Large-scale analysis by SAGE reveals new mechanisms of v-erbA oncogene action

<p>Abstract</p> <p>Background:</p> <p>The <it>v-erbA </it>oncogene, carried by the Avian Erythroblastosis Virus, derives from the <it>c-erbAα </it>proto-oncogene that encodes the nuclear receptor for triiodothyronine (T3R). v-ErbA transforms erythroid progenitors <it>in vitro </it>by blocking their differentiation, supposedly by interference with T3R and RAR (Retinoic Acid Receptor). However, v-ErbA target genes involved in its transforming activity still remain to be identified.</p> <p>Results:</p> <p>By using Serial Analysis of Gene Expression (SAGE), we identified 110 genes deregulated by v-ErbA and potentially implicated in the transformation process. Bioinformatic analysis of promoter sequence and transcriptional assays point out a potential role of c-Myb in the v-ErbA effect. Furthermore, grouping of newly identified target genes by function revealed both expected (chromatin/transcription) and unexpected (protein metabolism) functions potentially deregulated by v-ErbA. We then focused our study on 15 of the new v-ErbA target genes and demonstrated by real time PCR that in majority their expression was activated neither by T3, nor RA, nor during differentiation. This was unexpected based upon the previously known role of v-ErbA.</p> <p>Conclusion:</p> <p>This paper suggests the involvement of a wealth of new unanticipated mechanisms of v-ErbA action.</p

Étude du rôle du trafic intracellulaire dans la régulation de la signalisation Notch chez Drosophila melanogaster

La voie Notch est une voie de communication intercellulaire qui régule de nombreux processus développementaux chez les métazoaires, notamment la spécification des organes sensoriels chez la Drosophile. La voie est activée lorsque les ligands DSL présents à la surface de la cellule émettrice interagissent avec le récepteur Notch présent à la surface des cellules réceptrices. Le trafic membranaire et l endocytose jouent un rôle essentiel dans la régulation de la voie Notch. En particulier, l endocytose et le recyclage des ligands sont nécessaires à leur activation. L E3 Ubiquitine ligase Neuralized (Neur) régule l activité des ligands en promouvant leur ubiquitination et leur endocytose, toutefois le rôle exact de l ubiquitination et de l endocytose demeure mal compris. Nos résultats indiquent que Neur promeut la transcytose du ligand Delta depuis la membrane basolatérale vers la membrane apicale où réside Notch dans les cellules des organes sensoriels de Drosophile. Nous avons également identifié le complexe adaptateur-Clathrine AP-1 de Drosophile comme un nouveau régulateur négatif de la voie Notch. Chez les mammifères, AP-1 régule le recyclage basolatéral et le transport intracellulaire entre le réseau trans-golgien et les endosomes. Nous montrons que dans les organes sensoriels de Drosophile, AP-1 prévient l accumulation apicale du co-activateur de Notch, Sanpodo, et régule la stabilisation de Notch et Sanpodo au niveau des complexes jonctionnels riches en DE-Cadhérine, à l interface entre les cellules émettrice et réceptrice. L ensemble de nos résultats suggèrent que ce domaine jonctionnel pourrait être le site d interaction productif entre ligand et récepteur.Notch is a cell-cell communication pathway that regulates numerous developmental processes in metazoans. In Drosophila, sensory organ specification is governed by Notch signaling. Notch pathway is activated when DSL ligands from the signal-sending cell surface interact with Notch present at the signal-receiving cell surface. Membrane trafficking and endocytosis play a key role in the regulation of Notch signaling. In particular, ligands need to be endocytosed and recycled to be active. The E3 Ubiquitine ligase Neuralized (Neur) regulates ligands activity by promoting their ubiquitination and endocytosis. However, how ubiquitination and endocytosis contribute to ligands activation remains unknown. Our data show that Neur promotes the Delta ligand transcytosis from a basolateral to an apical membrane where Notch is enriched in Drosophila sensory organ cells. We also identified the Clathrin-adaptor complex AP-1 as a novel regulator of Notch signaling. Mammalian AP-1 complex regulates basolateral recycling and intracellular trafficking between trans-golgi network and endosomes. We show that in Drosophila sensory organ cells, AP-1 prevents the apical accumulation of the Notch co-activator Sanpodo, and regulates Notch and Sanpodo stabilization at the level of DE-Cadherin, at the interface between the sending-cell and the receiving-cell. Altogether, our results suggest that this junctional domain could be the site for productive interaction between the ligand and the receptor.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Numb inhibits the recycling of Sanpodo in Drosophila sensory organ precursor.

International audienceIn metazoans, unequal partitioning of the cell-fate determinant Numb underlies the generation of distinct cell fates following asymmetric cell division [1-5]. In Drosophila, during asymmetric division of the sensory organ precursor (SOP) cell, Numb is unequally inherited by the pIIb daughter cell, where it antagonizes Notch [1, 6-8]. Numb inhibits Notch partly through inhibiting the plasma membrane localization of Sanpodo (Spdo), a transmembrane protein required for Notch signaling during asymmetric cell division [9, 10]. Numb, by binding to Spdo and α-Adaptin, was proposed to mediate Spdo endocytosis alone or bound to Notch in the pIIb cell, thereby preventing Notch activation [11-16]. However, in addition to endocytosis, Numb also controls the postendocytic trafficking and degradation of Notch in mammals [17, 18] and negatively regulates basolateral recycling in C. elegans [19, 20]. Thus, whether Numb promotes the endocytosis of Spdo is a question that requires experimental demonstration and is therefore investigated in this article. Based on internalization assays, we show that Spdo endocytosis is restricted to cells in interphase and requires AP-2 activity. Surprisingly, the bulk endocytosis of Spdo occurs properly in numb mutant SOP, indicating that Numb does not regulate the steady-state localization of Spdo via Spdo internalization. We report that Numb genetically and physically interacts with AP-1, a complex regulating the basolateral recycling of Spdo [21]. In numb mutant organs, Spdo is efficiently internalized and recycled back to the plasma membrane. We propose that Numb acts in concert with AP-1 to control the endocytic recycling of Spdo to regulate binary-fate decisions

AP-1 controls the trafficking of Notch and Sanpodo toward E-cadherin junctions in sensory organ precursors.

International audienceIn Drosophila melanogaster, external sensory organs develop from a single sensory organ precursor (SOP). The SOP divides asymmetrically to generate daughter cells, whose fates are governed by differential Notch activation. Here we show that the clathrin adaptor AP-1 complex, localized at the trans Golgi network and in recycling endosomes, acts as a negative regulator of Notch signaling. Inactivation of AP-1 causes ligand-dependent activation of Notch, leading to a fate transformation within sensory organs. Loss of AP-1 affects neither cell polarity nor the unequal segregation of the cell fate determinants Numb and Neuralized. Instead, it causes apical accumulation of the Notch activator Sanpodo and stabilization of both Sanpodo and Notch at the interface between SOP daughter cells, where DE-cadherin is localized. Endocytosis-recycling assays reveal that AP-1 acts in recycling endosomes to prevent internalized Spdo from recycling toward adherens junctions. Because AP-1 does not prevent endocytosis and recycling of the Notch ligand Delta, our data indicate that the DE-cadherin junctional domain may act as a launching pad through which endocytosed Notch ligand is trafficked for signaling

New mechanisms of v-ErbA oncogene action revealed by SAGE analysis

National audienceThe v-erbA oncogene, carried by the Avian Erythroblastosis Virus, derives from the c-erbAalpha proto-oncogene which encodes the nuclear receptor for the thyroid hormone triiodothyronine (T3). In vitro, v-ErbA transforms erythroid progenitors by blocking their differentiation. It has been proposed that v-ErbA acts as a transcriptional repressor for genes normally activated by T3 and retinoic acid (RA), upregulated during the differentiation process. However, v-ErbA target genes responsible for transformation have yet to be identified. We used Serial Analysis of Gene Expression (SAGE) to analyze the transcriptome of avian erythroid progenitors (T2ECs), the natural target cells of v-erbA, expressing either an oncogenic form or a non-transforming form of verbA. The comparison of these two libraries revealed 83 genes differentially expressed between these two conditions. So far, the differential expression for 16 of them has been confirmed by real-time PCR on multiple independent repetitions. We observed that, among these v-ErbA target genes, some are activated by T3 and RA. This confirms that activation by T3 and RA receptors is indeed inhibited by v-ErbA. However, the expression of a vast majority of v-ErbA target genes did not vary in response to T3 and RA. These results suggest that v-ErbA must also act by T3- and RA-independent mechanisms in the transformation process. Furthermore, most v-erbA target genes do not vary during the differentiation process, in contrast to the expected role of v-erbA. In order to determine which major functions are deregulated by v-ErbA, we clustered the target genes identified according to the cellular function encoded by their corresponding proteins. We found that many of them are involved in the protein translation process. In order to understand the molecular mechanisms responsible for the coordinated variation of the v-ErbA target gene, we analyzed their promoter sequences and found the presence of c-myb binding sites as a signature motif of v-erbA target genes. This suggests a role for c-myb in the v-erbA-induced transformation process. Altogether, these studies demonstrate the involvement of new mechanisms pointing toward an unanticipated complexity of v-erbA oncogene action

New mechanisms of v-ErbA oncogene action revealed by SAGE analysis

National audienceThe v-erbA oncogene, carried by the Avian Erythroblastosis Virus, derives from the c-erbAalpha proto-oncogene which encodes the nuclear receptor for the thyroid hormone triiodothyronine (T3). In vitro, v-ErbA transforms erythroid progenitors by blocking their differentiation. It has been proposed that v-ErbA acts as a transcriptional repressor for genes normally activated by T3 and retinoic acid (RA), upregulated during the differentiation process. However, v-ErbA target genes responsible for transformation have yet to be identified. We used Serial Analysis of Gene Expression (SAGE) to analyze the transcriptome of avian erythroid progenitors (T2ECs), the natural target cells of v-erbA, expressing either an oncogenic form or a non-transforming form of verbA. The comparison of these two libraries revealed 83 genes differentially expressed between these two conditions. So far, the differential expression for 16 of them has been confirmed by real-time PCR on multiple independent repetitions. We observed that, among these v-ErbA target genes, some are activated by T3 and RA. This confirms that activation by T3 and RA receptors is indeed inhibited by v-ErbA. However, the expression of a vast majority of v-ErbA target genes did not vary in response to T3 and RA. These results suggest that v-ErbA must also act by T3- and RA-independent mechanisms in the transformation process. Furthermore, most v-erbA target genes do not vary during the differentiation process, in contrast to the expected role of v-erbA. In order to determine which major functions are deregulated by v-ErbA, we clustered the target genes identified according to the cellular function encoded by their corresponding proteins. We found that many of them are involved in the protein translation process. In order to understand the molecular mechanisms responsible for the coordinated variation of the v-ErbA target gene, we analyzed their promoter sequences and found the presence of c-myb binding sites as a signature motif of v-erbA target genes. This suggests a role for c-myb in the v-erbA-induced transformation process. Altogether, these studies demonstrate the involvement of new mechanisms pointing toward an unanticipated complexity of v-erbA oncogene action

Neuronal diversity and convergence in a visual system developmental atlas

International audienc

Large-scale analysis by SAGE reveals new mechanisms of oncogene action-3

<p><b>Copyright information:</b></p><p>Taken from "Large-scale analysis by SAGE reveals new mechanisms of oncogene action"</p><p>http://www.biomedcentral.com/1471-2164/8/390</p><p>BMC Genomics 2007;8():390-390.</p><p>Published online 26 Oct 2007</p><p>PMCID:PMC2194726.</p><p></p> the transforming form of v-ErbA. For each function, the annotations of all genes harbouring this function is represented (only the more precise term in the Gene Ontology and only the annotations concerning more than one half of genes are represented. GO MF: Gene Ontology molecular function, GO BP: Gene Ontology biological process, SP KW: Swissprot keyword, KEGG PW: KEGG pathway). A black square in the intersection of one line (one gene) and one column (one annotation) imply that the corresponding gene has the corresponding annotation. The gene names written in bold correspond to genes for which the differential expression has been confirmed by real-time PCR

Large-scale analysis by SAGE reveals new mechanisms of oncogene action-5

<p><b>Copyright information:</b></p><p>Taken from "Large-scale analysis by SAGE reveals new mechanisms of oncogene action"</p><p>http://www.biomedcentral.com/1471-2164/8/390</p><p>BMC Genomics 2007;8():390-390.</p><p>Published online 26 Oct 2007</p><p>PMCID:PMC2194726.</p><p></p>es. The Venn Diagrams represent a comparison between the list of v-ErbA repressed target genes (this study) and the genes upregulated during T2ECs differentiation [34] (Left part of the figure) or the list of v-ErbA activated target genes and the genes repressed during T2ECs differentiation (Right part of the figure). 6B – Comparison of v-ErbA, differentiation, T3 and RA target genes. The Venn diagram summarizes the overlap in the sets of genes that were activated or repressed in response to v-ErbA or/and T3 or/and RA or/and during the differentiation process in T2ECs