Fig 2: Osmotic stress causes unique changes in the nucleocytoplasmic distributions of ALS-linked RBPs. (A) Representative images of TDP-43, SFPQ, FUS, hnRNPA1 and hnRNPK in human motor neurons either untreated or treated with 0.4 M sorbitol treatment for 1 h to induce osmotic stress. Scale bars = 5 µm. (B) Quantification of the nuclear/cytoplasmic average intensity ratio for TDP-43, SFPQ, FUS, hnRNPA1 and hnRNPK in untreated (UT) conditions and upon osmotic stress (OSM) show a reduction in the nuclear/cytoplasmic ratio for each RBP upon osmotic stress. Data shown are mean nuclear/cytoplasmic ratios (±SD) per well, calculated from >10,000 cells from 3 control lines in 3 independent experimental repeats as a minimum. p-value from unpaired t-test; **** = p < 0.0001. (C) The fold change in the nucleocytoplasmic ratio was calculated upon osmotic stress for each ALS-linked RBP. Data are shown as mean fold change in comparison to untreated values in each experimental repeat. p-value is from a Brown–Forsythe ANOVA test; **** = p < 0.0001.

Fig 3: GFP-tagged FUS relocalisation to nucleoli is not affected in HeLa cells by ALS-associated mutations, following DRB-induced transcription stress.(A) GFP fluorescence was detected in HeLa cells expressing the ALS-related mutant proteins GFP-FUSR521C (GFP-R521C) (left) or GFP-FUSP525L (GFP-P525L) (right). The cells were treated with DMSO or with 100 µm DRB for 30 min. Anti-fibrillarin immunostaining was used to stain nucleoli. Scale bar, 10 µm.

Fig 4: FET-oncogene-induced changes in H3K27 trimethylation and downstream effects Immunoblot analysis (IB) of histone modifications in stably transfected HT1080 cell lines expressing EGFP, FUS-DDIT3-EGFP, or EWSR1-FLI1-EGFP. Antibodies against H3K27Ac, H3K27me3, H3K4me3, and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount. Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion oncoproteins and EGFP. One representative immunoblot is shown. More immunoblots including a second stable biological replicate and analysis of histone modifications after transient transfection (24 and 48 h) are shown in Fig EV2A–D.Graphs showing amount of protein (H3K27me3, H3K4me3, ratio H3K27me3/H3K4me3, H3K27Ac, and EZH2) quantified from immunoblots relative each corresponding loading control H4 or GAPDH, normalized to parental HT1080. Mean ± SEM is shown with individual replicates indicated by circles from two experiments with stable transfection (see Figs 4A, and EV2A and F) and by squares from two experiments with transient transfection (24 and 48 h, see Fig EV2B and G), n = 4. Student's t-test, ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Original data for all quantifications, including P-values, are shown as source data.SWI/SNF opposes the polycomb complex PRC2. The catalytic subunit of PRC2, EZH2, catalyzes the trimethylation of Lys27 on histone H3 (H3K27me3), a chromatin modification associated with closed chromatin, leading to downregulation of polycomb-regulated genes. FET fusion binding to SWI/SNF might compromise the function of SWI/SNF, including polycomb opposition. In particular, genes downregulated when FUS-DDIT3 is overexpressed in HT1080 cells (RNA-seq data, n = 3, compared to control, n = 4) overlap with upregulated gene-sets after EZH2 knockdown, NIPP1 knockdown and HDAC knockdown, and BAF57 (SMARCE1) reconstitution in a SMARCE1 null cell line. RNA-seq data of HT1080 wt (n = 4), HT1080 EGFP (n = 4), HT1080 FUS-DDIT3-EGFP (n = 3), and HT1080 EWSR1-FLI1-EGFP (n = 4) were generated and genes > twofold regulated were analyzed using the molecular signature database (MSigDB). Gene lists were compared to the gene-set collection “chemical and genetic perturbations” and top 20 gene-sets for each comparison are shown in the source data. q-values (FDR-adjusted P-values) are indicated in parentheses.Schematic presentation of FET family of fusion oncoproteins, targeted DNA sequences, and associated tumors. Left: FET N-terminal fusion partners and binding to SWI/SNF. Center: C-terminal transcription factor fusion partners and their DNA target sequences (JASPAR database). Right: Tumor types caused by respective fusion oncogene. Note tumor type specificity for each fusion oncoprotein and that the FET-NTDs replace each other as fusion partners in some entities. Only a selection of the FET family fusion oncoproteins and tumors is shown as more members are continuously discovered. Source data are available online for this figure.

Fig 5: FUS mutants induce increased FMRP puncta density in MN axons.(A) Representative images of primary MNs at 5 days in vitro (DIV 5). Wild-type (WT) FUS (green), detected with a C-terminal antibody, is primarily localized in the nucleus in Fus+/+ and Fus?14/+ neurons. ?14 FUS (magenta) is enriched in the cytoplasm of Fus?14/+ and Fus?14/?14 neurons. Nuclei are labeled with 4',6-diamidino-2-phenylindole (DAPI) (blue), and ß3-tubulin (cyan) is used as a neuronal marker. Low-intensity wild-type FUS-positive nuclear staining in Fus?14/?14 neurons is due to antibody cross-reactivity with another FET protein, likely EWSR1. (B) ?14 FUS distribution in a Fus?14/+ MN axon. ?14 FUS signal detected by confocal microscopy (top) and the deconvoluted signal (middle). Neurons were grown in microfluidic devices, and ß3-tubulin is used to identify axons. (C) Representative deconvolved images of FMRP axonal puncta in Fus+/+, Fus?14/+, and Fus?14/?14 MNs grown in microfluidic chambers (MFCs) (DIV 8). (D) Quantification of axonal FMRP puncta density in Fus+/+, Fus?14/+, and Fus?14/?14 MNs (n = 4, axons = 45 to 47, ****P < 0.0001; Kruskal-Wallis, followed by Dunn’s post hoc test). (E) Representative images showing axonal FMRP puncta either fully (white arrowheads) or partially (colored arrowheads) positive for ?14 FUS in Fus?14/+ and Fus?14/?14 MNs. (F) Segmentation of FMRP puncta density into fully ?14 positive and partially ?14 positive and negative. (G) Quantification of somatic FMRP fluorescence in Fus+/+, Fus?14/+, and Fus?14/?14 HB9::GFP (green fluorescent protein)–positive MNs (n = 4, MNs = 15 to 19). (H) Representative images of FMRP axonal puncta in FUS+/+ and FUSP525L/P525L iPSC-derived MNs grown in MFCs. (I) Quantification of axonal FMRP puncta density in FUS+/+ and FUSP525L/P525L iPSC-derived MNs as shown in (F) (n = 4, axons = 21 to 24; ****P < 0.0001, Student’s t test). Independent experiments are visualized in different shades of gray in the graphs. n.s., not significant.