Chromosomal locations of human <i>ZBED</i> genes, frog <i>ZBEDX</i> and human Buster1 (<i>ZBED5</i>).

a<p>Positions in the human genome (hg19 assembly), and the frog genome (<i>Xenopus tropicalis</i>, xenTro3 assembly).</p>b<p>Also referred to in the literature as hDREF, Tramp, and Human-<i>Ac</i>.</p>c<p>The frog <i>ZBEDX</i> gene.</p>d<p>This gene is also referred to as Buster1 of the <i>Buster</i> DNA transposon family in the literature, and is distinct from other ZBEDs.</p>e<p>Sequence conversion is not currently available for these genomes in the UCSC Genome Browser (<a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>).</p

Phylogenetic relationships of separate BED domains.

<p>Roman numerals refer to BED domain position within ZBED genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059940#pone-0059940-g001" target="_blank">Fig. 1</a>). Grey boxes on branches indicate hypothesized BED domain duplication events for the various <i>ZBED</i> genes. Posterior probabilities are provided next to tree nodes.</p

ZBED evolution.

<p>Phylogenetic tree for <i>ZBED</i> genes and related sequences from the <i>Ac</i> family. Two separate ZBED domestications are indicated. Numbers of included taxa are provided next to schematic clades. Active DNA transposons are marked with asterisks, and bold branches indicate posterior probabilities ≥95%.</p

Phylogenetic relationships of Buster1 (ZBED5).

<p>ZBED5 is identical to Buster1 and groups within the <i>Buster</i> family with strong support. Buster sequences are separate from collapsed clades representing the <i>Ac</i> family. Active DNA transposons are marked with asterisks, and bold branches indicate posterior probabilities ≥95%. Proposed nomenclature updates for ZBEDs 7, 8 and 9 are indicated next to branches ancestral to the respective ZBED (Buster) clade.</p

Small nucleolar RNA genes significantly up-regulated according to RNA-seq both on day 2 and day 4 after <i>Zbed6</i> silencing and qPCR validation.

a<p>Student's t-test, P<0.05).</p

Analysis of the correlation between differential gene expression (DE) after <i>Zbed6</i> silencing and the presence of ZBED6 binding sites within 5 kb of the transcription start site (TSS).

a<p>Overrepresentation of genes with ZBED6 binding sites among DE genes (Chi square test, two-tailed, d.f. = 1; Non DE genes vs All DE genes, P = 0.004; Non DE genes vs up-regulated DE genes, P = 0.002)</p

ZBED6 modulates the expression of Igf2 and Myogenin during differentiation of C2C12 cells.

<p>(A) Expression of <i>ZBED6</i>, <i>IGF2</i> and <i>Myogenin</i> mRNA monitored by qPCR before and after differentiation of control cells and transfected cells overexpressing ZBED6 from the pTRE-ZB vector. (B) Expression of <i>ZBED6</i>, <i>IGF2</i> and <i>Myogenin</i> mRNA monitored by qPCR before and after differentiation of control cells and cells in which ZBED6 has been silenced using siRNA. (C) Western blot analysis confirming altered expression of ZBED6 and Myogenin at the protein level. Actin was used as loading control.</p

RNA sequencing of <i>Zbed6</i>-silenced myoblast cells.

<p>(A) Quantitative PCR validation of <i>Zbed6</i>-silencing (light gray bar) and <i>Igf2</i> up-regulation (dark gray bar) two and four days post-<i>Zbed6</i> siRNA transfection. Biological triplicates were performed for both <i>Zbed6</i> and scrambled siRNA. Error bars, s.e.m. The asterisk indicates a significant difference (P<0.05) between control and <i>Zbed6</i>-silenced samples. (B) Western blot validation of <i>Zbed6</i>-silencing. The protein lysates from the pooled biological triplicates for each siRNA treatment were equally loaded to the protein gel after measuring the concentration. The specific band for ZBED6 protein (110 kDa) appeared above the 100-kDa band from the protein ladder and α-Tubulin was used as a reference protein. (C) Direct comparison of the read counts (y-axis) from <i>Zbed6</i>-silenced and negative control RNA-seq data across <i>Zbed6</i> and <i>Igf2</i> regions for both two and four days post siRNA transfection. The ChIP-seq data with ZBED6 antibody from Markljung et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094187#pone.0094187-Markljung1" target="_blank">[1]</a> are shown to pinpoint the binding sites of ZBED6.</p

Muscle proteins were significantly enriched among the differentially expressed (DE) genes.

<p>(A) The significantly enriched GO categories (y-axis) in the DE genes with FDR <0.05. (B) The Log2 fold changes from both RNA-seq (dark) and arrays (grey) of the DE genes associated with muscle proteins and contractile fiber, cytoskeleton protein binding and heart development. (C) The ZBED6 peak at about 1 kb upstream of <i>Twist2</i> gene (grey box) displayed together with placental mammal conservation score. The peak maxima overlapped with a highly conserved region (grey box), which contained the consensus motif GCTCGC of ZBED6 only in the placental mammals. A 10-bp insertion (+10) was present within the consensus motif in the opossum genome, indicating lack of ZBED6 binding site in this region. (D) The relative expression levels of <i>Twist2</i> from RNA-seq and array in either <i>Zbed6</i>-silenced or control C2C12 cells. (E) The relative luciferase activity (the ratio of firefly to <i>Renilla</i>) of <i>Igf2</i>, <i>Twist2</i> and empty pGL3 basic constructs in <i>Zbed6</i>-silenced or control C2C12 cells. **, P<0.01; ***, P<0.001.</p

Overrepresentation analysis of transcription factor binding sites within 2- or downstream of the transcription start site of genes showing significant differential expression after <i>Zbed6</i>-silencing in C2C12 myoblast cells.

a<p>Number of genes containing the transcription factor binding site.</p>b<p>Fisher's exact test.</p>c<p>M-values represent log2 transformation of fold changes between <i>Zbed6</i>-silenced samples and negative controls estimated by qPCR analysis. Triplicates for each treatment were used.</p>d<p>Student's t-test were used to calculate the statistical significance.</p