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