Journal: Molecular Oncology

Article Title: FET fusion oncoproteins interact with BRD4 and SWI/SNF chromatin remodelling complex subtypes in sarcoma

doi: 10.1002/1878-0261.13195

Figure Lengend Snippet: The interactomes of FET oncoproteins are enriched in phase separation propensity and transcriptional components. (A) Significantly enriched Panther protein class for FUS‐DDIT3‐interacting proteins. Percentage of proteins in protein class versus total of proteins matched to a protein class. (B) Significantly enriched gene sets from the “Reactome” and “Gene ontology (GO) biological processes” gene set collections for FUS‐DDIT3‐interacting proteins. Top 10 based on gene ratio is shown. Gene count is indicated by dot size and q‐value by colour. (C) Pie charts of FUS‐DDIT3 and EWSR1‐FLI1‐interacting proteins with phase separation propensity score (PScore) above or below the cutoff at 4. (D) Visualization of phase separation propensity score (PScore) of FET oncoproteins and their parental proteins. Pie chart shows proteins above or below the cutoff at 4. Blue dot indicates proteins with a high phase separation propensity, above the cutoff, visualized by black line. (E) Visualization of phase separation propensity score (PScore) of SWI/SNF components. Pie chart shows proteins above or below the cutoff at 4. Red dot indicates proteins with a high phase separation propensity, above the cutoff, visualized by black line. (F) Schematic visualization of potential BRD4, mediator, RNA polymerase II and FET‐FOP‐bound SWI/SNF complex interactions near chromatin. (G) Immunofluorescence staining of HT1080 cells transiently transfected with FUS‐DDIT3‐EGFP, probed with BRG1/BRD4, BRG1/MED1 and FUS‐DDIT3‐EGFP/BRD4 and analyzed with laser scanning microscopy. Representative images are shown. Scale bar: 5 µm. (H) Pearson’s correlation coefficient for co‐localization of BRG1 vs. BRD4, BRG1 vs. MED1, FUS‐DDIT3 vs. BRD4, FUS‐DDIT3 vs. BRG1 and FUS‐DDIT3 vs. MED1 in HT1080 cells transiently transfected with FUS‐DDIT3‐EGFP. (I) Manders’ split coefficients for co‐localization of BRG1 vs. BRD4, BRG1 vs. MED1, FUS‐DDIT3 vs. BRD4, FUS‐DDIT3 vs. BRG1 and FUS‐DDIT3 vs. MED1 in HT1080 cells transiently transfected with FUS‐DDIT3‐EGFP. The dark grey bar corresponds to the fraction of protein 1 (e.g. BRG1) overlapping with protein 2 (e.g. BRD4) and the light grey corresponds to the fraction of protein 2 (e.g. BRD4) overlapping with protein 1 (e.g. BRG1).

Article Snippet: After being washed in PBS, the cells were blocked in PBS with 2% bovine serum albumin and 0.5% Tween 20 for 20 min before incubation with antibodies specific for BRG1 (Santa Cruz Biotechnology, mouse, sc‐17796) diluted 1 : 100, MED1 (Atlas antibodies, Stockholm, Sweden; rabbit, HPA0552818) diluted 1 : 50, and BRD4 (Abcam, rabbit, ab128874) diluted 1 : 100.

Techniques: Immunofluorescence, Staining, Transfection, Laser-Scanning Microscopy