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.     

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.     

Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits

Protein complexes of the SWI/SNF family remodel nucleosome structure in an ATP-dependent manner. Each complex contains between 8 and 15 subunits, several of which are highly conserved between yeast, Drosophila, and humans. We have reconstituted an ATP-dependent chromatin remodeling complex using a subset of conserved subunits. Unexpectedly, both BRG1 and hBRM, the ATPase subunits of human SWI/SNF complexes, are capable of remodeling mono-nucleosomes and nucleosomal arrays as purified proteins. The addition of INI1, BAF155, and BAF170 to BRG1 increases remodeling activity to a level comparable to that of the whole hSWI/SNF complex. These data define the functional core of the hSWI/SNF complex.     

BAF57 governs androgen receptor action and androgen-dependent proliferation through SWI/SNF

Androgen receptor (AR) activity is required for prostate cancer development and progression. Thus, there is a major impetus to understand the regulation of AR action. We and others have previously shown that AR transactivation potential is dependent on the presence of an active SWI/SNF chromatin remodeling complex. However, the mechanisms underlying SWI/SNF regulation of the AR remained unsolved. We show here that the BAF57 subunit, an accessory component of the remodeling complex, is a critical regulator of AR function. We show that BAF57 is expressed in the luminal epithelia of the prostate and is required for AR-dependent transactivation in prostatic adenocarcinoma cells. Our data reveal that BAF57 can directly bind to the AR and is recruited to endogenous AR targets upon ligand activation. Loss of BAF57 or inhibition of BAF57 function severely compromised AR activity, as observed with both exogenous and endogenous AR targets. Rescue of BAF57 function restored AR activity, thus demonstrating a specific requirement of BAF57 for AR activity. This action of BAF57 proved to be dependent on SWI/SNF ATPase function. BAF57 has previously been implicated in nuclear receptor coactivator function, and we show that, although BAF57 facilitated coactivator activity, only a selected subset required BAF57 for coactivator function. Lastly, we demonstrate that both BAF57 and BRM are required for the proliferation of AR-dependent prostatic adenocarcinoma cells. In summary, these findings identify BAF57 as a critical modulator of the AR that is capable of altering AR activity, coactivator function, and AR-dependent proliferation.     

Differential expression of key subunits of SWI/SNF chromatin remodeling complexes in porcine embryos derived in vitro or in vivo

In vitro embryo production is an established method for both humans and animals, but is fraught with inferior development and health issues in offspring born after in vitro fertilization procedures. Analysis of epigenetic changes caused by exposure to in vitro conditions should shed light on potential sources of these phenotypes. Using immunocytochemistry, we investigated the localization and relative abundance of components associated with the SWI/SNF (Switch/Sucrose non‐fermentable) chromatin‐remodeling complex—including BAF155, BAF170, BAF180, BAF53A, BAF57, BAF60A, BAF45D, ARID1A, ARID1B, ARID2, SNF5, and BRD7—in oocytes and in in vitro‐produced and in vivo‐derived porcine embryos. Differences in the localization of BAF155, BAF170, BAF60A, and ARID1B among these sources indicate that improper timing of chromatin remodeling and cellular differentiation might occur in early preimplantation embryos produced and cultured in vitro.     

BAF155 methylation drives metastasis by hijacking super-enhancers and subverting anti-tumor immunity

Subunits of the chromatin remodeler SWI/SNF are the most frequently disrupted genes in cancer. However, how post-translational modifications (PTM) of SWI/SNF subunits elicit epigenetic dysfunction remains unknown. Arginine-methylation of BAF155 by coactivator-associated arginine methyltransferase 1 (CARM1) promotes triple-negative breast cancer (TNBC) metastasis. Herein, we discovered the dual roles of methylated-BAF155 (me-BAF155) in promoting tumor metastasis: activation of super-enhancer-addicted oncogenes by recruiting BRD4, and repression of interferon α/γ pathway genes to suppress host immune response. Pharmacological inhibition of CARM1 and BAF155 methylation not only abrogated the expression of an array of oncogenes, but also boosted host immune responses by enhancing the activity and tumor infiltration of cytotoxic T cells. Moreover, strong me-BAF155 staining was detected in circulating tumor cells from metastatic cancer patients. Despite low cytotoxicity, CARM1 inhibitors strongly inhibited TNBC cell migration in vitro, and growth and metastasis in vivo. These findings illustrate a unique mechanism of arginine methylation of a SWI/SNF subunit that drives epigenetic dysregulation, and establishes me-BAF155 as a therapeutic target to enhance immunotherapy efficacy.     

The chromatin remodeling factor SMARCB1 forms a complex with human cytomegalovirus proteins UL114 and UL44

Background

Human cytomegalovirus (HCMV) uracil DNA glycosylase, UL114, is required for efficient viral DNA replication. Presumably, UL114 functions as a structural partner to other factors of the DNA-replication machinery and not as a DNA repair protein. UL114 binds UL44 (HCMV processivity factor) and UL54 (HCMV-DNA-polymerase). In the present study we have searched for cellular partners of UL114.

Methodology/Principal Findings

In a yeast two-hybrid screen SMARCB1, a factor of the SWI/SNF chromatin remodeling complex, was found to be an interacting partner of UL114. This interaction was confirmed in vitro by co-immunoprecipitation and pull-down. Immunofluorescence microscopy revealed that SMARCB1 along with BRG-1, BAF170 and BAF155, which are the core SWI/SNF components required for efficient chromatin remodeling, were present in virus replication foci 24–48 hours post infection (hpi). Furthermore a direct interaction was also demonstrated for SMARCB1 and UL44.

Conclusions/Significance

The core SWI/SNF factors required for efficient chromatin remodeling are present in the HCMV replication foci throughout infection. The proteins UL44 and UL114 interact with SMARCB1 and may participate in the recruitment of the SWI/SNF complex to the chromatinized virus DNA. Thus, the presence of the SWI/SNF chromatin remodeling complex in replication foci and its association with UL114 and with UL44 might imply its involvement in different DNA transactions.     

A SWI/SNF-like factor from chicken liver that disrupts nucleosomes and transfers histone octamers in cis and trans

ATP-dependent chromatin remodeling factors have been implicated in nuclear processes involving DNA. Here we report partial purification and characterization of an ATP-dependent chromatin remodeling activity from chicken liver. Nuclear extract from chicken liver was fractionated chromatographically to enrich proteins immunoreacting to antibodies against components of human SWI/SNF, namely BRG1, BAF170, BAF155, and BAF57. Immunoreactivity to these antibodies elutes with a mass of about 2MDa on Sepharose CL-6B gel filtration, suggesting that they constitute a SWI/SNF-like complex (SLC). The SLC displays three chromatin-remodeling activities, viz. nucleosome disruption, octamer transfer, and nucleosome sliding (octamer transfer in cis). We further show that components of SLC, as revealed by immunoreactivity to the above antibodies, display a dynamic nucleocytoplasmic distribution and colocalize with RNA polymerase II in the liver nuclei. This report contributes to the understanding of phylogenetic generality of chromatin remodeling factors in eukaryotes.