Essential role of p18Hamlet/SRCAP-mediated histone H2A.Z chromatin incorporation in muscle differentiation
2010; Springer Nature; Volume: 29; Issue: 12 Linguagem: Inglês
10.1038/emboj.2010.85
ISSN1460-2075
AutoresAna Cuadrado, Nadia Corrado, Eusebio Perdiguero, Vanesa Lafarga, Pura Muñoz‐Cánoves, Ángel R. Nebreda,
Tópico(s)Fungal and yeast genetics research
ResumoArticle14 May 2010free access Essential role of p18Hamlet/SRCAP-mediated histone H2A.Z chromatin incorporation in muscle differentiation Ana Cuadrado Corresponding Author Ana Cuadrado Spanish National Cancer Center (CNIO), Madrid, Spain Search for more papers by this author Nadia Corrado Nadia Corrado Spanish National Cancer Center (CNIO), Madrid, Spain Search for more papers by this author Eusebio Perdiguero Eusebio Perdiguero University Pompeu Fabra (UPF) and ICREA, Barcelona, Spain Search for more papers by this author Vanesa Lafarga Vanesa Lafarga Spanish National Cancer Center (CNIO), Madrid, Spain Search for more papers by this author Pura Muñoz-Canoves Pura Muñoz-Canoves University Pompeu Fabra (UPF) and ICREA, Barcelona, Spain Search for more papers by this author Angel R Nebreda Corresponding Author Angel R Nebreda Spanish National Cancer Center (CNIO), Madrid, Spain Search for more papers by this author Ana Cuadrado Corresponding Author Ana Cuadrado Spanish National Cancer Center (CNIO), Madrid, Spain Search for more papers by this author Nadia Corrado Nadia Corrado Spanish National Cancer Center (CNIO), Madrid, Spain Search for more papers by this author Eusebio Perdiguero Eusebio Perdiguero University Pompeu Fabra (UPF) and ICREA, Barcelona, Spain Search for more papers by this author Vanesa Lafarga Vanesa Lafarga Spanish National Cancer Center (CNIO), Madrid, Spain Search for more papers by this author Pura Muñoz-Canoves Pura Muñoz-Canoves University Pompeu Fabra (UPF) and ICREA, Barcelona, Spain Search for more papers by this author Angel R Nebreda Corresponding Author Angel R Nebreda Spanish National Cancer Center (CNIO), Madrid, Spain Search for more papers by this author Author Information Ana Cuadrado 1,‡, Nadia Corrado1,‡, Eusebio Perdiguero2, Vanesa Lafarga1, Pura Muñoz-Canoves2 and Angel R Nebreda 1 1Spanish National Cancer Center (CNIO), Madrid, Spain 2University Pompeu Fabra (UPF) and ICREA, Barcelona, Spain ‡These authors contributed equally to this work *Corresponding authors. Spanish National Cancer Center (CNIO), Melchor Fernandez Almagro 3, Madrid 28029, Spain. Tel.: +34 91 732 8000; Fax: +34 91 732 8033; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2010)29:2014-2025https://doi.org/10.1038/emboj.2010.85 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 The chromatin-remodelling complex SNF2-related CBP activator protein (SRCAP) regulates chromatin structure in yeast by modulating the exchange of histone H2A for the H2A.Z variant. Here, we have investigated the contribution of H2A.Z-mediated chromatin remodelling to mammalian cell differentiation reprogramming. We show that the SRCAP subunit named ZNHIT1 or p18Hamlet, which is a substrate of p38 MAPK, is recruited to the myogenin promoter at the onset of muscle differentiation, in a p38 MAPK-dependent manner. We also show that p18Hamlet is required for H2A.Z accumulation into this genomic region and for subsequent muscle gene transcriptional activation. Accordingly, downregulation of several subunits or the SRCAP complex impairs muscle gene expression. These results identify SRCAP/H2A.Z-mediated chromatin remodelling as a key early event in muscle differentiation-specific gene expression. We also propose a mechanism by which p38 MAPK-mediated signals are converted into chromatin structural changes, thereby facilitating transcriptional activation during mammalian cell differentiation. Introduction Cell lineage specification from pluripotent stem cells is central for development and organ formation. This process requires modifications throughout the genome (Cui et al, 2009) to allow expression or silencing of particular sets of genes. At the molecular level, this choice is brought about by specific changes in chromatin (de la Serna et al, 2006; Kouzarides, 2007). In fact, defined epigenetic changes characterize terminally differentiated cell types. Skeletal muscle differentiation-specific gene expression is coordinately regulated by transcription factors of the MyoD and MEF2 families and by chromatin-remodelling factors (Sartorelli and Caretti, 2005; Perdiguero et al, 2009). In particular, recruitment of the SWI/SNF ATP-ase Brg1 and the trithorax (TrxG) methyltransferase Ash2L to the regulatory regions of muscle genes is necessary for their expression (Simone et al, 2004; Rampalli et al, 2007). Transcriptional activation may be also regulated by the replacement of canonical histones with histone variants. In yeast, the replacement of histone H2A by H2A.Z has been proposed to positively regulate gene transcription (Santisteban et al, 2000; Adam et al, 2001), and the majority of euchromatic gene promoters have been found to be enriched in H2A.Z (Raisner et al, 2005). In addition, yeast H2A.Z has been shown to antagonize the spread of the heterochromatin (mediated by Sir2 and Sir3), therefore acting as an anti-silencing factor (Meneghini et al, 2003). Genetic and biochemical approaches have further shown that the exchange of histone H2A.Z in yeast is specifically catalysed by the SWR1 chromatin-remodelling complex (Krogan et al, 2003; Meneghini et al, 2003; Kobor et al, 2004). Notably, H2A.Z is a highly conserved protein that has been found in many organisms from protozoan parasites to plants and humans. A role for H2A.Z in gene activation has also been documented in Caenorhabditis elegans, where H2A.Z is recruited to foregut promoters at the time of transcription onset and together with the transcription factor PHA-4 coordinates temporal gene expression (Updike and Mango, 2006). In mammals, H2A.Z is essential for embryonic development and chromosome segregation (Faast et al, 2001; Rangasamy et al, 2004). Interestingly, high-resolution profiling analysis of histone modifications in the human genome has shown the association of H2A.Z with functional regulatory elements, close to transcription start sites (Barski et al, 2007). There are two mammalian Swr1-like complexes that have been proposed to regulate H2A.Z deposition in cells, one is p400 (Gevry et al, 2007, 2009) and the other is Snf2-related CBP (CREB-binding protein) activator protein (SRCAP) (Wong et al, 2007). Moreover, SRCAP has been reported to catalyse the incorporation of H2A.Z into chromatin in vitro (Ruhl et al, 2006). However, little is known about the recruitment and function of H2A.Z at mammalian promoters during genomic reprogramming towards terminal cell differentiation. A recent report has shown increased levels of H2A.Z in genes that lose the H3K27me3 mark and become activated during differentiation of multipotent human primary hematopoietic stem cells into erythrocyte precursors (Cui et al, 2009). We have characterized a protein named ZNHIT1 or p18Hamlet as a substrate of p38α and p38β MAPKs, which mediates p53-dependent transcriptional responses to genotoxic stress (Cuadrado et al, 2007; Lafarga et al, 2007). ZNHIT1/p18Hamlet has been also identified as a subunit of the human SRCAP complex (Cai et al, 2005; Sardiu et al, 2008). The p38 MAPK pathway is critical for the activation of the muscle differentiation gene program (Lluis et al, 2006), which involves the p38 MAPK-regulated recruitment of the SWI/SNF and TrxG chromatin-remodelling complexes to muscle-specific loci (Simone et al, 2004; Rampalli et al, 2007). Here, we show that p18Hamlet and the SRCAP complex regulate muscle differentiation. Our results show an important role for SRCAP and histone H2A.Z incorporation in the initiation of the muscle-specific gene expression program, through the recruitment of the p38 MAPK-regulated p18Hamlet protein to muscle promoters, ensuring the changes in chromatin structure necessary for transcriptional activation. Results p18Hamlet is upregulated during muscle differentiation in a p38 MAPK-dependent manner To analyse the potential contribution of the p38 MAPK substrate p18Hamlet to skeletal muscle differentiation, we first investigated its expression pattern in C2C12 myoblasts. We found that p18Hamlet protein levels increased early during the differentiation process (Figure 1A), whereas p18Hamlet mRNA levels were very similar in undifferentiated and differentiated myoblasts (compare growth medium (GM) with differentiation medium (DM)) (Figure 1B). Moreover, the p38α and p38β chemical inhibitor SB203580 inhibited the accumulation of p18Hamlet (Figure 1C), confirming the relationship between p38 MAPK activation and the stabilization of the p18Hamlet protein (Cuadrado et al, 2007). Furthermore, p18Hamlet was phosphorylated during myoblast differentiation in a p38 MAPK-dependent manner (Figure 1C). Altogether, these data link p38 MAPK activation with the phosphorylation and accumulation of p18Hamlet during skeletal myogenesis. Figure 1.p18Hamlet protein levels increase during muscle differentiation in a p38 MAPK-dependent manner. (A) p18Hamlet, myogenin and MHC protein levels were analysed by immunoblotting in proliferating C2C12 myoblasts (GM) and during the differentiation process (DM). Tubulin was used as a loading control. (B) p18Hamlet mRNA levels were analysed by qRT–PCR and normalized to GAPDH at the indicated time points during C2C12 differentiation. Data are shown as means±s.d. of samples from a representative experiment assayed in triplicates. (C) C2C12 myoblasts were incubated in DM for the indicated times, either in the absence or presence of SB203580 (SB). Endogenous p18Hamlet was immunoprecipitated and analysed by immunoblotting with phospho-Thr and p18Hamlet antibodies. Myogenin levels in total lysates were analysed by immunoblotting. Tubulin and IgG were used as loading controls in total lysates and immunoprecipitates, respectively. Download figure Download PowerPoint Recruitment of p18Hamlet and H2A.Z to the myogenin promoter at early stages of muscle differentiation The yeast homolog of p18Hamlet, Vps71/Swc6, is essential for histone H2A.Z exchange catalysed by the SRW1 complex, enabling the association of the catalytic ATP-ase and histone H2A.Z interacting subunits (Wu et al, 2005). The Arabidopsis p18Hamlet homolog SEF is also required for the exchange of histone H2A for H2A.Z at the FLC promoter, which precedes FLC transcription (Deal et al, 2007; March-Diaz et al, 2007). However, the involvement of this chromatin-remodelling mechanism in mammalian cell differentiation remains unknown. Transcriptional activation of the myogenin gene is one of the earliest steps necessary for reprogramming undifferentiated myoblasts into fully differentiated muscle cells. We therefore investigated the potential binding of p18Hamlet and H2A.Z to the myogenin promoter at the onset of myoblast differentiation by using chromatin immunoprecipitation (ChIP) and quantitative PCR assays. First, we found that both proteins were highly enriched at the TATA box-containing region of the myogenin promoter compared with its binding to a non-coding DNA sequence located 18 kb upstream of the promoter, whereas histone H3 concentration was similar in the regions studied (Figure 2A and B). Moreover, the amount of p18Hamlet at the TATA box of the myogenin promoter substantially increased early in the differentiation process (Figure 2C). Importantly, the p38α and p38β inhibitor SB203580 impaired the recruitment of p18Hamlet to the myogenin TATA box (Figure 2C). Figure 2.p18Hamlet accumulates at the myogenin promoter at early stages of muscle differentiation. (A) Schematic representation of the mouse myogenin gene indicating the TATA box and the transcription start site. The positions of the DNA fragments amplified with three different pairs of primers are indicated. 'a' refers to a non-coding region located 18 kb upstream, 'b' to the TATA box-containing region, and 'c' to a region close to the TATA box of the myogenin promoter. (B) ChIP analysis of p18Hamlet, Histone H3 and H2A.Z binding to the genomic regions 'a', 'b' and 'c' of the myogenin promoter. qPCR data are shown as means±s.d. of samples from a representative experiment assayed in triplicates. In each case, relative binding to the 'b' and 'c' regions is referred to the binding to the 'a' region, which is given the value of 1. (C) p18Hamlet binding to the myogenin promoter (region 'b') was assayed by ChIP in proliferating C2C12 myoblasts (GM) and after 14 h in DM, either in the presence or absence of SB203580 (SB), as indicated. Both semiquantitative (left panel) and quantitative (right panel) PCRs were performed. Download figure Download PowerPoint We then analysed whether p18Hamlet recruitment correlated with H2A.Z incorporation at the myogenin promoter during the differentiation process. We found that H2A.Z was already detectable at the myogenin TATA box under basal (GM) conditions (Figures 3B and 4B). Furthermore, H2A.Z began to significantly accumulate at this genomic location as soon as 4 h after the induction of differentiation, and the accumulation was more evident after 14 h (Figure 3A and B; Supplementary Figure S1). As a control, histone H3 binding to the myogenin promoter remained constant during the same kinetics. Interestingly, the p38α and p38β inhibitor SB203580 impaired H2A.Z incorporation at the myogenin TATA box (Figure 3A and B). To determine the physiological relevance of these observations, we analysed the presence of H2A.Z at the myogenin TATA box of mouse primary myoblasts under both basal and differentiation conditions (Figure 3C). We detected a significant amount of H2A.Z bound to this genomic region under GM conditions, and this binding was abrogated by SB203580. Moreover, H2A.Z accumulated at the myogenin TATA box during the differentiation of primary myoblasts to the same extent as in C2C12 cells. Importantly, p38α−/− primary myoblasts lacked H2A.Z at the myogenin promoter (Figure 3C), and the reintroduction of p38α in p38α−/− myoblasts partially restored H2A.Z binding to the myogenin promoter under differentiation conditions, and subsequent myogenin gene expression (Supplementary Figure S2). Overall, these data strongly support that during the early phases of muscle differentiation, histone H2A.Z accumulates at the myogenin TATA box in a p38 MAPK-dependent manner. Figure 3.Histone H2A.Z accumulates at the myogenin promoter early during muscle differentiation in a p18Hamlet-dependent manner. (A) ChIP analysis of histones H2A.Z and H3 binding to the TATA box-containing region of the myogenin promoter in proliferating C2C12 myoblasts (GM) and at early times during muscle differentiation (DM) either in the presence or absence of SB203580 (SB). qPCR data are shown as means±s.d. of two independent experiments performed in triplicates. Histone-binding values are normalized to the respective input DNA and are referred to the binding of each histone under GM conditions, which is given the value of 1. (B) C2C12 myoblasts were cultured as indicated in (A), and the binding of H2A.Z, and H3 to the myogenin TATA box was analysed by ChIP followed by semiquantitative PCR. (C) Mouse wt and p38α−/− (KO) primary myoblasts were grown in GM or DM in the presence or absence of SB203580 (SB) for the indicated times. H2A.Z binding to the myogenin TATA box-containing region was analysed by ChIP and qPCR. Relative-binding values are referred to the binding in wt myoblasts under GM conditions, which is given the value of 1. (D) C2C12 myoblasts were incubated with either p18Hamlet or H2A.Z siRNAs and then cultured in GM or incubated in DM for 14 h. MyoD binding to the myogenin TATA box region was analysed by ChIP followed by semiquantitative PCR. Analysis by qPCR of the same samples is shown in Supplementary Figure S3. Download figure Download PowerPoint Figure 4.p18Hamlet mediates histone H2A.Z binding to chromatin and induces muscle-specific gene expression. (A) C2C12 myoblasts were cultured in growing conditions and transfected with control (C) or p18Hamlet siRNA, and 72 h later H2A.Z binding to the indicated regions of the myogenin promoter (see Figure 2A) was analysed by ChIP and qPCR. Semiquantitative PCR analysis was also performed with samples of the same regions. (B) C2C12 myoblasts were incubated in DM for 14 h and the effect of p18Hamlet downregulation on H2A.Z binding to the myogenin promoter was analysed by ChIP and both semiquantitative and quantitative PCRs. (C) C2C12 myoblasts were transfected with a Myc-tagged p18Hamlet expression construct and clones were selected with G418. Myogenin and both endogenous and Myc-tagged p18Hamlet protein levels were analysed by immunoblotting in the indicated clones. (D) Binding of H2A.Z to the myogenin TATA box-containing region was determined by ChIP assays and both semiquantitative and quantitative PCR in control (number 1) and p18Hamlet overexpressing (number 6) C2C12 clones (left and middle panels). Myogenin mRNA levels were estimated by semiquantitative RT–PCR (right panel). Download figure Download PowerPoint Finally, we investigated the importance of p18Hamlet recruitment and H2A.Z exchange for the assembly of the muscle-specific transcriptosome. One of the key pieces of this machinery is the transcription factor MyoD, which binds to the myogenin promoter at early stages during muscle differentiation (Berkes and Tapscott, 2005). Downregulation of either p18Hamlet or, specially, H2A.Z had a dramatic effect on MyoD binding to the myogenin promoter (Figure 3D; Supplementary Figure S3), suggesting that H2A.Z incorporation into chromatin is an essential step that precedes the transcriptional events taking place early during the process of muscle differentiation. It should be noted that p18Hamlet downregulation did not affect H2A.Z expression, and H2A.Z downregulation did not alter p18Hamlet protein levels (Supplementary Figure S4). p18Hamlet requirement for H2A.Z recruitment at the myogenin promoter Next, we investigated the relationship between p18Hamlet function and H2A.Z localization to chromatin using two different approaches. First, we designed siRNAs that reduced p18Hamlet levels in C2C12 cells by about 80% (Supplementary Figure S5). We observed that p18Hamlet downregulation abolished both basal (Figure 4A) as well as the differentiation-induced (Figure 4B) H2A.Z binding to the myogenin promoter. As a complementary experiment, we overexpressed p18Hamlet in C2C12 myoblasts grown in GM, and found a good correlation between p18Hamlet upregulation and myogenin induction (Figure 4C). Moreover, the myogenin protein levels in these clones were similar to those observed on the induction of differentiation in C2C12 myoblasts with DM (Supplementary Figure S6). Notably, forced expression of p18Hamlet promoted the recruitment of H2A.Z to the myogenin promoter in the differentiation-inhibitory conditions of GM, which correlated with enhanced transcription of myogenin (Figure 4D). These results indicate that p18Hamlet is required for H2A.Z recruitment to myogenin chromatin. p38 MAPK-mediated p18Hamlet phosphorylation is required for myogenin induction and H2A.Z deposition at the myogenin promoter To determine the importance of p18Hamlet phosphorylation for its ability to activate myogenin transcription, we compared the effect of expressing at similar levels either wild type (wt) or a non-phosphorylatable p18Hamlet mutant (4A) in C2C12 cells grown in GM. By using both biochemical (myogenin expression) and morphological (cell shape) parameters, we observed that the p18Hamlet 4A mutant was less competent to activate myogenesis than its wt counterpart (Figure 5A). Indeed, ectopic expression of p18Hamlet wt in C2C12 myoblasts induced the expression of myogenin in 47% of the transfected cells in GM. In contrast, when cells were transfected with the non-phosphorylatable form of p18Hamlet, <20% of the transfected cells were positive for myogenin (Figure 5A). Furthermore, the myoblast elongation typical of prefusing myocytes was exclusively observed in p18Hamlet wt-transfected cells in GM differentiation-restricting conditions (Figure 5A). To further show that p38 MAPK-mediated phosphorylation of p18Hamlet is required for H2A.Z recruitment at the myogenin promoter, we ectopically activated p38 MAPK signalling in C2C12 myoblasts by cotransfection of constitutively activated MKK6DD (a specific p38 MAPK activator) with either p18Hamlet wt or the 4A mutant. MKK6DD in combination with overexpressed wt p18Hamlet was sufficient to induce myogenin expression under GM conditions to a much higher extent than the p18Hamlet 4A mutant (Figure 5B). Moreover, the non-phosphorylable p18Hamlet mutant was unable to mediate p38 MAPK-dependent enrichment of H2A.Z at the myogenin promoter (Figure 5C). Altogether, these results strongly support that accumulation and phosphorylation of p18Hamlet is sufficient to induce histone H2A.Z recruitment into promoters of differentiation-regulated mammalian genes. Consistent with this idea, using a model of de novo induced myogenesis in adult mouse skeletal muscle, we found upregulation of p18Hamlet expression during myogenesis in vivo, correlating with p38 MAPK activation and the induction of myogenin (Figure 5D). Figure 5.Accumulation and phosphorylation of p18Hamlet are necessary for H2A.Z chromatin enrichment and myogenin expression. (A) C2C12 myoblasts were transfected with plasmids encoding either wt or a non-phosphorylatable version (4A) of Myc-tagged p18Hamlet. Expression of p18Hamlet, myogenin induction and myocyte morphological changes were analysed by immunofluorescence (left panel) in at least 300 cells. The efficiency of transfection was 28 and 20% for the wt and 4A p18Hamlet constructs, respectively. The percentage of myogenin-positive cells for each transfection is represented (right panel). (B) C2C12 myoblasts were transfected with either MKK6DD alone or in combination with Myc-tagged p18Hamlet wt and 4A mutant, and 24 h later, the total lysates were analysed by immunoblotting using the indicated antibodies. Tubulin was used as loading control. Transfection efficiency was about 50%. (C) Chromatin from C2C12 myoblasts transfected as in (B) was collected 24 h after transfection, and H2A.Z binding to the myogenin TATA box-containing region was assayed by ChIP. Both semiquantitative and quantitative PCRs are shown. (D) Upregulation of p18Hamlet expression during myogenic differentiation in vivo correlates with p38 MAPK activation and myogenin induction. Extracts of plantaris muscles either in basal state or undergoing de novo myogenesis (3 days after overloading) were analysed by immunoblotting using the indicated antibodies. Myogenin is a marker of differentiating satellite cells/myoblasts (left panel). Sections of mouse plantaris muscles as above were immunostained with the indicated antibodies and detected with peroxidase (H2A.Z and p18Hamlet) or immunofluorescence (Myogenin and Pax7). Nuclei were detected with DAPI. Pax7 identifies satellite cells/myoblasts present both in basal and overloading conditions. Myogenin-positive satellite cells are exclusively detected in muscle with ongoing myogenesis/differentiation (right panel). Bar=50 μm. Download figure Download PowerPoint p18Hamlet/SRCAP-mediated H2A.Z recruitment is necessary for muscle differentiation Mammalian SRCAP is a multiprotein complex whose components have been well characterized in yeast. Five of these components are essential for nucleosomal histone H2A for histone H2A.Z exchange as well as for complex integrity, including the catalytic subunit Swr1 (mammalian SRCAP), the H2A.Z-binding protein Swc2 (mammalian YL1), and two small proteins necessary for complex formation and nucleosome binding, Arp6 and Swc6 (p18Hamlet) (Wu et al, 2005). Although several groups have reported ZNHIT1/p18Hamlet as a member of the mammalian SRCAP complex, its specific role remains unclear. On the basis of the observed interactions between the essential components of the yeast SWR1 complex (Figure 6A), and due to the high homology of these proteins with their mammalian counterparts, we performed immunoprecipitation experiments to determine the ability of p18Hamlet to interact with other SRCAP complex members. We observed a clear interaction between p18Hamlet and H2A.Z when either both or only one of the proteins were overexpressed (Figure 6B). However, the interaction between the endogenous proteins, although detectable, was weak, consistent with the yeast model proposing that such interaction is not direct (Wu et al, 2005). Interestingly, we found that human Arp6 bound strongly to p18Hamlet (Figure 6C). To determine the importance of p18Hamlet phosphorylation for its interaction with Arp6, we transfected C2C12 cells with similar amounts of p18Hamlet wt or the mutant 4A. We observed that the interaction with Arp6 was markedly impaired in the case of the p18Hamlet 4A mutant (Figure 6D), indicating that p18Hamlet phosphorylation has an essential role in its interaction with Arp6 and presumably SRCAP complex integrity. According to the proposed model, H2A.Z recruitment to the SRCAP complex is mediated through its binding to the YL-1 subunit. To validate this hypothesis in the context of myogenesis, we analysed the coimmunoprecipitation of H2A.Z and YL-1 under GM and DM conditions. We found that H2A.Z binding to YL-1 was very low under proliferation conditions and increased at early stages of myogenesis, to dramatically decrease after 16 hours in differentiation media (Figure 6E). This result suggests that H2A.Z binding to the SRCAP complex occurs during muscle differentiation and that it is a dynamic process. To further support a role for SRCAP in H2A.Z recruitment at the myogenin promoter, we performed ChIP analysis of the SRCAP catalytic subunit in C2C12 cells under both proliferation and differentiation conditions, and found that SRCAP was already bound to chromatin in GM and accumulated in DM (Figure 6F). Interestingly, DM-induced binding of SRCAP to the myogenin TATA box-containing region was impaired both in the absence of p18Hamlet or upon p38 MAPK inhibition (Figure 6F). Figure 6.p18Hamlet phosphorylation is required for its interaction with Arp6, and regulation of H2A.Z binding to YL-1 during myogenesis. (A) Proposed interactions between essential components of SWR1, the yeast homolog of the SRCAP complex. (B) U2OS cells were transfected with 1 μg of HA-tagged H2A.Z and Myc-p18Hamlet expression vectors, and 48 h later, the total lysates were immunoprecipitated (IP) with HA antibodies to evaluate the interaction of H2A.Z with both overexpressed (Myc) and endogenous (End) p18Hamlet. Input represents 10% of the total lysate used for the IPs. (C) U2OS cells were transfected with 1 μg of either empty vector or a Myc-tagged Arp6 expression vector, and the total lysates were analysed by IP of p18Hamlet or H2A.Z endogenous proteins followed by Myc immunoblotting. Input corresponds to 5% of the total lysates used for the IPs. (D) C2C12 cells were transfected with p18Hamlet wt and mutant 4A alone or in combination with Myc-Arp6, and 48 h later, the total lysates were IP with a p18Hamlet antibody followed by Myc immunoblotting. Input corresponds to 10% of the total lysate used for IPs. (E) C2C12 cells were cultured in GM or in DM for the indicated times, and the total lysates were analysed by IP with an YL-1 antibody followed by H2A.Z immunoblotting. Input corresponds to 5% of the total lysates used for the IPs. (F) The presence of SRCAP in the myogenin TATA box-containing region was analysed by ChIP using chromatin obtained from C2C12 cells grown in GM or after 14 h in DM and either treated with p18Hamlet siRNA or SB203580 as indicated. Both quantitative and semiquantitative PCR analysis are shown. Download figure Download PowerPoint To assess the importance of the p18Hamlet/SRCAP-mediated chromatin remodelling in skeletal muscle differentiation, we analysed the consequences of downregulating p18Hamlet, YL-1, SRCAP and H2A.Z on muscle gene expression and myoblast fusion. Downregulation of p18Hamlet prevented the DM-induced myogenin expression (Figure 7A), in agreement with the impaired recruitment of H2A.Z observed at the myogenin promoter (see above, Figure 5A and B). Induction of late muscle differentiation-specific genes, such as myosin heavy chain (MHC) and muscle creatine kinase (MCK), was also impaired on p18Hamlet depletion in differentiation-promoting conditions (Figure 7B; Supplementary Figure S7). Moreover, myoblasts treated with two different p18Hamlet siRNAs remained undifferentiated, showing a severe defect in their ability to fuse into long multinucleated myotubes, even after 4 days in DM (Figure 7C). Conversely, overexpression of p18Hamlet sufficed to induce the expression of myogenin, MCK and MHC in proliferating C2C12 myoblasts (Figure 7D). These results indicate an essential role of the SRCAP component p18Hamlet in skeletal muscle differentiation. Figure 7.p18Hamlet downregulation prevents differentiation of C2C12 myoblasts, and its overexpression is sufficient to induce muscle-specific gene expression. (A) C2C12 myoblasts were transfected with control or two different p18Hamlet siRNAs, and 48 h later were seeded at 70% of confluency and incubated for up to 3 days in DM. Expression levels of p18Hamlet, myogenin and tubulin proteins were analysed by immunoblotting. (B) RNA was purified from C2C12 myoblasts that were transfected with control or p18Hamlet-oligo 1 siRNAs and then maintained in DM for the indicated times or in GM. Myogenin
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