The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C)1,2 analysis has revealed a complex genomic landscape of internal chromosomal structures in vertebrate cells3,4,5,6,7, but the identical sequence of sister chromatids has made it difficult to determine how they topologically interact in replicated chromosomes. Here we describe sister-chromatid-sensitive Hi-C (scsHi-C), which is based on labelling of nascent DNA with 4-thio-thymidine and nucleoside conversion chemistry. Genome-wide conformation maps of human chromosomes reveal that sister-chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister-chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired and are characterized by facultative heterochromatin and insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister-chromatid topologies and our scsHi-C technology will make it possible to investigate how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.
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All scsHi-C datasets generated in this study have been made available via the Gene Expression Omnibus (GEO) database under the series accession number GSE152373 and are also available from the authors upon request.
The ipython notebooks used to perform all the sequencing data analysis of data generated within this work are available at https://github.com/gerlichlab/scshic_analysis, along with a detailed description of each script and the figures they produce. The programing environment used to perform this analysis is provided as a docker container (https://hub.docker.com/repository/docker/gerlichlab/scshic_docker) under the tag ‘release-1.0’. All the versions of the software packages used are noted within the dockerfile.
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We acknowledge technical support by the IMBA/IMP/GMI BioOptics and Molecular Biology Services facilities, and the Vienna BioCenter Metabolomics and Next Generation Sequencing facilities. We thank I. Patten, P. Batty, M. W. G. Schneider, and M. Voichek for comments on the manuscript, and Life Science Editors for editing assistance. Research in the laboratory of D.W.G. is supported by the Austrian Academy of Sciences, an ERC Starting (Consolidator) Grant (281198), the Austrian Science Fund (FWF; Doktoratskolleg ‘Chromosome Dynamics’ DK W1238), and the Vienna Science and Technology Fund (WWTF; project LS17-003). Research in the laboratory of R.M. is supported by the Austrian Science Fund (P27947, P31691, F8011), the Austrian Research Promotion Agency FFG (West-Austrian BioNMR 858017), and the Vienna Science and Technology Fund (WWTF; project no. LS17-003). Research in the laboratory of S.L.A. is supported by the Austrian Academy of Sciences, the European Research Council (ERC-CoG-866166 and ERC-PoC-825710), the Vienna Science and Technology Fund (WWTF; project LS17-003), and the Austrian Science Fund (FWF; SFB F8002). Research in the laboratory of J.-M.P. is supported by Boehringer Ingelheim, the Austrian Research Promotion Agency (Headquarter grant FFG-852936), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme GA No 693949, the Human Frontiers Science Programme (RGP0057/2018) and the Vienna Science and Technology Fund (WWTF; grant LS19-029). M.M. received a PhD fellowship from the Boehringer Ingelheim Fonds. The VBCF Metabolomics Facility is funded by the City of Vienna through the Vienna Business Agency.
R.M. and C.G. are listed as inventors 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.
Peer review information Nature thanks David Gilbert, Benjamin Rowland and Hongtao Yu for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Synthetic hairpin-oligonucleotide used to probe 4sT conversion by OsO4. The theorized reaction educts and products are highlighted in red. b, High performance liquid chromatography (HPLC) trace at 260 nm of the oligos depicted in a before and after the conversion by OsO4/NH4Cl. The peak position of the oligo before conversion is indicated by a dashed line. c, Melting point analysis of a synthetic DNA hairpin containing 4sT or thymidine in the Watson–Crick base-paired stem (see Methods for details). d, Native mass spectrum of the oligonucleotide shown in a before and after OsO4/NH4Cl conversion measured in negative ion mode.
a, Percentage of live cells determined via Topro-3-Iodide staining of dead cells after 24 h incubation with the indicated compounds. Bars indicate mean of live cell percentages of individual wells from n = 2 biologically independent experiments. b, DNA damage assay performed after 24 h incubation with the indicated compounds. Scale bars indicate 5 μm. c, Quantification of mean fluorescence in cell nuclei stained by anti-p-γ-H2A.X antibody shown in b. Bars indicate mean of individual cells from n = 2 biologically independent experiments. d, Flow cytometry (FACS) analysis of cells progressing through S-phase in the presence or absence of 2 mM 4sT. DNA was stained using propidium iodide and kernel density estimation of signal in the PE-channel is shown. Cells were pre-synchronized to G1/S by thymidine and released into S-phase by removal of thymidine. The G2 sample was arrested by RO3306 and the G1 sample was arrested after progression through mitosis using aphidicolin. This experiment was repeated one more time with similar results.
a, Quantification of 4sT incorporation into genomic DNA of HeLa cells using mass spectrometry. Cells were grown in medium containing 4sT for 5 days and purified genomic DNA digested to single nucleotides. Indicated values reflect the percentage of 4sT in total measured thymidine. Bars indicate mean of n = 6 biologically independent experiments. b Quantification of 4sT incorporation using DNA sequencing. Cells were 4sT labelled as in a and purified genomic DNA chemically converted as in Fig. 1c. Indicated values are the sum of the A-to-G-mutation rate and the T-to-C mutation rate, normalized to the total amount of adenosine and thymidine measured respectively. Bars indicate mean of n = 3 biologically independent experiments. c, Overview of experimental procedure for differential labelling of sister chromatids using 4sT. d, Procedure of cell synchronization for scsHi-C sample preparation of cells synchronized to G2, prometaphase and the subsequent G1 phase. The different compounds were added to the cell culture medium as indicated by the colored bars. e, Cell cycle analysis of WT HeLa cells synchronized to G2, prometaphase and G1 as indicated in d. Anti-pH3S10 antibody was used to detect the mitotic state and propidium iodide to measure DNA content. Gates for different cell cycle stages are shown and the indicated numbers reflect percentage of cells that were measured. This experiment was repeated one time with similar results. See Supplementary Fig. 2 for the exact gating strategy. f, Average contact probability over different genomic distances of HeLa cells synchronized to G2 that were either labelled with 4sT or unlabelled. Experiment was repeated one time with similar results. g, Hi-C interaction matrices at example regions of HeLa cells synchronized to G2 that were either labelled with 4sT or unlabelled.
a, Depiction of all possible Hi-C ligation products of a sample where one strand of each sister chromatid has been labelled with a synthetic nucleotide. Only ligation products that carry two continuous halves that are labelled (“double-labelled”) can be used to discriminate cis sister contacts from trans sister contacts. This is because 4sT incorporation density is not high enough to allow detection of unlabelled reads based on the absence of signature mutations. If a given read exhibits signature mutations, however, it is possible to know with high confidence (see panel b and c) that it comes from the labelled strand. The ligation products that do not contain two halves with signature mutations are thus discarded during analysis. b, Histograms of signature point mutations per read (AG or TC) of conventional sequencing libraries constructed from cells that were grown for 5 days in the presence (“4sT-labelled”) or absence (“unlabelled”) of 4sT and treated with OsO4 as described in Fig. 1c. Note that at sites of 4sT-incorporation only 50% of PCR amplicons will contain the signature mutations, as 4sT base-pairs with unmodified A on the opposing DNA strand. Bar indicates mean of n = 3 biologically independent experiments. c, To assess how many signature mutations are required to confidently detect double-labelled reads, we analysed Hi-C libraries from cells grown in the absence of 4sT and calculated the false-positive rate of double-labelled read detection. Double-labelled reads were assigned based on different required signature point mutations on both read-halves. The samples shown was grown without 4sT, suggesting that every detected double-labelled read should be a false positive. Points indicate mean of n = 2 biologically independent experiments. When considering reads that contained at least two signature mutations, less than 0.2% were classified as “double-labelled”, indicating very low rates of misclassification. When cells were released into S-phase in the presence of 4sT, the percentage of double-labelled reads increased to 12% in the subsequent G2 phase (f). Thus, double-labelled reads are detected with a very low false positive rate and at sufficient yield to construct Hi-C-maps. d, Percentage of Hi-C contacts in HeLa wildtype cells synchronized to G2 in the presence of 4sT that can be used to assign sister-chromatid identity (“double-labelled reads”) based on a classification scheme that requires an equal amount or more than the shown number of signature mutations. The number used in this paper (2) is highlighted in red. Points indicate mean of n = 9 biologically independent experiments. e, Quantification of wrongly assigned trans sister contacts based on different signature mutation thresholds. To calculate the false-positive rate with which a cis sister contact is wrongly assigned as a trans sister contact, we adapted the scheme used to quantify the false-positive rate of trans-homologue contact assignment from ref. 35. Briefly, all contacts that exhibit a genomic separation smaller than 1 kb are assumed to be Hi-C artefacts that arise from uncut continuous pieces of chromatin. Such contacts should be exclusively classified as cis sister contacts and thus all trans sister contacts in this range are assumed to be false positives. To then quantify the percentage of incorrect trans sister Hi-C contacts among all trans sister Hi-C contacts, the calculated false-positive rate was multiplied by the number of cis sister contacts exhibiting separation larger than 1 kb and the resulting percentage of all trans sister contacts exhibiting separation larger than 1 kb plotted in this figure. Points indicate mean of n = 9 biologically independent experiments. We thus estimate that - at the number of required point mutations used in this paper (2) - the wrongly assigned trans sister contacts are below 2%. f, Quantification of Hi-C reads that are labelled on both sides for sister-specific contact classification, as a percentage of all reads. Cells were synchronized to the G1/S boundary and released into S-Phase in the presence of 4sT for the indicated times. The G2 sample was arrested using RO3306; the prometaphase sample was arrested using nocodazole; the control sample refers to unlabelled DNA and the G1/S 4sT sample refers to a sample that was treated with 4sT, but not released into S-Phase. Bars show the mean of n = 2 biologically independent replicates. g, Percentage of trans sister contacts based on all double-labelled reads that exhibit a genomic separation larger than 10 kb. Cells were released from G1/S block into medium containing 4sT and then arrested in G2 using RO3306, in prometaphase using nocodazole, or the following G1 using thymidine. Bars show mean of n = 2 biologically independent replicates.
a, Hi-C interaction matrices of the long arm of chromosome 1 of all contacts, cis sister, and trans sister contacts shown for two of the 11 G2 WT replicates. The all-contacts matrix was normalized to the total number of corrected contacts in the region of interest (ROI), whereas cis sister and trans sister contacts were normalized to the total amount of cis sister contacts and trans sister contacts in the ROI. Bin size of the matrix is 500 kb. b, HiCrep57 analysis of all, cis sister and trans sister contacts of all n = 11 biologically independent G2 replicates. Bars show the mean of all comparisons. c, Hi-C interaction matrix of the long arm of chromosome 1 of all, cis sister, and trans sister contacts of the two prometaphase replicates. Contacts were normalized as in a. d, HiCrep57 analysis of all, cis sister and trans sister contacts of n = 2 biologically independent prometaphase replicates. Bars show the mean of all comparisons.
a, All contacts, cis sister and trans sister contacts, as well as the ratio of trans sister observed/expected to cis sister observed/expected of n = 11 biologically independent, merged G2 samples at a representative region on chromosome 3 is displayed alongside the location of TAD boundaries and the trans sister pairing score (see Methods for details). Bin size is 30 kb. b, Comparison of sister-chromatid separation at 5 genomic loci measured by fluorescence in situ hybridization (FISH) and scsHi-C. Microscopy image shows examples for split and unsplit genomic sister loci, from G2-synchronized HeLa cell data reported in ref. 31. scsHi-C quantification of sister locus distance was done by calculating (1 – average trans sister contacts) in a region spanning 600 kb around each FISH target site and standardizing the resulting value (see Methods for more details). Each dot indicates one target locus, measured in n = 11 biologically independent HeLa WT G2 samples by scsHi-C. The points indicate the mean and the error indicates the standard deviation of the Hi-C measurements. Two-sided p-value for a Wald-test with t-distribution of the test statistic is shown with the null hypothesis being a zero slope (calculated using the scipy.stats.linregress function). P-value = 1 * 10−14. c, Comparison of sister-chromatid separation at 16 genomic loci measured by live cell microscopy and scsHi-C. Microscopy analysis was by live-cell imaging of 16 HeLa cell lines expressing dCas9-EGFP with different locus-specific gRNAs, using automated detection of merged or split sister loci in G2 cells, as reported in ref. 31. scsHi-C quantification of sister locus distance was done by calculating (1 – average trans sister contacts) in a region spanning 600 kb around each gRNA target site and standardizing the resulting value. Each dot indicates one target locus, measured in n = 11 biologically independent HeLa WT G2 samples by scsHi-C. The points indicate the mean and the error indicates the standard deviation of the Hi-C measurements. Two-sided p-value for a Wald-test with t-distribution of the test statistic is shown with the null hypothesis being a zero slope (calculated using the scipy.stats.linregress function). P-value = 3 * 10−27. d, Histogram of average trans sister contact frequency for annotated TADs (see Methods for details). Vertical lines indicate the cut-offs for “highly paired” and “highly unpaired” TADs. e, Average contact probability over different genomic distances for cis sister and trans sister contacts at “highly paired”, “highly unpaired” regions as well as the genome-wide average for the n = 11 biologically independent HeLa WT G2 samples. Trans sister contacts were evenly increased or decreased over variable genomic distances in these highly paired or unpaired domains, respectively. Curves were normalized to the contact frequency of doubly labelled reads below 1 kb. f, Average H3K27me3 ChIP-seq signal (fold-control) at “highly paired” domains. Within highly paired domains, H3K27me3 distributed relatively evenly, without marked enrichment at the edges. The domains of different size were scaled to range from arbitrary genomic units -0.5 to 0.5. The bars represent average ChIP-seq signal (fold-control). TAD boundaries are marked with dashed, grey vertical lines.
a, Cis sister- and trans sister contacts of n = 11 biologically independent, merged G2 samples at a representative region on chromosome 1 are displayed alongside the location of TAD boundaries (see Methods for details) and average trans sister and cis sister contact amount within a sliding window of 200 kb (see Methods for details). Bin size of matrix is 40 kb. b, Cis sister and trans sister contacts of n = 11 biologically independent, merged G2 samples at a representative region on chromosome 3 are displayed alongside the location of TAD boundaries (see Methods for details) and average trans sister and cis sister contact amount within a sliding window of 200 kb (see Methods for details). Bin size of matrix is 40 kb. c, Cis sister and trans sister contacts of n = 11 biologically independent, merged G2 samples at a representative region on chromosome 5 are displayed alongside the location of TAD boundaries (see Methods for details) and average trans sister and cis sister contact amount within a sliding window of 200 kb (see Methods for details). Bin size of matrix is 40 kb. These examples show that the high-resolution maps of G2 chromosomes reveal many fine structures inside TADs, which we currently cannot attribute to specific genomic features. d, Stack-up of corrected interaction frequency within sliding windows of 100 kb around CTCF Chip-seq peaks overlapping CTCF motifs of n = 11 biologically independent, merged G2 wildtype samples. The panel shows windows of 900 kb. The rows are sorted based on the centre enrichment. e, Quantification of trans-contact enrichment at CTCF Chip-seq peaks. The average observed/expected values for cis sister and trans sister contacts within a 80 kb window surrounding all (n = 60,929) annotated CTCF Chip-seq peaks overlapping a CTCF motif are displayed as a histogram. P-value was calculated using a two-sided Mann–Whitney U test. P-value = 2 * 10−296.
a, Western blot for Sororin and GAPDH of HeLa Sororin-AID cells synchronized to G2 and either treated with auxin (+) or H20 (-) as well as western blot for NIPBL and GAPDH of HeLa NIPBL-AID cells synchronized to G2 and either treated with auxin (+) or H2O (-). This experiment was repeated two (Sororin-AID samples) and three (NIPBL-AID samples) more times with similar results. Uncropped images are displayed in Supplementary Fig. 1. b, Cell cycle analysis of HeLa Sororin-AID and HeLa NIPBL-AID cells synchronized to G2 as indicated in Extended Data Fig. 3d, treated with auxin. Panel shows a FACS plot of cells stained for pH3S10 to mark mitotic cells and propidium iodide to measure DNA content. Gates for different cell cycle stages are shown and the indicated numbers reflect percentage of cells that were measured. This experiment was repeated three more times with similar results. c, Contact probability of all contacts at different genomic distances of HeLa NIPBL-AID cells synchronized to G2 (n = 4 biologically independent experiments) that were treated with auxin and HeLa WT cells synchronized to G2 (n = 11 biologically independent experiments). d, Chromosome congression analysis of NIPBL- and Sororin-depleted cells. AID-tagging often reduces protein levels already before the addition of auxin59,60, which for NIPBL might impair sister-chromatid cohesion establishment during S-phase. We therefore performed metaphase congression analysis by time-lapse microscopy of WT HeLa cells, HeLa Sororin-AID cells and HeLa NIPBL-AID cells stained with SiR-DNA. HeLa Sororin-AID cells were treated with auxin before the final S-phase and HeLa NIPBL-AID cells were treated with auxin after the final S-phase. Panel shows the cumulative frequency of cells congressing their chromosomes in metaphase after entering mitosis in a RO3306 wash-out. Pooled replicates are shown from n = 2 biologically independent experiments. e, Cis sister and trans sister contacts of n = 11 biologically independent, merged G2 wildtype samples, n = 4 biologically independent, merged G2 NIPBL-degraded samples and n = 3 biologically independent, merged Sororin-degraded samples at a representative region on chromosome 5 are displayed alongside the location of TAD boundaries (see Methods for details). The strong accumulation of trans sister contacts close to the diagonal in NIPBL-depleted cells indicates frequent contacts between sister chromatids and a tighter alignment. Owing to normalization of contacts to the marginal count per row, trans sister contacts appear less frequent at larger genomic distances. Bin size of matrix is 150 kb. f, HiCrep57 analysis of all, cis sister and trans sister contacts of all replicates of HeLa NIPBL-AID (n = 4 biologically independent experiments) or Sororin-AID (n = 3 biologically independent experiments) cells treated with auxin. Bars show the mean of all comparisons.
a, Stack-up of average cis sister and trans sister contacts for NIPBL-degraded cells (n = 4 biologically independent, merged experiments) within sliding windows of 100 kb along TADs; lines represent 6 Mb genomic windows of individual TADs, aligned at the centre and sorted by size. Compare Fig. 3e for WT data. b, Stack-up of average trans sister observed/expected values for NIPBL-degraded (n = 4 biologically independent, merged experiments) and WT cells (n = 11 biologically independent, merged experiments) within sliding windows of 100 kb along TADs, individual TADs aligned at centre and sorted by size. c, Stack-up of trans sister corrected interaction frequency (see Methods for details) along TADs that are defined as highly paired or highly unpaired in WT cells (Fig. 2f; Extended Data Fig. 6d), sorted by the size of TADs for NIPBL-degraded cells (n = 4 biologically independent, merged experiments) an WT cells (n = 11 biologically independent, merged experiments). Shown are windows of 6 Mb around the centre of the respective TADs. Pairing scores were calculated within a sliding window of 200 kb on a Hi-C matrix with 20 kb bin size. d, Stack-up of trans sister pairing score (see Methods for details) along TADs that are highly paired or highly unpaired in WT cells (Fig. 2f; Extended Data Fig. 6d), sorted by the size of TADs for NIPBL-degraded cells (n = 4 biologically independent, merged experiments). Shown are windows of 6 Mb around the centre of the respective TADs. Pairing scores were calculated within a sliding window of 200 kb on a Hi-C matrix with 20 kb bin size.
a, Synthesis of 5′-O-(4,4′-Dimethoxytrityl)-S-(2-cyanoethyl)-4-thiothymidine 3′-O-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. b, 1H-NMR (700 MHz, CDCl3) of 4sT phosphoramidite (diastereomeric mixture). c, 31P-NMR (282 MHz, CDCl3) of 4sT phosphoramidite (diastereomeric mixture).
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Mitter, M., Gasser, C., Takacs, Z. et al. Conformation of sister chromatids in the replicated human genome. Nature 586, 139–144 (2020). https://doi.org/10.1038/s41586-020-2744-4
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