Fig 2: SS18-SSX expression results in a functional PRC1–PRC2 complex uncoupling at its genomic binding sites.(A) Composite plots for RING1B, H2AK119ub, and H3K27me3 signals at 1338 SS18-SSX–binding sites, initially bearing the PRC2 repressive mark, showing that expression of the fusion protein in C3H10T1/2 cells results in an increase in the RING1B and H2AK119ub signals, and the removal of the H3K27me3 mark. (A, B) Heat maps for RING1B and H2AK119ub signals at the same 1338 SS18-SSX–binding sites as in (A), illustrating the reversibility of the chromatin remodeling pattern upon CRE-mediated SS18-SSX depletion in C3H10T1/2 cells. 20-kb windows centered on SS18-SSX–binding sites are shown. (C) Boxplot analysis of the RING1B, H2AK119ub, and H3K27me3 ChIP-seq signals in primary SyS 1 organoids, confirming the presence of PRC1 and the related H2AK119ub mark, but not the PRC2 mark H3K27me3, at broad BAF domains in primary SyS tumor models. (D) Representative example of RING1B recruitment and H2AK119ub deposition at a broad BAF complex domain in SyS organoid cultures. (E) Boxplot showing the distribution of peak widths for RING1B in control and SS18-SSX–expressing C3H10T1/2 cells. (F) Barplot depicting the percentage of RING1B-binding sites in each width quartile that overlap with the broadest SMARCA2/4 domains in SyS organoids. (G) Proximity ligation assay analyses demonstrate direct interactions between SS18-SSX and the PRC1 subunits RING1B and RYBP, but not the PRC2 core member EZH2, in C3H10T1/2 cells. (H) Proximity ligation assay analysis of SS18-SSX–expressing or control C3H10T1/2 cells confirming that the expression of the fusion protein enhances the assembly of the ncPRC1, as indicated by the increase in interactions between its core members RING1B and RYBP. (I) Heat map showing ChIP-seq signals for RYBP at SS18-SSX–binding sites in C3H10T1/2 cells. 20-kb windows centered on SS18-SSX–binding sites are shown. (J) Representative example of RING1B and RYBP recruitment at SS18-SSX–binding sites in C3H10T1/2 cells. (K) A mechanistic model of SS18-SSX chromatin remodeling activity in SyS. After SS18-SSX expression, the PRC2 repressive mark H3K27me3 is replaced by the active histone modifications H3K4me1 and H3K27ac. The concomitant recruitment of a non-canonical PRC1 by the fusion protein leads to PRC2-PRC1 uncoupling, H2AK119ub deposition and target gene activation. *** indicates P-value < 0.0001. Statistical analyses were performed by t test. See also Fig S7.

Fig 3: USP7 depletion constitutes an epigenetic vulnerability in synovial sarcoma.(A) Gene dependency score analysis for a panel of PRC1 members in SyS versus all other cell lines present in the DepMap database identifies USP7 as a selective vulnerability in SyS. Genes shown in red had an adjusted P-value < 0.05. (B) Heat map showing ChIP-seq signals for USP7 at 4877 SS18-SSX–binding sites in C3H10T1/2 cells. 20-kb windows centered on SS18-SSX–binding sites are shown. (C) Representative example of USP7 recruitment by SS18-SSX at its binding sites in C3H10T1/2 cells. (D, E) Proximity ligation assay analysis demonstrates direct interaction between USP7 and SS18-SSX in C3H10T1/2 and HSSYII SyS cells. (F) Proximity ligation assay shows a significant decrease in interactions between the ncPRC1 members RING1B and RYBP following USP7 removal in HSSYII cells. (G) Cell viability assays in SyS (HSSYII, SYO1) and EwS (A673, RDES) cells upon CRISPR-mediated USP7 KO confirm the selective detrimental effect of USP7 depletion in the SyS cells. *** indicates P-value < 0.0001. Statistical analyses were performed by t test. See also Fig S8.

Fig 4: Overexpression of RNF2 promotes proliferation, migration, and angiogenesis while inhibiting apoptosis in SW1353 cells. Note: pcDNA3.1-RNF2 and pcDNA3.1-NC were transfected into SW1353 cells, respectively. (A) RT-qPCR and (B) Western blot to detect RNF2 mRNA and protein expression in SW1353 cells; (C) CCK-8 to detect SW1353 cell viability; (D) EdU to detect SW1353 cell proliferation; (E) FITC/PI staining to calculate the number of early and late cell deaths for detecting SW1353 cell apoptosis; (F) Scratch assay to observe the migration distance of cells on the plate at 0 h and 24 h, respectively, for assessing SW1353 cell migratory capabilities; (G) After culture of HUVECs with conditioned media of SW1353 cells in different groups, tube formation assay to observe the number of tubules for evaluating SW1353 cell angiogenic capabilities. The cellular experiments were repeated three times, and the data were expressed as mean ± standard deviation. The independent sample t-test was used for the comparison between two groups. ** P < 0.01

Fig 5: High expression of RNF2 in CHS tissues and cells. Note: (A) RT-qPCR, (B) Western blot, and (C) immunohistochemistry (arrows indicate RNF2-positive cells) to detect RNF2 expression in CHS clinical samples (N = 25); **, P < 0.01 compared with the normal group; (D) RT-qPCR and (F) Western blot to detect RNF2 expression in CHS cell line. The cellular experiments were repeated three times, and the data were expressed as mean ± standard deviation. The independent samples t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups, with Tukey’s multiple comparisons test for post hoc analysis. **, P < 0.01, ***, P < 0.001 compared with the C-28/I2 group