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

Johnson, Kirsten M.; Mahler, Nathan; Saund, Ranajeet S.; Theisen, Emily R.; Taslim, Cenny; Callender, Nathan W.; Crow, Julie; Miller, Kyle R.; Lessnick, Stephen L.

doi:10.1073/pnas.1701872114

Cited by 66 publications

(115 citation statements)

References 29 publications

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“…Twenty-four hours after siRNA transfection, cells were seeded on soft agar and grown for 3 to 4 weeks. Quantifying the number of colonies formed revealed that the loss of EWS-FLI1 resulted in 60% fewer colonies compared with siSCR transfection, as had been previously reported (n = 3, p = 0.04, Student's t-test, Figure 2A and 2B) Johnson et al 2017). Cells with EWSR1 knocked down by siEWSR1 also produced 90% fewer colonies, revealing an EWSR1 dependency for growth on a soft agar substrate (n = 3, p = 0.004, Student's t-test).…”

Section: Ewsr1 Is Required For Anchorage-independent Growth In Ewing supporting

confidence: 81%

“…EWSR1 and EWS-FLI1 share the same LC domain, which undergoes oligomerization for the proteins to regulate transcription (Kwon et al 2013;Kato et al 2012;Boulay et al 2017). Previous studies have demonstrated that homotypic self-interactions by the EWS-FLI1 LC domain are required to affect transcription and transformation (Kwon et al 2013;Chong et al 2018;Johnson et al 2017). Additionally, EWS-FLI1 recruits EWSR1 to GGAArich sites along chromosomal DNA (Gorthi et al 2018;Boulay et al 2017).…”

Section: Transcript Levels In Ewing Sarcoma Are Affected By Both Ewsrmentioning

confidence: 99%

“…EWSR1 and EWS-FLI1 share the same low-complexity (LC) domain, which undergoes oligomerization for the proteins to regulate transcription 8,26,28 . Previous studies have demonstrated that homotypic selfinteractions by the EWS-FLI1 LC domain are required to affect transcription and transformation 8,34,38 .…”

Section: Transcript Levels In Ewing Sarcoma Are Affected By Both Ewsrmentioning

confidence: 99%

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Fusion protein EWS-FLI1 is incorporated into a protein granule in cells

Ahmed

Harrell

Schwartz

2020

Preprint

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Ewing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcription regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNAbinding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). The LC domain in EWS-FLI1 can bind RNA polymerase II (RNA Pol II) and can selfassemble through a process known as phase separation. The ability of EWSR1 and related RNA-binding proteins to assemble into ribonucleoprotein granules in cells has been vigorously studied and while many questions remain, the role of phase separation in EWS-FLI1 activity is less understood. We investigated the overlapping functions of EWSR1 and EWS-FLI1 in controlling gene expression and tumorigenic cell growth in Ewing sarcoma, which suggested these proteins function closely together. We then studied the nature of interactions between EWS-FLI1, EWSR1, and RNA Pol II. We observed EWSR1 and RNA Pol II to be present in protein granules in cells. We then identified protein granules in cells associated with the fusion protein, EWS-FLI1. The tyrosine residues in the LC domain are required for both the abilities of EWS-FLI1 to bind its partners, EWSR1 and RNA Pol II, and to incorporate into protein granules. These data suggest that interactions between EWS-FLI1, RNA Pol II, and EWSR1 in Ewing sarcoma can occur in the context of a molecular scaffold found within protein granules in the cell. 6 RNA SequencingA673 cells were transfected with 50 nM siRNA as described above. Cells were collected 72 hours posttransfection and total RNA was extracted using TRIzol reagent (ThermoFisher, Cat. #15596026). 1 µg of total RNA was prepared for sequencing using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, #E7490) to generate sequencing libraries according to manufacturer's instructions. Soft Agar Colony Formation AssayA673, SK-N-MC, and HEK293T/17 cells transfected with 50 nM siRNA were harvested 24 hours posttransfection. HEK293T/17 cells transfected with 50 nM siRNA and 2 µg of plasmid DNA were harvested 24 hours post-transfection. A673 and SK-N-MC cells were seeded at density of 1.0 x 10 5 cells. Cells were resuspended in 0.35% agarose in growth media and plated onto a bed of solidified 0.6% agarose in growth media. HEK293T/17 cells were seeded at a density of 20k to 30k cells. Cells were resuspended in 0.4% agarose in growth media onto a bed of solidified 0.6% agarose in growth media. A673 and SK-N-MC cells were grown at 37°C and 5% CO2 for 3-4 weeks, imaged, and then colonies were counted using ImageJ software. HEK293T/17 cells were grown at 37°C and 5% CO2 for 1-2 weeks, imaged, and then colonies were counted. Colonies with stained with 0.05% methylene blue. Cell Growth AssayA673 cells were reverse transfected at a density of 4.0 x 10 5 cells in 6-well dishes. Cells were collected by trypsinization and counted on a hemocytometer at 24, 48,...

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Section: Ewsr1 Is Required For Anchorage-independent Growth In Ewing supporting

confidence: 81%

Section: Transcript Levels In Ewing Sarcoma Are Affected By Both Ewsrmentioning

confidence: 99%

Section: Transcript Levels In Ewing Sarcoma Are Affected By Both Ewsrmentioning

confidence: 99%

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Fusion protein EWS-FLI1 is incorporated into a protein granule in cells

Ahmed

Harrell

Schwartz

2020

Preprint

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“…4C). The detected number of intranuclear hubs has the same order of magnitude as the number of EWS/FLI1-bound GGAA microsatellites across the human genome (~6000) estimated by ChIP-seq and bioinformatics analyses (34). To examine the spatial relationship between EWS/FLI1 hubs and GGAA microsatellites, we performed simultaneous confocal imaging of EWS/FLI1-Halo and 3D DNA FISH targeting genes adjacent to GGAA microsatellites that are regulated by EWS/FLI1 (Fig.…”

Section: Resultsmentioning

confidence: 90%

Dynamic and Selective Low-Complexity Domain Interactions Revealed by Live-Cell Single-Molecule Imaging

Chong

Dugast-Darzacq

et al. 2017

Preprint

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Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity domains (LCDs) but how they perform transactivation functions remains unclear. Recent studies report that TF-LCDs can undergo hydrogel formation or liquid-liquid phase separation in vitro. Here, livecell single-molecule imaging reveals that TF-LCDs form local high concentration interaction hubs at synthetic and endogenous genomic loci. TF-LCD hubs stabilize DNA binding, recruit RNA polymerase II (Pol II) and activate transcription. LCD-LCD interactions within hubs are highly dynamic, selective towards binding partners and differentially sensitive to disruption by hexanediols. These findings suggest that under physiological conditions, rapid reversible and multivalent LCD-LCD interactions occurring between TFs and the Pol II machinery underpin a central mechanism for transactivation and can play a key role in gene expression and disease.

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“…ChIP-Seq was performed as described(46). Chromatin was crosslinked with fresh 1% formaldehyde for 4 minutes and sheared by the Covaris ME220 for 12 minutes.…”

Section: Methodsmentioning

confidence: 99%

HOTAIRprimes the Ewing sarcoma family of tumors for tumorigenesis via epigenetic dysregulation involving LSD1

Siddiqui

Selich‐Anderson

Felgenhauer

et al. 2018

Preprint

Self Cite

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The EWS-FLI1 fusion protein drives oncogenesis in the Ewing sarcoma family of tumors (ESFT) in humans, but its toxicity in normal cells requires additional cellular events for oncogenesis. We show that the lncRNA HOTAIR maintains cell viability in the presence of EWS-FLI1 and redirects epigenetic regulation in ESFT. HOTAIR is consistently overexpressed in ESFTs and is not driven by EWS-FLI1. Repression of HOTAIR in ESFT cell lines significantly reduces anchorage-independent colony formation in vitro and impairs tumor xenograft growth in vivo. Overexpression of HOTAIR in human mesenchymal stem cells (hMSCs), a putative cell of origin of ESFT, and IMR90 cells induces colony formation. Critically, HOTAIR-expressing hMSCs and IMR90 cells remain viable with subsequent EWS-FLI1 expression. HOTAIR induces histone modifications and gene repression through interaction with the epigenetic modifier LSD1 in ESFT cell lines and hTERT-hMSCs. Our findings suggest that HOTAIR maintains ESFT viability through epigenetic dysregulation.SignificanceWhile the EWS-FLI1 fusion gene was determined to be the oncogenic driver in the overwhelming majority of ESFT, it is toxic to cell physiology and requires one or more additional molecular events to maintain cell viability. As these tumors have surprisingly few genetic mutations at diagnosis, epigenetic changes have been considered to be such an event, but the mechanism by which these changes are driven remains unclear. Our work shows that HOTAIR is consistently expressed among ESFT and induces epigenetic and gene expression changes that cooperate in tumorigenesis. Furthermore, expression of HOTAIR allows for cell viability in the setting of subsequent EWS-FLI1 expression. Our findings elucidate new steps of malignant transformation in this cancer and identify novel therapeutic targets.

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

Cited by 66 publications

References 29 publications

Fusion protein EWS-FLI1 is incorporated into a protein granule in cells

Fusion protein EWS-FLI1 is incorporated into a protein granule in cells

Dynamic and Selective Low-Complexity Domain Interactions Revealed by Live-Cell Single-Molecule Imaging

HOTAIRprimes the Ewing sarcoma family of tumors for tumorigenesis via epigenetic dysregulation involving LSD1

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