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Detecting chromatin interactions between and along sister chromatids with SisterC

Nature Methods volume 17, pages 1002–1009 (2020)Cite this article

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Abstract

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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Fig. 1: Outline of the SisterC chromosome conformation capture technique.
Fig. 2: SisterC allows differentiation of inter-sister and intra-sister interactions in mitotic chromosomes.
Fig. 3: SisterC data show that centromeres are more precisely and closely aligned than loci along chromosome arms.
Fig. 4: SisterC shows that cohesive cohesin mediates interactions between sister chromatids at shorter genomic distances than loops formed within sister chromatids by extruding cohesin.

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Data availability

All genomic data generated for this study are publicly available on the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE145695.

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.

Code availability

The Hi-C mapping pipeline distiller-nf is available at https://github.com/mirnylab/distiller-nf. The downstream analysis tools, pairtools and cooltools are available at https://github.com/mirnylab/pairtools and https://github.com/mirnylab/cooltools.

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Acknowledgments

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.

Author information

Authors and Affiliations

  1. Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA

    Marlies E. Oomen & Job Dekker

  2. RNA Therapeutics Institute and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA

    Adam K. Hedger & Jonathan K. Watts

  3. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    Job Dekker

Authors
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Contributions

M.E.O. and J.D. conceived and designed the project. M.E.O. cultured yeast, generated and analyzed the SisterC and Hi-C datasets and performed the flow cytometry experiments. A.K.H. and J.K.W. designed the HPLC and mass spectrometry experiments, A.K.H. performed and analyzed the HPLC and mass spectrometry experiments. M.E.O. and J.D. wrote the manuscript.

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Correspondence to Job Dekker.

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The authors declare no competing interests.

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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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Extended data

Extended Data Fig. 1 Outline of SisterC yeast culture conditions and flow cytometry analysis.

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.

Extended Data Fig. 2 Depletion of BrdU containing DNA molecules by PCR.

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.

Extended Data Fig. 3 Distance decay plots of all SisterC replicates.

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.

Source data

Extended Data Fig. 4 Distance decay plots of all SisterC control experiments.

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.

Source data

Extended Data Fig. 5 Observed/expected interaction heatmap of chrXIII:450,000–550,000.

Log2 observed over expected interaction frequency on region ChrXIII:450,000–550,000 (as shown in main Fig. 2) for all interactions a, inter-sister interactions b, and intra-sister interactions c.

Source data

Extended Data Fig. 6 Centromeric region of chrXII.

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.

Source data

Extended Data Fig. 7 Anchor plot on ChrXIII:469000.

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.

Source data

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.

Source data

Extended Data Fig. 9 SisterC pile up plots of origins of replication.

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.

Source data

Extended Data Fig. 10 Efficiency of SisterC inter- and intra-sister interaction assignment.

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.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–4.

Reporting Summary

Supplementary Table

Supplementary Table 1. SisterC and Hi-C mapping statistics across all DpnII and HindIII experiments

Source data

Source Data Fig. 1

HPLC datapoints.

Source Data Fig. 2

Datapoints of scaling plots and heatmaps

Source Data Fig. 3

Datapoints of scaling plots and heatmaps.

Source Data Fig. 4

Datapoints of heatmaps and anchorplot.

Source Data Extended Data Fig. 3

Datapoints of scaling plots.

Source Data Extended Data Fig. 4

Datapoints of scaling plots.

Source Data Extended Data Fig. 5

Datapoints of heatmaps.

Source Data Extended Data Fig. 6

Datapoints of heatmaps.

Source Data Extended Data Fig. 7

Datapoints of anchor plot.

Source Data Extended Data Fig. 8

Datapoints of heatmaps.

Source Data Extended Data Fig. 9

Datapoints of heatmaps.

Source Data Extended Data Fig. 10

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

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