Identification d’un nouveau rôle du facteur de transcription ERG dans la régulation de l’épissage alternatif en collaboration avec RBFOX2

Transcription factors (TFs) are usually defined as sequence-specific DNA-binding proteins that control transcription as a first step of gene expression. However, more and more evidence indicates that they are also involved in the regulation of posttranscriptional steps, in particular the splicing process. In our laboratory, we previously demonstrated that ERG protein, a member of Ets TFs has a role in the regulation of decay [1], a downstream mRNA processing event. In addition, it was suggested that TFs act directly on splicing through different mechanisms, such as by modifying RNA polymerase II elongation rates and altering the kinetics of exposure of splice sites, by the recruitment of transcriptional coactivators involved in splicing, or through the modulation of direct splicing factors expression. However, currently it was proposed that some TFs can control alternative splicing (AS) directly through their binding onto pre-mRNA, thus controlling AS via an unknown but direct mechanism [2]. In this work, we hypothesized that ERG TF can regulate AS process and this can be affected in cancers such as Ewing’s sarcoma (EwS). Thus, we reported for the first time that ERG can regulate AS of numerous splicing targets. This is probably by a direct mechanism. Indeed, first we showed that ERG associates with spliceosomal components, it is found on nascent pre-mRNA and induces AS through its CTAD domain, by inclusion or exclusion of an alternative exon when it is recruited onto a reporter. Second, transcriptomic analysis demonstrated that depletion of ERG affects a large number of AS events, mainly cassette exons, in a tissue-specific manner. Third, we observed that sequences of ERG-regulated cassette exons and their 200 adjacent intronic base pairs are enriched in binding motifs for RBFOX2, a tissue-specific splicing regulator and an important functional interactor of the LASR splicing complex. We demonstrated that ERG and RBFOX2 interact via their CTAD and C-terminal domains respectively, and both proteins collaborate to regulate a large set of cassette exons. Finally, our observations suggested that the splicing function of ERG is independent of its DNA-binding and transcriptional activity since we did not find any significant overlap between differentially expressed and differentially spliced genes in ERG-depleted cells, and a transcriptionally inactive ERG variant lacking the DNA-binding domain (ERG-ETS) presented full splicing activity in our reporter assay. Perturbation of the splicing program is a feature of EwS. This was previously attributed to the presence of FET-Ets fusions [3] and the splicing function of EWS-FLI1 was only attributed to its EWS moiety. In addition, the functional relevance of EWS-FLI1 in the EwS oncogenic process is still unknown. We have studied the role of the EWS-FLI1 fusion in the reporter assay and observed that its AS function is linked to its recruitment onto the reporter mRNA. In addition, our observations suggested that the FLI1-derived moiety can also be a major contributor to the fusion protein’s splicing function in EwS and that EWS-FLI1 also interacts with RFBOX2, suggesting that the EWS-FLI1 and RFBOX2 interaction could play a major role in EwS development. Altogether, our results support a model in which the ERG protein controls AS processes beyond its role as a TF. This new function of ERG in AS regulation should be considered for future cancer therapies

Insulin signaling controls the expression of O-GlcNAc transferase and its interaction with lipid microdomains

Lipid microdomains (rafts) are cholesterol-enriched dynamic ordered lipid domains belonging to cell membranes involved in diverse cellular functions, including signal transduction, membrane trafficking, and infection. Many studies have reported relationships between insulin signaling and lipid rafts. Likewise, links between insulin signaling and O-GlcNAcylation have also been described. However, the potential connection between O-GlcNAc and raft dynamics remains unexplored. Here we show that O-GlcNAc and the enzyme that creates this modification, O-GlcNAc transferase (OGT), are localized in rafts. On insulin stimulation, we observe time-dependent increases in OGT expression and localization within rafts. We show that these processes depend on activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Inhibition of OGT does not significantly affect cholesterol synthesis and raft building but decreases insulin receptor expression and PI3K and mitogen-activated protein kinase pathway activation. Taken together, these findings indicate that O-GlcNAcylation, lipid rafts, and signaling pathways are spatiotemporally coordinated to enable fundamental cellular functions

The transcription factor ERG recruits CCR4-NOT to control mRNA decay and mitotic progression

Control of mRNA levels, a fundamental aspect in the regulation of gene expression, is achieved through a balance between mRNA synthesis and decay. E26-related gene (Erg) proteins are canonical transcription factors whose previously described functions are confined to the control of mRNA synthesis. Here, we report that ERG also regulates gene expression by affecting mRNA stability and identify the molecular mechanisms underlying this function in human cells. ERG is recruited to mRNAs via interaction with the RNA-binding protein RBPMS, and it promotes mRNA decay by binding CNOT2, a component of the CCR4-NOT deadenylation complex. Transcriptome-wide mRNA stability analysis revealed that ERG controls the degradation of a subset of mRNAs highly connected to Aurora signaling, whose decay during S phase is necessary for mitotic progression. Our data indicate that control of gene expression by mammalian transcription factors may follow a more complex scheme than previously anticipated, integrating mRNA synthesis and degradation.status: publishe

The transcription factor ERG recruits CCR4-NOT to control mRNA decay and mitotic progression.

Control of mRNA levels, a fundamental aspect in the regulation of gene expression, is achieved through a balance between mRNA synthesis and decay. E26-related gene (Erg) proteins are canonical transcription factors whose previously described functions are confined to the control of mRNA synthesis. Here, we report that ERG also regulates gene expression by affecting mRNA stability and identify the molecular mechanisms underlying this function in human cells. ERG is recruited to mRNAs via interaction with the RNA-binding protein RBPMS, and it promotes mRNA decay by binding CNOT2, a component of the CCR4-NOT deadenylation complex. Transcriptome-wide mRNA stability analysis revealed that ERG controls the degradation of a subset of mRNAs highly connected to Aurora signaling, whose decay during S phase is necessary for mitotic progression. Our data indicate that control of gene expression by mammalian transcription factors may follow a more complex scheme than previously anticipated, integrating mRNA synthesis and degradation