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. 2005 Nov 25;354(2):246-57.
doi: 10.1016/j.jmb.2005.09.050. Epub 2005 Oct 4.

Nickel stimulates L1 retrotransposition by a post-transcriptional mechanism

Mohammed El-Sawy  1 , Shubha P Kale, Christine Dugan, Thuc Quyen Nguyen, Victoria Belancio, Heather Bruch, Astrid M Roy-Engel, Prescott L Deininger
Affiliations

Nickel stimulates L1 retrotransposition by a post-transcriptional mechanism

Mohammed El-Sawy et al. J Mol Biol. .

Abstract

Sequence studies of the human genome demonstrate that almost half of the DNA is derived from mobile elements. Most of the current retrotransposition activity arises from L1 and the L1-dependent, non-autonomous elements, such as Alu, contributing to a significant amount of genetic mutation and genomic instability. We present data demonstrating that nickel chloride, but not cobalt chloride, is able to stimulate L1 retrotransposition about 2.5-fold. Our data suggest that the stimulation occurs at a post-transcriptional level, possibly during the integration process. The effect of nickel on the cell is highly complex, limiting the determination of the exact mechanism of this stimulation. The observed stimulation of L1 retrotransposition is not due to a general increase in L1 transcription or an increase in the number of genomic nicks caused by nickel, but more likely caused by a decrease in DNA repair activities that influence the downstream events of retrotransposition. Our observations demonstrate the influence of environmental toxicants on human retroelement activity. We present an additional mechanism for heavy-metal carcinogenesis, where DNA damage through mobile element activation must be considered when dealing with genomic damage/instability in response to environmental agents.

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Figures

Figure 1
Figure 1
(a) Schematic of the L1 assay system. Top: RNA transcription is driven by the internal L1 promoter located in the 5′ untranslated region (UTR) or the CMV promoter. The construct contains the SV40 promoter in the 3′UTR in the “reverse” direction that will transcribe a neo gene containing a “forward” intron that blocks proper expression of the neomycin resistance. The intron interrupting the neomycin-resistance gene will be removed by splicing from RNA generated from the L1 or CMV promoter. In the L1 retrotransposition process, the RNA is reverse transcribed, followed by integration of the DNA into the genome. Bottom: The new L1 copy contains a functional neo gene. Only newly integrated copies that retrotransposed from the spliced L1 RNA will present neomycin resistance. Promoter and transcription orientations are indicated by black arrows. SD, splice donor; SA, splice acceptor; pA, SV40 polyadenylation signal. The neo gene in the opposite orientation relative to the L1 gene is shown as a hatched box. RNA is represented by thin lines with arrows to show the direction of transcription. Note that the Figure is not drawn to proportion. (b)Effect of NiCl2, (c) CoCl2 and (d) Paraquat dichloride on L1 retrotransposition activity in tissue culture. NeoR colonies from separate L1 transfections (black bar) treated with different doses of nickel chloride, cobalt chloride, or Paraquat dichloride are shown. An unrelated plasmid encoding neomycin-resistance was used as a transfection and toxicity control (open bar). The data are also shown adjusted for toxicity (hatched bar). Three independent assays in triplicate (n=9) were performed in HeLa cells and error bars indicate standard deviations. The treatment with 100 μM and 150 μM nickel showed a statistically significant difference from no treatment (Student’s t-test p<0.01(*)). Nickel stimulates L1 retrotransposition in a dose-dependent manner around 2.5-fold, but cobalt and Paraquat have no stimulatory effect.
Figure 2
Figure 2
Effect of NiCl2 on L1 promoter activity. (a) Schematic of the construct containing the L1.3 promoter (5′UTR sequence) cloned in front of the firefly luciferase gene. The L1 promoter is an internal promoter and transcription start is indicated by an arrow. (b) HeLa cells were transiently transfected with the L1 promoter or CMV-luciferase plasmid together with a plasmid expressing Renilla luciferase (pRL-CMV) used as a transfection control to which all results were normalized and expressed as percentage relative light units (RLU). The no-treatment control was used as 100%. Luciferase activity decreased about twofold when cells were treated with 150 μM NiCl2 (** Student’s paired t-test p< 0.00001 relative to no treatment control). (c) Effect of treatment with NiCl2 on L1 RNA levels. Evaluation of L1 expression levels was performed by Northern blot analysis of poly(A)-selected RNA from NIH 3T3 cells transiently transfected with the L1.3 Neo expression vector after treatment with 150 μM NiCl2 (Ni(+)). Untreated, transiently transfected cells were used as control (Ni(−)). Full-length L1.3 (FL1.3) and neomycin (Neo) mRNAs were detected by hybridization with randomly labeled Neo probe. Neomycin expression was used as an internal control to correct for transfection and loading variation. The ratio of the full-length L1 transcript/neo control transcript for treated and untreated cells is indicated.
Figure 3
Figure 3
(a) Evaluation of the effect of NiCl2 and CoCl2 on HeLa cell proliferation using the bromodeoxyuridine (BrdU) assay. Incorporation of BrdU was measured as relative light units (RLU) in response to various doses of NiCl2 (0, no treatment; 1, 50 μM; 2, 100 μM; 3, 150 μM; 4, 200 μM; 5, 250 μM; and 6, 300 μM) or CoCl2 (0, no treatment; 1, 12.5 μM; 2, 25 μM; 3, 50 μM; 4, 100 μM; and 5, 150 μM) as well as several control conditions (open bars) to evaluate whether cellular proliferation occurs in response to the heavy-metal treatments utilized. The no-treatment data were used to define 100% or baseline proliferation. Bars represent the averages of BrdU incorporation normalized relative to 100%, with the standard deviation shown as error bars. No significant increase in cell proliferation was seen in response to nickel or cobalt. (b) Cell-cycle distribution in response to NiCl2. HeLa cells were exposed to various doses of NiCl2, stained with propidium iodide and the cell-cycle was measured using fluorescence-activated cell sorting (FACS). Populations of cells in different stages of the cell-cycle (G1, G2 and S) are shown in the graph, together with the control. No effect on cell-cycle distribution in response to nickel was observed at doses below the cytotoxic threshold.
Figure 4
Figure 4
Effect of the presence of added MgCl2 on the NiCl2 retrotransposition activity. The L1 retrotransposition activity was evaluated after treatment for 48 h with: 0 or medium control; 150 μM NiCl2 (positive control); 150 μM MgCl2 and 1 mM MgCl2 (negative controls); 150 μM NiCl2+150 μM MgCl2; 150 μM MgCl2+1 mM MgCl2. NeoR colonies from separate L1 transfections are shown.
Figure 5
Figure 5
Endonuclease cleavage sites from L1 inserts. L1 inserts were recovered from the transiently transfected cells treated with 150 μM NiCl2 or 100 μM CoCl2 and from untreated cells (control). Comparison of the sequences of recovered L1 inserts and predicted pre-integration sites retrieved from the human genome database allowed for the characterization of the insertion site. The consensus for the L1 endonuclease site is shown in the box at the top. Note that the first cleavage (indicated by an arrow) occurs on the opposite strand shown in gray.
Figure 6
Figure 6
Model depicting the steps where nickel can potentially affect L1 retrotransposition intermediates. (a) Transcription: the first step in the retrotransposition process is the formation of a polyadenylated L1 transcript (broken line) from an L1 locus. It seems likely that increased expression of an L1 locus will result in a greater retrotransposition rate, although direct studies correlating transcript level and retrotransposition have not been carried out. Although heavy metals affect expression of many genes, L1 transcription is not increased by nickel exposure. (b) Generation of DNA nicks: it has been proposed that the L1 RNA, complexed with ORF2 protein that has endonuclease and reverse transcriptase activities, migrates to the genome where the endonuclease cleaves at the consensus 5′-TTAAAA-3′/3′-AA↑TTTT-5′, as shown. The T bases then prime reverse transcription of the RNA using the reverse transcriptase activity as shown. The generation of DNA nicks by nickel oxidation could potentially increase available priming sites for the L1 RNA. However, our data do not support this hypothesis. DNA repair processes are likely to be involved in repairing the DNA breaks generated by the L1 endonuclease preventing the L1 retrotransposition process. Inhibition of DNA repair enzymes at this step could favor the L1 insertion. (c) Reverse transcription, cDNA generation and integration. Recognition of the L1 complex during integration by the DNA repair machinery could result in its removal (a) and inhibition of the retrotransposition rate. In addition, completing the retrotransposition requires a second nick, caused by an unknown source, and linkage of the 3′ end of the cDNA to the chromosome. The cell must then complete second-strand synthesis and ligate the gaps. At least some of these steps must involve endogenous cellular activities. Potential nickel inhibition of DNA repair enzymes or other cellular proteins involved in this step that require Mg2+ could alter the equilibrium favoring the generation of new L1 inserts. Our data favor this hypothesis, where the increase in L1 activity is due to the nickel effect on cellular enzymes, in particular those involved in DNA repair.
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References

    1. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. International Human Genome Sequencing Consortium. Nature. 2001;409:860–921. - PubMed
    1. Chen JM, Stenson PD, Cooper DN, Ferec C. A systematic analysis of LINE-1 endonuclease-dependent retrotranspositional events causing human genetic disease. Hum Genet. 2005;117:411–427. - PubMed
    1. Gaudet F, Rideout WM, III, Meissner A, Dausman J, Leonhardt H, Jaenisch R. Dnmt1 expression in pre- and postimplantation embryogenesis and the maintenance of IAP silencing. Mol Cell Biol. 2004;24:1640–1648. - PMC - PubMed
    1. Yu F, Zingler N, Schumann G, Stratling WH. Methyl-CpG-binding protein 2 represses LINE-1 expression and retrotransposition but not Alu transcription. Nucl Acids Res. 2001;29:4493–4501. - PMC - PubMed
    1. Huang J, Fan T, Yan Q, Zhu H, Fox S, Issaq HJ, et al. Lsh, an epigenetic guardian of repetitive elements. Nucl Acids Res. 2004;32:5019–5028. - PMC - PubMed

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