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. 2020 Dec 23;4(2):e202000808. doi: 10.26508/lsa.202000808

Figure 6. SS18-SSX expression results in a functional PRC1–PRC2 complex uncoupling at its genomic binding sites.

Figure 6.

(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.