High Frequency Retrotransposition in Cultured Mammalian Cells

Moran, John V.; Holmes, Susan E.; Naas, Thierry; DeBerardinis, Ralph J.; Boeke, Jef D.; Kazazian, Haig H.

doi:10.1016/s0092-8674(00)81998-4

Cited by 947 publications

(1,466 citation statements)

References 57 publications

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“…In general, this expression decreases with the number of passages, and only 30% of RASFs in culture remained positive at passages 7-8. In cultures transfected with the pGL3-TS3 1-15 luciferase reporter plasmid, we observed an up to 2.4-fold increase in luciferase activity in hypomethylated RASFs after To confirm a direct and specific interaction between the TS3 sequence and transiently expressed L1-ORF1p in the cellular environment, we cotransfected the pGL3-TS3 1-15 luciferase reporter plasmid and the episomal mammalian expression plasmid pJM105 (14). In pJM105, a CMV promoter controls expression of the L1.2 element that is tagged with a retrotransposition reporter gene and carries a missense mutation in the RT domain of ORF2.…”

Section: Resultsmentioning

confidence: 88%

“…Such DNA intermediates are not exclusively generated during DNA replication. DNA viruses and retroviruses are additional sources of ssDNA species that could accumulate if a degrading enzyme is lacking (2,14). The accumulation of endogenous retroviruses triggers the elevated production of type I interferons, which are highly coexpressed with Toll-like receptor 3 (TLR-3)/TLR-7 in RA synovial tissue.…”

Section: Discussionmentioning

confidence: 99%

“…In the second experiment, we cotransfected the luciferase reporter plasmid pGL3-TS3 1-15 (in which luciferase expression is under control of the p38␦ MAPK promoter) with the L1-ORF1p expression plasmid pJM105 (14). In pJM105, the original retrotranspositioncompetent L1 element L1.2, which is characterized by a missense mutation in L1-ORF2, was set under transcriptional control of the cytomegalovirus (CMV) promoter of the mammalian episomal expression vector pCEP4 (Invitrogen) (14). Transfection was performed 1 day after cell passage (adjusted to 10 5 cells/TS45 culture flask), using 1 ml DMEM without FCS, a 1:1 ratio of pGL3 (in g) to CEP4 (in g), and a 1:3 ratio of vectors (in g) to FuGene 6 transfection reagent (in l).…”

Section: Methodsmentioning

confidence: 99%

“…Single-stranded DNA derived from endogenous retroelements, such as L1, was observed to accumulate in Trex-1-deficient cells. One role of Trex-1 is to prevent the accumulation of DNA derived from endogenous retroelements (3,14). We evaluated whether the expression levels of Trex-1 messenger RNA (mRNA) and protein are down-regulated in RASFs, which are characterized by an increased level of L1 gene products.…”

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confidence: 99%

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Trex‐1 deficiency in rheumatoid arthritis synovial fibroblasts

Neidhart

Karouzakis

Schumann

2010

Arthritis & Rheumatism

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Objective. To explore whether the increased expression of long interspersed nuclear element 1 (LINE-1; L1) messenger RNA (mRNA) and protein in rheumatoid arthritis synovial fibroblasts (RASFs) is associated with decreased expression of Trex-1, an exonuclease involved in the metabolization of L1 DNA: RNA hybrids.Methods. Chromatin immunoprecipitation was used to detect L1-related p40 protein (L1-ORF1p) binding sequences in RASFs. Luciferase activity was measured in the synovial fibroblasts following cotransfection of the episomal plasmid with pJM105 expressing L1-ORF1p and pGL3-TS3 carrying the target sequence for L1-ORF1p. This luciferase reporter assay was used to compare the activity between RASFs and osteoarthritis synovial fibroblasts (OASFs) and to assess correlations of luciferase activity with the expression of Trex-1 measured by flow cytometry. The expression of Trex-1 mRNA and protein was also compared using real-time polymerase chain reaction, immunohistochemistry, and Western blot analyses. The role of Trex-1 in the L1-ORF1p-mediated luciferase activity assay was studied using interfering RNAs (iRNA) and a Trex-1 expression vector.Results. Increased luciferase activity occurred after cotransfection of synovial fibroblasts with pJM105 and pGL3-TS3. L1-ORF1p activity was increased in RASFs as compared with OASFs, and this was correlated inversely with the expression of Trex-1. Levels of Trex-1 mRNA and protein were lower in RASFs than in OASFs. After transfection of the L1 expression plasmid, Trex-1 mRNA levels increased in OASFs, but not in RASFs. The addition of iRNA against Trex-1, however, resulted in an enhancement of L1-ORF1p activity in OASFs to the levels measured in RASFs. Overexpression of Trex-1 inhibited 5-azacytidine-induced expression of p38␦ MAPK, a gene carrying the TS3 sequence. Conclusion. The deficiency of Trex-1 in RASFs allows a longer half-life of gene products encoded by active endogenous L1 retrotransposons. This pathway may play a role in diseases in which the cells exhibit a "spontaneous" aggressive behavior.Trex-1 is the major 3Ј35Ј DNA exonuclease in mammalian cells, and mutations in the human trex-1 gene can cause Aicardi-Goutières syndrome (1), a disease that is characterized by perturbed immunity. Such mutations also appear to be involved in systemic lupus erythematosus (SLE) (2), in which the failure to process intermediates of nucleic acid metabolism can result in the activation of uncontrolled autoimmunity. Trex-1 prevents the accumulation of single-stranded DNA (ssDNA) fragments derived from endogenous long interspersed nuclear element 1 (LINE-1; L1) retrotransposons (3), the expression of which has been reported in rheumatoid arthritis synovial fibroblasts (RASFs) (4). The promoter of the L1 retroelement is hypomethylated in RASFs (5), reflecting global DNA hypomethylation. The L1-related p40 protein (L1-ORF1p) has nucleic acid binding properties and favors the transcription of specific genes (6). The L1-encoded p150 protein (L1-ORF2p) has a reverse transcription acti...

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Section: Resultsmentioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Trex‐1 deficiency in rheumatoid arthritis synovial fibroblasts

Neidhart

Karouzakis

Schumann

2010

Arthritis & Rheumatism

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“…Retrotransposition-competent cell lines have greatly assisted in understanding the mechanism of non-LTR retrotransposition in vivo [6][7][8][9][10] . Here we report the first such system in a parasitic protist E. histolytica.…”

Section: Discussionmentioning

confidence: 99%

Recombinant SINEs are formed at high frequency during induced retrotransposition in vivo

et al. 2012

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non-long terminal repeat Retrotransposons are referred to as long interspersed nuclear elements (LInEs) and their non-autonomous partners are short interspersed nuclear elements (sInEs). It is believed that an active sInE copy, upon retrotransposition, generates near identical copies of itself, which subsequently accumulate mutations resulting in sequence polymorphism. Here we show that when a retrotransposition-competent cell line of the parasitic protist Entamoeba histolytica, transfected with a marked sInE copy, is induced to retrotranspose, > 20% of the newly retrotransposed copies are neither identical to the marked sInE nor to the mobilized resident sInEs. Rather they are recombinants of resident sInEs and the marked sInE. They are a consequence of retrotransposition and not DnA recombination, as they are absent in cells not expressing the retrotransposition functions. This high-frequency recombination provides a new explanation for the existence of mosaic sInEs, which may impact on genetic analysis of sInE lineages, and measurement of phylogenetic distances.

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De novo retrotransposition of unbiased sequences in a human breast cancer cell clone

Werle-Schneider

Brevern

Sylla

et al. 1999

Genes Chromosom. Cancer

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It has been demonstrated recently that certain repetitive sequences and even expressed single‐copy genes are capable of retrotransposition, but little is known about the endogenous or exogenous modifiers of this process in human cells. Retrotransposition may contribute to gene inactivation and genetic instability in cancer development. We have used the human cell line MCF‐7 to generate a method for investigating de novo retrotransposition in breast cancer cells. The strategy employs a reporter construct transfected into MCF‐7 cells that encodes neomycin phosphotransferase gene (neoR) sequences interrupted by an intron derived from the gamma‐globin gene and sandwiched between two promoters in opposite orientation; the phosphotransferase is not produced in transfected cells expressing the plasmid until transposition via a spliced antisense neoR RNA intermediate has occurred, conferring a functional gene product and thereby resistance to G418. A stable transfectant line that showed presence of reporter plasmid DNA and expression of reporter antisense neoR was obtained and used to demonstrate spontaneous retrotransposition of neoR sequences: tester cells were subjected to selection in G418 medium, and neomycin‐resistant clones were isolated at a frequency of 10‐7. A simple PCR‐based prescreening of colonies fixed and stained in Petri dishes can be used to verify intronless neoR DNA. Expanded populations of G418‐resistant colonies were determined to be derived from reporter sequences that had transposed via an RNA intermediate by Southern blot genotyping. This experimental assay may be used for exploring endogenous and environmental factors that influence host cell‐mediated retrotransposition of unbiased cellular sequences in breast tumor cells. Genes Chromosomes Cancer 26:84–91, 1999. © 1999 Wiley‐Liss, Inc.

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High Frequency Retrotransposition in Cultured Mammalian Cells

Cited by 947 publications

References 57 publications

Trex‐1 deficiency in rheumatoid arthritis synovial fibroblasts

Trex‐1 deficiency in rheumatoid arthritis synovial fibroblasts

Recombinant SINEs are formed at high frequency during induced retrotransposition in vivo

De novo retrotransposition of unbiased sequences in a human breast cancer cell clone

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