Regulation of skeletal muscle gene expression by p38 MAP kinases

Review

TRENDS in Cell Biology

Vol.16 No.1 January 2006

Regulation of skeletal muscle gene expression by p38 MAP kinases Frederic Lluı´s1, Eusebio Perdiguero1, Angel R. Nebreda2 and Pura Mun˜oz-Ca´noves1 1 2

Center for Genomic Regulation (CRG), Program on Differentiation and Cancer, Barcelona, Spain Spanish National Cancer Center (CNIO), Madrid, Spain

The formation of skeletal muscle is a multistep process orchestrated by the basic helix–loop–helix myogenic regulatory factors (MRFs). A wide array of proteins can interact with the MRFs, resulting in either induction or repression of their myogenic potential and subsequent MRF-mediated muscle-specific transcription. Findings published over the past few years have unambiguously established a key role for the p38 MAP kinase pathway in the control of muscle gene expression at different stages of the myogenic process. Here, we discuss the mechanisms by which p38 MAP kinase controls skeletal muscle differentiation by regulating the sequential activation of MRFs and their transcriptional coactivators, including chromatin remodeling enzymes.

Introduction The regulation of skeletal muscle formation (myogenesis) is essential for normal development as well as in pathological conditions such as muscular dystrophies and inflammatory myopathies. Myogenesis is a dynamic process in which mononucleated undifferentiated myoblasts first proliferate, then withdraw from the cell cycle and finally differentiate and fuse to form the multinucleated mature muscle fiber. This process is controlled by members of a family of muscle-specific basic helix– loop–helix (bHLH) proteins that, in concert with members of the ubiquitous E2A and myocyte enhancer factor-2 (MEF2) families, activate the differentiation program by inducing transcription of regulatory and structural muscle-specific genes [1] (Figure 1). Additional levels of regulation impinge on this basic transcriptional model to provide further versatility to muscle gene expression. The question of how these signals are deciphered by the myogenic effectors has been the center of intensive investigation. A signaling pathway that plays a fundamental role in the transition of myoblasts to differentiated myocytes involves p38 mitogen-activated protein kinase (MAPK). Recent studies have demonstrated that p38 MAPK provides a link between the myogenic transcription factors that activate muscle genes directly and the chromatin remodeling activities associated with the muscle differentiation program. Here, we discuss how p38 MAPK controls the transcriptional circuitry that Corresponding author: Mun˜oz-Ca´noves, P. ([email protected]). Available online 1 December 2005

underlies tissue-specific gene expression, with particular emphasis on skeletal muscle. Regulation of skeletal muscle gene expression by myogenic regulatory factors Activation of muscle differentiation-specific genes is controlled by the myogenic regulatory factors (MRFs), which belong to the bHLH family of transcription factors (Figure 2). The MRF family consists of four members: Myf5, MyoD, myogenin and MRF4, all of which bind to sequence-specific DNA elements (E box: .CANNTG.) present in the promoters of muscle genes. Selective and productive recognition of E boxes on muscle promoters requires heterodimerization of MyoD with ubiquitously expressed bHLH E proteins, rendering the formation of this functional heterodimer the key event in skeletal myogenesis [2,3]. Preferences for specific sequences internal and external to the E box for MRF homodimers and MRF–E-protein heterodimers have been determined in vitro [4]. Nonetheless, this basic model of musclespecific transcription by MRF binding to E boxes is overly simplistic and encounters several pitfalls. For example, canonical E boxes are not exclusively found in the regulatory regions of muscle genes. Other transcription factors, including E protein homodimers and neurogenic bHLH transcription factors such as NeuroD utilize the same E box as MyoD on B cell- and neuron-specific promoters, respectively. However, skeletal myoblasts require MRFs, lymphocytes require E proteins, and neurons require NeuroD, to activate their corresponding tissue-specific genes. It seems likely that the cell-typerestricted expression of bHLH proteins might avoid the ectopic activation of bHLH target genes in the wrong lineage. Finally, different muscle genes are expressed at different times during myogenesis, despite all of them having E boxes in their promoter regions [1,5]. Thus, promoter-specific and temporal constraints are likely to be superimposed upon this basic model of myogenic transcriptional activation by MRFs. Interaction of MRFs with transcriptional cofactors The MRFs have the unique property of converting nonmuscle cells to the muscle lineage, strongly suggesting that they can, directly or indirectly, induce relaxation of the otherwise-repressed chromatin on their target genes. The potential of MyoD to stimulate muscle-specific gene transcription derives both from its intrinsic ability to

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Figure 1. Control of skeletal myogenesis by bHLH and MEF2 transcription factors. Myogenesis is a multi-step process by which new muscle fibers are formed from precursor muscle cells. Mononucleated undifferentiated myoblasts grow in proliferating conditions, characterized by a high mitogen content (proliferation); upon mitogen withdrawal, myoblasts differentiate into mononucleated myocytes (early differentiation) that subsequently start to fuse into multinucleated myotubes expressing muscle-specific proteins (late differentiation), to form the mature muscle fiber (terminal differentiation). Progression through the different myogenic stages is controlled by the sequential activation of four myogenic regulatory factors (MRFs) belonging to the basic helix–loop–helix (bHLH) family of transcription factors (Myf5, MyoD, myogenin and MRF4), which cooperate with the ubiquitously expressed E proteins (the E2A gene products E12 and E47, and HEB) and myocyte enhancer factor 2 (MEF2) transcriptional regulators to activate transcription of muscle-specific genes, coding for structural and enzymatic muscle proteins such as a-actin, myosin heavy chain (MHC) or muscle creatine kinase (MCK). Studies using both primary cultures of skeletal muscle as well as established muscle cell lines (which partially recapitulate myogenesis, thus being extensively used as myogenic model systems) have confirmed the expression of MyoD and Myf5 in undifferentiated myoblasts, while myogenin and MRF4 are activated at early and late differentiation stages, respectively [1]. In proliferating myoblasts, MRFs and E proteins associate with the HLH protein Id (inhibitor of differentiation). Since Id lacks the basic domain necessary for DNA binding, the resulting E protein–Id and MRF–Id heterodimers cannot bind the E box in the muscle promoters. Id expression is downregulated at the onset of differentiation, allowing the formation of the functional MRF–E-protein heterodimers (see text for details).

reorganize chromatin through a region rich in histidine and cysteine residues (H/C domain), which lies N-terminal to the basic region, and a potential amphipathic a-helix (helix III) in the C-terminal region [6–8] (Figure 2) and from its capacity to interact with histone acetyltransferases (HATs), especially p300/CBP and PCAF (p300/ CBP-associated factor) [9–14] (Box 1). Contrary to HATs, however, histone deacetylases (HDACs) can negatively regulate MyoD-dependent transcription by interacting directly with MyoD [15,16]. For example, in proliferating myoblasts, MyoD has been found on the myogenin promoter in association with HDAC1, acting as a transcriptional repressor [17] (Box 3). The class III HDAC Sir2 can also associate with, and deacetylate, both PCAF and MyoD, resulting in the inhibition of www.sciencedirect.com

muscle differentiation upon changes in the redox state of the cell [18]. Other studies have shown that the SWI/SNF ATP-dependent chromatin remodeling activity is necessary for MyoD-mediated activation of endogenous muscle differentiation-specific loci [19] (Box 1). Together, these data suggest that MyoD might direct the chromatinmodifying enzymes to muscle promoters and that, depending on the nature of the E-box-associated enzymatic activity, MyoD can activate or inhibit musclespecific gene transcription. Full activation of muscle gene expression by MRFs is also dependent on their association with members of the MEF2 family of transcription factors, MEF2A-D. MEF2 factors cannot activate muscle genes on their own, but they do potentiate the activity of the MRFs. Such

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Figure 2. Functional domains of the bHLH and MEF2 transcription factors. The MRFs share functionally distinct domains: The bHLH region is evolutionary conserved in the MRFs, being the HLH domain responsible for dimerization of these factors with the E proteins, whereas the basic domain is responsible for the binding to a canonical DNA sequence, CANNTG, called the E box, within the regulatory regions of muscle genes [1]. Importantly, MRF–E-protein heterodimers, but not the homodimers, are able to bind to the muscle E box [2]. The N- and C-terminal domains of the MRFs show sequence divergence and are important for transactivation, chromatin remodeling and protein– protein interactions. MyoD and Myf5 have a higher ability than myogenin to remodel the repressed chromatin at the target loci, owing to two domains conserved in the former, but not the latter, proteins: a region rich in histidine and cysteine residues (H/C domain), which lies N-terminal to the basic region, and a potential amphipathic a-helix (helix III) in the C-terminal region [1,5]. The H/C and helix 3 domains mediate interaction of MyoD with the Pbx transcription factor, which might be relevant for the initiation of transcription at the myogenin promoter [6,8,34] (see Box 3).

synergism requires the direct association between the bHLH domain of the MRF and the MADS (MCM1, agamous, deficiens, serum response factor) domain of the MEF2 protein [14,20] (Figure 2). The growing list of MRFassociated factors suggests that these intermolecular interactions are likely to regulate the specific and temporal association of MRFs with DNA-regulatory regions. Moreover, accumulating evidence indicates that the activities of the MRFs and associated cofactors might be subjected to posttranscriptional regulation by muscledifferentiation-induced signaling pathways.

Requirement for p38 MAPK activity in skeletal muscle differentiation Independent studies have unambiguously demonstrated that the p38 MAPK signaling pathway (Box 2) is a crucial regulator of skeletal muscle differentiation. Treatment with the p38a and p38b inhibitor SB203580 prevented the fusion of myoblasts into myotubes, as well as the induction of muscle-specific genes [21–24]. Importantly, a recent report has shown the requirement for p38a/p38b in the activation of the quiescent satellite cell (the muscle stem cell), although the mechanism underlying this effect remains unknown [25]. Forced activation of p38 MAPK by ectopic expression of a constitutively active mutant of MKK6 (MKK6-EE) is sufficient to override the inhibitory factors present in proliferating cells and to induce both the expression of differentiation markers and the appearance of multinucleated myotubes [23,26]. Furthermore, the ectopic expression of active MKK6-EE in rhabdomyosarcoma cells, which express MyoD but do not contain activated p38 MAPK, led to the induction of morphological and biochemical differentiation of the tumor cells [3], reinforcing the idea that p38 MAPK activity plays an essential role in muscle differentiation. Yet, the mechanisms that regulate p38 MAPK activity in differentiating muscle cells remain unidentified, although these appear to be different from those involved in the response to stress and cytokines. Thus, the kinetics of p38 MAPK activation in response to the later stimuli are fast and transient, whereas differentiation-induced p38 MAPK activation is www.sciencedirect.com

persistent, suggesting the need for constant p38 MAPK activity during myogenesis [3,21,23]. p38 MAPK-regulated mechanisms controlling musclespecific gene transcription While p38 MAPK plays an essential role in myoblast differentiation, the underlying molecular mechanisms of muscle-specific transcriptional control by p38 MAPKs remain largely unknown. Activation of MEF2 by p38 MAPK A potential explanation for the positive effect of p38 MAPK in myogenesis was provided by the finding that p38

Box 1. Chromatin modifying and remodeling activities The fundamental repeating unit of chromatin is the nucleosome, which consists of 146 base pairs of DNA wrapped around a histone octamer. Chromatin is generally repressive to extraneous access, owing to its compact and tight nucleosomal organization. Two main enzymatic activities induce chromatin modifications and regulate chromatin access: chromatin modifying complexes and chromatin remodeling complexes [40,46]. One specific modification of histones is acetylation, catalyzed by histone acetyltransferase (HAT) enzymes, which weakens the histone–DNA interaction and has been therefore associated with transcriptional activation. HAT activity is intrinsic to numerous transcriptional coactivators, including p300, a functional homolog of CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF). HATs can also acetylate certain transcription factors, thus influencing their activities [46]. Acetylation, however, is a reversible process, as deacetylation is regulated by histone deacetylases (HDACs), these being generally associated with transcriptional repression. Mammalian HDACs are grouped into three subclasses: class I, class II and class III HDACs [14,46]. On the other hand, chromatin-remodeling factors use the free energy freed by ATP hydrolysis to loosen DNA–histone contacts and thus facilitate the movement of the nucleosomes along a particular DNA sequence (originally termed ‘sliding’). Common to all chromatin-remodeling complexes is an ATPase subunit, the motor of the complex. One important chromatin-remodeling enzyme is SWI/SNF (switching/sucrose non-fermenting), which is a multisubunit complex that was first identified in yeast and is highly conserved among eukaryotes. The mammalian SWI/SNF family consists of complexes that contain one of two ATPases, either brahma (BRM) or brahma-related gene 1 (BRG1), as the catalytic subunit, which is associated with a variety of subunits called BRG1-associated factors (BAFs) [40].

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Box 2. The p38 MAP kinase signaling pathway The p38 mitogen-activated protein kinase (MAPK) pathway was initially described to be preferentially activated by different types of stress and cytokines, but numerous studies have since implicated this pathway in the regulation of a wide spectrum of cellular processes, including cell-cycle arrest, apoptosis, senescence, regulation of RNA splicing, tumorigenesis or differentiation of various cell types such as adipocytes, cardiomyocytes, neurons and myoblasts (reviewed in [47– 50]). In vertebrates, there are four p38 MAP kinases, p38a, p38b, p38g (SAPK3, ERK6) and p38d (SAPK4), which are all phosphorylated and activated by the MAPK kinase MKK6 (Figure I). Another p38 MAPK kinase is MKK3, which activates p38a, p38g and p38d, whereas MKK4 can also, in some cases, activate p38a. Once activated, p38 MAPKs phosphorylate serine/threonine residues of their substrates, which include transcription factors as well as protein kinases (see [49] and references therein). The identification of physiological substrates for p38a and p38b has been facilitated by the availability of specific pyridinyl imidazole inhibitors such as SB203580/SB202190 and the recently reported inhibitor of the four p38 isoforms (BIRB0796) [51] (Figure I). Knockout mice for p38a have been generated, but they die at midgestation [52–54], whereas tissue-specific knockouts have implicated p38a in cardiomyocyte proliferation and survival [55,56]. Recently, p38b, p38g, p38d and double p38g p38d knockout mice have been also generated, which appear to be viable and fertile [57,58].

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Other proteins Tau eEF2K SAKS1 Cdc25 SAP90 SAP97 α1-syntrophin

Phosphorylation of MEF2 by p38 MAPK The substrate specificity of the different p38 isoforms is known to overlap. Different reports have shown that p38a and p38b phosphorylate and enhance the transcriptional activities of MEF2A and MEF2C, but not MEF2D [27,28,59,60]. By contrast, p38g only weakly phosphorylates MEF2A, MEF2C and MEF2D in vitro and barely stimulates their transcriptional activities in vivo, whereas p38d does not phosphorylate any of them [23,61]. MEF2C can be phosphorylated by p38 MAPKs in the transactivation domain residues Thr293, Thr300 and Ser387 [27]. Phosphorylation of all these residues is important for MEF2C activation by p38 MAPK in lymphoid cells [27]. The p38 MAPK phosphorylation sites in MEF2C are conserved in MEF2A (amino acid relationship between MEF2C–MEF2A: Thr293–Thr312; Thr300–Thr319; Ser387– Ser453). However, Ser453 did not play any regulatory role in CHO

MAPK induces the transcriptional activity of MEF2 proteins (Box 2). Han and Ulevitch [27] were the first to demonstrate that, in lipopolysaccharide-stimulated macrophages, p38 MAPK directly phosphorylated the transactivation domain of MEF2C on Thr293, Thr300 and Ser387. Importantly, p38 MAPK was also shown to phosphorylate endogenous MEF2A and MEF2C in muscle cells in vivo [22,23,28]. However, Thr293 was the only crucial MEF2C regulatory phosphorylation site in skeletal muscle [23], suggesting that MEF2 regulation by p38 MAPK might involve selective phosphorylation of distinct residues in a tissue-restricted manner. The interactions between MEF2 transcription factors and MRFs during muscle differentiation [20] raise the possibility that p38-MAPK-mediated phosphorylation of MEF2 family members might contribute to the transcriptional synergy between MyoD and MEF2. However, several pieces of evidence argue against this possibility. Indeed, the interaction between MEF2C and MyoD in a mammalian two-hybrid system is not affected by p38 MAPK [23], and mutation of Thr293 to alanine, which prevents phosphorylation and activation of MEF2C in muscle cells, does not affect MyoD–MEF2C functional synergism [23,29]. Moreover, a Gal4–MyoD fusion protein that is impaired in its association with MEF2 proteins can still be activated by MKK6 [3,23]. These findings suggest that distinct www.sciencedirect.com

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Figure I.

and 293 cells, suggesting the existence of cell-type-dependent and isoform-specific phosphorylation events [59]. In agreement with this, Thr293 appears to be the only p38 phosphorylation site that is important for MEF2C regulation in differentiating myocytes [22,23,28]. Recently, abrogation of p38 MAPK signaling was shown to block MEF2 activation in a MEF2 transgenic ‘sensor’ mouse, leading to the inhibition myogenic differentiation in somite cultures and in embryos in vivo [62].

mechanisms might control the stimulation of the intrinsic MEF2C transcriptional activity and the induction of functional synergism between MEF2C and MyoD. Regulation of the MRF transcriptional activity by p38 MAPK The requirement for p38 MAPK in the activation of MyoD-dependent muscle promoter transcription in a MEF2-independent manner suggests that MyoD (and other MRFs) are likely to be direct targets of p38 MAPK. Accordingly, p38 MAPK was able to efficiently phosphorylate Ser5 of MyoD both in vitro and in vivo [23]. However, mutation of Ser5 to alanine did not alter significantly MyoD transcriptional activity [23], indicating that p38 MAPK stimulates MyoD-dependent transcription by, as yet unidentified, indirect mechanisms. One possibility would be that p38 MAPK stimulates myogenic transcription by targeting the MRF-associated cofactors and/or chromatin-modifying enzymes. Consistent with this prediction, phosphorylation of the obligate MyoD partner E47 by p38 MAPK has been shown to have important consequences for muscle gene transcription [30] (Box 3). In particular, p38 MAPK-mediated phosphorylation of E47 at Ser140 induced MyoD–E47 heterodimer formation, subsequent binding to the E box on muscle promoters and activation of muscle-specific

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gene transcription. By contrast, non-phosphorylatable E47 (Ser140 mutated to Ala) failed to associate with MyoD, displaying reduced myogenic potential. Elegant studies by Dilworth et al. [31], using forced dimers of MyoD and/or E47 fusion proteins, have demonstrated that the role of E proteins is not limited to providing a dimerization partner that facilitates MyoD–DNA binding. Thus, forced MyoD homodimers were able to bind to a muscle E box in the context of nucleosomes, but, contrarily to a forced MyoD–E7 heterodimer, they could not drive transcription from the E-box-containing promoter owing to its inability to recruit the necessary p300 HAT activity to the promoter region [9,10]. The authors propose that the heterodimers might provide a scaffolding that is conformationally preferable over the homodimers for establishing a coactivator bridge between the proximal E boxes and the minimal promoter to activate transcription [31]. In this scenario, p38 MAPK might be favoring both the formation of the functional MyoD–E47 transcription factor as well as the subsequent interaction with DNA and the chromatinassociated proteins. Nevertheless, some issues remain unclear: should all MyoD-dependent promoters be regulated alike? Does p38 regulate the expression of all MyoD-dependent genes? In this regard, a recent chromatin immunoprecipitation (ChIP) coupled with mouse promoter DNA microarray hybridization (‘ChIP-on-chip’) analysis has led to the identification of w100 novel genes bound by MyoD both in myoblasts and in differentiated myocytes [32]; intriguingly, activation of the target genes did not always correlate with MyoD binding, consequently raising additional fundamental questions: what dictates the preferential binding of MyoD to different target promoters?; what leads to transcriptional activation or inactivation of MyoD-bound genes in myoblasts? In a different study, Bergstrom et al. [33] applied expression arrays and ChIP analysis to a cellular model system of

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MyoD-mediated myogenesis, showing that MyoD was bound to the regulatory regions of all the muscle genes studied, whether expressed early or late during muscle differentiation. Importantly, these authors identified specific loci where MyoD was stably bound but did not activate transcription without p38 MAPK signaling. Thus, the p38 MAPK signaling pathway, rather than acting globally on all MyoD-regulated genes, can apparently modulate the activity of MyoD at a restricted subset of promoters, establishing dynamic modulation of the MyoD-induced programs of gene expression. Future investigations should clarify whether the MyoD–E47 heterodimer is active on all MyoDresponsive promoters and whether the p38-MAPKmediated phosphorylation of E47 affects its dimerization with other MRFs, and hence the expression of the corresponding target genes. Regulation of chromatin remodeling activities on muscle-specific promoters by p38 MAPK Although all MRFs can bind to the muscle E box with similar affinities, their efficiencies in initiating transcription from normally silent genes differ significantly, with myogenin being less effective than Myf5 and MyoD. The main explanation for this might be that MyoD and Myf5, but not myogenin, contain the H/C and helix III domains endowed with chromatin-remodeling activities (Figure 2). Notably, these two domains of MyoD have been shown to mediate interaction with the transcription factor Pbx on the myogenin promoter, providing stable binding of MyoD to this promoter [6]. A joint effort between the groups of Tapscott and Imbalzano has subsequently uncovered a two-step connection between Pbx/MyoD and the chromatin remodeling complex SWI/SNF, which might underlie the initiation of myogenin gene transcription. According to this model, Pbx is constitutively bound to the myogenin promoter through a Pbx-binding site, located adjacent to a non-canonical E box, and associates with MyoD in a SWI/

Box 3. p38 MAP kinases in the control of muscle-specific gene expression During the proliferation stage, in undifferentiated myoblasts, the activity of MyoD is repressed by the association of E proteins with Id (and to a lesser extent by MyoD–Id association), preventing the formation of functional MyoD–E-protein heterodimers and further binding to the E box on the muscle promoters [1]. Notably, on the myogenin promoter, the activity of MyoD can also be repressed by its association with HDAC1 in myoblasts [16,17]. The transcription factor Pbx has also been found on the myogenin promoter in undifferentiated myoblasts, next to a non-canonical E box [6]. These latter results, concerning exclusively data for the myogenin promoter, are shown inside a dashed-line box (Figure I). At early stages of myogenic differentiation, Id levels are transcriptionally downregulated, allowing the potential formation of MyoD–Eprotein heterodimers [1]. Importantly, p38 MAPK is activated at the onset of muscle differentiation and phosphorylates E47 at Ser140, promoting the formation of the functional MyoD–E47 heterodimer rather than the nonfunctional homodimers, and subsequent binding of the heterodimer to the E box on different muscle promoters [30]. HDAC1 is dissociated from MyoD, whereas HATs (p300/PCAF) are recruited to regulatory regions through association with MyoD–E47 [9– 13]. Phosphorylation by p38 MAPKs also allows targeting of the SWI/SNF chromatin-remodeling complex to the muscle promoters, as well as increased transcriptional activity of MEF2, which, through www.sciencedirect.com

interaction with MyoD, contributes to the overall induction of muscle gene transcription [3,20,22,23,28,29,36]. Finally, p38 also facilitates the phosphorylation and progression of RNA polymerase II, in a MyoDmediated feed-forward circuit [42]. On the myogenin promoter (see dashed-line box), MyoD–E47 heterodimers have been proposed to interact with Pbx, through a Pbx-binding site and a non-canonical E box. Pbx-bound MyoD recruits HATs and the SWI/SNF complex (in a p38-MAPK-dependent manner), and subsequently MyoD–E47 can access the canonical E box in the promoter [6,34]. Whether this Pbxbased model applies to the activation of other muscle genes, in addition to myogenin, awaits investigation. In terminal-differentiation stages, the expression of MRF4 is induced (see also Figure 1). It should be emphasized that MRF4 is the most abundant MRF in adult muscle tissue. As E47 can heterodimerize with MRF4, it is proposed that p38 phosphorylation of E47 at Ser140 will promote the formation of MRF4–E47 heterodimers (as it does with MyoD–E47 [30]) and the subsequent binding to the E boxes on target genes. Upon p38 MAPK phosphorylation of Ser31 and Ser42 in the Nterminal transactivating domain of MRF4, the overall MRF4-mediated transcription is reduced [26]. While the effect of p38 MAPK on Eprotein–MRF activity can occur throughout myogenesis, the p38MAPK–MRF4-mediated repressive mechanism is gene selective and specifically occurs only at terminal stages of differentiation.

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SNF-independent manner. Then, Pbx-associated MyoD recruits SWI/SNF, which in turn will facilitate direct binding of MyoD to the canonical E box of the myogenin promoter [6,8,34] (Box 3). Thus, binding sites for cofactors such as MEF2 or Pbx might help to expose a crucial E box or substitute for an E box and facilitate the formation of a stable and functional MyoD transcriptional complex [1,8,35]. Given the known ability of p38 MAPK to phosphorylate some of these cofactors (i.e. MEF2) as well as particular bHLH proteins (i.e. E47, MRF4 – see below), www.sciencedirect.com

the regulation of the homo- or heterotypic interactions by p38 MAPK-mediated posttranslational modification cannot be discarded. A breakthrough in these studies came from Simone et al. [36], who showed that, in the absence of p38 MAPK signaling, the chromatin of the myogenin and MCK promoters was not remodeled under differentiationpromoting conditions. Most interestingly, pharmacological inhibition of p38a and p38b activity prevented the association, on these muscle promoters, between MyoD

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and the ATPase subunits of the SWI/SNF complex, BRG1 and BRM, although neither the acetylation status of histones nor the recruitment of p300 and PCAF were affected. These results demonstrate a crucial and specific role for p38 MAPK in the recruitment of SWI/SNF to muscle gene promoters, providing an additional mechanism to account for the positive effect of p38 MAPK in myogenesis [36]. The SWI/SNF subunit BAF60 could be phosphorylated by p38 MAPK in vitro, although the functional relevance has yet to be established. It is noteworthy, however, that several BAF60 isoforms have been implicated as the surface for interactions between the SWI/SNF complex and transcription factors [37,38]. Further studies by de la Serna et al. [34] have shown that BRG1 can interact with both MyoD and Pbx on the myogenin promoter. Taken together, it is reasonable to suggest that p38 MAPK might control not only the BRG1based association of SWI/SNF to the myogenin promoter but also its interactions with acetylated chromatin, MyoD and Pbx (Box 3). The work by Simone et al. [36] further showed that the p38 MAPK-mediated activation of a chromatin-integrated Gal4-responsive promoter by a Gal4–MyoD(N-terminal) fusion protein required BRG1 and BRM. Intriguingly, the Gal4–MyoD(N-terminal) neither binds MEF2 nor contains any of the domains previously reported to mediate activation of genes in repressive chromatin [5,20]. It seems therefore unlikely that regulation of MyoD-dependent transcription by the p38-MAPK–SWI/SNF pathway relies exclusively on p38-MAPK-mediated phosphorylation of MEF2 or on MyoD–Pbx interactions. These results are in conflict, at least in part, with the model in which the interaction of MyoD with Pbx (through the MyoD C/H and helix3 domains) would be necessary for SWI/SNF recruitment [6,34]. As a reconciling alternative, SWI/SNF could interact with the acidic transactivation domains or other regions of sequence-specific transcription factors on the target loci [39,40]. Whether the effects of the p38 MAPK pathway on E47–MyoD heterodimer formation and on the SWI/SNF complex recruitment are exerted exclusively on MyoD-dependent promoters or also affect promoters regulated by other MRFs, and whether they are mediated by interactions with Pbx or other cofactors, awaits further investigation. Regulation of RNA polymerase II recruitment to muscle-specific promoters by p38 MAPK Studies from several groups have indicated that p38 MAPK might contribute to the recruitment of the active RNA polymerase II to muscle-specific promoters through at least three different mechanisms. First, recruitment of the SWI/SNF complex has been linked to the engagement of the active fraction of RNA polymerase II [41]. Accordingly, the inhibition of BRG1/ BRM recruitment to muscle promoters by treatment with the p38a/p38b inhibitor SB203580 correlated with reduced levels of active RNA polymerase II at the myogenin and MCK promoters [36]. Second, a muscle gene transcription circuit initiated by MyoD in association with MEF2 and RNA polymerase II is facilitated by p38 MAPK [42]. MyoD has been shown to www.sciencedirect.com

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initiate the expression of specific MEF2 isoforms at the onset of differentiation and to activate (through an unknown mechanism) the p38 MAPK pathway, which in turn will facilitate MyoD and MEF2 binding at genes expressed late in the myogenic program. Importantly, the binding of MEF2D has been shown to recruit RNA polymerase II, correlating with the transcription of these genes. Conversely, expression of late-activated genes could be advanced by precocious activation of p38 MAPK and expression of MEF2D, demonstrating a mechanism for temporally patterning muscle gene expression through a MyoD-mediated feed-forward circuit involving p38 MAPK [42]. Two distinct roles have been proposed for p38 MAPK in this circuit. On one side, p38 MAPK might function as a rate-limiting factor in promoting the binding of MEF2D and MyoD to muscle promoters, most likely through an effect on chromatin – i.e. through targeting the SWI/SNF complex to muscle loci. Furthermore, p38 MAPK might facilitate the progression of RNA polymerase II, probably through the phosphorylation of MEF2D. Despite the original nature of this model, the latter assumption is in discrepancy with results from other groups showing that MEF2D, in particular, is not a good phosphorylation substrate for p38a and p38b (see Box 2). These differences might be explained by the use of different experimental approaches, strongly suggesting the need to reconfirm the phosphorylation of the MEF2 isoforms by p38 MAPK in the muscle context. Nonetheless, the proposal of a MyoD-initiated feed-forward circuit involving p38 MAPK and MEF2 is appealing. Third, the yeast p38 MAPK, Hog1, has been shown to interact with, and recruit, the RNA polymerase II complex to yeast stress-responsive promoters such as STL1, by association with the Hot1 transcription factor [43]. Taken together, these studies demonstrate that p38 MAPK plays a role in chromatin remodeling through its action on different transcriptionally competent molecules, and these mechanisms might be shared by different organisms ranging from mammals to yeast. Moreover, these findings establish a link between differentiationactivated p38 MAPK and recruitment of chromatinremodeling complexes to transcriptionally active loci during skeletal myogenesis. This also extends the function of p38 MAPK beyond its ability to activate gene expression by direct phosphorylation of transcription factors. Selective repression of myogenesis by p38 MAPK A novel and unexpected inhibitory function of p38 MAPK at late stages of myogenesis has been recently reported in two independent studies. Weston et al. [44] showed that treatment of primary limb mesenchymal cultures, which should differentiate to cartilage, with p38a/p38b inhibitors enhances muscle formation rather than promoting chondrogenesis. In addition, G8 and C2C12 muscle cells co-cultured with the primary limb mesenchymal cells also showed enhanced expression of myogenic markers and myotube formation upon treatment with the p38 MAPK inhibitors. Likewise, the transcriptional activity of MEF2–GAL4 fusion proteins expressed in primary cultures was also enhanced by treatment with the p38a/ p38b inhibitors [44]. Using a different experimental

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approach, Suelves et al. [26] showed that inhibition of p38 MAPK activity at late stages of C2C12 cell differentiation resulted in increased expression of certain skeletal muscle genes. In particular, p38 MAPK phosphorylated in vitro and in vivo Ser31 and Ser42 located in the N-terminal transactivating domain of MRF4, leading to downregulation of its transcriptional activity, which induced a repressive, but selective, effect on the expression of muscle genes during terminal differentiation [26] (Box 3). Recently, elegant studies by Kassar-Duchossoy et al. [45], using Myf5:MyoD double-null mutants whose MRF4 expression is unaffected, have demonstrated an unexpected role for MRF4 in the early stages of mouse myogenesis, challenging its established role in terminal differentiation. Thus, the specific function played by MRF4 during myogenesis is more complex than previously anticipated – MyoD and Myf5 seem to be required for commitment to the myogenic lineage, whereas myogenin plays a role in the expression of the differentiated phenotype, and MRF4 can partly exert both functions. Concluding remarks The formation of skeletal muscle is a well-orchestrated multistep process controlled by the MRF family of transcription factors. Several cofactors enhance or repress the myogenic potential of the MRFs, either directly or indirectly, thus influencing the expression of musclespecific genes. Phosphorylation and activation of MEF2, a co-activator of the MRF family member MyoD, was for many years the sole explanation for the promyogenic effect of p38 MAPK. However, it is now clear that the stimulation of MyoD-dependent transcription by p38 MAPK also involves MEF-2-independent mechanisms. These include the p38-MAPK-mediated phosphorylation of the MyoD dimerization partner E47, which promotes formation of functional MyoD–E47 heterodimers and initiation of muscle-specific transcription, as well as the recruitment of chromatin-remodeling SWI/SNF activity and RNA polymerase II to muscle gene promoters. Overall, a new model for myogenic differentiation is emerging in which the MRFs display unique functions and activate different subsets of muscle genes in a distinct spatial and temporal fashion. In addition to the promyogenic role of p38 MAPK at early myogenic stages described above, an unexpected repressive p38 MAPK function has also been identified, which operates selectively at late stages of muscle differentiation. Although p38 MAPK emerges as a pivotal molecule orchestrating sequential events in the myogenic pathway, many details of p38-MAPK-induced myogenesis remain to be elucidated – for example, the relative contribution of the four p38 MAPK family members to muscle differentiation. Most of the work that demonstrates the requirement for p38 MAPK in myogenesis is based on the use of synthetic compounds such as SB203580, which only inhibit the activity of p38a and p38b. Whether different p38 MAPK family members specifically regulate the expression of particular subsets of genes, at different stages of differentiation, and whether they possess inducing or repressing activities, remain to be determined. The identification of new myogenic substrates for www.sciencedirect.com

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the different p38 MAPK family members as well as the upstream signaling mechanisms will undoubtedly increase our understanding of how p38 MAPK regulates myogenesis. Finally, it is important to note that the cellculture studies outlined here require verification using in vivo models, including the generation of mice with muscle-specific inactivation of the individual p38 MAPK family members. These mice should provide powerful biological models to address the regulation of muscle formation by p38 MAPK. Acknowledgements We apologize to the authors whose original work is not included in the references owing to space limitations. Work in the authors’ laboratories is supported by the Spanish Ministry of Education and Science (SAF2004– 06983, HF2004–0185), the Muscular Dystrophy Association, Marato-TV3 and AFM. E.P. was a recipient of a Novartis postdoctoral fellowship.

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