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Series GSE94502 Query DataSets for GSE94502
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 [RNA-Seq]
Organism Homo sapiens
Experiment type Expression 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.
 
Overall design The treatments applied to our RNA-seq A673 samples were for EWS/FLI knockdown/rescue experiments. Briefly, A673 cells were stably infected and selected for expression of a control Luc-RNAi or the EF-2-RNAi to knock-down EWS/FLI. Following 4 days of selection, cells were infected with either an empty pMSCV-hygro vector or vector expressing full length type IV EWS/FLI, the Ä22 EWS/FLI mutant, or mut9 EWS/FLI mutant to rescue EWS/FLI (or respective mutant EWS/FLI) expression in these cells.
 
Contributor(s) Johnson KM
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
 
Platforms (1)
GPL20301 Illumina HiSeq 4000 (Homo sapiens)
Samples (21)
GSM2483525 del22-1
GSM2483526 del22-2
GSM2483527 del22-3
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 PRJNA374499
SRA SRP099405

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE94502_20160322_RNA-seq_normalized_cts.csv.gz 6.5 Mb (ftp)(http) CSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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