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• W2149282942 abstract "Article1 July 2015free access Brahma is required for cell cycle arrest and late muscle gene expression during skeletal myogenesis Sonia Albini Sonia Albini Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Paula Coutinho Toto Paula Coutinho Toto Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Alessandra Dall'Agnese Alessandra Dall'Agnese Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Barbora Malecova Barbora Malecova Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Carlo Cenciarelli Carlo Cenciarelli CNR-Istituto di Farmacologia Traslazionale, Rome, Italy Search for more papers by this author Armando Felsani Armando Felsani CNR-Istituto di Biologia Cellulare e Neurobiologia, Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Maurizia Caruso Maurizia Caruso CNR-Istituto di Biologia Cellulare e Neurobiologia, Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Scott J Bultman Scott J Bultman Department of Genetics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Pier Lorenzo Puri Corresponding Author Pier Lorenzo Puri Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA IRCCS Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Sonia Albini Sonia Albini Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Paula Coutinho Toto Paula Coutinho Toto Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Alessandra Dall'Agnese Alessandra Dall'Agnese Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Barbora Malecova Barbora Malecova Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Carlo Cenciarelli Carlo Cenciarelli CNR-Istituto di Farmacologia Traslazionale, Rome, Italy Search for more papers by this author Armando Felsani Armando Felsani CNR-Istituto di Biologia Cellulare e Neurobiologia, Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Maurizia Caruso Maurizia Caruso CNR-Istituto di Biologia Cellulare e Neurobiologia, Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Scott J Bultman Scott J Bultman Department of Genetics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Pier Lorenzo Puri Corresponding Author Pier Lorenzo Puri Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA IRCCS Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Author Information Sonia Albini1,‡, Paula Coutinho Toto1,‡, Alessandra Dall'Agnese1, Barbora Malecova1, Carlo Cenciarelli2, Armando Felsani3, Maurizia Caruso3, Scott J Bultman4 and Pier Lorenzo Puri 1,5 1Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA 2CNR-Istituto di Farmacologia Traslazionale, Rome, Italy 3CNR-Istituto di Biologia Cellulare e Neurobiologia, Fondazione Santa Lucia, Rome, Italy 4Department of Genetics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA 5IRCCS Fondazione Santa Lucia, Rome, Italy ‡These authors contributed equally to this work *Corresponding author. Tel: +1 858 646 3161; E-mail: [email protected] EMBO Reports (2015)16:1037-1050https://doi.org/10.15252/embr.201540159 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Although the two catalytic subunits of the SWI/SNF chromatin-remodeling complex—Brahma (Brm) and Brg1—are almost invariably co-expressed, their mutually exclusive incorporation into distinct SWI/SNF complexes predicts that Brg1- and Brm-based SWI/SNF complexes execute specific functions. Here, we show that Brg1 and Brm have distinct functions at discrete stages of muscle differentiation. While Brg1 is required for the activation of muscle gene transcription at early stages of differentiation, Brm is required for Ccnd1 repression and cell cycle arrest prior to the activation of muscle genes. Ccnd1 knockdown rescues the ability to exit the cell cycle in Brm-deficient myoblasts, but does not recover terminal differentiation, revealing a previously unrecognized role of Brm in the activation of late muscle gene expression independent from the control of cell cycle. Consistently, Brm null mice displayed impaired muscle regeneration after injury, with aberrant proliferation of satellite cells and delayed formation of new myofibers. These data reveal stage-specific roles of Brm during skeletal myogenesis, via formation of repressive and activatory SWI/SNF complexes. Synopsis The two catalytic subunits of the SWI/SNF chromatin-remodeling complex have distinct functions during skeletal muscle differentiation. Brm promotes cell cycle arrest by repressing cyclin D1, controls satellite cell proliferation and activates late muscle genes, while Brg1 activates muscle genes at early stages of myogenesis. Brm and Brg1 regulate distinct stages of muscle differentiation during skeletal myogenesis. Brm-mediated repression of cyclin D1 is required for myoblast cell cycle arrest prior to differentiation. Brm is required for activation of late muscle differentiation and proper muscle regeneration. Introduction Developmental and adult skeletal myogenesis are activated by the basic helix-loop-helix (bHLH) family of myogenic regulatory factors (MRFs), MyoD, Myf5, MRF4, and myogenin, which share the ability to promote transcription from E-box sequences (CANNTG) found in the regulatory region of many muscle-specific genes 123. MRF competence to promote transcription of muscle genes relies on the interaction with the SWI/SNF chromatin-remodeling complex 45. SWI/SNF complex appears to mediate the unique ability of MyoD and Myf5 to remodel the chromatin and activate transcription at previously silent muscle loci 67, but is also required for the maintenance of muscle gene expression at later stages of skeletal myogenesis 8. SWI/SNF complexes are composed of two mutually exclusive enzymatic subunits (the ATPases Brahma (Brm) and Brm-related gene 1 (Brg1)) and a number of structural subunits, collectively referred to as Brg1/Brm-associated factors (BAFs) 910. Because of the variable, cell type-specific assembly of distinct subunits and their alternative variants, SWI/SNF complexes are heterogeneous in their composition and function 111213. This structural and functional heterogeneity suggests that distinct SWI/SNF complexes might simultaneously exist in the same cell type to perform specialized functions depending on the cell context and differentiation stage. One major determinant of SWI/SNF variability is conferred by the mutually exclusive incorporation of the catalytic ATPase, Brg1 and Brm. Several studies have shown the importance of Brg1- and Brm-based complexes in the control of gene expression, cell proliferation, differentiation and transformation 1415. Both Brg1 and Brm are also known to interact with and stimulate the activity of several transcription factors, including the glucocorticoid receptor and C/EBP 1617. Other studies have suggested a functional redundancy between Brm and Brg1 18192021. Collectively, the information reported above seems in apparent conflict with the mutually exclusive presence of Brg1 and Brm in individual SWI/SNF complex and prompted an interest toward elucidating specific functions of these two proteins in various cellular processes. Previous attempts to address the individual role of Brg1 and Brm during skeletal myogenesis, by using dominant negative mutants, revealed an essential role for both proteins in the activation of the myogenic program 62223. However, the conclusions from these studies were limited by the use of an experimental system in which the myogenic program was activated in fibroblasts by tetracycline-inducible MyoD, and Brg1 and Brm were functionally inactivated by enzymatically defective dominant negative mutants 22 or neutralizing antibodies 23. Moreover, while these seminal studies clearly indicated the importance of both Brg1 and Brm in the activation of the myogenic program, the individual role of each subunit in the control of discrete transcriptional networks could not be established. More recently, Imbalzano's laboratory has exploited RNAi to knock down the levels of Brg1, showing that Brg1 controls muscle genes and muscle-specific microRNAs (myomiRs) expression in skeletal muscle cells 24. Likewise, we have shown by RNAi-mediated knockdown the essential role of Brg1/BAF60C-based SWI/SNF complex in the activation of the myogenic program in C2C12 muscle cells 25. Still, the specific function of Brm in skeletal myogenesis remains obscure to date. Gene knockout studies in mice have demonstrated that inactivating mutations in Brg1 are embryonic lethal, whereas Brm-inactivated mice are viable and fertile, suggesting that Brg1 may functionally replace Brm within the SWI/SNF complexes during development 262728. However, Brm−/− mice show increased body weight and alteration of cellular growth control 2628, indicating the requirement of Brm in the control of tissue growth, differentiation, and homeostasis. Despite the high degree of homology between the two subunits and their partial overlapping role, different expression profiles were reported by Muchardt et al 29 showing that Brg1 is expressed constitutively, whereas Brm levels fluctuate with increased expression in G0-arrested cells and in cells induced to differentiate; furthermore, the expression of Brm, but not Brg1, was negatively regulated upon mitogenic stimulation as well as in ras-transformed cells 29. Moreover, Brm and Brg1 appear to direct distinct cellular pathways, by recruitment to specific promoters through preferential interaction with certain classes of transcription factors. Brg1 binds to zinc finger protein through a unique N-terminal domain not present in Brm, while Brm interacts with two ankyrin-repeats proteins that are crucial in the Notch signal transduction 30. More recently, studies have investigated the individual roles of Brg1 and Brm in various cellular processes, revealing again individual, cooperating, and redundant activities of these two proteins depending on the cell type and the specific context 18192021. In the present study, we have used an integrated genome wide analysis of gene expression and gene knockdown with in vitro and in vivo studies to systematically address the role of Brg1 and Brm during skeletal myogenesis. Results Differential expression profiles and function of Brg1 and Brm during C2C12 skeletal muscle differentiation We compared the expression levels of Brg1 and Brm in C2C12 myoblasts during proliferation (growth medium, GM) and differentiation into myotubes (differentiation medium, DM). This transition is well illustrated by the relative expression levels of cyclin D1 (detected in proliferating myoblasts and downregulated during differentiation) and myosin heavy chain (MyHC), which is specifically induced during C2C12 differentiation (Fig 1). While the same levels of expression of Brg1 protein were detected in proliferating myoblasts and during the whole differentiation process, Brm protein and RNA levels were progressively upregulated during C2C12 differentiation (Fig 1A and C). Consistently, immunofluorescence analysis revealed nuclear expression of Brm detectable in few undifferentiated myoblasts, while a higher signal was detected in all the nuclei of MyHC-expressing myotubes (Fig 1B). By contrast, Brg1 showed a uniform nuclear expression in both undifferentiated myoblasts and differentiated myotubes (Fig 1B). These data indicate that Brg1 and Brm are differentially regulated during skeletal muscle differentiation. Figure 1. Brm and Brg1 show specific profiles of expression and activities during skeletal muscle differentiation Time course of protein expression during terminal differentiation of C2C12 myoblasts representative of three independent experiments. Myoblasts were cultured in growth medium (GM) until they reached confluence, and then shifted to differentiate in differentiation medium (DM) for 48 h. Cellular extracts were analyzed by Western blot with antibodies against BRG1, Brm, myosin heavy chain (MyHC), and cyclin D1. Cdk4 probing was used to check for equal loading of the samples. Immunofluorescence analysis of Brm and Brg1 expression in C2C12 cells cultured in GM or DM conditions. Scale bar, 50 μm. Efficiency of BRM and BRG1 knockdown at 48 h post-transfection performed in C2C12 cells using siRNAs (control interference is a scrambled sequence and referred as siScr) was monitored by qRT–PCR. Data are presented as average ± SEM (n > 3). Immunofluorescence for Brm or Brg1 performed in proliferating myoblasts upon siRNA against Brg1 (siBrg1), Brm (siBrm) or scrambled (siScr) to check for efficient depletion of the proteins. Scale bar, 50 μm. Brightfield images and MyHC staining were performed at various time points of differentiation in C2C12 cells in which siRNAs were delivered in GM as depicted in the scheme above. Scale bar, 50 μm. Quantification of fusion index of three independent experiments calculated as percentage of nuclei within MyHC-expressing myotubes. Data are presented as average ± SEM (n > 3). *P < 0.05; ***P < 0.001 (unpaired Student's t-test). Download figure Download PowerPoint To gain further insight into the specific role of Brg1 and Brm at discrete stages of skeletal myogenesis, we individually downregulated their expression by small interfering RNA (siRNA)-mediated knockdown in undifferentiated myoblasts, followed by a phenotypic analysis of the derived populations of myoblasts. Knockdown of each protein resulted in a uniform and persistent depletion of Brg1 or Brm in C2C12 myoblasts, with at least 70% reduction in both transcripts and protein levels after 48 h of DM, as compared to scramble (siScr) controls (Fig 1C and D; see also Fig 3C). Interestingly, two distinct phenotypes were observed in Brm- or Brg1-downregulated muscle cells, as compared to the control cells. Both phase contrast and immunofluorescence images (Fig 1E) documented that while Brg1-depleted cultures showed a complete absence of myotubes, Brm-depleted cells displayed a severe impairment in the formation of myotubes, which appeared reduced in number and size, with a lower fusion index as compared to control (siScr) cells (Fig 1E and F). During these experiments, we consistently observed a higher number of myoblasts in siBrm-treated myoblasts following induction of differentiation, as compared to siBrg1 and control samples, suggesting an increased proliferative activity possibly derived from an impaired cell cycle arrest that typically precedes the activation of the differentiation program upon mitogen withdrawal. Indeed, EdU incorporation experiments revealed that the large majority (~80%) of siBrm myoblasts continued to proliferate after 48 h, as compared to control samples and siBrg1 myoblasts (Fig 2A and B, top panel). The effect of Brm on cell proliferation was further monitored by manual cell counting at several time points after differentiation (Fig 2B, middle panel) and by FACS-assisted count of EdU-positive cells (Fig 2B, bottom panel). All these analyses demonstrated that siBrm C2C12 cells retained proliferative activity in DM, while siScr and siBrg1 C2C12 cells ceased dividing (Fig 2B). Of note, a small fraction of siBrm myoblasts could differentiate, but failed to form multinucleated myotubes with the size that is typically observed in control cells (Fig 2A). This evidence indicates the presence of two populations in siBrm myoblasts exposed to differentiation conditions: one large population that escaped the differentiation-induced G0/G1 cell cycle arrest and continued to proliferate instead of differentiating, and another, smaller population, which could exit the cell cycle, but failed to complete the differentiation process. While Brm was not detectable by immunofluorescence in the sporadic siBrm MyHC-positive cells (data not shown), it remains formally possible that the latter population derives from cells in which Brm was not efficiently depleted. Alternatively, these cells might have initiated the differentiation program prior to the downregulation of Brm or a redundant, Brm-independent, cell cycle arrest can be activated in a small fraction of cultured myoblasts. By contrast, siBrg1 myoblasts uniformly failed to differentiate, despite their ability to withdraw from cell cycle arrest in response to differentiation conditions, as no EdU-positive cells were detected when cells were incubated in DM (Fig 2A and B). Collectively, these data indicate that Brg1 and Brm perform essential functions during skeletal myogenesis, likely through separable mechanisms. Figure 2. Downregulation of Brm or Brg1 leads to specific alterations of cell cycle and differentiation of C2C12 myoblasts A, B. C2C12 were depleted for Brm (siBrm), Brg1 (siBrg1), or a scrambled (siScr) sequence by small interfering RNA (siRNA) during proliferation (GM), and samples were analyzed at different time points during differentiation (DM 18 h and DM 48 h). Double EdU/MyHC staining was performed after incubation of EdU 12 h before fixing the cells (A). Scale bar, 50 μm. Percentage of EdU-positive cells was calculated counting 10 fields of EdU-positive cells (B, top graph). Proliferation analysis was performed by counting the number/field of siRNA-treated C2C12 at the time point indicated (B, middle graph) and by flow cytometry by BrdU incorporation (B, bottom graph) as percentage of BrdU+ cells. Data are presented as average ± SEM (n = 3). C. Heat map showing the expression profiles of transcripts in siRNA-treated C2C12 collected at 18 h and 48 h of differentiation. D. Venn diagram showing overlap between genes downregulated in C2C12 depleted for Brm and Brg1 at early (18 h) and late (48 h) differentiation time points. The percentage of skeletal muscle genes annotated in each category is indicated. Download figure Download PowerPoint Figure 3. Brm controls muscle differentiation-associated cell cycle arrest by repressing cyclin D1 expression A, B. Immunofluorescence analysis of cyclin D1 expression in C2C12 cells depleted for Brm (siBrm) or Brg1 (siBrg1) in DM 48 h (A) and relative quantification reporting the percentage (%) of cyclin D1-positive cells (B). Scale bar, 50 μm. Data are presented as average ± SEM (n > 3). P-value was calculated using unpaired Student's t-test, ***P < 0.001. Experiments were performed at least three times. C. Western blot analysis performed in siBrm, siBrg1, and siScr C2C12 cells cultured in GM or DM, using antibodies against Brm, Brg1, cyclin D1, myogenin, and Actn3. α-actin was used as a loading control. Experiments were performed at least three times. D. Recruitment of Brg1 and Brm and analysis of H3K27me3 on a promoter sequence of the Ccnd1 gene in GM and DM. Arrows indicate the regions amplified by the primers used. Protein recruitment is expressed as relative enrichment of each factor compared to IgG after normalization for total input control (n = 3, error bars represent SEM). P-value was calculated using unpaired Student's t-test, *P < 0.05; ***P < 0.001. Download figure Download PowerPoint Brg1 and Brm regulate distinct and overlapping clusters of genes during C2C12 myoblast differentiation To further elucidate the individual roles of Brm and Brg1 during skeletal myogenesis, we performed a gene expression microarray in siBrg1 and siBrm myoblasts (with siScr as control) at two sequential stages of differentiation—18 and 48 h after incubation in DM—that were selected to reveal the relative impact of Brg1 or Brm on gene expression at early and late stages of muscle differentiation, respectively. The complete list of modulated genes is available and can be accessed through GEO Series accession number GSE44993. (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE44993). A list of the up- and downregulated genes is also shown in Table EV1. Interestingly, the gene expression profiles of C2C12 cells depleted of Brm or Brg1 showed distinct and overlapping clusters of up- and downregulated genes (Fig 2C and D). The largest fraction of downregulated genes was observed in siBrg1 C2C12 cells (Fig 2D) and was enriched in genes implicated in skeletal muscle differentiation, and other general aspect of cellular differentiation, such as tissue morphology and development, cell signaling, cell cycle, and cell death (Fig EV1). This is consistent with an essential role of Brg1 in the activation of early genes that promote skeletal muscle differentiation such as myogenin, as predicted by the phenotype of siBrg1 myoblasts (Figs 1 and 3C) and previous studies 82225. Conversely, downregulated genes in siBrm1 myoblasts showed only a modest enrichment in a subset of skeletal muscle late genes, which were also found downregulated in siBrg1 myoblasts (overlapping cluster of genes), indicating a possible cooperation of Brg1 and Brm in the activation of a cluster of common downstream muscle differentiation genes (Figs 2D, EV1 and EV2). Among the upregulated genes, we noted enrichment in genes belonging to the cell cycle and proliferation networks in both siBrg1 and siBrm myoblasts (Fig EV1). These genes are likely to be repressed, either directly or indirectly, through a Brg1- and/or Brm-mediated mechanism. Interestingly, at early differentiation stages (DM 18 h), the timing when myoblasts exit the cell cycle prior to differentiating, we observed upregulation of cell cycle-related genes (Figf, Vegfc, Ccng, Ccnd1) only in siBrm myoblasts (Figs 2D and EV1). Among these genes, we annotated one key activator of cell cycle progression—the cyclin D1 gene Ccnd1—that was specifically upregulated in siBrm myoblasts. At 48 h of DM, cyclin D1 continued to be upregulated in siBrm C2C12 cells, although it was also annotated among the upregulated genes in siBrg1 C2C12 cells (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE44993). By using qRT–PCR, we confirmed the presence of elevated levels of cyclin D1 transcripts in siBrm C2C12 cells, during proliferation (GM) and differentiation conditions (Fig EV2). The elevated mRNA level of cyclin D1 was also confirmed in Brm−/− MEFs compared to WT MEFs during a myogenic conversion assay at several time points of differentiation (Fig EV4E). By contrast, qRT–PCR analysis confirmed that the early muscle differentiation gene myogenin was downregulated only in siBrg1 C2C12 cells, while the expression of four commonly downregulated genes (EzH1, Actn3, MEF2c, and Actc1) was reduced in both siBrg1 and siBrm cells (Fig EV2). Click here to expand this figure. Figure EV1. Gene ontologyGene ontology analysis of C2C12 that were interfered for Brm (siBrm), Brg1 (siBrg1), or a scrambled sequence (siScr) by small interfering RNA (siRNA) analyzed during proliferation (GM) and at different time points during differentiation (DM 18 h and DM 48 h). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Gene expression analysis of differentially expressed genes for microarray validationList of selected genes differentially expressed in differentiating siBrm and siBrg1 C2C12 cells as compared to siScr C2C12 cells and relative expression profile monitored by qPCR. Data are presented as average ± SEM of three independent experiments, and P-value was calculated using the unpaired Student's t-test (*P < 0.05; **P < 0.01). Download figure Download PowerPoint Brm controls muscle differentiation-associated cell cycle arrest by repressing Ccnd1 expression Previous works established a critical, unique role of cyclin D1 in the regulation of myoblasts proliferation and inhibition of differentiation 3132333435, indicating that Ccnd1 repression is important for cell cycle exit and activation of the myogenic program at early stages of myoblast differentiation. Given the proliferative phenotype observed only in siBrm myoblasts, we decided to focus on Ccnd1, as a potential Brm-repressed gene that mediates the proliferative phenotype of siBrm myoblasts. We evaluated the expression of cyclin D1 in siBrm and siBrg1 C2C12, as compared to siScr C2C12 cells. Immunofluorescence and Western blot analysis showed a large proportion of siBrm myoblasts continue to express cyclin D1, as a reflection of their failure to withdraw from the cell cycle (Fig 3A–C). By contrast, cyclin D1 was downregulated in siBrg1 cells placed in DM (Fig 3A–C). We further investigated the specific role of Brm vs. Brg1 in the repression of Ccnd1 transcription by using chromatin immunoprecipitation (ChIP) experiments. This analysis demonstrated that Brm, and not Brg1, bound the regulatory elements of Ccnd1 with an increased chromatin binding along with myoblast differentiation observed at −591 bp from the transcription start sites that coincided with an accumulation of the repressive histone mark H3K27 tri-methylation (H3K27me3) (Fig 3D), which has been previously detected by ChIP-seq studies 36. This evidence indicates that Brm directly mediates Ccnd1 repression at the early onset of muscle differentiation. To establish a causal relationship between Ccnd1 expression, failure to arrest the cell cycle and defective formation of terminally differentiated myotubes in siBrm myoblasts, we downregulated Ccnd1 by siRNA and evaluated the effect on cell cycle arrest (as assessed by EdU incorporation) and on the expression of markers of terminal differentiation (indicated by expression of myogenin and MyHC) in siBrm, siBrg1, and siScr myoblasts (Fig 4A). siRNA efficiently reduced cyclin D1 transcripts (Fig 4C), leading to uniform depletion of cyclin D1 protein in C2C12 at all experimental points (Fig EV3), and effectively restored the ability of siBrm myoblasts to arrest the cell cycle in response to differentiation signals (DM) (Fig 4B, compare right and left panels, and Fig 4D). Interestingly, the recovery of cell cycle arrest in siBrm myoblasts was not sufficient to resume the expression of late muscle differentiation proteins, such as MyHC (Fig 4B (right panel), D and E). No effect was observed on the cell cycle profile and expression of differentiation markers in siBrg1 and siScr myoblasts that were depleted of Ccnd1 (Fig 4B–E). These data support the essential role of Ccnd1 repression in Brm-mediated arrest of cell cycle during muscle differentiation. However, the failure to complete the differentiation program of Ccnd1-depleted siBrm myoblasts indicates that cell cycle arrest and terminal differentiation are dissociated in siBrm myoblasts and suggests an additional role of Brm in the activation of muscle gene expression that is independent of its ability to arrest the cell cycle. Click here to expand this figure. Figure EV3. Efficient Cyclin D1 downregulation upon siRNA transfectionImmunofluorescence analysis of cyclin D1 expression in C2C12 cells depleted for Brm (siBrm) or Brg1 (siBrg1) in GM or DM 48 h upon delivery of siRNA against cyclin D1 or control sequence. Note that cyclin D1 is effectively depleted (bottom panel) and is specifically upregulated in DM condition only in Brm-depleted cells (upper panel). Scale bar, 50 μm. Download figure Download PowerPoint Figure 4. Stage-specific requirement of Brg1 or Brm for the activation of the differentiation program in C2C12 myoblasts Schematic representation of the experimental setting, with siRNA delivered to C2C12 cells in GM and EdU pulses in GM or DM 6 h before collecting cells. Cyclin D1 (or control Scr) was downregulated by siRNA in C2C12 cells, which were subsequently interfered for Brm, Brg1, or scrambled sequences (siScr) by small interfering RNA (siRNA). Cells were then cultured in DM for 48 h. Immunofluorescence analysis of EdU incorporation, myogenin and MyHC in C2C12 collected from experimental conditions indicated in (A). Percentages of positive nuclei or cells are indicated in the top right corner of each panel. Nuclei are counterstained with DAPI. The effect of siBrg1, siBrm, or siScr on EdU incorporation, myogenin and MyHC expression was evaluated in siScr (left panels) or siCyclinD1 (right panels) C2C12 cells. Scale bar, 50 μm. Relative expression levels of Ccnd1 transcripts were monitored by qRT–PCR in siScr, siBrg1, and siBrm C2C12 cells in GM and DM (48 h). Data are presented as average ± SEM (n = 3). Quantification of EdU incorporation in nuclei, as percentage of EdU-positive nuclei/total nuclei in randomly selected fields, in siScr, siBrg1, and siBrm C2C12 cells in GM and DM (48 h), in the presence or absence of siCyclinD1. Data are presented as average ± SEM (n > 3). ***P < 0.01 (unpaired Student's t-test). Fusion index was calculated by immunofluorescence staining, as percentage of nuclei within MyHC-expressing myotubes, performed in siScr, siBrg1, and siBrm C2C12 cells cultured in DM (48 h), in the presence or absence of siCyclinD1. Error bars represent average ± SEM (n = 3). Download figure Download PowerPoint Brm but not Brg1 is required for the completion of muscle differentiation The lack of multinucleated myotubes in siBrm cells upon do" @default.
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• W2149282942 title "Brahma is required for cell cycle arrest and late muscle gene expression during skeletal myogenesis" @default.
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