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EWS::FLI cooperatively binds at GGAA microsatellites via DBD-a4 helix in TTC-466 [Micro-C]

ABSTRACT: 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

ORGANISM(S): Homo sapiens

PROVIDER: GSE268935 | GEO | 2025/02/27

REPOSITORIES: GEO

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EWS::FLI cooperatively binds at GGAA microsatellites via DBD-a4 helix in TTC-466 [CTCF_CnT]

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

EWS::FLI cooperatively binds at GGAA microsatellites via DBD-a4 helix in TTC-466 [H3K27ac_CnT]

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

EWS::FLI cooperatively binds at GGAA microsatellites via DBD-a4 helix in TTC-466 [RNA-seq]

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

EWS::FLI cooperatively binds at GGAA microsatellites via DBD-a4 helix in TTC-466 cells [FLAG_CnT]

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

EWS::FLI cooperatively binds at GGAA microsatellites via DBD-a4 helix

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: A673

2024-02-01 | GSE249578 | GEO

Homo sapiens strain:TTC-466 cell line

Project description:LSD1 inhibitor HCI-2509 and gene expression in Ewing sarcoma (TTC-466)

| PRJNA213799 | ENA

Emergent Properties of EWS/FLI Regulation via GGAA Microsatellites in Ewing's Sarcoma.

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

The DBD-<i>α</i>4 helix of EWS::FLI is required for GGAA microsatellite binding that underlies genome regulation in Ewing sarcoma.

Project description:Ewing sarcoma is the second most common bone cancer in children and young adults. In 85% of patients, a translocation between chromosomes 11 and 22 results in a potent fusion oncoprotein, EWS::FLI. EWS::FLI is the only genetic alteration in an otherwise unaltered genome of Ewing sarcoma tumors. The EWS portion of the protein is an intrinsically disordered domain involved in transcriptional regulation by EWS::FLI. The FLI portion of the fusion contains a DNA binding domain shown to bind core GGAA motifs and GGAA repeats. A small alpha-helix in the DNA binding domain of FLI, DBD-α4 helix, is critical for the transcription function of EWS::FLI. In this study, we aimed to understand the mechanism by which the DBD-α4 helix promotes transcription, and therefore oncogenic transformation. We utilized a multi-omics approach to assess chromatin organization, active chromatin marks, genome binding, and gene expression in cells expressing EWS::FLI constructs with and without DBD-α4 helix. Our studies revealed DBD-α4 helix is crucial for cooperative binding of EWS::FLI at GGAA microsatellites. This binding underlies many aspects of genome regulation by EWS::FLI such as formation of TADs, chromatin loops, enhancers and productive transcription hubs.

| S-EPMC10862889 | biostudies-literature

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

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. 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 genome-wide 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.

| S-EPMC5603999 | biostudies-literature

Identification of two types of GGAA-microsatellites and their roles in EWS/FLI binding and gene regulation in Ewing sarcoma.

Project description:Ewing sarcoma is a bone malignancy of children and young adults, frequently harboring the EWS/FLI 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 gene 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. We identify two distinct classes of GGAA-microsatellites and demonstrate that EWS/FLI responsiveness is dependent on microsatellite length. At close range "promoter-like" microsatellites, EWS/FLI binding and subsequent target gene activation is highly dependent on number of GGAA-motifs. "Enhancer-like" microsatellites demonstrate length-dependent EWS/FLI binding, but minimal correlation for activated and none for repressed targets. Our data suggest 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 unique perspective on the role of non-coding DNA in cancer susceptibility and therapeutic development.

| S-EPMC5665490 | biostudies-literature

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