BRCA1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells

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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Fig. 1: A whole-genome siRNA knockdown screen for L1 regulators.
Fig. 2: Secondary validations of the DNA repair factors.
Fig. 3: Evidence that L1 uses and induces stalled replication forks.
Fig. 4: Re-expression of wild-type BRCA1 represses L1 retrotransposition in UWB1.289 ovarian carcinoma cells.
Fig. 5: HR/FA pathways repress L1 endonuclease-dependent and independent retrotransposition through BRCA1-mediated DNA resection.
Fig. 6: Mechanism of BRCA1-mediated repression of L1 retrotransposition.
Fig. 7: BRCA1 inhibits ORF2 translation in the cytoplasm.
Fig. 8: Model of BRCA1 inhibition of L1 retrotransposition.

Data availability

All the raw data of the primary and secondary screens are provided in the Supplementary Tables.

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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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Authors

Contributions

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.

Corresponding authors

Correspondence to Paolo Mita or Jef D. Boeke.

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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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Supplementary information

41594_2020_374_MOESM10_ESM.avi

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).

41594_2020_374_MOESM11_ESM.avi

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 Information

Supplementary Information including Supplementary Note and 10 Supplementary Figures

Reporting Summary

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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