Cell division promotes efficient retrotransposition in a stable L1 reporter cell line
© Xie et al.; licensee BioMed Central Ltd. 2013
Received: 8 November 2012
Accepted: 8 February 2013
Published: 6 March 2013
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.
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.
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.
KeywordsCell-cycle arrest Cell-cycle synchronization Cell division Dual-luciferase assay LINE-1 Non-LTR retrotransposon Nuclear import Tetracycline-inducible promoter Transcription Stable cell line
A comparison of the methods and findings from three studies
Kubo et al.
Shi et al.
Embedded in a helper-dependent adenovirus
Embedded in an SB DNA transposon
Mouse phosphoglycerate kinase-1
A native human L1 promoter (5′UTR)
Bi-directional tetracycline-inducible promoter
Human L1 RP
Human L1 LRE3
Synthetic mouse L1 ORFeus
Transient transfection with nucleofector
Stably integrated by SB100X
L1 expression detection
L1 RNA (RT-PCR)
Co-expressed Rluc; L1 ORF1p
Cells for cell-cycle arrest assay
Human glioma (Gli36)
Human fetal lung fibroblast (IMR-90); human cervical carcinoma (HeLa)
HeLa Tet-ORFeus stable cell line
Cell-cycle arrest experiments and observed effects on retrotransposition
(i) G0 arrest ➜ complete inhibitiona;
(i) G1, S, G2, or M arrest ➜ strong inhibitionc
(i) S, or S+G2/M arrest ➜ strong inhibition;
(ii) G1/S arrest ➜ partial inhibitionb
(ii) Cell-cycle synchronized cells ➜reduced retrotransposition if cells divided one fewer cycle
Conclusion(s) regarding to the role of cell division
L1 retrotransposition can occur in non-dividing cells
Cell division is required for L1 retrotransposition; L1 transcription is the limiting step
Cell division promotes efficient L1 retrotransposition; the inhibitory effect of cell-cycle arrest on retrotransposition cannot be explained by reduced L1 transcription alone
Role of active nuclear import
L1 RNP can be actively imported into the nucleus
An active nuclear import mechanism is a possible explanation for residual retrotransposition in cell-cycle arrested cells
Development of a stable HeLa Tet-ORFeus cell line
Control of L1 retrotransposition in HeLa Tet-ORFeus cells by doxycycline
Cell-cycle arrest inhibits L1 retrotransposition
L1 retrotransposition in synchronized HeLa Tet-ORFeus cells
In summary, our data provide additional support for an important role of cell division in L1 retrotransposition and argue that restricting the accessibility of L1 RNP to nuclear DNA could be a post-translational regulatory mechanism for retrotransposition (Table 1). As compared with the two previous studies[8, 9], our experimental approach has several advantages for assessing the role of cell division in retrotransposition. First, the dual-luciferase system provided an efficient means of simultaneous quantification of both L1 expression and L1 retrotransposition. Second, the use of an inducible, integrated reporter not only allowed us to avoid variation in gene transfer efficiency between experimental conditions but also to better resemble the replication cycle of endogenous L1 elements, which express from chromosomal rather than episomal DNA. Indeed, it allowed us to separate two layers of regulation in cell-cycle arrested cells: one layer is at the transcriptional level, which was highlighted by Shi et al.; the other layer is downstream and independent of L1 transcription, as indicated by both Rluc signals and ORF1p levels (discussed below). Lastly, our inducible system permitted the comparison of retrotransposition in synchronized cell populations where the major difference was the number of mitoses completed.
Integrating our data and those of previous studies[8, 9], we propose that active cell division promotes retrotransposition. All three studies showed strong inhibition of retrotransposition when cells were arrested. Shi et al. analyzed L1 RNA levels in their assay system and attributed the inhibitory effect on retrotransposition largely to reduced L1 transcription (Table 1). The assay systems used by this study and Kubo et al. enabled retrotransposition at larger dynamic ranges, permitting the evaluation of additional layers of regulation. In this study, after Fluc signals were normalized to the co-expressed Rluc, we observed an additional 10-fold reduction in retrotransposition in cell-cycle arrested HeLa Tet-ORFeus cells and a 2.6-fold reduction in synchronized cells undergoing one fewer round of cell division. Thus, after factoring in the effect of drug treatment on L1 expression, our data support an important role of cell division in promoting efficient L1 retrotransposition in a manner independent of L1 expression. It is noteworthy that, even when the variable infection rate was not taken into consideration, Kubo et al. found a three-fold reduction of retrotransposition in G1/S arrested cells in addition to a 16-fold reduction of retrotransposition in G0 arrested Gli36 cells (Table 1). On the other hand, both Kubo et al. and the current study showed substantial retrotransposition in cell-cycle arrested cells (for two of the three inhibitors tested, we observed statistically significant Fluc signals at approximately 10-fold above the assay background). Currently, we cannot exclude the possibility that the residual retrotransposition observed in arrested cell populations in both studies originates from a minor population of cycling cells. An alternative explanation for such residual retrotransposition is that L1 retrotransposition may also be facilitated by a yet uncharacterized active nuclear import mechanism (Table 1). Indeed, the control experiments performed by Kubo et al. in G1/S arrested cells, showing differential transduction by retroviral and lentiviral vectors, support the presence of an active nuclear import mechanism for L1 retrotransposition. It is noteworthy that some non-LTR (long terminal repeat) retrotransposons have evolved active nuclear import strategies for their propagation in respective host species. A precedent of active nuclear import has been reported for the telomeric repeat-specific SART1 retrotransposon from Bombyx mori: its ORF1p contains functional nuclear localization signals (NLSs), which are required for active retrotransposition. Thus far, no NLS has been reported in mammalian L1 proteins although both ORF1 and ORF2 proteins have highly basic regions, which is a common feature of nuclear localized proteins. Alternatively, it is possible active nuclear import is mediated by other host-derived components of L1 RNP. Recently, two poly(A) binding proteins, PABPN1 and PABPC1, were found to be associated with L1 RNP. Of particular interest, PABC1 was found to be critical for RNP formation; as it can shuttle between the cytoplasm and the nucleus, it would be interesting to determine whether PABPC1 mediates RNP nuclear import.
A caveat shared by all three studies is that the role of cell division in retrotransposition was mainly assessed in cancerous cell lines (Table 1; but note Shi et al. also tested normal human fetal lung fibroblasts). Additionally, two of these studies ( and the current study) used non-native promoters to drive L1 expression (Table 1), which precludes the study of the endogenous transcriptional regulation of L1 with these systems. Nevertheless, a unifying view from these and other studies of L1 variants, mutations, and host factors ([15–19] and citations therein) is that retrotransposition is not a simple function of L1 expression. By extension, the level of L1 expression cannot be equated with the frequency of retrotransposition and the evaluation of retrotransposition should take into consideration the cell-cycle status. For example, in normal individuals, endogenous L1 expression has only been confirmed at protein level in testicular and ovarian tissues ([20–22]; reviewed in). It is noteworthy that L1 ORF1p is detected in two distinct stages of male germ cell development, namely, in gonocytes (embryonic stage) and in meiotic/post-meiotic germ cells (prepuberal and through adulthood)[20–22]. However, during both stages cell division is limited: gonocytes are mitotically arrested in G0 phase while spermatocytes only divide twice before becoming haploid spermatids. Similarly, in the female germline, L1 ORF1p is detected during the meiotic prophase I in embryonic oocytes, which are subsequently arrested in the diplotene stage of the meiotic prophase I and do not divide until puberty. Therefore, both male and female germline development may have been programmed in a way that restricts excessive retrotransposition by avoiding frequent nuclear membrane breakdown when L1 is expressed. Thus, to understand the developmental timing of retrotransposition, it is imperative to measure the level of retrotransposition directly.
Fluc disrupted by an antisense intron
Long interspersed element type one
Nuclear localization signal
Open reading frame 1 protein
Quantitative polymerase chain reaction
Sleeping Beauty DNA transposon
Tetracycline-controlled transactivator advanced
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.
- Cordaux R, Batzer MA: The impact of retrotransposons on human genome evolution. Nat Rev Genet. 2009, 10: 691-703. 10.1038/nrg2640.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- Martin SL: Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells. Mol Cell Biol. 1991, 11: 4804-4807.PubMed CentralView ArticlePubMedGoogle Scholar
- Hohjoh H, Singer MF: Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA. EMBO J. 1996, 15: 630-639.PubMed CentralPubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- Han JS, Boeke JD: A highly active synthetic mammalian retrotransposon. Nature. 2004, 429: 314-318. 10.1038/nature02535.View ArticlePubMedGoogle Scholar
- Xie 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.PubMed CentralView ArticlePubMedGoogle Scholar
- 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 Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009, 41: 753-761. 10.1038/ng.343.View ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- Rosser JM: An W: L1 expression and regulation in humans and rodents. Front Biosci (Elite Ed). 2012, 4: 2203-2225.PubMed CentralView ArticleGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- Lesch BJ, Page DC: Genetics of germ cell development. Nat Rev Genet. 2012, 13: 781-794. 10.1038/nrg3294.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.