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Epigenetic silencing of engineered L1 retrotransposition events in human embryonic carcinoma cells

Abstract

Long interspersed element-1 (LINE-1 or L1) retrotransposition continues to affect human genome evolution1,2. L1s can retrotranspose in the germline, during early development and in select somatic cells3,4,5,6,7,8; however, the host response to L1 retrotransposition remains largely unexplored. Here we show that reporter genes introduced into the genome of various human embryonic carcinoma-derived cell lines (ECs) by L1 retrotransposition are rapidly and efficiently silenced either during or immediately after their integration. Treating ECs with histone deacetylase inhibitors rapidly reverses this silencing, and chromatin immunoprecipitation experiments revealed that reactivation of the reporter gene was correlated with changes in chromatin status at the L1 integration site. Under our assay conditions, rapid silencing was also observed when reporter genes were delivered into ECs by mouse L1s and a zebrafish LINE-2 element, but not when similar reporter genes were delivered into ECs by Moloney murine leukaemia virus or human immunodeficiency virus, suggesting that these integration events are silenced by distinct mechanisms. Finally, we demonstrate that subjecting ECs to culture conditions that promote differentiation attenuates the silencing of reporter genes delivered by L1 retrotransposition, but that differentiation, in itself, is not sufficient to reactivate previously silenced reporter genes. Thus, our data indicate that ECs differ from many differentiated cells in their ability to silence reporter genes delivered by L1 retrotransposition.

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Figure 1: L1 expression and retrotransposition in EC cells.
Figure 2: Engineered L1 retrotransposition events are efficiently silenced in EC cells.
Figure 3: Analyses of L1 silencing in a clonal (pk-5) cell line.
Figure 4: Analysis of L1 silencing in differentiating cells.

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Acknowledgements

We thank P. W. Andrews for providing human EC lines, discussing unpublished data from his laboratory and giving advice during the course of this study. We thank A. Macia and M. Munoz-Lopez (Andalusian Stem Cell Bank) for sharing unpublished data; A. V. Furano, H. Kazazian, J. K. Kim, H. Kopera, A. Muotri, and members of the Moran and Garcia-Perez laboratories for critical reading of the manuscript; and G. Smith and L. Villa for help in creating the time-lapsed movie. We thank M. Velkey for providing plasmid pBSSK-pgk; H. Kazazian for providing plasmid pJCC5/LRE3; J. Boeke for providing synthetic mouse LINE-1 constructs; M. Kajikawa and N. Okada for providing the zebrafish LINE-2 expression plasmids; T. Fanning for providing the polyclonal ORF1 antibody; I. Damjanov for comments on the teratoma characterization; T. Lanigan for preparing Moloney murine leukaemia virus retroviral supernatants; C. Pigott for EC culture advice; and T. de la Cueva, P. Catalina and A. Nieto (Andalusian Stem Cell Bank) for their help with mouse experimentation, SKY-FISH and pathology analyses, respectively. J.V.M. is supported by the National Institutes of Health (NIH) (GM060518 and GM082970) and the Howard Hughes Medical Institute. J.L.G.-P. is supported by the Instituto de Salud Carlos III - Consejeria de Salud Junta de Andalucia (ISCIII-CSJA) (EMER07/056), by a Marie Curie International Reintegration Grant action (FP7-PEOPLE-2007-4-3-IRG), by CICE (P09-CTS-4980) and Proyectos en Salud (PI0002/2009) from Junta de Andalucia (Spain) and through the Spanish Ministry of Health (FIS PI08171 and Miguel Servet CP07/00065). M.M. is supported by the ISCIII-CSJA (EMER07/056). P.M. is supported by the Spanish Ministry of Science and Innovation MICINN-PLANE (PLE-2009-0111), by CICE (P08-CTS-3678) from Junta de Andalucia (Spain) and by the Spanish Ministry of Health (FIS PI070026). K.S.O’S. is supported by the NIH (NS-048187 and GM-069985). K.L.C. is supported by the Burroughs Wellcome Foundation and by an NIH Research Project Grant (R01) (AI051198). G.D.H is supported by a National Institute of Diabetes and Digestive and Kidney Diseases NIH R01 (DK62027). J.O.S. is supported by a Cellular and Molecular Approaches to Systems and Integrative Biology Training Grant (T32-GM08322). D.A.K. is supported by The Irvington Institute Fellowship Program of the Cancer Research Institute. S.M. is supported by a CICE (P08-CTS-3678) scholarship from Junta de Andalucia, Spain. C.C.C. is supported by a Rackham Predoctoral Fellowship from the University of Michigan. We defrayed the costs of DNA sequencing in part with the University of Michigan's Cancer Center Support Grant (NIH 5 P30 CA46592).

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Contributions

J.V.M. and J.L.G.-P. directed the project, designed experiments and drafted the manuscript. J.L.G.-P. performed experiments with the assistance of M.M. and K.S.O’S. (cell cycle experiments), J.O.S. and G.D.H (chromatin immunoprecipitation experiments), D.A.K., C.C.C. and K.L.C. (human immunodeficiency virus-based experiments), and S.M. and P.M. (teratoma assays). All the authors commented on the manuscript.

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Correspondence to Jose L. Garcia-Perez or John V. Moran.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, legend for Supplementary Movie 1, Supplementary Tables 1-2, Supplementary Figures 1-10 with legends and References. (PDF 17434 kb)

Supplementary Movie 1

This movie shows reactivation of L1-retro-EGFP expression in pk-5 Cells (see page 17 of the Supplementary Information file for full legend). (MOV 962 kb)

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Garcia-Perez, J., Morell, M., Scheys, J. et al. Epigenetic silencing of engineered L1 retrotransposition events in human embryonic carcinoma cells. Nature 466, 769–773 (2010). https://doi.org/10.1038/nature09209

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