Council United Kingdom (BBSRC UK) Grant ref. BB/J018554/1 (S.A.S., M.Y.), the Royal Society UK (J.B.), NIH U54 4D Nucleome grant (DK107980) and NIH R01 (HG003143) (A.G., G.F., J.M.B., L.M., and J.D.). J.D. in an investigator of the Howard Hughes Medical Institute.
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S.A.S. performed all cell culture and generated Hi-C libraries. A.G. analysed sequenced libraries and Hi-C data sets. G.F. modelled chromosome conformation of the budding yeast nucleus with help from A.G. J.M.B. guided Hi-C library instruction and analysed sequenced libraries. M.Y. and C.M. constructed and characterized the inducible condensin alleles. J.D. advised on study construction and guided processing and analysis of Hi-C data sets. L M. guided the modelling of the Hi-C data. J.B. conceived and coordinated the study. J.B., G.F. and A.G. wrote the manuscript with input from S.A.S., L.M. and J.D.
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Supplementary Figure 3 Experimental set up and confirmation of Rabl conformation of yeast nucleus arrested in G1 and M.
(a) At collection for Hi-C analysis an aliquot of cells was fixed and DNA stained with DAPI. Only experiments that had >94% large budded cells were taken for further processing. Budding index demonstrates CDK activation in yeast cells. Mitotic cells were then assessed as to whether they had maintained the pre-anaphase arrest—as indicated by a single nucleus. Or had proceeded into anaphase—as indicated by 2 split nuclei. Abbreviations for states are as used in text specifically M - cdc20 arrested, MH—cohesin depleted (scc1-73) cdc20 arrested, MD – condensin depleted (smc2td GAL1-smc2K38I) cdc20 arrested. R1 and R2 refer to replicate 1 and replicate 2, respectively. Therefore two independent experiments were conducted for each state. (b) Telomeres (40 kb) of all chromosome arms have been grouped according to arm length. The interaction frequency between the 8 shortest and 8 longest arms relative to each other has been analyzed. (c) Zoom into contact heatmaps from G1 and M datasets for selected regions on (top) ChrXV (0-330 kb (CENXV is at 330 kb)) and (bottom) the post-rDNA region of ChrXII (660 kb to 940 kb). Each block represents 10 kb bin. (d) Zoom into log2 ratio of M over G1 contact maps for selected regions on (top) ChrXV (0-330 kb (CENXV is at 330 kb)) and (bottom) the post-rDNA region of ChrXII (660 kb to 940 kb). (e) Overlaid P(s) curves for each individual chromosome arm taken from G1 (green) or M (blue) cells. All contact maps shown were assembled from two independent experiments.
Supplementary Figure 4 Mitotic chromosome conformation can be accounted for by addition of intra-chromosomal loops, but not by sister-crosslinks.
(a) The family of P(s) curves for 150 loops, and a range of different coverage levels (left). And the family of P(s) curves for coverage = 0.4, and a range of number of loops (right). (b) Simulations with sister-crosslinks imposed with the indicated frequency (12 kb, 192 kb, none) at random positions along chromosome arms in different simulations. Importantly, sister-crosslink simulations do not display two phases in their P(s), unlike experimental M-phase P(s) curves (grey, two replicas).
Supplementary Figure 5 Mitotic chromosome compaction requires cohesin function.
(a) (Top) Zoom into contact heatmaps from MH data for selected regions on (left) ChrXV (0-330 kb (CENXV is at 330 kb)) and (right) the post-rDNA region of ChrXII (660 kb to 940 kb). (Bottom) Zoom into log2 ratio of MH over M contact maps for selected regions on (left) ChrXV (0-330 kb (CENXV is at 330 kb)) and (right) the post-rDNA region of ChrXII (660 kb to 940 kb). (b) (Left) Log2 (MH/M) ratio of contacts for ChrXII. Regions where contact frequency was higher in MH (-cohesin) than M (wt cohesin) are shown in red, regions where contact frequency was lower in MH than M in blue. The post-rDNA region is highlighted by the orange bar. (Right) Contact probability, P(s), as a function of genomic separation, s, specifically for the post-rDNA region of ChrXII for the replicate experiments of MH and M. All contact maps shown were assembled from two independent experiments.
Supplementary Figure 6 Cohesin-dependent compaction is independent of sister chromatid cohesion.
(a) FACS for DNA content (left) and Western blotting (right) showing that cdc45 and cd45 scc1-73 cells enter mitosis without DNA replication, with CDK phosphorylating condensin on Smc4 Serine 4 (Smc4 S4P) with the same kinetics as wildtype cells (∗ unspecific band). Picture of Ponceau stained blot confirms equal loading of Western (right, bottom). Western blotting for to confirm CDK activation in cells was from one experiment. (b) Plot of nuclear morphology examining number of DAPI stained cells from the indicated timepoints that have undergone nuclear division. % of single nucleus (rectangles), double nuclei (checked line with squares) and cells with an anaphase nucleus are shown (grey line). (c) (Top) Zoom into contact heatmaps from cdc45 mitotically arrested cells, C, (top) and cohesin depleted mitotically arrested cells, CH, (bottom), for selected regions on (left) ChrXV (0-330 kb (CENXV is at 330 kb)) and (right) the post-rDNA region of ChrXII (660 kb to 940 kb). (d) Zoom into log2 ratio of CH over C contact maps for selected regions on (left) ChrXV (0-330 kb (CENXV is at 330 kb)) and (right) the post-rDNA region of ChrXII (660 kb to 940 kb). Arrows indicate a prominent track of cohesin-dependent contacts seen also in Supplementary Fig. 3a). All contact maps shown were assembled from two independent experiments.
Supplementary Figure 7 Mitotic conformation following depletion of Smc2 and characterization of smc2K38I allele.
(a) (Left) Hi-C data collected from M phase cells following disruption of conDensin with smc2td allele to deplete Smc2 (MDsmc2). Chromosomes XIII to XVI are shown as representative of the whole genome. (Middle) Log2 ratio of smc2 depleted M dataset over wt M dataset (MD smc2/M), respectively. (Right) P(s) of M versus MDsmc2. Data set assembled from one Hi-C data set. (b) Description and characterization of the smc2td GAL1smc2K38I allele used in Figs 5 and 6. (i) Western blot showing degradation of the degron tagged Smc2 protein and the concurrent GAL1 induced expression of Smc2K38I mutant in both nocodazole and cdc20 arrested metaphase state. Expression examined by Western blot in one experiment (ii) FACS analysis of DNA content following degradation of smc2td with/without expression of Smc2K38I. Representative profiles shown from one of two independent experiments. Expression of Smc2K38I increases the aneuploidy of cells generated following one cell division (right) as shown by increased number of cells with more than 2C and less than 1C DNA content. Profiled cells also contain V5-tagged Brn1 (as in (iii)). (iii) ChIP analysis of condensin complex enrichment as assayed by ChIP with Brn1-V5 at CEN4, in wildtype cells (wt), smc2 degron cells (smc2-td), or smc2-td combined with expression of smc2K38I shown in a boxplot format with all data points shown (sample size n = 11, 5, 3 experiments, respectively). Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, data points are plotted as open circles. The increased penetrance of the smc2K38I phenotype with regard to aneuploidy and chromatin binding suggests that this allele approximates the null state. (c) (Left) Zoom into contact heatmaps from condensin depleted mitotically arrested cells, MD, for ChrXV (0-330 kb (CENXV is at 330 kb)). (Right) Zoom into log2 ratio of MD over M contact maps for ChrXV (0-330 kb (CENXV is at 330 kb))
Supplementary Figure 8 Changes between in cis and in trans tRNA-tRNA loci contacts in the different datasets.
(a) The map of average contact probability between tRNA pairs located on the same chromosomal arm and separated by 80 kb–120 kb. To avoid indirect clustering effects, we selected tRNA-tRNA pairs located more than 100 kb away from a centromere or a telomere (90 pairs in total). (b) Same as in (a), but for tRNA pairs located on the same chromosomal arm, but separated by 180 kb–220 kb (50 pairs in total). (c) Same as in (a) and (b), but for tRNA pairs located on different chromosomes (8290 pairs in total). (d) Speculative models of how cohesin complexes have a dual role in both generating chromatin loops in cis and sister chromatid cohesion in trans. Cohesin complexes act in chromatin loop formation and sister chromatid cohesion independently. In this model distinct populations of cohesin complexes are engaged in chromatin loop formation and sister chromatid cohesion. We speculate that loop forming complexes will exhibit dynamic binding of chromatin whereas cohesive cohesin complexes will be stably bound to chromatin. (e) Cohesin complexes could simultaneously act in sister chromatid cohesion and in loop formation. This model would require that both cohesive and non-cohesive complexes could promote chromatin loops. In the ‘handcuff’ model of cohesive cohesin this would require two loop-promoting cohesin complexes being brought together.
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Schalbetter, S., Goloborodko, A., Fudenberg, G. et al. SMC complexes differentially compact mitotic chromosomes according to genomic context. Nat Cell Biol 19, 1071–1080 (2017). https://doi.org/10.1038/ncb3594
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DOI: https://doi.org/10.1038/ncb3594
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