ABSTRACT
Ewing sarcoma (EwS) is a cancer that arises in the bones and soft tissues, typically driven by the Ewing's sarcoma breakpoint region 1-Friend leukemia virus integration 1 (EWS-FLI) oncogene. Implementation of genetically modified animal models of EwS has proved difficult largely owing to EWS-FLI's high toxicity. The EWS-FLI1FS frameshift variant that circumvents toxicity but is still able to perform key oncogenic functions provided the first study model in Drosophila. However, the quest for Drosophila lines expressing full-length, unmodified EWS-FLI remained open. Here, we show that EWS-FLI1FS's lower toxicity is owed to reduced protein levels caused by its frameshifted C-terminal peptide, and report new strategies through which we have generated Drosophila lines that express full-length, unmodified EWS-FLI. Using these lines, we have found that the upregulation of transcription from GGAA-microsatellites (GGAAµSats) presents a positive linear correlation within a wide range of EWS-FLI protein concentrations. In contrast, rather counterintuitively, GGAAµSats-independent transcriptomic dysregulation presents relatively minor differences across the same range, suggesting that GGAAµSat-dependent and -independent transcriptional upregulation present different kinetics of response with regards to changing EWS-FLI protein concentration. Our results underpin the functional relevance of varying EWS-FLI expression levels and provide experimental tools to investigate, in Drosophila, the effect of the EWS-FLI 'high' and 'low' states that have been reported and are suspected to be important for EwS in humans.
Subject(s)
Oncogene Proteins, Fusion , Proto-Oncogene Protein c-fli-1 , RNA-Binding Protein EWS , Animals , RNA-Binding Protein EWS/metabolism , RNA-Binding Protein EWS/genetics , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Protein c-fli-1/metabolism , Proto-Oncogene Protein c-fli-1/genetics , Humans , Sarcoma, Ewing/genetics , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/pathology , Animals, Genetically Modified , Drosophila/genetics , Drosophila/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolismABSTRACT
High RanGTP around chromatin is important for governing spindle assembly during meiosis and mitosis by releasing the inhibitory effects of importin α/ß. Here we examine how the Ran gradient regulates Kinesin-14 function to control spindle organization. We show that Xenopus Kinesin-14, XCTK2, and importin α/ß form an effector gradient that is highest at the poles and diminishes toward the chromatin, which is opposite the RanGTP gradient. Importin α and ß preferentially inhibit XCTK2 antiparallel microtubule cross-linking and sliding by decreasing the microtubule affinity of the XCTK2 tail domain. This change in microtubule affinity enables RanGTP to target endogenous XCTK2 to the spindle. We propose that these combined actions of the Ran pathway are critical to promote Kinesin-14 parallel microtubule cross-linking to help focus spindle poles for efficient bipolar spindle assembly. Furthermore, our work illustrates that RanGTP regulation in the spindle is not simply a switch, but rather generates effector gradients where importins α and ß gradually tune the activities of spindle assembly factors.
