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The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct from its DNA-binding function in Ewing sarcoma

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Homo sapiens

PROVIDER: GSE160898 | GEO | 2021/06/15

REPOSITORIES: GEO

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The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct and separable from its DNA-binding function in Ewing sarcoma.

Project description:Ewing sarcoma is an aggressive bone cancer of children and young adults defined by the presence of a chromosomal translocation: t(11;22)(q24;q12). The encoded protein, EWS/FLI, fuses the amino-terminal domain of EWS to the carboxyl-terminus of FLI. The EWS portion is an intrinsically disordered transcriptional regulatory domain, while the FLI portion contains an ETS DNA-binding domain and two flanking regions of unknown function. Early studies using non-Ewing sarcoma models provided conflicting information on the roles of each domain of FLI in EWS/FLI oncogenic function. We therefore sought to define the specific contributions of each FLI domain to EWS/FLI activity in a well-validated Ewing sarcoma model and, in doing so, to better understand Ewing sarcoma development mediated by the fusion protein. We analyzed a series of engineered EWS/FLI mutants with alterations in the FLI portion using a variety of assays. Fluorescence anisotropy, CUT&RUN, and ATAC-sequencing experiments revealed that the isolated ETS domain is sufficient to maintain the normal DNA-binding and chromatin accessibility function of EWS/FLI. In contrast, RNA-sequencing and soft agar colony formation assays revealed that the ETS domain alone was insufficient for transcriptional regulatory and oncogenic transformation functions of the fusion protein. We found that an additional alpha-helix immediately downstream of the ETS domain is required for full transcriptional regulation and EWS/FLI-mediated oncogenesis. These data demonstrate a previously unknown role for FLI in transcriptional regulation that is distinct from its DNA-binding activity. This activity is critical for the cancer-causing function of EWS/FLI and may lead to novel therapeutic approaches.

| S-EPMC8298202 | biostudies-literature

The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct from its DNA-binding function in Ewing sarcoma [RNA-seq]

Project description:The purpose of this study was to determine the roles of the flanking regions of the FLI ETS DNA-binding domain in gene regulation of Ewing sarcoma cells. EWS/FLI cDNA constructs including various deletions of the FLI portion were virally transduced into cells and we sought to determine the transcriptional profile of each.

2021-06-15 | GSE160897 | GEO

The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct from its DNA-binding function in Ewing sarcoma [CUT&RUN]

Project description:The purpose of this study was to compare the relevance of the FLI portion for genomic localization of the EWS/FLI protein in Ewing sarcoma cells. EWS/FLI cDNA constructs including various deletions of the FLI portion were virally transduced into cells and we sought to determine the ATAC profile of each.

2021-06-15 | GSE160896 | GEO

The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct from its DNA-binding function in Ewing sarcoma [ATAC-seq]

Project description:The purpose of this study was to compare the relevance of the FLI portion of EWS/FLI in the ability of the protein to act as a pioneer factor in Ewing sarcoma cells. EWS/FLI cDNA constructs including various deletions of the FLI portion were virally transduced into cells and we sought to determine the ATAC profile of each.

2021-06-15 | GSE160895 | GEO

The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct from its DNA-binding function in Ewing sarcoma [RNA-seq III]

Project description:The purpose of this study was to determine transcriptional regulatory activity of EWS/FLI mutants in an alternative, non-Ewing sarcoma cell line. EWS/FLI cDNA constructs including various deletions of the FLI portion were transfected in HEK-293EBNA cells and we sought to determine the transcriptional profile of each through RNA-sequencing.

2021-06-15 | GSE173414 | GEO

The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct from its DNA-binding function in Ewing sarcoma [RNA-seq II]

Project description:The purpose of this study was to determine the roles of the alpha-helical region just downstream of the FLI ETS DNA-binding domain in EWS/FLI-driven gene regulation of Ewing sarcoma cells. EWS/FLI cDNA constructs including various deletions of the FLI portion were virally transduced into cells and we sought to determine the transcriptional profile of each.

2021-06-15 | GSE173376 | GEO

The role of FLI-1-EWS, a fusion gene reciprocal to EWS-FLI-1, in Ewing sarcoma.

Project description:Ewing sarcoma is a cancer of bone and soft tissue in children that is characterized by a chromosomal translocation involving EWS and an Ets family transcription factor, most commonly FLI-1. The EWS-FLI-1 fusion oncogene is widely believed to play a central role in Ewing sarcoma. The EWS-FLI-1 gene product regulates the expression of a number of genes important for cancer progression, can transform mouse cells such as NIH3T3 and C3H10T1/2, and is necessary for proliferation and tumorigenicity of Ewing sarcoma cells, suggesting that EWS-FLI-1 is the causative oncogene. However, a variety of evidence also suggest that EWS-FLI-1 alone cannot fully explain the Ewing sarcomagenesis. Here we report that FLI-1-EWS, a fusion gene reciprocal to EWS-FLI-1, is frequently expressed in Ewing sarcoma. We present evidence suggesting that endogenous FLI-1-EWS is required for Ewing sarcoma growth and that FLI-1-EWS cooperates with EWS-FLI-1 in human mesenchymal stem cells, putative cells of origin of Ewing sarcoma, through abrogation of the proliferation arrest induced by EWS- FLI-1.

| S-EPMC4701224 | biostudies-literature

BCL11B is up-regulated by EWS/FLI and contributes to the transformed phenotype in Ewing sarcoma.

Project description:The EWS/FLI translocation product is the causative oncogene in Ewing sarcoma and acts as an aberrant transcription factor. EWS/FLI dysregulates gene expression during tumorigenesis by abnormally activating or repressing genes. The expression levels of thousands of genes are affected in Ewing sarcoma, however, it is unknown which of these genes contribute to the transformed phenotype. Here we characterize BCL11B as an up-regulated EWS/FLI target that is necessary for the maintenance of transformation in patient derived Ewing sarcoma cells lines. BCL11B, a zinc finger transcription factor, acts as a transcriptional repressor in Ewing's sarcoma and contributes to the EWS/FLI repressed gene signature. BCL11B repressive activity is mediated by the NuRD co-repressor complex. We further demonstrate that re-expression of SPRY1, a repressed target of BCL11B, limits the transformation capacity of Ewing sarcoma cells. These data define a new pathway downstream of EWS/FLI required for oncogenic maintenance in Ewing sarcoma.

| S-EPMC3601955 | biostudies-literature

Ewing sarcoma cells secrete EWS/Fli-1 fusion mRNA via microvesicles.

Project description:Tumours defined as Ewing sarcoma (ES) constitute a group of highly malignant neoplasms that most often affect children and young adults in the first 2 decades of life. The EWS/Fli-1 fusion gene, a product of the translocation t(11;22) (q24; 12), is detected in 95% of ES patients. Recently, it was validated that cells emit a heterogeneous mixture of vesicular, organelle-like structures (microvesicles, MVs) into their surroundings including blood and body fluids, and that these MVs contain a selected set of tumor-related proteins and high levels of mRNAs and miRNAs. In this present study, we detected the Ewing sarcoma-specific EWS/Fli-1 mRNA in MVs from the culture medium of ES cell lines carrying t(11;22) (q24; 12). Also, we detected this fusion gene in approximately 40% of the blood samples from mice inoculated with xenografts of TC135 or A673 cells. These findings indicate the EWS/Fli-1 mRNA in MVs might be a new non-invasive diagnostic marker for specific cases of Ewing sarcoma.

| S-EPMC3790721 | 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

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