EloC and Corto bind <i>rho</i> in wing imaginal discs.

<p>(A, B): Wing phenotypes induced by <i>rho</i> loss-of-function (A) or over-expression (B). Asterisks mark truncated L5 (in A) or ectopic veins (in B). (C): Schematic structure of <i>rho</i> with exons represented by boxes and introns by lines. Black arrows show primer pairs used for ChIP experiments. (D): Binding of Corto chromodomain (FH-CortoCD) and EloC (FH-EloC) on <i>rho</i>. For each genotype, the mean of two independent experiments is shown. Error bars correspond to standard deviations.</p

Down-regulation of <i>EloC</i> by RNA interference impairs both cell proliferation and cell differentiation in wing imaginal discs.

<p>(A): Clones expressing the <i>ValEloC</i> transgene (GFP⁺ cells, shown by white arrows) are located at the periphery of the disc and are very small compared to control clones. (B, C, D): Wings from pharates in which <i>ValEloC</i> is driven by <i>nub::Gal4</i> (C) or <i>rn::Gal4</i> (D), both expressed in the wing pouch <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077592#pone.0077592-StPierre1" target="_blank">[66]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077592#pone.0077592-Ng1" target="_blank">[67]</a> are small compared to wild-type pharate wings (B) and exhibit severe wing blade defects. By contrast, longitudinal veins (shown by asterisks) are formed in the proximal-most part of the wing blade where <i>nub::Gal4</i> and <i>rn::Gal4</i> are not expressed.</p

<i>Elo</i> genes control wing cell identity.

<p>(A): Wing from control <i>w¹¹¹⁸</i> fly (L1-L5: longitudinal veins; ACV and PCV: anterior and posterior cross-veins). (B, C): Wings from <i>+/EloB^EP3132</i> and <i>EloB^EP3132/Df(3R)BSC518</i> flies exhibit truncated L5. (D): Wings from <i>+/sd::Gal4</i> flies have a very faint ectopic vein phenotype and no margin phenotype. (E, F): Wings from flies over-expressing <i>EloA</i> exhibit ectopic vein and margin phenotypes. (G, H, I): <i>EloC^SH1520</i> and <i>EloC^SH1299</i> loss-of-function alleles diminish expressivity of the ectopic vein phenotype induced by the <i>bs^EY23316</i> loss-of-function allele. Strong phenotype: ectopic veins everywhere in the wing (shown in G). Mild phenotype: ectopic veins under the posterior cross-vein only (shown in H).</p

Decreasing <i>EloC</i> expression suppresses ectopic veins induced by <i>blistered</i> loss-of-function.

<p>The upper allele was brought by the mother. The number of <i>EloC</i>/<i>bs^EY23316</i> females with ectopic veins was compared to the number of <i>+/bs^EY23316</i> females with ectopic veins (z-test, ^a p<0.001). The mild ectopic vein phenotype corresponds to presence of ectopic veins distal to the posterior cross-vein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077592#pone-0077592-g005" target="_blank">Figure 5H</a>), whereas the strong ectopic vein phenotype corresponds to presence of ectopic veins everywhere in the wing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077592#pone-0077592-g005" target="_blank">Figure 5G</a>).</p

Can pre-implantation biopsies predict renal allograft function in paediatric renal transplant recipients ?

<p>The upper allele was brought by the mother. The number of females with ectopic veins among flies transheterozygous for <i>Elo</i> and <i>corto</i> mutations was compared to the number of females with ectopic veins among flies with a <i>corto</i> mutation only (z-test, ^a p<0.001).</p

<i>corto</i> and several TrxG genes control wing cell identity.

<p>(A, B): Ectopic vein phenotypes induced by the <i>corto⁰⁷¹²⁸</i> loss-of-function allele (A) or by <i>corto⁴²⁰</i> loss-of-function clones (B). (C): <i>corto⁴²⁰</i> homozygous clones (GFP^- cells) in wing imaginal discs. (D, E, F): Ectopic vein phenotypes induced by <i>mor</i>, <i>kis</i> or <i>trx</i> loss-of-function alleles. In A, B, D, E, F, asterisks mark ectopic veins.</p

Deregulation of <i>EloA, EloB</i> or <i>EloC</i> expression using <i>P</i>-element insertion lines.

<p>(A): Structure of <i>EloA</i>, <i>EloB</i> and <i>EloC</i> genes showing localization of the <i>P</i>-elements used in this study. Exons are represented by boxes, and introns by lines. Black arrowheads show positions of primer pairs used to quantify <i>Elo</i> gene expression. (B): Quantification of <i>Elo</i> gene expression in <i>EloA^G4930</i>, <i>EloB^EP3132</i>, <i>EloC^SH1520</i> or <i>EloC^SH1299</i> homozygous or heterozygous larvae. (C): Quantification of <i>Elo</i> gene expression in <i>da::Gal4>>EloA^G4930</i> or <i>da::Gal4>>EloB^EP3132</i> larvae. (D): Quantification of <i>EloC</i> expression in <i>da::Gal4</i>>><i>ValEloC</i> embryos. Relative <i>Elo</i> expression levels were obtained by normalization to <i>Rp49</i> (black bars, B to D), <i>RpL12</i> (grey bars, B, C) or <i>eIF-2α</i> (grey bars, D).</p

Comparison of genes deregulated by <i>cortoCD</i> and <i>RpL12</i> over-expression.

<p>(A) Scatter plot of log₂ fold changes (FC) showing (<i>sd::Gal4>UAS::FH-cortoCD vs sd::Gal4/+</i>) on X-axis and (<i>sd::Gal4>UAS::RpL12-Myc vs sd::Gal4/+</i>) on Y-axis before cutoff. Note the high correlation coefficient (R² = 0.634). (B) Venn diagrams showing the intersection of genes deregulated in <i>FH-cortoCD</i> and <i>RpL12-Myc</i> over-expressions after cutoff [<i>P</i>-value<4.10⁻¹⁸; absolute log₂(assay/control)>1]. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003006#pgen.1003006.s007" target="_blank">Tables S3</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003006#pgen.1003006.s008" target="_blank">S4</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003006#pgen.1003006.s009" target="_blank">S5</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003006#pgen.1003006.s010" target="_blank">S6</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003006#pgen.1003006.s011" target="_blank">S7</a> for detailed gene lists.</p

Preferential binding of Corto chromodomain to RPL12 trimethylated on lysine 3.

<p>(A) Biacore sensorgrams showing binding of either RPL12 unmethylated peptide (RPL12 um, left panel) or RPL12 peptide trimethylated on lysine 3 (RPL12K3me3, right panel) to CortoCD. Increasing concentrations of RPL12 um or RPL12K3me3 peptides were used [from 0 (light grey lines) to 10 µM (darker grey to black lines]. Binding (Y-axis, Response) is expressed in Resonance Units (RU) relative to time (X-axis). Note the response due to end of injection of the peptides at 300 s. (B) Kinetic parameters of interaction between CortoCD and RPL12 um, RPL12K3me2 or RPL12K3me3 peptides. Note that CortoCD interacts specifically with RPL12 trimethylated on lysine 3 (K_D = 8 µM). (C) Equilibrium dissociation constant (K_D) calculated for CortoCD or HP1CD in interaction with RPL12 um peptide, RPL12 methylated peptides, or RPL12K3A peptide. Note that CortoCD specifically binds to RPL12K3me3 (K_D<100 µM). (D) Equilibrium dissociation constant (K_D) calculated from CortoCD or HP1CD interacting with unmethylated or trimethylated histone H3 peptides. For H3K9me3, peptide concentration was increased from 0 to 1 µM. For RPL12 and H3K27me3 peptide concentration was increased from 0 to 10 µM. NB: no binding; nd: not determined.</p

Corto interacts with nuclear ribosomal proteins and co-immunoprecipitates with RPL12 <i>via</i> its chromodomain.

<p>(A) Silver stained polyacrylamide gel showing polypeptides pulled-down by GST-CortoCD covalently linked on agarose beads from nuclear or cytoplasmic embryonic extracts. Four bands consistently appearing after incubation are enriched in nuclear extracts are shown by asterisks (P30, P21, P20 and P15). Line 0: no extract, 1: cytoplasmic extract, 2: nuclear extract. (B, C, D) Co-immunoprecipitation experiments. S2 cells were co-transfected with plasmids expressing FLAG-CortoCD, FLAG-Corto or FLAG-CortoΔCD and Myc-RPL12. Immunoprecipitations were performed with either anti-FLAG (α-FLAG) or anti-Myc (α-Myc) and revealed by Western blot with the same antibodies. Spnt: supernatant, IP: immunoprecipitation. (B) FLAG-CortoCD co-immunoprecipitated with Myc-RPL12 and conversely. Extracts were run on a 15% acrylamide gel. (C) FLAG-Corto co-immunoprecipitated with Myc-RPL12 and conversely. Extracts were run on a 12% acrylamide gel. (D) FLAG-CortoΔCD did not co-immunoprecipitate with Myc-RPL12 and conversely. Extracts were run on a 12% acrylamide gel.</p