Epigenetic regulation of myogenesis - Taylor & Francis Online

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Epigenetics 4:8, 541-550; November 16, 2009; © 2009 Landes Bioscience

Epigenetic regulation of myogenesis Eusebio Perdiguero,1,* Pedro Sousa-Victor,1 Esteban Ballestar2 and Pura Muñoz-Cánoves1,3,* 1 Cell Biology Group; Department of Experimental and Health Sciences; Pompeu Fabra University (UPF); CIBER on Neurodegenerative diseases (CIBERNED); Barcelona, Spain; 2Chromatin and Disease Group; Cancer Epigenetics and Biology Programme (PEBC); Bellvitge Biomedical Research Institute (IDIBELL); Barcelona, Spain; 3Institució Catalana de Recerca i Estudis Avançats (ICREA); Barcelona, Spain

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dult skeletal muscle provides a unique paradigm for studying stem to differentiated cell transitions. In response to environmental stress, quiescent muscle stem cells (satellite cells) are activated and proliferative, at which stage they can either differentiate and fuse to form new muscle fibers or alternatively self-renew and maintain the muscle stem cell reservoir. This multi-step myogenic process is orchestrated by muscle regulatory proteins such as Pax3/Pax7 and members of the MyoD family of transcription factors. Findings published over the past few years have uncovered that epigenetic mechanisms critically repress, maintain or induce muscle-specific transcriptional programs during myogenesis. These studies are increasing our understanding of how muscle lineage-specific information encoded in chromatin merges with muscle regulatory factors to drive muscle stem cells through transitions during myogenesis.

Key words: myogenesis, satellite cell, epigenetics, transcription factors, microRNAs Submitted: 10/01/09 Accepted: 10/05/09 Previously published online: www.landesbioscience.com/journals/ epigenetics/article/10258 *Correspondence to: Eusebio Perdiguero and Pura Muñoz-Cánoves; Email: eusebio. [email protected] and [email protected]

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Skeletal muscle formation (myogenesis) in the vertebrate embryo relies on myogenic progenitor cells, which support developmental muscle growth and give rise to nearly all muscle tissue cell descendants. During the embryonic stage, early muscle progenitor cells express Pax3 and Pax7, two paired-homeobox transcription factors responsible for cell survival and myogenic lineage specification. After birth, myogenic progenitors adopt a satellite position outside the myofiber (under the basal lamina), entering a quiescent state.1 Upon environmental activating signals derived from injury or stress, satellite cells undergo apico-basal asymmetric cell divisions to

Epigenetics

both maintain their population through self-renewal and to give rise to committed myogenic cells; the latter cells (myoblasts) proliferate, migrate, differentiate and fuse into new fibers,1-3 thereby sustaining postnatal muscle repair and growth. Classically, Pax3/7 transcription factors are considered the major regulators of muscle cell specification and tissue formation during development, playing a similar role during adult muscle regeneration. Interestingly, recent in vivo studies have challenged this view, by demonstrating that their transcriptional activity is dispensable for de novo myogenesis in the adult.4 This discovery illustrates that distinct molecular players govern embryonic and postnatal myogenesis: as opposed to developmentally driven processes, where Pax3/7 play a predominant role, in adulthood, muscle fibers and their associated satellite cells respond predominantly to environmental signals, sustaining regeneration in response to injury or enlargement in response to workload through mechanisms which do not rely on these transcription factors. Many environmental cues can activate satellite cells, e.g., adhesion molecules, growth factors and cytokines released by the neighboring cells—a local milieu composed of fibroblasts, interstitial cells, resident macrophages and microvasculature-related cells, namely endothelial cells, pericytes and mesoangioblasts.5 Extracellular cues are transmitted to the muscle cell nucleus through signaling cascades, among which the p38 MAPK and the IGF1-AKT pathways are thought to play a major role.6-15 In particular, signaling pathways will induce the downregulation

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of quiescence-associated genes in satellite cells in response to external activating signals and will regulate the subsequent activation of a muscle-specific transcription factor network, composed of four muscle-specific regulatory factors (MRFs) which belong to the basic helix-loop-helix (bHLH) family of transcription factors (Myf5, MyoD, Myogenin and MRF4). The MRFs cooperate with ubiquitous E proteins (the E2A gene products, E12 and E47, and HEB) and MEF2 transcriptional regulators16-18 through association with E-boxes and MEF2-boxes, respectively, on muscle loci, thereby inducing musclespecific gene transcription. Recent findings have uncovered an epigenetic control layer over the basal muscle-specific transcriptional machinery during myogenesis. Indeed, switching the satellite cell from a repressive to an activating state is caused by a combination of genetic and epigenetic events ranging from covalent modification of histones and transcription factors to chromatin remodeling activities which, together, will lead to full expression of the muscle-specific gene program.1-3,18 Notably, in response to environmental signals, the epigenetic machinery active during myogenesis appears to use predominantly p38 MAPK and AKT signaling cascades to impinge on the classical transcriptional machinery,8 suggesting an interconnection between transcription factors and chromatin-associated activities through signaling pathways on muscle loci. In this review, we will discuss recent advances in the epigenetic regulation of postnatal myogenesis and its cross-talk with the basic muscle-specific transcriptional machinery, including the impact of microRNAs on the myogenic scenario which, together, have shed some light to our understanding of how myogenesis is controlled. Transcriptional Regulation of Myogenesis The ordered and sequential expression of MRFs upon activation of satellite cells, and their underlying mechanisms of action, is beginning to be elucidated. MyoD and Myf5 transcription factors are expressed in undifferentiated proliferating

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myoblasts, while Myogenin and MRF4 are expressed subsequently at early and late differentiation stages, respectively.17,18 The Pax7 transcription factor is expressed in quiescent satellite cells, serving as a bona fide marker for their localization beneath the basal lamina.1-3 Interestingly, recent studies have demonstrated that Myf5 is a Pax7 target gene in satellite cells upon activation.19 Activated satellite cells expressing Pax7 and Myf5 upregulate MyoD entering into a proliferative state.20,21 A model has been proposed whereby a cross-inhibitory interaction between Pax7 and the MRFs controls satellite cell fate decisions (proliferation, differentiation or self-renewal) with the Pax7/MyoD ratio being the critical modulator of this choice.22 Consistent therewith, in vivo lineage tracing experiments for Myf5 have demonstrated that satellite cells which have never expressed Myf5 constitute the self-renewing population of stem cells23 (Fig. 1). Given the recent finding that satellite cell-dependent de novo myogenesis in the adult can be Pax3/Pax7-independent, the proposed model needs to be rethought.4 Other transcription factors and layers of transcriptional-epigenetic control are thus needed for satellite cell activation in adult muscle. In this context, it has been recently demonstrated that FoxO3 binds to the Myod promoter regulating its transcription in proliferating myoblasts.24 Proliferation and differentiation are mutually exclusive processes in myogenesis. Indeed, cessation of proliferation by downregulation of cyclin D1 and dephosphorylation of pRb are required for initiation of muscle differentiation-specific gene expression. The p38 MAPK pathway, by antagonizing the proliferation-promoting cJun N-terminal kinase (JNK) pathway, leads to downregulation of cyclin D1 expression, thus promoting cell cycle-exit and allowing muscle differentiation to commence.11 Initiation of the differentiation program requires the association of MyoD with E proteins and the binding of the MyoD/E protein heterodimers to the E boxes of the muscle gene promoters. A major mechanism for maintaining a proliferative state in myoblasts in the presence of Myf5 and MyoD is through Id (inhibitor of differentiation), an HLH protein lacking the basic

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DNA-binding domain, which is expressed at high levels in proliferating myoblasts. Association of Id to MyoD or E proteins results in the formation of non-functional E protein/Id and MyoD/Id heterodimers that cannot bind to E boxes, thus preventing anticipated myogenic differentiation. The downregulation of Id expression when myoblasts exit the cell cycle at the onset of differentiation thus allows the formation of functional heterodimers and muscle differentiation-specific gene expression to proceed.16-18 Other proteins (like the HLH factors Twist, MyoR and Myst-1, and ZEB and I-mfa proteins) are also repressors of the MRFs through direct association or sequestration of the E boxes.16 In addition to its role in the myoblast cell cycle exit, several studies have unambiguously shown that the p38 MAPK pathway is essential for the onset of differentiation14 by modulating the activity of transcription factors and epigenetic regulators on muscle loci: (1) induction of MyoD/E47 heterodimer formation by phosphorylation of E47; (2) recruitment of the SWI/SNF chromatin remodeling complex—probably via phosphorylation of the BAF60c subunit; (3) induction of MEF2 transcriptional activity by direct phosphorylation; (4) recruitment of the Trithorax group TrxG/Ash2L complex through p38-phosphorylated MEF2D8,14,25 (see below). Transcription factors other than MRFs and MEF2 proteins have also been shown to contribute to the activation of muscle-specific transcription. Pbx/Meis transcription factors are constitutively bound to E-boxes at muscle gene promoters and mediate the initial binding of MyoD/E47.26 Recently, the serum response factor (SRF), known to promote the transcriptional activation of muscle genes like Acta1,27 has been shown to interact with the transcriptional co-factor enhancer of polycomb1 (Epc1), providing a recruiting complex for histone acetyltransferase p300 to muscle specific genes,28 favoring muscle gene transcription. Moreover, changes in the general transcription machinery have also proven to be essential for the initiation of muscle-specific gene expression. A newly-described mechanism involves the disruption of the canonical holo-TFIID complex and its replacement by a novel

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TRF3/TAF3 (TBP-related factor 3/ TATA-binding protein-associated factor 3) complex.29,30 This switch has been postulated as regulating spatial and temporal patterns of gene expression. Epigenetic Control of Muscle Gene Expression The progressive dissection of the transcription factor network involved in myogenesis has revealed the participation of various elements of the epigenetic and chromatin machinery. Chromatin is generally repressive and changes in its structure are necessary not only to allow access to target sequences but also to locally lead the chromatin to specific states of transcriptional competence. Two main enzymatic activities induce chromatin modifications and regulate chromatin access: chromatin modifying complexes and chromatin remodeling complexes.31,32 Chromatin modifying complexes contain catalytic subunits of different histone modifying enzymes that catalyze the reversible posttranslational modification of histones and other chromatin factors. Histone modifications are associated with both active gene expression, such as acetylation of histones H3 and H4 (acetyl H3, acetyl H4) and trimethylation of lysine 4 of histone H3 (H3K4me3), and gene repression, including trimethylation of lysines 9 and 27 of histone H3 (H3K9me3; H3K27me3) and trimethylation of lysine 20 of histone H4 (H4K20me3). Combinations or sequential addition of various post-translational modifications to histone-tail amino acid residues have different functional consequences for gene activity and chromatin organization and are thought to form a histone code.33 The modification status of a specific residue is maintained through the activity of various enzymes. For instance, the acetylation status of histones is maintained by the antagonic action of histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes. HAT activity is intrinsic to numerous transcriptional coactivators, including p300, a functional homologue of the CREBbinding protein (CBP), and the p300/ CBP-associated factor (PCAF). HATs can also acetylate certain transcription factors thus influencing their activities.31


Conversely, histone deacetylation is generally associated with transcriptional repression. Mammalian HDACs are grouped into three subclasses: Class I, Class II and Class III HDACs (also called Sirtuins).31,34 Additionally, chromatin-remodeling factors use the free energy released by ATP hydrolysis to loosen DNA-histone contacts and thus facilitate the movement of the nucleosomes along a particular DNA sequence. Common to all chromatinremodeling complexes is an ATPase subunit, the motor of the complex. Among a variety of chromatin-remodeling enzymes, an important one 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).32,35 In addition to post-translational modification of histones, methylation of CpG dinucleotides constitutes a major source of epigenetic information. Specifically, methylation at CpG-containing promoters constitutes a mechanism to repress specific sets of genes. This mechanism is closely linked to elements of the histone modifying machinery and, for instance, it is accepted that Polycomb group proteins, that catalyze trimethylation of K27 H3, target DNA methylation at specific sites.36-38 Maintenance of quiescence and proliferation. Epigenetic regulatory events play an essential role in maintaining quiescence and proliferation states in satellite cells, thus preventing their ectopic differentiation. DNA methylation has classically been postulated as one of the major repressive systems acting on muscle gene loci; indeed, pioneering experiments showed that DNA methyltransferase inhibitors (like 5-azacytidine) were capable of inducing the transdifferentiation of fibroblasts into myoblasts.39,40 Demethylation of the Myod promoter, leading to its derepression, underlay this mind-challenging effect (reviewed in ref. 41). Specifically, demethylation including the distal enhancer of Myod and the

Epigenetics

Myog promoter appears necessary for the differentiation program to proceed.41,42 However, the precise mechanisms regulating methylation/demethylation during adult myogenesis are still far from being understood; it still presently unknown whether such methylation/demethylation changes imply a specific event for a subset of genes or precedes/follows general gene expression. Future studies should investigate which mechanism and the specific DNA methyltransferases and demethylases acting during myogenesis. Repressive epigenetic mechanisms acting on chromatin-associated histones have been more thoroughly investigated. In quiescent and proliferating satellite cells, the histones of muscle differentiationspecific gene promoters are hypoacetylated and contain H3K9me2 and H3K27me3 residues. This repressive environment is catalyzed by HDACs and histone lysine methyl-transferases (HKMT) from the Polycomb group (PcG) and Suv39H1 families.18,41 Repression of transcription by PcG proteins is thought to be the gene default state, as PcG proteins can be found in both active and inactive genes, being repression counteracted by TrxG proteins which are specifically targeted to active genes.43,44 The HKMT Ezh2, the catalytic activity of the Polycomb repressive complex 2 (PRC2) is recruited to inactive muscle gene promoters by the transcriptional regulator Ying Yang 1 (YY1) which promotes transcriptional repression through H3K27 trimethylation.45 However, further studies are needed to demonstrate whether PRC2-mediated repression is a general mechanism for all muscle-specific genes or whether different methylation complexes regulate distinct sets of genes. For instance, some promoters are methylated at H3K9 by the HKMT Suv39H1, which associates with Heterochromatin protein 1 (HP1) and HDAC4/5 proteins.46,47 HP1α and HP1β isoforms associate in turn with MyoD in proliferating myoblasts, leading to inhibition of their transcriptional activity.48 Lastly, methylated residues recruit MBD2, a methyl-CpG binding domain (MBD) protein, which has been shown to act as a repressor in the Myog promoter.49 HDACs do not bind specific DNA elements but interact with chromatin through

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association with transcription factors and other histone-modifying proteins and MBDs. In proliferating myoblasts, several members of class I, II HDACs (HDAC 1, 2, 3, 4 and 5) and Sirtuins (also called class III HDACs; Sir2), interact with MyoD and MEF2, and, in the absence of differentiation-promoting signals, not only remove acetyl groups at histone tails but also maintain transcription factors in a deacetylated state. Interestingly, HATs p300 and the CREB-binding protein or pCAF are found in complexes which include both repressive and activating factors.18,41,50 HDAC1 is recruited to muscle gene promoters by YY1, as part of the complex that contains Ezh2,45 while other studies have shown that HDAC1/2 can be recruited to muscle loci by MyoD.51 Interestingly, Sir2 (whose enzymatic activity is regulated by the availability of NAD +) is found in a protein complex containing pCAF and MyoD, and therefore represses MyoD bound promoters,52 thus suggesting that Sir2 behaves as a redox-sensor in response to metabolic changes regulating an adaptive muscle-specific gene response. HDAC3 and HDAC4 are, in turn, recruited by MEF2 in a complex that also contains the nuclear receptor corepressor NCOR/SMRT (a silencing mediator of retinoic acid and the thyroid hormone receptor53), while, as described above, Suv39H1 can also recruit HADC4/5 to the Myog promoter.47 Very recent studies have shown that the recruitment of HDAC complexes can be also mediated by MBD2.49 Another way of modifying chromatin to control gene transcription involves the replacement of canonical histones with histone variants or the expression of particular histone isoforms in a cell state- or cell type-specific manner.54,55 Expression of the MyoD gene is repressed by the homeobox protein Msx1, which binds to the Histone 1b (H1b) isoform, which is enriched at the core enhancer region of MyoD,56 implicating a yet-to-be understood downregulation of H1b during gene activation. Additional histone replacing mechanisms impacting on the control of myogenesis will likely be deciphered in the future. In summary, the following model can be outlined: in transcriptionally inactive

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muscle regulatory regions, YY1 recruits Ezh2 and HDACs (putatively the full Polycomb repressive complex 2 PRC2), MyoD recruits both pCAF and Sirtuins, and MEF2 recruits HDACs and NCOR/ SMRT; consequently, DNA is methylated and chromatin is hypoacetylated with histones containing H3K27me3 and H3K9me2 marks (Fig. 2A). Overall, YY1, MyoD and MEF2 recruit chromatin modifiers such as HDACs, HKMTs and HP1 when they are bound to the regulatory elements of target genes in proliferating myoblasts. Cell cycle regulation. Activation of the muscle differentiation program in satellite cells requires irreversible cell cycle withdrawal. However, the precise kinetics of the underlying molecular events remain unclear. Indeed, several mechanisms of cell cycle and S-phase genes’ inactivation coexist temporally with mechanisms inducing early activation of the muscle differentiation-specific gene program. The pocket protein pRb regulates cell cycle exit through repression of the E2F transcription factors and recruitment of a still unknown methyltransferase activity promoting H3K9me3 and H3K27me3 marks to cell cycle-associated genes.57,58 Also, Suv39H1 HKMT has been shown to silence S-phase genes.47 Moreover, a satellite cell quiescence-induced gene, the TrxG mixed lineage leukemia 5 (MLL5), has been found to suppress inappropriate expression of S-phase promoting genes and maintain expression of muscle determination genes Pax7 and Myf5 in quiescent cells, thus providing a novel understanding of the mechanism regulating maintenance/transition of satellite cells in their quiescent/active states.59 Activation of the muscle differentiation gene program. Upon reception of the differentiation-promoting signals, the repressive landscape changes rapidly, with PRC2 H3K27me3 repressive marks being substituted by TrxG H3K4me3. It has been proposed that genes poised for transcription are marked by H3K4me2, whereas those actively transcribing are marked by H3K4me3.60 In activated satellite cells, Pax7 binds to H3K4me2 regulatory elements in target genes (such as Myf5), leading to the recruitment of the TRxG histone methyltransferase complex

Epigenetics

that includes Ash2L and Wdr5 subunits, which will in turn induce strong H3K4 trimethylation around the transcription start site, establishing a transcriptionally active domain.19,25 Furthermore, the TrxG complex can be recruited to muscle-specific promoters such as muscle creatine kinase (MCK) through its association with p38phosphorylated MEF2D.25 Interestingly, new evidence indicates that the histone demethylase UTX targets muscle-specific genes leading to a localized demethylation of H3K27me3 within their promoter/ enhancer (Rampalli S and Dilworth FJ, personal communication). Interesting studies in recent years have shown that, in proliferating myoblasts, muscle gene promoters can be partially loaded by muscle transcription factors in an inactive state, in a complex including HDACs/HATs. In this scenario, active HDACs and Sirtuins will deacetylate HATs and inhibit their acetyltransferase activity, as demonstrated for Sir2 and pCAF.52 Moreover, additional studies have shown that non-acetylated MyoD has less affinity for interaction with p300 and CREB-binding protein and retains no transcriptional activity.61,62 As mentioned above, the cells’ redox balance regulates Sir2 activity. Upon receiving differentiation signals, the [NAD +]/[NADH] ratio decreases in myoblasts, with subsequent inhibition of Sir2 activity.18,52 The Sir2/pCAF balance is therefore quickly altered, the deacetylase activity of Sir2 declines and pCAF induces acetylation of several proteins including histones, MyoD and MEF2 and, moreover, it autoacetylates.18 In line with this, the IGF1/AKT signaling pathway has been shown to phosphorylate p300, promoting its association with MyoD and consequently increasing the acetylation of muscle gene promoters.12 This mechanism fits with a previous finding showing that the combined association of p300 and pCAF with MyoD promotes a strong activation of transcription, whereas pCAF alone is only a moderate inducer.62 Altogether, inactivation of HDACs and Sirtuins is coupled with activation of HATs which will promote activation of transcription factors and nucleosomes though a mechanism activated in response to extracellular (for

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Figure 1. Satellite cell-driven myogenesis. Quiescent satellite cells in adult muscles are characterized by Pax7 expression. Upon injury or workload, satellite cells are activated and divide asymmetrically, thereby generating a self-renewing cell and a committed progenitor which begins to express the muscle regulatory factor Myf5. Myf5-expressing cells enter the cell cycle, undergo rounds of proliferation turning into MyoD-expressing myoblasts, which later on will express Myogenin and downregulate Pax7. This pool of cells will differentiate and fuse to form new myofibers during adult muscle regeneration.

example, inflammatory and metabolic) cues. Chromatin remodeling activities have also been shown to play an essential role in the activation of muscle gene promoters, with the recruitment of the SWI/SNF (BAF) chromatin remodeling complexes to myogenic loci being of particular relevance. The SWI/SNF complex facilitates the binding and formation of the RNA polymerase II (Pol II) preinitiation complex and promotes transcriptional elongation. SWI/SNF ATPase subunits BRG1 or BRM contain bromodomains, which recognize acetylated lysines on histone tails,35,63 and are responsible for nucleosome remodeling. Recruitment of SWI/SNF to chromatin occurs through interactions with Pol II, through the bromodomain, and with sequence-specific transcriptional activators.35,63 In the context of muscle-specific gene loci, the SWI/


SNF recruitment is dependent on p38 MAPK activity, since its inhibition could prevent the association of the BRG1 catalytic subunit of SWI/SNF to muscle gene promoters,64 probably through a mechanism involving MyoD/E47 and Pbx.65 Direct phosphorylation of the BAF60c subunit of SWI/SNIF by p38 MAPK has been demonstrated,64 although in vivo evidence of this has yet to be furnished. Supporting the increasing evidence implicating protein kinases in recruitment of chromatin-associated activities and transcriptional induction, ERK and Msk1 kinases have also been reported to recruit nuclear receptors and BRG1 to hormone regulated promoters.66 Further chromatin modifications have been shown to be essential for SWI/SNF recruitment. Arginine methyltransferases Prmt5 and Carm1/Prmt4 induce dimethylation of H3R8 and H3R17 residues

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in muscle promoters.67,68 H3R8me2 is necessary for BRG1 interaction with early muscle differentiation-specific gene promoters like Myog 67 and H3R17me2 for late promoters like MCK.68 Finally, SWI/SNF recruitment could be also mediated by DPF3, a PHD finger protein which binds acetylated and methylated histone residues,69 though this has not yet been demonstrated in mammalian satellite cells. Recently, several mechanisms very distinct and apart in their nature have been proposed as participating in the activation of the muscle gene program. Expression of the Myod gene in Xenopus embryos involves H3.3 variant accumulation, through an unknown remodeling mechanism which could be mediated by methylation of H3.3 at lysine 4;70,71 this latter event remains to be validated in mammalian muscle cells. Additionally,

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Figure 2. Transcriptional and epigenetic control of muscle-specific gene expression. (A) In quiescent and proliferating satellite cells, muscle specific gene promoters are repressed through a combination of mechanisms. MyoD (and E proteins, not shown) is repressed by the association with Id (among other transcriptional repressors) preventing the formation of functional MyoD-E-protein heterodimers. MyoD is also repressed by its association with Sir2 in a complex that also contains pCAF and HP1. YY1 recruits Ezh2 (and the PRC2 complex) and class I HDACs, and MEF2 recruits class II HDACs and NCOR/SMRT. DNA is methylated and chromatin is hypoacetylated, with histones containing H3K27me3 and H3K9me2 marks which may bind other MDB proteins like MBD2. (B) Once extracellular cues are transmitted inside the nucleus through signaling cascades, transcriptionally active muscle regulatory regions will contain phosphorylated/acetylated MyoD/E protein heterodimers, MEF2 dimers and SRF transcription factors. In collaboration with DPF3 and arginine methyltransferases Prmt4/5, the SWI/SNF remodeling complex will be recruited. Transcription will be initiated by Pol II and TRF3/TAF3 transcription complexes. Overall, DNA is demethylated and chromatin is hyperacetylated, with histones harboring H3K9me3 and H3R8/H3R17me2 marks. Proteins targeted by miRNAs are indicated.

oxidative stress-activated focal adhesion kinase (FAK) is necessary for the displacement of MBD2 from muscle-gene loci,49 suggesting that activation of oxidative stress and cell-cell contact cascades may coexist with p38 MAPK and AKT in the induction and transmission of signals to the myoblast nucleus. Taken together, a model can be envisioned where transcriptionally active muscle regulatory regions would contain phosphorylated/acetylated MRF/E protein heterodimer, MEF2 dimers and SRF, which in concert with DPF3 and arginine

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methyltransferases will recruit SWI/SNF remodeling activities, Pol II and TRF3/ TAF3 transcription complexes; subsequently, DNA is demethylated and chromatin is hyperacetylated, with histones harboring H3K9me3 and H3R8/H3R17 marks (Fig. 2B). Epigenetic Regulation of Muscle Fiber Type Skeletal muscles have a heterogeneous fiber-type composition. The fiber-type profile of the different muscles is established

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during development independently of neural control, but postnatal innervation has an essential role in the maintenance and modulation of fiber-type properties in adult muscle. Differences in the speed of adult muscle fiber contraction are related to the expression of different myosin heavy chain (MHC) isoforms (MHC I slow and IIa, IIx, IIb fast), and it has been thoroughly demonstrated that muscle-unloading results in a switch from slow to fast MHC gene expression.72 Moreover, once terminally differentiated muscle fibers are formed, they can differ markedly with

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regard to their metabolic properties (oxidative vs. glycolytic), due to the expression of different metabolic gene programs.72 The Calcineurin-NFAT pathway appears as a major regulator of the maintenance and induction of the slow gene program in adult muscles.73 Interestingly, the transcription factor NFAT is a p38 phosphorylation target in distinct cell types, and is also an effector pathway of cardiac hypertrophy.74 It is therefore tempting to hypothesize that the p38 MAPK pathway could also control particular muscle fiber type gene programs in adult muscles. Metabolic changes in the distinct fiber types have also been shown to be epigenetically regulated. Indeed, the interplay between HATs/HDACs in adult muscle fibers is essential for the regulation of their metabolic attributes, since Class II HDACs are selectively degraded by the proteasome in slow, oxidative myofibers, enabling the MEF2 transcription factor to activate this specific gene program.75 In line with this, HDAC4 is preferentially expressed in fast oxidative fibers and its depletion enhances a glycolytic metabolism.76 This switch is accompanied by type I MHC deacetylation, and fast type IIx and IIb MHCs hyperacetylation and enrichment in H3K4me3.77 Interestingly, the mammalian target of rapamycin (mTOR) was shown to be necessary for the maintenance of mitochondrial oxidative function in skeletal muscle cells through the targeting of YY1 and peroxisome proliferator-activated receptor coactivator (PGC)-1α.78 In addition, some microRNAs have been implicated in fiber type determination (see below). Regulation of Myogenesis by MicroRNAs MicroRNAs (miRNAs) are non-coding single-stranded RNAs of 18–24 nucleotides in length that constitute a new class of post-transcriptional negative regulators of gene expression.79 miRNAs are initially transcribed by RNA polymerase II as long transcripts known as primary-miRNAs (pri-miRNAs),80 that are processed by a nuclear multiprotein complex which contains Drosha and DGCR8/Pasha, generating precursor miRNAs (pre-miRNAs) with stem-loop structures.81,82 These pre-


miRNAs are then transported into the cytoplasm by the nuclear export factor Exportin 5, where they serve as substrates of RNase Dicer to generate ~22 nt miRNA duplexes.82-84 miRNA molecules assemble together with Argonaute proteins to form the RNA-induced silencing complex (RISC), which acts to silence miRNA specific target genes.82 Gene silencing is elicited by base pairing—typically with the 3' untranslated region (UTR) of their target mRNAs—that results in mRNA degradation (if the pairing is perfect) or in inhibition of translation (if there is imperfect matching).82,85 Some studies have addressed miRNA global functions in tissue development by analyzing the effects of Dicer loss of function, which results in the accumulation of unprocessed pre-miRNAs. During muscle embryogenesis, specific inactivation of Dicer results in perinatal lethality, reduced muscle mass and abnormal myofiber morphology, placing miRNAs as critical regulators of embryonic muscle development.86 Many of the miRNAs identified so far are regulators of multiple processes in different tissues and lineages. However, some miRNAs function in a lineage-specific manner. These include miR-133, miR-1 and its variant miR-206, that together constitute the “myomiRs” or muscle-specific miRNAs. miR-1 and miR-133 are clustered on the same chromosomal loci and transcribed together. Further processing yields two independent mature miRNAs with opposing roles in the modulation of skeletal muscle proliferation and differentiation. While miR-1 promotes myogenesis by targeting HDAC4, miR-133 enhances myoblast proliferation by repressing SRF.87 Similarly to miR-1, miR-206 promotes muscle differentiation by targeting the p180 subunit of DNA polymerase alpha, which downregulates its expression, with the consequent inhibition of DNA synthesis, allowing cell cycle withdrawal and commitment to terminal differentiation.88 In addition, miR-1 and miR-206 are essential for the regulation of connexin 43 (Cx43) protein levels, ensuring the proper development of singly innervated muscle fibers through the downregulation of Cx43-dependent gap junctional communication.89

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Interestingly, miRNAs have been implicated in fiber type determination during muscle growth, since miR-1 and 133 are downregulated during functional overload of the muscle and miR-206 is upregulated in the same process, being associated with slow fiber type formation.90 In addition to the myomiRs previously mentioned, ubiquitously expressed miRNAs are also involved in the regulation of skeletal muscle development and function. miR-27 is expressed both in the differentiating skeletal muscle of the embryonic myotome and in activated satellite cells of the adult muscle. Pax3 mRNA is specifically targeted by miR-27, downregulating its protein levels and ensuring normal entry into the myogenic differentiation program.91 miR-181 cooperates in the differentiation program by targeting Hox-A11, a repressor of myoblast terminal differentiation, which results in MyoD induction and the consequent expression of muscle markers.92 Also, miR-29 functions as a positive regulator of myogenesis through feedback inhibition of the transcription factor YY1 which, in combination with Ezh2, functions as a repressor of muscle-specific gene expression,93 as described above. Ezh2 is itself targeted by miR-26a, contributing to its downregulation and allowing terminal differentiation.94 Some of the described miRNAs are themselves regulated by the myogenic transcription factors that are required for skeletal muscle formation. MyomiR genes are direct transcriptional targets of SRF, MyoD and MEF2,95 MyoD directly activates the expression of miR-206,96 and Myogenin and MyoD occupancy was found in regions upstream of miR-1, miR133 and miR-206.97 Conclusions and Perspectives Understanding the regulation of myogenesis and, in particular, how muscle stem cells change their behavior during this process, has been a major aim for years in the skeletal muscle research area. Myogenesis is a well-orchestrated multistep process controlled by the muscle regulatory factor family of transcription factors (MRFs). Indeed, all skeletal muscles can be eliminated by functionally inactivating three MRF genes (Myf5, Mrf4 and MyoD).

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Beyond that, the temporal expression of these factors and their association with the Pax3, Pax7, SRF, MEF2, Pbx and other regulators, introduces a greater complexity to the overall muscle gene regulatory program. However, this view is necessarily over-simplistic, and it is clear now that these regulatory transcription factors do not act in isolation. There is increasing evidence that the transition from the muscle precursor cell leading to the establishment of fully developed mammalian muscle tissue, as well as its capacity to repair in response to damage, involves interactions between genetic factors and epigenetic modulators of chromatin, in addition to microRNAs. Chromatin endows genes with the ability to be programmed in multiple ways in order to function within a complex genome for the specialized purpose of the cell, helping in establishing the distinct cell lineages. Significant progress has been made in defining the diverse enzymatic complexes that control chromatin assembly and the regulated activity of this structure. However, in the skeletal muscle field, only during these last years epigenetic studies have really emerged, thereby increasing our understanding of the mechanisms regulating muscle gene expression. Although many black boxes remain in the story, these mechanisms are revealed to be increasingly sophisticated and elegant. We have got further insight into the distinct functions of complexes such as ATP-dependent remodelers, HATs, HDACs and DNMTs, and how they are targeted to specific muscle genes by chromatin-bound regulatory transcription factors. We also have a much better picture of how different combinations of enzymatic complexes act together in an ordered manner to regulate muscle gene expression and how specific histone modifications affect this process. We have gained understanding of the critical function of signaling pathways (in particular that of p38 MAPK) in connecting and transmitting extracellular signals to satellite cell nuclei, in order for the cell to adapt to the demands of the environment. We have also learnt that muscle- and non-musclespecific microRNAs can modulate distinct myogenic steps. Much work remains to be done in this area. It is important to note that most findings outlined here are based

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on cell-culture studies—using immortalized myogenic cell lines—and therefore they require validation using in vivo models, including the generation of mice with muscle-specific inactivation of the individual transcriptional and epigenetic regulators. Genetic studies that define the role of chromatin remodeling and modifying complexes in specifying developmental or differentiation programs, and in regulating the capacity of adult muscle to undergo efficient regeneration, will be of great interest. With so many unresolved issues, some of which were touched upon only briefly in this review, the coming years of epigenetic research in myogenesis promise to be exciting. Acknowledgements

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