The opposition between polycomb repressive complexes (PRC) and BAF (mSWI/SNF) complexes plays critical roles in development and disease. Mutations in the genes encoding BAF subunits contribute to over 20% of human malignancy, yet the underlying mechanisms remain unclear owing largely to a lack of assays to assess BAF function in vivo. To address this, we have developed a widely applicable recruitment assay system and find that BAF opposes PRC by rapid, ATP-dependent eviction, leading to the formation of accessible chromatin. Reversing this process results in reassembly of facultative heterochromatin. Surprisingly, BAF-mediated PRC eviction occurs in the absence of PolII occupancy, transcription, and replication. Further, we find that tumor suppressor and oncogenic BAF complex mutations result in differential effects on PRC eviction. These studies define a mechanistic sequence underlying the resolution and formation of facultative heterochromatin and demonstrate that BAF opposes polycomb complexes on a minute-by-minute basis to provide epigenetic plasticity.

Perturbations to mammalian SWI/SNF (BAF) complexes contribute to over 20% of human cancers, with driving roles first identified in malignant rhabdoid tumor (MRT), an aggressive pediatric cancer characterized by biallelic inactivation of the core BAF complex subunit SMARCB1 (BAF47). However, the mechanism by which this alteration contributes to tumorigenesis remains poorly understood. We find that BAF47 loss destabilizes BAF complexes on chromatin, absent significant changes in intra-complex integrity. Rescue of BAF47 in BAF47-deficient sarcoma cell lines results in increased genome-wide BAF complex occupancy, facilitating widespread enhancer activation and opposition of polycomb-mediated repression at bivalent promoters. We demonstrate differential regulation by BAF and PBAF complexes at enhancers and promoters, respectively, suggesting distinct functions of each complex which are perturbed upon BAF47 loss. Our results demonstrate collaborative mechanisms of mSWI/SNF-mediated gene activation, identifying functions that are coopted or abated to drive human cancers and developmental disorders.

Synovial sarcoma (SS) is defined by the hallmark SS18-SSX fusion oncoprotein, which renders BAF complexes aberrant in two manners: gain of SSX to the SS18 subunit and concomitant loss of BAF47 subunit assembly. Here we demonstrate that SS18-SSX globally hijacks BAF complexes on chromatin to activate an SS transcriptional signature that we define using primary tumors and cell lines. Specifically, SS18-SSX retargets BAF complexes from enhancers to broad polycomb domains to oppose PRC2-mediated repression and activate bivalent genes. Upon suppression of SS18-SSX, reassembly of BAF47 restores enhancer activation, but is not required for proliferative arrest. These results establish a global hijacking mechanism for SS18-SSX on chromatin, and define the distinct contributions of two concurrent BAF complex perturbations.

Highlights d DDX6-depleted PSCs lose P-bodies and cannot exit pluripotency d DDX6 depletion perturbs adult stem potency in a contextdependent manner d DDX6 suppresses the translation of P-body enriched TFs and chromatin regulators d P-body loss profoundly alters chromatin organization in stem cells

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.

Mammalian SWI/SNF (BAF) chromatin remodeling complexes play critical roles in maintaining chromatin architecture and gene expression. Genomic sequencing efforts over the past several years have unveiled a major role for these complexes in the development of human cancer as well as neurologic disease, prompting the need to interrogate underlying mechanisms and to develop new methods to comprehensively understand mSWI/SNF complex function. Here we discuss the emerging insights from genetic, biochemical, and functional genomic studies in the field and suggest approaches toward further basic investigations, as well as therapeutic targeting of chromatin remodeling machinery.

Synovial sarcoma (SS) is a soft-tissue malignancy driven by a recurrent chromosomal translocation (t(X;18)) that uniformly produces the SS18-SSX oncogenic fusion protein. SS18 is a core subunit of the mammalian SWI/SNF (BAF) complexes, which remodel nucleosomes in an ATP-dependent manner and antagonistically oppose gene-silencing activity of polycomb complexes to maintain transcriptional control throughout development and differentiation. We previously discovered that in SS, incorporation of the oncogenic SS18-SSX fusion into BAF complexes leads to eviction of the tumor-suppressor BAF47 (INI1/SMARCB1) subunit, and aberrant activation of polycomb target genes by displacement of H3K27me3-mediated repression. However, uncoupling the oncogenic consequences of two co-occurrent BAF complex perturbations, gain of 78- amino acids to SS18 and loss of BAF47 has remained a challenge for the field; understanding gain- versus loss-of-function properties of these molecular events is critical to the identification of effective targeted therapeutics for this patient population. Here we show that synovial sarcoma primary tumors and cell lines harbor a transcriptional signature markedly distinct from sarcomas such as malignant rhabdoid tumors, which are driven solely by biallelic loss of BAF47. Indeed, we show that SS18-SSX-containing BAF complexes exhibit distinct chromatin localization in that suppression of SS18-SSX results in a near complete genome-wide retargeting of BAF complexes. We find that SS18-SSX directs BAF complexes to polycomb-repressed sites to activate embryonic development and neuronal gene pathways hallmark to SS primary tumors. Strikingly, using biochemical affinity assays, we demonstrate that the SSX 78aa tail dramatically increases the affinity of BAF complexes for chromatin as well as their genomic footprint sizes upon ChIP-seq analyses. This is in stark contrast to the demonstrated decrease in chromatin affinity and genomic occupancy resulting from the loss of function of BAF47 in MRT. Moreover, using CRISPR/Cas9-mediated KO of BAF47 in SS cell lines, we show that the proliferative arrest of SS cell lines upon suppression of SS18-SSX is independent of the BAF47 reassembly into BAF complexes, thereby demonstrating a unique SSX-driven oncogenic mechanism distinct from BAF47 loss. Taken together, these studies uncover a novel functionality of the SSX tail that is required for SS oncogenesis, and inform the selection of appropriate targeted therapeutic agents for this gain-of-function BAF complex-driven cancer. Citation Format: Matthew J. McBride, John L. Pulice, Robert T. Nakayama, Nazar Mashtalir, Davis R. Ingram, Jack F. Shern, Javed Khan, Jason L. Hornick, Alexander J. Lazar, Cigall Kadoch. SSX drives gain-of-function BAF complex chromatin affinity and genomic targeting in synovial sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3875. doi:10.1158/1538-7445.AM2017-3875

Synovial sarcoma (SS) is a soft-tissue malignancy driven by a recurrent chromosomal translocation (t(X;18)) that uniformly produces the SS18-SSX oncogenic fusion protein. SS18 is a core subunit of the mammalian SWI/SNF (BAF) complexes, which remodel nucleosomes in an ATP-dependent manner and antagonistically oppose gene-silencing activity of polycomb-repressive complexes to maintain transcriptional control throughout development and differentiation. We previously discovered that in SS, incorporation of the oncogenic SS18-SSX fusion into BAF complexes leads to eviction of the tumor-suppressor BAF47 (INI1/SMARCB1) subunit, and aberrant activation of polycomb target genes by displacement of H3K27me3-mediated repression. However, uncoupling the oncogenic consequences of two co-occurrent BAF complex perturbations, gain of 78- amino acids of SSX to SS18 and loss of BAF47, has remained a challenge for the field. To identify effective targeted therapeutics for this patient population, it is critical that we understand the contribution of the gain- versus loss-of-function properties of these molecular events in this malignancy. Here we demonstrate that the SSX 78aa tail engages mononucleosomes and targeted, quantitative mass spectrometry proteomics reveals preferential engagement to nucleosomes decorated with histone modifications associated with transcriptional repression. Using biochemical affinity assays, we find that SSX dramatically increases the affinity of SS18-SSX-containing BAF complexes for chromatin, thereby decreasing the dynamic mobility of BAF complexes. Furthermore, we show that SS18-SSX-containing BAF complexes possess a broader genomic footprint and exhibit distinct chromatin localization in that expression of SS18-SSX drives a near complete retargeting of BAF complexes genome-wide. SS18-SSX directs BAF complexes to polycomb-repressed sites to activate embryonic development and neuronal gene pathways hallmark to SS primary tumors. This targeting by SSX results in a transcriptional signature markedly distinct from sarcomas such as malignant rhabdoid tumors, which are driven solely by biallelic loss of BAF47. Moreover, using CRISPR/Cas9-mediated KO of BAF47 in SS cell lines, we show that the proliferative arrest of SS cell lines upon suppression of SS18-SSX is independent of BAF47 reassembly into BAF complexes, thereby demonstrating that SSX targeting of BAF complexes drives oncogenesis in a manner distinct from BAF47 loss. Taken together, these studies uncover a novel functionality of the SSX tail that is required for SS oncogenesis, and inform the selection of appropriate targeted therapeutic agents for this gain-of-function BAF complex-driven cancer. This abstract is also being presented as Poster B25. Citation Format: Matthew J. McBride, John L. Pulice, Robert T. Nakayama, Nazar Mashtalir, Davis R. Ingram, Jacob D. Jaffe, Jack F. Shern, Javed Khan, Jason L. Hornick, Alexander J. Lazar, Cigall Kadoch. SSX-mediated chromatin engagement and targeting of BAF complexes activates oncogenic transcription in synovial sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr PR11.