The FET family of RNA-binding proteins, FUS, EWSR1, and TAF15, contribute to transcriptional regulation and RNA maturation, but their core functions remain unclear. Chromosomal rearrangements involving FUS, EWSR1, or TAF15 drive multiple cancers, and mutations in the genes encoding the FET proteins are associated with neurodegenerative disease. Here, using nanoscale imaging, we show that endogenous EWSR1 and newly synthesized RNA exhibit a network-like organization with EWSR1 foci forming the nodes of this ribonucleoprotein network. Acute depletion of EWSR1 causes a rapid but transient reduction in nascent RNA levels and cellular metabolic activity without affecting active transcription. Notably, loss of EWSR1 induces a compensatory mechanism involving the reorganization of FUS and TAF15 to closely resemble that of EWSR1, including enhanced clustering with newly synthesized RNA. Together, our findings reveal functional redundancy within the FET protein family that is critical for the homeostatic regulation of nascent RNA levels.

Depletion of TNS3 in EWS cells expressing ETS1 results in changes in the distribution of vinculin and F-actin

Ectopic expression of ETS1 has transcriptome-wide effects.

The genome-wide binding of EWSR1::FLI1 and associated epigenetic marks.

Several cancers arise because of chromosomal rearrangements that result in a fusion oncoprotein which includes the N-terminus of FUS, EWSR1, or TAF15, members of the FET family of RNA-binding proteins. Examples of fusion oncoproteins involving the FET proteins, include FUS::ATF1, FUS::CREB3L1, FUS::DDIT3, EWSR1::CREB1, EWSR1::ERG, EWSR1::FLI1, EWSR1::PATZ1, EWSR1::VEZF1, EWSR1::WT1, TAF15::ZNF384, and TAF15::NR4A3. The FET proteins have similar protein domains, specifically, a N-terminal low complexity domain (LCD), an RNA-recognition motif (RRM), a Zn-finger domain, and a C-terminal nuclear localization signal. Study of the LCDs of the FET proteins has proven essential to determining the biophysical properties of the interactions that define biocondensates, and functional studies have implicated FET proteins as regulators of transcription and RNA maturation. However, the exact functions of the FET proteins remain poorly defined. An enhanced understanding of FET protein function is critical to understanding how the biocondensate properties of these proteins and their functions may contribute to the regulation of gene expression and to tumorigenesis. To investigate the function of the FET proteins, we are using high-resolution microscopy to examine the cellular localization of these proteins at endogenous levels of expression, with an initial focus on EWSR1. To generate endogenous EWSR1 reporter cell lines, we used CRISPR-Cas9 gene editing to introduce a DNA cassette at the 5’ end of EWSR1 exon 1. The DNA cassette expressed a FLAG-FKBP12F36V (FF) peptide that enables the nanoscale imaging of EWSR1 using immunofluorescence (IF) and its depletion via compound-regulated (dTAG-13) proteasomal degradation. Using these modified EWSR1 reporter cell lines, we show that EWSR1 interacts with nascent RNA to form an essential, DNA-independent, non-random ribonucleoprotein network (RNP). Depletion of EWSR1 results in a rapid, but transient reduction in nascent RNA and metabolic activity. Loss of EWSR1 mediates no changes in a marker of transcriptional elongation. However, as EWSR1 is depleted, the expression of FUS and TAF15 increase and concurrently exhibit nuclear reorganization. The observed change in the localizations of FUS and TAF15 reestablishes the formation of non-random RNP networks and restores nascent RNA accumulation. We conclude that the EWSR1 RNP forms an essential scaffold for nascent RNA and in the absence of EWSR1, FUS and TAF15 safeguard this critical function. Soumya Sundara Rajan, Imran Khan, Tamara L. Jones, Langston Lim, Andy Tran, Michael J. Kruhlak, Natasha J. Caplen. Biocondensates in action: The formation and reformation of EWSR1, FUS and TAF15 ribonucleoprotein networks [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr IA004.

Supplementary Materials and Methods - RNA sequencing, Chromatin Immunoprecipitation (ChIP) and CUT&RUN analysis, Immunoblotting and Immunofluorescence

EWSR1::FLI1-regulated transcription factor genes

ETS1 promotes the migration of EWS cells and regulates the expression of TENSIN3

EWS cells expressing ETS1 exhibit TENSIN3-dependent changes in the organization of vinculin

HNRNPH1 is a regulator of alternative splicing, but few studies have defined the splicing events it mediates. Here, we used short- and long-read RNA sequencing to interrogate the transcriptome-wide effects of HNRNPH1 depletion and its regulation of specific splicing events. Differential alternative splicing analysis revealed effects on the transcriptome that involved all splice event categories. We confirmed HNRNPH1's regulation of a splicing event involving TCF3-exons 18a and 18b that encode distinct TCF3 transcription factor isoforms. Extending this finding, we present evidence that in neuroblastoma, HNRNPH1 is a MYCN target, potentially explaining the higher levels of HNRNPH1 and TCF3-exon 18a transcript variants in this tumor type. Analysis of two skipped exon events determined that HNRNPH1 regulates the splicing of exons encoding part of the EIF4G1 translation initiation factor's N-terminus and an exon included in the 5'UTR of specific transcript variants encoding the mitotic kinase AURKA. Using reporter constructs, we show this AURKA 5'UTR exon enhances expression, suggesting HNRNPH1 could contribute to regulating AURKA protein levels. Our findings highlight HNRNPH1's roles in regulating the expression of proteins with diverse cellular functions.

Abstract Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor that affects children and young adults. Ewing sarcomas harbor few mutations beyond the chromosomal translocation that initiates disease, and the mechanistic basis for the metastasis of these tumors remains poorly understood. The epigenome of EWS cells reflects the regulatory state of genes associated with the DNA binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG’s repression of transcription factor genes, particularly those encoding transcriptional regulators of cell differentiation. To examine EWSR1::FLI1/ERG’s regulation of gene expression, we assayed DNA binding using ChIP-seq or CUT&RUN, RNA using RNA-sequencing (RNA-seq) or qRT-PCR, and protein using immunoblotting or immunofluorescence. We depleted the expression of EWSR1::FLI1/ERG proteins using RNAi and overexpressed ETS1 using a full-length cDNA or CRISPR activation.RNA-seq analysis of control and EWSR1::FLI1-silenced TC-32, TC-71, and A673 EWS cells identified 67 genes encoding proteins with DNA binding activity that exhibited significant increases in expression following depletion of EWSR1::FLI1. Comparison of the expression of these 67 transcription factor genes in EWS cell lines (n=41) and tumors (n=79) showed 37 expressed at a wider range in tumors relative to cell lines (p <0.01). This more variable expression in EWS tumors suggests that one or more of these transcription factors could exert a phenotypic effect. Focusing on one of the EWSR1::FLI1-repressed target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3 repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1’s binding of promoter regions. Analysis of EWS cells in which we profiled ETS1 binding and assessed the effects of ectopically expressed ETS1 defined 265 genes as positively regulated by ETS1 of which 103 also exhibited an increase in expression following depletion of EWSR1::FLI1. Of these 103 genes, we focused on ETS1’s regulation of TNS3, as assessment of multiple EWS tumor expression profiles, showed positive correlation of ETS1 and TNS3 expression. TNS3 encodes TENSIN3, a focal adhesion protein that regulates cytoskeletal reorganization and contributes to cell migration by connecting the cytoplasmic tail of integrins to the actin cytoskeleton. EWS cell lines (SK-N-MC (EWSR1::FLI1) and ES-5838 (EWSR1::ERG), in which we activated ETS1 expression (CRISPRa) exhibited a migratory phenotype and increased TNS3 expression. Critically, the activated ETS1 EWS cell lines show TNS3 accumulation at leading cell edges, with F-actin cytoskeletal reorganization, a phenotype associated with cell migration. Our study demonstrates that EWS cells expressing ETS1 exhibit a more migratory phenotype, which, has the potential to promote the dissemination of cells and, thus, metastasis. Citation Format: Vernon Justice Ebegboni, Tamara L. Jones, Tayvia Brownmiller, Erica Pehrsson, Patrick Zhao, Soumya Sundara Rajan, Natasha J. Caplen. ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2855.

Abstract The RNA binding protein HNRNPH1 is a critical regulator of alternative splicing (AS). We have previously demonstrated that HNRNPH1 mediates an exon exclusion event required for the expression of the mRNA encoding the fusion oncoprotein expressed in a subset (∼37%) of Ewing sarcomas. Furthermore, studies by others have shown that HNRNPH1 regulates a mutually exclusive exon (MXE) AS event (exon-18a versus exon-18b) that defines the expression of TCF3 transcript variants, which, when dysregulated, promotes tumorigenesis in Burkitt’s lymphoma. In this study, we have interrogated the transcriptome-wide effects of depleting HNRNPH1 by RNAi to further explore its regulation of transcripts expressed by cancer-associated genes. We performed whole transcriptome RNA sequencing (RNAseq; short-read, Illumina; long-read, PacBio IsoSeq) of two cell lines (HEK-293T and HT-1080) post 48-hour transfection with a control siRNA (siNeg) or an siRNA targeting HNRNPH1 (siHNRNPH1). We then assembled a workflow to quantify changes in individual AS events (percent-spliced-in, ΔPSI=±0.1) using rMATS and MAJIQ, and differential transcript expression (DTE) using RSEM (pValue≤0.05) and EBSeq (FDR≤0.05). RNAseq analyses were validated using PCR assays. The rMATS analysis revealed skipped exons (SE) as the most frequent event effected in HEK-293T and HT-1080 cells following HNRNPH1 silencing, with 1069 SE or MXE events from 607 genes common to both. Gene annotation (Metascape) of these 607 genes identified 49 as cancer-associated. Consistent with previous studies, we detected a significant change in the TCF3-exon-18a/18b MXE event using rMATS (decreased PSI for 18a) and MAJIQ (negative ΔPSI for 18a and positive ΔPSI for 18b) following HNRNPH1 silencing in both cell lines. Furthermore, DTE revealed a corresponding decrease in 18a-containing TCF3-transcripts and an increase in 18b-containing TCF3-transcripts, validating our analytical pipeline. Next, we extended our analysis to uncharacterized HNRNPH1-regulated AS events, beginning with examination of transcripts encoding the mitotic kinase AURKA. Previous studies have shown that AS of AURKA transcripts localizes to the untranslated regions (UTRs), particularly the 5’UTR. However, regulators of these AS events are unknown, and it is unclear how these UTR variants affect AURKA protein expression. Long-read RNAseq of AURKA mRNAs from both cell lines revealed extensive 5’UTR AS, and the rMATS and MAJIQ analyses showed decreased PSI and negative ΔPSI for a specific 5’UTR-exon following HNRNPH1 silencing. DTE revealed corresponding decreases in abundance of AURKA transcripts containing this exon (AURKA-203, -205, -209, and -213) upon HNRNPH1 depletion, which we confirmed with variant-specific RT-PCR. Furthermore, immunoblot analysis showed a decrease in AURKA protein expression following the silencing of HNRNPH1. Our assessment of AURKA AS suggests HNRNPH1 mediates the inclusion of a specific AURKA-5’UTR-exon, providing evidence for a previously uncharacterized post-transcriptional regulatory mechanism of AURKA expression. Citation Format: Tayvia Brownmiller, Patricio Pichling, Tamara L Jones, Ioannis Grammatikakis, Soumya Sundara Rajan, Erica C Pehrsson. HNRNPH1 regulates the alternative splicing of transcripts expressed by cancer-associated genes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A001.

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor that affects children and young adults. Ewing sarcomas harbor few mutations beyond the chromosomal translocation that initiates disease, and the mechanistic basis for the metastasis of these tumors remains poorly understood. The epigenome of EWS cells reflects the regulatory state of genes associated with the DNA binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG’s repression of transcription factor genes, particularly those encoding transcriptional regulators of cell differentiation. To examine EWSR1::FLI1/ERG’s regulation of gene expression, we assayed DNA binding using ChIP-seq or CUT&RUN, RNA using RNA-sequencing (RNA-seq) or qRT-PCR, and protein using immunoblotting or immunofluorescence. We depleted the expression of EWSR1::FLI1/ERG proteins using RNAi and overexpressed ETS1 using a full-length cDNA or CRISPR activation.RNA-seq analysis of control and EWSR1::FLI1-silenced TC-32, TC-71, and A673 EWS cells identified 67 genes encoding proteins with DNA binding activity that exhibited significant increases in expression following depletion of EWSR1::FLI1. Comparison of the expression of these 67 transcription factor genes in EWS cell lines (n=41) and tumors (n=79) showed 37 expressed at a wider range in tumors relative to cell lines (p <0.01). This more variable expression in EWS tumors suggests that one or more of these transcription factors could exert a phenotypic effect. Focusing on one of the EWSR1::FLI1-repressed target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3 repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1’s binding of promoter regions. Analysis of EWS cells in which we profiled ETS1 binding and assessed the effects of ectopically expressed ETS1 defined 265 genes as positively regulated by ETS1 of which 103 also exhibited an increase in expression following depletion of EWSR1::FLI1. Of these 103 genes, we focused on ETS1’s regulation of TNS3, as assessment of multiple EWS tumor expression profiles, showed positive correlation of ETS1 and TNS3 expression. TNS3 encodes TENSIN3, a focal adhesion protein that regulates cytoskeletal reorganization and contributes to cell migration by connecting the cytoplasmic tail of integrins to the actin cytoskeleton. EWS cell lines (SK-N-MC (EWSR1::FLI1) and ES-5838 (EWSR1::ERG), in which we activated ETS1 expression (CRISPRa) exhibited a migratory phenotype and increased TNS3 expression. Critically, the activated ETS1 EWS cell lines show TNS3 accumulation at leading cell edges, with F-actin cytoskeletal reorganization, a phenotype associated with cell migration. Our study demonstrates that EWS cells expressing ETS1 exhibit a more migratory phenotype, which, has the potential to promote the dissemination of cells and, thus, metastasis. Citation Format: Vernon Justice Ebegboni, Tamara L. Jones, Tayvia Brownmiller, Erica Pehrsson, Patrick Zhao, Soumya Sundara Rajan, Natasha J. Caplen. ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2855.

Abstract The mechanistic basis for the metastasis of Ewing sarcomas remains poorly understood, as these tumors harbor few mutations beyond the chromosomal translocation that initiates the disease. Instead, the epigenome of Ewing sarcoma cells reflects the regulatory state of genes associated with the DNA-binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG's repression of transcription factor genes, concentrating on those that exhibit a broader range of expression in tumors than in Ewing sarcoma cell lines. Focusing on one of these target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3-repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1’s binding of promoter regions, substantially altering the transcriptome of Ewing sarcoma cells, including the upregulation of the gene encoding TENSIN3 (TNS3), a focal adhesion protein. Ewing sarcoma cell lines expressing ETS1 (CRISPRa) exhibited increased TNS3 expression and enhanced movement compared with control cells. Visualization of control Ewing sarcoma cells showed a distributed vinculin signal and a network-like organization of F-actin; in contrast, ETS1-activated Ewing sarcoma cells showed an accumulation of vinculin and F-actin toward the plasma membrane. Interestingly, the phenotype of ETS1-activated Ewing sarcoma cell lines depleted of TNS3 resembled the phenotype of the control cells. Critically, these findings have clinical relevance as TNS3 expression in Ewing sarcoma tumors positively correlates with that of ETS1. Implications: ETS1’s transcriptional regulation of the gene encoding the focal adhesion protein TENSIN3 in Ewing sarcoma cells promotes cell movement, a critical step in the evolution of metastasis.

Abstract The initiating genetic event in several pediatric tumor-types, including Ewing sarcoma (EWS), involve translocations affecting the EWSR1 locus. The EWSR1 gene encodes EWSR1, a transcriptional regulator. In EWS cells expressing the fusion oncoprotein EWSR1::FLI1, studies have shown disrupted function of EWSR1 function and that this contributes to the deregulated gene expression observed in these tumors. Recently, we reported the nucleoplasmic organization of endogenous EWSR1 in EWS cells. In brief, we determined that at a resolution of ∼120 nm, EWSR1 is present in two states, a distributed state which is present throughout the nucleoplasm and as foci. Both EWSR1 states localize with nascent RNA, while foci also significantly colocalize with phosphorylated RNA polymerase II (p-RNA pol II). In this study, we have examined EWSR1’s localization at a higher resolution (40 nm) in EWS and non-EWS cells. For this study, we used two EWS cell lines, A673 and TC-32, and a non-EWSR1 driven sarcoma cell line, HT-1080 that expresses EWSR1 and FLI1. Using gene editing, we generated EWSR1 reporter cell lines that express either mNeonGreen or the FLAG peptide fused to the N-terminus of EWSR1. In A673 cells, we also generated isogenic cell lines that express an 11 amino acid peptide (HiBiT) fused to either EWSR1 or EWSR1::FLI1. Following validation of successfully modified clones, we used fluorescence or immunofluorescence (IF) and super-resolution confocal and stimulated emission depletion microscopy to assess protein localization. We observed that EWSR1’s overall organization in A673, TC-32 and HT-1080 cells is similar with over 95% of nuclear localization and its existence in two states, distributed and foci. At a resolution of 40 nm or less, we found that EWSR1 foci in EWS and non-EWS cells are composed of 3 to 6 distinct IF signals. Furthermore, in all three cell lines, we observed that the distributed EWSR1 signal exists as a fibro-granular network that colocalizes with nascent RNA. While these features proved similar between the three cell lines, we detected some differences in EWSR1’s organization in EWS and non-EWS cells. Specifically, in the HT-1080 cells, EWSR1 foci are smaller (∼80 nm) than that seen in EWS cells (A673: ∼247 nm, TC-32: ∼578 nm). Also, EWS cells exhibit minimal localization of EWSR1 to nucleoli, but in HT-1080 cells, we observed EWSR1 in one or more nucleoli of ∼30% of cells. In addition, we observed significantly less colocalization of EWSR1 foci with p-RNA pol II in the HT-1080 cells (Pearson’s correlation coefficient (PCC) = 0.47) compared to EWS cells (PCC, A673 = 0.85; TC-32 = 0.87). One reason for these differences could be the interaction of EWSR1 with EWSR1::FLI1 affecting EWSR1’s localization. To begin to evaluate this hypothesis, we examined the nuclear localizations of EWSR1::FLI1 and EWSR1 and observed colocalization of these proteins (PCC=0.45). Collectively, our study of endogenous EWSR1 shows its localization in EWS cells differs from that of non-EWS cells, which could affect the regulation of gene expression in EWS. Citation Format: Soumya Sundara Rajan, Tamara L. Jones, Vernon J. Ebegboni, Langston Lim, Michael J. Kruhlak, Natasha J. Caplen. EWSR1 in Ewing sarcoma cells exhibits enhanced recruitment to sites of active transcription compared to that observed in non-Ewing sarcoma cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A016.

EWSR1 is a member of the FET family of nucleic acid binding proteins that includes FUS and TAF15. Here, we report the systematic analysis of endogenous EWSR1's cellular organization in human cells. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.