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| Status |
Public on Oct 22, 2017 |
| Title |
A novel role for the EWS portion of EWS/FLI in binding GGAA-microsatellites required for oncogenic transformation in Ewing sarcoma [ChIP-Seq] |
| Organism |
Homo sapiens |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
|
| Summary |
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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| Overall design |
Examination of EWS/FLI binding in A673 cells subjected to various treatments
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| Contributor(s) |
Johnson KM, Lessnick SL |
| Citation(s) |
28847958 |
| Submission date |
Feb 03, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Kirsten Johnson |
| E-mail(s) |
kirsten.johnson@nationwidechildrens.org
|
| Phone |
6143552989
|
| Organization name |
Nationwide Childrens Hospital
|
| Department |
Childhood Cancer and Blood Disorders
|
| Lab |
Stephen Lessnick
|
| Street address |
The Research Institute, WA5110, 700 Childrens Drive
|
| City |
Columbus |
| State/province |
Ohio |
| ZIP/Postal code |
43205 |
| Country |
USA |
| |
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| Platforms (1) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
|
| Samples (4)
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| This SubSeries is part of SuperSeries: |
| GSE94503 |
A novel role for the EWS portion of EWS/FLI in binding GGAA-microsatellites required for oncogenic transformation in Ewing sarcoma |
|
| Relations |
| BioProject |
PRJNA369808 |
| SRA |
SRP098815 |
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