The pro-apoptotic activity of Drosophila Rbf1 involves dE2F2-dependent downregulation of diap1 and buffy mRNA

International audienceThe retinoblastoma gene, rb, ensures at least its tumor suppressor function by inhibiting cell proliferation. Its role in apoptosis is more complex and less described than its role in cell cycle regulation. Rbf1, the Drosophila homolog of Rb, has been found to be pro-apoptotic in proliferative tissue. However, the way it induces apoptosis at the molecular level is still unknown. To decipher this mechanism, we induced rbf1 expression in wing proliferative tissue. We found that Rbf1-induced apoptosis depends on dE2F2/dDP heterodimer, whereas dE2F1 transcriptional activity is not required. Furthermore, we highlight that Rbf1 and dE2F2 downregulate two major anti-apoptotic genes in Drosophila: buffy, an anti-apoptotic member of Bcl-2 family and diap1, a gene encoding a caspase inhibitor. On the one hand, Rbf1/dE2F2 repress buffy at the transcriptional level, which contributes to cell death. On the other hand, Rbf1 and dE2F2 upregulate how expression. How is a RNA binding protein involved in diap1 mRNA degradation. By this way, Rbf1 downregulates diap1 at a post-transcriptional level. Moreover, we show that the dREAM complex has a part in these transcriptional regulations. Taken together, these data show that Rbf1, in cooperation with dE2F2 and some members of the dREAM complex, can downregulate the anti-apoptotic genes buffy and diap1, and thus promote cell death in a proliferative tissue

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

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

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

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

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

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