Expression (A) and kinetic (B) of <i>PB</i> transposase by type and quantity of nucleic acid.

<p>HeLa cells were transfected with indicated amounts (A) or 187.5 ng (B) of <i>PB</i> mRNA or pDNA and total protein extraction was performed 24 h (A) or at indicated times (B) post-transfection. Transposase (V5PB Tp) expression was determined by Western-Blot and protein quantification was normalized to the endogenous Menin protein. Values represent the average of 3 experiments done in triplicate. Mock: untransfected cells. * Indicates statistically significant differences between mRNA and pDNA (p<0.05).</p

Transposition assay in mammalian cells.

<p>(A) Donor plasmid harboring the transposable element. This vector is composed of the neomycin phosphotransferase gene (Neo^R) flanked by the <i>PB</i> ITRs. (B) Transposition efficiency by type and quantity of nucleic acid. Cells were transfected with indicated amounts of <i>PB</i> mRNA or pDNA alongside with 187.5 ng of donor plasmid. GFP mRNA or pDNA (500 ng) served as negative controls of transposition (Mock: recombination events). To consider only transposition events, the number of colonies observed in the presence of the transposase source was subtracted by the number of colonies obtained in the respective negative control performed without transposase for each quantity. Values represent the average of 3 experiments done in triplicate. * Represents statistically significant difference between mRNA and pDNA (p<0.05).</p

<i>piggyBac</i> transposase bioavailability.

<p>(<b>A</b>) <b>Half-life of the <i>PB</i> mRNA</b>. Cells were transfected with 187.5 ng of <i>PB</i> mRNA. Total RNA was extracted at indicated times, reverse transcribed and subjected to qPCR. 18S RNA served as an internal standard to normalize the data. (<b>B</b>) <b>Persistence of <i>PB</i> pDNA after transfection</b>. Cells were transfected with 187.5 ng of <i>PB</i> pDNA and plasmid rescue was performed at 0 to 20 days. Ampicillin-resistant colonies were selected to evaluate the persistence of the plasmid. (<b>C</b>) <b>Half-life of the <i>PB</i> transposase (V5PB Tp)</b>. Cells were transfected with 187.5 ng of <i>PB</i> mRNA, incubated 18 h to reach the peak of transposase expression and treated with cycloheximide (t0=100). Total protein extraction was done at the indicated times from t0. The transposase half-life was determined by Western-Blot and quantification was normalized to the endogenous actin protein.</p

Detection of H2AX phosphorylation following dose-dependent <i>PB</i> mRNA or pDNA transfection.

<p>Cells were transfected with indicated amounts of <i>PB</i> mRNA or pDNA and total protein extraction was performed 24 h post-transfection. γ-H2AX expression was determined by Western-Blot and protein quantification was normalized to the endogenous Menin protein. Mock: untransfected cells. T-_GFP: cells transfected with 500 ng of GFP mRNA or pDNA. T+: untransfected cells treated with 2 µg/mL doxorubicin (positive control). * Indicates statistically significant differences between treated and untreated cells (p<0.05). Values represent the average of 3 experiments done in triplicate. The signal between the dotted line (mock control) and solid line (T-_GFP control) is considered to be due to the “transfection effect”. Above the solid line, the signal is due to the “transposase effect”.</p

Location of regions essential to silencing activity within the Δ8 segments of <i>MOS1</i>.

<p>(<b>a</b>) Variant fragments within the sequences of the Δ8-<i>MOS1</i>. Dashes indicate positions that are present in each fragment. (<b>b</b>) Expression of the <i>Firefly</i> and the <i>Renilla</i> luciferase marker genes using transient expression assays in HeLa cells. The assays were performed with Δ8-<i>MOS1</i> and five variants cloned in + orientation. (<b>c</b>) and (<b>d</b>) Impact of the Δ7 segments of <i>MOS1</i> on the expression of the <i>Firefly</i> and the <i>Renilla</i> luciferase marker genes using transient expression assays in cell lineages H4 (TARBP2+/NRSF-/YY1+/NFAT-5-) (<b>c</b>) and DT40 (TARBP2+/NRSF+/YY1+/NFAT-5-) (<b>d</b>). In (<b>b</b>), (<b>c</b>) and (<b>d</b>), each histogram bar corresponds to the median value obtained from three experiments done in triplicate. Bars correspond to quartiles 1 and 3. The median ratio RLU from <i>Firefly</i>/RLU from <i>Renilla</i> were calculated as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005902#pgen.1005902.g002" target="_blank">Fig 2</a>. The area where the ratios “RLU from <i>Firefly</i>/RLU from <i>Renilla</i>” were above 1 (i.e. where no strong silencer effect is observed) is coloured in grey. In (<b>b</b>) * and ** indicate a significant difference (p<0.05) with the P_Luc controls. ** also indicates a significant difference (p<0.05)with HS2_P_Luc_Δ8-<i>MOS1</i>.</p

Activity of the <i>Mos1</i> and <i>Hsmar1</i> promoter in presence or absence of their Δ7 segment.

<p>(<b>a</b>) Schematic representation of expression cassettes containing a luciferase reporter gene that were used to evaluate the impact of the DNA element under investigation (EUI; here Δ7 segment) on their promoter pMos or pHsmar1 that are composed of the 5’ inverted terminal repeat plus the 5’ untranslated terminal region (black arrows). The DNA sequences of pMos and pHsmar1 are supplied in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005902#pgen.1005902.s003" target="_blank">S3 Fig</a>. The assay is based on the transient expression of two plasmids: (i) the pRL-Tk plasmid that expresses the <i>Renilla</i> luciferase under control of a Thymidine kinase promoter as a control for transfection efficiency, (ii) a derivative of the pGL3 plasmid that expresses the <i>Firefly</i> luciferase under control of an early SV40 promoter. Effect of Δ7-<i>MOS1</i> (<b>b</b>) and Δ7-<i>HSMAR1</i> (<b>c</b>) DNA segments on their own promoter in HeLa cells. Results are represented by median values from three experiments done in triplicate. Bars correspond to quartiles 1 and 3. * indicates significant differences (p<0.05) between the pMos1-Luc-Δ7, pHsmar1-Luc-Δ7, and pMos1-Luc or pHsmar1-Luc, respectively.</p

<i>Mariner</i> Transposons Contain a Silencer: Possible Role of the Polycomb Repressive Complex 2 - Fig 11

<p><b>Counting of YY1 (a) and NFAT-5 (b) binding sites along the sequence of genomic Δ7 <i>Hsmar2</i> segments.</b> Areas filled in grey located the Δ8 segment within the Δ7 <i>Hsmar2</i> segment.</p

Ubiquitous functioning of Δ7-<i>MOS1</i> segment and Δ7 segments originating from 3 other distantly related <i>mariner</i> elements in animal cells.

<p>Stable expression assays were performed in (<b>a</b>) amphibian Speedy cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005902#pgen.1005902.ref013" target="_blank">13</a>] and (<b>b</b>) Insect (Lepidoptera) Sf21 cells. The average number of colonies obtained with the control [<i>NeoR</i>] construct in <b>a</b>, and [pIE1-<i>NeoR</i>] in <b>b</b>, were about 50 and 1100, respectively. (<b>c</b>) Comparison of orthologous Δ7 segments from <i>Mos1</i> (light grey bars), <i>Mcmar1</i> (mid-grey bars), <i>Himar1</i> (dark grey bars) and <i>Hsmar1</i> (black bars) cloned downstream of the marker gene in positive or negative orientations in HeLa cells. Each histogram bar corresponds to the median value obtained from three experiments done in triplicate. Bars correspond to quartiles 1 and 3. The [<i>NeoR</i>] construct serves as positive reference and was set to 100%. The Δ1[<i>NeoR</i>]Δ2 construct serves as the negative control. * indicates a significant difference (p<0.05) compared to the [<i>NeoR</i>] control.</p

Impact of PRC2 inhibition with DZNep on the silencer of <i>Mos1</i>.

<p>(<b>a</b>) Expression of the <i>Firefly</i> luciferase marker gene from P_Luc and HS2_P_Luc in absence or presence of 5 μM DZNep using transient expression assays in HeLa cells. (<b>b</b>) Expression of the <i>Firefly</i> luciferase gene from 4 different HS2_P_Luc plasmid constructs containing Δ8-<i>MOS1</i> variants cloned downstream of the marker gene. In (<b>a</b>) and (<b>b</b>), each histogram bar corresponds to the median value obtained from three experiments done in triplicate. Bars correspond to quartiles 1 and 3. * indicates a significant difference (p<0.05) between cells treated or not with 5 μM DZNep. In (<b>a</b>), the ratio “RLU from <i>Firefly</i>/RLU from <i>Renilla</i>” for the P_Luc transfection in the absence of DZNep is used as a reference and fixed at 1 arbitrary unit. All other ratios were calculated taking this reference into account. In (<b>b</b>), the ratios “RLU from <i>Firefly</i>/RLU from <i>Renilla</i>” for the HS2_P_Luc transfection done in absence or presence of 5 μM DZNep are used as references and fixed at 1 arbitrary unit. They were respectively used to calculate the ratios of each construct assayed in absence or in presence of 5 μM DZNep.</p

Characterization of the YY1 binding sites within the Δ8 segments.

<p>(<b>a</b>) Schematic representation of the <i>Mos1</i> transposon and its 10 deletion derivatives used to define a minimal silencer (Δ8 and Δ81 to Δ89). Δ8 spanned from positions 903 to 1212 of the <i>Mos1</i> sequence. Five derivatives of ~100 bp were tiled over ~50 bp (Δ81 to Δ85). Blank rectangle: <i>Mos1</i> transposase (Tpase ORF). Black arrow: 5’ ITR fused to the 5’ UTR. Grey arrow: 3’ UTR fused to the 3’ ITR. (<b>b</b>) Nucleic acid sequence of the Δ8-<i>MOS1</i> segment and putative TF binding sites frequently found within PRE in drosophila. Blue: <i>YY1</i>, red: <i>NRSF</i>, green: <i>Zeste</i>, pink: <i>GAGA</i> factor, turquoise: <i>GTGT</i> factor. Arrows indicate their orientation. The amino acid sequence of the C-terminal region of MOS1 is indicated using the one-letter code. (<b>c</b>) DNA binding activity of the YY1 factor to the Δ8-<i>MOS1</i>, Δ8-<i>HIMAR1</i>, Δ8-<i>MCMAR1</i>, and Δ8-<i>HSMAR1</i> segments. HeLa cells nuclear extracts were incubated with the ATTO-labelled Δ8 segments of the four transposases and the DNA-protein complexes were visualized by EMSA photography. Specificity was assayed using an anti-YY1 serum and super-shifted YY1/Δ8 complexes (one or two bands) are indicated by an asterisk. The content of each sample is shown above each lane: NE: nuclear extract, Anti-YY1: anti-YY1 serum. (<b>d</b>) Detection of YY1 binding sites within the Δ81 to Δ85 segments. The specificity of the shifted complex observed in lane 2, 8 and 11 was verified in lane 3, 9 and 12 in which the anti-YY1 serum allows obtaining super-shifted complexes (indicated by *). All these experiments were done in triplicate and representative pictures are shown.</p