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Cell fitness screens reveal a conflict between LINE-1 retrotransposition and DNA replication

Abstract

LINE-1 retrotransposon overexpression is a hallmark of human cancers. We identified a colorectal cancer wherein a fast-growing tumor subclone downregulated LINE-1, prompting us to examine how LINE-1 expression affects cell growth. We find that nontransformed cells undergo a TP53-dependent growth arrest and activate interferon signaling in response to LINE-1. TP53 inhibition allows LINE-1+ cells to grow, and genome-wide-knockout screens show that these cells require replication-coupled DNA-repair pathways, replication-stress signaling and replication-fork restart factors. Our findings demonstrate that LINE-1 expression creates specific molecular vulnerabilities and reveal a retrotransposition–replication conflict that may be an important determinant of cancer growth.

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Fig. 1: Heterogeneous LINE-1 expression in colon cancer.
Fig. 2: LINE-1 inhibits cell growth in RPE by activating the p53-p21 pathway.
Fig. 3: LINE-1 activates a p53 and IFN response.
Fig. 4: Mapping LINE-1 fitness interactions in TP53-deficient cells.
Fig. 5: The Fanconi anemia pathway is essential in p53-deficient cells.
Fig. 6: LINE-1 activity induces replication stress.
Fig. 7: Model of LINE-1-induced replication stress.

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Data availability

MAGeCK-normalized sgRNA read counts from CRISPR knockout screens and RNA-seq counts and differential expression values have been deposited in the GEO database under accession number GSE119999. Source data for Figs. 2b, 5c,e,f and 6d,e are available online. Requests for resources and reagents should be directed to and will be fulfilled by K.H.B.. Select plasmids created in the Burns Lab can be accessed at Addgene (https://www.addgene.org/Kathleen_Burns/).

References

  1. Mathias, S. L., Scott, A. F., Kazazian, H. H. Jr., Boeke, J. D. & Gabriel, A. Reverse transcriptase encoded by a human transposable element. Science 254, 1808–1810 (1991).

    Article  CAS  PubMed  Google Scholar 

  2. Feng, Q., Moran, J. V., Kazazian, H. H. Jr. & Boeke, J. D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell 87, 905–916 (1996).

    Article  CAS  PubMed  Google Scholar 

  3. Hohjoh, H. & Singer, M. F. Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA. EMBO J. 15, 630–639 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Woodcock, D. M., Lawler, C. B., Linsenmeyer, M. E., Doherty, J. P. & Warren, W. D. Asymmetric methylation in the hypermethylated CpG promoter region of the human L1 retrotransposon. J. Biol. Chem. 272, 7810–7816 (1997).

    Article  CAS  PubMed  Google Scholar 

  5. Liu, N. et al. Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators. Nature 553, 228–232 (2018).

    Article  CAS  PubMed  Google Scholar 

  6. Haoudi, A., Semmes, O. J., Mason, J. M. & Cannon, R. E. Retrotransposition-competent human LINE-1 induces apoptosis in cancer cells with intact p53. J. Biomed. Biotechnol. 2004, 185–194 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Belgnaoui, S. M., Gosden, R. G., Semmes, O. J. & Haoudi, A. Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells. Cancer Cell Int. 6, 13 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Gasior, S. L., Wakeman, T. P., Xu, B. & Deininger, P. L. The human LINE-1 retrotransposon creates DNA double-strand breaks. J. Mol. Biol. 357, 1383–1393 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wallace, N. A., Belancio, V. P. & Deininger, P. L. L1 mobile element expression causes multiple types of toxicity. Gene 419, 75–81 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kines, K. J. et al. The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations. Mob. DNA 7, 8 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Luan, D. D., Korman, M. H., Jakubczak, J. L. & Eickbush, T. H. Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72, 595–605 (1993).

    Article  CAS  PubMed  Google Scholar 

  12. Rodic, N. et al. Long interspersed element-1 protein expression is a hallmark of many human cancers. Am. J. Pathol. 184, 1280–1286 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ardeljan, D., Taylor, M. S., Ting, D. T. & Burns, K. H. The human long interspersed element-1 retrotransposon: an emerging biomarker of neoplasia. Clin. Chem. 63, 816–822 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Iskow, R. C. et al. Natural mutagenesis of human genomes by endogenous retrotransposons. Cell 141, 1253–1261 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lee, E. et al. Landscape of somatic retrotransposition in human cancers. Science 337, 967–971 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Shukla, R. et al. Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma. Cell 153, 101–111 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Tubio, J. M. C. et al. Mobile DNA in cancer. Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes. Science 345, 1251343 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Rodic, N. et al. Retrotransposon insertions in the clonal evolution of pancreatic ductal adenocarcinoma. Natt. Med. 21, 1060–1064 (2015).

    Article  CAS  Google Scholar 

  19. Ewing, A. D. et al. Widespread somatic L1 retrotransposition occurs early during gastrointestinal cancer evolution. Genome Res. 25, 1536–1545 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Doucet-O’Hare, T. T. et al. LINE-1 expression and retrotransposition in Barrett’s esophagus and esophageal carcinoma. Proc. Natl Acad. Sci. USA 112, E4894–E4900 (2015).

    PubMed  PubMed Central  Google Scholar 

  21. Doucet-O’Hare, T. T. et al. Somatically acquired LINE-1 insertions in normal esophagus undergo clonal expansion in esophageal squamous cell carcinoma. Hum. Mutat. 37, 942–954 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Scott, E. C. et al. A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer. Genome Res. 26, 745–755 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Tang, Z. et al. Human transposon insertion profiling: Analysis, visualization and identification of somatic LINE-1 insertions in ovarian cancer. Proc. Natl Acad. Sci. USA 114, E733–E740 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Burns, K. H. Transposable elements in cancer. Nat. Rev. Cancer 17, 415–424 (2017).

    Article  CAS  PubMed  Google Scholar 

  25. Jung, H., Choi, J. K. & Lee, E. A. Immune signatures correlate with L1 retrotransposition in gastrointestinal cancers. Genome Res. 28, 1136–1146 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Schauer, S. N. et al. L1 retrotransposition is a common feature of mammalian hepatocarcinogenesis. Genome Res. 28, 639–653 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wylie, A. et al. p53 genes function to restrain mobile elements. Genes Dev. 30, 64–77 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kawano, K. et al. HIV-1 Vpr and p21 restrict LINE-1 mobility. Nucleic Acids Res. 46, 8454–8470 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ruscetti, M. et al. NK cell-mediated cytotoxicity contributes to tumor control by a cytostatic drug combination. Science 362, 1416–1422 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Yu, Q. et al. Type I interferon controls propagation of long interspersed element-1. J. Biol. Chem. 290, 10191–10199 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bregnard, C. et al. Upregulated LINE-1 activity in the fanconi anemia cancer susceptibility syndrome leads to spontaneous pro-inflammatory cytokine production. EBioMedicine 8, 184–194 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  32. De Cecco, M. et al. L1 drives IFN in senescent cells and promotes age-associated inflammation. Nature 566, 73–78 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Thomas, C. A. et al. Modeling of TREX1-dependent autoimmune disease using human stem cells highlights L1 accumulation as a source of neuroinflammation. Cell Stem Cell 21, 319–331 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Simon, M. et al. LINE1 derepression in aged wild-type and SIRT6-deficient mice drives inflammation. Cell Metab. 29, 871–885 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Pfeffer, L. M. The role of nuclear factor kappaB in the interferon response. J. Interferon Cytokine Res. 31, 553–559 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Dai, L., Huang, Q. & Boeke, J. D. Effect of reverse transcriptase inhibitors on LINE-1 and Ty1 reverse transcriptase activities and on LINE-1 retrotransposition. BMC Biochem. 12, 18 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Smith, G. et al. Mutations in APC, Kirsten-ras, and p53–alternative genetic pathways to colorectal cancer. Proc. Natl Acad. Sci. USA 99, 9433–9438 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Miki, Y. et al. Disruption of the APC gene by a retrotransposal insertion of L1 sequence in a colon cancer. Cancer Res. 52, 643–645 (1992).

    CAS  PubMed  Google Scholar 

  39. Goodier, J. L., Cheung, L. E. & Kazazian, H. H. Jr. Mapping the LINE1 ORF1 protein interactome reveals associated inhibitors of human retrotransposition. Nucleic Acids Res. 41, 7401–7419 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Taylor, M. S. et al. Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition. Cell 155, 1034–1048 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Moldovan, J. B. & Moran, J. V. The Zinc-finger antiviral protein ZAP inhibits LINE and Alu retrotransposition. PLoS Genet. 11, e1005121 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Taylor, M. S. et al. Dissection of affinity captured LINE-1 macromolecular complexes. Elife 7, e30094 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Tchasovnikarova, I. A. et al. GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells. Science 348, 1481–1485 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Robbez-Masson, L. et al. The HUSH complex cooperates with TRIM28 to repress young retrotransposons and new genes. Genome Res. 28, 836–845 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Adamson, B., Smogorzewska, A., Sigoillot, F. D., King, R. W. & Elledge, S. J. A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. Nat. Cell Biol. 14, 318–328 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Lubas, M. et al. Interaction profiling identifies the human nuclear exosome targeting complex. Mol. Cell 43, 624–637 (2011).

    Article  CAS  PubMed  Google Scholar 

  47. Benitez-Guijarro, M. et al. RNase H2, mutated in Aicardi–Goutieres syndrome, promotes LINE-1 retrotransposition. EMBO J. 37, e98506 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Gannon, H. S. et al. Identification of ADAR1 adenosine deaminase dependency in a subset of cancer cells. Nat. Commun. 9, 5450 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Nalepa, G. & Clapp, D. W. Fanconi anaemia and cancer: an intricate relationship. Nat. Rev. Cancer 18, 168–185 (2018).

    Article  CAS  PubMed  Google Scholar 

  50. Richardson, C. D. et al. CRISPR–Cas9 genome editing in human cells occurs via the Fanconi anemia pathway. Nat. Genet. 50, 1132–1139 (2018).

    Article  CAS  PubMed  Google Scholar 

  51. Moran, J. V. et al. High frequency retrotransposition in cultured mammalian cells. Cell 87, 917–927 (1996).

    Article  CAS  PubMed  Google Scholar 

  52. Ward, I. M. & Chen, J. Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J. Biol. Chem. 276, 47759–47762 (2001).

    Article  CAS  PubMed  Google Scholar 

  53. Her, J., Ray, C., Altshuler, J., Zheng, H. & Bunting, S. F. 53BP1 mediates ATR–Chk1 signaling and protects replication forks under conditions of replication stress. Mol. Cell Biol. 38, e00472-17 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Shigechi, T. et al. ATR–ATRIP kinase complex triggers activation of the Fanconi anemia DNA repair pathway. Cancer Res. 72, 1149–1156 (2012).

    Article  CAS  PubMed  Google Scholar 

  55. Cortez, D., Guntuku, S., Qin, J. & Elledge, S. J. ATR and ATRIP: partners in checkpoint signaling. Science 294, 1713–1716 (2001).

    Article  CAS  PubMed  Google Scholar 

  56. Cimprich, K. A. & Cortez, D. ATR: an essential regulator of genome integrity. Nat. Rev. Mol. Cell Biol. 9, 616–627 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Bhat, K. P. & Cortez, D. RPA and RAD51: fork reversal, fork protection, and genome stability. Nat. Struct. Mol. Biol. 25, 446–453 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Feeney, L. et al. RPA-mediated recruitment of the E3 ligase RFWD3 is vital for interstrand crosslink repair and human health. Mol. Cell 66, 610–621.e4 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018).

    Article  CAS  PubMed  Google Scholar 

  60. Pisanic, T. R., 2nd et al. Long interspersed element 1 retrotransposons become deregulated during the development of ovarian cancer precursor lesions. Am. J. Pathol. 189, 513–520 (2018).

  61. Zhouchunyang, X. et al. Expression of L1 retrotransposon open reading frame protein 1 (L1ORF1p) in gynecologic cancers. Hum. Pathol. 92, 39–47 (2019).

    Article  CAS  Google Scholar 

  62. Hamperl, S. & Cimprich, K. A. Conflict resolution in the genome: how transcription and replication make it work. Cell 167, 1455–1467 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Mita, P. et al. LINE-1 protein localization and functional dynamics during the cell cycle. Elife 7, e30058 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  64. Flasch, D. A. et al. Genome-wide de novo L1 retrotransposition connects endonuclease activity with replication. Cell 177, 837–851 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Sultana, T. et al. The landscape of L1 retrotransposons in the human genome is shaped by pre-insertion sequence biases and post-insertion selection. Mol. Cell 74, 555–570 (2019).

    Article  CAS  PubMed  Google Scholar 

  66. Rodriguez-Martin, B. et al. Pan-cancer analysis of whole genomes reveals driver rearrangements promoted by LINE-1 retrotransposition in human tumours. Preprint at bioRxiv https://doi.org/10.1101/179705 (2018).

  67. Mita, P. et al. BRCA1 mediated homologous recombination and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells. Nat. Struct. Mol. Biol. https://doi.org/10.1038/s41594-020-0374-z (2020).

  68. Lecona, E. & Fernandez-Capetillo, O. Targeting A. T. R. in cancer. Nat. Rev. Cancer 18, 586–595 (2018).

    Article  CAS  PubMed  Google Scholar 

  69. Chan, E. M. et al. WRN helicase is a synthetic lethal target in microsatellite unstable cancers. Nature 568, 551–556 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Ishizuka, J. J. et al. Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade. Nature 565, 43–48 (2019).

    Article  CAS  PubMed  Google Scholar 

  71. Fischer, M. Census and evaluation of p53 target genes. Oncogene 36, 3943–3956 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Fischer, M., Quaas, M., Steiner, L. & Engeland, K. The p53–p21–DREAM–CDE/CHR pathway regulates G2/M cell cycle genes. Nucleic Acids Res. 44, 164–174 (2016).

    Article  CAS  PubMed  Google Scholar 

  73. Lambrus, B. G. et al. A USP28–53BP1–p53–p21 signaling axis arrests growth after centrosome loss or prolonged mitosis. J. Cell Biol. 214, 143–153 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Lambrus, B. G. et al. p53 protects against genome instability following centriole duplication failure. J. Cell Biol. 210, 63–77 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Grabundzija, I. et al. Comparative analysis of transposable element vector systems in human cells. Mol. Ther. 18, 1200–1209 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR–Cas9. Nat. Biotechnol 34, 184–191 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  78. Hart, T. et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell 163, 1515–1526 (2015).

    Article  CAS  PubMed  Google Scholar 

  79. Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  80. Wang, J., Vasaikar, S., Shi, Z., Greer, M. & Zhang, B. WebGestalt 2017: a more comprehensive, powerful, flexible and interactive gene set enrichment analysis toolkit. Nucleic Acids Res. 45, W130–W137 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Szklarczyk, D. et al. The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res. 45, D362–D368 (2017).

    Article  CAS  PubMed  Google Scholar 

  82. Santos, A., Wernersson, R. & Jensen, L. J. Cyclebase 3.0: a multi-organism database on cell-cycle regulation and phenotypes. Nucleic Acids Res. 43, D1140–D1144 (2015).

    Article  CAS  PubMed  Google Scholar 

  83. An, W. et al. Characterization of a synthetic human LINE-1 retrotransposon ORFeus-Hs. Mob. DNA 2, 2 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Kowarz, E., Loscher, D. & Marschalek, R. Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines. Biotechnol. J. 10, 647–653 (2015).

    Article  CAS  PubMed  Google Scholar 

  85. Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Ostertag, E. M., Prak, E. T., DeBerardinis, R. J., Moran, J. V. & Kazazian, H. H. Jr. Determination of L1 retrotransposition kinetics in cultured cells. Nucleic Acids Res. 28, 1418–1423 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Wu, P. H. et al. Evolution of cellular morpho-phenotypes in cancer metastasis. Sci. Rep. 5, 18437 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Wu, P. H., Hung, S. H., Ren, T., Shih Ie, M. & Tseng, Y. Cell cycle-dependent alteration in NAC1 nuclear body dynamics and morphology. Phys. Biol. 8, 015005 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  89. Steranka, J. P. et al. Transposon insertion profiling by sequencing (TIPseq) for mapping LINE-1 insertions in the human genome. Mob. DNA 10, 8 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Human Brunello CRISPR knockout pooled library was a gift from D. Root and J. Doench (Addgene no. 73178). pSBtet-RN and pSBtet-GN were gifts from E. Kowarz (Addgene plasmid no. 60501 and 60503). pCMV(CAT)T7-SB100 was a gift from Z. Izsvak (Addgene plasmid no, 34879). JM111 was a gift from H. Kazazian. pSicoR-mCh_empty was a gift from M. Ramalho-Santos (Addgene no. 219070). LentiGuide-Puro was a gift from F. Zhang (Addgene no. 52963). We thank J. Gucwa at the Sidney Kimmel Flow Cytometry Core and the staff of the NYU Genome Technology center. We thank J. S. Bader for his statistical expertise. We thank B. A. Bari, R. M. Hughes, B. Vogelstein, J. V. Moran and H. H. Kazazian for helpful discussion and review of the manuscript. We thank J. Fairman of the Department of Art as Applied to Medicine at Johns Hopkins University School of Medicine for illustrations. This study was funded by F30CA221175 (D.A.), P50GM107632 (K.H.B., J.D.B., D.F.), U54CA210173 (P.W.) and the Sol Goldman Pancreatic Research Center (K.H.B., R.H.H.).

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Authors

Contributions

D.A. and K.H.B. conceptualized this work and wrote the manuscript. Experiments were performed by D.A., J.P.S., C.L., P.-H.W., J.S.S., M.G. and Z.L. All data were primarily analyzed by D.A. Key resources were provided by A.J.H. (RPE cells), A.S. (FANCI mAb), and M.S.T. and V.D. (deidentified colorectal cancer samples). All authors participated in manuscript revisions. Funding was acquired by D.A., J.D.B. and K.H.B.

Corresponding authors

Correspondence to Daniel Ardeljan or Kathleen H. Burns.

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The authors declare no competing interests.

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Peer review information Beth Moorefield 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 LINE-1 heterogeneity in colon cancer.

(a) Tissues collected for transposon insertion profiling by sequencing (TIP-seq) mapping of tumor-specific LINE insertions. Fresh frozen tissue was collected from two sites in the primary tumor (P1, P2) in the colon and one site in the metastatic tumor (M) in the liver. Normal tissue was collected from the liver. The liver metastasis exhibited ORF1p immunoreactivity as well (data not shown). (b) Circos plot detailing TIP-seq results and whether insertions were found in the primary (P only), metastasis (M only) or in both (P & M). In the validation process, we identified 11 3′ transduction events, 6 of which mapped to two LINE-1 sequences on Xp22.2 and one on 3q21.1 that are known to be highly active tumor alleles. As expected, the majority of this tumor’s de novo insertions were intronic or intergenic and not near known tumor suppressors or oncogenes. (c) We genotyped the insertions using hemi-specific PCR in genomic DNA obtained from dissected histology slides and compared to the allele’s presence in bulk frozen tissue used for TIP-seq. In all samples, we detected an inherited LINE-1 on 1q42.3, indicating that our PCR conditions were sufficient to genotype LINE-1 alleles. An early de novo insertion on 10q26.3 was found in all frozen tissue samples (primary and metastasis) and both CDX2high and CDX2dim slide-dissected samples. An insertion on 3q22.2 is present in the primary tumor subclonally and in the metastasis and therefore occurred before metastasis but after dedifferentiation of the CDX2dim clone. An insertion on 18q22.1 occurred late, after metastasis to the liver had occurred, since it was found in the primary CDX2high clone and not in the metastasis.

Extended Data Fig. 2 TP53 effects on LINE-1+ cell growth and retrotransposition.

(a) Demonstration of effective TP53 knockdown. RPE cells were treated with TP53 shRNA lentivirus (pDA079) or control lentivirus (pDA081). The Western blot shows the p53 response to treatment with the DNA intercalator doxorubicin (200 ng ml–1 for 24 h). (b) Left, the retrotransposition reporter assay. LINE-1 is expressed from a plasmid with an antisense eGFP in the 3′UTR that is interrupted by a sense intron. During transcription, the intron is spliced, reconstituting the coding potential of the eGFP reporter. The eGFP reporter carries with it a CMV promoter and is inserted into the genome by LINE-1. Expression of eGFP from the genome allows for fluorescence-based quantification of retrotransposition rate by flow cytometry. Right, reporter assay performed in RPE with TP53 knockdown or control ± s.e.m., n = 3 independent experiments. P value was calculated by two-sided t-test. (c) Normalized median read counts of sgRNAs targeting TP53 and CDKN1A in cells expressing either LINE-1 (navy blue) or eGFP (green) control compared to non-targeting-controls (NTC). Individual sgRNAs are indicated by circles or triangles. Results from two biological replicates are depicted.

Extended Data Fig. 3 LINE-1 RNAseq analysis.

(a) Genes regulated by cell cycle were curated from CycleBase v3.072 and differential expression values were plotted. S, G2, and M phase genes were significantly downregulated in LINE-1+ cells. Unpaired two-sided t-tests were used for statistical testing. N/A = not applicable. *p-values vs. N/A: G1 = not significant (n.s.), G1/S = 1.7e-9, S = 1.5e-2, G2 = 2.1e-13, G2/M = 5.2e-6, M = 3.4e-10. (b) Flow cytometry was used to assess cell cycle by quantifying DNA content using a PI DNA stain in Tet-On LINE-1 or Tet-On luciferase cells induced with 1 µg ml–1 doxycycline for 48 h. LINE-1+ cells with wild-type (WT) p53 accumulated in G1 phase (2n DNA copy number), whereas TP53KD resulted in more even cell cycle proportions. These data are from one experiment. (c) Relative fold-change of interferon-stimulated genes in LINE-1 compared to luciferase-expressing cells measured by RNAseq. Error bars indicate s.e.m. (d) RNAseq analysis revealed upregulation of NF-kB and several target genes in LINE-1+ cells. Error bars indicate s.e.m. (e) Differential expression of IFNB1 (right) and interferon-stimulated genes (left) in p53-knockdown cells expressing LINE-1 or luciferase for 72 h. Measured by qRT-PCR. Error bars indicate s.d., n = 3 biological replicates. * p < 0.05, ** p < 0.001. (f) Differential expression of TLR3, IFIT1, and IFIT2 with the addition of 5μM zalcitabine (ddC) or 5μM didanosine (ddI) in p53-knockdown cells expressing LINE-1 or luciferase for 72 h. Measured by qRT-PCR, n = 3 independent experiments. P values indicated within the plots.

Extended Data Fig. 4 TP53-Knockdown Screen Supplement.

(a) Behavior of non-targeting-control sgRNAs in the screen over time. Data points indicate the median sgRNA count per replicate and error bars the 95% confidence interval. (b) Behavior of TP53- and CDNK1A-targeting sgRNAs. Median values are depicted with 95% Confidence Intervals. There is no appreciable change in TP53 sgRNA representation between LINE-1+ and luciferase control cells, indicating loss of p53 function due to the shRNA. CDNK1A sgRNAs do not differ between groups as well, suggesting that CDKN1A effects are contingent on p53 function. (c) Examples of essential gene knockouts that deplete from both LINE-1+ and luciferase + cells. Median values are depicted with 95% Confidence Intervals. (d) Knockout of APC provides a growth advantage to LINE-1+ cells. Median values are depicted with 95% Confidence Intervals. (e) Knockout of the interferon alpha and beta receptor subunit 1 (IFNAR1) but not subunit 2 (IFNAR2) provides a growth advantage in LINE-1+ cells. Median values are depicted with 95% Confidence Intervals.

Extended Data Fig. 5 HUSH knockout is synthetic lethal due to derepression of the LINE-1 transgene.

(a) Gene screen ranks by Zs scores. HUSH genes are in blue. (b) HUSH complex sgRNA performance during the screen. All knockouts drop out early from LINE-1+ cells (red) and do not affect growth of luciferase+ cells (black). Median values are depicted with 95% Confidence Intervals. (c) 12 d clonogenic growth assay in cells expressing LINE-1 (doxycycline-induced) with targeted knockouts of HUSH components compared to non-targeting-control (NTC). n = 3 independent experiments. Error bars indicate ± s.e.m. P values calculated by one-sided t-test. (d) Western blot comparing ORF1p and ORF2p expression in HUSH knockout cells or non-target-controls (NTC) that have not been treated with doxycycline compared to NTC with 24 h of 1 µg ml–1 doxycycline treatment. ORF1p and ORF2p expression are only detected in NTC-treated cells with doxycycline added to the culture media. The double banding pattern for ORF1p is consistently seen with codon-optimized LINE-1. (e) Western blot comparing ORF1p and ORF2p expression 24 h after 1 µg ml–1 doxycycline treatment in HUSH knockouts compared to NTC. The ORF2p antibody cannot distinguish between endogenous or transgenic LINE-1 expression. (f) qRT-PCR analysis of LINE-1 transgene expression in HUSH knockouts compared to NTC (induced with 1 µg ml–1 doxycycline). Because the LINE-1 transgene is codon-optimized, qRT-PCR is specific for the transgene and does not amplify endogenous LINE-1 sequences. *p < 0.001. (g) Linear regression plot of LINE-1 transgene expression and ORF1p and ORF2p expression in HUSH knockouts compared to NTC. Shaded area indicates 95% confidence interval for regression line. Both ORF1p and ORF2p increase in expression with higher transgene mRNA expression, although the increase in ORF1p is minimal compared to that observed with ORF2p. (h) Heatmap of immunofluorescence imaging depicting the proportion of cells expressing ORF1p and ORF2p at different levels in HEK293T cells expressing Tet-On LINE-1 (pDA055) at increasing doses of doxycycline.

Extended Data Fig. 6 RNA processing gene knockouts sensitize cells to LINE-1.

(a) StringDB network plot of the 81 mRNA processing genes identified by this screen. Edges indicate known protein-protein interactions. This network is enriched for spliceosome machinery (green nodes). (b) Screen behavior of significant genes belonging to the spliceosome KEGG GO term. Median sgRNA counts are depicted with 95% Confidence Intervals. (c) Clonogenic assay (12 d) comparing growth of luciferase+ and LINE-1+ cells (induced with 1 µg ml–1 doxycycline) treated with 1 nM pladienolide B (PLA-B) or vehicle (DMSO). n = 3 independent experiments. Error bars indicate s.e.m. P value calculated by unpaired one-sided t-test. (d) Behavior of nuclear exosome complex genes in the screen. Median values are depicted with 95% Confidence Intervals. (e) Behavior of RNASEH2 component sgRNAs in the screen. Median values are depicted with 95% Confidence Intervals. (f) Behavior of ADAR1 sgRNAs in the screen. Median values are depicted with 95% Confidence Intervals.

Extended Data Fig. 7 The Fanconi Anemia Pathway is required for growth of LINE-1+ cells.

(a) Behavior of sgRNAs targeting Fanconi Anemia pathway genes in the screen. Median values are depicted with 95% Confidence Intervals. (b) Western blot of DNA damage marker γH2A.X in chromatin-bound protein fractions of LINE-1+ cells with or without perturbations to the FA pathway. H3 was used as loading control. γH2A.X levels were quantified and graphed relative to NTC-treated, LINE-1+ cells. (c) Clonogenic assay (10 d). TP53KD cells constitutively expressing Cas9 are treated with lentivirus encoding non-targeting-control (NTC) or FANCD2 sgRNA and then transfected with eGFP (pDA083) or the native LINE-1 sequence L1RP (pDA077). Left, representative images of colonies. Scale bar = 1 cm. Right, data are presented as the rate of LINE-1 per 100 eGFP colonies ± s.d. to control for transfection efficiency across samples, n = 3 independent experiments. P value obtained by unpaired two-sided t-test. (d) Quantification of FANCD2 foci in G1 and G2 phase (EdU-) HeLa cells. Number of cells per group: G1 untreated (n = 104), G1 HU (n = 352), G1 wildtype LINE-1 (n = 186), G1 RT (D702Y) (n = 138), G2 untreated (n = 60), G2 HU (n = 58), G2 wildtype LINE-1 (n = 42), G2 RT (D702Y) (n = 32). Two-sided t-tests were used for statistical comparisons. HU = hydroxyurea. RT = reverse transcriptase. ns = not significant.

Extended Data Fig. 8 Viability assays with LINE-1 mutants.

(a) Tet-On constructs for wild-type and mutant LINE-1 expression. (b) Viability of HEK293T cells after 4 days expressing wild-type or a mutant at increasing doxycycline doses. A multivariate ANOVA (Viability ~ ORF2 * doxycycline) was performed in R to calculate p values for ORF2 mutant status and doxycycline dose. Tests of viability differences among ORF2 mutants were further performed using two-sided t-tests at the 1000 ng ml–1 doxycycline dose. N = 6 replicates per doxycycline dose. (c) Western blot of ORF1p and ORF2p 24 hours after inducing protein expression with 1000 ng ml–1 doxycycline.

Supplementary information

Supplementary Information

Supplementary Tables 1, 4, 5 and 6 and Supplementary Methods.

Reporting Summary

Supplementary Table 2

Ranking of KD screen results. Columns include the gene symbol, fitness interaction (rescue, synthetic lethal). Ranks are indicated for genome-wide significant genes that demonstrate either rescue or synthetic lethal interactions.

Supplementary Table 3

Analysis of fitness interactions among previously known LINE-1 interactors (modify retrotransposition and/or physically bind LINE-1 proteins).

Supplementary Data 1

Unprocessed Western blots for Figs. 2b, 5c,e,f and 6d,e.

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Ardeljan, D., Steranka, J.P., Liu, C. et al. Cell fitness screens reveal a conflict between LINE-1 retrotransposition and DNA replication. Nat Struct Mol Biol 27, 168–178 (2020). https://doi.org/10.1038/s41594-020-0372-1

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