Cycline G, contrôle de la transcription et stabilité du développement

.During development, cells progressively acquire their identity by establishing specifictranscriptional profiles that will be maintained throughout successive cell divisions byepigenetic mechanisms. Despite numerous sources of developmental variability - whetherenvironmental, genetic or stochastic, living organisms exhibit uncannily stereotypedphenotypes, suggesting the existence of regulation processes tending to developmentalhomeostasis. In Drosophila, Cyclin G could participate in developmental stability, whichbuffers stochastic developmental variation. Moreover, this cyclin is a transcriptionalregulator and interacts with two Enhancers of Trithorax and Polycomb (ETP): ASX and Corto,both involved in transcriptional gene silencing and activation. By studying the transcriptomeof Drosophila imaginal wing discs overexpressing Cyclin G, we have identified itstranscriptional targets. We determined that the ETP-interacting domain of Cyclin G (whichbinds ASX and Corto) may be involved in developmental stability. Our results show that afunctional interaction between Cyclin G and two Polycomb group complexes involved intranscriptional gene silencing (PR-DUB and PRC1), may control the expression of genesrequired for developmental stability. Additionally, Cycling G might participate indevelopmental stability through functional interactions with splicing factors. Altogether, ourresults suggest that Cyclin G deregulation may induce an increase in transcriptional noise,resulting in heightened developmental variation.Au cours du développement, les cellules acquièrent progressivement leur identité enétablissant des profils transcriptionnels spécifiques qui seront maintenus à travers lesdivisions cellulaires successives par des mécanismes dits épigénétiques. En dépit denombreuses sources de variations de l’environnement, génétiques ou aléatoires, lesorganismes vivants présentent des phénotypes très stéréotypés. Cela suggère l’existence deprocessus de régulation assurant l’homéostasie du développement. Chez la drosophile, laprotéine Cycline G jouerait un rôle dans la stabilité du développement, processus quitamponne les variations aléatoires du développement. De plus, cette cycline est unrégulateur trancriptionnel et interagit avec deux Enhancers de Trithorax et Polycomb (ETP) :ASX et Corto, impliqués dans l’activation et la répression de nombreux gènes. L’analyse dutranscriptome des disques imaginaux d’ailes de larves de drosophile qui surpexpriment CycGnous a permis d’identifier ses cibles transcriptionnelles. Nous avons montré que le domained’interaction avec les ETP (ASX et Corto) de Cycline G pourrait être impliqué dans la stabilitédu développement. Nos résultats suggèrent qu’une interaction fonctionnelle entre Cycline Get deux complexes Polycomb répresseurs (PR-DUB et PRC1), contrôlerait l’expression degènes importants pour la stabilité du développement. Par ailleurs, l’interaction fonctionnelleentre des facteurs d’épissage et Cycline G serait également impliquée dans la stabilité dudéveloppement. Nos résultats suggèrent que la dérégulation de CycG induirait uneaugmentation du bruit transcriptionnel, ayant des répercussions sur la stabilité dudéveloppement

Drosophila Cyclin G and epigenetic maintenance of gene expression during development

Background: Cyclins and cyclin-dependent kinases (CDKs) are essential for cell cycle regulation and are functionally associated with proteins involved in epigenetic maintenance of transcriptional patterns in various developmental or cellular contexts. Epigenetic maintenance of transcription patterns, notably of Hox genes, requires the conserved Polycomb-group (PcG), Trithorax-group (TrxG), and Enhancer of Trithorax and Polycomb (ETP) proteins, particularly well studied in Drosophila. These proteins form large multimeric complexes that bind chromatin and appose or recognize histone post-translational modifications. PcG genes act as repressors, counteracted by trxG genes that maintain gene activation, while ETPs interact with both, behaving alternatively as repressors or activators. Drosophila Cyclin G negatively regulates cell growth and cell cycle progression, binds and co-localizes with the ETP Corto on chromatin, and participates with Corto in Abdominal-B Hox gene regulation. Here, we address further implications of Cyclin G in epigenetic maintenance of gene expression. Results: We show that Cyclin G physically interacts and extensively co-localizes on chromatin with the conserved ETP Additional sex combs (ASX), belonging to the repressive PR-DUB complex that participates in H2A deubiquitination and Hox gene silencing. Furthermore, Cyclin G mainly co-localizes with RNA polymerase II phosphorylated on serine 2 that is specific to productive transcription. CycG interacts with Asx, PcG, and trxG genes in Hox gene maintenance, and behaves as a PcG gene. These interactions correlate with modified ectopic Hox protein domains in imaginal discs, consistent with a role for Cyclin G in PcG-mediated Hox gene repression. Conclusions: We show here that Drosophila CycG is a Polycomb-group gene enhancer, acting in epigenetic maintenance of the Hox genes Sex combs reduced (Scr) and Ultrabithorax (Ubx). However, our data suggest that Cyclin G acts alternatively as a transcriptional activator or repressor depending on the developmental stage, the tissue or the target gene. Interestingly, since Cyclin G interacts with several CDKs, Cyclin G binding to the ETPs ASX or Corto suggests that their activity could depend on Cyclin G-mediated phosphorylation. We discuss whether Cyclin G fine-tunes transcription by controlling H2A ubiquitination and transcriptional elongation via interaction with the ASX subunit of PR-DUB.Science, Faculty ofZoology, Department ofReviewedFacult

Single amino-acid mutation in a Drosoph ila melanogaster ribosomal protein: An insight in uL11 transcriptional activity.

The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes (RPGs). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes (RiBis) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co-localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D. melanogaster uL11. Unexpectedly, the uL11K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11K3A but not in uL11K3Y. Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. However, Histone Association Assays indicate that the mutant proteins still bind chromatin. RNA-seq analyses from wing imaginal discs show that Corto represses RPG expression whereas very few genes are deregulated in uL11 mutants. We propose that Corto, by repressing RPG expression, ensures that all ribosomal proteins are present at the correct stoichiometry, and that uL11 fine-tunes its transcriptional regulation of RPGs

Single amino-acid mutation in a Drosoph ila melanogaster ribosomal protein: An insight in uL11 transcriptional activity

International audienceThe ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes ( RPGs ). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes ( RiBis ) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co-localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D . melanogaster uL11 . Unexpectedly, the uL11 K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11 K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11 K3A but not in uL11 K3Y . Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. However, Histone Association Assays indicate that the mutant proteins still bind chromatin. RNA-seq analyses from wing imaginal discs show that Corto represses RPG expression whereas very few genes are deregulated in uL11 mutants. We propose that Corto, by repressing RPG expression, ensures that all ribosomal proteins are present at the correct stoichiometry, and that uL11 fine-tunes its transcriptional regulation of RPGs

Cyclin G and the Polycomb Repressive complexes PRC1 and PR-DUB cooperate for developmental stability

International audienc

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

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

<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