Suicidal Autointegration of <i>Sleeping Beauty</i> and <i>piggyBac</i> Transposons in Eukaryotic Cells

<div><p>Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. ‘Cut and paste’ DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, <i>Sleeping Beauty</i> (<i>SB</i>) and <i>piggyBac (PB)</i> that are widely used for genome manipulation in vertebrate species. <i>SB</i> originates from fish and <i>PB</i> from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither <i>SB</i> nor <i>PB</i> is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the <i>SB</i> transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both <i>SB</i> and <i>PB</i> were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, <i>SB</i> is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for <i>PB</i>.</p></div

Bimolecular transposition events generated by <i>PB</i>.

<p>Transposition assay was performed by using ‘solo’ transposon substrates, either alone or mixed in equimolar ratios, in the present of the mPB transposase. The statistical significance of differences is shown by asterisk above the bars, *P<0.05. Molecular analysis identified no transposase-mediated integration events in the resistant colonies using <i>PBΔright</i> (background). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen-1004103-t001" target="_blank">Table 1</a>.</p

Comparing autointegration profile to the predicted, close-to-random target site distribution of <i>SB</i> transposition.

<p>A. Distribution of 53 <i>de novo</i> autointegration events (triangles) detected by the assay shown in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen-1004103-g001" target="_blank">Figure 1B</a>). Autointegration products were isolated from individual bacterial clones, sequenced and mapped to the <i>SBrescue</i> construct. The thin arrow indicates the location of the sequencing primer on the left IR. B. Comparison of the predicted and experimental insertion events. The <i>SBrescue</i> construct is shown in a linear mode. The <i>SB Vstep</i> scores and experimental insertion events were shown below. Un, undetectable.</p

The cellular factor of BANF1 interferes with autointegration.

<p>A. Relative autointegration frequencies of <i>SB</i> (<i>SB7K</i>, left panel) and PB (<i>PB7K</i>, right panel) in HeLa cells, where BANF1 was either knocked-down or overexpressed. Knocking down of BANF1 stimulated, whereas overexpressing of BANF1 inhibited autointegration of both <i>SB</i> and <i>PB</i> transposons (n = 3). The statistical significance of differences is shown by asterisk above the bars *P<0.05. B. A SILAC pull-down experiment using anti-HA resin to investigate interaction partners of HMGXB4 in the presence/absence of <i>SB10</i> transposase in transiently transfected HEK293T cells. Schematic representation of the SILAC/pull-down experimental approach in which stable isotope labeled amino acids [Light (L) or Medium heavy (M)] are added in the form of medium supplement to culture HEK293T cells. Detection of interaction partners is performed by mass spectrometry. Scatter plot displays the normalized log2 SILAC ratio M/L values (X-axis) versus log2 intensity (Y-axis) of proteins detected in the interactome around HMGXB4⁻ in presence of the SB transposase. Each dot represents an individual protein, while their position indicates their abundance in the complex pulled down by the bait of HMGXB4. Proteins with a positive log2 SILAC M/L ratio, including BANF1 and <i>SB</i> are enriched in the protein complex around HMGXB4. C. Co-immunoprecipitation assay to investigate the interaction partners of HMGXB4, a physical interaction partner of the <i>Sleeping Beauty</i> transposase, SB10 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen.1004103-Walisko2" target="_blank">[13]</a>. <i>SB10</i> and HA-tagged HMGXB4 were transiently transfected into HEK293T cells (see Methods). In comparison to negative control, BANF1 and <i>SB</i> are enriched in the pull-down by HMGXB4-HA.</p

Autointegration properties of <i>PiggyBac</i> transposition.

<p>A. Frequency of <i>PB</i> autointegration events in HeLa cells using the PB2K construct. PBase, m<i>PB</i> transposase <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen.1004103-Cadinanos1" target="_blank">[50]</a>. B. The structure of the <i>PB2K</i> construct. For explanation, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen-1004103-g001" target="_blank">Figure 1A</a>. Distribution of <i>de novo PB</i> insertions indicated by black triangles (n = 22) on the <i>PB2K</i> construct. C. Sequence of three (3/22) representative single-ended transposition events mapped to the B, C and rpsL regions of <i>PB2K</i>. Sequences flanking the right inverted repeat of the <i>PB</i> transposon in <i>PB2K</i>. Original sequences (bold); <i>de novo</i> integration events (normal); target site of PB transposition, TTAA (italic). D. Distribution of six single-ended transposition events on the <i>PBsingle</i> construct. <i>Kan</i>: kanamycin resistant gene (<i>Kan</i>). Dark bars indicate the control experiment with only transposon vector; light bars indicate the experiment with both transposon vector and transposase expressing vector. E. Sequence of the six individual single-ended transposition shown on <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen-1004103-g003" target="_blank">Figure 3D</a>. The <i>PB</i> transposon is shown as a two-headed arrow, representing the IRs (black). Frequencies are shown in parentheses.</p

Transposase-mediated integration events of ‘solo’ substrates.

<p>HeLa cells were co-transfected with the ‘solo’ transposon constructs in the present of either mPB or SB100X transposases, while a catalytically inactive <i>SB</i> transposase, D3 was used as a control. Frequency of substrate integration was calculated as a ratio of colony numbers in the presence vs absence of transposases. Colonies were picked and analysed for transposase-mediated integration events. Transposase-mediated integration is defined when the IR of the transposon is integrated into a respective target site in the genome (see also Supporting <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004103#pgen.1004103.s006" target="_blank">Text S1</a>). ND: not detected.</p

Both <i>SB</i> and <i>PB</i> transposons are sensitive to the size of the transposon.

<p>A. Excision, autointegration and transposition profiles of <i>SB</i> (left panel) and <i>PB</i> (right panel) transposons. The name and the size of the various constructs are shown below the plots. The values using the smallest constructs (SB2K or PB2K) were set to 100% (n = 3). B. Transposition assay performed by using <i>SB</i> (upper panel) and <i>PB</i> (lower panel) transposon constructs of various sizes.</p

AKIR-1 binds preferentially to <i>nlp</i> gene promoters in the absence of infection.

<p><b>A.</b> Specific binding of AKIR-1::GFP onto promoters (left panels) or 3’ UTR (right panel) of <i>act-1</i> (left panel; 2 different PCR amplicons, A and B), and <i>nlp-29</i>, <i>nlp-31</i> and <i>nlp-34</i>, represented as the fold enrichment of the specific ChIP signal obtained using an anti-GFP antibody for immunoprecipitation relative to that when blocked beads were used, measured by quantitative PCR. Data is normalised to input; the average (and standard error) from three independent experiments is shown. ***, p < 0.0001; **, p < 0.001; ns, p > 0.1; paired 2-tail Student’s t test. <b>B.</b> Model for the role of AKIR-1 in the regulation of <i>nlp</i> AMP gene expression upon infection. Under normal conditions (left), the AKIR-1/NuRD complex is recruited to the <i>nlp-29</i> locus, leading to modification (red stars) of histones (ovoids), and formation of an open chromatin structure. Upon infection, STA-2 is activated and, following removal of the AKIR-1/NuRD complex, is responsible for expression of the <i>nlp</i> genes. Infection could impact chromatin structure, but here we assume that it does not. When AKIR-1 is absent (right), an open chromatin structure cannot be formed, precluding STA-2-dependent expression of the <i>nlp</i> genes following infection, but not affecting the low basal STA-2-independent gene expression. The images are adapted, with permission, from <a href="https://www.activemotif.com" target="_blank">https://www.activemotif.com</a>.</p

Evolutionary plasticity in the innate immune function of Akirin

<div><p>Eukaryotic gene expression requires the coordinated action of transcription factors, chromatin remodelling complexes and RNA polymerase. The conserved nuclear protein Akirin plays a central role in immune gene expression in insects and mammals, linking the SWI/SNF chromatin-remodelling complex with the transcription factor NFκB. Although nematodes lack NFκB, Akirin is also indispensable for the expression of defence genes in the epidermis of <i>Caenorhabditis elegans</i> following natural fungal infection. Through a combination of reverse genetics and biochemistry, we discovered that in <i>C</i>. <i>elegans</i> Akirin has conserved its role of bridging chromatin-remodellers and transcription factors, but that the identity of its functional partners is different since it forms a physical complex with NuRD proteins and the POU-class transcription factor CEH-18. In addition to providing a substantial step forward in our understanding of innate immune gene regulation in <i>C</i>. <i>elegans</i>, our results give insight into the molecular evolution of lineage-specific signalling pathways.</p></div

Validation of AKIR-1 interactors by Western blotting.

<p><b>A.</b> Complexes immunopurified using an anti-GFP antibody from control or infected worms with a single copy <i>AKIR-1</i>::<i>GFP</i> insertion (<i>wt; frSi12[pNP157(akir-1p</i>::<i>AKIR-1</i>::<i>GFP)] II)</i> were probed with specific antibodies. The results for two independent pull-downs are shown. The presence of HDA-1 and LET-418 (NuRD complex components) could be confirmed. Anti-ACT-1 was used to control the total input for each immunoprecipitation. <b>B.</b> Complexes immunopurified using an anti-FLAG antibody, from a strain co-expressing AKIR-1::GFP and FLAG-tagged CEH-18 (<i>wt; frSi12[pNP157(akir-1p</i>::<i>AKIR-1</i>::<i>GFP)] II; wgIs533[CEH-18</i>::<i>TY1</i>::<i>GFP</i>::<i>3xFLAG + unc-119(+)]</i>), were probed with anti-FLAG (top panel) and anti-GFP (bottom) antibodies. In addition to the immunopurified complex (IP), the extract before immunopurification (Input), the unbound fraction (flow-through: FT) and proteins immunopurified using an unrelated antibody (Mock) were also analysed.</p