<i>CycG</i> interacts with several <i>PcG</i> and <i>ETP</i> genes for developmental stability.

<p>Centroid size FA (FA10) of <i>ETP</i> or <i>PcG</i> heterozygous mutant females combined with <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i> (dark orange; <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i>; <i>PcG/+</i> or <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i>; <i>ETP/+</i>) and <i>ETP</i> or <i>PcG</i> heterozygous mutant females combined with <i>da-Gal4</i> (blue; <i>da-Gal4/+; PcG/+</i> or <i>da-Gal4</i>; <i>ETP/+</i>). The grey dashed line shows FA of <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i><i>/+</i> females. (F-tests, *p-value<0.05; ** p-value<0.01; *** p-value<0.001, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s007" target="_blank">S5 Table</a>). Source data are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s008" target="_blank">S6 Table</a>.</p

The ETP interacting domain limits <i>CycG</i>-induced FA.

<p><b>A–</b>Map of the 566 amino-acid Cyclin G protein showing the ETP interacting and PEST domains. <b>B–</b>Wing centroid size FA (FA10) of females <i>da-Gal4</i>/+ (+), <i>+/UAS-CycG</i>^<i>FL</i><i>; da-Gal4/+</i>, (<i>CycG</i>^<i>FL</i>) and <i>+/UAS-CycG</i>^<i>ΔE</i>; <i>da-Gal4</i>, (<i>CycG</i>^<i>ΔE</i>). <b>C–</b>Wing centroid size FA (FA10) of females <i>da-Gal4</i>/+ (+), <i>+/ UAS-CycG</i>^<i>ΔP</i><i>; da-Gal4/+</i> (<i>CycG</i>^<i>ΔP</i>) and <i>+/UAS-CycG</i>^{<i>ΔEΔP</i>}<i>; da-Gal4/+</i> (<i>CycG</i>^{<i>ΔEΔP</i>}). (F-tests, *** p-value<0.001, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s005" target="_blank">S3 Table</a>). Source data are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s006" target="_blank">S4 Table</a>.</p

Genes deregulated in wing imaginal discs expressing <i>CycG</i>^<i>ΔP</i>.

<p><b>A–</b>RT-qPCR analysis of endogenous <i>CycG</i> expression in <i>da-Gal4</i>,<i>UAS-CycG</i>^<i>ΔP</i><i>/+</i> and <i>da-Gal4/+</i> wing imaginal discs. Expression of <i>CycG</i> was normalized on the geometric mean of <i>Lam</i> and <i>rin</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s010" target="_blank">S8 Table</a>). t-tests, ** p-value<0.01. Error bars correspond to standard deviations. <b>B–</b>Ontology of up-regulated and down-regulated genes in <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i><i>/+ vs da-Gal4/+</i> wing imaginal discs. Gene ontology analysis was performed with DAVID (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s011" target="_blank">S9 Table</a>). <b>C–</b>RT-qPCR analysis of <i>RPL15</i>, <i>RPL7</i> and <i>Rack1</i> expression in <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i><i>/+</i> and <i>da-Gal4/+</i> wing imaginal discs. Expression of <i>RPL15</i>, <i>RPL7</i> and <i>Rack1</i> were normalized on the geometric mean of <i>Lam</i> and <i>rin</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s012" target="_blank">S10 Table</a>). t-tests, ** p-value<0.01. Error bars correspond to standard deviations. t-tests, ** p-value<0.01; *** p-value<0.001.</p

Functional subnetwork identified in wing imaginal discs expressing <i>CycG</i>^<i>ΔP</i>.

<p>Schematic representation of a sub-network of 222 genes centred on Cyclin G (CycG_subnetwork.xmml) and identified using JactiveModules (Z score 48.53). In this sub-network, 65 genes were up-regulated in <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i> <i>vs da-Gal4/+</i> wing imaginal discs (green gradient), 124 genes were down-regulated (red gradient), and 33 genes were not significantly deregulated (grey). Genes bound by Cyclin G are circled in blue. Transcription factor genes are represented by squares. Genes were clustered depending on their function. Black edges correspond to interactions discovered in the present study. Grey edges correspond to interactions described in the literature and imported into the WID network using DroID.</p

Snapshots illustrated Cyclin G ChIP-seq.

<p>Focus on <i>RPL7</i> (A), <i>RPL5</i> (B), <i>Rack1</i> (C) and <i>CycG</i> (D).</p

Cyclin G shares target genes with PRC1, Asx and RNAPolII but not with Calypso.

<p>Venn diagrams showing the intersection between Cyclin G-bound genes in <i>+/ UAS-CycG</i>^<i>ΔP</i><i>; da-Gal4/+</i> wing imaginal discs with Pc and RNAPlII (A), Asx and Calypso (B), and K3K27me3 (C) in wild-type wing imaginal discs.</p

Local deregulation of <i>CycG</i> induces high FA.

<p>Wing length FA (FA10) of females bearing a Gal4 driver either associated with <i>UAS-CycG</i>^<i>ΔP</i> (dark orange) or alone (light orange). Wing length was measured as the distance between landmarks 3 and 13. (F-tests, *** p-value<0.001, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s003" target="_blank">S1 Table</a>). Source data are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s004" target="_blank">S2 Table</a>.</p

Polycomb and Enhancer of Polycomb and Trithorax alleles used in this study.

<p>Polycomb and Enhancer of Polycomb and Trithorax alleles used in this study.</p

Genes bound by Asx, Calypso, Pc, Ph, Psc, or RNAPolII, or enriched in H3K27me3 in the sub-network of 222 genes centred on Cyclin G.

<p>Bound genes are represented in blue.</p

Cyclin G co-localizes with H2AK118ub at many sites on polytene chromosomes but overexpression of <i>CycG</i> does not modify global H2AK118ub.

<p><b>A, A’, A”–</b>Immunostaining of polytene chromosomes from <i>w</i>^<i>1118</i> third instar larvae. H2AK118ub (red), Cyclin G (green), DAPI (blue). <b>A”‘</b>–Close-up of the box showed in A”. <b>B, B’–</b>Wing imaginal discs of 3^rd instar larvae expressing <i>CycG</i>^<i>ΔP</i> in the posterior compartment under control of the <i>en-Gal4</i> driver, stained with anti-Cyclin G (green) and anti-H2AK118ub (red). <b>C, C’–</b>Wing imaginal discs of 3^rd instar larvae expressing <i>CycG</i>^{<i>ΔEΔP</i>} in the posterior compartment under control of the <i>en-Gal4</i> driver, stained with anti-Cyclin G (green) and anti-H2AK118ub (red). <b>D, D’, D”, D”’</b>–GFP clones in wing imaginal discs stained with anti-H2AK118ub (red). D’, D” and D”’ are close-up views of the yellow rectangle shown in D. <b>E, E’, E”, E”’</b>–<i>CycG</i>^<i>ΔP</i> clones marked by GFP in wing imaginal discs stained with anti-H2AK118ub (red). E’, E” and E”’ are close-up views of the yellow rectangle shown in E. <b>F, F’, F”, F”’</b>–<i>CycG</i>^{<i>ΔEΔP</i>} clones marked by GFP in wing imaginal discs stained with anti-H2AK118ub (red). F’, F” and F”’ are close-up views of the yellow rectangle shown in F. Scale bars: 50 μm.</p