SUMMARY Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes—canonical BRGI/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes—and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.

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Mammalian SWI/SNF chromatin remodeling complexes exist in three distinct, final-form assemblies: canonical BAF (cBAF), PBAF, and a newly-characterized non-canonical complex, ncBAF. However, their complex-specific targeting on chromatin, functions and roles in disease remain largely undefined. Here, we comprehensively mapped complex assemblies on chromatin and found that ncBAF complexes uniquely localize to CTCF sites and promoters. We identified ncBAF subunits as synthetic lethal targets specific to synovial sarcoma (SS) and malignant rhabdoid tumor (MRT), which share in common cBAF complex (SMARCB1 subunit) perturbation. Chemical and biological depletion of the BRD9 subunit of ncBAF rapidly attenuates SS and MRT cell proliferation. Notably, in cBAF-perturbed cancers, ncBAF complexes maintain gene expression at retained CTCF-promoter sites, and function in a manner distinct from fusion oncoprotein-bound complexes. Taken together, these findings unmask the unique chromatin targeting and function of ncBAF complexes and present new cancer-specific therapeutic targets.

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.

Summary Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9- based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks which recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex. Finally, by integrating functional networks with large protein-protein interaction networks, we discovered novel protein complexes involving recently-evolved genes of unknown function. Taken together, these findings demonstrate the utility of genetic perturbation screens alone and in combination with large-scale biophysical data to enhance our understanding of mammalian protein complexes in normal and disease states.

In order to characterize the specificity of expression of the neurotransmitter biosynthetic gene dopamine beta-hydroxylase (DBH), the identification of proteins that interact with the DB1 enhancer was initiated. A homeobox-containing cDNA was isolated from a PC12 expression cDNA library screened with the DB1 enhancer. The homeodomain is a member of the paired-like class, and is encoded by several nonidentical cDNAs. The cDNAs contain the same sequence in the homeodomain and 3' coding and noncoding sequences, but diverge in sequence 5' to the homeodomain. This family of homeobox-containing cDNAs is named Arix. Arix mRNA transcripts are found only in noradrenergic, DBH-positive tissues, and in cell lines derived from those tissue. The DB1 enhancer contains two binding sites for the Arix homeodomain, and both sites contribute to basal activity of the DBH promoter. When introduced into tissue culture, Arix regulates the transcriptional activity from the DBH promoter, and also from the promoter of the tyrosine hydroxylase gene, encoding the initial enzyme of the catecholamine biosynthetic pathway. The pattern of expression of the Arix transcripts, the presence of the homeodomain, and the transcriptional regulatory properties suggest that this family of proteins may be involved in the specificity of expression of the catecholamine biosynthetic genes.

In T lymphocytes WASP and WIP regulate TCR signaling but their role in lymphoma is largely unknown. Here we show that the expression of WASP and WIP is frequently low or absent in anaplastic large cell lymphoma (ALCL) compared to other T cell lymphomas. In ALK+ ALCL, WASP and WIP expression is regulated by ALK oncogenic activity via its downstream mediators STAT3 and C/EBP-β. ALK+ lymphomas were accelerated in WASP- and WIP-deficient mice. In the absence of WASP, active GTP-bound CDC42 was increased and the genetic deletion of one CDC42 allele was sufficient to impair lymphoma growth. WASP-deficient lymphoma showed increased MAPK pathway activation that could be exploited as a therapeutic vulnerability. Our findings demonstrate that WASP and WIP are tumor suppressors in T cell lymphoma and suggest that MEK inhibitors combined with ALK inhibitors could achieve a more potent therapeutic effect in ALK+ ALCL.

Preclinical and clinical evidence implicates N-methyl-D-aspartate receptor (NMDAr) signaling in early embryological development. However, the role of NMDAr signaling in early development has not been well studied. Here, we use a mouse embryonic stem cell model to perform a step-wise exploration of the effects of NMDAr signaling on early cell fate specification. We found that antagonism of the NMDAr impaired specification into the neuroectodermal and mesoendodermal cell lineages, with little or no effect on specification of the extraembryonic endoderm cell lineage. Consistent with these findings, exogenous NMDA promoted neuroectodermal differentiation. Finally, NMDAr antagonism modified expression of several key targets of TGF-β superfamily signaling, suggesting a mechanism for these findings. In summary, this study shows that NMDAr antagonism interferes with the normal developmental pathways of embryogenesis, and suggests that interference is most pronounced prior to neuroectodermal and mesoendodermal cell fate specification.