Differential requirements for different subfamilies of the mammalian SWI/SNF chromatin remodeling enzymes in myoblast cell cycle progression and expression of the Pax7 regulator

Abstract

The mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) families of ATP-dependent chromatin remodeling enzymes are established co-regulators of gene expression. mSWI/SNF complexes can be assembled into three major subfamilies: BAF (BRG1 or BRM-Associated Factor), PBAF (Polybromo containing BAF), or ncBAF (non-canonical BAF) that are distinguished by the presence of mutually exclusive subunits. The mechanisms by which each subfamily contributes to the establishment or function of specific cell lineages are poorly understood. Here, we determined the contributions of the BAF, ncBAF, and PBAF complexes to myoblast proliferation via knock down (KD) of distinguishing subunits from each complex. KD of subunits unique to the BAF or the ncBAF complexes reduced myoblast proliferation rate, while KD of PBAF-specific subunits did not affect proliferation. RNA-seq from proliferating KD myoblasts targeting Baf250A (BAF complex), Brd9 (ncBAF complex), or Baf180 (PBAF complex) showed mis-regulation of a limited number of genes. KD of Baf250A specifically reduced the expression of Pax7, which is required for myoblast proliferation, concomitant with decreased binding of Baf250A to and impaired chromatin remodeling at the Pax7 gene promoter. Although Brd9 also bound to the Pax7 promoter, suggesting occupancy by the ncBAF complex, no changes were detected in Pax7 gene expression, Pax7 protein expression or chromatin remodeling at the Pax7 promoter upon Brd9 KD. The data indicate that the BAF subfamily of the mSWI/SNF enzymes is specifically required for myoblast proliferation via regulation of Pax7 expression.

Keywords: Baf250A; Myoblasts; Pax7; Proliferation; SWI/SNF.

Figure 1.. Baf250A, Brd9 and Baf180 expression in proliferating wild type (WT), shRNA scrambled sequence (scr) controls, and shRNA knockdown C2C12 myoblasts.

Representative immunoblot (top) and quantification (bottom) of Baf250A (A), Brd9 (B), and Baf180 (C) levels in proliferating cells after 48 h of proliferation. For all samples, data are the mean ± SE of three independent biological replicates. Immunoblots against GAPDH were used as loading controls. Samples were compared to the corresponding wild type sample. ***P < 0.001

Figure 2.. Knockdown of Baf250A impairs myoblast proliferation while Brd9 knockdown has a minor effect.

Cell counting assay of proliferating wild type (WT) myoblasts, myoblasts transduced with scrambled shRNA (shRNA scr), Baf250A (A), Brd9 (B) or Baf180 (C) shRNAs. (D) Trypan blue staining showed no changes in viability upon knockdown of Baf250A, Brd9 or Baf180. (E) Representative immunoblot of pro-caspase 3 and activated Caspase 3 in control and knockdown proliferating myoblasts after 2 days of proliferation. Vinculin was used as loading control and treatment with 1 μM staurosporine for 24 h was used as a positive control for apoptosis induction. (F) Quantification of activated caspase 3 protein expression. All quantified data are the mean ± SE for three independent experiments. *****p < 0.00001.

Figure 3.. Knockdown of Baf250A or Brd9 alters progression of cell cycle.

Representative histograms of cell cycle progression (left panels) and percentage of cells in each cell cycle phase (right panel) for asynchronous myoblasts (A), cells arrested in mitosis with nocodazole at the time of release (B), and after 16 h (C) and 20 h post-release (D). The data are representative of 4 independent biological experiments, *****P < 0.00001. (E) Representative immunoblot of pro-caspase 3 and activated caspase 3 in control and Baf250A or Brd9 KD cells knockdown 20 h after nocodoazole release. Vinculin was used as loading control. Treatment with 1 μM staurosporine for 24 h was used as a positive control for apoptosis induction. (F) Quantification of activated caspase 3 protein expression. Bar graphs show the mean ± SE for three independent experiments, * P < 0.05; ***** P < 0.00001.

Figure 4.. Baf250A, Brd9, and Baf180 KD myoblasts re-enter the cell cycle with equivalent kinetics following a cycloheximide-induced G1 block.

Representative histograms of cell cycle progression (left panels) and the percentage of cells in each cell cycle phase (right panel) for myoblasts arrested in G1/S with cycloheximide at the time of release (A), and after 8 (B), 16 (C), 20 (D), and 30 h post-release (E). Bar graphs show the mean ± SE for three independent biological experiments.

Figure 5.. Changes in gene expression dependent on Baf250A or Brd9 knockdown.

Volcano plots displaying differentially expressed genes between scr control and Baf250A knockdown (A) or Brd9 knockdown (C) C2C12 cells. The y-axis corresponds to the mean log10 expression levels (P values). The red and blue dots represent the up- and down-regulated transcripts in Baf250A knockdown (false-discovery rate [FDR] of <0.05), respectively. The gray dots represent the expression levels of transcripts that did not reach statistical significance (FDR of >0.05). (B,D) GO term analysis of differentially expressed, up-regulated genes induced by the KD of Baf250A (B) or of Brd9 (D) in proliferating myoblasts. Cut-off was set at 2.0 of the - log(adjusted P value). No significant enrichment of downregulated GO categories were identified under these conditions. See Supp. Tables 4 and 5 for the complete list of genes.

Figure 6.. Baf250A knockdown affects Pax7 expression.

(A) Steady state mRNA levels of Pax7 determined by qRT-PCR from proliferating C2C12 myoblasts. (B) Representative western blots showing Pax7 expression in Baf250A and Brd9 knockdown proliferating myoblasts. (C) Representative light micrographs of proliferating untreated C2C12 myoblasts (Wild type) and proliferating cells transduced with either scr, Baf250A, Brd9 or Baf180 shRNAs 48 h prior and immunostained for Pax7. Data are the mean ± SE for three independent experiments. ** P < 0.01, *** P < 0.001, *****P < 0.00001.

Fig 7.. Baf250A and Brd9 bind to the Pax7 promoter, but only Baf250A contributes to chromatin remodeling at the Pax7 promoter.

ChIP-qPCR showing binding of Baf250A (A) and Brd9 (B) binding to the Pax7 promoter. (C) The IgG control for the Pax7 promoter. (D-F) Amplification of the IgH enhancer as negative sequence control for Baf250A, Brd9, and IgG pulldowns. (G) Left panel: Schematic representation of the location of PvuII restriction enzyme sites used for restriction enzyme accessibility assays (REAA) relative to the Pax7 mRNA start site. Right panel: The accessibility of PvuII sites near the Pax7 promoter was measured by REAA relative to the accessibility of the the PvuII site proximal to the Pax7 mRNA start site in wildtype (WT) cells, which was normalized to 1. The data represent 3 independent biological experiments. *P ≤ 0.05, **P < 0.01, ***P < 0.001.

Figure 8.. Reduced proliferation in Baf250A KD cells is rescued by re-introduction of Pax7.

(A) Relative Pax7 mRNA levels determined by qRT-PCR from the indicated proliferating myoblasts. Values are relative to the amount in the wild-type (WT) control cells, which were set at 1. (B) Representative western blots showing Pax7 protein levels in the indicated proliferating myoblasts. (C) Representative light micrographs of the indicated proliferating myoblasts stained for Pax7. (D) Cell counting assay results for the indicated myoblasts. Data are the mean ± SE for three independent experiments. * P, 0.05, ** P < 0.01, *** P < 0.001, *****P < 0.00001. NT, non-transduced, EV, empty vector, Dox, doxycycline.