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.
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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.
The authors declare no competing interests.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information including Supplementary Note and 10 Supplementary Figures
Raw data and hit lists for the genome-wide siRNA knockdown screen
Enrichment analysis results for the L1 supporters identified in our screen
log2 fold changes of the DNA repair factors in 96-well validation screen
L1 insertion sequences recovered in control cells and cells depleted of BRCA1 or FANCM
List and description of DNA constructs used in this study
Oligos and primers used in this study
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.
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).
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).
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
Nature Structural & Molecular Biology (2020)