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Chromosomal fragile site breakage by EBV-encoded EBNA1 at clustered repeats

Nature volume 616, pages 504–509 (2023)Cite this article

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Abstract

Epstein–Barr virus (EBV) is an oncogenic herpesvirus associated with several cancers of lymphocytic and epithelial origin1,2,3. EBV encodes EBNA1, which binds to a cluster of 20 copies of an 18-base-pair palindromic sequence in the EBV genome4,5,6. EBNA1 also associates with host chromosomes at non-sequence-specific sites7, thereby enabling viral persistence. Here we show that the sequence-specific DNA-binding domain of EBNA1 binds to a cluster of tandemly repeated copies of an EBV-like, 18-base-pair imperfect palindromic sequence encompassing a region of about 21 kilobases at human chromosome 11q23. In situ visualization of the repetitive EBNA1-binding site reveals aberrant structures on mitotic chromosomes characteristic of inherently fragile DNA. We demonstrate that increasing levels of EBNA1 binding trigger dose-dependent breakage at 11q23, producing a fusogenic centromere-containing fragment and an acentric distal fragment, with both mis-segregated into micronuclei in the next cell cycles. In cells latently infected with EBV, elevating EBNA1 abundance by as little as twofold was sufficient to trigger breakage at 11q23. Examination of whole-genome sequencing of EBV-associated nasopharyngeal carcinomas revealed that structural variants are highly enriched on chromosome 11. Presence of EBV is also shown to be associated with an enrichment of chromosome 11 rearrangements across 2,439 tumours from 38 cancer types. Our results identify a previously unappreciated link between EBV and genomic instability, wherein EBNA1-induced breakage at 11q23 triggers acquisition of structural variations in chromosome 11.

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Fig. 1: Sequence-specific DNA-binding domain mediates enrichment of EBNA1 at a single genomic locus in the endogenous human genome.
Fig. 2: A cluster of regularly interspersed copies of an 18-bp imperfect palindromic sequence at 11q23 mediates sequence-specific enrichment of EBNA1.
Fig. 3: Repetitive DNA containing the 18-bp imperfect palindromes forms aberrant structures characteristic of fragile DNA and undergoes breakage and micronucleation after induced EBNA1 expression.
Fig. 4: Elevated abundance of EBNA1 triggers 11q23 breakage and micronucleation in cells latently infected with EBV.

Data availability

The PacBio sequence datasets used in this study are publicly available and can be found at the following ftp links: HG003, ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/AshkenazimTrio/HG003_NA24149_father/PacBio_MtSinai_NIST/PacBio_minimap2_bam/HG003_PacBio_GRCh38.bam; HG004, ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/AshkenazimTrio/HG004_NA24143_mother/PacBio_MtSinai_NIST/PacBio_minimap2_bam/HG004_PacBio_GRCh38.bam; HG006, ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/ChineseTrio/HG006_NA24694-huCA017E_father/PacBio_MtSinai/PacBio_minimap2_bam/HG006_PacBio_GRCh38.bam; and HG007, ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/ChineseTrio/HG007_NA24695-hu38168_mother/PacBio_MtSinai/PacBio_minimap2_bam/HG007_PacBio_GRCh38.bam. All datasets used for the structural variation (SV) analyses are publicly available. For the 78 NPC samples, the SV calls are available through Supplementary Data Table 6 in ref. 46 and Supplementary Data Table 5 in ref. 45. For the PCAWG samples, the consensus SV calls were downloaded from the ICGC data portal (https://dcc.icgc.org/releases/PCAWG/consensus_sv). The following two files, which are both open access, were downloaded and used for the downstream SV analyses: final_consensus_sv_bedpe_passonly.icgc.public.tgz (https://dcc.icgc.org/api/v1/download?fn=/PCAWG/consensus_sv/final_consensus_sv_bedpe_passonly.icgc.public.tgz) and final_consensus_sv_bedpe_passonly.tcga.public.tgz (https://dcc.icgc.org/api/v1/download?fn=/PCAWG/consensus_sv/final_consensus_sv_bedpe_passonly.tcga.public.tgz). Source data are provided with this paper.

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Acknowledgements

This work was financially supported by grants from the US National Institutes of Health (R35 GM122476 to D.W.C. and R01ES030993-01A1, R01ES032547 and R01CA269919 to L.B.A.). J.S.Z.L. is supported by a postdoctoral fellowship from the Damon Runyon Cancer Research Foundation. L.B.A. is supported by a Packard Fellowship for Science and Engineering. D.W.C. receives salary support from the Ludwig Institute for Cancer Research. We thank S. P. Nandi for help with sequencing. The computational analyses reported in this manuscript have utilized the Triton Shared Computing Cluster at the San Diego Supercomputer Center of UC San Diego.

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Author notes

  1. Dong Hyun Kim

    Present address: Oncology Research Unit, Pfizer Inc., San Diego, CA, USA

Authors and Affiliations

  1. Ludwig Cancer Research, UC San Diego, La Jolla, CA, USA

    Julia Su Zhou Li, Dong Hyun Kim & Don W. Cleveland

  2. Department of Cellular and Molecular Medicine, UC San Diego, La Jolla, CA, USA

    Julia Su Zhou Li, Ammal Abbasi, Dong Hyun Kim, Ludmil B. Alexandrov & Don W. Cleveland

  3. Moores Cancer Center, UC San Diego, La Jolla, CA, USA

    Ammal Abbasi, Scott M. Lippman, Ludmil B. Alexandrov & Don W. Cleveland

  4. Department of Bioengineering, UC San Diego, La Jolla, CA, USA

    Ammal Abbasi & Ludmil B. Alexandrov

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Contributions

J.S.Z.L. designed and carried out the experiments. J.S.Z.L. and D.W.C. analyzed data. A.A. carried out the bioinformatics and cancer genomics analyses. D.H.K. carried out microscopy of live cells. S.M.L. provided expertise in NPC. L.B.A. oversaw the bioinformatics and cancer genomics analyses. J.S.Z.L. and D.W.C. wrote the manuscript with input from all authors.

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Correspondence to Julia Su Zhou Li or Don W. Cleveland.

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Competing interests

L.B.A. is a compensated consultant and has equity interest in io9, LLC. His spouse is an employee of Biotheranostics, Inc. L.B.A. is an inventor of US patent 10,776,718 and also declares US provisional patent applications with serial numbers 63/289,601, 63/269,033 and 63/412,835. L.B.A. and A.A. declare US provisional patent applications with serial numbers 63/366,392 and 63/367,846. S.M.L. is a co-founder of io9, LLC and declares a provisional patent application for Methods and Biomarkers in Cancer (US provisional application serial number 63/179,215). All other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 EBNA1 localization is enriched at a single genomic locus in the endogenous human genome.

(a) Schematic representation of the Flag-tagged allele of full length EBNA1. (b) anti-EBNA1 (Santa Cruz sc-81581) immunoblot of the indicated cell lines. Immunoblots were repeated 3 times with similar results. (c) Representative anti-Flag immunofluorescence images of Flag-EBNA1FL foci in the indicated cell lines as quantified in Figure 1d from three independent experiments. TK6 cells are established from B-lymphocytes immortalized with EBV. Raji cells and Daudi cells are derived from EBV-infected Burkitt’s Lymphoma. RPEs are hTERT-immortalized primary retinal pigment epithelial cells. DLD1s are derived from colon cancer. HeLas are derived from cervical cancer. U2OS cells are derived from osteosarcoma. MEFs are mouse embryonic fibroblasts. (d) Schematic representation of sequence-specific enrichment of dCas9 (yellow) in complex with a single sgRNA (blue) targeting a sequence (grey) clustered at a repetitive site.

Extended Data Fig. 2 The human genome contains a cluster of EBV-like 18bp imperfect palindromic sequences at 11q23.

Number of copies (y-axis) of 18bp imperfect palindromic sequences with up to 6 variant and 2 asymmetric nucleotides as plotted per 0.1 mega-base region across the human reference genome GRCh38 (a) and number of copies of 18bp imperfect palindromic sequences with up to 5 variant and 2 asymmetric nucleotides as plotted per 10 kilo-base region at 11q23 region for long-read sequenced genomes of two individuals of Ashkenazi descent (b) and two individuals of Chinese descent (c).

Extended Data Fig. 3 11q23 repetitive site containing 18bp imperfect palindromes is evolutionarily conserved amongst the Great Apes.

(a) Pairwise sequence alignment of the 11q23 repetitive site (chr11:114,604,212-114,625,620) in the human genome against 30 mammalian genomes. Vertical lines with darker grayscale color represent higher alignment quality, whereas a horizontal single or double line represents a lack of sequence homology between the human and the aligned genome. (b) Comparison of the consensus motif logos of the repeat sequence from the human, chimpanzee, or EBV genome showing that while the 18bp palindromes are conserved amongst all three species, the interspersed sequences are distinct between the primates and EBV.

Extended Data Fig. 4 CRISPR labeling and CRISPR cutting approaches demonstrating localization of EBNA1 at the cluster of 18bp imperfect palindromic sequences at 11q23.

(a) Schematic of the consensus sequence of the 42bp repeat unit showing the sgRNA sequence (sgPalindrome) used in the CRISPR labeling approach. (b) Schematic of the consensus sequence of the 42bp repeat unit showing the sgRNA sequences (sgNon-Targeting and sgPalindrome) used in the CRISPR cutting approach. (c) Representative anti-Flag (in green) and anti-Myc (in red) IF of U2OS cells stably expressing Flag-dCas9 directed by the indicated sgRNAs with or without colocalization with Myc-EBNA1DBD as quantified in (d). (e) Representative anti-Flag IF of Flag-EBNA1DBD in U2OS cells treated with Cas9 and the indicated sgRNAs as quantified in (f).

Extended Data Fig. 5 Repetitive DNA at 3q29 forms aberrant structures on mitotic chromosomes.

(a) Schematic representation of the oligo-FISH approach using a fluorescently labeled oligo (in green) to target a portion of the repeat sequence clustered at 3q29. (b) Representative oligo-FISH (c) Zoomed-in representative oligo-FISH showing normal appearance (two dots) or fragile appearance (single dot, multiple dots) on mitotic chromosomes in the indicated cell lines as quantified in (d) with or without 0.2uM aphidicolin treatment for 24 h.

Extended Data Fig. 6 Dox-inducible expression of EBNA1DBD in DLD1 cells.

(a) Schematic of the dox-inducible Flag-EBNA1DBD allele encoding the indicated amino acid residues of EBNA1 stably integrated in DLD1 cells. (b) Anti-Flag and anti-GADPDH western blots showing expression of Flag-EBNA1DBD induced with 200ng/ml doxycycline at the indicated days post induction. For gel source data, see Supplementary Fig. 1. (c) Representative anti-Flag immunofluorescence images showing nuclear foci on day 1 and appearance of micronuclear foci on Day 4 as quantified in (d-e). A total of 830 cells were quantified. Data are presented as bars representing mean values from two independent experiments.

Extended Data Fig. 7 Live imaging of the inheritance of EBNA1 foci through cell division.

(a) Schematic of the clover-tagged allele of EBNA1 used for live-imaging at 10min intervals for 48 h starting at day 3 following transduced expression in Dld1s or HeLas. Still images capturing either symmetric inheritance (b) or asymmetric inheritance of EBNA1 foci into primary nuclei (c-d) or micronuclei (e) of daughter cells as quantified in (f). 64 mitotic events were scored for DLD1 cells, 24 mitotic events for HeLa cells.

Extended Data Fig. 8 EBNA1-induced breakage of chromosome 11 produces an ATM-containing proximal fragment that undergoes micronucleation.

(a) Schematic of dual-colored FISH using whole chromosome 11 probe (green) and probe against ATM (red). (b) Representative FISH images of DLD1 cells showing intact chromosome 11’s (Day 0) and broken chromosome 11 fragments containing ATM proximal to the site of breakage at day 1 to 4 post dox induction. (c) Data represent the percentage of spreads with broken chromosome 11 from two independent experiments. 62 mitotic spreads were quantified on Day 0, 53 on Day 1, 48 on Day2, 58 on Day 3, and 61 on Day 4. (d) Representative FISH images showing micronucleation of chromosome 11 fragments with or without ATM foci. (f) Stacked bars represent the percentage of cells with chromosome 11 micronuclei with the indicated number of ATM foci from two independent experiments. 310 cells were quantified on Day 0, 220 cells on Day 1, 190 on Day 2, 214 on Day 3, and 198 on Day 4. (e) Representative FISH images showing ATM foci in primary nuclei. (g) Stacked bars represent the percentage of primary nuclei with the indicated number of ATM foci from two independent experiments. 800 cells were quantified on Day 0, 900 cells on Day 1, 750 on Day 2, 950 on Day 3, and 780 on Day 4.

Extended Data Fig. 9 EBNA1-induced breakage of chromosome 11 produces an MLL-containing distal fragment that undergoes micronucleation.

(a) Schematic of dual-colored FISH using whole chromosome 11 probe (green) and probe against MLL (red). (b) Representative FISH images of DLD1 cells showing intact chromosome 11’s (Day 0) and broken chromosome 11 fragments containing MLL distal to the site of breakage at day 1 to 4 post dox induction. (c) Data represent the percentage of spreads with broken chromosome 11 from two independent experiments. 71 mitotic spreads were quantified on Day 0, 49 on Day 1, 50 on Day2, 53 on Day 3, and 71 on Day 4. (d) Representative FISH images showing micronucleation of chromosome 11 fragments with or without MLL foci. (f) Stacked bars represent the percentage of cells with chromosome 11 micronuclei with the indicated number of MLL foci from two independent experiments. 400 cells were quantified on Day 0, 210 cells on Day 1, 235 on Day 2, 190 on Day 3, and 180 on Day 4. (e) Representative FISH images showing MLL foci in primary nuclei. (g) Stacked bars represent the percentage of primary nuclei with the indicated number of MLL foci. 950 cells were quantified on Day 0, 880 cells on Day 1, 980 on Day 2, 1100 on Day 3, and 950 on Day 4.

Extended Data Fig. 10 EBNA1-induced micronucleation of chromosome 11 includes the p-arm.

(a) Schematic of dual-colored FISH using whole chromosome 11 probe (green) and probe against 11p15 (red) on the p-arm. (b) Representative FISH images of DLD1 cells showing 11p15 on the p-arm of either intact chromosome 11’s (Day 0) or broken chromosome 11 fragments (Day 4) upon induced expression of EBNA1. (c) Representative FISH images showing that EBNA1-induced micronucleation of chromosome 11 (Day 4) includes 11p15 on the p-arm. (d) Stacked bars represent the percentage of cells with chromosome 11 micronuclei with or without 11p15. 400 cells were quantified on Day 0, 310 cells on Day 2 and 380 cells on Day 4.

Extended Data Fig. 11 EBNA1-induced breakage at 11q23 is dependent on the DNA binding domain.

(a) Schematic of the Flag-tagged alleles of EBNA1 expressed in HeLa cell that harbor two copies of chromosome 11 and a derivative chromosome 11 lacking 11q23. (b) anti-Flag immunoblot of cells expressing the indicated alleles. For gel source data, see Supplementary Fig. 1. (c) Anti-Flag immunofluorescence showing localization of the indicated EBNA1 alleles. Numbers indicate the percentage of nuclei with flag foci. (d) Representative dual colored FISH showing chromosome 11’s in HeLa cells, including two copies of chromosome 11 (in green) harboring MLL (in red) and one derivative chromosome 11 lacking MLL. White arrows indicate the site of breakage proximal to MLL as quantified in (f). 48 mitotic spreads were quantified for non-transduced HeLas, 65 for HeLas expressing EBNA1FL, 98 for HeLas expressing EBNA1ΔGAGR, 40 for HeLas expressing EBNA1ΔDBD, 69 for HeLas expressing EBNA1DBD, and 82 for HeLas expressing EBNA1DBDΔmid. Data are presented as bars representing mean values from two independent experiments. (e) Representative FISH images of interphase nuclei showing micronucleation of chromosome 11 fragments containing MLL as quantified in (g). 210 cells were quantified for non-transduced HeLas, 150 for HeLas expressing EBNA1FL, 195 for HeLas expressing EBNA1ΔGAGR, 170 for HeLas expressing EBNA1ΔDBD, 230 for HeLas expressing EBNA1DBD, and 210 for HeLas expressing EBNA1DBDΔmid. Data are presented as bars representing mean values from two independent experiments.

Extended Data Fig. 12 EBNA1 abundance has a dose-dependent effect on the frequency of breakage at 11q23 in DLD1 cells.

(a) anti-Flag immunoblot of dox-induced Flag-EBNA1DBD expression levels at the indicated doxycycline concentrations as quantified in (b). For gel source data, see Supplementary Fig. 1. Data are presented as mean values +/- SEM from three independent experiments. (c) Dual colored FISH of mitotic chromosome 11 (in green) and MLL (in red) showing breakage proximal to MLL at day two following induction with the indicated doxycycline concentrations as quantified in (d). A total of 786 mitotic spreads were counted. Data are presented as mean values +/- SEM from three independent experiments. (e) Representative anti-Flag immunofluorescence images of Flag-EBNA1DBD expression at 20ng/ml or 200ng/ml. (f) Percentage of cells with flag foci. (g) Intensity of flag foci of 90 signals are quantified with horizontal bars representing the mean values.

Extended Data Fig. 13 Cell lines latently infected with EBV express EBNA1 levels slightly below the levels required to induce 11q23 breakage.

(a) Schematic of the antibodies used to quantify EBNA1. (b) Coomassie blue staining of recombinant BSA next to recombinant EBNA1 (Abcam 138345). (c) anti-EBNA1 (Santa Cruz sc-81581) immunoblot of recombinant EBNA1 next to whole cell lysates of the indicated cell lines. Anti-GAPDH immunoblot shows variation amongst the EBV-infected cell lines when loading the same number of cells. (d) anti-EBNA1 immunoblot of EBNA1 in the indicated EBV-infected cell lines next to HeLa cells expressing Flag-EBNA1ΔGAGR loaded at the indicated dilutions. (e) Bar graph represents signal intensity values normalized to number of cells loaded showing that the 1x Flag-EBNA1ΔGAGR in HeLa cells is expressed at 14-fold relative to baseline EBNA1 in Daudi cells and 8-fold in Raji cells and Tk6 cells. Immunoblots were repeated 3 times where S.D. values were below +/- 10%. (f-g) Anti-Flag immunoblot of HeLa cells, DLD1 cells, Raji cells, and Tk6 cells expressing the indicated Flag-tagged EBNA1 alleles. Bar graph represents fold relative to latent EBNA1 in Raji or Tk6 cells, calculated using the Flag-EBNA1ΔGAGR allele as 8-fold. Immunoblots were each repeated 3 times, S.D. values were below +/- 10%. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 14 Elevated levels of EBNA1 trigger breakage at 11q23 in cells latently infected with EBV.

(a) anti-EBNA1 immunoblot verifies the presence of latent EBV genomes in Raji and Tk6 cells where baseline endogenous EBNA1 levels are largely unaffected by induced expression of Flag-EBNA1DBD. Anti-Flag immunoblot shows doxycycline-inducible expression of Flag-EBNA1DBD. For gel source data, see Supplementary Fig. 1. Numbers (in red) indicate fold relative to baseline endogenous EBNA1 levels, calculated using Flag-EBNA1DBD levels in DLD1 cells previously determined to be 2, 8, 15, 20, and 24-fold. Immunoblots were repeated 3 times, S.D. Values were below +/- 10%. Representative dual-colored FISH images of chromosome 11 (in green) and MLL (in red) showing 11q23 breakage proximal to MLL (indicated by the white arrows) in either (b) Raji cells or Tk6 cells (c) as quantified in (d) following two days of treatment with the indicated dox concentrations. A total of 228 mitotic spreads were quantified for Raji cells. A total of 284 spreads were quantified for Tk6 cells. Data are presented as horizontal bars representing means from two independent experiments.

Extended Data Fig. 15 Prevalence of chromosome 11 structural variations across EBV-infected Nasopharyngeal Carcinoma (NPC) and Pan-Cancer Analysis of Whole Genomes (PCAWG).

Average number of structural variations (SV) per Mb (a) and translocations (b) of n = 78 EBV-infected NPC genomes. Bars represent the minimum, median and the maximum values observed in each violin plot (Two-sided Mann-Whitney U rank test, P-value is indicated, not corrected for multiple hypothesis testing). (c-d) Schematic of clustered structural variations (deletions, inversions, duplications, inverted translocations) on chromosome 11 in NPC genomes. (e-f) Proportion of samples with or without detectable EBV that harbor at least two chromosome 11 structural variations (Two-sided Fisher’s exact test, p-value is indicated, not corrected for multiple hypothesis testing; corrected p-values are 0.08 for pan-cancer and 0.05 for Head-SCC). (g-h) Proportion of structural variations (SV) that are on chromosome 11 in samples with or without detectable EBV. Bars represent the minimum, median and the maximum values observed in each violin plot (Two-sided Mann-Whitney U rank test. P-value is indicated, not corrected for multiple hypothesis testing).

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Supplementary Fig. 1

Uncropped western blots and protein gels. The black box indicates the area cropped for the corresponding figure.

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Li, J.S.Z., Abbasi, A., Kim, D.H. et al. Chromosomal fragile site breakage by EBV-encoded EBNA1 at clustered repeats. Nature 616, 504–509 (2023). https://doi.org/10.1038/s41586-023-05923-x

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