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Sister chromatid–sensitive Hi-C to map the conformation of replicated genomes

Abstract

Chromosome conformation capture (Hi-C) techniques map the 3D organization of entire genomes. How sister chromatids fold in replicated chromosomes, however, cannot be determined with conventional Hi-C because of the identical DNA sequences of sister chromatids. Here, we present a protocol for sister chromatid–sensitive Hi-C (scsHi-C) that enables the distinction of DNA contacts within individual sister chromatids (cis sister contacts) from those between sister chromatids (trans sister contacts), thereby allowing investigation of the organization of replicated genomes. scsHi-C is based on live-cell labeling of nascent DNA by the synthetic nucleoside 4-thio-thymidine (4sT), which incorporates into a distinct DNA strand on each sister chromatid because of semi-conservative DNA replication. After purification of genomic DNA and in situ Hi-C library preparation, 4sT is chemically converted into 5-methyl-cytosine in the presence of OsO4/NH4Cl to introduce T-to-C signature point mutations on 4sT-labeled DNA. The Hi-C library is then sequenced, and ligated fragments are assigned to sister chromatids on the basis of strand orientation and the presence of signature mutations. The ensemble of scsHi-C contacts thereby represents genome-wide contact probabilities within and across sister chromatids. scsHi-C can be completed in 2 weeks, has been successfully applied in HeLa cells and can potentially be established for any cell type that allows proper cell cycle synchronization and incorporation of sufficient amounts of 4sT. The genome-wide maps of replicated chromosomes detected by scsHi-C enable investigation of the molecular mechanisms shaping sister chromatid topologies and the relevance of sister chromatid conformation in crucial processes like DNA repair, mitotic chromosome formation and potentially other biological processes.

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Fig. 1: Genome organization throughout the cell cycle.
Fig. 2: Overview of scsHi-C methodology.
Fig. 3: Assessing 4sT toxicity in HeLa cells.
Fig. 4: Quality control measurements during scsHi-C library preparation.
Fig. 5: Genome-wide maps of sister chromatid contacts.
Fig. 6: Average contact probabilities over different genomic intervals.
Fig. 7: Average contact probability maps around sets of genomic loci.

Data availability

All datasets used in the preparation of this paper were published in ref. 33 and are available from Gene Expression Omnibus under accession number GSE152373 and from the authors upon request.

Code availability

The code used to generate the figures in this manuscript was originally published in Mitter et al.33. Specifically, the ipython notebooks to generate all the plots shown in this manuscript can be found at https://github.com/gerlichlab/scshic_analysis78. A programming environment to perform all analyses shown within this manuscript is provided as a docker container at https://hub.docker.com/repository/docker/gerlichlab/scshic_docker71. The preprocessing pipeline that can be used to convert raw data to .mcool files is available at https://github.com/gerlichlab/scshic_pipeline72.

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Acknowledgements

The authors acknowledge technical support by the IMBA/IMP/GMI BioOptics and Molecular Biology Services facilities and the Vienna BioCenter Metabolomics and Next Generation Sequencing facilities. Research in the laboratory of D.W.G. is supported by the Austrian Academy of Sciences, the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 101019039), the Austrian Science Fund (FWF; Doktoratskolleg ‘Chromosome Dynamics’ DK W1238) and the Vienna Science and Technology Fund (WWTF; projects LS17-003 and LS19-001). Research in the laboratory of R.M. is supported by the Austrian Science Fund (P31691 and F8011), the Austrian Research Promotion Agency FFG (West-Austrian BioNMR 858017) and the WWTF (project nr. LS17-003). M.M. received a PhD fellowship from the Boehringer Ingelheim Fonds. Z.T. received a Hertha Firnberg Programme fellowship of the FWF (T 1246). The VBCF Metabolomics Facility is funded by the City of Vienna through the Vienna Business Agency.

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Contributions

M.M. developed the protocol for scsHi-C, with help from R.M. (4sT conversion chemistry), T.K. (mass spectrometry), C.C.H.L. (data processing) and D.W.G. (biological interpretation). M.M., Z.T. and D.W.G. wrote the manuscript, except the procedures section on mass spectrometry, which was written by T.K. D.W.G., M.M., Z.T. and R.M. acquired funding.

Corresponding authors

Correspondence to Michael Mitter or Daniel W. Gerlich.

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Competing interests

R.M. is listed as inventor on a patent application that has been filed concerning the nucleoside conversion chemistry of this work (Osmiumtetroxide-based conversion of RNA and DNA containing thiolated nucleotides, US Patent App. 16/533,988). The other authors declare no competing interests.

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Mitter, M. et al. Nature 586, 139–144 (2020): https://doi.org/10.1038/s41586-020-2744-4

Extended data

Extended Data Fig. 1 Yield of scsHi-C using HeLa Kyoto cells and 4sT incorporation in other cell lines.

a, Yield of scsHi-C performed by using HeLa Kyoto cells at different steps of the scsHi-C protocol. b, Quantification of 4sT incorporation into genomic DNA of HCT116, HEK293 and RPE1 cells. Cells were cultured in the presence of 2 mM 4sT for 5 d, and genomic DNA was purified, digested into nucleosides and analyzed by mass spectrometry. The percentage of 4sT over total thymidine is shown from n = 2 biologically independent experiments for each cell line.

Extended Data Fig. 2 HPLC-tandem MS chromatograms of a separated standard mixture of 4sT and thymidine.

Top panels, SRM (selected reaction monitoring) traces of dT (m/z 243.1 to m/z 127.1) and of its in-source fragmentation product (m/z 127 to m/z 54) are shown. Lower panels, SRM traces of thio-dT (m/z 259.1 to m/z 143.1) and the respective in-source fragmentation product (m/z 127.1 to m/z 54.1) are depicted. Both nucleosides generate only one fragment ion (neutral loss of the sugar); therefore, we recommend recording the SRM traces of the in-source products, which can either be used for quantification (if of significant signal intensity) or as a qualifier for confirming the respective nucleoside.

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Mitter, M., Takacs, Z., Köcher, T. et al. Sister chromatid–sensitive Hi-C to map the conformation of replicated genomes. Nat Protoc 17, 1486–1517 (2022). https://doi.org/10.1038/s41596-022-00687-6

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