Abstract 1469: Visualization of endogenous EWSR1 in Ewing sarcoma cells reveals a regular nucleoplasmic organization that includes concentration at regions of active transcription
Authors: Rajan Soumya Sundara , Ebegboni Vernon , Jones Tamara L. , Pichling Patrick , Ludwig Katelyn R. , Chari Raj , Kruhlak Michael , Loncarek Jadranka , Caplen Natasha J. 
Published: June 2023

Abstract
EWSR1, a member of the FET protein family, contains low complexity and nucleic acid binding domains and regulates transcription. Biochemical and depletion studies have demonstrated that EWSR1 regulates basal transcription, and that in Ewing sarcoma (EWS) cells expressing the fusion oncoprotein EWSR1::FLI1, EWSR1 may contribute to the deregulation of gene expression observed in this cancer. To further study EWSR1’s function in Ewing sarcoma, we generated reporter cell lines to visualize and quantify its endogenous expression within EWS cells. To generate EWSR1-fluorescent reporter A673 and TC-32 EWS cell lines, we used CRISPR-Cas9 and sgRNAs targeting the 5’ end of the first exon of EWSR1 and a donor plasmid consisting of the mNeonGreen (mNG) reporter gene, a G4S3 linker, and EWSR1 homology arm templates. Upon validation of successfully modified clones (A673 and TC-32), we used confocal microscopy to confirm EWSR1’s localization to the nucleoplasm. Also, consistent with previous analysis of EWSR1’s protein-protein interactions, we confirmed using immunoprecipitation that the mNG-EWSR1 protein retains its interaction with EWSR1::FLI1 and RNA polymerase II (RNA pol II) - total and phosphorylated (pS5 and pS2). FRAP analysis (Zeiss Airyscan) showed that in cells, EWSR1 exists in two fractions, one that rapidly exchanges (A673 T½: 9.47 secs; TC-32 T½: 6.13 secs) and one that remains bound for a longer duration (A673 T½: 92.15 secs; TC-32; T½: 52.63 secs). We next used structured illumination microscopy to interrogate EWSR1’s spatial organization in EWS cells at a high resolution (~100 nm). First, we determined that EWSR1’s organization within the nucleoplasm is regular as measured by a signal-to-signal distance of ~300 nm in both cell lines (A673 cells 297±6.71 nm; TC-32 cells 304±8.59 nm). Analysis of EWSR1’s spatial organization relative to nucleic acids and proteins that function in gene expression indicates that EWSR1 partially co-localizes with DNA (visualized using DAPI), Histone 3, BRD4 and MED1 (visualized using immunofluorescence - IF). In contrast, we observed EWSR1 colocalizes completely with nascent RNA (visualized using Click-IT RNA Alexa Fluor 488, Invitrogen) (Manders overlap coefficient (MOC) A673: 0.96±0.005; TC-32: 0.97±0.005). Furthermore, we detected over 90% colocalization of concentrated EWSR1 signals (defined by specific fluorescent intensities) with RNA pol II (IF) in states associated with transcriptional initiation (pS5) (MOC A673: 0.94±0.011; TC-32: 0.93±0.02) and elongation (pS2) (MOC A673: 0.95±0.02; TC-32: 0.97±0.06). These studies will aid analysis of changes in EWSR1’s localization within the nucleus and relative to proteins that regulate gene expression following inhibition of transcription, including the application of compounds that disrupt EWSR1::FLI1’s transcriptional activity.
Citation Format: Soumya Sundara Rajan, Vernon Ebegboni, Tamara L. Jones, Patrick Pichling, Katelyn R. Ludwig, Raj Chari, Michael Kruhlak, Jadranka Loncarek, Natasha J. Caplen. Visualization of endogenous EWSR1 in Ewing sarcoma cells reveals a regular nucleoplasmic organization that includes concentration at regions of active transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1469.