Fig 2: PBAF loss impairs REST binding at repressed chromatin regions.(A-B) Western blot analysis of ARID2 WT and KO chromatin extracts in (A) NHEM and (B) 4L melanoma cells. NTC, non-targeting control sgRNA. LAMIN A/C was used as loading control. (C) Metagene profile of REST and CTCF ChIP-seq in ARID2 WT and KO SKmel147 melanoma cells. (D) Metagene profile of REST ChIP-seq in ARID2 WT and KO NHEMs. (E) MA plot displaying differentially bound REST peaks upon ARID2 KO, as determined by DiffBind analysis. Peaks are plotted according to the extent of the change (fold change KO-WT) and their normalized read density. (F) Heatmap showing REST occupancy in NHEM at differentially bound (Up or Down peaks) or constant REST regions upon ARID2 KO. ChIP-seq signal for ARID2, SS18 and the histone marks H3K27ac and H3K27me3 is also shown at the same regions.

Fig 3: PBAF-enriched regions present a lower level of remodeling activity.(A) Metagene plots displaying ATAC-seq levels at PBAF-, Shared, or BAF-enriched regions in 4L melanoma cells and NHEM (B) Box plot representing chromatin accessibility levels in 4L melanoma cells (left panel) or NHEMs (right panel) at PBAF-, Shared or BAF-enriched regions. Control (CTRL) sites include ATAC peaks not overlapping with any SWI/SNF region. Significance was calculated by Mann-Whitney U test. (C) Scatter plot showing correlation levels between SS18 or ARID2 levels and ATAC signal at their respective regions. To ensure that only fully assembled SWI/SNF complexes were represented in this analysis, only SS18 and BRG1 or ARID2, PBRM1 and BRG1 overlapping peaks were included as BAF and PBAF regions, respectively. (D) Biological Processes GO analysis of genes localized at repressed PBAF chromatin regions (cluster #1 of Fig. S2A, B) in melanoma cells or NHEMs. (E) Genome browser snapshot showing occupancy of SWI/SNF and PRC2 components, as well as histone marks at ACTL6B genomic locus in 4L melanoma cells.

Fig 4: The recognition of H2AK119ub is crucial for the condensates function of onco-fusion.A Schematic illustration for SS18-SSX onco-fusion protein. The C-terminal acidic region (AR) DPEEDDE → AAAAAAA was highlighted. B Representative image of immunofluorescence for H2AK119ub in BJ fibroblasts with the lentiviral expression of wild-type or AR mutant SS18-SSX1-EGFP. Scale bars, 5 μm. C Histogram shows the number of condensates in BJ fibroblasts with lentiviral expression of wild-type or AR mutant SS18-SSX1-EGFP in (B). Data are mean±s.d., two-sided, unpaired t test; n = 8 nuclei. D Pull-down assays of the SS18-SSX1-EGFP-Flag (SS-Flag) and SS18-SSX1(AR Mut)-EGFP-Flag (AR Mut-Flag) protein. Binding to the H2AK119ub was shown by immunoblotting. E The expression of representative downstream genes of SS18-SSX in BJ fibroblasts with lentiviral expression of EGFP, SS18, wild-type and AR mutant SS18-SSX1. Data are mean±s.d., n = 3 independent experiments. F Representative image of EdU assay in synovial sarcoma cell line SW982 with the lentiviral expression of EGFP, wild-type and AR mutant SS18-SSX1. Scale bars, 200 μm. G Histogram shows the ratio of EdU-positive cells in (E). Data are mean±s.d., two-sided, unpaired t test, **P < 0.01. n = 3 independent experiments.

Fig 5: SS18 forms condensates.a Representative images of FRAP experiments with lentiviral SS18-EGFP expressed in mESCs. The droplet undergone bleaching was indicated in the red box. The blue dotted line represents the nuclear boundary. Scale bars,3 μm. laser intensity 2.5%, detector gain 500 V, background subtraction treatment. Seven condensates from two independent experiments. b Time course microscopy images of fluorescence recovery in SS18-EGFP condensates after partial photobleaching at the edge (case 1) and middle (case 2). SS18-EGFP was expressed by transient transfection in 293 cells to obtain the large enough condensates. The bleached regions were marked by yellow asterisks. Scale bars, 1 μm. laser intensity 2.5%, detector gain 500 V, background subtraction treatment. Eight condensates from two independent experiments. c Kymography for the time course of pixel intensity along the white line in the condensate of case 2 in (b). The intensity recovered in the bleached region inside the condensate as the unbleached part decreased. Pixel number = 103. d The intensity dynamics inside and outside of the bleached regions within condensates (n = 8) was fit to an exponential function. t = 0 was the bleached time point. τ(inside) = 1.841 s, τ(outside) = 1.905 s; Plateau(inside) = 66.18%, Plateau(outside) = 79.91%, diffusion coefficient on the order of D ~ L2/τ ~5 μm2/s, L is the diameter of the bleached region. Data are mean ± s.d., two-sided, unpaired t test; n = 8 condensates. e Representative droplet fusion events are shown by time course images. SS18-EGFP was expressed by transient transfection to obtain the large condensates as in (b). Scale bars, 2 μm. laser intensity 2.5%, detector gain 500 V, background subtraction treatment. Nine fusion/fission cases.