BAF Subunits Research Articles

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

GENE-50. SWI/SNF COMPLEX HETEROGENEITY RELATES WITH POLYPHENOTYPIC DIFFERENTIATION, PROGNOSIS AND IMMUNE RESPONSE IN RHABDOID TUMORS

Abstract PURPOSE Rhabdoid tumors (RTs) arise within (Atypical Teratoid/Rhabdoid Tumor-AT/RT) or outside the brain (extraCNS-RT-eCNS-RT) and are driven mainly by inactivation of the SWI/SNF complex subunit SMARCB1. A pathognomonic hallmark of RTs is heterogeneity characterized by multilineage differentiation of tumor cells including anomalous neuronal differentiation in a subset of eCNS-RT. The mechanisms that regulate heterogeneity in RTs are unknown. Because remodeling of the SWI/SNF complex regulates differentiation, we hypothesized that SWI/SNF-BAF and PBAF complex heterogeneity correlates with both multilineage differentiation and clinical outcome. EXPERIMENTAL DESIGN: We performed an integrated analysis of SWI/SNF complex subunit alterations in the developing kidney and cerebellum (most common regions of origin for RT) in comparison to eCNS-RT (n=14) and AT/RT (n=25) tumors. RT samples were interrogated using immunohistochemistry, DNA methylation and gene expression analyses. RESULTS (A) The SWI/SNF-BAF paralogs ACTL6A/ACTL6B were expressed in a mutually exclusive manner in the developing cerebellum and kidney. In contrast, a subset of eCNS-RTs lost mutual exclusivity and co-expressed both subunits. These tumors showed aberrant DNA methylation of genes that regulate neuronal and renal development and demonstrated immunohistochemical evidence of neuronal differentiation. (B) Low expression of the PBAF subunit-PBRM1 identified a group of AT/RTs in younger children with better overall prognosis. PBRM1-low AT/RT and eCNS-RTs showed altered DNA methylation and gene expression in immune-related genes accompanied by increased CD8 cytotoxic T-cell infiltration. CONCLUSIONS Heterogeneity in SWI/SNF BAF (ACTL6A/ACTL6B) and PBAF (PBRM1) subunits correlates with histogenesis, contributes to the immune microenvironment and prognosis in RTs and may inform opportunities to develop immunotherapies.

The ATPase BRG1/SMARCA4is a protein interaction platform that recruits BAF subunits and the transcriptional repressor REST/NRSF in neural progenitor cells.

The BAF complex (SWI/SNF) is an ATP-dependent chromatin remodeler that adapts the structural organization of the chromatin. Despite a growing understanding of the composition of BAF in different cell types, the interaction network within the BAF complex is poorly understood. Here, we characterized an isoform of the BRG1/SMARCA4 ATPase expressed in human neural progenitor cells. By electron microscopy and image processing, the neural BRG1/SMARCA4 shows an elongated globular structure, which provides a considerably larger surface than anticipated. We show that neural BRG1/SMARCA4 binds to BAF57/SMARCE1 and BAF60A/SMARCD1, two further components of BAF. Moreover, we demonstrate an interaction between the neural BRG1/SMARCA4 isoform and the central neurodevelopmental transcriptional repressor REST/NRSF. Our results provide insights into the assembly of a central transcriptional repressor complex, link the structure of the neural BRG1/SMARCA4 to its role as a protein-protein interaction platform and suggest BRG1/SMARCA4 as a key determinant that directs the BAF complex to specific DNA sites by interacting with transcription factors and regulators.

EWS-FLI1 modulated alternative splicing of ARID1A reveals novel oncogenic function through the BAF complex.

Connections between epigenetic reprogramming and transcription or splicing create novel mechanistic networks that can be targeted with tailored therapies. Multiple subunits of the chromatin remodeling BAF complex, including ARID1A, play a role in oncogenesis, either as tumor suppressors or oncogenes. Recent work demonstrated that EWS–FLI1, the oncogenic driver of Ewing sarcoma (ES), plays a role in chromatin regulation through interactions with the BAF complex. However, the specific BAF subunits that interact with EWS–FLI1 and the precise role of the BAF complex in ES oncogenesis remain unknown. In addition to regulating transcription, EWS–FLI1 also alters the splicing of many mRNA isoforms, but the role of splicing modulation in ES oncogenesis is not well understood. We have identified a direct connection between the EWS–FLI1 protein and ARID1A isoform protein variant ARID1A-L. We demonstrate here that ARID1A-L is critical for ES maintenance and supports oncogenic transformation. We further report a novel feed-forward cycle in which EWS–FLI1 leads to preferential splicing of ARID1A-L, promoting ES growth, and ARID1A-L reciprocally promotes EWS–FLI1 protein stability. Dissecting this interaction may lead to improved cancer-specific drug targeting.

Aspects morphologiques et moléculaires des tumeurs malignes associées à une dérégulation des complexes BAF

BAF complexes are chromatin remodelling complexes made up of 15 subunits which overview transcription regulation. A subset of their subunits are notoriously linked to cancer, with the examples of SMARCB1, SMARCA4, ARID1A/1B and PBRM1. The complexes act as tumor suppressor genes, commonly mutated in a wide array of malignancies with an overrepresentation of sarcomas and tumors of the central nervous system. The recurrent inactivation of their genes points towards their driving role in the tumorigenesis of SMARCB1 in malignant rhabdoid tumors and SMARCA4 in small cell carcinoma of the ovary, hypercalcemic type. These tumors are morphologically similar composed of solid sheets of cells displaying vesicular nuclei dotted with clear chromatin and conspicuous nucleoli. Genomically, they share simple diploid profiles with no other alterations than in the culprit gene. Other mesenchymal tumors, distinct from malignant rhabdoid tumors are associated with BAF alterations, namely epithelioid sarcomas, SMARCA4-deficient thoracic sarcomas. BAF subunits are mostly inactivated through mutations or deletions but also occur through translocations in medullary carcinoma of the kidney and synovial sarcomas. Apart from tumors displaying recurrent alterations of the complexes, some variants or tumor variants display BAF alterations, including epithelioid malignant peripheral nerve sheet tumors and poorly differentiated chordomas. Lastly, some malignancies display low frequency of BAF alterations, in keeping with their passenger role in tumorigenesis with the example of dedifferentiated carcinomas, especially in colon, lung and uterus. BAF complexes alterations correlate with morphological features recognizable by microscopy, paving the way for their routine diagnosis and potential therapeutic prospects.

The BAF and PRC2 Complex Subunits Dpf2 and Eed Antagonistically Converge on Tbx3 to Control ESC Differentiation.

SummaryBAF complexes are composed of different subunits with varying functional and developmental roles, although many subunits have not been examined in depth. Here we show that the Baf45 subunit Dpf2 maintains pluripotency and ESC differentiation potential. Dpf2 co-occupies enhancers with Oct4, Sox2, p300, and the BAF subunit Brg1, and deleting Dpf2 perturbs ESC self-renewal, induces repression of Tbx3, and impairs mesendodermal differentiation without dramatically altering Brg1 localization. Mesendodermal differentiation can be rescued by restoring Tbx3 expression, whose distal enhancer is positively regulated by Dpf2-dependent H3K27ac maintenance and recruitment of pluripotency TFs and Brg1. In contrast, the PRC2 subunit Eed binds an intragenic Tbx3 enhancer to oppose Dpf2-dependent Tbx3 expression and mesendodermal differentiation. The PRC2 subunit Ezh2 likewise opposes Dpf2-dependent differentiation through a distinct mechanism involving Nanog repression. Together, these findings delineate distinct mechanistic roles for specific BAF and PRC2 subunits during ESC differentiation.

Characterizing the role of SWI/SNF-related chromatin remodeling complexes in planarian regeneration and stem cell function

SWI/SNF-related chromatin remodeling complexes, including the human BAF and PBAF complexes, are involved in controlling stem cell pluripotency and differentiation in many species. However, these complexes have not been fully characterized in planarians, an emerging model for the in vivo study of stem cells. These flatworms have the ability to regenerate following injury or amputation, and we sought to investigate the role of chromatin remodeling in this process through bioinformatic and genetic characterization of the SWI/SNF-like complexes in Schmidtea mediterranea. We identified planarian homologs of all human BAF and PBAF subunits, and then examined their expression patterns and RNAi phenotypes. We found that the genes are expressed in both stem cells and differentiated tissues, and their knockdown results in impaired regeneration and other phenotypes indicating stem cell dysfunction. Knockdown of core complex members and Smed-ARID1 led to an increase in steady-state mitotic cell number, however, the stereotypical proliferative response that follows amputation was reduced following Smed-BRG1/BRM-2 RNAi. The number of differentiating epidermal lineage cells and expression of epidermal and muscle lineage markers were also reduced following SWI/SNF knockdown. Our findings provide insight into the importance of the SWI/SNF complex in stem cell function and cellular differentiation in planarians.

Glioma tumor suppressor candidate region gene 1 (GLTSCR1) and its paralog GLTSCR1-like form SWI/SNF chromatin remodeling subcomplexes

The mammalian SWI/SNF chromatin remodeling complex is a heterogeneous collection of related protein complexes required for gene regulation and genome integrity. It contains a central ATPase (BRM or BRG1) and various combinations of 10-14 accessory subunits (BAFs for BRM/BRG1 Associated Factors). Two distinct complexes differing in size, BAF and the slightly larger polybromo-BAF (PBAF), share many of the same core subunits but are differentiated primarily by having either AT-rich interaction domain 1A/B (ARID1A/B in BAF) or ARID2 (in PBAF). Using density gradient centrifugation and immunoprecipitation, we have identified and characterized a third and smaller SWI/SNF subcomplex. We termed this complex GBAF because it incorporates two mutually exclusive paralogs, GLTSCR1 (glioma tumor suppressor candidate region gene 1) or GLTSCR1L (GLTSCR1-like), instead of an ARID protein. In addition to GLTSCR1 or GLTSCR1L, the GBAF complex contains BRD9 (bromodomain-containing 9) and the BAF subunits BAF155, BAF60, SS18, BAF53a, and BRG1/BRM. We observed that GBAF does not contain the core BAF subunits BAF45, BAF47, or BAF57. Even without these subunits, GBAF displayed in vitro ATPase activity and bulk chromatin affinity comparable to those of BAF. GBAF associated with BRD4, but, unlike BRD4, the GBAF component GLTSCR1 was not required for the viability of the LNCaP prostate cancer cell line. In contrast, GLTSCR1 or GLTSCR1L knockouts in the metastatic prostate cancer cell line PC3 resulted in a loss in proliferation and colony-forming ability. Taken together, our results provide evidence for a compositionally novel SWI/SNF subcomplex with cell type-specific functions.

Modeling cancer driver events in vitro using barrier bypass-clonal expansion assays and massively parallel sequencing

The information on candidate cancer driver alterations available from public databases is often descriptive and of limited mechanistic insight, which poses difficulties for reliable distinction between true driver and passenger events. To address this challenge, we performed in-depth analysis of whole-exome sequencing data from cell lines generated by a barrier bypass-clonal expansion (BBCE) protocol. The employed strategy is based on carcinogen-driven immortalization of primary mouse embryonic fibroblasts and recapitulates early steps of cell transformation. Among the mutated genes were almost 200 COSMIC Cancer Gene Census genes, many of which were recurrently affected in the set of 25 immortalized cell lines. The alterations affected pathways regulating DNA damage response and repair, transcription and chromatin structure, cell cycle and cell death, as well as developmental pathways. The functional impact of the mutations was strongly supported by the manifestation of several known cancer hotspot mutations among the identified alterations. We identified a new set of genes encoding subunits of the BAF chromatin remodeling complex that exhibited Ras-mediated dependence on PRC2 histone methyltransferase activity, a finding that is similar to what has been observed for other BAF subunits in cancer cells. Among the affected BAF complex subunits, we determined Smarcd2 and Smarcc1 as putative driver candidates not yet fully identified by large-scale cancer genome sequencing projects. In addition, Ep400 displayed characteristics of a driver gene in that it showed a mutually exclusive mutation pattern when compared with mutations in the Trrap subunit of the TIP60 complex, both in the cell line panel and in a human tumor data set. We propose that the information generated by deep sequencing of the BBCE cell lines coupled with phenotypic analysis of the mutant cells can yield mechanistic insights into driver events relevant to human cancer development.

Smarca4 ATPase mutations disrupt direct eviction of PRC1 from chromatin.

Trithorax-group proteins and their mammalian homologs, including those in BAF (mSWI/SNF) complexes, are known to oppose the activity of Polycomb repressive complexes (PRCs). This opposition underlies the tumor-suppressive role of BAF subunits and is expected to contribute to neurodevelopmental disorders. However, the mechanisms underlying opposition to Polycomb silencing are poorly understood. Here we report that recurrent disease-associated mutations in BAF subunits induce genome-wide increases in PRC deposition and activity. We show that point mutations in SMARCA4 (also known as BRG1) mapping to the ATPase domain cause loss of direct binding between BAF and PRC1 that occurs independently of chromatin. Release of this direct interaction is ATP dependent, consistent with a transient eviction mechanism. Using a new chemical-induced proximity assay, we find that BAF directly evicts Polycomb factors within minutes of its occupancy, thereby establishing a new mechanism for the widespread BAF-PRC opposition underlying development and disease.

Dynamics of BAF-Polycomb complex opposition on heterochromatin in normal and oncogenic states.

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.

Abstract 2658: Genome-wide mistargeting of oncogenic SWI/SNF(BAF) complexes in SMARCB1(BAF47)-deficient sarcomas

Abstract SMARCB1/BAF47/INI1 is a core subunit of the mammalian SWI/SNF (BAF) family of ATP-dependent chromatin remodeling complexes, which remodel nucleosome architecture to achieve coordinated regulation of gene expression. Genetic loss of SMARCB1 has been identified in several cancer types, including malignant rhabdoid tumor (MRT, 98%) and epithelioid sarcoma (EpS, 90%), strongly implicating this event as the oncogenic driver in these malignancies. However, the precise mechanism underpinning the tumor suppressive function of BAF47 to date remains unclear. In order to elucidate the underlying mechanism and to identify direct genetic targets of aberrant BAF complexes in this context, we comprehensively evaluated the effects of BAF47 reintroduction in BAF47-deficient sarcomas with respect to complex subunit and associated protein factor composition and stability, global chromatin structure, and gene regulation. Reintroduced BAF47 stably integrated into BAF complexes, and remarkably, stabilized a highly specific set of BAF subunits, resulting in an increased complex molecular weight and stoichiometric nuclear abundance. These biochemical changes inducing the formation of wild-type complexes in MRT and EpS cell settings were directly linked to reproducible changes in BAF complex localization genome-wide, particularly, in the targeting to H3K4me3-marked promoter regions of direct target genes to establish DNA accessibility. Importantly, changes in BAF47-dependent BAF complex targeting between oncogenic and induced wild-type conditions were reproducibly associated with differential chromatin architecture and gene expression signatures hallmark to both MRT and EpS, and uniformly resulted in proliferative senescence of MRT and EpS cell lines in culture. These studies highlight, for the first time, the full spectrum of structural and functional contributions of the BAF47 subunit, implicating its role as a keystone in heteromorphic BAF complex assembly; BAF47 is required for the stable integration of several BAF subunits and novel interacting factors, which we determine govern specific genome-wide targeting mechanisms and chromatin-templated activities. These results reveal the mechanisms underlying the oncogenesis of BAF47-deficient sarcomas and point toward novel therapeutic strategies for this group of human sarcomas. Citation Format: Robert Nakayama, Robert T. Williams, Seth H. Cassel, Mingxiang Teng, Rafael A. Irizarry, George D. Demetri, Cigall Kadoch. Genome-wide mistargeting of oncogenic SWI/SNF(BAF) complexes in SMARCB1(BAF47)-deficient sarcomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2658.

Abstract NG05: Chromatin accessibility underlies the tumor-suppression role of BAF (mSWI/SNF) complexes in many malignancies

Abstract The mammalian SWI/SNF (mSWI/SNF or BAF) complex is an ATP-dependent chromatin remodeler long thought to be a tumor suppressor because it is frequently inactivated in cell lines. It was definitively identified as a tumor suppressor in malignant rhabdoid tumors (MRTs), an aggressive pediatric cancer. In most of these tumors, the dedicated BAF subunit SMARCB1 (BAF47) undergoes loss-of-heterozygosity, with no other large-scale chromosomal instability, leading to the conclusion that the tumor-suppression mechanism in MRTs is “epigenetic” in nature, meaning that it leads to changes in gene expression without necessarily altering other sequences of DNA. However, these tumors account for a small fraction of all cancers containing mutations in BAF subunits, and other evidence implicates the BAF complex in DNA repair processes, making its tumor-suppression mechanism uncertain. We sought to determine the applicability of this epigenetic model of tumor suppression in the large number of other cancers bearing BAF subunit mutations. To answer this question, we analyzed exome sequencing and genomic copy-number data derived from 5,659 patient samples, together representing 22 cancer types and over 4,500,000 genetic alterations, to compare the genomic states of tumors with BAF subunit defects against similar tumors that lacked such defects. We find that BAF subunits collectively are mutated significantly more frequently than expected in 19 of the 22 cancer types examined, far more than expected. The increase in BAF subunit mutations was statistically robust, both in samples with low mutation rates (e.g., lower-grade glioma), as well as those with high rates (e.g., melanoma, tumors of the lung), indicating that mutation of the complex is under positive selection in the vast majority of cancers. We also performed an analysis of copy-number variation of BAF subunits in each cancer, and find recurrent patterns of biallelic deletions and amplifications. In many malignancies, mutation and biallelic deletion of BAF subunits occurs at rates comparable to or higher than well-known tumor suppressors, such as TP53, PTEN, and RB1. In the majority of cancer types, tumors with BAF subunit defects have higher mutational rates than cancers of the same tissue lacking such defects. By reconstructing the mutational processes active in each patient sample, we find that excess mutations in BAF-deficient cancers represent the signatures of defective mismatch repair, cytidine deamination, UV-damage response, DNA end joining, Pol epsilon function, and other enzymatic processes acting on DNA. In four cancer types where ChIP-seq tracks were available for the healthy cell type of origin, we tracked the locations of over 280,000 mutations, and find that excess mutations in BAF-deficient tumors are biased towards regions of elevated H3K36me3 and low H3K27me3 in their corresponding non-cancerous tissues. We hypothesized that these mutations resulted from inaccessibility for repair and maintenance factors when a BAF subunit is defective. We tested this hypothesis using topoisomerase II alpha binding as representative of enzymes that require access to DNA, and find that rapid conditional deletion of the BAF ATPase Brg led to an increase in genome-wide deposition of H3K27me3 at Brg-dependent topoisomerase II alpha sites from an initial low state, contributing to loss of enzyme accessibility at its target sites. Our data indicate that the tumor suppressive functions of BAF complexes arise from their contribution to maintaining genomic stability by providing access to DNA for repair and maintenance mechanisms. Consistent with this conclusion, we examined levels of P53 and phospho-P53 in mouse embryonic fibroblasts expressing a tamoxifen-inducible deletion of Brg, the ATPase of the BAF complex. Deletion of Brg causes a significant increase in the levels of P53 and phospho-P53 following DNA damage by a panel of conditions, confirming that genomic stability and DNA repair is compromised upon loss of BAF activity. Our data are consistent with a general model, wherein DNA accessibility is maintained at a steady state arising from a balance between silencing factors and factors (including the BAF complex) that maintain accessibility. Many repair and maintenance factors operate on DNA that require at least transient accessibility, making the BAF complex an upstream contributor to several activities. When BAF activity is compromised, the equilibrium is shifted towards a state that is less accessible to repair enzymes. This disruption of normal repair and maintenance function leads to accumulation of mutations. Our analysis of 5,659 patient genomes indicates that this is the mechanism of tumor suppression under positive selection in the majority of human malignancies. Citation Format: Courtney Hodges, Diana Hargreaves, Erik Miller, Gerald R. Crabtree. Chromatin accessibility underlies the tumor-suppression role of BAF (mSWI/SNF) complexes in many malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr NG05. doi:10.1158/1538-7445.AM2015-NG05

Abstract 3023: Chromatin accessibility underlies the tumor suppressor role of BAF (mSWI/SNF) complexes in many human cancers

Abstract Recent exome sequencing studies of human cancer indicate that ∼20% of malignancies have mutations in the subunits of the BAF (mSWI/SNF) chromatin remodeling complex, implicating the complex as a major tumor suppressor. However, the mechanisms of tumor suppression remain uncertain. To understand how defective BAF subunits contribute to cancer, we analyzed the genomic states of 5,659 patient samples derived from 22 tumor types, and compared samples containing genetic defects of the BAF complex against samples containing no such defects. We show that in the majority of cancer types, tumors with BAF subunit defects have significantly higher mutation counts than cancers of the same tissue lacking BAF defects. By reconstructing the mutational processes active in each patient sample, we find that excess mutations in BAF-deficient cancers represent the signatures of defective mismatch repair, cytidine deamination, UV-damage response, DNA end joining, Pol epsilon function, and other enzymatic processes acting on DNA. In four cancer types where ChIP-seq tracks were available for the healthy cell type of origin, we tracked the locations of over 280,000 mutations, and find that excess mutations in BAF-deficient tumors are biased towards regions of elevated H3K36me3 and low H3K27me3 in their corresponding non-cancerous tissues. We hypothesized that these mutations resulted from inaccessibility for repair and maintenance factors when a BAF subunit is defective. We tested this hypothesis using topoisomerase IIa binding as model for processes that require enzymatic access to DNA, and found that rapid conditional deletion of the ATPase Brg led to an increase in genome-wide deposition of H3K27me3 at Brg-dependent topoisomerase IIa sites from an initial low state. Our data indicate that the tumor-suppressive functions of BAF complexes arise largely from the requirement for BAF complexes to maintain genomic stability by providing access to DNA for repair and maintenance mechanisms. Note: This abstract was not presented at the meeting. Citation Format: Courtney Hodges, Diana C. Hargreaves, Erik L. Miller, Gerald R. Crabtree. Chromatin accessibility underlies the tumor suppressor role of BAF (mSWI/SNF) complexes in many human cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3023. doi:10.1158/1538-7445.AM2015-3023

Abstract SY24-03: Chromatin regulation in cancer

Abstract Recent exom sequencing studies have discovered that a wide variety of human malignancies appear to have driving mutations in genes encoding subunits of chromatin regulatory complexes. In particular mutations in the subunits of the BAF (mSWI/SNF) complex (Khavari, P. et al. Nature 1993; Wu, J.I. et al. Cell 2009) are most common and found in over 20% of all human malignancies (Kadoch, C et al. Nature Genetics 2013). Mutations in subunits of the polycomb repressive complex 2 (PRC2) are also frequent and are found most commonly in the catalytic EZH2 subunit (Garroway and Lander Cell 2013; Dawson MA, and Kouzarides T. Cell. 2012). These two activities oppose one another and appear to form a dynamic balance that controls expression of genes involved in cell proliferation, genome protection and development. BAF complexes can function as either onocgenes or as tumor suppressors (Kadoch, C. and Crabtree Cell 2013; Kadoch et al Nature Genetics 2013, Dunaief JL et al Cell 1994). (see also the Abstract by Cigall Kadoch). In general these subunits behave as tumor suppressors. We find that BAF complexes are necessary for the binding and function of TopoIIa over the genome (Dykhuzien et al Nature 2013). Deletion of subunits such as Brg, BAF250a, BAF47 or BAF57 leads to the presence of anaphase bridges reflecting a failure of decatenation during mitosis. Deletion of several BAF subunits also evokes the decatenation checkpoint with cell cycle arrest that can be specifically repressed with the TopoIIa S1524A mutation that is unable to initiate the decatenation checkpoint. TopoIIa directly interacts with BAF250a through a region that contains several dozen mutations in human cancer. A small molecule screen for inhibitors that block the ability of BAF complexes to suppress the PRC1 subunit, BMI revealed several TopoIIa inhibitors (Dykhuzien et al Nature 2013). Surprisingly these TopoIIa inhibitors mimic both the repression of genes such as Bmi1, Ring1a and other polycomb subunits and also the activation of genes such as FGF4. These studies suggest that TopoIIa and BAF function together to both control transcription and also to resolve decatenated DNA during mitosis. The later may contribute to the role of BAF complexes as tumor suppressors. Interestingly, the most frequently mutated subunits in human disease, BAF250a, SS18, Bcl7 and others are not required for in vitro chromatin remodeling, ATP activity or complex assembly and have no yeast homologues. In addition, we find that deletion of subunits of the complexes do not change nucleosome positioning over the mammalian genome. These unexpected findings underline the importance of developing new techniques to study chromatin regulation. With this goal in mind we have developed strains of mice that allow one to direct a specific chromatin regulatory activity to one allele of a marked gene using small molecule inducers of proximity. Using this approach we have explored the kinetics and stability of heterchromatin formation (Hathaway, Bell et al Cell 2012; Hodges et al PNAS 2013) revealing that H3K9Me3 induced heterochromatin is limited by intrinsic reaction rates rather than borders (Hodges et al PNAS 2012). Additional studies directed at the mechanism of action of the oncogenic MLL-fusion proteins and the ATP-dependent remodeling complexes will be discussed. Citation Format: Gerald Crabtree. Chromatin regulation in cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY24-03. doi:10.1158/1538-7445.AM2014-SY24-03

The role of BAF (mSWI/SNF) complexes in mammalian neural development.

The BAF (mammalian SWI/SNF) complexes are a family of multi-subunit ATP-dependent chromatin remodelers that use ATP hydrolysis to alter chromatin structure. Distinct BAF complex compositions are possible through combinatorial assembly of homologous subunit families and can serve non-redundant functions. In mammalian neural development, developmental stage-specific BAF assemblies are found in embryonic stem cells, neural progenitors and postmitotic neurons. In particular, the neural progenitor-specific BAF complexes are essential for controlling the kinetics and mode of neural progenitor cell division, while neuronal BAF function is necessary for the maturation of postmitotic neuronal phenotypes as well as long-term memory formation. The microRNA-mediated mechanism for transitioning from npBAF to nBAF complexes is instructive for the neuronal fate and can even convert fibroblasts into neurons. The high frequency of BAF subunit mutations in neurological disorders underscores the rate-determining role of BAF complexes in neural development, homeostasis, and plasticity.

A Chromatin Remodeling Complex Regulates Topoisomerase IIα Function

TOP2A-mediated DNA decatenation during mitosis requires the BAF subunits BRG1 and BAF250A.

Kinetic Analysis of npBAF to nBAF Switching Reveals Exchange of SS18 with CREST and Integration with Neural Developmental Pathways

During the development of the vertebrate nervous system, neural progenitors divide, generate progeny that exit mitosis, and then migrate to sites where they elaborate specific morphologies and synaptic connections. Mitotic exit in neurons is accompanied by an essential switch in ATP-dependent chromatin regulatory complexes from the neural progenitor Brg/Brm-associated factor (npBAF) to neuron-specific nBAF complexes that is in part driven by miR-9/9* and miR-124. Recapitulating this microRNA/chromatin switch in fibroblasts leads to their direct conversion to neurons. We have defined the kinetics of neuron-specific BAF complex assembly in the formation of induced neurons from mouse embryonic stem cells, human fibroblasts, and normal mouse neural differentiation and, using proteomic analysis, found that this switch also includes the removal of SS18 and its replacement by CREST at mitotic exit. We found that switching of chromatin remodeling mechanisms is highly correlated with a broad switch in the use of neurogenic transcription factors. Knock-down of SS18 in neural stem cells causes cell-cycle exit and failure to self-renew, whereas continued expression of SS18 in neurons blocks dendritic outgrowth, underlining the importance of subunit switching. Because dominant mutations in BAF subunits underlie widely different human neurologic diseases arising in different neuronal types, our studies suggest that the characteristics of these diseases must be interpreted in the context of the different BAF assemblies in neurons rather than a singular mammalian SWItch/sucrose nonfermentable (mSWI/SNF) complex.

BAF complexes facilitate decatenation of DNA by topoisomerase IIα

Recent exon sequencing studies of human tumors have revealed that subunits of mSWI/SNF or BAF complexes are mutated in more than 20% of all human malignancies,1,2 yet the mechanisms involved in tumor suppression are unclear. BAF chromatin remodeling complexes are polymorphic assemblies that use energy provided by ATP hydrolysis to regulate transcription through the control of chromatin structure3 and the placement of Polycomb (PcG) across the genome4,5. Several proteins dedicated to this multi-subunit complex, including SMARCA4 (BRG1) and BAF250A (ARID1A), are mutated at frequencies similar to that of recognized tumor suppressors. In particular, the core ATPase BRG1 is mutated in 5-10% of childhood medulloblastoma6-9 and greater than 15% of Burkitt's Lymphoma.10,11 Here we find a novel function of BAF complexes in decatenating newly replicated sister chromatids, a requirement for proper chromosome segregation during mitosis. We find that deletion of Brg1, as well as the expression of Brg1 point mutants identified in human tumors leads to anaphase bridge formation (sister chromatids linked by catenated strands of DNA), and a G2/M phase block characteristic of the decatenation checkpoint. Endogenous BAF complexes directly interact with endogenous TopoIIα through BAF250a and are required for TopoIIα binding to about 12,000 sites over the genome. Our results demonstrate that TopoIIα's chromatin binding is dependent on the ATPase activity of Brg1, which is compromised in oncogenic Brg1 mutants. These studies indicate that the ability of TopoIIα to prevent DNA entanglement at mitosis requires BAF complexes and suggest that this activity contributes to the role of BAF subunits as tumor suppressors.

Screening for Inhibitors of an Essential Chromatin Remodeler in Mouse Embryonic Stem Cells by Monitoring Transcriptional Regulation

The SWI/SNF-like adenosine triphosphate (ATP)–dependent chromatin remodeling complex, esBAF, is both necessary and, in some contexts, sufficient to induce the pluripotent state. Furthermore, mutations in various BAF subunits are associated with cancer. Little is known regarding the precise mechanism(s) by which this complex exerts its activities. Thus, it is unclear which protein interactions would be important to disrupt to isolate a relevant readout of mechanism. To address this, we developed a gene expression–based assay to identify inhibitors of the native esBAF complex. Specifically, a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay was developed in mouse embryonic stem (ES) cells to monitor expression of Bmi1, a developmentally important gene repressed by the esBAF complex. The assay was miniaturized to a 384-well format and used to screen a diverse collection of compounds, including novel products of diversity-oriented synthesis (DOS). Confirmed hits were validated using a knock-in ES cell reporter line in which luciferase is inserted into the Bmi1 locus. Several of the validated hits regulate a panel of target genes in a manner similar to the BAF chromatin-remodeling complex. Together these data indicate that expression-based screening using qRT-PCR is a successful approach to identify compounds targeting the regulation of key developmental genes in ES cells.