Enhancer switching in cell lineage priming is linked to eRNA, Brg1's AT-hook, and SWI/SNF recruitment.

RNA transcribed from enhancers, i.e., eRNA, has been suggested to directly activate transcription by recruiting transcription factors and co-activators. Although there have been specific examples of eRNA functioning in this way, it is not clear how general this may be. We find that the AT-hook of SWI/SNF preferentially binds RNA and, as part of the esBAF complex, associates with eRNA transcribed from intronic and intergenic regions. Our data suggest that SWI/SNF is globally recruited in cis by eRNA to cell-type-specific enhancers, representative of two distinct stages that mimic early mammalian development, and not at enhancers that are shared between the two stages. In this manner, SWI/SNF facilitates recruitment and/or activation of MLL3/4, p300/CBP, and Mediator to stage-specific enhancers and super-enhancers that regulate the transcription of metabolic and cell lineage priming-related genes. These findings highlight a connection between ATP-dependent chromatin remodeling and eRNA in cell identity and typical- and super-enhancer activation.

Brahma-related gene 1 has time-specific roles during brain and eye development.

During development, gene expression is tightly controlled to facilitate the generation of the diverse cell types that form the central nervous system. Brahma-related gene 1 (Brg1, also known as Smarca4) is the catalytic subunit of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex that regulates transcription. We investigated the role of Brg1 between embryonic day 6.5 (E6.5) and E14.5 in Sox2-positive neural stem cells (NSCs). Being without major consequences at E6.5 and E14.5, loss of Brg1 between E7.5 and E12.5 resulted in the formation of rosette-like structures in the subventricular zone, as well as morphological alterations and enlargement of neural retina (NR). Additionally, Brg1-deficient cells showed decreased survival in vitro and in vivo. Furthermore, we uncovered distinct changes in gene expression upon Brg1 loss, pointing towards impaired neuron functions, especially those involving synaptic communication and altered composition of the extracellular matrix. Comparison with mice deficient for integrase interactor 1 (Ini1, also known as Smarcb1) revealed that the enlarged NR was Brg1 specific and was not caused by a general dysfunction of the SWI/SNF complex. These results suggest a crucial role for Brg1 in NSCs during brain and eye development.

Small Cell Carcinoma of the Ovary, Hypercalcemic Type, in a 12-Month-Old Girl.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a highly aggressive malignant tumor affecting predominantly young adults and adolescents with an average age of 23.9 at time of diagnosis. Up to two thirds of patients have paraneoplastic hypercalcemia. The molecular signature of these tumors is SMARCA4 mutations, with somatic and germline pathogenic variants previously described. We report a case of a previously healthy one-year-old girl who was noticed to have mild anemia and an abdominal mass during a well-child visit. Further laboratory testing revealed hypercalcemia. A computerized tomography scan showed a left-sided ovarian mass (9.3 x 7.3 x 7 cm). The resection specimen showed a large ovarian tumor with solid tan-yellow cut surfaces and small foci of necrosis. Microscopically, the tumor was composed of sheets of small, hyperchromatic epithelioid cells with focal rhabdoid large cell morphology. The tumor cells were strongly and diffusely positive for WT1 (N-terminal antibodies) with focal EMA and Pan-keratin positivity. Absent SMARCA4 (BRG1) protein expression by immunohistochemistry ultimately established the diagnosis of small cell carcinoma of the ovary, hypercalcemic type. To our knowledge, this is the youngest patient reported in the literature.

Degron mediated BRM/SMARCA2 depletion uncovers novel combination partners for treatment of BRG1/SMARCA4-mutant cancers.

Recent studies have highlighted that cancer cells with a loss of the SWI/SNF complex catalytic subunit BRG1 are dependent on the remaining ATPase, BRM, making it an attractive target for cancer therapy. However, an understanding of the extent of target inhibition required to arrest cell growth, necessary to develop an appropriate therapeutic strategy, remains unknown. Here, we utilize tunable depletion of endogenous BRM using the SMASh degron, and interestingly observe that BRG1-mutant lung cancer cells require near complete depletion of BRM to robustly inhibit growth both in vitro and in vivo. Therefore, to identify pathways that synergize with partial BRM depletion and afford a deeper response, we performed a genome-wide CRISPR screen and discovered a combinatorial effect between BRM depletion and the knockout of various genes of the oxidative phosphorylation pathway and the anti-apoptotic gene MCL1. Together these studies provide an important framework to elucidate the requirements of BRM inhibition in the BRG1-mutant state with implications on the feasibility of targeting BRM alone, as well as reveal novel insights into pathways that can be exploited in combination toward deeper anti-tumor responses.

The ATPase BRG1/SMARCA4 is 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.

SWI/SNF-dependent genes are defined by their chromatin landscape.

SWI/SNF complexes are evolutionarily conserved, ATP-dependent chromatin remodeling machines. Here, we characterize the features of SWI/SNF-dependent genes using BRM014, an inhibitor of the ATPase activity of the complexes. We find that SWI/SNF activity is required to maintain chromatin accessibility and nucleosome occupancy for most enhancers but not for most promoters. SWI/SNF activity is needed for expression of genes with low to medium levels of expression that have promoters with (1) low chromatin accessibility, (2) low levels of active histone marks, (3) high H3K4me1/H3K4me3 ratio, (4) low nucleosomal phasing, and (5) enrichment in TATA-box motifs. These promoters are mostly occupied by the canonical Brahma-related gene 1/Brahma-associated factor (BAF) complex. These genes are surrounded by SWI/SNF-dependent enhancers and mainly encode signal transduction, developmental, and cell identity genes (with almost no housekeeping genes). Machine-learning models trained with different chromatin characteristics of promoters and their surrounding regulatory regions indicate that the chromatin landscape is a determinant for establishing SWI/SNF dependency.

Baf45a Mediated Chromatin Remodeling Promotes Transcriptional Activation for Osteogenesis and Odontogenesis.

Chromatin remodeling, specifically the tissue-specific regulation in mineralized tissues, is an understudied avenue of gene regulation. Here we show that Baf45a and Baf45d, two Baf45 homologs belong to ATPase-dependent SWI/SNF chromatin remodeling complex, preferentially expressed in osteoblasts and odontoblasts compared to Baf45b and Baf45c. Recently, biochemical studies revealed that BAF45A associates with Polybromo-associated BAF (PBAF) complex. However, the BAF45D subunit belongs to the polymorphic canonical BRG1-associated factor (cBAF) complex. Protein profiles of osteoblast and odontoblast differentiation uncovered a significant increase of BAF45A and PBAF subunits during early osteoblast and odontoblast maturation. Chromatin immunoprecipitation sequencing (ChIP-seq) during the bone marrow stromal cells (BMSCs) differentiation showed higher histone H3K9 and H3K27 acetylation modifications in the promoter of Baf45a and Baf45d and increased binding of bone and tooth specific transcription factor RUNX2. Overexpression of Baf45a in osteoblasts activates genes essential for the progression of osteoblast maturation and mineralization. Furthermore, shRNA-mediated knockdown of Baf45a in odontoblasts leads to markedly altered genes responsible for the proliferation, apoptosis, DNA repair, and modest decrease in dentinogenic marker gene expression. Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) assay in Baf45a knockout osteoblasts revealed a noticeable reduction in chromatin accessibility of osteoblast and odontoblast specific genes, along with transcription factor Atf4 and Klf4. Craniofacial mesenchyme-specific loss of Baf45a modestly reduced the mineralization of the tooth and mandibular bone. These findings indicated that BAF45A-dependent mineralized tissue-specific chromatin remodeling through PBAF-RUNX2 crosstalk results in transcriptional activation is critical for early differentiation and matrix maturation of mineralized tissues.

Neuronal activity-induced BRG1 phosphorylation regulates enhancer activation.

Neuronal activity-induced enhancers drive gene activation. We demonstrate that BRG1, the core subunit of SWI/SNF-like BAF ATP-dependent chromatin remodeling complexes, regulates neuronal activity-induced enhancers. Upon stimulation, BRG1 is recruited to enhancers in an H3K27Ac-dependent manner. BRG1 regulates enhancer basal activities and inducibility by affecting cohesin binding, enhancer-promoter looping, RNA polymerase II recruitment, and enhancer RNA expression. We identify a serine phosphorylation site in BRG1 that is induced by neuronal stimulations and is sensitive to CaMKII inhibition. BRG1 phosphorylation affects its interaction with several transcription co-factors, including the NuRD repressor complex and cohesin, possibly modulating BRG1-mediated transcription outcomes. Using mice with knockin mutations, we show that non-phosphorylatable BRG1 fails to efficiently induce activity-dependent genes, whereas phosphomimic BRG1 increases enhancer activity and inducibility. These mutant mice display anxiety-like phenotypes and altered responses to stress. Therefore, we reveal a mechanism connecting neuronal signaling to enhancer activities through BRG1 phosphorylation.

Brg1 inhibits E-cadherin expression in lung epithelial cells and disrupts epithelial integrity.

Brahma-related gene-1 (Brg1), a key chromatin remodeling factor, is associated with cell proliferation and migration in kidney and heart cells, but few reports have examined its role in airway epithelial cell. Airway epithelial injury, which is involved in the entire pathological process of asthma, is an important cause of recurrent asthma. Here, we studied the function of Brg1 in an ovalbumin (OVA)-induced asthma model (lung-specific conditional Brg1 (Brg1-/-) knockdown mice) and human bronchial epithelial 16HBE cells stably expressing Brg1 shRNA. Our results showed that high expression of Brg1 was detected in asthmatic children and in mouse models. Brg1-/- mice showed improved airway hyperresponsiveness (AHR) and bronchial epithelial integrity, along with reduced inflammatory cell infiltration and airway mucus secretion, when challenged with OVA. Furthermore, cell proliferation, migration, and expression of E-cadherin increased in 16HBE cells in which Brg1 was silenced. We further demonstrated that Brg1 bound to and inactivated a critical region (-86/+60 bp) within the E-cadherin promoter in bronchial epithelial cells. Thus, Brg1 might act as an important regulator of airway epithelial integrity in asthma progression and might be a novel therapeutic target. KEY MESSAGES: • Depletion of Brg1 improves the integrity of airway epithelium in asthma by regulating E-cadherin expression in lung epithelial cells. • Knockdown of Brg1 increased the cell proliferation and migration by human bronchial epithelial 16HBE cells. • Brg1 might bLLe a novel therapeutic target in asthma.

Camk2a-Cre-mediated conditional deletion of chromatin remodeler Brg1 causes perinatal hydrocephalus.

Mammalian SWI/SNF-like BAF chromatin remodeling complexes are essential for many aspects of neural development. Mutations in the genes encoding the core subunit Brg1/SmarcA4 or other complex components cause neurodevelopmental diseases and are associated with autism. Congenital hydrocephalus is a serious brain disorder often experienced by these patients. We report a role of Brg1 in the pathogenesis of hydrocephalus disorder. We discovered an unexpected early activity of mouse Camk2a-Cre transgene, which mediates Brg1 deletion in a subset of forebrain neurons beginning in the late embryonic stage. Brg1 deletion in these neurons led to severe congenital hydrocephalus with enlargement of the lateral ventricles and attenuation of the cerebral cortex. The Brg1-deficient mice had significantly smaller subcommissural organs and narrower Sylvian aqueducts than mice that express normal levels of Brg1. Effects were non-cell autonomous and may be responsible for the development of the congenital hydrocephalus phenotype. Our study provides evidence indicating that abnormalities in Brg1 function result in defects associated with neurodevelopmental disorders and autism.