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Characterization of Engineered L1 Retrotransposition Events: The Recovery Method

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Transposons and Retrotransposons

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1400))

Abstract

Long Interspersed Element class 1 retrotransposons (LINE-1 or L1) are abundant Transposable Elements in mammalian genomes and their mobility continues to impact the human genome. The development of engineered retrotransposition assays has been instrumental to understand how these elements are regulated and to identify domains involved in the process of retrotransposition. Additionally, the modification of a retrotransposition indicator cassette has allowed developing straightforward approaches to characterize the site of new L1 insertions in cultured cells. In this chapter, we describe a method termed “L1-recovery” that has been used to characterize the site of insertion on engineered L1 retrotransposition events in cultured mammalian cells. Notably, the recovery assay is based on a genetic strategy and avoids the use of PCR and thus reduces to a minimum the appearance of false positives/artifacts.

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References

  1. Beck CR, Garcia-Perez JL, Badge RM, Moran JV (2011) LINE-1 elements in structural variation and disease. Annu Rev Genomics Hum Genet 12:187–215. doi:10.1146/annurev-genom-082509-141802

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Levin HL, Moran JV (2011) Dynamic interactions between transposable elements and their hosts. Nat Rev Genet 12(9):615–627. doi:10.1038/nrg3030

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Macia A, Blanco-Jimenez E, Garcia-Perez JL (2015) Retrotransposons in pluripotent cells: impact and new roles in cellular plasticity. Biochim Biophys Acta 1849(4):417–426. doi:10.1016/j.bbagrm.2014.07.007

    Article  CAS  PubMed  Google Scholar 

  4. de Koning AP, Gu W, Castoe TA, Batzer MA, Pollock DD (2011) Repetitive elements may comprise over two-thirds of the human genome. PLoS Genet 7(12):e1002384. doi:10.1371/journal.pgen.1002384

    Article  PubMed Central  PubMed  Google Scholar 

  5. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, Stange-Thomann N, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, Gibbs RA, Muzny DM, Scherer SE, Bouck JB, Sodergren EJ, Worley KC, Rives CM, Gorrell JH, Metzker ML, Naylor SL, Kucherlapati RS, Nelson DL, Weinstock GM, Sakaki Y, Fujiyama A, Hattori M, Yada T, Toyoda A, Itoh T, Kawagoe C, Watanabe H, Totoki Y, Taylor T, Weissenbach J, Heilig R, Saurin W, Artiguenave F, Brottier P, Bruls T, Pelletier E, Robert C, Wincker P, Smith DR, Doucette-Stamm L, Rubenfield M, Weinstock K, Lee HM, Dubois J, Rosenthal A, Platzer M, Nyakatura G, Taudien S, Rump A, Yang H, Yu J, Wang J, Huang G, Gu J, Hood L, Rowen L, Madan A, Qin S, Davis RW, Federspiel NA, Abola AP, Proctor MJ, Myers RM, Schmutz J, Dickson M, Grimwood J, Cox DR, Olson MV, Kaul R, Raymond C, Shimizu N, Kawasaki K, Minoshima S, Evans GA, Athanasiou M, Schultz R, Roe BA, Chen F, Pan H, Ramser J, Lehrach H, Reinhardt R, McCombie WR, de la Bastide M, Dedhia N, Blocker H, Hornischer K, Nordsiek G, Agarwala R, Aravind L, Bailey JA, Bateman A, Batzoglou S, Birney E, Bork P, Brown DG, Burge CB, Cerutti L, Chen HC, Church D, Clamp M, Copley RR, Doerks T, Eddy SR, Eichler EE, Furey TS, Galagan J, Gilbert JG, Harmon C, Hayashizaki Y, Haussler D, Hermjakob H, Hokamp K, Jang W, Johnson LS, Jones TA, Kasif S, Kaspryzk A, Kennedy S, Kent WJ, Kitts P, Koonin EV, Korf I, Kulp D, Lancet D, Lowe TM, McLysaght A, Mikkelsen T, Moran JV, Mulder N, Pollara VJ, Ponting CP, Schuler G, Schultz J, Slater G, Smit AF, Stupka E, Szustakowski J, Thierry-Mieg D, Thierry-Mieg J, Wagner L, Wallis J, Wheeler R, Williams A, Wolf YI, Wolfe KH, Yang SP, Yeh RF, Collins F, Guyer MS, Peterson J, Felsenfeld A, Wetterstrand KA, Patrinos A, Morgan MJ, de Jong P, Catanese JJ, Osoegawa K, Shizuya H, Choi S, Chen YJ (2001) Initial sequencing and analysis of the human genome. Nature 409(6822):860–921

    Google Scholar 

  6. Beck CR, Collier P, Macfarlane C, Malig M, Kidd JM, Eichler EE, Badge RM, Moran JV (2010) LINE-1 retrotransposition activity in human genomes. Cell 141(7):1159–1170

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Brouha B, Schustak J, Badge RM, Lutz-Prigge S, Farley AH, Moran JV, Kazazian HH Jr (2003) Hot L1s account for the bulk of retrotransposition in the human population. Proc Natl Acad Sci U S A 100(9):5280–5285

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Luan DD, Korman MH, Jakubczak JL, Eickbush TH (1993) Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72(4):595–605

    Article  CAS  PubMed  Google Scholar 

  9. Macia A, Munoz-Lopez M, Cortes JL, Hastings RK, Morell S, Lucena-Aguilar G, Marchal JA, Badge RM, Garcia-Perez JL (2011) Epigenetic control of retrotransposon expression in human embryonic stem cells. Mol Cell Biol 31(2):300–316

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Speek M (2001) Antisense promoter of human L1 retrotransposon drives transcription of adjacent cellular genes. Mol Cell Biol 21(6):1973–1985

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Swergold GD (1990) Identification, characterization, and cell specificity of a human LINE-1 promoter. Mol Cell Biol 10(12):6718–6729

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Boeke JD (1997) LINEs and Alus--the polyA connection. Nat Genet 16(1):6–7

    Article  CAS  PubMed  Google Scholar 

  13. Moran JV, DeBerardinis RJ, Kazazian HH Jr (1999) Exon shuffling by L1 retrotransposition. Science 283(5407):1530–1534

    Article  CAS  PubMed  Google Scholar 

  14. Moran JV, Holmes SE, Naas TP, DeBerardinis RJ, Boeke JD, Kazazian HH Jr (1996) High frequency retrotransposition in cultured mammalian cells. Cell 87(5):917–927

    Article  CAS  PubMed  Google Scholar 

  15. Scott AF, Schmeckpeper BJ, Abdelrazik M, Comey CT, O’Hara B, Rossiter JP, Cooley T, Heath P, Smith KD, Margolet L (1987) Origin of the human L1 elements: proposed progenitor genes deduced from a consensus DNA sequence. Genomics 1(2):113–125

    Article  CAS  PubMed  Google Scholar 

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

    PubMed Central  CAS  PubMed  Google Scholar 

  17. Hohjoh H, Singer MF (1997) Sequence-specific single-strand RNA binding protein encoded by the human LINE-1 retrotransposon. EMBO J 16(19):6034–6043

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Khazina E, Weichenrieder O (2009) Non-LTR retrotransposons encode noncanonical RRM domains in their first open reading frame. Proc Natl Acad Sci U S A 106(3):731–736

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Martin SL, Bushman FD (2001) Nucleic acid chaperone activity of the ORF1 protein from the mouse LINE-1 retrotransposon. Mol Cell Biol 21(2):467–475

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Feng Q, Moran JV, Kazazian HH Jr, Boeke JD (1996) Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell 87(5):905–916

    Article  CAS  PubMed  Google Scholar 

  21. Martin F, Maranon C, Olivares M, Alonso C, Lopez MC (1995) Characterization of a non-long terminal repeat retrotransposon cDNA (L1Tc) from Trypanosoma cruzi: homology of the first ORF with the ape family of DNA repair enzymes. J Mol Biol 247(1):49–59

    Article  CAS  PubMed  Google Scholar 

  22. Mathias SL, Scott AF, Kazazian HH Jr, Boeke JD, Gabriel A (1991) Reverse transcriptase encoded by a human transposable element. Science 254(5039):1808–1810

    Article  CAS  PubMed  Google Scholar 

  23. Dmitriev SE, Andreev DE, Terenin IM, Olovnikov IA, Prassolov VS, Merrick WC, Shatsky IN (2007) Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5′ untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated. Mol Cell Biol 27(13):4685–4697

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Alisch RS, Garcia-Perez JL, Muotri AR, Gage FH, Moran JV (2006) Unconventional translation of mammalian LINE-1 retrotransposons. Genes Dev 20(2):210–224

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Esnault C, Maestre J, Heidmann T (2000) Human LINE retrotransposons generate processed pseudogenes. Nat Genet 24(4):363–367

    Article  CAS  PubMed  Google Scholar 

  26. Wei W, Gilbert N, Ooi SL, Lawler JF, Ostertag EM, Kazazian HH, Boeke JD, Moran JV (2001) Human L1 retrotransposition: cis preference versus trans complementation. Mol Cell Biol 21(4):1429–1439

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Doucet AJ, Hulme AE, Sahinovic E, Kulpa DA, Moldovan JB, Kopera HC, Athanikar JN, Hasnaoui M, Bucheton A, Moran JV, Gilbert N (2010) Characterization of LINE-1 ribonucleoprotein particles. PLoS Genet 6(10):e1001150

    Article  PubMed Central  PubMed  Google Scholar 

  28. Goodier JL, Zhang L, Vetter MR, Kazazian HH Jr (2007) LINE-1 ORF1 protein localizes in stress granules with other RNA-binding proteins, including components of RNA interference RNA-induced silencing complex. Mol Cell Biol 27(18):6469–6483

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Taylor MS, Lacava J, Mita P, Molloy KR, Huang CR, Li D, Adney EM, Jiang H, Burns KH, Chait BT, Rout MP, Boeke JD, Dai L (2013) Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition. Cell 155(5):1034–1048. doi:10.1016/j.cell.2013.10.021

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Gilbert N, Lutz S, Morrish TA, Moran JV (2005) Multiple fates of l1 retrotransposition intermediates in cultured human cells. Mol Cell Biol 25(17):7780–7795

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Coufal NG, Garcia-Perez JL, Peng GE, Marchetto MC, Muotri AR, Mu Y, Carson CT, Macia A, Moran JV, Gage FH (2011) Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells. Proc Natl Acad Sci U S A 108(51):20382–20387. doi:10.1073/pnas.1100273108

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Ostertag EM, Kazazian HH Jr (2001) Twin priming: a proposed mechanism for the creation of inversions in L1 retrotransposition. Genome Res 11(12):2059–2065

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Freeman JD, Goodchild NL, Mager DL (1994) A modified indicator gene for selection of retrotransposition events in mammalian cells. Biotechniques 17(1):46, 48-49, 52

    CAS  PubMed  Google Scholar 

  34. Moran JV (1999) Human L1 retrotransposition: insights and peculiarities learned from a cultured cell retrotransposition assay. Genetica 107(1-3):39–51

    Article  CAS  PubMed  Google Scholar 

  35. Rangwala SH, Kazazian HH (2009) The L1 retrotransposition assay: a retrospective and toolkit. Methods 49(3):219–226

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Wei W, Morrish TA, Alisch RS, Moran JV (2000) A transient assay reveals that cultured human cells can accommodate multiple LINE-1 retrotransposition events. Anal Biochem 284(2):435–438

    Article  CAS  PubMed  Google Scholar 

  37. Cost GJ, Feng Q, Jacquier A, Boeke JD (2002) Human L1 element target-primed reverse transcription in vitro. EMBO J 21(21):5899–5910

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Coufal NG, Garcia-Perez JL, Peng GE, Yeo GW, Mu Y, Lovci MT, Morell M, O’Shea KS, Moran JV, Gage FH (2009) L1 retrotransposition in human neural progenitor cells. Nature 460(7259):1127–1131

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Gilbert N, Lutz-Prigge S, Moran JV (2002) Genomic deletions created upon LINE-1 retrotransposition. Cell 110(3):315–325

    Article  CAS  PubMed  Google Scholar 

  40. Morrish TA, Garcia-Perez JL, Stamato TD, Taccioli GE, Sekiguchi J, Moran JV (2007) Endonuclease-independent LINE-1 retrotransposition at mammalian telomeres. Nature 446(7132):208–212

    Article  CAS  PubMed  Google Scholar 

  41. Morrish TA, Gilbert N, Myers JS, Vincent BJ, Stamato TD, Taccioli GE, Batzer MA, Moran JV (2002) DNA repair mediated by endonuclease-independent LINE-1 retrotransposition. Nat Genet 31(2):159–165

    Article  CAS  PubMed  Google Scholar 

  42. Muotri AR, Chu VT, Marchetto MC, Deng W, Moran JV, Gage FH (2005) Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition. Nature 435(7044):903–910

    Article  CAS  PubMed  Google Scholar 

  43. Symer DE, Connelly C, Szak ST, Caputo EM, Cost GJ, Parmigiani G, Boeke JD (2002) Human l1 retrotransposition is associated with genetic instability in vivo. Cell 110(3):327–338

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

David Cano, Santiago Morell, and Andres J. Pulgarin have contributed equally to this study and are listed alphabetically. We acknowledge current members of the J.L.G.-P. lab (Genyo) for valuable input during the course of the project. We also acknowledge Dr. John Moran (University of Michigan, USA) for helpful discussions. D.C. is supported by a FPU fellowship from the Government of Spain (MINECO, Ref AP2010-0135). J.L.G.P’s lab is supported by CICE-FEDER-P09-CTS-4980, CICE-FEDER-P12-CTS-2256, Plan Nacional de I+D+i 2008-2011 and 2013-2016 (FIS-FEDER-PI11/01489 and FIS-FEDER-PI14/02152), PCIN-2014-115-ERA-NET NEURON II, the European Research Council (ERC-Consolidator ERC-STG-2012-233764) and by an International Early Career Scientist grant from the Howard Hughes Medical Institute (IECS-55007420).

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    Authors and Affiliations

    1. Department of Human DNA Variability, Pfizer/University of Granada and Andalusian Regional Government Center for Genomics and Oncology (GENYO), Avda Ilustracion 114, PTS Granada, 18016, Granada, Spain

      David Cano, Santiago Morell, Andres J. Pulgarin, Suyapa Amador & Jose L. Garcia-Pérez Ph.D.

    2. Genyo (Center for Genomics and Oncological Research), Pfizer/Universidad de Granada/Junta de Andalucia, PTS Granada, Spain

      Jose L. Garcia-Pérez Ph.D.

    3. Institute of Genetics and Molecular Medicine (IGMM), University of Edinburgh, Edinburgh, UK

      Jose L. Garcia-Pérez Ph.D.

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    1. David Cano
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    2. Santiago Morell
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    3. Andres J. Pulgarin
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    4. Suyapa Amador
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    5. Jose L. Garcia-Pérez Ph.D.
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    Correspondence to Jose L. Garcia-Pérez Ph.D. .

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    1. Institute of Genetics and Molecular Medi, Genyo (Center for Genomics and Oncologic, Granada, Spain

      Jose L. Garcia-Pérez

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    Cano, D., Morell, S., Pulgarin, A.J., Amador, S., Garcia-Pérez, J.L. (2016). Characterization of Engineered L1 Retrotransposition Events: The Recovery Method. In: Garcia-Pérez, J. (eds) Transposons and Retrotransposons. Methods in Molecular Biology, vol 1400. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3372-3_12

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    • DOI: https://doi.org/10.1007/978-1-4939-3372-3_12

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