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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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/).
Mathias, S. L., Scott, A. F., Kazazian, H. H. Jr., Boeke, J. D. & Gabriel, A. Reverse transcriptase encoded by a human transposable element. Science 254, 1808–1810 (1991).
Feng, Q., Moran, J. V., Kazazian, H. H. Jr. & Boeke, J. D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell 87, 905–916 (1996).
Hohjoh, H. & Singer, M. F. Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA. EMBO J. 15, 630–639 (1996).
Woodcock, D. M., Lawler, C. B., Linsenmeyer, M. E., Doherty, J. P. & Warren, W. D. Asymmetric methylation in the hypermethylated CpG promoter region of the human L1 retrotransposon. J. Biol. Chem. 272, 7810–7816 (1997).
Liu, N. et al. Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators. Nature 553, 228–232 (2018).
Haoudi, A., Semmes, O. J., Mason, J. M. & Cannon, R. E. Retrotransposition-competent human LINE-1 induces apoptosis in cancer cells with intact p53. J. Biomed. Biotechnol. 2004, 185–194 (2004).
Belgnaoui, S. M., Gosden, R. G., Semmes, O. J. & Haoudi, A. Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells. Cancer Cell Int. 6, 13 (2006).
Gasior, S. L., Wakeman, T. P., Xu, B. & Deininger, P. L. The human LINE-1 retrotransposon creates DNA double-strand breaks. J. Mol. Biol. 357, 1383–1393 (2006).
Wallace, N. A., Belancio, V. P. & Deininger, P. L. L1 mobile element expression causes multiple types of toxicity. Gene 419, 75–81 (2008).
Kines, K. J. et al. The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations. Mob. DNA 7, 8 (2016).
Luan, D. D., Korman, M. H., Jakubczak, J. L. & Eickbush, T. H. Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72, 595–605 (1993).
Rodic, N. et al. Long interspersed element-1 protein expression is a hallmark of many human cancers. Am. J. Pathol. 184, 1280–1286 (2014).
Ardeljan, D., Taylor, M. S., Ting, D. T. & Burns, K. H. The human long interspersed element-1 retrotransposon: an emerging biomarker of neoplasia. Clin. Chem. 63, 816–822 (2017).
Iskow, R. C. et al. Natural mutagenesis of human genomes by endogenous retrotransposons. Cell 141, 1253–1261 (2010).
Lee, E. et al. Landscape of somatic retrotransposition in human cancers. Science 337, 967–971 (2012).
Shukla, R. et al. Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma. Cell 153, 101–111 (2013).
Tubio, J. M. C. et al. Mobile DNA in cancer. Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes. Science 345, 1251343 (2014).
Rodic, N. et al. Retrotransposon insertions in the clonal evolution of pancreatic ductal adenocarcinoma. Natt. Med. 21, 1060–1064 (2015).
Ewing, A. D. et al. Widespread somatic L1 retrotransposition occurs early during gastrointestinal cancer evolution. Genome Res. 25, 1536–1545 (2015).
Doucet-O’Hare, T. T. et al. LINE-1 expression and retrotransposition in Barrett’s esophagus and esophageal carcinoma. Proc. Natl Acad. Sci. USA 112, E4894–E4900 (2015).
Doucet-O’Hare, T. T. et al. Somatically acquired LINE-1 insertions in normal esophagus undergo clonal expansion in esophageal squamous cell carcinoma. Hum. Mutat. 37, 942–954 (2016).
Scott, E. C. et al. A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer. Genome Res. 26, 745–755 (2016).
Tang, Z. et al. Human transposon insertion profiling: Analysis, visualization and identification of somatic LINE-1 insertions in ovarian cancer. Proc. Natl Acad. Sci. USA 114, E733–E740 (2017).
Burns, K. H. Transposable elements in cancer. Nat. Rev. Cancer 17, 415–424 (2017).
Jung, H., Choi, J. K. & Lee, E. A. Immune signatures correlate with L1 retrotransposition in gastrointestinal cancers. Genome Res. 28, 1136–1146 (2018).
Schauer, S. N. et al. L1 retrotransposition is a common feature of mammalian hepatocarcinogenesis. Genome Res. 28, 639–653 (2018).
Wylie, A. et al. p53 genes function to restrain mobile elements. Genes Dev. 30, 64–77 (2016).
Kawano, K. et al. HIV-1 Vpr and p21 restrict LINE-1 mobility. Nucleic Acids Res. 46, 8454–8470 (2018).
Ruscetti, M. et al. NK cell-mediated cytotoxicity contributes to tumor control by a cytostatic drug combination. Science 362, 1416–1422 (2018).
Yu, Q. et al. Type I interferon controls propagation of long interspersed element-1. J. Biol. Chem. 290, 10191–10199 (2015).
Bregnard, C. et al. Upregulated LINE-1 activity in the fanconi anemia cancer susceptibility syndrome leads to spontaneous pro-inflammatory cytokine production. EBioMedicine 8, 184–194 (2016).
De Cecco, M. et al. L1 drives IFN in senescent cells and promotes age-associated inflammation. Nature 566, 73–78 (2019).
Thomas, C. A. et al. Modeling of TREX1-dependent autoimmune disease using human stem cells highlights L1 accumulation as a source of neuroinflammation. Cell Stem Cell 21, 319–331 (2017).
Simon, M. et al. LINE1 derepression in aged wild-type and SIRT6-deficient mice drives inflammation. Cell Metab. 29, 871–885 (2019).
Pfeffer, L. M. The role of nuclear factor kappaB in the interferon response. J. Interferon Cytokine Res. 31, 553–559 (2011).
Dai, L., Huang, Q. & Boeke, J. D. Effect of reverse transcriptase inhibitors on LINE-1 and Ty1 reverse transcriptase activities and on LINE-1 retrotransposition. BMC Biochem. 12, 18 (2011).
Smith, G. et al. Mutations in APC, Kirsten-ras, and p53–alternative genetic pathways to colorectal cancer. Proc. Natl Acad. Sci. USA 99, 9433–9438 (2002).
Miki, Y. et al. Disruption of the APC gene by a retrotransposal insertion of L1 sequence in a colon cancer. Cancer Res. 52, 643–645 (1992).
Goodier, J. L., Cheung, L. E. & Kazazian, H. H. Jr. Mapping the LINE1 ORF1 protein interactome reveals associated inhibitors of human retrotransposition. Nucleic Acids Res. 41, 7401–7419 (2013).
Taylor, M. S. et al. Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition. Cell 155, 1034–1048 (2013).
Moldovan, J. B. & Moran, J. V. The Zinc-finger antiviral protein ZAP inhibits LINE and Alu retrotransposition. PLoS Genet. 11, e1005121 (2015).
Taylor, M. S. et al. Dissection of affinity captured LINE-1 macromolecular complexes. Elife 7, e30094 (2018).
Tchasovnikarova, I. A. et al. GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells. Science 348, 1481–1485 (2015).
Robbez-Masson, L. et al. The HUSH complex cooperates with TRIM28 to repress young retrotransposons and new genes. Genome Res. 28, 836–845 (2018).
Adamson, B., Smogorzewska, A., Sigoillot, F. D., King, R. W. & Elledge, S. J. A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. Nat. Cell Biol. 14, 318–328 (2012).
Lubas, M. et al. Interaction profiling identifies the human nuclear exosome targeting complex. Mol. Cell 43, 624–637 (2011).
Benitez-Guijarro, M. et al. RNase H2, mutated in Aicardi–Goutieres syndrome, promotes LINE-1 retrotransposition. EMBO J. 37, e98506 (2018).
Gannon, H. S. et al. Identification of ADAR1 adenosine deaminase dependency in a subset of cancer cells. Nat. Commun. 9, 5450 (2018).
Nalepa, G. & Clapp, D. W. Fanconi anaemia and cancer: an intricate relationship. Nat. Rev. Cancer 18, 168–185 (2018).
Richardson, C. D. et al. CRISPR–Cas9 genome editing in human cells occurs via the Fanconi anemia pathway. Nat. Genet. 50, 1132–1139 (2018).
Moran, J. V. et al. High frequency retrotransposition in cultured mammalian cells. Cell 87, 917–927 (1996).
Ward, I. M. & Chen, J. Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J. Biol. Chem. 276, 47759–47762 (2001).
Her, J., Ray, C., Altshuler, J., Zheng, H. & Bunting, S. F. 53BP1 mediates ATR–Chk1 signaling and protects replication forks under conditions of replication stress. Mol. Cell Biol. 38, e00472-17 (2018).
Shigechi, T. et al. ATR–ATRIP kinase complex triggers activation of the Fanconi anemia DNA repair pathway. Cancer Res. 72, 1149–1156 (2012).
Cortez, D., Guntuku, S., Qin, J. & Elledge, S. J. ATR and ATRIP: partners in checkpoint signaling. Science 294, 1713–1716 (2001).
Cimprich, K. A. & Cortez, D. ATR: an essential regulator of genome integrity. Nat. Rev. Mol. Cell Biol. 9, 616–627 (2008).
Bhat, K. P. & Cortez, D. RPA and RAD51: fork reversal, fork protection, and genome stability. Nat. Struct. Mol. Biol. 25, 446–453 (2018).
Feeney, L. et al. RPA-mediated recruitment of the E3 ligase RFWD3 is vital for interstrand crosslink repair and human health. Mol. Cell 66, 610–621.e4 (2017).
Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018).
Pisanic, T. R., 2nd et al. Long interspersed element 1 retrotransposons become deregulated during the development of ovarian cancer precursor lesions. Am. J. Pathol. 189, 513–520 (2018).
Zhouchunyang, X. et al. Expression of L1 retrotransposon open reading frame protein 1 (L1ORF1p) in gynecologic cancers. Hum. Pathol. 92, 39–47 (2019).
Hamperl, S. & Cimprich, K. A. Conflict resolution in the genome: how transcription and replication make it work. Cell 167, 1455–1467 (2016).
Mita, P. et al. LINE-1 protein localization and functional dynamics during the cell cycle. Elife 7, e30058 (2018).
Flasch, D. A. et al. Genome-wide de novo L1 retrotransposition connects endonuclease activity with replication. Cell 177, 837–851 (2019).
Sultana, T. et al. The landscape of L1 retrotransposons in the human genome is shaped by pre-insertion sequence biases and post-insertion selection. Mol. Cell 74, 555–570 (2019).
Rodriguez-Martin, B. et al. Pan-cancer analysis of whole genomes reveals driver rearrangements promoted by LINE-1 retrotransposition in human tumours. Preprint at bioRxiv https://doi.org/10.1101/179705 (2018).
Mita, P. et al. BRCA1 mediated homologous recombination and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells. Nat. Struct. Mol. Biol. https://doi.org/10.1038/s41594-020-0374-z (2020).
Lecona, E. & Fernandez-Capetillo, O. Targeting A. T. R. in cancer. Nat. Rev. Cancer 18, 586–595 (2018).
Chan, E. M. et al. WRN helicase is a synthetic lethal target in microsatellite unstable cancers. Nature 568, 551–556 (2019).
Ishizuka, J. J. et al. Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade. Nature 565, 43–48 (2019).
Fischer, M. Census and evaluation of p53 target genes. Oncogene 36, 3943–3956 (2017).
Fischer, M., Quaas, M., Steiner, L. & Engeland, K. The p53–p21–DREAM–CDE/CHR pathway regulates G2/M cell cycle genes. Nucleic Acids Res. 44, 164–174 (2016).
Lambrus, B. G. et al. A USP28–53BP1–p53–p21 signaling axis arrests growth after centrosome loss or prolonged mitosis. J. Cell Biol. 214, 143–153 (2016).
Lambrus, B. G. et al. p53 protects against genome instability following centriole duplication failure. J. Cell Biol. 210, 63–77 (2015).
Grabundzija, I. et al. Comparative analysis of transposable element vector systems in human cells. Mol. Ther. 18, 1200–1209 (2010).
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR–Cas9. Nat. Biotechnol 34, 184–191 (2016).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Hart, T. et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell 163, 1515–1526 (2015).
Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).
Wang, J., Vasaikar, S., Shi, Z., Greer, M. & Zhang, B. WebGestalt 2017: a more comprehensive, powerful, flexible and interactive gene set enrichment analysis toolkit. Nucleic Acids Res. 45, W130–W137 (2017).
Szklarczyk, D. et al. The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res. 45, D362–D368 (2017).
Santos, A., Wernersson, R. & Jensen, L. J. Cyclebase 3.0: a multi-organism database on cell-cycle regulation and phenotypes. Nucleic Acids Res. 43, D1140–D1144 (2015).
An, W. et al. Characterization of a synthetic human LINE-1 retrotransposon ORFeus-Hs. Mob. DNA 2, 2 (2011).
Kowarz, E., Loscher, D. & Marschalek, R. Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines. Biotechnol. J. 10, 647–653 (2015).
Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).
Ostertag, E. M., Prak, E. T., DeBerardinis, R. J., Moran, J. V. & Kazazian, H. H. Jr. Determination of L1 retrotransposition kinetics in cultured cells. Nucleic Acids Res. 28, 1418–1423 (2000).
Wu, P. H. et al. Evolution of cellular morpho-phenotypes in cancer metastasis. Sci. Rep. 5, 18437 (2015).
Wu, P. H., Hung, S. H., Ren, T., Shih Ie, M. & Tseng, Y. Cell cycle-dependent alteration in NAC1 nuclear body dynamics and morphology. Phys. Biol. 8, 015005 (2011).
Steranka, J. P. et al. Transposon insertion profiling by sequencing (TIPseq) for mapping LINE-1 insertions in the human genome. Mob. DNA 10, 8 (2019).
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.).
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.
(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.
(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.
(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.
(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.
(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.
(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.
(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.
(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 Tables 1, 4, 5 and 6 and Supplementary Methods.
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.
Analysis of fitness interactions among previously known LINE-1 interactors (modify retrotransposition and/or physically bind LINE-1 proteins).
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
Mobile DNA (2020)