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

Phenotype of transgenic flies over-expressing <i>cortoCD</i>.

<p>Scanning electron microscopy images of male genitalia (A), aristae (B) and male sex combs (C). On the left, control <i>da::Gal4/+</i> flies. On the right, <i>da::Gal4>UAS::FH-cortoCD-231</i> flies. Males over-expressing <i>FH-cortoCD</i> present rotated genitalia and sex combs with a reduced number of teeth. Aristae of flies over-expressing <i>FH-cortoCD</i> are partially transformed into leg, a phenotype called <i>aristapedia</i>. Strengths of these phenotypes are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003006#pgen.1003006.s005" target="_blank">Table S1</a>.</p

Ontology of genes deregulated in wing imaginal discs over-expressing <i>cortoCD</i> or <i>RpL12</i>.

<p>Gene Ontology (GO) term enrichment of genes deregulated by CortoCD and RPL12 considering DAVID identification <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003006#pgen.1003006-Huang2" target="_blank">[67]</a>. Highly represented, non-redundant categories were selected according to the hypergeometric test adjusted <i>P</i>-values. The number of genes in each category is shown on the X-axis. GO of biological process (A), molecular function (B) and cellular component (C) of genes upregulated (left) and down-regulated (right) by over-expression of <i>RpL12</i> (black) or <i>cortoCD</i> (grey). Full GO data (GO ID, description, number of genes in each category, enrichment and adjusted <i>P</i>-values) for each category are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003006#pgen.1003006.s012" target="_blank">Tables S8</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003006#pgen.1003006.s013" target="_blank">S9</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003006#pgen.1003006.s014" target="_blank">S10</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003006#pgen.1003006.s015" target="_blank">S11</a>.</p

Chromatin environment of RPL12.

<p>Co-immunostainings of salivary gland polytene chromosomes from <i>esg::Gal4>UAS::RpL12-Myc</i> larvae using anti-Myc antibodies (green) and anti-H3K27me3 (A), anti-H3K4me3 (B), anti-RNAPolIIS5p (C) or anti-RNAPolIIS2p (D) antibodies (red). Some co-localizations between RPL12-Myc and H3K27me3, H3K4me3 or RNAPolIIS5p are shown with yellow arrows on the merged pictures. (E) Close-up showing few co-localizations of RPL12 and RNAPolIIS5p. (F) Close-up showing numerous co-localizations of RPL12 and RNAPolIIS2p.</p

Occupancy of the <i>hsp70</i> gene by CortoCD and RPL12 is enhanced upon heat shock.

<p>(A) General structure of <i>hsp70</i> genes. qPCR amplicons are indicated. Coordinates of qPCR amplicons relative to <i>hsp70</i> transcriptional start site are indicated (from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003006#pgen.1003006-Boehm1" target="_blank">[28]</a>). (B) ChIP-qPCR analysis of <i>hsp70</i> from <i>w¹¹¹⁸</i> wing imaginal discs using mock or anti-RNAPolIISer2p antibodies. (C) ChIP-qPCR analysis of <i>hsp70</i> from <i>sd::Gal4>UAS-FH-CortoCD</i> wing imaginal discs using mock, anti-HA, or anti-RNAPolIISer2p antibodies. (D) ChIP-qPCR analysis of <i>hsp70</i> from <i>sd::Gal4>UAS-RpL12-Myc</i> wing imaginal discs using mock, anti-Myc, or anti-RNAPolIISer2p antibodies. White and black histograms represent ChIP-qPCR data without or with 5 minutes heat shock, respectively. 2L: negative control amplified from an intergenic region located on the left arm of chromosome 2 (coordinates 11.413.862 to 11.413.946) that does not present any read in our RNA-seq analysis and is enriched in H3K27me3 in wing imaginal dics <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003006#pgen.1003006-PrezLluch1" target="_blank">[64]</a>. Error bars represent coefficients of variation obtained from 4 independent experiments.</p

Chromatin environment of Corto.

<p>Co-immunostainings of salivary gland polytene chromosomes from <i>w¹¹¹⁸</i> larvae using anti-Corto antibodies (green) and anti-H3K27me3 (A), anti-H3K4me3 (B), anti-RNAPolIIS5p (C) or anti-RNAPolIIS2p (D) antibodies (red). The two arrows in A point to X chromosome tips from two nuclei. Some co-localizations between Corto and H3K4me3 or RNAPolIIS2p are shown with yellow arrows on the merged pictures. (E) Close-up showing co-localizations of Corto and RNAPolIIS5p. (F) Close-up showing rare co-localizations of Corto and RNAPolIIS2p.</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

The Corto chromodomain is a chromatin-targeting module.

<p>(A) Squash of salivary gland polytene chromosomes from an <i>esg::Gal4>UAS::FH-cortoCD-231</i> third instar larva. Anti-FLAG immunostaining shows that FH-CortoCD binds chromatin at many sites (left: DAPI, middle: anti-FLAG, right: merge). Arrow-heads point to DAPI-free Corto bands. (B) Magnifications of the tip of polytene chromosome 3L (bands 61 to 63) from third instar larvae, either <i>esg::Gal4>UAS::FH-cortoCD</i> immunostained with anti-FLAG antibody or <i>w¹¹¹⁸</i> immunostained with anti-Corto antibody (DAPI, immunostaining, merge). Bottom: conformity between endogenous Corto and FH-CortoCD binding sites.</p

Lysine 3 of RPL12 is essential for interaction with Corto.

<p>(A) PRALINE multiple sequence alignment of the 100 first residues of RPL12 from different eukaryotes showing conserved lysines (highlighted in orange) or arginines (highlighted in yellow) known to be methylated in several species. <i>Drosophila melanogaster</i> (<i>D.m.</i>; AE013599), <i>Arabidopsis thaliana</i> (<i>A.t.</i>; AAD18140), <i>Homo sapiens</i> (<i>H.s.</i>; NM_000976), <i>Saccharomyces cerevisiae</i> (<i>S.c.</i>; NP_010860), <i>Schizosaccharomyces pombe</i> (<i>S.p.</i>; NP_587897), <i>Cænorhabditis elegans</i> (<i>C.e.</i>; NP_502542), <i>Danio rerio</i> (<i>D.r.</i>; AAI65413) and <i>Mus musculus</i> (<i>M.m.</i>; CAM22324). Lysine 3 can be trimethylated in <i>H.s</i>, <i>S.c</i>, <i>S.p.</i> and <i>A.t.</i>; lysine 10 can be trimethylated in <i>S.c.</i>; lysine 39 can be dimethylated in <i>S.p.</i>; lysine 83 can be monomethylated in <i>E. coli</i>, arginine 67 can be δ-monomethylated in <i>S.c.</i> and <i>S.p.</i> (see references in the text). (B) <i>Drosophila</i> S2 cells expressing either EGFP-CortoCD (top) or RPL12-mRFP (bottom). CortoCD was provided with a nuclear localization signal to force its entry into the nucleus. Note its punctuated pattern in the nucleus. RPL12-mRFP was present in both nuclear and cytoplasmic compartments. (C) Simultaneous expression of EGFP-CortoCD and wild-type or mutant RPL12-mRFP in <i>Drosophila</i> S2 cells. EGFP-CortoCD perfectly co-localized with wild-type RPL12-mRFP, as well as with RPL12K10A, RPL12K39A, RPL12R67A and RPL12K83A mutants within nuclei, exhibiting a punctuated pattern. Note that wild-type RPL12 and these mutant forms were not detected in the cytoplasm. RPL12K3A did not present a punctuated nuclear pattern and was detected in the cytoplasm. Scale bar: 2.5 µm.</p