Brahma is required for proper expression of the floral repressor FLC in Arabidopsis

Background

BRAHMA (BRM) is a member of a family of ATPases of the SWI/SNF chromatin remodeling complexes from Arabidopsis. BRM has been previously shown to be crucial for vegetative and reproductive development.

Methodology/Principal Findings

Here we carry out a detailed analysis of the flowering phenotype of brm mutant plants which reveals that, in addition to repressing the flowering promoting genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), BRM also represses expression of the general flowering repressor FLOWERING LOCUS C (FLC). Thus, in brm mutant plants FLC expression is elevated, and FLC chromatin exhibits increased levels of histone H3 lysine 4 tri-methylation and decreased levels of H3 lysine 27 tri-methylation, indicating that BRM imposes a repressive chromatin configuration at the FLC locus. However, brm mutants display a normal vernalization response, indicating that BRM is not involved in vernalization-mediated FLC repression. Analysis of double mutants suggests that BRM is partially redundant with the autonomous pathway. Analysis of genetic interactions between BRM and the histone H2A.Z deposition machinery demonstrates that brm mutations overcome a requirement of H2A.Z for FLC activation suggesting that in the absence of BRM, a constitutively open chromatin conformation renders H2A.Z dispensable.

Conclusions/Significance

BRM is critical for phase transition in Arabidopsis. Thus, BRM represses expression of the flowering promoting genes CO, FT and SOC1 and of the flowering repressor FLC. Our results indicate that BRM controls expression of FLC by creating a repressive chromatin configuration of the locus.     

Coordination of the Vernalization Response through a VIN3 and FLC Gene Family Regulatory Network in Arabidopsis

Vernalization is an environmentally induced epigenetic switch in which winter cold triggers epigenetic silencing of floral repressors and thus provides competence to flower in spring. Vernalization triggers the recruitment of chromatin-modifying complexes to a clade of flowering repressors that are epigenetically silenced via chromatin modifications. In Arabidopsis thaliana, VERNALIZATION INSENSITIVE3 (VIN3) and its related plant homeodomain finger proteins act together with Polycomb Repressive Complex 2 to increase repressive histone marks at floral repressor loci, including FLOWERING LOCUS C (FLC) and its related genes, by vernalization. Here, we show that VIN3 family of proteins nonredundantly functions to repress different subsets of the FLC gene family during the course of vernalization. Each VIN3 family protein binds to modified histone peptides in vitro and directly associates with specific sets of FLC gene family chromatins in vivo to mediate epigenetic silencing. In addition, members of the FLC gene family are also differentially regulated during the course of vernalization to mediate proper vernalization response. Our results show that these two gene families cooperated during the course of evolution to ensure proper vernalization response through epigenetic changes.     

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.     

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.     

1H, 15N, and 13C resonance assignments and secondary structure of the SWIRM domain of human BAF155, a chromatin remodeling complex component

Mammalian SWI/SNF complexes are evolutionary conserved, ATP-dependent chromatin remodeling units. BAF155 in the SWI/SNF complex contains several highly conserved domains, including SANT, SWIRM, and leucine zipper domains. The biological roles of the SWIRM domain remain unclear; however, both structural and biochemical analyses of this domain have suggested that it could mediate protein-protein or protein-DNA interactions during the chromatin remodeling process. The human BAF155 SWIRM domain was cloned into the Escherichia coli expression vector pMAL-c2X and purified using affinity chromatography for structural analysis. We report the backbone ¹H, ¹⁵N, and ¹³C resonance assignments and secondary structure of this domain using nuclear magnetic resonance (NMR) spectroscopy and the TALOS+ program. The secondary structure consists of five α-helices that form a typical histone fold for DNA interactions. Our data suggest that the BAF155 SWIRM domain interacts with nucleosome DNA (K _d = 0.47 μM).     

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.     

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.     

FLC: A key regulator of flowering time in Arabidopsis

The timing of floral transition has significant consequences for reproductive success in plants. The molecular genetic dissection of flowering time control in Arabidopsis identified an integrated network of pathways that quantitatively control this developmental switch. A central player in this process is the FLOWERING LOCUS C gene (FLC), which blocks flowering by inhibiting the genes required to switch the meristem from vegetative to floral development. Three systems (the FRIGIDA gene, vernalization, and the autonomous pathway) all influence the state of FLC. Last years many new genes have been identified that regulate FLC expression, and most of them are involved in the modification of FLC chromatin. This review focuses on recent insights in FLC regulation.