Role for the EWS domain of EWS/FLI in binding GGAA-microsatellites required for Ewing sarcoma anchorage independent growth

• Published in: Proceedings of the National Academy of Sciences - PNAS 2017-09, Vol.114 (37), p.9870-9875
• Main Authors: Johnson, Kirsten M., Mahler, Nathan R., Saund, Ranajeet S., Theisen, Emily R., Taslim, Cenny, Callender, Nathan W., Crow, Jesse C., Miller, Kyle R., Lessnick, Stephen L.
• Format: Article
• Language: English
• Subjects: Affinity; Binding; Biological Sciences; Cancer; Cell Line, Tumor; Cell Proliferation - genetics; Children & youth; CRISPR-Cas Systems; DAX-1 Orphan Nuclear Receptor - genetics; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Ewing's sarcoma; Gene expression; Gene sequencing; Genes; Genomes; HEK293 Cells; Humans; In vivo methods and tests; Microfilament Proteins - genetics; Microfilament Proteins - metabolism; Microsatellite Repeats - genetics; Microsatellites; Polymorphism; Protein Domains - genetics; Receptors, Cytoplasmic and Nuclear - genetics; Receptors, Cytoplasmic and Nuclear - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Regulatory sequences; Response Elements - genetics; Ribonucleic acid; RNA; RNA-Binding Protein EWS - genetics; RNA-Binding Protein EWS - metabolism; Sarcoma; Sarcoma, Ewing - genetics; Sarcoma, Ewing - metabolism; Studies
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1701872114

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. These sequences provide EWS/FLI-mediated activation to reporter constructs, suggesting that they function as EWS/FLI-response elements. We now demonstrate the critical role of an EWS/FLI-bound GGAA-microsatellite in regulation of the NR0B1 gene as well as for Ewing sarcoma proliferation and anchorage-independent growth. Clinically, 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 remains unknown. Our biochemical studies, using recombinant Δ22 (a version of EWS/FLI containing only the FLI portion), demonstrate a stoichiometry of one Δ22-monomer binding to every two consecutive GGAA-repeats on shorter microsatellite sequences. Surprisingly, the affinity for Δ22 binding to GGAA-microsatellites significantly decreased, and ultimately became unmeasureable, when the size of the microsatellite was increased to the sweet-spot length. In contrast, a fully functional EWS/FLI mutant (Mut9, which 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 genomewide ChIP-sequencing (ChIP-seq) and RNA-seq studies extended these findings to the in vivo setting. Together, these data demonstrate the critical requirement of GGAA-microsatellites as EWS/FLI activating response elements in vivo and reveal an unexpected role for the EWS portion of the EWS/FLI fusion in binding to sweet-spot GGAA-microsatellites.