EWS::FLI cooperatively binds at GGAA microsatellites via DBD-a4 helix
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE249578 | GEO | 2024/02/01
REPOSITORIES: GEO
Similar Datasets
Project description:Multi-omics study of DBD-a4 helix of EWS::FLI to understand the underlying mechanism of transcriptional regulation in Ewing sarcoma cell line: TTC-466
2025-02-27 | GSE268942 | GEO
Project description:Multi-omics study of DBD-a4 helix of EWS::FLI to understand the underlying mechanism of transcriptional regulation in Ewing sarcoma cell line: TTC-466
2025-02-27 | GSE268941 | GEO
Project description:Multi-omics study of DBD-a4 helix of EWS::FLI to understand the underlying mechanism of transcriptional regulation in Ewing sarcoma cell line: TTC466
2025-02-27 | GSE268940 | GEO
Project description:Multi-omics study of DBD-a4 helix of EWS::FLI to understand the underlying mechanism of transcriptional regulation in Ewing sarcoma cell line: TTC-466
2025-02-27 | GSE268944 | GEO
Project description:Multi-omics study of DBD-a4 helix of EWS::FLI to understand the underlying mechanism of transcriptional regulation in Ewing sarcoma cell line: TTC-466
2025-02-27 | GSE268935 | GEO
Project description:EWS-FLI-1 was silenced by an shRNA in A673 Ewing sarcoma cells and the resulting alterations in the secretome was analyzed by GeLC-MS/MS approach (six gel slices for each sample, luciferase shRNA-expressing cell secretome as control)
2021-07-06 | PXD016052 | Pride
Project description:Ewing sarcoma is a bone malignancy of children and young adults, frequently harboring the EWS/FLI t(11;22)(q24;q12) chromosomal translocation. The resulting fusion protein is an aberrant transcription factor that uses highly repetitive GGAA-containing elements (microsatellites) to activate and repress thousands of target genes mediating oncogenesis. However, the mechanisms of EWS/FLI interaction with microsatellites and regulation of target genes expression is not clearly understood. Here, we profile genome-wide protein binding and gene expression. Using a combination of unbiased genome-wide computational and experimental analysis, we define GGAA-microsatellites in a Ewing sarcoma context. Our study identifies two distinct classes of GGAA-microsatellites and demonstrates that EWS/FLI responsiveness is dependent on microsatellite length. At close range (within 5 kb) “promoter-like” microsatellites, EWS/FLI binding and subsequent target genes activation is highly dependent on the number of GGAA-motifs. “Enhancer-like” microsatellites demonstrate a positive correlation with length-dependent EWS/FLI binding, but minimal correlation for activated and none for repressed target genes. Our data suggest that EWS/FLI binds to “promoter-like” and “enhancer-like” microsatellites to mediate activation and repression of target genes through different regulatory mechanisms. Such characterization contributes valuable insight to EWS/FLI transcription factor biology and clarifies the role of GGAA-microsatellites on a global genomic scale. This may provide a unique perspective on the role of non-coding DNA in cancer susceptibility and therapeutic development.
2017-11-09 | GSE99959 | GEO
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.
2017-10-22 | GSE94502 | GEO
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.
2017-10-22 | GSE94480 | GEO
Project description:ETS proteins are a family of transcription factors that play important roles in the development of cancer. The Ewing's sarcoma EWS/ETS fusion oncoproteins control a number of cancer-relevant phenotypes in that disease. We recently demonstrated that EWS/FLI, the most common EWS/ETS fusion in Ewing's sarcoma, regulates a portion of its target genes, including the critical target NR0B1, via GGAA-containing microsatellites in their promoters. Given the unusual nature of microsatellites as EWS/FLI response elements, we sought to elucidate the mechanism of EWS/FLI activity at these sites. We found that the ability to bind GGAA microsatellites is shared by multiple ETS family members from distinct phylogenetic subfamilies. Importantly, however, only EWS/ETS-containing fusions are capable of mediating transcriptional activation via these elements, highlighting a neomorphic function of the Ewing's sarcoma fusion proteins. Additional analysis revealed that the GGAA microsatellite binds EWS/FLI with an affinity that is 2 to 3 orders of magnitude lower than previously identified high-affinity consensus/redundant binding sites. The stoichiometry of this interaction is 2 protein molecules for each DNA molecule, suggesting that EWS/FLI binds these elements as a homodimer. The isolated FLI ETS domain bound microsatellite sequences in a nearly identical fashion to full-length EWS/FLI, thus indicating that residues required for homodimeric binding are localized to the ETS domain. These data suggest a new paradigm for an ETS family member binding to DNA at cancer-relevant genetic loci and highlight emergent properties of EWS/FLI that are required for the development of Ewing's sarcoma.
| S-EPMC2935179 | biostudies-literature