The BAF60 Subunit of the SWI/SNF Chromatin-Remodeling Complex Directly Controls the Formation of a Gene Loop at FLOWERING LOCUS C in Arabidopsis

SWI/SNF complexes mediate ATP-dependent chromatin remodeling to regulate gene expression. Many components of these complexes are evolutionarily conserved, and several subunits of Arabidopsis thaliana SWI/SNF complexes are involved in the control of flowering, a process that depends on the floral repressor FLOWERING LOCUS C (FLC). BAF60 is a SWI/SNF subunit, and in this work, we show that BAF60, via a direct targeting of the floral repressor FLC, induces a change at the high-order chromatin level and represses the photoperiod flowering pathway in Arabidopsis. BAF60 accumulates in the nucleus and controls the formation of the FLC gene loop by modulation of histone density, composition, and posttranslational modification. Physiological analysis of BAF60 RNA interference mutant lines allowed us to propose that this chromatin-remodeling protein creates a repressive chromatin configuration at the FLC locus.     

SWI/SNF protein component BAF250a regulates cardiac progenitor cell differentiation by modulating chromatin accessibility during second heart field development

ATP-dependent SWI/SNF chromatin remodeling complexes alter the structure of chromatin at specific loci and facilitate tissue-specific gene regulation during development. Several SWI/SNF subunits are required for cardiogenesis. However, the function and mechanisms of SWI/SNF in mediating cardiac progenitor cell (CPC) differentiation during cardiogenesis are not well understood. Our studies of the SWI/SNF chromatin remodeling complex identified that BAF250a, a regulatory subunit of the SWI/SNF, plays a key role in CPC differentiation. BAF250a ablation in mouse second heart field (SHF) led to trabeculation defects in the right ventricle, ventricular septal defect, persistent truncus arteriosus, reduced myocardial proliferation, and embryonic lethality around E13. Using an embryonic stem cell culture system that models the formation and differentiation of SHF CPCs in vivo, we have shown that BAF250a ablation in CPCs specifically inhibits cardiomyocyte formation. Moreover, BAF250a selectively regulates the expression of key cardiac factors Mef2c, Nkx2.5, and Bmp10 in SHF CPCs. Chromatin immunoprecipitation and DNase I digestion assays indicate that BAF250a regulates gene expression by binding selectively to its target gene promoters and recruiting Brg1, the catalytic subunit of SWI/SNF, to modulate chromatin accessibility. Our results thus identify BAF250a-mediated chromatin remodeling as an essential epigenetic mechanism mediating CPC differentiation.     

Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3?×?FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individual proteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3?×?FLAG tag, Twin-Strep tag, or CBP-3?×?FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.     

Specialized RSC: Substrate Specificities for a Conserved Chromatin Remodeler

The remodel the structure of chromatin (RSC) nucleosome remodeling complex is a conserved chromatin regulator with roles in chromatin organization, especially over nucleosome depleted regions therefore functioning in gene expression. Recent reports in Saccharomyces cerevisiae have identified specificities in RSC activity toward certain types of nucleosomes. RSC has now been shown to preferentially evict nucleosomes containing the histone variant H2A.Z in vitro. Furthermore, biochemical activities of distinct RSC complexes has been found to differ when their nucleosome substrate is partially unraveled. Mammalian BAF complexes, the homologs of yeast RSC and SWI/SNF complexes, are also linked to nucleosomes with H2A.Z, but this relationship may be complex and extent of conservation remains to be determined. The interplay of remodelers with specific nucleosome substrates and regulation of remodeler outcomes by nucleosome composition are tantalizing questions given the wave of structural data emerging for RSC and other SWI/SNF family remodelers.     

Analysis of the SWI/SNF chromatin-remodeling complex during early heart development and BAF250a repression cardiac gene transcription during P19 cell differentiation

The regulatory networks of differentiation programs and the molecular mechanisms of lineage-specific gene regulation in mammalian embryos remain only partially defined. We document differential expression and temporal switching of BRG1-associated factor (BAF) subunits, core pluripotency factors and cardiac-specific genes during post-implantation development and subsequent early organogenesis. Using affinity purification of BRG1 ATPase coupled to mass spectrometry, we characterized the cardiac-enriched remodeling complexes present in E8.5 mouse embryos. The relative abundance and combinatorial assembly of the BAF subunits provides functional specificity to Switch/Sucrose NonFermentable (SWI/SNF) complexes resulting in a unique gene expression profile in the developing heart. Remarkably, the specific depletion of the BAF250a subunit demonstrated differential effects on cardiac-specific gene expression and resulted in arrhythmic contracting cardiomyocytes in vitro. Indeed, the BAF250a physically interacts and functionally cooperates with Nucleosome Remodeling and Histone Deacetylase (NURD) complex subunits to repressively regulate chromatin structure of the cardiac genes by switching open and poised chromatin marks associated with active and repressed gene expression. Finally, BAF250a expression modulates BRG1 occupancy at the loci of cardiac genes regulatory regions in P19 cell differentiation. These findings reveal specialized and novel cardiac-enriched SWI/SNF chromatin-remodeling complexes, which are required for heart formation and critical for cardiac gene expression regulation at the early stages of heart development.     

Residual Complexes Containing SMARCA2 (BRM) Underlie the Oncogenic Drive of SMARCA4 (BRG1) Mutation

Collectively, genes encoding subunits of the SWI/SNF (BAF) chromatin remodeling complex are mutated in 20% of all human cancers, with the SMARCA4 (BRG1) subunit being one of the most frequently mutated. The SWI/SNF complex modulates chromatin remodeling through the activity of two mutually exclusive catalytic subunits, SMARCA4 and SMARCA2 (BRM). Here, we show that a SMARCA2-containing residual SWI/SNF complex underlies the oncogenic activity of SMARCA4 mutant cancers. We demonstrate that a residual SWI/SNF complex exists in SMARCA4 mutant cell lines and plays essential roles in cellular proliferation. Further, using data from loss-of-function screening of 165 cancer cell lines, we identify SMARCA2 as an essential gene in SMARCA4 mutant cancer cell lines. Mechanistically, we reveal that Smarca4 inactivation leads to greater incorporation of the nonessential SMARCA2 subunit into the SWI/SNF complex. Collectively, these results reveal a role for SMARCA2 in oncogenesis caused by SMARCA4 loss and identify the ATPase and bromodomain-containing SMARCA2 as a potential therapeutic target in these cancers.