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Cohesin maintains replication timing to suppress DNA damage on cancer genes

Nature Genetics volume 55, pages 1347–1358 (2023)Cite this article

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Abstract

Cohesin loss-of-function mutations are frequently observed in tumors, but the mechanism underlying its role in tumorigenesis is unclear. Here, we found that depletion of RAD21, a core subunit of cohesin, leads to massive genome-wide DNA breaks and 147 translocation hotspot genes, co-mutated with cohesin in multiple cancers. Increased DNA damages are independent of RAD21-loss-induced transcription alteration and loop anchor disruption. However, damage-induced chromosomal translocations coincide with the asymmetrically distributed Okazaki fragments of DNA replication, suggesting that RAD21 depletion causes replication stresses evidenced by the slower replication speed and increased stalled forks. Mechanistically, approximately 30% of the human genome exhibits an earlier replication timing after RAD21 depletion, caused by the early initiation of >900 extra dormant origins. Correspondingly, most translocation hotspot genes lie in timing-altered regions. Therefore, we conclude that cohesin dysfunction causes replication stresses induced by excessive DNA replication initiation, resulting in gross DNA damages that may promote tumorigenesis.

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Fig. 1: RAD21 depletion causes DNA breaks and translocations in K562 cells.
Fig. 2: RAD21 depletion leads to translocations on cohesin co-mutated genes.
Fig. 3: Loop anchors and transcription alterations are not the main factors in RAD21-depletion-induced DSBs.
Fig. 4: RAD21-depletion-induced translocations are correlated with DNA replication.
Fig. 5: RAD21 depletion reprograms genome-wide replication timing.
Fig. 6: Dormant origin firing leads to alteration in replication timing.
Fig. 7: Cohesin preserves genome integrity through repression of dormant origin firing in the middle of loops.

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Data availability

All sequencing data generated in the present study were deposited at the NCBI Gene Expression Omnibus (GEO) under the accession number: GSE198285. The data generated and analyzed in this study are based on a human reference (GRCh37/hg19) and a mouse reference (GRCm38/mm10). All public data used in this study have been listed in Supplementary Table 3. Source data are provided with this paper.

Code availability

Publicly available software and packages were used in this study. All software and packages used in this study are listed in Supplementary Table 6 with descriptions in the Methods section.

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Acknowledgements

We acknowledge the members of the Hu laboratory for their helpful discussions. The checkpoint kinase antibodies were gifts from H. Wu at Peking University. The plasmid backbones for the degron system and mES cells with OsTIR expression were gifts from X. Ji at Peking University. Fruit fly S2 cells were gifts from A. J. Zhu at Peking University. We thank S. Qin and J. Ren at the Core Facilities of the School of Life Sciences, Peking University. We also thank L. Du from the Flow Cytometry Core at the National Center for Protein Sciences, Peking University. This work was supported by the National Key R&D Program of China (grant nos. 2022YFC3400201 and 2017YFA0506700 to J. Hu) and the NSFC (grant no. 32122018 to J. Hu). J. Hu is an investigator at the PKU-TSU Center for Life Sciences. Y. Liu is supported by the Boehringer Ingelheim–Peking University Postdoctoral Program.

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  1. These authors contributed equally: Jinchun Wu, Yang Liu, Zhengrong Zhangding, Xuhao Liu.

Authors and Affiliations

  1. The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Center for Life Sciences, Genome Editing Research Center, Peking University, Beijing, China

    Jinchun Wu, Yang Liu, Zhengrong Zhangding, Xuhao Liu, Chen Ai, Tingting Gan, Haoxin Liang, Yuefeng Guo, Mohan Chen, Yiyang Liu, Jianhang Yin, Weiwei Zhang & Jiazhi Hu

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Contributions

J.H. conceived and supervised the project. J.W., Y.L., X.L. and J.H. designed the experiments. J.W., Y.L., X.L., T.G., H.L., Y.G., M.C., Y-Y.L., J.Y. and W.Z. performed the experiments. J.W., Y.L., Z.Z., X.L., C.A. and J.H. analyzed the data. J.W., Y.L., Z.Z. and J.H. wrote the paper.

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Correspondence to Jiazhi Hu.

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Extended data

Extended Data Fig. 1 Cohesin mutations in cancers and construction of RAD21 degron system in K562 cells.

a Mutation frequency of cohesin subunits in dozens of cancer types. Cancer types in the TCGA database with more than 2000 cases and over 0.1% RAD21 mutation are displayed and ranked according to the mutation frequency of RAD21. b Proportions of four types of mutations in cohesin subunits. Mutations are obtained from the Catalogue of Somatic Mutations In Cancer (COSMIC) database. The SIFT tool is used to predict the functional consequence of cohesin mutations, including amino acid insertions, deletions, and substitutions, which identifies deleterious and neutral mutations (See Method for details). The case number of mutations in each subunit is labeled above each pie chart. Subs., substitutions; Indels, insertions, and deletions. c Knock-in and PCR validation strategy for RAD21 tagged with mAID-mClover in K562 cells. The representative PCR plot is shown at the bottom, the result was confirmed by two independent replicates. d Flow cytometry analyzing mClover fluorescence of two RAD21-mAC K562 clones after IAA treatment for the indicated time. e Quantification of γH2A.X foci in WT and RAD21-mAC cells without IAA treatment. The red lines indicate the median number of γH2A.X foci and the examined cell numbers are labeled at the bottom. Two-sided Mann–Whitney test; n.s., no significance.

Source data

Extended Data Fig. 2 RAD21 depletion causes DNA breaks in mES and K562 cells.

a Western blotting for validation of Rad21-mAC (1C4 or 1D2) mES cells. The result was confirmed by two independent replicates. b Flow cytometry analyzing mClover fluorescence of WT and Rad21-mAC mES clones with or without IAA treatment. The percentage of mClover-positive cells is labeled. c Number of broken chromatids in metaphase mES cells. Red lines, medium; n, examined cell number; two-sided t-test; ****, p < 0.0001. d Examples of chromosomes and chromatids that were intact or broken (red triangles). Telomeres are red dots. Two independent replicates. Bar (white lines), 10 μm. The exemplified chromosome and chromatids are zoom-in with 3.5x magnification. e Percentage of four types of metaphases in IAA-treated WT and Rad21-mAC mES cells. The four states of cohesion phenotypes were classified as previously24: Unresolved, unresolved sister chromatid arms; Arm-resolved, resolved arms of sister chromatids; Separated, separated sister chromatids that remain closed; Scattered, scattered chromatids. f Translocation frequency in IAA-untreated cells. Mean ± SD from three biological replicates; two-sided t-test. g Bar plots exhibiting translocation junctions captured from MYC (left) and TP53 (right) bait in IAA-treated cells. The number of translocations was normalized to 100,000 editing events. The red arrow and dashed line indicate the bait site. Numbers of junctions in + (red) or – (blue) orientation are shown. h Protein level of ectopically expressed RAD21 and mutants (Flag-tagged, red triangles). Relative amounts of RAD21 variants were indicated at the bottom. Two independent replicates. i Flow cytometry analyzing mClover fluorescence of RAD21-mAC cells with shRNA treatment for 2 days. j Protein level of RAD21 in WT cells after shRNA treatment for 2 days. Numbers show the relative amount of remaining RAD21 to that of WT cells. Two independent replicates. k Frequency of translocations after RAD21 knock-down in WT K562 cells. Mean ± SD from three biological replicates; two-sided t-test; **, p < 0.01; ***, p < 0.001.

Source data

Extended Data Fig. 3 RAD21 depletion in K562 cells leads to recurrent breaks on cancer-related genes.

a Flow diagram for the identification of translocation hotspot genes. See Methods for details. b Circos plots showing the genome-wide translocations and translocation hotspot genes cloned from the TP53 locus with RAD21 depletion. Legends are depicted as described in Fig. 2a. c Translocation frequencies of RAD21-depletion-induced translocation hotspot genes in RAD21-mAC 4# captured by MYC DSBs or RAD21-mAC 1# and 4# captured by TP53 DSBs. Legends are depicted as described in Fig. 2b. d Circos plots exhibiting the distribution of genome-wide DSBs in IAA-treated WT and RAD21-mAC cells. Histogram exhibits genome-wide DNA breaks with 1-Mb bin size and with a minimum of 10 in the log scale. Numbers in the circos plots represent the relative levels of DSBs in comparison to that of WT. e Representative profile of END-seq-captured DNA breaks on the BCR gene. The number of breaks was normalized to the same number of spike-in DSBs. See Methods for details. f The permutation test of PEM-seq-captured hotspot genes and fusion genes annotated in the Atlas of Genetics and Cytogenetics in Oncology and Haematology database. The red line indicates the overlapped number (Nreal) between hotspot genes and fusion genes. Histograms show the distribution of overlapped numbers between hotspot genes and randomized genes from 1000 draws. The black dashed line indicates the upper bound of 95% confidence interval of the simulation. Nnull indicates the mean value of the simulation. See Methods for details. g Mutation frequency of PEM-seq-identified translocation hotspot genes in the non-small cell lung cancer (top), glioma (middle), and colorectal cancer (bottom) patients with (red bars) or without (black bars) cohesin mutations. Legends are depicted as described in Fig. 2d. The genes on the left side of the dashed lines have p-values < 0.05 with the two-sided Chi-square test for independence of cohesin mutations.

Extended Data Fig. 4 Loop anchors and transcription alterations are not the main driven factors for RAD21 depletion-induced DSBs.

a RAD21 ChIP-seq signals in WT and RAD21-mAC cells with and without IAA treatment for 6 hours. mClover-negative RAD21-mAC cells were sorted for ChIP-seq analysis. b Profiles of RAD21 distribution in WT and RAD21-mAC cells with IAA treatment for 6 hours. mClover-negative cells of RAD21-mAC were sorted for ChIP-seq analysis. c A representative profile of PRO-seq signals and translocations in IAA-treated WT and RAD21-mAC cells at a hotspot gene AGAP1. Translocations were presented as dot plots with the legend indicated below.

Extended Data Fig. 5 RAD21 depletion-induced translocations are associated with DNA replication.

a Representative profile of translocation junction bias in IAA-treated WT and RAD21-mAC K562 cells from TP53 locus. The legends of indicated panels are described in Fig. 4a. b The correlation between PEM-seq-identified translocation junction bias and Okazaki fragments from OK-seq. Each point indicates a unique peak within regions displayed in Fig. 4a or Extended Data Fig. 5a. The two-sided Pearson’s correlation coefficients (PCC) and p-values (p) are marked. The grey shadow indicates 95% confidence interval of a linear regression model. c Percentages of genome-wide translocation junctions within the Watson or Crick peaks. Mean ± SD from three biological replicates; two-sided t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001. d Orientations of PEM-seq-captured translocation and Okazaki fragments. The leftward fork contains Okazaki fragments on the Watson strand. DNA breaks occurring on either the template DNA of leading or lagging strands lead to the translocation junctions with a positive or Watson orientation. The rightward fork contains Okazaki fragments on the Crick strand and DNA breaks on the leading or lagging strand form translocation junctions with a negative or Crick orientation. e Representative profile of PEM-seq-captured translocations in K562 cells under HU or APH treatment.

Extended Data Fig. 6 RAD21 depletion reprograms genome-wide replication timing.

a FACS sorting strategy for replication timing analysis. The black and green circles gate the mClover negative and positive cells, respectively. The red rectangles indicate the six fractions of sorted cells according to DNA content. b A representative profile of replication timing in WT and RAD21-mAC cells (clone 4#) with IAA treatment for 6 hours or 24 hours. c Scatter plot of S50 values between WT K562 replicates (clone 4#) with IAA treatment for 24 hours. The grey region between dashed lines indicates the 95% quantile interval of WT replicates. Legends are described in Fig. 5b and the computation details are described in Methods. d Scatter plot of S50 values between WT and RAD21-mAC cells (clone 4#) with IAA treatment for 6 hours. Legends are described in Fig. 5b. e Distribution of S50 values in IAA-treated WT and RAD21-mAC cells. The p-value from the two-sided Mann–Whitney test is labeled on the top of the plots. f Scatter plot of S50 values between WT mES replicates with IAA treatment for 3 hours. Legends are described in Fig. 5b.

Extended Data Fig. 7 Dormant origin firing leads to alteration in replication timing.

a Boxplots showing the number of RAD21 foci per cell in IAA-treated WT and RAD21-mAC cells. Middle lines of boxplots indicating the median, upper and lower hinges indicate 25% and 75% quartiles, respectively, and upper or down whiskers indicate the extremum value no larger or smaller than the 1.5x IQR (inter-quartile range), respectively. Two-sided Mann–Whitney test; ****, p < 0.0001. b Schematic for NAIL-seq analysis to identify ERIZs (details in Methods). c Release of G1-arrested WT and RAD21-depleted cells. K562 cells were synchronized in G1 phase for 6 hours of IAA treatment, and released into a fresh medium with IAA. Cells were labeled with BrdU for 30 minutes before harvest. Rx, release for x hours. d Western blotting showing the amounts of chromatin-bound RAD21, CTCF, ORC2, and MCM6 in G1-arrested cells. Two independent replicates. e Pearson’s correlation between biological replicates of EdU/HU signals. f Venn plot showing the overlap of ERIZs identified from two clones after RAD21 depletion. g Heatmap showing the intensity of EdU/HU signals in disappeared ERIZs. h PRO-seq signals from cells with or without RAD21 depletion in non-transcribed regions (non-TR) adjacent to each class of ERIZs. i Western blotting showing the level of phosphorylated-CHK1 and -CHK2 in IAA-treated WT and RAD21-mAC cells. WT cells with 10 mM HU treatment for 6 hours were used as positive control. Two independent replicates.j Representative profiles of EdU/HU signals and peaks in Rad21-mAC mES cells (clone 1C4) with or without IAA treatment. The OK-seq signal was from GSE11727464. k Venn plot showing the overlap of EdU/HU peaks identified from mES cells with or without IAA treatment. l Percentage of each class of ERIZs in RAD21-depleted mES cells. m A representative profile of replication timing in RAD21-depleted mES cells. Grey shadows highlight two new EdU/HU peaks. n Density plots showing the distribution of S50 values of four classes of ERIZs identified from RAD21-depleted mES cells in the WT or RAD21-depletion replication timelines, respectively.

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Extended Data Fig. 8 Cohesin represses dormant origin firing in the middle of chromatin loops.

a Model of loop formation. b Top: CTCF degron system in K562 cells. Bottom: Western blotting of the CTCF-mAC K562 cell line (clone 5C9). Two independent replicates. c Violin plots showing distances between ERIZ centers and the nearest loop anchor in K562 cells. P-values from two-sided Mann–Whitney test are labeled on top. Red lines and numbers indicate median distances. d Distributions of distances between the neighbor loop anchor to the center of ERIZs in mES cells. Loop list of mES cells were from GSE8082065. Anchors and adjacent 100-kb regions within each loop are aligned at the center of anchors with loop regions on the right. ERIZs outside loops were excluded for analysis. Dashed lines and numbers mark median distances. e Violin plots for statistics of the distances between ERIZ centers and the nearest loop anchor in mES cells. P-values from the two-sided Mann–Whitney test are labeled on the top. Red lines and numbers indicate the median. f Knockout strategy (left) and PCR validation (right) indicating the CBE heterozygous knockout cell line. Two independent replicates. g Number of PEM-seq-captured translocations within or out of new ERIZ regions. New, new ERIZs; Ctrl., random genomic regions locate outside of ERIZs but harboring equal widths to new ERIZ regions. Mean ± SD, three biological replicates; two-sided paired t-test; *, p < 0.05; ***, p < 0.001. h Distribution of MCM5 ChIP-seq signals in the treatment (blue) or control (black) of α-amanitin within loop domains. The fold-change of MCM5 is defined as the ratio of ChIP-ed over input signals and normalized to z-scores. P-value is calculated by the two-sided Kolmogorov-Smirnov test. i Working model of cohesin-mediated loop extrusion in preserving early replication initiation near loop anchors. Distribution of MCM double hexamers undergoes regulation mediated by cohesin-driven loop extrusion, transcription, and stalls at loop boundaries, resulting in early replication adjacent to loop boundaries. Loss of cohesin induces dormant origin firing away from loop boundaries in the early S phase and results in elevated DNA damage and cancer-related gene vulnerability.

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Supplementary information

Supplementary Information

Supplementary methods for ChIP–seq, PRO-seq, replication foci immunofluorescence, fusion gene analysis and mutation predictions.

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Supplementary Table 1

Translocation hotspot gene list identified by PEM-seq in RAD21-depleted cells.

Supplementary Table 2-7

Materials, cell lines and pipelines used in this study.

Source data

Source Data Fig. 1

Uncropped gels and western blots for Fig. 1a and Extended Data Figs 1c, 2a,h,j, 7d,i and 8b,f.

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Wu, J., Liu, Y., Zhangding, Z. et al. Cohesin maintains replication timing to suppress DNA damage on cancer genes. Nat Genet 55, 1347–1358 (2023). https://doi.org/10.1038/s41588-023-01458-z

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