SWI/SNF Chromatin-Remodeling Factor Smarcd3/Baf60c Controls Epithelial-Mesenchymal Transition by Inducing Wnt5a Signaling

We previously identified a gene signature predicted to regulate the epithelial-mesenchymal transition (EMT) in both epithelial tissue stem cells and breast cancer cells. A phenotypic RNA interference (RNAi) screen identified the genes within this 140-gene signature that promoted the conversion of mesenchymal epithelial cell adhesion molecule-negative (EpCAM⁻) breast cancer cells to an epithelial EpCAM^+/high phenotype. The screen identified 10 of the 140 genes whose individual knockdown was sufficient to promote EpCAM and E-cadherin expression. Among these 10 genes, RNAi silencing of the SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c in EpCAM⁻ breast cancer cells gave the most robust transition from the mesenchymal to epithelial phenotype. Conversely, expression of Smarcd3/Baf60c in immortalized human mammary epithelial cells induced an EMT. The mesenchymal-like phenotype promoted by Smarcd3/Baf60c expression resulted in gene expression changes in human mammary epithelial cells similar to that of claudin-low triple-negative breast cancer cells. These mammary epithelial cells expressing Smarcd3/Baf60c had upregulated Wnt5a expression. Inhibition of Wnt5a by either RNAi knockdown or blocking antibody reversed Smarcd3/Baf60c-induced EMT. Thus, Smarcd3/Baf60c epigenetically regulates EMT by activating WNT signaling pathways.     

Ubiquitin-dependent and Ubiquitin-independent Control of Subunit Stoichiometry in the SWI/SNF Complex

The mammalian SWI/SNF chromatin remodeling complex is a key player in multiple chromatin transactions. Core subunits of this complex, including the ATPase, Brg-1, and various Brg-1-associated factors (BAFs), work in concert to maintain a functional remodeling complex. This intra-complex regulation is supervised by protein-protein interactions, as stoichiometric levels of BAF proteins are maintained by proteasomal degradation. We show that the mechanism of BAF155-mediated stabilization of BAF57 involves blocking its ubiquitination by preventing interaction with TRIP12, an E3 ubiquitin ligase. Consequently, as opposed to complexed BAF57, whose principal lysines are unavailable for ubiquitination, uncomplexed BAF57 can be freely ubiquitinated and degraded by the proteasome. Additionally, a BAF57 mutant, which contains no lysine residues, was found to retain its ability to be stabilized by interaction with BAF155, suggesting that in addition to the ubiquitin-dependent mechanism of BAF57 degradation, there exists a ubiquitin-independent mechanism that may involve the direct interaction of BAF57 with the proteasome. We propose that this regulatory mechanism exists to ensure functional fidelity of the complex and prevent the accumulation of uncomplexed proteins, which may disrupt the normal activity of the complex.     

Repression of FLOWERING LOCUS C and FLOWERING LOCUS T by the Arabidopsis Polycomb repressive complex 2 components

Polycomb group (PcG) proteins are evolutionarily conserved in animals and plants, and play critical roles in the regulation of developmental gene expression. Here we show that the Arabidopsis Polycomb repressive complex 2 (PRC2) subunits CURLY LEAF (CLF), EMBRYONIC FLOWER 2 (EMF2) and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) repress the expression of FLOWERING LOCUS C (FLC), a central repressor of the floral transition in Arabidopsis and FLC relatives. In addition, CLF directly interacts with and mediates the deposition of repressive histone H3 lysine 27 trimethylation (H3K27me3) into FLC and FLC relatives, which suppresses active histone H3 lysine 4 trimethylation (H3K4me3) in these loci. Furthermore, we show that during vegetative development CLF and FIE strongly repress the expression of FLOWERING LOCUS T (FT), a key flowering-time integrator, and that CLF also directly interacts with and mediates the deposition of H3K27me3 into FT chromatin. Our results suggest that PRC2-like complexes containing CLF, EMF2 and FIE, directly interact with and deposit into FT, FLC and FLC relatives repressive trimethyl H3K27 leading to the suppression of active H3K4me3 in these loci, and thus repress the expression of these flowering genes. Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that the CLF-containing PRC2-like complexes play a significant role in control of flowering in Arabidopsis.     

Role of the SWI/SNF Chromatin Remodeling Complex in Regulation of Inflammation Gene Expression

Molecular Biology - The process of inflammation is the body’s natural defense response to the penetration of foreign substances and molecules from the outside. Many proteins, signaling...     

The SWI/SNF Subunit/Tumor Suppressor BAF47/INI1 Is Essential in Cell Cycle Arrest upon Skeletal Muscle Terminal Differentiation

Myogenic terminal differentiation is a well-orchestrated process starting with permanent cell cycle exit followed by muscle-specific genetic program activation. Individual SWI/SNF components have been involved in muscle differentiation. Here, we show that the master myogenic differentiation factor MyoD interacts with more than one SWI/SNF subunit, including the catalytic subunit BRG1, BAF53a and the tumor suppressor BAF47/INI1. Downregulation of each of these SWI/SNF subunits inhibits skeletal muscle terminal differentiation but, interestingly, at different differentiation steps and extents. BAF53a downregulation inhibits myotube formation but not the expression of early muscle-specific genes. BRG1 or BAF47 downregulation disrupt both proliferation and differentiation genetic programs expression. Interestingly, BRG1 and BAF47 are part of the SWI/SNF remodeling complex as well as the N-CoR-1 repressor complex in proliferating myoblasts. However, our data show that, upon myogenic differentiation, BAF47 shifts in favor of N-CoR-1 complex. Finally, BRG1 and BAF47 are well-known tumor suppressors but, strikingly, only BAF47 seems essential in the myoblasts irreversible cell cycle exit. Together, our data unravel differential roles for SWI/SNF subunits in muscle differentiation, with BAF47 playing a dual role both in the permanent cell cycle exit and in the regulation of muscle-specific genes.     

Establishment of the Winter-Annual Growth Habit via FRIGIDA-Mediated Histone Methylation at FLOWERING LOCUS C in Arabidopsis

In Arabidopsis thaliana, flowering-time variation exists among accessions, and the winter-annual (late-flowering without vernalization) versus rapid-cycling (early flowering) growth habit is typically determined by allelic variation at FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). FRI upregulates the expression of FLC, a central floral repressor, to levels that inhibit flowering, resulting in the winter-annual habit. Here, we show that FRI promotes histone H3 lysine-4 trimethylation (H3K4me3) in FLC to upregulate its expression. We identified an Arabidopsis homolog of the human WDR5, namely, WDR5a, which is a conserved core component of the human H3K4 methyltransferase complexes called COMPASS-like. We found that recombinant WDR5a binds H3K4-methylated peptides and that WDR5a also directly interacts with an H3K4 methyltransferase, ARABIDOPSIS TRITHORAX1. FRI mediates WDR5a enrichment at the FLC locus, leading to increased H3K4me3 and FLC upregulation. WDR5a enrichment is not required for elevated H3K4me3 in FLC upon loss of function of an FLC repressor, suggesting that two distinct mechanisms underlie elevated H3K4me3 in FLC. Our findings suggest that FRI is involved in the enrichment of a WDR5a-containing COMPASS-like complex at FLC chromatin that methylates H3K4, leading to FLC upregulation and thus the establishment of the winter-annual growth habit.     

Subunit Rtt102 Controls the Conformation of the Arp7/9 Heterodimer and Its Interactions with Nucleotide and the Catalytic Subunit of SWI/SNF Remodelers

Chromatin-remodeling complexes are assembled around a catalytic subunit that contains a central ATPase domain and flanking sequences that recruit auxiliary subunits. The catalytic subunits of SWI/SNF remodelers recruit Arp7/9 through a helicase/SANT-associated (HSA) domain N-terminal to the ATPase domain. Arp7/9-containing remodelers also carry the auxiliary subunit Rtt102, but the role of this subunit is poorly understood. Here, we show that Rtt102 binds with nanomolar affinity to the Arp7/9 heterodimer and modulates its conformation and interactions with the ATPase subunit and nucleotide. When bound to Rtt102, Arp7/9 interacts with a shorter segment of the HSA domain. Structural analysis by small-angle x-ray scattering further shows that when bound to Rtt102, the complex of Arp7/9 with the catalytic subunit assumes a more stable compact conformation. We also found that Arp7, Arp9, and Arp7/9 interact very weakly with ATP, but Rtt102 promotes high-affinity ATP binding to a single site in the heterodimer. Collectively, the results establish a function for subunit Rtt102 as a stabilizing factor for the Arp7/9 heterodimer, enhancing its interaction with nucleotide and controlling the conformation of SWI/SNF remodelers in an Arp7/9-dependent manner.     

The Arabidopsis SWI2/SNF2 Chromatin Remodeler BRAHMA Regulates Polycomb Function during Vegetative Development and Directly Activates the Flowering Repressor Gene SVP

The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development.