Abstract
Background
Long interspersed element type one (L1) actively modifies the human genome by inserting new copies of itself. This process, termed retrotransposition, requires the formation of an L1 ribonucleoprotein (RNP) complex, which must enter the nucleus before retrotransposition can proceed. Thus, the nuclear import of L1 RNP presents an opportunity for cells to regulate L1 retrotransposition post-translationally. The effect of cell division on L1 retrotransposition has been investigated by two previous studies, which observed varied degrees of inhibition in retrotransposition when primary cell strains or cancer cell lines were experimentally arrested in different stages of the cell cycle. However, seemingly divergent conclusions were reached. The role of cell division on retrotransposition remains highly debated.
Findings
To monitor both L1 expression and retrotransposition quantitatively, we developed a stable dual-luciferase L1 reporter cell line, in which a bi-directional tetracycline-inducible promoter drives the expression of both a firefly luciferase-tagged L1 element and a Renilla luciferase, the latter indicative of the level of promoter induction. We observed an additional 10-fold reduction in retrotransposition in cell-cycle arrested cells even after retrotransposition had been normalized to Renilla luciferase or L1 ORF1 protein levels. In synchronized cells, cells undergoing two mitoses showed 2.6-fold higher retrotransposition than those undergoing one mitosis although L1 expression was induced for the same amount of time.
Conclusions
Our data provide additional support for an important role of cell division in retrotransposition and argue that restricting the accessibility of L1 RNP to nuclear DNA could be a post-translational regulatory mechanism for retrotransposition.
Findings
Long interspersed elements type one (LINE-1; L1), the only active autonomous transposable element in the human genome, have played a major role in human genome evolution and are also responsible for an increasing number of sporadic human genetic diseases[1–3]. To make new copies, a source L1 element must successfully navigate through every stage of the retrotransposition process (that is, transcription, translation, and target-primed reverse transcription). An essential intermediate step is the formation of an L1 ribonucleoprotein (RNP) complex between L1 mRNA and proteins[4–6]. L1 RNP must enter the nucleus before a new copy is made via target-primed reverse transcription[7]. Therefore, the nuclear import of L1 RNP presents an opportunity for cells to regulate L1 retrotransposition post-translationally. As nuclear import can occur passively when nuclear envelope breaks down during cell division, the efficiency of retrotransposition is predicted to be higher in actively dividing cells. Indeed, the effect of cell division has been investigated by two previous studies, which compared the level of L1 retrotransposition in cell-cycle arrested primary cell strains and cancer cell lines[8, 9]. Although both observed varied degrees of inhibition in retrotransposition when cells were experimentally arrested in different stages of the cell cycle, one study concluded that cell division was required for retrotransposition and the other determined that L1 retrotransposition could occur in non-dividing cells (Table 1). Hence, the role of cell division on retrotransposition remains highly debated to date.
Development of a stable HeLa Tet-ORFeus cell line
To investigate the effect of cell-cycle arrest on L1 retrotransposition, we wished to establish an assay system that meets the following criteria: (1) It must be a stable cell line with an integrated L1 reporter. Having an integrated L1 reporter eliminates variation in transfection efficiency that is inherent in transient assays. However, this requirement necessitates the use of an inducible promoter because, otherwise, L1 insertions will accumulate while the cell line is being established. (2) The promoter activity (that is, transcription) can be conveniently monitored in parallel to L1 retrotransposition. (3) Both the promoter activity and L1 retrotransposition can be measured with high sensitivity and in a wide dynamic range. Accordingly, we designed an inducible dual-luciferase L1 assay vector, pYX056 (Figure 1A; detailed in Additional file1). The design combined a gene regulation and a gene delivery system. First, the Tet-Off Advanced Inducible Gene Expression System allows stringent control of L1 expression. The bi-directional PTight inducible promoter drives expression of Renilla luciferase (Rluc) and a hyperactive synthetic mouse L1, ORFeus[10]. The latter is tagged with a firefly luciferase/antisense intron (FlucAI) reporter cassette[12] (Figure 1B). Single cell clones were acquired by limiting dilution method and screened for the lack of Rluc expression in doxycycline-supplemented medium but high levels of Rluc expression upon doxycycline withdrawal (Figure 1C; detailed in Additional file1).
Abbreviations
- Fluc:
-
Firefly luciferase
- FlucAI:
-
Fluc disrupted by an antisense intron
- L1:
-
Long interspersed element type one
- NLS:
-
Nuclear localization signal
- ORF1p:
-
Open reading frame 1 protein
- qPCR:
-
Quantitative polymerase chain reaction
- Rluc:
-
Renilla luciferase
- RNP:
-
Ribonucleoprotein
- SB:
-
Slee** Beauty DNA transposon
- tTA:
-
Tetracycline-controlled transactivator advanced
References
Cordaux R, Batzer MA: The impact of retrotransposons on human genome evolution. Nat Rev Genet. 2009, 10: 691-703. 10.1038/nrg2640.
Hancks DC, Kazazian HH: Active human retrotransposons: variation and disease. Curr Opin Genet Dev. 2012, 22: 191-203. 10.1016/j.gde.2012.02.006.
Belancio VP, Hedges DJ, Deininger P: Mammalian non-LTR retrotransposons: for better or worse, in sickness and in health. Genome Res. 2008, 18: 343-358. 10.1101/gr.5558208.
Martin SL: Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells. Mol Cell Biol. 1991, 11: 4804-4807.
Hohjoh H, Singer MF: Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA. EMBO J. 1996, 15: 630-639.
Kulpa DA, Moran JV: Ribonucleoprotein particle formation is necessary but not sufficient for LINE-1 retrotransposition. Hum Mol Genet. 2005, 14: 3237-3248. 10.1093/hmg/ddi354.
Luan DD, Korman MH, Jakubczak JL, Eickbush TH: Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell. 1993, 72: 595-605. 10.1016/0092-8674(93)90078-5.
Kubo S, Seleme MC, Soifer HS, Perez JL, Moran JV, Kazazian HH, Kasahara N: L1 retrotransposition in nondividing and primary human somatic cells. Proc Natl Acad Sci U S A. 2006, 103: 8036-8041. 10.1073/pnas.0601954103.
Shi X, Seluanov A, Gorbunova V: Cell divisions are required for L1 retrotransposition. Mol Cell Biol. 2007, 27: 1264-1270. 10.1128/MCB.01888-06.
Han JS, Boeke JD: A highly active synthetic mammalian retrotransposon. Nature. 2004, 429: 314-318. 10.1038/nature02535.
**e Y, Rosser JM, Thompson TL, Boeke JD, An W: Characterization of L1 retrotransposition with high-throughput dual-luciferase assays. Nucleic Acids Res. 2011, 39: e16-10.1093/nar/gkq1076.
Mates L, Chuah MK, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, Grzela DP, Schmitt A, Becker K, Matrai J, Ma L, Samara-Kuko E, Gysemans C, Pryputniewicz D, Miskey C, Fletcher B, VandenDriessche T, Ivics Z, Isvask Z: Molecular evolution of a novel hyperactive Slee** Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009, 41: 753-761. 10.1038/ng.343.
Rosser JM, An W: Repeat-induced gene silencing of L1 transgenes is correlated with differential promoter methylation. Gene. 2010, 456: 15-23. 10.1016/j.gene.2010.02.005.
Matsumoto T, Takahashi H, Fujiwara H: Targeted nuclear import of open reading frame 1 protein is required for in vivo retrotransposition of a telomere-specific non-long terminal repeat retrotransposon, SART1. Mol Cell Biol. 2004, 24: 105-122. 10.1128/MCB.24.1.105-122.2004.
Dai L, Taylor MS, O'Donnell KA, Boeke JD: Poly(A) binding protein C1 is essential for efficient L1 retrotransposition and affects L1 RNP formation. Mol Cell Biol. 2012, 32: 4323-4336. 10.1128/MCB.06785-11.
Peddigari S, Li PW, Rabe JL: Martin SL: hnRNPL and nucleolin bind LINE-1 RNA and function as host factors to modulate retrotransposition. Nucleic Acids Res. 2012, 41: 575-585.
Coufal NG, Garcia-Perez JL, Peng GE, Marchetto MC, Muotri AR, Mu Y, Carson CT, Macia A, Moran JV, Gage FH: Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells. Proc Natl Acad Sci U S A. 2011, 108: 20382-20387. 10.1073/pnas.1100273108.
Evans JD, Peddigari S, Chaurasiya KR, Williams MC, Martin SL: Paired mutations abolish and restore the balanced annealing and melting activities of ORF1p that are required for LINE-1 retrotransposition. Nucleic Acids Res. 2011, 39: 5611-5621. 10.1093/nar/gkr171.
Martin SL, Bushman D, Wang F, Li PW, Walker A, Cummiskey J, Branciforte D, Williams MC: A single amino acid substitution in ORF1 dramatically decreases L1 retrotransposition and provides insight into nucleic acid chaperone activity. Nucleic Acids Res. 2008, 36: 5845-5854. 10.1093/nar/gkn554.
Branciforte D, Martin SL: Developmental and cell type specificity of LINE-1 expression in mouse testis: implications for transposition. Mol Cell Biol. 1994, 14: 2584-2592. 10.1128/MCB.14.4.2584.
Trelogan SA, Martin SL: Tightly regulated, developmentally specific expression of the first open reading frame from LINE-1 during mouse embryogenesis. Proc Natl Acad Sci U S A. 1995, 92: 1520-1524. 10.1073/pnas.92.5.1520.
Ergun S, Buschmann C, Heukeshoven J, Dammann K, Schnieders F, Lauke H, Chalajour F, Kilic N, Stratling WH, Schumann GG: Cell type-specific expression of LINE-1 open reading frames 1 and 2 in fetal and adult human tissues. J Biol Chem. 2004, 279: 27753-27763. 10.1074/jbc.M312985200.
Rosser JM: An W: L1 expression and regulation in humans and rodents. Front Biosci (Elite Ed). 2012, 4: 2203-2225.
Western PS, Miles DC, van den Bergen JA, Burton M, Sinclair AH: Dynamic regulation of mitotic arrest in fetal male germ cells. Stem Cells. 2008, 26: 339-347. 10.1634/stemcells.2007-0622.
Lesch BJ, Page DC: Genetics of germ cell development. Nat Rev Genet. 2012, 13: 781-794. 10.1038/nrg3294.
Acknowledgements
We thank all the An lab members for helpful discussions. This work was funded by the College of Veterinary Medicine of Washington State University, which did not have any role in the study design, data collection, analysis and interpretation of data, or in the writing of the article and the decision to submit it for publication.
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Authors’ contributions
YX performed the studies and drafted the manuscript. LM, ZIv, ZIz, and SM contributed reagents and provided scientific consultation. WA directed the studies and finalized the manuscript. All authors read and approved the final manuscript.
Electronic supplementary material
13100_2012_73_MOESM2_ESM.pdf
Additional file 2: Figure S1.: Dose-dependent induction of L1 retrotransposition in HeLa Tet-ORFeus cells. HeLa Tet-ORFeus cells were seeded in 96-well plate at 3,000 cells/well and cultured in the presence of different concentrations of doxycycline. Fluc and Rluc were measured after 48 h incubation. Error bars represent mean±SE (n=4). At high doses in the range of 6.3 to 100 ng/mL, both Rluc and Fluc showed no deviation from background readings. Retrotransposition, as indicated by the Fluc signal, was detected in cells treated with lower doses of doxycycline. In particular, retrotransposition reached 120-fold above background under 0.8 ng/mL of doxycycline (P <0.001) and 3,600-fold above background in doxycycline-free medium (P <0.001). As expected, the level of retrotransposition was correlated with PTight promoter activity, which was measured by Rluc. At 0.8 ng/mL of doxycycline, Rluc was induced to five-fold above background (P <0.05); in doxycycline-free medium, Rluc was induced to 620-fold above background (P <0.001). It should be noted that Fluc signal had increased above background at 1.6 to 3.2 ng/mL concentrations while Rluc activity remained undetectable. This discrepancy is likely due to the known higher sensitivity of Fluc than Rluc. Thus, our data showed that L1 retrotransposition efficiency in HeLa Tet-ORFeus cells could be induced by reducing or eliminating doxycycline from the culture medium. (PDF 57 kb) (PDF 58 KB)
13100_2012_73_MOESM3_ESM.pdf
Additional file 3: Figure S2.: Induction of L1 retrotransposition in HeLa Tet-ORFeus cells after multiple passages. HeLa Tet-ORFeus cells were maintained in the presence of 100 ng/mL doxycycline and passaged in approximately every 3 days. Aliquots of cells from each of the 10 continuous passages (P0 to P9) were seeded in the presence (Dox+, shown in panel A) or absence (Dox-, shown in panel B) of 100 ng/mL doxycycline. Fluc and Rluc were measured 48 h after seeding. Note very different scales are used for the two panels. Panel A shows that Fluc and Rluc signals from uninduced cells are always below 1,000 relative light units, which represent the assay background and are comparable to readings from empty wells. Cells from most passages were seeded at the density of 3,000 to 5,000 cells/well in 96-well plates. The only exception was cells from P2, which were seeded at a much higher density (40,000 cells/well) in a 96-well plate; this suboptimal seeding density may explain the much reduced Fluc and Rluc signals in P2 cells in the absence of doxycycline (panel B). Error bars represent mean±SE (n=4 or 6). In summary, for cells from all passages tested, Fluc and Rluc were completely inhibited by doxycycline but were consistently induced upon doxycycline withdrawal. (PDF 67 kb) (PDF 67 KB)
13100_2012_73_MOESM4_ESM.pdf
Additional file 4: Figure S3.: Dose-dependent inhibition of L1 retrotransposition by 3TC in HeLa Tet-ORFeus cells. HeLa Tet-ORFeus cells were seeded in a 96-well plate at 3,000 cells/well and cultured in the presence of different concentrations of 2′,3′-dideoxy-3′-thiacytidine (3TC; 0, 0.016, 0.08, 0.4, 2, or 10 μM) and with (Dox+) or without (Dox-) 100 ng/mL doxycycline. Fluc signals were measured after 48 h incubation with Promega ONE-Glo Luciferase Assay System. Error bars represent mean±SE (n=8). Two-tailed Student’s t-test was used to compare Fluc signals from 3TC-treated cells to non-3TC-treated cells, respectively, for Dox+ and Dox- conditions; resulting P values are indicated (**P <0.01, ***P <0.001). (PDF 51 kb) (PDF 52 KB)
13100_2012_73_MOESM5_ESM.pdf
Additional file 5: Figure S4.: Effect of cell-cycle arrests on Rluc and Fluc activities in HeLa Tet-ORFeus cells. The underlying data are the same as in Figure 3B but Rluc and Fluc data are separately graphed to highlight the difference among experimental conditions. Raw Rluc (panel A) and Fluc (panel B) readings are shown underneath the x-axis labels. They were normalized by cell viability first and then to those from Dox- cells and plotted. Error bars represent mean±SE (n=6). Pairwise two-tailed Student’s t-test was used to compare Rluc or Fluc signals between treatment groups; resulting P values are indicated (*P <0.05, **P <0.01, ***P <0.001). (PDF 91 kb) (PDF 92 KB)
13100_2012_73_MOESM6_ESM.pdf
Additional file 6: Figure S5.: Cell-cycle progression after HeLa Tet-ORFeus cells released from double-thymidine block. HeLa Tet-ORFeus cells were synchronized at G1/S phase and subsequently allowed to cycle by incubating in complete medium in the absence of thymidine and doxycycline. The time of release from thymidine block was designated as time 0. Cells were collected every 4 h and subjected to cell-cycle analysis. The distribution of cell-cycle phases (G1, S, and G2/M) was plotted over time. The first column ‘C’ denotes a control population of unsynchronized cells. Note cells progressed through the first full cycle (from S, G2/M, G1 to the next S) within the first 20 h relatively synchronously but the second cycle was not as synchronous as the first cycle. (PDF 75 kb) (PDF 76 KB)
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**e, Y., Mates, L., Ivics, Z. et al. Cell division promotes efficient retrotransposition in a stable L1 reporter cell line. Mobile DNA 4, 10 (2013). https://doi.org/10.1186/1759-8753-4-10
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DOI: https://doi.org/10.1186/1759-8753-4-10