In Research Project 1 (RP1) of our FusOnc2 U54, we aim to understand the mechanisms underpinning SS18-SSX-mediated guidance of BAF ATP-dependent chromatin remodeling complexes to target loci on chromatin. Specifically, we aim to define the specific chromatin (i.e. nucleosome/histone) features and gene targets of SS18-SSX fusion oncoprotein-bound BAF complexes. Further, we aim to link these findings with synthetic lethal dependencies, such as polycomb repressive complex 1 (PRC1) and ncBAF complex components, that we and others have identified to be specific for synovial sarcoma (SS) (Michel et al., 2018; McBride et al., 2020; McBride et al., 2018). Over the course of the first two years supported by the FusOnc2 U54, we have made exciting progress in identifying the mechanism of engagement of the SS18-SSX fusion oncoprotein and chromatin, specifically, that a basic region on the SSX 78aa component of the SS18-SSX fusion protein binds to the acidic patch of the nucleosome, and an acidic C-terminal region mediates the selective binding preference for H2AUb-decorated nucleosomes (McBride et al., 2020) (Figure 1A-B). This SSX-specific engagement results in aberrant protein subunit composition, the orientation of BAF complex modules around their nucleosome substrates, as well as chromatin remodeling activities. These findings provide a mechanistic basis for SS-specific synthetic lethal dependencies on PRC1 components, particularly the RING1A/B enzymatic components, which place ubiquitination marks on histones (H2A K119Ub) (McBride et al., 2020). With these unexpected and impactful new biochemical and genomic findings, we now aim to achieve a 3D atomic-level understanding of the interaction between the SS18-SSX fusion oncoprotein-bound BAF complexes and their nucleosome substrates. These studies are within scope of all three RP1 Specific Aims, with particular relevance to Aim 2 (Biochemically and biophysically characterize chromatin landscape features that recruit the SS18-SSX fusion) and Aim 3 (Discover and mechanistically interrogate synovial sarcoma-specific synthetic lethal dependencies). To accomplish this, we have recently formed a new collaborative partnership with Dr. Yuan He and his team at Northwestern University. Specifically, Dr. He’s structural biology team has developed approaches to solve structures of chromatin-bound complexes in their most native configurations, via advances in large protein-DNA complex assembly, cryo-EM sample preparation, data processing methods, and model building tools. His team carries a unique, highly specialized set of expertise, that is particularly enabling for the study of SS18-SSX and its endogenous chromatin interactions. Of note, SS18-SSX-bound BAF complexes are bound to chromatin at extremely high affinity, making biochemical purification and isolation of complexes very challenging (Figure 2A). Further, while we determined that SS18-SSX-bound BAF complexes exhibit biochemical preference for H2A monoubiquitinated nucleosomes (H2A K119Ub) (Figure 2B-C), other histone mark and histone mark combinations may contribute to binding and activity and may direct divergent BAF-nucleosome (chromatin) interactions. Therefore, strategies to purify native BAF-nucleosome complexes for structural determination directly from synovial sarcoma cell lines and/or in overexpression settings in human cells, adapting strategies that we have recently developed and applied in other settings (Mashtalir et al., 2020), represent major advantages to the study of the SS18-SSX fusion oncoprotein in human synovial sarcoma.
|Effective start/end date||9/10/21 → 6/30/23|
- Dana-Farber Cancer Institute (132501//3U54CA231638-01S1)
- National Cancer Institute (132501//3U54CA231638-01S1)
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