Signal-dependent incorporation of MyoD-BAF60c into Brg1-based SWI/SNF chromatin-remodelling complex
2011; Springer Nature; Volume: 31; Issue: 2 Linguagem: Inglês
10.1038/emboj.2011.391
ISSN1460-2075
AutoresSonia Vanina Forcales, Sonia Albini, Lorenzo Giordani, Barbora Malecová, Luca Cignolo, Andrei V. Chernov, Paula Coutinho, Valentina Saccone, Silvia Consalvi, Roy Williams, Kepeng Wang, Zhenguo Wu, Svetlana Baranovskaya, Andrew Miller, F. Jeffrey Dilworth, Prem Puri,
Tópico(s)Protein Degradation and Inhibitors
ResumoArticle8 November 2011free access Signal-dependent incorporation of MyoD–BAF60c into Brg1-based SWI/SNF chromatin-remodelling complex Sonia V Forcales Sonia V Forcales Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USAPresent address: Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Spain Search for more papers by this author Sonia Albini Sonia Albini Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Lorenzo Giordani Lorenzo Giordani Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Dulbecco Telethon Institute (DTI), IRCCS Fondazione Santa Lucia and European Brain Research Institute, Rome, Italy Search for more papers by this author Barbora Malecova Barbora Malecova Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Luca Cignolo Luca Cignolo Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Andrei Chernov Andrei Chernov Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Paula Coutinho Paula Coutinho Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Valentina Saccone Valentina Saccone Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Dulbecco Telethon Institute (DTI), IRCCS Fondazione Santa Lucia and European Brain Research Institute, Rome, Italy Search for more papers by this author Silvia Consalvi Silvia Consalvi Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Dulbecco Telethon Institute (DTI), IRCCS Fondazione Santa Lucia and European Brain Research Institute, Rome, Italy Search for more papers by this author Roy Williams Roy Williams Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Kepeng Wang Kepeng Wang The Hong Kong University of Science and Technology, Kowloon, Hong Kong Search for more papers by this author Zhenguo Wu Zhenguo Wu The Hong Kong University of Science and Technology, Kowloon, Hong Kong Search for more papers by this author Svetlana Baranovskaya Svetlana Baranovskaya Agilent Technologies, La Jolla, CA, USA Search for more papers by this author Andrew Miller Andrew Miller Agilent Technologies, La Jolla, CA, USA Search for more papers by this author F Jeffrey Dilworth F Jeffrey Dilworth Sprott Center for Stem Cell Research, Ottawa Hospital Research Institute, Regenerative Medicine Program, Ottawa, Ontario, Canada Search for more papers by this author Pier Lorenzo Puri Corresponding Author Pier Lorenzo Puri Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Dulbecco Telethon Institute (DTI), IRCCS Fondazione Santa Lucia and European Brain Research Institute, Rome, Italy Search for more papers by this author Sonia V Forcales Sonia V Forcales Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USAPresent address: Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Spain Search for more papers by this author Sonia Albini Sonia Albini Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Lorenzo Giordani Lorenzo Giordani Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Dulbecco Telethon Institute (DTI), IRCCS Fondazione Santa Lucia and European Brain Research Institute, Rome, Italy Search for more papers by this author Barbora Malecova Barbora Malecova Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Luca Cignolo Luca Cignolo Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Andrei Chernov Andrei Chernov Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Paula Coutinho Paula Coutinho Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Valentina Saccone Valentina Saccone Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Dulbecco Telethon Institute (DTI), IRCCS Fondazione Santa Lucia and European Brain Research Institute, Rome, Italy Search for more papers by this author Silvia Consalvi Silvia Consalvi Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Dulbecco Telethon Institute (DTI), IRCCS Fondazione Santa Lucia and European Brain Research Institute, Rome, Italy Search for more papers by this author Roy Williams Roy Williams Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Search for more papers by this author Kepeng Wang Kepeng Wang The Hong Kong University of Science and Technology, Kowloon, Hong Kong Search for more papers by this author Zhenguo Wu Zhenguo Wu The Hong Kong University of Science and Technology, Kowloon, Hong Kong Search for more papers by this author Svetlana Baranovskaya Svetlana Baranovskaya Agilent Technologies, La Jolla, CA, USA Search for more papers by this author Andrew Miller Andrew Miller Agilent Technologies, La Jolla, CA, USA Search for more papers by this author F Jeffrey Dilworth F Jeffrey Dilworth Sprott Center for Stem Cell Research, Ottawa Hospital Research Institute, Regenerative Medicine Program, Ottawa, Ontario, Canada Search for more papers by this author Pier Lorenzo Puri Corresponding Author Pier Lorenzo Puri Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA Dulbecco Telethon Institute (DTI), IRCCS Fondazione Santa Lucia and European Brain Research Institute, Rome, Italy Search for more papers by this author Author Information Sonia V Forcales1, Sonia Albini1, Lorenzo Giordani1,2, Barbora Malecova1, Luca Cignolo1, Andrei Chernov1, Paula Coutinho1, Valentina Saccone1,2, Silvia Consalvi1,2, Roy Williams1, Kepeng Wang3, Zhenguo Wu3, Svetlana Baranovskaya4, Andrew Miller4, F Jeffrey Dilworth5 and Pier Lorenzo Puri 1,2 1Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, La Jolla, CA, USA 2Dulbecco Telethon Institute (DTI), IRCCS Fondazione Santa Lucia and European Brain Research Institute, Rome, Italy 3The Hong Kong University of Science and Technology, Kowloon, Hong Kong 4Agilent Technologies, La Jolla, CA, USA 5Sprott Center for Stem Cell Research, Ottawa Hospital Research Institute, Regenerative Medicine Program, Ottawa, Ontario, Canada *Corresponding author. Muscle Development and Regeneration Program, Sanford-Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Tel.: +1 858 646 3161; Fax: +1 858 795 5412; E-mail: [email protected] or [email protected] The EMBO Journal (2012)31:301-316https://doi.org/10.1038/emboj.2011.391 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 Figures & Info Tissue-specific transcriptional activators initiate differentiation towards specialized cell types by inducing chromatin modifications permissive for transcription at target loci, through the recruitment of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodelling complex. However, the molecular mechanism that regulates SWI/SNF nuclear distribution in response to differentiation signals is unknown. We show that the muscle determination factor MyoD and the SWI/SNF subunit BAF60c interact on the regulatory elements of MyoD-target genes in myoblasts, prior to activation of transcription. BAF60c facilitates MyoD binding to target genes and marks the chromatin for signal-dependent recruitment of the SWI/SNF core to muscle genes. BAF60c phosphorylation on a conserved threonine by differentiation-activated p38α kinase is the signal that promotes incorporation of MyoD–BAF60c into a Brg1-based SWI/SNF complex, which remodels the chromatin and activates transcription of MyoD-target genes. Our data support an unprecedented two-step model by which pre-assembled BAF60c–MyoD complex directs recruitment of SWI/SNF to muscle loci in response to differentiation cues. Introduction The muscle-specific basic helix-loop-helix (bHLH) transcription factor (TF) MyoD provides a general paradigm to elucidate the physical and functional interactions between tissue-specific TFs and the machinery that promotes chromatin modifications at specific loci (Sartorelli and Caretti, 2005; Tapscott, 2005). Among muscle-specific bHLH TFs, MyoD (and its functional paralogue Myf5) possesses the ability to activate transcription at previously silent muscle loci, via domains that mediate chromatin remodelling and nucleosome displacement at target genes (Gerber et al, 1997). Nucleosome disruption is frequently carried out by the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodelling complexes, which use energy from ATP hydrolysis for disruption of interactions between DNA and histone octamers. SWI/SNF-mediated chromatin remodelling permits the loading of the ‘transcriptosome’ on the regulatory elements of tissue-specific genes and the activation of gene transcription (de la Serna et al, 2006; Cairns, 2009). SWI/SNF complexes are composed of an ATPase subunit (either Brg1 or Brm) and a variable number of structural subunits assembled in different combinations (Clapier and Cairns, 2009; Wu et al, 2009). The involvement of the SWI/SNF complex in a variety of cell activities, including proliferation (Muchardt and Yaniv, 2001), transformation (Reisman et al, 2009), DNA damage signalling and repair (Gong et al, 2006; Park et al, 2006; Sinha et al, 2009), pluripotency and lineage determination (Lessard and Crabtree, 2010; Singhal et al, 2010) indicates a widespread function of SWI/SNF in the regulation of gene expression. Such a versatile activity appears to be conferred on SWI/SNF by a heterogeneous subunit composition and signal-dependent regulation (Wu et al, 2009). However, the mechanism that regulates SWI/SNF composition and chromatin distribution in response to specific cues, such as those that activate muscle-specific gene expression, remains unsolved (Albini and Puri, 2010). MyoD is expressed in myoblasts well before the activation of muscle gene transcription (Weintraub, 1993), possibly in a conformation (homodimers) that precludes ‘productive’ binding to DNA-responsive elements (the E-Box sequence, CANNTG) (Puri and Sartorelli, 2000). On induction of differentiation, MyoD interactions with E2A gene products (e.g., E12 or E47) leads to the formation of heterodimers with an increased affinity for E-Box elements and ability to activate transcription (Tapscott, 2005). In myoblasts, MyoD interaction with chromatin has been the object of controversy (Bergstrom et al, 2002; Berkes et al, 2004; Simone et al, 2004; de la Serna et al, 2005; Mal, 2006). The recent advent of chromatin immunoprecipitation (ChIP)-based technologies for genome-wide analysis has indicated the existence of previously unappreciated interactions between MyoD and chromatin in undifferentiated myoblasts (Blais et al, 2005; Cao et al, 2006, 2010). Still, the function and regulation of MyoD in myoblasts remain obscure. In particular, it is unknown how MyoD recognizes and accesses target genes before induction of differentiation. In undifferentiated myoblasts, the expression of most of MyoD-target genes is prevented by the repressive chromatin conformation imposed by the nucleosomes, which typically preclude the access to transcriptional activators. Thus, a current gap of knowledge concerns the mechanism by which MyoD gains access to target genes in myoblasts, and how differentiation signals confer on MyoD the ability to recruit the machinery that reconfigures the chromatin into a conformation permissive for transcription from target genes. Results Physical interactions between MyoD and BAF60c To elucidate the regulation of MyoD function in myoblasts, we carried out a two-hybrid screen, in which MyoD was used as bait. This system has the advantage of identifying potential proteins that interact with MyoD in the monomer/homodimer conformation predicted in myoblasts (Li et al, 1996), prior to the activation of muscle genes. Using the C-terminal domain of MyoD that contains a chromatin-remodelling domain (Gerber et al, 1997) (Supplementary Figure S1A), we identified a number of potential interacting proteins. From these we focused on BAF60b (SMARCD2) and BAF60c (SMARCD3)—two structural components of the SWI/SNF chromatin-remodelling complex, which is required for the activation of muscle gene transcription (de la Serna et al, 2001, 2005; Simone et al, 2004). Previous work established the essential role of BAF60c in cardiac and skeletal muscle development (Lickert et al, 2004; Takeuchi and Bruneau, 2009). However, the relative role of BAF60 variants a (SMARCD1), b and c in skeletal myogenesis has not been systematically addressed by previous studies. BAF60 variants show a tissue-specific distribution, with BAF60b and c being abundantly expressed in skeletal muscles (Supplementary Figure S1B), as also originally reported by Crabtree and colleagues (Wang et al, 1996). When we monitored the expression levels of BAF60 variants in mouse primary satellite cells, we noted that only BAF60c was upregulated during the differentiation process (Supplementary Figure S1C and F). The selective upregulation of BAF60c was also observed during differentiation of human primary myoblasts (Supplementary Figure S1D) and established skeletal muscle cell lines, such as mouse C2C12 myoblasts (Supplementary Figure S1E and F, right panel). This evidence indicates a relationship between BAF60c levels and myoblast differentiation. Among the two human BAF60c alternative variants—BAF60c1 and 2—BAF60c2 was the only form expressed in human skeletal myoblasts and upregulated in differentiated myotubes (Supplementary Figure S1G). We therefore decided to restrict our analysis to the BAF60c2 variant, which corresponds to mouse BAF60c and is hereafter referred to as BAF60c. In-vitro pull-down experiments using GST–MyoD full length or a mutant lacking the C-terminal domain confirmed the essential role of the C-terminal domain of MyoD for the interaction with BAF60c (Supplementary Figure S1H). The same interaction and requirement of MyoD C-terminus was also confirmed by co-immunoprecipitation of exogenously expressed Flag-tagged MyoD and Xp-tagged BAF60c (Supplementary Figure S1I). Stage-specific interactions between MyoD, BAF60c and Brg1 during myogenic differentiation We further examined whether BAF60c could directly associate with MyoD in vitro. For this purpose, we performed in-vitro interaction studies with purified proteins, using GST–BAF60c in combination with either Flag-tagged purified MyoD, forced MyoD∼MyoD homodimer or MyoD∼E12 heterodimer. Pull-down assay showed that BAF60c interacted efficiently with MyoD and forced MyoD∼MyoD homodimers, while we observed a reduced ability of BAF60c to interact with the forced MyoD∼E12 heterodimers (Figure 1A). Because MyoD homodimers could only form in undifferentiated myoblasts (Li et al, 1996), these data suggest that an interaction between BAF60c and MyoD can take place in undifferentiated myoblasts, prior to the activation of the differentiation programme. Figure 1.BAF60c and MyoD interact in vitro and in vivo. (A) Pull-down assay was performed using GST–BAF60c and baculovirus-purified Flag-tagged MyoD, MyoD∼MyoD and MyoD∼E12 forced dimmers. (B) Co-immunoprecipitations from nuclear extracts of myoblasts and myotubes, with anti-MyoD and anti-BAF60c antibodies. (C–E) PLA was used to monitor nuclear ‘in situ’ interactions between MyoD, Brg1 and Flag–BAF60c during C2C12 differentiation. Each fluorescent dot, ‘blob’, represents the co-localization of the indicated proteins in myoblasts and myotubes. The quantification of the blobs is represented in the adjacent graphic. The BlobFinder software (Allalou and Wählby, 2009) was used to localize and quantify the blobs from images acquired with fluorescent microscopy. The average of blobs/nuclei in the graphic corresponds to the quantification of several images from three different experiments in myoblasts and myotubes. Download figure Download PowerPoint Previous studies have shown both physical and functional interactions between the SWI/SNF enzymatic subunit Brg1 and the muscle regulatory factors MyoD and MEF2 only in differentiating muscle cells (Simone et al, 2004; de La Serna et al, 2005; Rampalli et al, 2007; Serra et al, 2007). Thus, we asked whether the interaction between BAF60c and MyoD detected in myoblasts could precede the association between MyoD and Brg1. Co-immunoprecipitation studies were used to monitor the combinatorial interactions between endogenous MyoD, Brg1 and BAF60c in undifferentiated myoblasts (cultured in growth medium—GM) and differentiating myoblasts (cultured for 18 h in differentiation medium—DM) (Figure 1B). For this purpose, we generated an antibody against BAF60c that specifically recognizes a sequence within the C-terminal region that is not conserved in BAF60a and b—see detailed description in Supplementary data. Immunoprecipitation with anti-MyoD antibodies revealed an interaction between MyoD and BAF60c in both undifferentiated myoblasts and differentiating myoblasts, while the interaction between MyoD and Brg1 was restricted to differentiating myoblasts (Figure 1B, upper panel). The other SWI/SNF catalytic subunit Brm did not interact with MyoD in any of the above conditions (data not shown). Immunoprecipitation with anti-BAF60c antibodies revealed an interaction between BAF60c and MyoD, and between BAF60c and Brg1 at both stages (Figure 1B, lower panel). Collectively, these results show physical interactions between MyoD and BAF60c in myoblasts in a complex distinct from the Brg1- and Brm-based SWI/SNF complexes. Moreover, these results indicate that in myoblasts, a fraction of BAF60c does not bind MyoD and is part of Brg1-based SWI/SNF complexes, which are involved in MyoD-independent regulation of gene expression. Importantly, the reciprocal interactions between Brg1, BAF60c and MyoD detected on induction of differentiation indicate the formation of an MyoD-associated BAF60c/Brg1-based SWI/SNF complex, which coincides with the activation of muscle gene expression. To further substantiate this finding, we used a proximity ligation assay (PLA) that detects the fluorescence signal generated by two antibodies when bound to proteins in such close proximity (at least 30 nm) that indicates a physical interaction (Fredriksson et al, 2002; Allalou and Wählby, 2009; see also Materials and methods). We monitored the interactions between endogenous MyoD, Brg1 and Flag–BAF60c at sequential stages of skeletal muscle differentiation. Flag–BAF60c was used in this assay because the Flag antibody generates an optimal signal for PLA, as compared with the endogenous BAF60c, and permits a direct comparison with mutated forms of BAF60c (see Supplementary Figure S5). The nuclear signal reflecting interactions between MyoD and BAF60c showed an intensity and number of foci (‘blobs’) that were comparable in undifferentiated and differentiating myoblasts (Figure 1C). In contrast, MyoD and Brg1 co-localization was restricted to the nuclei of differentiating C2C12 cells (Figure 1D). Likewise, co-localization of BAF60c and Brg1 dramatically increases in differentiating myoblasts, although interactions between Brg1 and BAF60c could also be detected (albeit to a much lesser extent) in undifferentiated myoblasts (Figure 1E). Interestingly, in differentiating cells, interactions between these proteins were predominantly detected in the nuclear periphery, which is enriched in transcriptionally active SWI/SNF complexes (Brickner, 2009). These results are consistent with the co-immunoprecipitation data shown in Figure 1B, and indicate that BAF60c can be present in distinct complexes during skeletal myogenesis. In undifferentiated myoblasts, BAF60c can be found in Brg1-based SWI/SNF complexes, which do not contain MyoD, and also associated with MyoD in a separate complex that does not contain the Brg1 and Brm ATPases. In differentiating myoblasts, the formation of a complex containing MyoD, BAF60c and Brg1 suggests that differentiation cues promote the incorporation of MyoD–BAF60c into a Brg1-based SWI/SNF complex. This evidence indicates a signal-dependent exchange in composition of MyoD-associated SWI/SNF complexes, and suggests that distinct BAF60c-containing complexes are involved in the regulation of gene expression at different stages of skeletal myogenesis. This notion is consistent with specialized functions of distinct SWI/SNF subcomplexes, as recently reported for the regulation of neurogenesis (Wu et al, 2007) and more generally for a versatile control of gene expression (Wu et al, 2009). BAF60c and Brg1 co-regulate the expression of MyoD-target genes during myoblast differentiation To gain functional insight into the specific role of BAF60c in skeletal myogenesis, we performed gene expression profiling on C2C12 myoblasts that were subjected to siRNA-mediated knockdown of either one of each BAF60 variants or the SWI/SNF catalytic subunits Brg1 and Brm. In an initial phenotypical screening, we noted that only downregulation of Brg1 and BAF60c prevented the formation of multinucleated myotubes (Supplementary Figure S2A and B). In contrast, in BAF60b- (Supplementary Figure S2A and B), BAF60a- or Brm-depleted C2C12 cells (data not shown), the formation of multinucleated myotubes proceeded normally. Interestingly, downregulation of BAF60b (but not BAF60a and Brm) could also reduce the expression of certain muscle genes, such as muscle creatine kinase (MCK) (Supplementary Figure S2C). Based on this evidence, as well as on our initial identification by two-hybrid system that both BAF60b and c are MyoD-interacting proteins (Supplementary Figure S1A, H and I), and the partial, functional redundancy between BAF60b and c (Takeuchi and Bruneau, 2009), we decided to restrict our analysis to C2C12 cells in which Brg1, BAF60b or c were individually knocked down. We performed gene expression profile analysis in differentiating C2C12 cells that were depleted of Brg1, BAF60b or BAF60c, as compared with the expression profile of control C2C12 cells (scrambled RNAi). We first determined the genes that were induced during muscle differentiation, by comparing the gene expression profile of undifferentiated myoblasts (GM) and differentiating myocytes (DM, 18 h). The large majority of the genes induced in DM coincided in the populations of native C2C12 cells or scrRNAi C2C12 cells, and were enriched in genes implicated in muscle differentiation and other specialized functions of differentiated skeletal muscles, such as contraction, cytoskeleton and metabolism. Gene expression microarray analysis of C2C12 cells in which Brg1, BAF60b or c were downregulated by RNAi showed unique and overlapping roles of these SWI/SNF components in the activation of genes involved in muscle differentiation, contractile activity and metabolism (the complete list of genes downregulated by siRNA-mediated depletion of each of these SWI/SNF subunits is accessible through GEO Series accession number GSE24573; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE24573). Depletion of Brg1 or BAF60c led to the downregulation of the large majority of muscle differentiation genes; however, some of the genes downregulated in BAF60b-depleted C2C12 were also included within the category of muscle genes. Intersections of the genes downregulated by independent knockdown of Brg1, BAF60b or BAF60c identified categories of genes that can be co-regulated by combinations of these SWI/SNF components (Supplementary Figure S2D; Supplementary Table SI). Specifically, among the genes induced during C2C12 myoblast differentiation, we identified genes that were downregulated by Brg1, BAF60b and BAF60c (denominated common downregulated genes -Brg1/BAF60b/BAF60c- in Supplementary Figure S2D and listed in Supplementary Table SI). These genes required the expression of each of these SWI/SNF components and contained a significant number of muscle-specific genes previously identified as MyoD-target genes (Sartorelli et al, 1997; Bergstrom et al, 2002; Blais et al, 2005; Cao et al, 2006, 2010; Di Padova et al, 2007). Importantly, analysis of C2C12 cells depleted of either Brg1 or BAF60c identified a large number of downregulated genes that were not affected by BAF60b downregulation (unique downregulated genes—Brg1/BAF60c-, Supplementary Figure S2D; Supplementary Table SI). Those genes were also enriched with previously identified MyoD-target genes. By contrast, very few downregulated genes were identified in C2C12 cells depleted of either Brg1 or BAF60b, but not affected by BAF60c depletion (unique downregulated genes—Brg1/BAF60b-, in Supplementary Figure S2D and listed in Supplementary Table SI). From these genes, only two MyoD-target genes were annotated in this list (Supplementary Table SI). Gene ontology and process network analysis (Supplementary Figure S3A–C) confirmed that the vast majority of genes co-regulated by Brg1 and BAF60c were involved in muscle differentiation and related functions. When we considered all the genes downregulated by Brg1 and BAF60c (by merging the list of common downregulated genes Brg1/BAF60b/BAF60c and unique downregulated genes Brg1/BAF60c—see the ‘enriched list’ in Supplementary Table SI), we found that most of the muscle genes induced during C2C12 differentiation were included in this list. These data indicate a unique, essential role of BAF60c in the activation of muscle differentiation and suggests that Brg1 and BAF60c are essential components of the SWI/SNF complex that promotes muscle gene expression during skeletal myogenesis. These data also indicate an ‘ancillary’ role for BAF60b in the expression of certain muscle genes. Interestingly, a functional redundancy between BAF60b and c has previously been described during cardiomyogenesis (Lickert et al, 2004; Takeuchi and Bruneau, 2009), but the modality of regulation of common genes by BAF60b and c has to be determined. However, the present and previous studies (Lickert et al, 2004; Takeuchi and Bruneau, 2009) indicate a functional hierarchy between BAF60b and BAF60c, with BAF60c playing a more essential role in the activation of muscle genes. This critical role of BAF60c was further confirmed by independent experiments in which BAF60c knockdown inhibited morphological and biochemical differentiation in clones of mouse C2C12 cells in which BAF60c was stably downregulated by retroviral delivery of shRNA (Supplementary Figure S4A–C) and in rat L8 myoblasts (Supplementary Figure S4D and E) that were transiently transfected with BAF60c siRNA. Importantly, depletion of BAF60c in all these muscle cells led to an impaired formation of myotubes and reduced expression of differentiation markers, such as myogenin and myosin heavy chain (MyHC) (Supplementary Figure S4). BAF60c/MyoD chromatin binding precedes Brg1-based SWI/SNF recruitment to MyoD-target genes The enrichment in known MyoD-target genes within the category of genes co-regulated by Brg1 and BAF60c suggested that a Brg1/BAF60c-based SWI/SNF complex is recruited to the regulatory sequences of MyoD-target genes. From these genes, we selected myogenin for a detailed analysis of the chromatin recruitment of MyoD, Brg1 and BAF60c on the promoter elements. Myogenin promoter has been extensively analysed by previous studies, which have identified specific elements that mediate chromatin interactions of distinct TFs, including MyoD, the homodomain protein Pbx1, MEF2 and Six proteins (Edmondson et al, 1992; Knoepfler et al, 1999; Bergstrom et al, 2002; Berkes et al, 2004; Simone et al, 2004; de la Serna et al, 2005; Mal, 2006; Berghella et al, 2008; Palacios et al, 2010; Seenundun et al, 2010). ChIP analysis of the myogenin promoter revealed the presence of endogenous MyoD and BAF60c, but not Brg1, in proliferating myoblasts (Figure 2A), in which myogenin is not expressed. The structural SWI/SNF subunit Ini1 (BAF47), which is typically present in Brg1-based complexes, was also not detected on the myogenin promoter (Figure 2A). This evidence is consistent with the selective interaction of MyoD and BAF60c in undifferentiated myoblasts (Figure 1C) and their presence on myogenin promoter without Brg1-based SWI/SNF complex, prior to myogenin transcription. On induction of differentiation (differentiation medium—DM), which coincides with myogenin transcription, Brg1 and Ini1 were detected on the myogenin promoter, together with pre-
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