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Cell fitness screens reveal a conflict between LINE-1 retrotransposition and DNA replication

Abstract

LINE-1 retrotransposon overexpression is a hallmark of human cancers. We identified a colorectal cancer wherein a fast-growing tumor subclone downregulated LINE-1, prompting us to examine how LINE-1 expression affects cell growth. We find that nontransformed cells undergo a TP53-dependent growth arrest and activate interferon signaling in response to LINE-1. TP53 inhibition allows LINE-1+ cells to grow, and genome-wide-knockout screens show that these cells require replication-coupled DNA-repair pathways, replication-stress signaling and replication-fork restart factors. Our findings demonstrate that LINE-1 expression creates specific molecular vulnerabilities and reveal a retrotransposition–replication conflict that may be an important determinant of cancer growth.

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Fig. 1: Heterogeneous LINE-1 expression in colon cancer.
Fig. 2: LINE-1 inhibits cell growth in RPE by activating the p53-p21 pathway.
Fig. 3: LINE-1 activates a p53 and IFN response.
Fig. 4: Mapping LINE-1 fitness interactions in TP53-deficient cells.
Fig. 5: The Fanconi anemia pathway is essential in p53-deficient cells.
Fig. 6: LINE-1 activity induces replication stress.
Fig. 7: Model of LINE-1-induced replication stress.

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Data availability

MAGeCK-normalized sgRNA read counts from CRISPR knockout screens and RNA-seq counts and differential expression values have been deposited in the GEO database under accession number GSE119999. Source data for Figs. 2b, 5c,e,f and 6d,e are available online. Requests for resources and reagents should be directed to and will be fulfilled by K.H.B.. Select plasmids created in the Burns Lab can be accessed at Addgene (https://www.addgene.org/Kathleen_Burns/).

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Acknowledgements

Human Brunello CRISPR knockout pooled library was a gift from D. Root and J. Doench (Addgene no. 73178). pSBtet-RN and pSBtet-GN were gifts from E. Kowarz (Addgene plasmid no. 60501 and 60503). pCMV(CAT)T7-SB100 was a gift from Z. Izsvak (Addgene plasmid no, 34879). JM111 was a gift from H. Kazazian. pSicoR-mCh_empty was a gift from M. Ramalho-Santos (Addgene no. 219070). LentiGuide-Puro was a gift from F. Zhang (Addgene no. 52963). We thank J. Gucwa at the Sidney Kimmel Flow Cytometry Core and the staff of the NYU Genome Technology center. We thank J. S. Bader for his statistical expertise. We thank B. A. Bari, R. M. Hughes, B. Vogelstein, J. V. Moran and H. H. Kazazian for helpful discussion and review of the manuscript. We thank J. Fairman of the Department of Art as Applied to Medicine at Johns Hopkins University School of Medicine for illustrations. This study was funded by F30CA221175 (D.A.), P50GM107632 (K.H.B., J.D.B., D.F.), U54CA210173 (P.W.) and the Sol Goldman Pancreatic Research Center (K.H.B., R.H.H.).

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Authors

Contributions

D.A. and K.H.B. conceptualized this work and wrote the manuscript. Experiments were performed by D.A., J.P.S., C.L., P.-H.W., J.S.S., M.G. and Z.L. All data were primarily analyzed by D.A. Key resources were provided by A.J.H. (RPE cells), A.S. (FANCI mAb), and M.S.T. and V.D. (deidentified colorectal cancer samples). All authors participated in manuscript revisions. Funding was acquired by D.A., J.D.B. and K.H.B.

Corresponding authors

Correspondence to Daniel Ardeljan or Kathleen H. Burns.

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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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Extended data

Extended Data Fig. 1 LINE-1 heterogeneity in colon cancer.

(a) Tissues collected for transposon insertion profiling by sequencing (TIP-seq) mapping of tumor-specific LINE insertions. Fresh frozen tissue was collected from two sites in the primary tumor (P1, P2) in the colon and one site in the metastatic tumor (M) in the liver. Normal tissue was collected from the liver. The liver metastasis exhibited ORF1p immunoreactivity as well (data not shown). (b) Circos plot detailing TIP-seq results and whether insertions were found in the primary (P only), metastasis (M only) or in both (P & M). In the validation process, we identified 11 3′ transduction events, 6 of which mapped to two LINE-1 sequences on Xp22.2 and one on 3q21.1 that are known to be highly active tumor alleles. As expected, the majority of this tumor’s de novo insertions were intronic or intergenic and not near known tumor suppressors or oncogenes. (c) We genotyped the insertions using hemi-specific PCR in genomic DNA obtained from dissected histology slides and compared to the allele’s presence in bulk frozen tissue used for TIP-seq. In all samples, we detected an inherited LINE-1 on 1q42.3, indicating that our PCR conditions were sufficient to genotype LINE-1 alleles. An early de novo insertion on 10q26.3 was found in all frozen tissue samples (primary and metastasis) and both CDX2high and CDX2dim slide-dissected samples. An insertion on 3q22.2 is present in the primary tumor subclonally and in the metastasis and therefore occurred before metastasis but after dedifferentiation of the CDX2dim clone. An insertion on 18q22.1 occurred late, after metastasis to the liver had occurred, since it was found in the primary CDX2high clone and not in the metastasis.

Extended Data Fig. 2 TP53 effects on LINE-1+ cell growth and retrotransposition.

(a) Demonstration of effective TP53 knockdown. RPE cells were treated with TP53 shRNA lentivirus (pDA079) or control lentivirus (pDA081). The Western blot shows the p53 response to treatment with the DNA intercalator doxorubicin (200 ng ml–1 for 24 h). (b) Left, the retrotransposition reporter assay. LINE-1 is expressed from a plasmid with an antisense eGFP in the 3′UTR that is interrupted by a sense intron. During transcription, the intron is spliced, reconstituting the coding potential of the eGFP reporter. The eGFP reporter carries with it a CMV promoter and is inserted into the genome by LINE-1. Expression of eGFP from the genome allows for fluorescence-based quantification of retrotransposition rate by flow cytometry. Right, reporter assay performed in RPE with TP53 knockdown or control ± s.e.m., n = 3 independent experiments. P value was calculated by two-sided t-test. (c) Normalized median read counts of sgRNAs targeting TP53 and CDKN1A in cells expressing either LINE-1 (navy blue) or eGFP (green) control compared to non-targeting-controls (NTC). Individual sgRNAs are indicated by circles or triangles. Results from two biological replicates are depicted.

Extended Data Fig. 3 LINE-1 RNAseq analysis.

(a) Genes regulated by cell cycle were curated from CycleBase v3.072 and differential expression values were plotted. S, G2, and M phase genes were significantly downregulated in LINE-1+ cells. Unpaired two-sided t-tests were used for statistical testing. N/A = not applicable. *p-values vs. N/A: G1 = not significant (n.s.), G1/S = 1.7e-9, S = 1.5e-2, G2 = 2.1e-13, G2/M = 5.2e-6, M = 3.4e-10. (b) Flow cytometry was used to assess cell cycle by quantifying DNA content using a PI DNA stain in Tet-On LINE-1 or Tet-On luciferase cells induced with 1 µg ml–1 doxycycline for 48 h. LINE-1+ cells with wild-type (WT) p53 accumulated in G1 phase (2n DNA copy number), whereas TP53KD resulted in more even cell cycle proportions. These data are from one experiment. (c) Relative fold-change of interferon-stimulated genes in LINE-1 compared to luciferase-expressing cells measured by RNAseq. Error bars indicate s.e.m. (d) RNAseq analysis revealed upregulation of NF-kB and several target genes in LINE-1+ cells. Error bars indicate s.e.m. (e) Differential expression of IFNB1 (right) and interferon-stimulated genes (left) in p53-knockdown cells expressing LINE-1 or luciferase for 72 h. Measured by qRT-PCR. Error bars indicate s.d., n = 3 biological replicates. * p < 0.05, ** p < 0.001. (f) Differential expression of TLR3, IFIT1, and IFIT2 with the addition of 5μM zalcitabine (ddC) or 5μM didanosine (ddI) in p53-knockdown cells expressing LINE-1 or luciferase for 72 h. Measured by qRT-PCR, n = 3 independent experiments. P values indicated within the plots.

Extended Data Fig. 4 TP53-Knockdown Screen Supplement.

(a) Behavior of non-targeting-control sgRNAs in the screen over time. Data points indicate the median sgRNA count per replicate and error bars the 95% confidence interval. (b) Behavior of TP53- and CDNK1A-targeting sgRNAs. Median values are depicted with 95% Confidence Intervals. There is no appreciable change in TP53 sgRNA representation between LINE-1+ and luciferase control cells, indicating loss of p53 function due to the shRNA. CDNK1A sgRNAs do not differ between groups as well, suggesting that CDKN1A effects are contingent on p53 function. (c) Examples of essential gene knockouts that deplete from both LINE-1+ and luciferase + cells. Median values are depicted with 95% Confidence Intervals. (d) Knockout of APC provides a growth advantage to LINE-1+ cells. Median values are depicted with 95% Confidence Intervals. (e) Knockout of the interferon alpha and beta receptor subunit 1 (IFNAR1) but not subunit 2 (IFNAR2) provides a growth advantage in LINE-1+ cells. Median values are depicted with 95% Confidence Intervals.

Extended Data Fig. 5 HUSH knockout is synthetic lethal due to derepression of the LINE-1 transgene.

(a) Gene screen ranks by Zs scores. HUSH genes are in blue. (b) HUSH complex sgRNA performance during the screen. All knockouts drop out early from LINE-1+ cells (red) and do not affect growth of luciferase+ cells (black). Median values are depicted with 95% Confidence Intervals. (c) 12 d clonogenic growth assay in cells expressing LINE-1 (doxycycline-induced) with targeted knockouts of HUSH components compared to non-targeting-control (NTC). n = 3 independent experiments. Error bars indicate ± s.e.m. P values calculated by one-sided t-test. (d) Western blot comparing ORF1p and ORF2p expression in HUSH knockout cells or non-target-controls (NTC) that have not been treated with doxycycline compared to NTC with 24 h of 1 µg ml–1 doxycycline treatment. ORF1p and ORF2p expression are only detected in NTC-treated cells with doxycycline added to the culture media. The double banding pattern for ORF1p is consistently seen with codon-optimized LINE-1. (e) Western blot comparing ORF1p and ORF2p expression 24 h after 1 µg ml–1 doxycycline treatment in HUSH knockouts compared to NTC. The ORF2p antibody cannot distinguish between endogenous or transgenic LINE-1 expression. (f) qRT-PCR analysis of LINE-1 transgene expression in HUSH knockouts compared to NTC (induced with 1 µg ml–1 doxycycline). Because the LINE-1 transgene is codon-optimized, qRT-PCR is specific for the transgene and does not amplify endogenous LINE-1 sequences. *p < 0.001. (g) Linear regression plot of LINE-1 transgene expression and ORF1p and ORF2p expression in HUSH knockouts compared to NTC. Shaded area indicates 95% confidence interval for regression line. Both ORF1p and ORF2p increase in expression with higher transgene mRNA expression, although the increase in ORF1p is minimal compared to that observed with ORF2p. (h) Heatmap of immunofluorescence imaging depicting the proportion of cells expressing ORF1p and ORF2p at different levels in HEK293T cells expressing Tet-On LINE-1 (pDA055) at increasing doses of doxycycline.

Extended Data Fig. 6 RNA processing gene knockouts sensitize cells to LINE-1.

(a) StringDB network plot of the 81 mRNA processing genes identified by this screen. Edges indicate known protein-protein interactions. This network is enriched for spliceosome machinery (green nodes). (b) Screen behavior of significant genes belonging to the spliceosome KEGG GO term. Median sgRNA counts are depicted with 95% Confidence Intervals. (c) Clonogenic assay (12 d) comparing growth of luciferase+ and LINE-1+ cells (induced with 1 µg ml–1 doxycycline) treated with 1 nM pladienolide B (PLA-B) or vehicle (DMSO). n = 3 independent experiments. Error bars indicate s.e.m. P value calculated by unpaired one-sided t-test. (d) Behavior of nuclear exosome complex genes in the screen. Median values are depicted with 95% Confidence Intervals. (e) Behavior of RNASEH2 component sgRNAs in the screen. Median values are depicted with 95% Confidence Intervals. (f) Behavior of ADAR1 sgRNAs in the screen. Median values are depicted with 95% Confidence Intervals.

Extended Data Fig. 7 The Fanconi Anemia Pathway is required for growth of LINE-1+ cells.

(a) Behavior of sgRNAs targeting Fanconi Anemia pathway genes in the screen. Median values are depicted with 95% Confidence Intervals. (b) Western blot of DNA damage marker γH2A.X in chromatin-bound protein fractions of LINE-1+ cells with or without perturbations to the FA pathway. H3 was used as loading control. γH2A.X levels were quantified and graphed relative to NTC-treated, LINE-1+ cells. (c) Clonogenic assay (10 d). TP53KD cells constitutively expressing Cas9 are treated with lentivirus encoding non-targeting-control (NTC) or FANCD2 sgRNA and then transfected with eGFP (pDA083) or the native LINE-1 sequence L1RP (pDA077). Left, representative images of colonies. Scale bar = 1 cm. Right, data are presented as the rate of LINE-1 per 100 eGFP colonies ± s.d. to control for transfection efficiency across samples, n = 3 independent experiments. P value obtained by unpaired two-sided t-test. (d) Quantification of FANCD2 foci in G1 and G2 phase (EdU-) HeLa cells. Number of cells per group: G1 untreated (n = 104), G1 HU (n = 352), G1 wildtype LINE-1 (n = 186), G1 RT (D702Y) (n = 138), G2 untreated (n = 60), G2 HU (n = 58), G2 wildtype LINE-1 (n = 42), G2 RT (D702Y) (n = 32). Two-sided t-tests were used for statistical comparisons. HU = hydroxyurea. RT = reverse transcriptase. ns = not significant.

Extended Data Fig. 8 Viability assays with LINE-1 mutants.

(a) Tet-On constructs for wild-type and mutant LINE-1 expression. (b) Viability of HEK293T cells after 4 days expressing wild-type or a mutant at increasing doxycycline doses. A multivariate ANOVA (Viability ~ ORF2 * doxycycline) was performed in R to calculate p values for ORF2 mutant status and doxycycline dose. Tests of viability differences among ORF2 mutants were further performed using two-sided t-tests at the 1000 ng ml–1 doxycycline dose. N = 6 replicates per doxycycline dose. (c) Western blot of ORF1p and ORF2p 24 hours after inducing protein expression with 1000 ng ml–1 doxycycline.

Supplementary information

Supplementary Information

Supplementary Tables 1, 4, 5 and 6 and Supplementary Methods.

Reporting Summary

Supplementary Table 2

Ranking of KD screen results. Columns include the gene symbol, fitness interaction (rescue, synthetic lethal). Ranks are indicated for genome-wide significant genes that demonstrate either rescue or synthetic lethal interactions.

Supplementary Table 3

Analysis of fitness interactions among previously known LINE-1 interactors (modify retrotransposition and/or physically bind LINE-1 proteins).

Supplementary Data 1

Unprocessed Western blots for Figs. 2b, 5c,e,f and 6d,e.

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Ardeljan, D., Steranka, J.P., Liu, C. et al. Cell fitness screens reveal a conflict between LINE-1 retrotransposition and DNA replication. Nat Struct Mol Biol 27, 168–178 (2020). https://doi.org/10.1038/s41594-020-0372-1

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