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. 2008 Feb;36(2):648-65.
doi: 10.1093/nar/gkm1045. Epub 2007 Dec 10.

Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells

Alexander Kirilyuk  1 , Genrich V Tolstonog, Annette Damert, Ulrike Held, Silvia Hahn, Roswitha Löwer, Christian Buschmann, Axel V Horn, Peter Traub, Gerald G Schumann
Affiliations

Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells

Alexander Kirilyuk et al. Nucleic Acids Res. 2008 Feb.

Abstract

LINE-1 (L1) is a highly successful autonomous non-LTR retrotransposon and a major force shaping mammalian genomes. Although there are about 600 000 L1 copies covering 23% of the rat genome, full-length rat L1s (L1Rn) with intact open reading frames (ORFs) representing functional master copies for retrotransposition have not been identified yet. In conjunction with studies to elucidate the role of L1 retrotransposons in tumorigenesis, we isolated and characterized 10 different cDNAs from transcribed full-length L1Rn elements in rat chloroleukemia (RCL) cells, each encoding intact ORF1 proteins (ORF1p). We identified the first functional L1Rn retrotransposon from this pool of cDNAs, determined its activity in HeLa cells and in the RCL cell line the cDNAs originated from and demonstrate that it is mobilized in the tumor cell line in which it is expressed. Furthermore, we generated monoclonal antibodies directed against L1Rn ORF1 and ORF2-encoded recombinant proteins, analyzed the expression of L1-encoded proteins and found ORF1p predominantly in the nucleus. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia.

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Figures

Figure 1.
Figure 1.
Full-length L1Rn elements are transcribed in RCL cells. (A) Structure of L1Rn. A functional full-length L1Rn element is characterized by two ORFs flanked by 5′ and 3′ UTRs. The bipartite 5′ UTR consists of a monomer, which can be tandemly repeated, and a non-repeated tether (t). The most 5′ monomer is only partially duplicated (black truncated box with arrowhead) in all genomic rat elements identified so far. Horizontal arrows indicate the binding sites of the oligonucleotides Pr-L1Rn1 and Pr-L1Rn2 used to amplify cDNAs generated from full-length transcripts. The binding site of the 500-bp digoxigenin-labeled probe used to detect L1-specific transcripts is localized at the 5′ end of ORF2 (black bar). Open bars represent ORF1- and ORF2-encoded polypeptides against which monoclonal antibodies were raised. The polypeptides are covering amino acid positions 144–402 and 292–480 of ORF1p and ORF2p, respectively (accession no. DQ100480). LPR, length polymorphism region; GrPPT, G-rich polypurine tract; An, A-rich 3′ tract; EN, endonuclease; RT, reverse transcriptase; C, cysteine-rich motif. (B) L1Rn transcriptional products in RCL cells. PolyA+ RNA was isolated from RCL cells that had reached the maximum population density of ∼106 cells/ml (lane 2) and from cells that were UV-irradiated (lane 1). Two microgram of each RNA were separated by agarose gel electrophoresis and subjected to northern blot analysis using a 500-bp probe (Figure 1A). (C) Schematic structures of 10 L1Rn cDNAs synthesized from poly(A)+ RNA from UV-irradiated RCL cells. cDNAs are flanked by primer sequences Pr-L1Rn1 and Pr-L1Rn2. Names of the resulting cDNAs are listed on the left, while accession numbers are specified on the right. Termination codons within ORF2 sequences are indicated by vertical arrows. Two deletions in the ORF2-coding region of cDNA L1-17 covering 103 and 220 nts, respectively, are indicated by interrupted bars.
Figure 2.
Figure 2.
Analyses of cDNA-encoded ORF1p classes and intergenic regions (IGR). (A) Alignment of the N-terminal third of the cDNA-encoded ORF1p sequences including the hypervariable region. Amino acid substitutions and insertions relative to the L1mlvi2-rn14 reference sequence (accession no. U87602) are in bold and underlined. Lysine deletion at position 59 (asterisk) and a seven-amino acids insertion (black bar) are hallmarks diagnostic for the I-21pmod-type class. Twenty-two-amino acids tandem repetitions are indicated by arrows. cDNA names and ORF1 classes to which the encoded protein sequences can be allocated, are listed. (B) Alignment of the non-coding IGR of the functional L1Rn-cDNA L1-3 with IGRs of functional L1Md (accession no. AY053455) and L1Hs (accession no. M80343) elements. The sequences start in the poorly conserved C-terminal regions of ORF1 and extend through the first ATG of ORF2. ORF1 stop codons as well as stop codons that are localized in the IGRs and are in frame with ORF1 are underlined; ATG start codons of each ORF2 are italicized.
Figure 3.
Figure 3.
The L1Rn element L1-3 retrotransposes at high frequency in HeLa cells. (A) L1Rn retrotransposition reporter constructs. The human L1.3 sequence of pJM101/L1.3 (flanked by NotI and BstZ17I sites) was replaced by the L1-3 and L1-13 cDNAs (flanked by NotI and EcoRV sites), respectively. The resulting reporter constructs are pAD5/L1-3 and pAD6/L1-13. (B) Results of the retrotransposition reporter assay. G418R foci were fixed to 6-well dishes and stained with Giemsa solution. At the timepoint of transfection with pAD5/L1-3 (well # 2, 3, 5, 6), pAD6/L1-13 (well # 8, 9) and the control constructs pJM101/L1RP (+C, well # 4, 7) and pJM101/L1RPH230A (–C, well # 1), respectively, each well was containing 2 × 105 HeLa cells. The negative control construct pJM101/L1RPH230A is expressing an EN-defective allele of L1RP. (C) Structures of three L1-3-derived genomic de novo insertions. Both post-integration sites and pre-integration sequences are shown. The nucleotide position of the 5′ truncation within the L1 reporter cassette is indicated. Numbering corresponds to the nucleotide position in the active L1-3 element (accession no. DQ100473). Insertion lengths range from 1.47 to 4.01 kb. All insertions are demonstrating the structural hallmarks of retrotransposition by TPRT as they are truncated at their 5′ ends, end in a poly(A)+ tail, and are flanked by short TSDs (red) of 3–15 bp. All three insertions integrated into sequences (blue) that are preferred sites for mammalian L1 integration in vivo (as denoted by the vertical arrows). The L1 endonuclease cleavage site on the bottom strand is indicated in blue. Notably, insertion #1 is truncated 15 nucleotides upstream of the 3′ end of the mneoI indicator cassette (pos. 5976). This insertion also resulted in five extra nucleotides (indicated in green) at the left junction between L1-mneo and genomic DNA.
Figure 4.
Figure 4.
Functional L1Rn elements retrotranspose in RCL suspension cells. (A) Growth curves of differently transfected RCL cells. Cells were transfected with pJM101/L1RP, pJM101/L1RPH230A, pAD5/L1-3 and pAD6/L1-13, separately, and were grown in hygromycin-containing medium for the following 29 days. Transfection experiments were performed in triplicate. In each of these experiments identical numbers of hygromycin-resistant cells (HygR) expressing the different L1 retrotransposition reporter constructs were then plated in medium containing G418 separately, and selected for G418 resistance for another 28 days. Live cells were counted by trypan blue exclusion. Transfection of RCL cells with pAD6/L1-13 or with the negative control construct pJM101/L1RPH230A led to the elimination of RCL cells after 18-25 days of G418 selection. By contrast, transfection with pJM101/L1RP and pAD5/L1-3 was leading to G418R cells that continued to grow at a density of 1.3–1.5 × 106 cells/ml. Each curve represents arithmetic means of cell densities resulting from three independent transfection experiments performed with the same reporter plasmid. Error bars were calculated but are barely visible because the courses of each of the three growth curves are almost identical. (B) When cell densities of pJM101/L1RP- and pAD5/L1-3-transfected suspension cultures reached their maximum after 26–30 days of G418 selection in all six independent transfection experiments, neoR-cells of each suspension culture were harvested to inoculate G418-containing medium at a density of 2 × 103 cells/ml in order to monitor growth curves of the differently transfected RCL cells. Rapid exponential growth without cell loss is demonstrating proliferation of pJM101/L1RP- and pAD5/L1-3-transfected and neoR-selected RCL cells in all three independent experiments. Error bars were calculated but are not visible as the courses of all six growth curves are almost identical. (C) PCR assay for correct splicing of the artificial intron in the neoR gene after retrotransposition from the reporter plasmids pJM101/L1RP and pAD5/L1-3. Genomic DNA was extracted from the differentially transfected RCL cells after 28 days of G418 selection, and used as template for PCR (lanes 3 and 4). This strategy allows distinction of the spliced and reverse-transcribed form of the neoR gene (870 bp PCR product) from the original unspliced form (1773 bp PCR product) expressed from the reporter construct and confirmed integration into the genome via authentic retrotransposition (80,81). PCR was performed on pAD5/L1-3 plasmid DNA mixed with genomic RCL DNA (lane 1), pSV2neo (BD Biosciences) mixed with genomic RCL DNA (lane 2), genomic DNA from pJM101/L1RP–transfected RCL cells (lane 3), and genomic DNA from pAD5/L1-3 –transfected RCL cells (lane 4); lanes 5 and 6, negative control genomic DNAs from untransfected RCL and HeLa cells, respectively; lane 7, minus template control; lane M, 1-kb Plus DNA ladder (Invitrogen).
Figure 5.
Figure 5.
Monoclonal anti-ORF1pΔ1–143 antibodies (rG24) specifically recognize ORF1-encoded proteins. (A) Recombinant 33-kDa ORF1pΔ1–143 was used for the generation of monoclonal antibodies. MW, molecular weight marker. (B) Immunoblot detection of 10 ng of purified His-tagged ORF1pΔ1–143 with rG24 (lane 1) and anti-His-tag antibodies (lane 2). (C) Immunoblot analysis examining the specificity of the generated monoclonal anti-ORF1p antibody. Sixty microgram whole cell extracts from pORF1pΔ1–143-IRESpuro (lane 2)- or pIRESpuro-transfected (lane 3) REF cells were loaded on a 12% SDS–PAA gel. In contrast to the parental empty expression vector pIRESpuro, pORF1pΔ1–143-IRESpuro is expressing the N-terminally truncated 33-kDa L1Rn-ORF1 protein, which is detected by the rG24 antibody. As a loading control the membrane was stripped and incubated with an anti-β-actin antibody; lane 1, 10 ng purified ORF1pΔ1–143 protein; (D–F) Confocal images of REF cells transfected with ORF1p- (D, E) and ORF1pΔ1–143- (F) expressing constructs. ORF1-encoded proteins and vimentin were stained using monoclonal rG24 (green) and polyclonal anti-vimentin antibodies (red), respectively. The co-localization channel (yellow) was generated using the ImarisColoc module. Cytoplasmic ORF1p aggregates co-localize with vimentin filaments partially. Scale bar—10 µm.
Figure 6.
Figure 6.
L1Rn ORF1p is able to form filaments that co-align with vimentin filaments. (A and B) In situ overlay of recombinant L1-21 ORF1p onto PFA-fixed and Triton X-100-extracted MEFs. Immunofluorescence images of L1Rn ORF1p (A) and vimentin (B) are shown in green and red colors, respectively. Each panel represents the 3D reconstruction of xy confocal sections. (C) ORF1p and vimentin filaments co-align partially. The co-localization channel (yellow) was generated using the ImarisColoc module. Scale bar—10 µm. (D) Dot blot overlay assay demonstrating interaction of ORF1p with vimentin. ORF1p was immobilized onto a nitrocellulose membrane (dots I–IV). Each ORF1p dot contained 2 μg of protein. After blocking, immobilized ORF1p was overlaid with an excess of soluble vimentin, α-tubulin or β-actin, respectively. Incubation was carried out for 60 min. In order to control for successful immobilization and immunoblotting procedure, 50 ng of soluble vimentin, α-tubulin and β-actin were immobilized on the nitrocellulose membrane in parallel (dots V–VII). Membranes were washed subsequently and submitted to immunoblot analyses with anti-vimentin, anti-α-tubulin or anti-β-actin antibodies, respectively, due to the schematic shown. In contrast to α-tubulin and β-actin, only vimentin can be detected with its specific antibody on the nitrocellulose membrane, after overlaying ORF1p dots with the respective IF protein (dots I–III). This is indicating that only vimentin is binding to ORF1p.
Figure 7.
Figure 7.
Endogenous L1Rn ORF1-encoded proteins are predominantly localized to the nucleus of RCL cells. (A) Silver staining of L1Rn ORF1-encoded proteins immunoprecipitated from RCL cell lysates with RG24 IgG. Molecular weights of the two proteins of ∼46 and ∼40 kDa correspond to the theoretical masses of L1Rn-ORF1 protein classes I-21a and I-21p, respectively. (B) Confocal images of RCL cells immunostained with the rG24 antibody (red). (C) Secondary antibody control. Confocal images were merged with a differential interference contrast (DIC) micrograph to show the outlines and nuclei of cells. Outlines of nuclear envelopes are emphasized in yellow. Scale bar—10 µm. (D) Immunoblot analysis of subcellular fractions of RCL cells. Three different protein fractions (C, cytosolic; N, nuclear; CS, cytoskeletal matrix proteins) were separated by SDS–PAGE and blotted onto nitrocellulose. The membrane was incubated consecutively with anti-ORF1p- antibody (upper panel) and with an antibody directed against the nucleus-specific laminB protein (lower panel), which shows efficient separation of the subcellular fractions.
Figure 8.
Figure 8.
The monoclonal anti-ORF2pM292–480 antibody 2H9 detects endogenous ORF2-encoded proteins. (A and B) Confocal images of HeLa cells transfected with either ORF2p-expressing pEF6-rORF2 (A) or the parental empty vector pEF6V5-His (B) and stained with the 2H9 IgM (red). (C) Immunoblot analysis examining the specificity of the generated monoclonal antiORF2p antibody. Fifty micrograms of whole cell extract from pEF6-rORF2 (lane 2)- or pEF6V5-His-transfected (lane 1) REF cells were loaded on a 6% SDS–PAA gel. In contrast to the parental empty expression vector pEF6V5-His, pEF6-rORF2 is expressing intact L1Rn ORF2p. As a loading control the membrane was stripped and incubated with an anti-α1-catenin antibody (lower panel). (D) Immunoblot analysis of RCL cell extract with monoclonal 2H9 antibody reveals two bands representing 150- and 90-kDa polypeptides. The 150-kDa band corresponds to the theoretical molecular mass of L1Rn ORF2p. (E) Silver staining of L1Rn ORF2-encoded proteins immunoprecipitated from RCL cell lysates with 2H9 IgM. Two polypeptides with apparent molecular masses of 150 and 90 kDa were detected and are consistent with the proteins detected by immunoblot analyses. Scale bar—10 µm.
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