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Transcriptomic analysis to functionally map the intrinsically disordered domain of EWS/FLI [Experiment 2]

ABSTRACT: The purpose of this study was to define unique classes of EWS/FLI target genes and the distinct set of structural features present in the EWS domain required for target gene regulation at different EWS/FLI response elements. This study reports the first genome-wide transcriptomic description of a partially-functional EWS/FLI mutant . We report new structure-function dependencies across different classes of response element and tie these dependencies to the ability of EWS/FLI to transform cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE122536 | GEO | 2019/02/21

REPOSITORIES: GEO

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Transcriptomic analysis to functionally map the intrinsically disordered domain of EWS/FLI [Experiment 1]

Project description:The purpose of this study was to define unique classes of EWS/FLI target genes and the distinct set of structural features present in the EWS domain required for target gene regulation at different EWS/FLI response elements. This study reports the first genome-wide transcriptomic description of a partially-functional EWS/FLI mutant . We report new structure-function dependencies across different classes of response element and tie these dependencies to the ability of EWS/FLI to transform cells.

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Transcriptomic analysis to functionally map the intrinsically disordered domain of EWS/FLI

Project description:This SuperSeries is composed of the SubSeries listed below. The data represent functional transcriptomic profiles of the oncogenic fusion transcription factor EWS/FLI.

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Transcriptomic analysis to functionally map the intrinsically disordered domain of EWS/FLI

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A novel role for the EWS portion of EWS/FLI in binding GGAA-microsatellites required for oncogenic transformation in Ewing sarcoma [RNA-Seq]

Project description:Ewing sarcoma usually expresses the EWS/FLI fusion transcription factor oncoprotein. EWS/FLI regulates myriad genes required for Ewing sarcoma development. EWS/FLI binds GGAA-microsatellite sequences in vivo and in vitro, and these sequences provide EWS/FLI-mediated activation to reporter constructs, suggesting that they function as EWS/FLI-response elements. Genomic GGAA-microsatellites are highly variable and polymorphic. Current data suggest that there is an optimal “sweet-spot” GGAA-microsatellite length (of 18-26 GGAA repeats) that confers maximal EWS/FLI-responsiveness to target genes, but the mechanistic basis for this was not known. We now demonstrate the absolute necessity of an EWS/FLI-bound GGAA-microsatellite in regulation of the NR0B1 gene, as well as for Ewing sarcoma proliferation and oncogenic transformation. Biochemical studies, using recombinant Δ22 (a version of EWS/FLI containing only the FLI portion) demonstrated a stoichiometry of one Δ22-monomer binding to every two consecutive GGAA-repeats on shorter GGAA-microsatellite sequences. Surprisingly, the affinity for Δ22 binding to GGAA-microsatellites significantly decreased, and ultimately became unmeasureable, when the size of the GGAA-microsatellite was increased to the “sweet-spot” length. In contrast, a fully-functional EWS/FLI mutant (Mut9) that retains approximately half of the EWS portion of the fusion showed low affinity for smaller GGAA-microsatellites, but instead significantly increased its affinity at “sweet-spot” microsatellite lengths. Single-gene ChIP and genome-wide ChIP-seq and RNA-seq studies extended these findings to the in vivo setting. Taken together, these data demonstrate the absolute requirement of GGAA-microsatellites as EWS/FLI activating response elements in vivo and reveal an unsuspected novel role for the EWS portion of the EWS/FLI fusion in binding to optimal-length GGAA-microsatellites.

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