Abstract
Long interspersed element-1 (LINE-1, or L1) is the only autonomous retrotransposon that is active in human cells. Different host factors have been shown to influence L1 mobility; however, systematic analyses of these factors are limited. Here, we developed a high-throughput microscopy-based retrotransposition assay that identified the double-stranded break (DSB) repair and Fanconi anemia (FA) factors active in the S/G2 phase as potent inhibitors and regulators of L1 activity. In particular, BRCA1, an E3 ubiquitin ligase with a key role in several DNA repair pathways, directly affects L1 retrotransposition frequency and structure and plays a distinct role in controlling L1 ORF2 protein translation through L1 mRNA binding. These results suggest the existence of a ‘battleground’ at the DNA replication fork between homologous recombination (HR) factors and L1 retrotransposons and reveal a potential role for L1 in the genotypic evolution of tumors characterized by BRCA1 and HR repair deficiencies.
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All the raw data of the primary and secondary screens are provided in the Supplementary Tables.
References
Burns, K. H. & Boeke, J. D. Human transposon tectonics. Cell 149, 740–752 (2012).
Huang, C. R. L., Burns, K. H. & Boeke, J. D. Active transposition in genomes. Annu. Rev. Genet. 46, 651–675 (2012).
Sassaman, D. M. et al. Many human L1 elements are capable of retrotransposition. Nat. Genet. 16, 37–43 (1997).
Martin, S. L. & Bushman, F. D. Nucleic acid chaperone activity of the ORF1 protein from the mouse LINE-1 retrotransposon. Mol. Cell Biol. 21, 467–475 (2001).
Cost, G. J., Feng, Q., Jacquier, A. & Boeke, J. D. Human L1 element target-primed reverse transcription in vitro. EMBO J. 21, 5899–5910 (2002).
Feng, Q., Moran, J. V., Kazazian, H. H. & Boeke, J. D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell 87, 905–916 (1996).
Mita, P. et al. LINE-1 protein localization and functional dynamics during the cell cycle. eLife 7, 210–210 (2018).
Alisch, R. S., Garcia-Perez, J. L., Muotri, A. R., Gage, F. H. & Moran, J. V. Unconventional translation of mammalian LINE-1 retrotransposons. Genes Dev. 20, 210–224 (2006).
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).
Jurka, J. & Klonowski, P. Integration of retroposable elements in mammals: selection of target sites. J. Mol. Evol. 43, 685–689 (1996).
Gilbert, N., Lutz, S., Morrish, T. A. & Moran, J. V. Multiple fates of L1 retrotransposition intermediates in cultured human cells. Mol. Cell. Biol. 25, 7780–7795 (2005).
Moran, J. V. et al. High frequency retrotransposition in cultured mammalian cells. Cell 87, 917–927 (1996).
Symer, D. E. et al. Human l1 retrotransposition is associated with genetic instability in vivo. Cell 110, 327–338 (2002).
Goodier, J. L. Restricting retrotransposons: a review. Mob. DNA 7, 16–16 (2016).
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).
Coufal, N. G. et al. Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells. PNAS 108, 20382–20387 (2011).
Suzuki, J. et al. Genetic evidence that the non-homologous end-joining repair pathway is involved in LINE retrotransposition. PLoS Genet. 5, e1000461–e1000461 (2009).
Morrish, T. A. et al. DNA repair mediated by endonuclease-independent LINE-1 retrotransposition. Nat. Genet. 31, 159–165 (2002).
Servant, G. et al. The nucleotide excision repair pathway limits L1 retrotransposition. Genetics 205, 139–153 (2017).
Brégnard, 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).
Liu, N. et al. Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators. Nature 553, 228–232 (2017).
Hampf, H. & Gossen, M. Promoter crosstalk effects on gene expression. J. Mol. Biol. 365, 911–920 (2007).
Taylor, M. S. et al. Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition. Cell 155, 1034–1048 (2013).
An, W. et al. Characterization of a synthetic human LINE-1 retrotransposon ORFeus -Hs. Mob. DNA 2, 1–1 (2011).
Szklarczyk, D. et al. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 47, D607–D613 (2019).
Ardeljan, D. et al. Cell fitness screens reveal a conflict between LINE-1 retrotransposition and DNA replication. Nat. Struct. Mol. Biol. https://doi.org/10.1038/s41594-020-0372-1 (2020).
Krejci, L., Altmannova, V., Spirek, M. & Zhao, X. Homologous recombination and its regulation. Nucleic Acids Res. 40, 5795–5818 (2012).
Li, X. & Heyer, W.-D. Homologous recombination in DNA repair and DNA damage tolerance. Cell Res. 18, 99–113 (2008).
Ceccaldi, R., Sarangi, P. & D’Andrea, A. D. The Fanconi anaemia pathway: new players and new functions. Nat. Rev. Mol. Cell 17, 337–349 (2016).
Dungrawala, H. & Cortez, D. Purification of proteins on newly synthesized DNA using iPOND. Methods Mol. Biol. 1228, 123–131 (2015).
Michl, J., Zimmer, J. & Tarsounas, M. Interplay between Fanconi anemia and homologous recombination pathways in genome integrity. EMBO J. 35, 909–923 (2016).
Schlacher, K., Wu, H. & Jasin, M. A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2. Cancer Cell 22, 106–116 (2012).
DelloRusso, C. et al. Functional characterization of a novel BRCA1-null ovarian cancer cell line in response to ionizing radiation. Mol. Cancer Res. 5, 35–45 (2007).
Morrish, T. A. et al. Endonuclease-independent LINE-1 retrotransposition at mammalian telomeres. Nature 446, 208–212 (2007).
Ruffner, H., Joazeiro, C. A., Hemmati, D., Hunter, T. & Verma, I. M. Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity. Proc. Natl Acad. Sci. USA 98, 5134–5139 (2001).
Shen, S. X. et al. A targeted disruption of the murine Brca1 gene causes gamma-irradiation hypersensitivity and genetic instability. Oncogene 17, 3115–3124 (1998).
Huertas, P. & Jackson, S. P. Human CtIP mediates cell cycle control of DNA end resection and double strand break repair. J. Biol. Chem. 284, 9558–9565 (2009).
You, Z. & Bailis, J. M. DNA damage and decisions: CtIP coordinates DNA repair and cell cycle checkpoints. Trends Cell Biol. 20, 402–409 (2010).
Przetocka, S. et al. CtIP-mediated fork protection synergizes with BRCA1 to suppress genomic instability upon DNA replication stress. Mol. Cell 72, 568–582 e6 (2018).
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. https://doi.org/10.1128/MCB.00472-17 (2018).
Villa, M., Bonetti, D., Carraro, M. & Longhese, M. P. Rad9/53BP1 protects stalled replication forks from degradation in Mec1/ATR-defective cells. EMBO Rep. 19, 351–367 (2018).
Bunting, S. F. et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243–254 (2010).
Polato, F. et al. CtIP-mediated resection is essential for viability and can operate independently of BRCA1. J. Exp. Med. 211, 1027–1036 (2014).
Beyer, A., Bandyopadhyay, S. & Ideker, T. Integrating physical and genetic maps: from genomes to interaction networks. Nat. Rev. Genet. 8, 699–710 (2007).
Higgs, M. R. et al. BOD1L is required to suppress deleterious resection of stressed replication forks. Mol. Cell 59, 462–477 (2015).
Xu, S. et al. Abro1 maintains genome stability and limits replication stress by protecting replication fork stability. Genes Dev. 31, 1469–1482 (2017).
Poole, L. A. & Cortez, D. Functions of SMARCAL1, ZRANB3, and HLTF in maintaining genome stability. Crit. Rev. Biochem. Mol. Biol. 52, 696–714 (2017).
Fungtammasan, A., Walsh, E., Chiaromonte, F., Eckert, K. A. & Makova, K. D. A genome-wide analysis of common fragile sites: What features determine chromosomal instability in the human genome? Genome Res. 22, 993–1005 (2012).
Arlt, M. F. et al. BRCA1 is required for common-fragile-site stability via its G2/M checkpoint function. Mol. Cell. Biol. 24, 6701–6709 (2004).
Gilbert, N., Lutz-Prigge, S. & Moran, J. V. Genomic deletions created upon LINE-1 retrotransposition. Cell 110, 315–325 (2002).
Jensen, S., Gassama, M. P. & Heidmann, T. Retransposition of the drosophilia LINE I element can induce deletion in the target DNA: A simple model also accounting for the variability of the normally observed target site duplications. Biochem. Biophys. Res. Commun. 202, 111–119 (1994).
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).
Dacheux, E. et al. BRCA1-dependent translational regulation in breast cancer cells. PLoS One 8, e67313–e67313 (2013).
Mao, Z., Bozzella, M., Seluanov, A. & Gorbunova, V. DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells. Cell Cycle 7, 2902–2906 (2014).
Dai, L., Taylor, M. S., O’Donnell, K. A. & Boeke, J. D. Poly(A) binding protein C1 Is essential for efficient L1 retrotransposition and affects L1 RNP formation. Mol. Cell Biol. 32, 4323–4336 (2012).
Kalva, S., Boeke, J. D. & Mita, P. Gibson Deletion: a novel application of isothermal in vitro recombination. Biol. Proced. Online 20, 2 (2018).
Xie, Y., Rosser, J. M., Thompson, T. L., Boeke, J. D. & An, W. Characterization of L1 retrotransposition with high-throughput dual-luciferase assays. Nucleic Acids Res. 39, e16 (2011).
Xie, Y. et al. Cell division promotes efficient retrotransposition in a stable L1 reporter cell line. Mob. DNA 4, 10 (2013).
Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).
Hasson, S. A. et al. High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy. Nature 504, 291–295 (2013).
Pasetto, M. et al. Whole-genome RNAi screen highlights components of the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity. Proc. Natl Acad. Sci. USA 112, E1135–E1142 (2015).
Grohar, P. J. et al. Identification of an inhibitor of the EWS-FLI1 oncogenic transcription factor by high-throughput screening. J. Natl Cancer Inst. 103, 962–978 (2011).
Vassilev, A. et al. Identification of genes that are essential to restrict genome duplication to once per cell division. Oncotarget 7, 34956–34976 (2016).
Xiao, S. et al. Genome-scale RNA interference screen identifies antizyme 1 (OAZ1) as a target for improvement of recombinant protein production in mammalian cells. Biotechnol. Bioeng. 113, 2403–2415 (2016).
Sivan, G., Ormanoglu, P., Buehler, E. C., Martin, S. E. & Moss, B. Identification of restriction factors by human genome-wide RNA interference screening of viral host range mutants exemplified by discovery of SAMD9 and WDR6 as inhibitors of the vaccinia virus K1L-C7L- mutant. MBio 6, e01122 (2015).
Rodić, N. et al. Long interspersed element-1 protein expression is a hallmark of many human cancers. Am. J. Pathol. 184, 1280–1286 (2014).
Grimm, J. B. et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Methods 12, 244–250 (2015).
Acknowledgements
We thank T. Huang and K. H. Burns for helpful discussions and comments on the manuscript. This work was supported by NIH grants P50GM107632 to J.D.B. and P01AG051449 to J. Sedivy.
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P.M., X.S. and J.D.B. conceived the project; P.M. and X.S. performed experiments; P.M., D.J.K. and C.Y. conducted the primary screen; D.L., N.A. and A.W. contributed new reagents/approaches; P.M., X.S., D.F., D.J.K., S.K., J.S.B, C.Y. and J.D.B. analyzed results; P.M., X.S. and J.D.B. wrote the manuscript; All authors read and commented on the manuscript.
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Supplementary information
Supplementary Information
Supplementary Information including Supplementary Note and 10 Supplementary Figures
Supplementary Table 1
Raw data and hit lists for the genome-wide siRNA knockdown screen
Supplementary Table 2
Enrichment analysis results for the L1 supporters identified in our screen
Supplementary Table 3
log2 fold changes of the DNA repair factors in 96-well validation screen
Supplementary Table 4
L1 insertion sequences recovered in control cells and cells depleted of BRCA1 or FANCM
Supplementary Table 5
List and description of DNA constructs used in this study
Supplementary Table 6
Oligos and primers used in this study
Supplementary Table 7
Summary table of the epistasis analysis reported in Supplementary Figure 8. Cells are colored red if depletion of the considered gene induced a significant increase of L1 retrotransposition compared to siCtrl (in single knockdowns) or siBRCA1 (in double knockdowns). Cells are colored blue if the depletion of the considered gene induced a significant decrease of L1 retrotransposition compared to siCtrl (in single knockdowns) or siBRCA1 (in double knockdowns). The number of + indicate the magnitude of decrease or increase. X means that depletion of the considered gene did not induce any significant alteration in L1 retrotransposition compared to siCtrl (in single KDs) or siBRCA1 (in double KDs) treatments.
Supplementary Video 1
Live-cell imaging of FUCCI cells expressing ORF2 in G1. FUCCI cells expressing L1 were imaged every 30 min for 48 h. Geminin and Cdt1 peptides are visualized in green and red, respectively. Merged channels, ORF2p (cy5 channel) and bright field are shown as a movie. The merged channel (left panel) shows a cluster of cells in the center of the field starting to express ORF2p in G1 phase (red nuclei).
Supplementary Video 2
Live-cell imaging of FUCCI cells expressing ORF2 in S/G2. FUCCI cells expressing L1 were imaged every 30 min for 48 h. Geminin and Cdt1 peptides are visualized in green and red, respectively. Merged channels, ORF2p (cy5 channel) and bright field are shown as a movie. The merged channel (left panel) shows two cells in the center of the field starting to express ORF2p in S/G2 phase (green nuclei).
Supplementary Data 1
Unprocessed western blots for Figs. 2 and 7 and Supplementary Figs. 3 and 10
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Mita, P., Sun, X., Fenyö, D. et al. BRCA1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells. Nat Struct Mol Biol 27, 179–191 (2020). https://doi.org/10.1038/s41594-020-0374-z
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DOI: https://doi.org/10.1038/s41594-020-0374-z
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