Protein subnetwork of human orthologs of genes parasitized by EnSpm-N6_DR elements in zebrafish classified by their involvement in specific developmental pathways, such as neurogenesis, synaptic transmission and regulation of programmed cell death.

<p>The network is visualized in STRING action view with lines and arrows of different colors indicating diverse types of interaction: binding (blue), activation (green), inhibition (red), post-translational modifications (violet) and co-expression (yellow). Of note the recently published interaction between TRIM8 and p53 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046642#pone.0046642-Caratozzolo1" target="_blank">[34]</a> was added explicitly as it is not yet annotated within the STRING database.</p

Functional p53 responsive elements map within the zebrafish-specific EnSpm-N6_DR transposon.

<p>(<b>A</b>) Predicted p53 responsive elements (REs) and their respective sequences in the first intron of <i>Danio rerio trim8a</i> long allele, <i>trim8a</i> short allele and <i>trim8b</i>. The indicated positions for REs are calculated from the annotated transcription start site. (<b>B</b>) Responsiveness to <i>Danio rerio</i> p53 transactivation of the first intron of <i>trim8a</i> engineered to contain different combinations of REs. The assessed mutant constructs are schematically represented on the left. (<b>C</b>) Zebrafish p53-dependent transactivation assessment in luciferase reporter assays of <i>Danio rerio trim8a</i> long allele, <i>trim8a</i> short allele, <i>trim8b</i> and <i>Homo sapiens p21</i> and <i>TRIM8</i>.</p

Transposon-embedded p53REs show p53-mediated transactivation.

<p>(<b>A</b>) Zebrafish p53-dependent transactivation assessment in luciferase reporter assays of 27 sequences containing EnSpm-N6_DR elements and mapping in close proximity to the indicated genes. Tested sequences are detailed in <b>Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046642#pone.0046642.s010" target="_blank">Table S2</a></b>, They are categorized by mapping location within gene loci, i.e. 5′, 3′, within the first or other more downstream exons. The names of the human orthologs are used for simplicity. Note that <i>atp2b1</i>, <i>cacna1d</i>, <i>dach2</i>, <i>drd2</i>, <i>drd4</i>, <i>fibcd1</i>, <i>grin2a</i>, <i>itga8</i>, <i>lamb2</i>, <i>pgbd1</i>, <i>rics</i>, <i>sema3a</i>, <i>sh2d3c</i>, <i>ski</i>, <i>tnfsf10</i>, <i>ttll9</i> and <i>wfikkn2</i> correspond to zebrafish <i>atp2b1a</i>, <i>cacna1da</i>, <i>dacha</i>, <i>drd4-rs</i>, <i>dkeyp-51b7.3</i>, <i>grin2aa</i>, <i>zgc:172265</i>, <i>lamb2l</i>, <i>si:ch73-353p21.4</i>, <i>dkey-269g4.4</i>, <i>sema3aa</i>, <i>sh2d3ca</i>, <i>skia</i>, <i>tnfsf10l4</i>, <i>si:dkey-211h10.2</i> and LOC564992, respectively. The percentage of transactivated targets per mapping class is shown in panel (<b>B</b>).</p

trim8a and trim8b expression in zebrafish embryos.

<p>(<b>A</b>) Expression pattern of <i>trim8b</i> at 18 hpf determined by whole mount ISH. Left panel: lateral view; right panel: dorsal view. Of note <i>trim8a</i> is not detected at this stage. (<b>B</b>) Comparison of the expression patterns of <i>trim8a</i> (top row) and <i>trim8b</i> (bottom row) after 30 to 72 hours of development. Embryos are in lateral (first three columns) or dorsal views (far right column). <i>Trim8b</i> expression at 48 hpf and 72 hpf (lateral view) is shown in de-yolked and flat mounted embryos (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046642#s4" target="_blank">Methods</a>). Anterior to the left. AD, anterior diencephalon; C, cerebellum; DD, dorsal diencephalon; DH, dorsal hindbrain; H, hindbrain; OT, optic tectum; R, retina; SCN, ventral spinal chord neurons; T, tegmentum; TG, trigeminal ganglia; VMB ventro-lateral midbrain; VZ, brain ventricular zone.</p