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

<div><p>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.</p></div

Downregulation of BAF47 alters muscle terminal differentiation and cell cycle exit.

<p><b>A.</b> BAF47 and BRG1 occupancy at <i>cyclin D1</i> promoter. ChIP-qPCR analyses of BAF47 and BRG1 in myoblast in proliferating C2C12 cells and at 24 h of differentiation. The immunoprecipitated material was quantified by qPCR, and results are expressed as fold enrichment of the % of Input of BAF47 or BRG1 ChIP over % of Input of the IgG average. Data are represented as mean ±SEM, n = 3. <b>B.</b> Scheme of the timing for samples collection. C2C12 myoblasts were transfected with control, BRG1, BAF53a or BAF47 siRNAs and samples prepared either 24 h after transfection (0) or after 72 h in DM (72) or 10 h after the switch back to GM (72+10 h GM). <b>C.</b> Total protein extracts were analyzed by WB with the indicated antibodies. <b>D.</b> Quantification of cyD1 levels from 3 to 8 independent WB. Data are expressed compared to control 0 h. Error bars represent SEM. For each time point, statistics were calculated compare to the same time point from control. Only significative p-values (Student T-test, two tailed, unpaired) are indicated ** = <0.01. <b>E</b>. Total RNA was isolated, reverse transcribed using random primers and used as templates for PCR amplification with cyclin D1-specific primers and normalized to cyclophilin A specific primers. Data are expressed compared to control 0 h.</p

BRG1 and BAF47 interact with SWI/SNF and N-CoR-1 complex components in a differentiation-dependent manner.

<p><b>A.</b> Nuclear extracts from proliferating (GM) or differentiating C2C12 myoblasts (24 h DM) were used for immunoprecipitation (IP) with antibodies against BRG1 or BAF47, or with normal rabbit IgG as a negative control. The resulting precipitates were analyzed by WB with the indicated antibodies. Nuclear extracts (Inputs, 1% of input extracts) were loaded to assess endogenous protein levels. *: non-specific IgG band. <b>B.</b> Nuclear extracts from proliferating (GM) or differentiating C2C12 myoblasts (24 h DM) were fractionated on glycerol gradient ranging from 11% (fraction 1) to 33% (fraction 11); (-) empty lane. Fractions were collected and analyzed by WB using the indicated antibodies. Nuclear extracts (Inputs, 2.5% of input extracts) were loaded to assess endogenous protein levels.</p

Effect of SWI/SNF subunits BAF47, BAF53a and BRG1 knockdown on the irreversible cell cycle exit.

<p>C2C12 myoblasts were transfected with control, BRG1, BAF53a or BAF47 siRNAs and cells were labeled with BrdU either after 96 h in DM or 12 h after the switch back to GM (96+12 h GM). We show one representative of 3 independent experiments. Left panel: BrdU indexes ratio of BrdU positive nuclei/DAPI labeled nuclei) are shown in the histogram, error bars represent SEM from 3 quantifications of the presented experiment (*p<10⁻⁵ according to Student test (two tailed, unpaired)). Right panel: A: DAPI staining (blue) (x20) and B: BrdU immunostaining (green) (x20).</p

SWI/SNF complex subunits are differently involved in skeletal muscle terminal differentiation.

<p><b>A.</b> C2C12 myoblasts were transfected with control, BRG1, BAF53a or BAF47 siRNAs. Immunofluorescence analyses using anti-MCK antibody were performed after 72h in DM. Cells were DAPI-stained prior to fluorescent microscopy analyses (magnification x20). <b>B and C.</b> C2C12 myoblasts were transfected with control, BRG1, BAF53a or BAF47 siRNAs. 24 h post-transfection (0) cells were placed in DM and harvested after 24 h, 48 h or 72 h. <b>B.</b> Total protein extracts were analyzed by WB with the indicated antibodies. *: non-specific bands. For each time point, % of protein siRNA-induced downregulation compared to the same time from control scrambled siRNA is indicated at the bottom of the western blot (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108858#pone.0108858.s002" target="_blank">Figure S2</a> for additional WB analyses). <b>C.</b> Quantification of MCK levels from 3 to 8 independent western blot. Error bars represent standard error of the mean (SEM). For each time point, statistics were calculated compared to the same time point from control. Significative p-values (Student T-test, two tailed, unpaired) are indicated * = <0.05; ** = <0.01.</p