Chromosome segregation requires both compaction and disentanglement of sister chromatids. We describe SisterC, a chromosome conformation capture assay that distinguishes interactions between and along identical sister chromatids. SisterC employs 5-bromo-2′-deoxyuridine (BrdU) incorporation during S-phase to label newly replicated strands, followed by Hi-C and then the destruction of 5-bromodeoxyuridine-containing strands via Hoechst/ultraviolet treatment. After sequencing of the remaining intact strands, this allows assignment of Hi-C products as inter- and intra-sister interactions based on the strands that reads are mapped to. We performed SisterC on mitotic Saccharomyces cerevisiae cells. We find precise alignment of sister chromatids at centromeres. Along arms, sister chromatids are less precisely aligned, with inter-sister connections every ~35 kilobase (kb). Inter-sister interactions occur between cohesin binding sites that are often offset by 5 to 25 kb. Along sister chromatids, cohesin results in the formation of loops of up to 50 kb. SisterC allows study of the complex interplay between sister chromatid compaction and their segregation during mitosis.
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In Fig. 1, Scc1-calibrated ChIP–seq tracks from Hu et al.22 were used for the cohesin pile-up SisterC heatmaps and ChIP–seq tracks. This dataset is available on GEO under accession number GSM1712309. The peaks were called on this dataset using MACS2. The pairwise cohesin interactions were compiled by listing all possible pairwise combinations of cohesin peak sites in the same chromosome, followed by separation based on distance between cohesin pairs (smaller than 10 kb, 10 to 20 kb, 20 to 35 kb and 35 to 50 kb). The cohesin sites in a 50-kb window around the centromeres and on all of chrXII and chrIV were removed from the dataset. Additionally, to investigate distance decay, Hi-C samples from cdc45 mutant cells were used from Schalbetter et al.7. This dataset is available on GEO under accession number GSM2327664. This data was processed identically to the Hi-C libraries produced for this study. The sites of origin of replication were downloaded from OriDB (http://www.oridb.org/)44. Source data are provided with this paper.
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We thank all current and former Dekker lab members for helpful discussions, in particular S. V. Venev for advice on data analysis. We thank J. Benanti, H. Arsenault and N. Rhind for advice on culturing and synchronizing yeast and providing the yeast strain used in this study. We thank S. Schalbetter, N. Minchell and M. Naele for providing their yeast Hi-C protocol and suggestions. We also thank B. van Steensel for advice and suggestions. This work was supported by grants from the National Institutes of Health (no. HG003143 to J.D. and no. NS111990 to J.K.W.). J.D. is an investigator of the Howard Hughes Medical Institute.
The authors declare no competing interests.
Peer review information Lei Tang was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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a, Asynchronous yeast cultures are cultured and synchronized in late G1 using alpha factor. Cells are released in media containing BrdU or Thymidine, followed by an arrest in mitosis using nocodazole or G1 using a second alpha factor arrest. Cells are harvested and prepared for Hi-C or SisterC library production or processed for BrdU detection using HPLC. b, Flow cytometry analysis of cell cycle profile and BrdU incorporation of harvested yeast cultures for preparation of SisterC libraries.
DNA fragments were amplified in presence of 0%, 10%, 50%, 90% and 100% BrdU to allow for incorporation in both strands (first 5 lanes). This was followed by treatment of UV only (second 5 lanes), Hoechst only (third 5 lanes) or treatment with both UV and Hoechst (last 5 lanes). Fragments containing more than 10% BrdU did not get amplified after UV/Hoechst treatment. This experiment was repeated twice with highly similar results.
Distance decay plots of all SisterC mitotic libraries: DpnII replicate 1 a, DpnII replicate 2 b, DpnII replicate 3 c, and HindIII replicate 1 d.
Distance decay plots of all SisterC control libraries: a-b, G1 (a) and mitotic (b) arrested cells grown in BrdU and treated with UV/Hoechst. c-d, G1 (c) and mitotic (d) arrested cells grown in BrdU, not treated with UV/Hoechst. e-f, G1 (e) and mitotic (f) arrested cells grown in Thymidine, treated with UV/Hoechst. g-h, G1 (g) and mitotic (h) arrested cells grown in Thymidine, not treated with UV/Hoechst.
a-c, Interaction heatmaps of 50 kb window around the centromeric region of chrXII, for all interactions (a), inter-sister interactions (b) and intra-sister interactions (c). d-f, Log2 observed over expected interaction heatmaps of all interactions (d), inter-sister interactions (e) and intra-sister interactions (f) on the centromeric region on chrXII.
Interaction frequency of inter-sister and intra-sister interactions anchored on chrXIII:469,000 shows higher frequency of inter-sister interactions at 25 kb distance from the anchor (highlighted in green). Annotated below the plot are the positions of Scc1 peaks.
Extended Data Fig. 8 Ranking of SisterC signal on pairs of cohesin binding sites at different genomic distances.
a, b, Inter-sister interaction (a) and intra-sister interaction (b) frequency of pairs of cohesin binding sites separated by 10 to 20 kb were ranked by inter-sister interaction intensity for 10 kb window around the cohesin binding site. c, Interaction pile up plots of inter-sister and intra-sister interactions of the top 10 percent sites that were ranked in (a). d-e, Inter-sister interaction (d) and intra-sister interaction (e) frequency of pairs of cohesin binding sites separated by 35 to 50 kb were ranked by intra-sister interaction intensity for 10 kb window around the cohesin binding site. f, Interaction pile up plots of inter-sister and intra-sister interactions of the top 10 percent sites that were ranked in (e). g, 284 cohesin sites that anchor the top 10% inter-sister interacting cohesin pairs at 10 to 20 kb distance were identified as well as again 284 cohesin sites that anchor the top 10% intra-sister interacting cohesin pairs at 35 to 50 kb distance. 65 cohesin sites are found to mediate both top 10% inter-sister cohesin-cohesin interactions and top 10% intra-sister cohesin-cohesin interactions.
a, b, Mitotic SisterC inter-sister interactions (a) and intra-sister interactions (b) plotted on replication origins. c, Pile up plot of Hi-C data of G1 alpha arrested cells on replication origins.
a, b, Normalized frequency SisterC G1 (a) and mitotic (b) inter-sister and intra-sister interactions as a function of AT percentage. c, d, Normalized frequency SisterC G1 (c) and mitotic (d) inter-sister and intra-sister interactions as a function of distance to DpnII digestion site. e, f, Normalized frequency SisterC G1 (e) and mitotic (f) inter-sister and intra-sister interactions as a function of distance to origins of replication.
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Datapoints of anchor plot.
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Datapoints of frequency plots.
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Oomen, M.E., Hedger, A.K., Watts, J.K. et al. Detecting chromatin interactions between and along sister chromatids with SisterC. Nat Methods 17, 1002–1009 (2020). https://doi.org/10.1038/s41592-020-0930-9
Nature Reviews Molecular Cell Biology (2021)