BRG1/BRM-associated Factor Complex Research Articles

Arid1a-dependent canonical BAF complex suppresses inflammatory programs to drive efficient germinal center B cell responses.

The mammalian Brg1/Brm-associated factor (BAF) complexes are major regulators of nucleosomal remodeling that are commonly mutated in several cancers, including germinal center (GC)-derived B cell lymphomas. However, the specific roles of different BAF complexes in GC B cell biology are not well understood. Here we show that the AT-rich interaction domain 1a (Arid1a) containing canonical BAF (cBAF) complex is required for maintenance of GCs and high-affinity antibody responses. While Arid1a-deficient B cells undergo initial activation, they fail to sustain the GC program. Arid1a establishes permissive chromatin landscapes for B cell activation and is concomitantly required to suppress inflammatory gene programs. The inflammatory signatures instigated by Arid1a deficiency promoted the recruitment of neutrophils and inflammatory monocytes. Dampening of inflammatory cues through interleukin-1β blockade or glucocorticoid receptor agonist partially rescued Arid1a-deficient GCs, highlighting a critical role for inflammation in impeding GCs. Our work reveals essential functions of Arid1a-dependent cBAF in promoting efficient GC responses.

Alternative splicing of a chromatin modifier alters the transcriptional regulatory programs of stem cell maintenance and neuronal differentiation

Development of embryonic stem cells (ESCs) into neurons requires intricate regulation of transcription, splicing, and translation, but how these processes interconnect is not understood. We found that polypyrimidine tract binding protein 1 (PTBP1) controls splicing of DPF2, a subunit of BRG1/BRM-associated factor (BAF) chromatin remodeling complexes. Dpf2 exon 7 splicing is inhibited by PTBP1 to produce the DPF2-S isoform early in development. During neuronal differentiation, loss of PTBP1 allows exon 7 inclusion and DPF2-L expression. Different cellular phenotypes and gene expression programs were induced by these alternative DPF2 isoforms. We identified chromatin binding sites enriched for each DPF2 isoform, as well as sites bound by both. In ESC, DPF2-S preferential sites were bound by pluripotency factors. In neuronal progenitors, DPF2-S sites were bound by nuclear factor I (NFI), while DPF2-L sites were bound by CCCTC-binding factor (CTCF). DPF2-S sites exhibited enhancer modifications, while DPF2-L sites showed promoter modifications. Thus, alternative splicing redirects BAF complex targeting to impact chromatin organization during neuronal development.

Abstract 5591: ARID domain containing BAF subunits exhibit neomorphism: Have we identified the new Achilles heel in cancer

Abstract The SWI/SNF chromatin remodeler utilizes energy of ATP hydrolysis to slide or evict nucleosomes or histones, respectively, thus enabling nuclear processes by driving an ‘open’ chromatin architecture. The mammalian SWI/SNF, also termed the BrG1/Brm associated factor (BAF) complex, is the major chromatin remodeler in ontogeny and adult life. Three major BAF complexes namely canonical (cBAF), polybromo (pBAF), and non-canonical (ncBAF) have been studied. BAF components, classified as a tumor suppressors, exhibit frequent mutations in cancers. Though, studies have evaluated functional roles of BAF subunits, very few attempted to study BAF mutations identified across cancer types. In our previous studies, we identified tumor specific mutations in the gene encoding ‘A-T rich interaction domain containing 1B’ (ARID1B), within its nuclear localization signal (NLS). NLS-inactivated ARID1B exhibited cytoplasmic localization and promoted tumorigenesis by activating the ERK and Wnt/β-catenin pathways. More recently, we detected inactivating mutations in the gene encoding ARID2, a component of the pBAF complex and an ARID1B paralogue. We now present detailed characterization of tumor-specific ARID2 truncations that were presumed to be inactivating in nature. One such truncation mutation viz. p.Ser989fsArg21*, identified in our previous study, is expected to yield an ARID2 protein truncating at amino acid position 1010 (as against the full length of 1835 amino acids). Scrutiny of The Cancer Genome Atlas cBioPortal and Cancer Cell Line Encyclopedia mutation data bases revealed several ARID2 mutations expected to truncate the protein around the 1010 position. Surprisingly, ectopic expression of truncated ARID2 forms promoted oncogenic function in both ARID2 proficient and deficient backgrounds, thus validating a neomorphic gain of function for the truncated protein. Fluorescence based intracellular localization assays revealed a surprising cytoplasmic localization of truncated ARID2. Further, we mapped the ARID2 NLS by in silico analysis followed by evaluation of several deletion constructs, to be located at 1488-1518 position; thus, explaining the cytoplasmic localization of ARID2 forms truncating around 1010 position. Further, truncated ARID2 was unable to bind other pBAF components and did not sequester other pBAF components to the cytoplasm, in both ARID2 proficient and deficient cells. Tandem Affinity Purification Mass-spectrometry revealed putative interaction partners for truncated ARID2; analysis is currently underway to reveal the mechanism of tumorigenic induction exhibited by truncated ARID2. Immunohistochemistry on colorectal and breast cancer tissue microarrays revealed ARID2 cytoplasmic localization in a significant fraction of samples. We have discovered a novel YIN-YANG function of BAF components expected to yield efficient therapeutic options in cancer. Citation Format: Sanjana Sarkar, Murali Dharan Bashyam. ARID domain containing BAF subunits exhibit neomorphism: Have we identified the new Achilles heel in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5591.

BRG1/BRM inhibitor targets AML stem cells and exerts superior preclinical efficacy combined with BET or menin inhibitor

AbstractBRG1 (SMARCA4) and BRM (SMARCA2) are the mutually exclusive core ATPases of the chromatin remodeling BAF (BRG1/BRM-associated factor) complexes. They enable transcription factors/cofactors to access enhancers/promoter and modulate gene expressions responsible for cell growth and differentiation of acute myeloid leukemia (AML) stem/progenitor cells. In AML with MLL1 rearrangement (MLL1r) or mutant NPM1 (mtNPM1), although menin inhibitor (MI) treatment induces clinical remissions, most patients either fail to respond or relapse, some harboring menin mutations. FHD-286 is an orally bioavailable, selective inhibitor of BRG1/BRM under clinical development in AML. Present studies show that FHD-286 induces differentiation and lethality in AML cells with MLL1r or mtNPM1, concomitantly causing perturbed chromatin accessibility and repression of c-Myc, PU.1, and CDK4/6. Cotreatment with FHD-286 and decitabine, BET inhibitor (BETi) or MI, or venetoclax synergistically induced in vitro lethality in AML cells with MLL1r or mtNPM1. In models of xenografts derived from patients with AML with MLL1r or mtNPM1, FHD-286 treatment reduced AML burden, improved survival, and attenuated AML-initiating potential of stem-progenitor cells. Compared with each drug, cotreatment with FHD-286 and BETi, MI, decitabine, or venetoclax significantly reduced AML burden and improved survival, without inducing significant toxicity. These findings highlight the FHD-286–based combinations as a promising therapy for AML with MLL1r or mtNPM1.

Abstract C061: The impact of the BRG1/BRM-associated factor (BAF) complex on the androgen receptor cistrome in African American prostate cancer

Abstract We aimed to establish in prostate cancer (PCa) how and why the androgen receptor displays differential functions in a manner driven by genomic ancestry, namely between African American (AA) and European American (EA) PCa, with the goal to assess how this may contribute to PCa health disparities. We utilized EA and AA non-malignant prostate and PCa cell models with confirmed genomic ancestry and defined the basal and dihydrotestosterone (DHT)-regulated AR and H3K27ac cistromes using CUT&RUN, and integrated these data with publicly available genomic counterparts. The DHT-dependent AR and H3K27ac cistromes significantly overlapped within AA cells and EA cells but there was significantly less overlap between AA and EA cells. For example, H3K27ac distributions were frequent (~15-20k sites) in each of non-malignant AA RC43N and EA HPr1AR, and malignant RC43T and LNCaP, and the significance of the overlaps were comparable for basal and DHT treated cells, but this was not significantly impacted by ancestry. By contrast the AR cistrome was highly DHT-dependent, with ~2k sites notably in LNCaP and RC43T, which very significantly overlapped with H3K27ac in the same cells but significantly less so across cells. Thus, although the H3K27ac cistrome was comparable across cells, the overlap with AR was driven by genomic ancestry. To investigate this, we focused on the BAF complexes as previously we identified reduced expression of a key component, SMARCA5, in AA PCa. These large multimeric complexes regulate nucleosome positioning during gene transcription and DNA repair. In PCa, the cBAF complex impacts different transcription factors, including ERG, and it is tempting to speculate that the corruption of cBAF identified in AA PCa phenocopies the impact of TMPRSS2-ETS impact observed in EA PCa. To test the possibility that the cBAF complex corrupts AR genomic functions in AA PCa we exploited scRNA-Seq data from PCa organoids from the Rb and Pten double knockout (PBCre4:Rb1f/f:Pten f/f ) to establish that expression of genes in the cBAF genes complex were collectively significantly altered in neuroendocrine PCa (NEPC) and Pouf23 androgen indifferent lineages. Secondly, knockdown of another cBAF component, SMARCC1, enhanced Enzalutamide sensitivity in same organoids. Finally, to test the impact of cBAF complex on AR signaling we have generated AA and EA PCa cell lines that stably express dCRISPR-activator (VP64) or repressor (KRAB) proteins and designed gRNAs to regulate expression of, for example, SMARCA5 and measure the impact on the AR cistrome and function. In summary, these findings identified that the shared AR and H3K27ac cistromes are driven by genomic ancestry, and that reduced expression of SMARCA5 and other components of the cBAF complex are significant in AA and advanced PCa, and impact AR signaling. Our future directions are focused on how SMARCA5 expression disrupts the balance between transcription factor functions, such as for the AR and other transcription factors, and effective DNA repair. Citation Format: Shahid S. Hussain, Debasis Nayak, David W. Goodrich, Clayton Yates, Moray Campbell. The impact of the BRG1/BRM-associated factor (BAF) complex on the androgen receptor cistrome in African American prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C061.

Selective Globin Gene Regulation By the Non-Canonical Baf Chromatin Remodeling Complex

The regulation of the beta-type globin genes has served as a powerful model for developmental transcriptional control, with humans expressing embryonic (HBE), two fetal (HBG1/2) and two adult (HBB and HBD) versions at the corresponding developmental stages. Reactivation of HBG in adult erythroid cells is of benefit to patients with sickle cell disease and β-thalassemia. While past efforts focused on the identification of repressors of the HBG genes, including BCL11A, ZBTB7A, and NFI-A/X, to date no transcriptional activators have been identified with selectivity for any of the globin genes. Using a CRISPR-Cas9 screen in the HUDEP2 erythroid cell line, we recently identified BRG1/SMARCA4 as a potential activator of HBG production. BRG1 is a core ATPase component of the multi-subunit BAF (BRG1/BRM-associated factor) complex; different subtypes of this large chromatin remodeler family are specific to particular cell types or developmental stages. BRG1 was previously implicated in globin gene regulation but its molecular composition and whether it has selectivity for any of the genes in the cluster is unknown. Notably, we found that loss of BRG1 in both HUDEP2 and primary erythroid cells impairs transcription of HBG to a much greater extent than that of HBB. Here we functionally dissected BAF subunits to decipher composition of the relevant BAF complex and the mechanism for this unexpected HBG gene selectivity. We used CRISPR-Cas9 in HUDEP2 cells and human primary erythroid cultures to test the requirement for 14 different BAF subunits for selective HBG transcription. Among these, the BAF60A component emerged as critically required for the normal production of HBG but not HBB in HUDEP2 cells. Depletion of BAF60A in human primary erythroid cultures reduced HBG by up to 80% with no reduction in HBB as assessed by western blot, flow cytometry, HPLC, RT-PCR, and droplet digital RT-PCR. We also observed a reduction in HBD expression, suggesting that BAF60A functions in a gene selective manner but not necessarily favoring the fetal over the adult-type globin genes. α-type globin genes were unaffected by BAF60A loss, further demonstrating the gene selectivity of BAF60A action. RNA-seq of BAF60A-targeted cells showed that the most downregulated transcripts were from the two HBG genes and the BGLT3 long non-coding RNA, which typically parallels HBG in expression. These results revealed an unexpected gene selectivity of the BAF complex, conveyed at least in part by BAF60A. To assess global chromatin accessibility upon BAF60A loss in primary erythroid cells we utilized ATAC-seq. Among ~132,000 accessibility peaks, ~10,000 were reduced in BAF60A targeted cells. Interestingly, we saw a strong decrease in chromatin accessibility at the HBG and HBD genes with minimal to no effect on the HBB or α-globin genes, in line with gene expression data, again reflecting the gene specificity of BAF60A. BAF60A can be found in different classes of BAF complexes, but is absolutely required for the non-canonical BAF (ncBAF) subtype. ncBAF is a relatively recently described BAF complex that is distinguished from other classes of BAF complexes by the presence of the bromodomain protein BRD9. Therefore, we hypothesized that ncBAF is the relevant BAF complex required for selective HBG transcription. Genetic or pharmacologic disruption of BRD9 in HUDEP2 cells recapitulated the BAF60A phenotype, with decreased HBG and HBD expression but sparing of the HBB and α-globin genes. This is the first study of the role of the non-canonical BAF complex in hemoglobin gene regulation. Our results demonstrate remarkable selectivity of this subtype of a broadly active chromatin remodeling complex for specific hemoglobin genes in adult erythroid cells. Further work on the ncBAF complex will focus on rerouting the BAF complex to promote the transcription of HBG and HBD in hemoglobinopathies.

Dissecting the role of SWI/SNF component ARID1B in steady-state hematopoiesis

AbstractThe adenosine triphosphate (ATP)–dependent chromatin remodeling complex, SWItch/Sucrose Non-Fermentable (SWI/SNF), has been implicated in normal hematopoiesis. The AT-rich interaction domain 1B (ARID1B) and its paralog, ARID1A, are mutually exclusive, DNA-interacting subunits of the BRG1/BRM-associated factor (BAF) subclass of SWI/SNF complex. Although the role of several SWI/SNF components in hematopoietic differentiation and stem cell maintenance has been reported, the function of ARID1B in hematopoietic development has not been defined. To this end, we generated a mouse model of Arid1b deficiency specifically in the hematopoietic compartment. Unlike the extensive phenotype observed in mice deficient in its paralog, ARID1A, Arid1b knockout (KO) mice exhibited a modest effect on steady-state hematopoiesis. Nonetheless, transplantation experiments showed that the reconstitution of myeloid cells in irradiated recipient mice was dependent on ARID1B. Furthermore, to assess the effect of the complete loss of ARID1 proteins in the BAF complex, we generated mice lacking both ARID1A and ARID1B in the hematopoietic compartment. The double-KO mice succumbed to acute bone marrow failure resulting from complete loss of BAF–mediated chromatin remodeling activity. Our Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analyses revealed that >80% of loci regulated by ARID1B were distinct from those regulated by ARID1A; and ARID1B controlled expression of genes crucial in myelopoiesis. Overall, loss of ARID1B affected chromatin dynamics in murine hematopoietic stem and progenitor cells, albeit to a lesser extent than cells lacking ARID1A.

Examining the Role of Individual Baf Chromatin Remodeling Complex Components in Globin Gene Regulation

Regulation of the switch from fetal (HbF, α2γ2) to adult (HbA,α2β2) hemoglobin production is an important area of study as reversal of the switch is a key therapeutic approach for treatment of sickle cell disease and β-thalassemia. While multiple specific repressors of γ-globin gene transcription have been identified, there has not been a parallel finding of a γ-globin specific transcriptional activator. The mammalian BAF (BRG1/BRM-associated factor) complex is a multi-subunit, ATP-dependent, chromatin remodeling complex of the SWI/SNF family, with diverse composition and roles in regulation of gene expression. BRG1/SMARCA4 is a core component of the complex with ATPase catalytic activity. Prior studies showed that BRG1 is essential for proper β-globin expression and regulates long-range chromatin interactions between the locus control region (LCR) and the β-globin gene promoter. Studies in K562 cells, where γ-globin expression is predominant, have similarly shown the requirement for BRG1 for transcriptional activation. To date, the role of the BAF complex in regulation of γ-globin versus β-globin gene expression has not been well characterized. Moreover, the considerable diversity in the subtypes of this complex (such as polybromo-associated BAF or PBAF and non-canonical BAF), with subunit composition and functions that differ across cell types and developmental stages, suggests the hypothesis that specific subtypes or subunits of the BAF complex have specific roles in γ-globin gene regulation. We recently identified BRG1 as a potential activator of γ-globin production in a HUDEP2 CRISPR screen targeting ATPase and helicase domains. In validation experiments in HUDEP2 and primary human erythroid cultures, loss of BRG1 led to a marked decrease in γ-globin containing F-cells by flow cytometry and γ-globin protein by western blot, along with a smaller decrease in β-globin production. Because the BAF subunits have significant heterogeneity in expression and function, we asked whether any components may have specificity for γ-globin versus β-globin gene transcription in adult erythroid cells. Using CRISPR-Cas9 targeting in a HUDEP2 sub-clone with elevated fetal hemoglobin levels, we systematically ablated 12 individual BAF/PBAF complex components and assayed fetal hemoglobin by flow cytometry, HPLC, and quantitative western blots. We identified 5 components whose loss of function led to a reduction in γ-globin relative to β-globin: BAF53A, BAF155, BAF60A, BAF60B, and BAF57. We subsequently depleted these five components in primary human erythroid cultures from three independent donors using CRISPR-Cas9 targeting. While loss of BAF53A impaired cell proliferation, loss of remaining components was well tolerated. Loss of BAF155 and BAF60B had minimal effect on globin chain production. Loss of BAF60A had the most potent effect on γ-globin, with up to 80% reduction in γ-globin with no decrease in β-globin production. BAF57 knockdown led to a more modest decrease in γ-globin, but with some decrease in total globin production. Together these data suggest that the requirements for some specific components of the BAF complex differ for activation of fetal or adult globin gene transcription. Additional studies are under way to characterize chromatin accessibility changes at the β-globin locus in response to alterations in the BAF complex, as well as the response to loss of additional BAF complex components.

Loss of BAF (mSWI/SNF) chromatin-remodeling ATPase Brg1 causes multiple malformations of cortical development in mice.

Mutations in genes encoding subunits of the BAF (BRG1/BRM-associated factor) complex cause various neurodevelopmental diseases. However, the underlying pathophysiology remains largely unknown. Here, we analyzed the function of Brahma-related gene 1 (Brg1), a core ATPase of BAF complexes, in the developing cerebral cortex. Loss of Brg1 causes several morphological defects resembling human malformations of cortical developments (MCDs), including microcephaly, cortical dysplasia, cobblestone lissencephaly and periventricular heterotopia. We demonstrated that neural progenitor cell renewal, neuronal differentiation, neuronal migration, apoptotic cell death, pial basement membrane and apical junctional complexes, which are associated with MCD formation, were impaired after Brg1 deletion. Furthermore, transcriptome profiling indicated that a large number of genes were deregulated. The deregulated genes were closely related to MCD formation, and most of these genes were bound by Brg1. Cumulatively, our study indicates an essential role of Brg1 in cortical development and provides a new possible pathogenesis underlying Brg1-based BAF complex-related neurodevelopmental disorders.

Postnatal Cranial Characterization in a Mouse Model of Cortex‐Specific Overexpression of BAF170

The BRG1/BRM-associated factor (BAF) chromatin remodeling complexes are composed of the mutually exclusive brahma or brahma-related gene 1 ATPases, along with several other structural subunits and their isoforms. BAF complexes serve specific functions in widely different cell types and developmental processes. Mammalian BAF170, a subunit of chromatin remodeling complex mSWI/SNF, is an intrinsic factor controlling cortical size. It was previously reported that conditional deletion of BAF170 promotes indirect neurogenesis by increasing the pool of intermediate progenitors and results in an enlarged cortex, whereas cortex specific BAF170 overexpression results in a smaller cortex. However, the influence of cortex specific BAF170 overexpression on size and shape of the craniofacial complex, specifically the skull, is not well understood. We hypothesized that the skull phenotype of mice with cortex specific BAF170 overexpression may include indirect effects of the reduced cortex including reduced intracranial volume and linear distance measures of the skull relative to unaffected littermates. To further understand the influence of cortex-specific conditional overexpression of BAF170 on the cranial phenotype of young mice, we examined two postnatal ages of the cortex specific BAF170 overexpression mouse model. Here, we combine measures of gross observation (body weight, body length, brain weight) and of 3D micro-computed tomography imaging of the head (overall skull length, cranial length, cranial height, cranial width, intracranial volume) at 28 (P28; N=6) and 42 (P42; N=12) days of age to assess the influence of BAF170 overexpression on skull morphology as compared to unaffected littermates. In our small sample, gross examination of the brain indicated an obviously distinctive brain morphological phenotype at both ages, including a visibly smaller cortex, in mice with overexpression of BAF170. Results showed few other differences at P28, although brain weight and skull width were reduced in mice with cortex specific BAF170 overexpression as compared to unaffected littermates. By P42, brain weight was no longer different between the genotypes, but cranial length, width, and height were all smaller. Additionally, intracranial volume was reduced in mice overexpressing BAF170 at P42. These results suggest that BAF170 overexpression in mice, which specifically supervises development of a smaller cortex, affects their skull phenotype in terms of size and possibly shape at P28 and P42, with potentially increased differences occurring with age. Further morphometric exploration is necessary to determine whether additional craniofacial differences exist at other ages.

Acute BAF perturbation causes immediate changes in chromatin accessibility.

Cancer-associated, loss-of-function mutations in genes encoding subunits of the BRG1/BRM-associated factor (BAF) chromatin-remodeling complexes1-8 often cause drastic chromatin accessibility changes, especially in important regulatory regions9-19. However, it remains unknown how these changes are established over time (for example, immediate consequences or long-term adaptations), and whether they are causative for intracomplex synthetic lethalities, abrogating the formation or activity of BAF complexes9,20-24. In the present study, we use the dTAG system to induce acute degradation of BAF subunits and show that chromatin alterations are established faster than the duration of one cell cycle. Using a pharmacological inhibitor and a chemical degrader of the BAF complex ATPase subunits25,26, we show that maintaining genome accessibility requires constant ATP-dependent remodeling. Completely abolishing BAF complex function by acute degradation of a synthetic lethal subunit in a paralog-deficient background results in an almost complete loss of chromatin accessibility at BAF-controlled sites, especially also at superenhancers, providing a mechanism for intracomplex synthetic lethalities.

SWI/SNF (BAF) complexes: From framework to a functional role in endothelial mechanotransduction.

Endothelial cells (ECs) are constantly subjected to an array of mechanical cues, especially shear stress, due to their luminal placement in the blood vessels. Blood flow can regulate various aspects of endothelial biology and pathophysiology by regulating the endothelial processes at the transcriptomic, proteomic, miRNomic, metabolomics, and epigenomic levels. ECs sense, respond, and adapt to altered blood flow patterns and shear profiles by specialized mechanisms of mechanosensing and mechanotransduction, resulting in qualitative and quantitative differences in their gene expression. Chromatin-regulatory proteins can regulate transcriptional activation by modifying the organization of nucleosomes at promoters, enhancers, silencers, insulators, and locus control regions. Recent research efforts have illustrated that SWI/SNF (SWItch/Sucrose Non-Fermentable) or BRG1/BRM-associated factor (BAF) complex regulates DNA accessibility and chromatin structure. Since the discovery, the gene-regulatory mechanisms of the BAF complex associated with chromatin remodeling have been intensively studied to investigate its role in diverse disease phenotypes. Thus far, it is evident that (1) the SWI/SNF complex broadly regulates the activity of transcriptional enhancers to control lineage-specific differentiation and (2) mutations in the BAF complex proteins lead to developmental disorders and cancers. It is unclear if blood flow can modulate the activity of SWI/SNF complex to regulate EC differentiation and reprogramming. This review emphasizes the integrative role of SWI/SNF complex from a structural and functional standpoint with a special reference to cardiovascular diseases (CVDs). The review also highlights how regulation of this complex by blood flow can lead to the discovery of new therapeutic interventions for the treatment of endothelial dysfunction in vascular diseases.

Requisite Chromatin Remodeling for Myeloid and Erythroid Lineage Differentiation from Erythromyeloid Progenitors

SUMMARYThe mammalian SWitch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodeling BAF (BRG1/BRM-associated factor) complex plays an essential role in developmental and pathological processes. We show that the deletion of Baf155, which encodes a subunit of the BAF complex, in the Tie2(+) lineage (Baf155 (CKO) leads to defects in yolk sac myeloid and definitive erythroid (EryD) lineage differentiation from erythromyeloid progenitors (EMPs). The chromatin of myeloid gene loci in Baf155 CKO EMPs is mostly inaccessible and enriched mainly by the ETS binding motif. BAF155 interacts with PU.1 and is recruited to PU.1 target gene loci together with p300 and KDM6a. Treatment of Baf155 CKO embryos with GSK126, an H3K27me2/3 methyltransferase EZH2 inhibitor, rescues myeloid lineage gene expression. This study uncovers indispensable BAF-mediated chromatin remodeling of myeloid gene loci at the EMP stage. Future studies exploiting epigenetics in the generation and application of EMP derivatives for tissue repair, regeneration, and disease are warranted.

Structural Insights into the Evolutionarily Conserved BAF Chromatin Remodeling Complex.

The switch/sucrose nonfermentable (SWI/SNF) family of proteins acts to regulate chromatin accessibility and plays an essential role in multiple cellular processes. A high frequency of mutations has been found in SWI/SNF family subunits by exome sequencing in human cancer, and multiple studies support its role in tumor suppression. Recent structural studies of yeast SWI/SNF and its human homolog, BAF (BRG1/BRM associated factor), have provided a model for their complex assembly and their interaction with nucleosomal substrates, revealing the molecular function of individual subunits as well as the potential impact of cancer-associated mutations on the remodeling function. Here we review the structural conservation between yeast SWI/SNF and BAF and examine the role of highly mutated subunits within the BAF complex.

HRP2-DPF3a-BAF complex coordinates histone modification and chromatin remodeling to regulate myogenic gene transcription.

Functional crosstalk between histone modifications and chromatin remodeling has emerged as a key regulatory mode of transcriptional control during cell fate decisions, but the underlying mechanisms are not fully understood. Here we discover an HRP2–DPF3a–BAF epigenetic pathway that coordinates methylated histone H3 lysine 36 (H3K36me) and ATP-dependent chromatin remodeling to regulate chromatin dynamics and gene transcription during myogenic differentiation. Using siRNA screening targeting epigenetic modifiers, we identify hepatoma-derived growth factor-related protein 2 (HRP2) as a key regulator of myogenesis. Knockout of HRP2 in mice leads to impaired muscle regeneration. Mechanistically, through its HIV integrase binding domain (IBD), HRP2 associates with the BRG1/BRM-associated factor (BAF) chromatin remodeling complex by interacting directly with the BAF45c (DPF3a) subunit. Through its Pro-Trp-Trp-Pro (PWWP) domain, HRP2 preferentially binds to H3K36me2. Consistent with the biochemical studies, ChIP-seq analyses show that HRP2 colocalizes with DPF3a across the genome and that the recruitment of HRP2/DPF3a to chromatin is dependent on H3K36me2. Integrative transcriptomic and cistromic analyses, coupled with ATAC-seq, reveal that HRP2 and DPF3a activate myogenic genes by increasing chromatin accessibility through recruitment of BRG1, the ATPase subunit of the BAF complex. Taken together, these results illuminate a key role for the HRP2-DPF3a-BAF complex in the epigenetic coordination of gene transcription during myogenic differentiation.

Structure of nucleosome-bound human BAF complex.

Mammalian SWI/SNF family chromatin remodelers, BRG1/BRM-associated factor (BAF) and polybromo-associated BAF (PBAF), regulate chromatin structure and transcription, and their mutations are linked to cancers. The 3.7-angstrom-resolution cryo-electron microscopy structure of human BAF bound to the nucleosome reveals that the nucleosome is sandwiched by the base and the adenosine triphosphatase (ATPase) modules, which are bridged by the actin-related protein (ARP) module. The ATPase motor is positioned proximal to nucleosomal DNA and, upon ATP hydrolysis, engages with and pumps DNA along the nucleosome. The C-terminal α helix of SMARCB1, enriched in positively charged residues frequently mutated in cancers, mediates interactions with an acidic patch of the nucleosome. AT-rich interactive domain-containing protein 1A (ARID1A) and the SWI/SNF complex subunit SMARCC serve as a structural core and scaffold in the base module organization, respectively. Our study provides structural insights into subunit organization and nucleosome recognition of human BAF complex.

Systematic characterization of BAF mutations provides insights into intracomplex synthetic lethalities in human cancers.

Aberrations in genes coding for subunits of the BAF chromatin remodeling complexes are highly abundant in human cancers. Currently, it is not understood how these loss-of-function mutations contribute to cancer development and how they can be targeted therapeutically. The cancer-type-specific occurrence patterns of certain subunit mutations suggest subunit-specific effects on BAF complex function, possibly by the formation of aberrant residual complexes. Here, we systematically characterize the effects of individual subunit loss on complex composition, chromatin accessibility and gene expression in a panel of knock-out cell lines deficient for 22 BAF subunits. We observe strong, specific and sometimes discordant alterations dependent on the targeted subunit and show that these explain intra-complex co-dependencies, including the synthetic lethal interactions SMARCA4-ARID2, SMARCA4-ACTB and SMARCC1-SMARCC2. These data provide insights into the role of different BAF subcomplexes in genome-wide chromatin organization and suggest approaches to therapeutically target BAF mutant cancers.

Dynamic BAF chromatin remodeling complex subunit inclusion promotes temporally distinct gene expression programs in cardiogenesis.

ABSTRACTChromatin remodeling complexes instruct cellular differentiation and lineage specific transcription. The BRG1/BRM-associated factor (BAF) complexes are important for several aspects of differentiation. We show that the catalytic subunit gene Brg1 has a specific role in cardiac precursors (CPs) to initiate cardiac gene expression programs and repress non-cardiac expression. Using immunopurification with mass spectrometry, we have determined the dynamic composition of BAF complexes during mammalian cardiac differentiation, identifying several cell-type specific subunits. We focused on the CP- and cardiomyocyte (CM)-enriched subunits BAF60c (SMARCD3) and BAF170 (SMARCC2). Baf60c and Baf170 co-regulate gene expression with Brg1 in CPs, and in CMs their loss results in broadly deregulated cardiac gene expression. BRG1, BAF60c and BAF170 modulate chromatin accessibility, to promote accessibility at activated genes while closing chromatin at repressed genes. BAF60c and BAF170 are required for proper BAF complex composition, and BAF170 loss leads to retention of BRG1 at CP-specific sites. Thus, dynamic interdependent BAF complex subunit assembly modulates chromatin states and thereby participates in directing temporal gene expression programs in cardiogenesis.

Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

Chromatin Remodeling BAF (SWI/SNF) Complexes in Neural Development and Disorders.

The ATP-dependent BRG1/BRM associated factor (BAF) chromatin remodeling complexes are crucial in regulating gene expression by controlling chromatin dynamics. Over the last decade, it has become increasingly clear that during neural development in mammals, distinct ontogenetic stage-specific BAF complexes derived from combinatorial assembly of their subunits are formed in neural progenitors and post-mitotic neural cells. Proper functioning of the BAF complexes plays critical roles in neural development, including the establishment and maintenance of neural fates and functionality. Indeed, recent human exome sequencing and genome-wide association studies have revealed that mutations in BAF complex subunits are linked to neurodevelopmental disorders such as Coffin-Siris syndrome, Nicolaides-Baraitser syndrome, Kleefstra's syndrome spectrum, Hirschsprung's disease, autism spectrum disorder, and schizophrenia. In this review, we focus on the latest insights into the functions of BAF complexes during neural development and the plausible mechanistic basis of how mutations in known BAF subunits are associated with certain neurodevelopmental disorders.