MyoD and the transcriptional control of myogenesis

Seminars in Cell & Developmental Biology 16 (2005) 585–595

Review

MyoD and the transcriptional control of myogenesis Charlotte A. Berkes a,∗ , Stephen J. Tapscott b b

a Colorado College, Colorado Springs, CO 80903, USA Fred Hutchinson Cancer Research Center, Mailstop C3-168, 1100 Fairview Avenue North, Seattle, WA 98109-1024, USA

Available online 11 August 2005

Abstract The basic helix-loop-helix myogenic regulatory factors MyoD, Myf5, myogenin and MRF4 have critical roles in skeletal muscle development. Together with the Mef2 proteins and E proteins, these transcription factors are responsible for coordinating muscle-specific gene expression in the developing embryo. This review highlights recent studies regarding the molecular mechanisms by which the muscle-specific myogenic bHLH proteins interact with other regulatory factors to coordinate gene expression in a controlled and ordered manner. © 2005 Elsevier Ltd. All rights reserved. Keywords: Gene expression; Myogenesis; Development; Chromatin

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The specification and differentiation of skeletal muscle by the MyoD family of bHLH proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of E proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibitors of myogenic transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other myogenic activators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional domains of MyoD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Myogenic targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of co-activators and co-repressors in controlling myogenic transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Control of myogenesis via signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Putting together pieces of the puzzle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The myogenic regulatory factors (MRFs) are critical for the determination and terminal differentiation of skeletal muscle. In the two decades since their discovery, in vivo studies have elucidated the specific roles of MyoD and its relatives ∗ Corresponding author. Present address: Department of Microbiology and Immunology, 513 Parnassus, S472, University of California, San Francisco, CA 94143-0414, USA. Tel.: +1 415 502 4810; fax: +1 415 502 8201. E-mail address: [email protected] (C.A. Berkes).

1084-9521/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2005.07.006

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Myf5, myogenin, and MRF4, and cell culture studies have uncovered the basic mechanisms by which they function in transcription. The MRFs, together with Mef2 family proteins and other general and muscle-specific factors, coordinate the activities of a host of co-activators and co-repressors, resulting in tight control of gene expression during myogenesis. The events occurring at muscle-specific promoters have been dissected in molecular detail, uncovering a multitude of functional and direct interactions between MRFs and signaling proteins, chromatin modifying factors, and other transcriptional regulators. Information regarding the genetic

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networks controlling myogenesis, the signaling networks that are deployed to initiate myogenesis in the developing somites, and the molecular mechanisms that mediate activation of muscle-specific genes has expanded considerably in the past several years. In this review, we will focus on the latter of these three subjects, with particular attention to the MyoD family of transcription factors.

2. The specification and differentiation of skeletal muscle by the MyoD family of bHLH proteins Nearly 20 years ago, subtractive hybridization experiments were performed to identify and isolate myoblastspecific transcripts that were capable of orchestrating myogenic conversion of 10T1/2 fibroblasts [1,2]. This work led to the identification of a single cDNA, named MyoD, which was capable of converting a variety of cell types (e.g., fibroblasts, chondrocytes, neurons, amniocytes) to myoblasts [3–5]. MyoD belongs to a much larger class of DNA-binding proteins containing a basic helix-loop-helix (bHLH) domain. Soon after the discovery of MyoD, three closely related genes were identified: Myf5, myogenin, and MRF4 (reviewed in [6]). In vitro, each MRF efficiently binds to consensus CANNTG sites (E boxes), which are present in the promoters and enhancers of muscle-specific genes [7,8]. Forced expression of MRF proteins in non-muscle cells in culture can induce myogenic differentiation, albeit with varying efficiency [5,9–11]. These similarities in the basic modes of MRF function have enabled us to use the inaugural member of the MRF family, MyoD, as the primary paradigm in molecular studies of transcriptional regulation. In vivo, however, the distinct yet overlapping roles played by the four MRFs become more apparent; these roles can be partially attributed to differences in temporal expression patterns as well as protein sequence. In the developing mouse embryo, Myf5 expression is induced in the dorsal-medial somites (which later gives rise to trunk and intercostal muscles), and is followed by expression of MyoD in the dorsal-lateral somites (which later gives rise to body wall and limb muscles). The Wnt, Sonic hedgehog (Shh), and other signaling pathways have been shown to contribute to muscle determination by inducing expression of Myf5 and MyoD (reviewed in [12]). Expression of both MyoD and Myf5 is the key step which results in commitment of multipotential somite cells to the myogenic lineage, since disruption of both genes results in the absence of skeletal myoblasts [13]. Null mutations in either MyoD or Myf5 result in apparently normal muscle development, demonstrating a degree of genetic redundancy in the MyoD family. However, upon careful examination, mild defects in trunk skeletal muscle are observed in Myf5 null embryos, whereas early limb and branchial arch muscle development is delayed in MyoD null embryos, demonstrating that these genes control early specification of epaxial versus hypaxial muscle lineages [14]. Mice lacking myogenin have very poorly developed skeletal muscle tissue even though myoblasts are present, sug-

gesting that myogenin plays a critical role in terminal differentiation of myoblasts, but is dispensable for establishing the myogenic lineage[15,16]. Furthermore, myogenin cannot efficiently mediate myogenesis in the developing mouse embryo when substituted into the Myf5 locus, suggesting that the ability to establish the muscle lineage is not simply a matter of the timing of expression in the embryo, but is an inherent property within the protein itself [17]. In support of this in vivo evidence, although each of the myogenic bHLH proteins can initiate myogenesis when expressed in non-muscle cells in vitro, myogenin is not nearly as efficient as MyoD or Myf5 in initiating expression of some muscle-specific genes. The ability of MyoD and Myf5 to initiate previously silent muscle-specific genes has been mapped to a C-terminal region of MyoD, which forms a putative ␣-helix [18]. The specific role played by MRF4 during myogenesis is somewhat more complex. MRF4 is expressed transiently in the mouse myotome at embryonic day 9.0 (E9.0), immediately following Myf5 expression. Its expression tapers by E11.5, and is reinitiated at E16.0 in differentiating muscle fibers. Thus, its complex temporal expression pattern suggests potential roles in both muscle determination and terminal differentiation. Myogenesis can be partially rescued in myogenin−/− embryos by a myogenin promoter-MRF4 transgene [19], supporting a role for MRF4 in terminal differentiation. Furthermore, in embryonic stem (ES) cells lacking myogenin, fully differentiated muscle fibers can be generated by overexpression of MRF4 [20], but not MyoD [21]. Analysis of the role played by MRF4 in vivo has been complicated by the fact that MRF4 and Myf5 are located in tandem and expression of each gene is not completely independent of the other. Recently, a sophisticated series of Myf5 mutants have been generated, some of which express MRF4 in the absence of Myf5. In Myf5:MyoD double-null mice whose MRF4 expression is unaffected, skeletal muscle is indeed present [22]; this work clearly demonstrates a role for MRF4 in the early stages of myogenesis, in addition to its role in terminal differentiation. Taken together, these studies suggest that MyoD and Myf5 are required for commitment to the myogenic lineage, whereas myogenin plays a critical role in the expression of the terminal muscle phenotype previously established by MyoD and Myf5, and MRF4 partly subserves both roles. Thus, MyoD and Myf5, and to an extent, MRF4, can be considered “commitment” or “specification” factors, whereas myogenin is a “differentiation” factor, and MRF4 has aspects of both functions. Although the expression patterns and general functions of each of the MRFs are clearly distinct from one another, the specific target genes of each factor in vivo are not known. As reviewed in Chanoine et al., recent work in the Xenopus model system shows that each MRF has a specific subset of target genes which can also be activated by other MRFs, albeit less efficiently, thus supporting the “distinct but overlapping roles” model [23]. Whether or not similar mechanisms apply in mammalian systems is yet to be fully determined; however, studies of specific promoters

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indicate sequence-specific functions of each factor can occur [24,25]. The genomes of vertebrate species contain homologs of all four MRF genes, with some amphibians and fish possessing duplicate copies of MRF genes. For example, two distinct MyoD genes have been identified in Xenopus laevis, one of which is expressed maternally and the other zygotically [26]. In invertebrate species such as flies [27], sea urchins[28], and nematodes [29], only one MyoD family gene is present, suggesting that the gene duplications that gave rise to MyoD, myogenin, Myf5, and MRF4 must have occurred prior to the divergence of chordates. If MyoD, Myf5, myogenin, and MRF4 each play critical roles in either muscle determination or differentiation in vertebrates, how can one single MyoD family gene suffice in invertebrates? Ascidians seem to have solved this problem by having two developmentally regulated splice forms of MyoD: CiMDFa and CiMDFb. CiMDFa is expressed both maternally and zygotically and lacks a portion of the C-terminus that is present in CiMDFb [30]. Interestingly, this C-terminal portion is homologous to the C-terminal portion of murine MyoD, which is required for initial expression of myogenic markers [18]. 3. Role of E proteins MRFs are class II (tissue-specific) bHLH transcription factors and are capable of either homo-dimerization with themselves, or heterodimerization with class I bHLH factors. Class I factors, which include the E proteins HEB/HTF4, E2-2/ITF-2, and E12/E47, are ubiquitously expressed in various tissues and at different times during development. The basic region is required for DNA-binding, whereas the HLH domain mediates dimerization with other bHLH proteins. All bHLH dimers bind to a consensus sequence called an E box, comprised of the sequence CANNTG, where each half of the dimer recognizes one half-site. Any of the MRFs can bind E boxes as heterodimers with any of the E proteins in vitro, although preferences for specific sequences flanking and internal to the E box have been determined for MRF homodimers and MRF/E protein heterodimers in vitro [7]. However, the biologically relevant partners for each MRF have been difficult to determine. The E protein HEB is constitutively expressed and localized to the nucleus in developing L6 and C2C12 cells, as well as rat primary myotubes, suggesting that this E protein may play an especially important role in myogenesis [31]. It is interesting that skeletal muscle is formed in all of the knock-out mouse models of the individual E proteins, suggesting some redundancy of function within the family.

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premature activity of MyoD is prevented: post-translational modification, association with co-repressor proteins, and association with proteins which titrate MyoD away from the DNA. Post-translational modifications and co-repressors of MyoD will be addressed under “The Role of Coactivators and Corepressors in Controlling Myogenic Transcription”. A number of proteins have been identified which act as myogenic antagonists by directly binding to E proteins and/or MyoD family proteins, and blocking their ability to bind E boxes and/or activate transcription at muscle-specific promoters. Many of these inhibitors are themselves helix-loop-helix domain proteins, and include Id, Twist, MyoR and Mist-1. Id comprises a family of HLH proteins whose expression is upregulated under high-serum conditions. A high level of Id protein in the cell is inhibitory for MRF activity because Id is capable of efficiently heterodimerizing with E proteins, sequestering them and preventing their interaction with the MRFs [32]. Id proteins may also heterodimerize with MRFs, albeit with lower efficiency. Since Id lacks the basic region required for DNA-binding, MRF/Id heterodimers are thought to be devoid of transcriptional activity. A “forced” MyoDE47 dimer in which MyoD and E47 are contained within a single polypeptide is resistant to inhibition by Id, providing evidence in support of this mechanism [33]. Similar to Id, Twist is also an HLH protein which inhibits myogenic differentiation by dimerization with E proteins, sequestering them from MRFs in inactive complexes [34]. Unlike Id, Twist possesses a basic region which functions not by binding to DNA, but by binding to the basic region in MRFs, thus preventing the interaction between MRFs and their cognate E boxes in muscle-specific promoters [35]. Twist is also capable of inhibiting myogenesis via direct interaction with Mef2 proteins [34]. In addition to Id and Twist, MyoR and Mist-1 are inhibitors of myogenesis. These factors contain basic regions and form dimers with the MRFs. MRF/MyoR and MRF/Mist-1 heterodimers are competent to bind E boxes; however, these dimers are unable to activate transcription when bound to DNA [36,37]. Mdfi (formerly known as I-mfa) is a negative regulator of MRFs, but is not itself a member of the HLH family and functions quite differently than the HLH family negative regulators. Mdfi is a cytoplasmic protein which functions by binding to and sequestering MRFs within the cytoplasm, preventing their translocation into the nucleus [38]. Mdfi is also a potential regulator of the Wnt signaling pathway because it can interact with Tcf/Lef proteins and prevent DNA-binding [39]. Together with the observation that it is expressed in some stem cell populations [40] suggests that it might have an important role in integrating wnt signaling and bHLH activity in early events of cells specification or differentiation.

4. Inhibitors of myogenic transcription 5. Other myogenic activators MyoD is expressed in myoblasts well before activation of its target genes, both in vivo and in tissue culture systems. There are several well-described mechanisms by which

The MRFs are assisted by the myocyte enhancer factor 2 (Mef2) family of transcription factors in order to mediate

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expression of muscle-specific genes (reviewed in [41]). Mef2 proteins belong to the MADS (MCM1, agamous, deficiens, serum response factor) box-containing transcription factor family. The Mef2 family consists of four members, Mef2AD, each of which is encoded on a separate gene. While expression of MRFs is restricted to muscle, Mef2 genes are expressed widely during development. Mef2 proteins bind to an A/T-rich DNA sequence element (C/TTA(A/T)4 TAG/A) which is found in the promoters of many muscle-specific genes [42]. On its own, Mef2 does not possess the ability to recapitulate the myogenic differentiation program when expressed in cell lines in vitro, nonetheless, several pieces of evidence support a critical role for Mef2 in assisting the MRFs. Mef2 and MyoD interact directly in vitro and synergistically activate transfected reporters driven by E boxes and Mef2 binding sites [43]. In the promoters and enhancers of muscle-specific genes, E boxes and Mef2 binding sites are often located within close proximity to one another, providing further support for a model in which MyoD and Mef2 bind DNA and activate transcription in a cooperative fashion [44]. In Drosophila, mutation of the single Mef2 gene inhibits differentiation of muscle cells [45]. While inactivation of the Mef2C gene in mice results in embryonic lethality due to a defect in cardiac morphogenesis, no defects were observed in skeletal muscle, possibly due to functional redundancy amongst the Mef2 family [46]. Indeed, in addition to Mef2C, Mef2A, and Mef2D are expressed in skeletal muscle, and expression and splicing of these isoforms is altered in response to MyoD [47]. Another notable family of transcription factors critical for myogenesis is the Six family of homeodomain proteins. There are six members of the Six family in mammals, each playing different roles in various developmental contexts [48]. The Drosophila ortholog of mouse Six family proteins, sine oculis, interacts with two coactivator proteins (eyes absent and daschund) to regulate gene expression during eye development [49,50]. A parallel mechanism has been demonstrated in a mouse somite explant culture system in which Six1 interacts with the orthologs of eyes absent and dachshund (Eya2 and Dach2, respectively) to synergistically regulate myogenic gene expression [51]. mRNA for Six1 and Six4 is detected specifically in skeletal muscle, and Six1 is especially abundant in the developing somites. Six1 and Six4 bind to the Mef3 site of the myogenin promoter in vitro, and mutation of this site prevents muscle-specific expression of a myogenin-lacZ transgene in mice, demonstrating the importance of this site in regulating myogenin expression during muscle development [52]. The Six4 knockout mouse develops normally [53], but mice lacking Six1 have impaired primary myogenesis and delayed expression of myogenin and MyoD in limb buds, indicating a positive role for Six1 during muscle development in vivo [54]. Mef3 sites have been identified in the promoters of other muscle-specific genes such as cardiac troponin C [55], aldolase A [56–58], and muscle creatine kinase [59].

Two domains of MyoD that had previously been shown to be necessary to initiate chromatin remodeling at the myogenin promoter [60], the H/C domain and helix III domain, are required for cooperation with Pbx and Meis in the context of the myogenin promoter [61]. Pbx and Meis are homeodomain family transcription factors which were originally identified as genes involved in pattern formation during embryogenesis [62–65]. Rearrangement and/or mutation of Pbx genes has been linked to leukemia [66,67]. Pbx and meis function as co-factors for Hox proteins; whereas Hox dimers normally display little binding specificity in isolation [68–71], binding specificity and affinity can be modulated through cooperative binding with Pbx family proteins [72]. Molecular interactions between Pbx and Meis homeodomain proteins and bHLH transcription factors have been described previously. The Hox-like pancreas/duodenal homeobox-1 protein (Pdx1) forms a trimeric complex with Pbx1b and Meis2b. This trimeric complex cooperates with a heterodimer of the pancreas-specific bHLH protein PTF1 (p48) and the E protein HEB to activate gene expression through the pancreas-specific elastase enhancer [73]. PbxMeis complexes have also been shown to bind cooperatively with MyoD family proteins in vitro on a synthetic DNA element containing a consensus Pbx-Meis site and E box, and cooperative binding requires a conserved tryptophan residue within the H/C domain of the MyoD family proteins [74]. Recently, our laboratory has described a biological role for Pbx-Meis family proteins in myogenesis. Pbx and Meis bind to an evolutionarily conserved site within the myogenin promoter which is flanked by a non-canonical E box, and this site is crucial for full activation of the myogenin promoter. Pbx appears to be constitutively bound to the myogenin promoter, as well as other muscle-specific promoters. There are four Pbx family members in mice, and a specific muscle phenotype in Pbx knockout mice has not been described, possibly due to a high level of genetic redundancy [75–78]. However, it is interesting to note that loss of CEH-20, the C. elegans ortholog of Pbx, results in abnormal mesodermal differentiation and absence of muscle cells [79]. Further studies are imperative to understand the roles of Pbx family members in vertebrate skeletal muscle development. A number of other factors have been shown to cooperate with MyoD family proteins to activate expression of musclespecific genes. For example, the muscle LIM protein (MLP) physically interacts with MRFs via the bHLH domain and is required for differentiation of C2C12 myoblasts in culture [80,81]. In addition, muscle-specific gene expression often requires cooperation of ubiquitously expressed DNA-binding factors such as Sp1 and AP1 [82,83].

6. Functional domains of MyoD The MRFs are distinct from all other bHLH proteins in that each contains a conserved muscle recognition motif within the basic domain comprised of three amino acids; this myo-

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genic “code”, comprised of the amino acids ATK, confers the ability to activate muscle-specific genes [84,85]. These amino acid residues are required for cooperativity between MyoD and Mef2 on both endogenous muscle genes [43] and transfected reporters [86], providing strong evidence that at least part of the function of the myogenic code is to mediate interaction with Mef2 proteins. There is also evidence that one of the myogenic code residues, A-114, is critical for proper conformation of its activation domain [87]. Further investigation of the function(s) of the myogenic code residues may uncover important interactions with additional co-activators. Deletion and mutation analyses have previously identified three domains of MyoD, in addition to the bHLH region, which perform critical functions in muscle-specific gene regulation: the N-terminal acidic domain, the histidine and cysteine-rich domain, and helix III. The N-terminal domain, including amino acids 3–56, is highly acidic and can mediate activation of a GAL reporter when tethered to the yeast GAL4 DNA-binding domain [88]. The N-terminal acidic domain of MyoD is required for activation of a transfected reporter driven by multimerized E boxes, suggesting it functions as a classical transcriptional activation domain (TAD) and it is required for cooperative binding to adjacent E boxes in a DNA fragment [89]. In addition to the N-terminal “TAD”, two other conserved motifs have been identified—one N-terminal to the bHLH domain rich in histidine and cysteine residues (H/C), and another domain C-terminal to the bHLH containing a putative ␣-helical structure referred to as helix III. A MyoD deletion mutant lacking the H/C region is unable to initiate chromatin remodeling at the myogenin locus [60], possibly because it does not interact with the resident Pbx-Meis complex and therefore does not initially bind to the myogenin locus. The helix III motif has similarly been shown to be necessary for the activation of endogenous myogenin and some other transcriptionally repressed genes during myogenesis [18] and is also necessary for binding to the myogenin promoter in the context of the Pbx-Meis complex. Studies using chimeras of MyoD and other myogenic bHLH proteins have shown that the H/C and helix III domains of MyoD are functionally conserved between MyoD and Myf5, but not myogenin, thus establishing a key molecular distinction between the myogenic specification factors (MyoD and Myf5) and myogenin.

7. Myogenic targets MyoD activation leads to robust expression of several well-characterized target genes such as myogenin, Mcadherin, myosin heavy and light chains, and muscle creatine kinase. In addition to these muscle-specific genes, it has also been well-established that MyoD up-regulates expression of the cyclin-dependent kinase inhibitor p21Waf/Cip1 , causing an irreversible exit of the differentiating cells from the cell cycle [90,91]. We and others have characterized expression of genes during myogenesis on a genome-wide level [92–95].

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Upon induction of MyoD activity in a model system of MyoD-mediated myogenesis, the expression of a wide range of gene targets are modulated with varying kinetics [92]. Genes regulated by MyoD serve a wide range of biological functions, such as transcription factors (Six family proteins, Mef2), muscle structural genes, and cell cycle regulators. Although clusters of genes are expressed at different times following MyoD induction, chromatin immunoprecipitation experiments indicate that MyoD directly binds to the regulatory elements of genes expressed throughout the differentiation program. Temporal specificity is achieved, at least in part, through a feed-forward mechanism wherein some genes are activated immediately by MyoD and later activated genes need both MyoD and the additional participation of one of the earlier MyoD targets [47]. Although the vast majority of MyoD target genes are up-regulated, there are a number of genes whose expression decreases upon MyoD induction and it will be interesting to determine the mechanism and whether this is mediated by the miRNAs induced during myogenesis [96].

8. The role of co-activators and co-repressors in controlling myogenic transcription Three classes of co-activators are known to cooperate with transcription factors to mediate specific and patterned gene expression: histone modifying proteins such as histone acetylases and methylases, SWI/SNF family chromatin remodeling factors, and proteins in the TRAP/Mediator family. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) both interact with MyoD and have opposing activities that might be critical to switch MyoD from a repressor to an activator at some loci, which might be aided by the subsequent recrutment of SWI/SNF remodeling complexes. HAT proteins function by transferring acetyl groups from acetyl-coA to the lysines residues in histone proteins (namely, H3 and H4), and in some cases, to non-histone proteins. Histone acetylation promotes transfer of histones H2A/H2B from the DNA to chaperone proteins [97], thus increasing access of transcription factors to the DNA, and MyoD binding to specific promoters has been shown to occur concomitantly with histone acetylation [92]. HAT activity increases during the course of myogenic differentiation [98], and the HAT protein p300/CBP is required for expression of muscle-specific genes [99] In differentiating C2C12 myotubes, MyoD is found in a complex with p300 and another HAT-containing protein, PCAF. This trimeric complex is able to stably associate with an E box in vitro, suggesting that MyoD recruits p300 and PCAF to muscle-specific promoters [100]. The interaction between p300 and MyoD requires the N-terminal activation domain of MyoD, and the association between PCAF and MyoD is less robust in vitro [101]. Injection of anti-p300 or anti-PCAF antisera into C2C12 and 10T1/2 fibroblasts inhibited myogenic differentiation, however, the histone acetyltransferase domain of p300 is dispensible for

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MyoD activity in transient transfection experiments [100]. These data supported a model in which MyoD recruits p300 and PCAF to muscle-specific genes, leading to histone acetylation, chromatin remodelling, and subsequent activation of transcription. In addition to histones, MyoD is also a substrate for acetylation by both p300 and PCAF in vitro; acetylation of MyoD on three lysine residues enhances its ability to bind DNA and activate transcription [102,103]. However, the precise roles of p300 and PCAF have been difficult to define using tissue culture models. Recently, the specific roles played by both p300/CBP and PCAF have been determined using an in vitro transcription system [104]. Rather than playing redundant roles, p300 and PCAF act sequentially to increase transactivation from chromatinized muscle gene templates in vitro. MyoD recruits p300 to the promoter, which acetylates histones H3 and H4 within the nucleosomes. p300 is necessary to recruit pCAF, which acetylates three lysine residues within MyoD itself, thus increasing its transacriptional activity, possibly by strengthening the interaction between the acetylated lysines of MyoD and the bromodomain of CBP [105]. This in vitro myogenic transcription system will provide an invaluable tool for understanding the interactions between MyoD, other activators, and the chromatin modifying and remodeling proteins. Histone deacetylases have been shown to negatively regulate expression of muscle-specific genes via interactions with both MyoD and Mef2 proteins. The class II histone deacetylases (HDAC4 and HDAC5) interact with Mef2 proteins and repress activation of transcription from promoters containing Mef2 sites [106–109]. The class II HDACs can also inhibit MyoD-mediated transcription, but only in promoters which contain a Mef2 site in addition to an E box, suggesting that this inhibition is mediated indirectly through Mef2 [110]. The class I histone deacetylase HDAC1 associates directly with MyoD, is capable of deacetylating MyoD in vitro and inhibits the ability of PCAF to enhance MyoD-dependent transcription in cell culture experiments [111]. Furthermore, MyoD is found in a complex with HDAC1 in undifferentiated myoblasts, but not in differentiating myotubes [111]. MyoD also interacts with the nuclear receptor co-repressor/silencing mediator of retinoic acid and thyroid hormone receptor (NCoR/SMRT), which has been shown to recuit HDAC1 to target promoters [112]. There is a growing body of evidence that the interactions between MyoD family proteins and Mef2 family proteins with either HATs or HDACs is a regulated process, and that the balance between HAT-associated activators and HDACassociated activators plays an important role in controlling the onset of differentiation. For example, upon differentiation, the retinoblastoma protein (pRb) can displace HDAC1 from MyoD, freeing MyoD to activate transcription [109]. Release of MyoD from its interaction with HDAC1 is required for its association with PCAF, which is a critical step in MyoD activation [111]. A regulated transition between HAT and HDAC interactions has also been reported for Mef2 proteins.

The MEF2/HDAC complex is sensitive to calcium-dependent kinase (CamK) activity, which mediates the muscle differentiation promoting effect of insulin-like growth factor 1(IGF1). CamK phosphorylates HDAC5 on two serine residues, which stimulates binding of the chaperone protein 14-3-3, leading to exposure of the nuclear export signal in HDAC5 and subsequent removal from the nucleus [113]. Several histone methylases have been isolated and characterized, however, little is known about their role in myogenesis. The coactivator-associated arginine methyltransferase1 (CARM1) protein has recently been shown to play a positive role in myogenesis. CARM1 is a member of the protein-arginine N-methyltransferase (PRMT) family, which catalyzed the methylation of arginine residues on a broad range of protein substrates. CARM1 can methylate histone H3 in vitro, and is required for myogenic differentiation in cell culture systems. CARM1 potentiates myogenesis by a mechanism in which it associates directly with the steroid receptor coactivator GRIP1 and Mef2C in a ternary complex. Members of the SWI/SNF family of chromatin remodeling transcription factors play critical roles in transcriptional control. SWI/SNF proteins are normally components of multi-subunit complexes which change chromatin structure by altering DNA-histone contacts within a nucleosome in an ATP-dependent manner (reviewed in [114]). All SWI/SNF family members contain a motif found commonly in transcription factors called a bromodomain, which is capable of binding to acetylated lysine residues within histone Nterminal tails in vitro [115–117]. An examination of polytene chromosomes in Drosophila showed that the fly SWI/SNF homolog, BRM, is associated with nearly all transcriptionally active chromatin [118]. The mammalian homologs of the yeast SWI/SNF proteins, BRG1 and BRM, are required for myogenesis in cells expressing MyoD. Microinjection of anti-BRG1 or anti-BRM antisera [119] or expression of dominant negative forms of these enzymes inhibits musclespecific, MyoD-mediated gene expression [120], demonstrating a critical role for ATP-dependent chromatin remodeling enzymes in myogenesis. A logical assumption is that SWI/SNF complexes are recruited to promoters by MyoD or Mef2 family proteins, which is supported by recent data showing co-immunoprecipitation of MyoD, p300, PCAF, and BRG1 from nuclear extracts of differentiating C2C12 myotubes [119]. However, it is not known whether the interactions between MyoD and BRG1 are direct. In differentiation conditions, p300, PCAF, and BRG1 are recruited to the myogenin and muscle creatine kinase promoters along with MyoD. BRG recruitment and subsequent chromatin remodeling can be uncoupled from histone acetylation by treatment of cells with SB203580, an inhibitor of the p38 pathway. In vitro, p38 is capable of phosphorylating BAF60, a subunit of the SWI/SNF complex which has previously been shown to mediate interactions between the SWI/SNF complex and transcriptional activators [121,122]. Inhibition of the p38 signaling pathway blocks recruitment of SWI/SNF to muscle promoters in vivo without affecting recruitment of

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MyoD, HAT proteins, or histone acetylation at the promoter [119]. Generally, proteins in the TRAP/Mediator family interact with both sequence-specific transcription factors and components of the general transcription machinery, forming a molecular bridge between the two (reviewed in [123]), however, the role of the mediator complex in muscle gene expression remains largely unexplored. The TRAP220 subunit of the TRAP/mediator complex has been shown to enhance activation by several transcription factors [124,125]. In fact, while TRAP220 is required for adipogenesis mediated by PPAR␥2, it is dispensible for myogenesis in mouse embryonic fibroblasts [126]. These results do not rule out a role for TRAP/mediator in myogenic gene regulation, but rather suggest that MyoD and/or Mef2 may be interacting primarily with other members of the mediator complex; these details remain to be established.

9. Control of myogenesis via signaling pathways The p38 kinase has a particularly robust role in expression of muscle-specific genes, and the specific mechanisms by which p38 impinges upon the muscle gene regulatory pathway have been well-described in recent papers. p38 kinase activity increases over the course of skeletal muscle differentiation and its activity is required for terminal differentiation [127]. p38 functions, in part, by phosphorylating the transactivation domain of Mef2 [128–131] and inhibition of p38 stifles the transcriptional activation potential of Mef2 factors, whereas expression of a constitutively activated allele of a p38 upstream regulator promotes myogenesis [127,132,133]. p38 activity is required most stringently at genes expressed in the late phases of myogenesis; these genes include a preponderance of muscle structural genes and contractile proteins which are crucial for adoption of muscle morphology and function [92]. Penn et al. have recently demonstrated a clear mechanism by which p38 increases muscle-specific gene expression. Binding of both MyoD and Mef2 to lateactivated promoters in vivo is stimulated by p38 activity, which, in turn, leads to recruitment of Pol II. The role of p38 in facilitating binding of MyoD and Mef2 is likely to involve chromatin, as binding of these factors to DNA is not enhanced by p38 activity in in vitro assays [47]. Although the mechanism remains unknown, this could be mediated by the p38 dependent recruitment of SWI/SNF by MyoD [119] or through a p38-dependent phosporylation of histones as has been shown for some immediate-early genes [134,135].

10. Putting together pieces of the puzzle In the past several years, we have learned a great deal regarding the factors associating with muscle-specific promoters and enhancers, and have described gene expression during myogenesis on a genome-wide level [92,94]. The

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mechanisms by which chromatin structure regulate gene expression have been well-described in many other systems. The remaining challenge will be to put the pieces together to synthesize a coherent picture of myogenic transcriptional regulation. It is well-established that SWI/SNF factors and histone acetyltransferase co-activators play an indispensable role in activation of muscle-specific genes. However, little is known regarding the temporal sequence of events that occurs at a muscle-specific promoter preceding and during activation. In some cases, such as at the yeast PHO8 promoter [136] and the mammalian interferon-␤ promoter [137], the precise order of recruitment of DNA-binding factors, histone modifying proteins, and nucleosome remodeling factors has been described in great detail (reviewed in [138]). These studies have provided valuable information, but it is not clear whether the mechanisms described can be applied generally. It is likely that promoter-specific activity of MyoD is achieved by promoter-specific requirements for co-activators. For example, a global analysis of transcript levels has shown that the mammalian SWI/SNF proteins are only required for expression of roughly half of all MyoD targets (Berkes and Bergstrom, unpublished data). It will be interesting to examine the temporal sequence of events occurring at muscle-specific promoters/enhancers for sets of genes that show different temporal regulation. The myogenin promoter makes a particularly good system for such experiments because it contains a region rich in cis elements bound by MRFs, Mef2, Six1, and Pbx/Meis; these binding proteins are likely to comprise an “enhanceosome” analogous to the IFN␤ enhanceosome described previously [137]. The work described in this review also brings us to the cusp of understanding a question that has long eluded us: how do the MRFs initially “find” the appropriate E boxes within a repressive chromatin context in order to initiate transcription? A simple calculation tells us that the E box sequence CANNTG occurs approximately once in every 256 bases in the genome. Even if one accounts for E box preferences of MRF/E protein heterodimers [7], there are still thousands of E boxes throughout the genome near genes which are certainly not activated by MyoD. One notable example of this is found in the IgH promoter, which contains a MyoD consensus site which is bound by MyoD in vitro, yet is not activated by MyoD in a cellular context [139]. How does MyoD distinguish developmentally appropriate binding sites from those which are identical in sequence, yet irrelevant? The answer to such a question is likely to be complex and involve a number of different mechanisms. While nucleosome packaging may “hide” many of these developmentally irrelevant E boxes and prevent inappropriate MyoD binding, a repressive chromatin structure also initially blocks access to the E boxes within muscle-specific promoters [60]. Our recent finding that the ubiquitously expressed homeodomain protein Pbx is constitutively associated with myogenin and some other muscle-specific promoters suggests that it may act as an accessory factor to MyoD, helping it recognize these promoters prior to the onset of differentiation. In support of

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this theory, the same regions of MyoD which are required for MyoD to bind to the myogenin promoter and initiate chromatin remodeling are also required for cooperation with Pbx/Meis heterodimers [60,61]. By integrating the data summarized in this review, several models can be put forth regarding the events controlling expression of muscle-specific genes. In one model, differentiation cues stimulate dissociation of MyoD from inhibitory proteins, allowing it to associate with E proteins and contact muscle promoter E boxes. Whether MyoD forms a complex with HATs and SWI/SNF complexes prior to promoter binding, or recruits these factors following association with an E box remains unknown. Binding of other activators such as Mef2 family proteins to the promoter, activation of the p38 pathway, and possibly other events then enables chromatin remodeling at the promoter, formation of a pre-initiation complex, and initiation of transcription. At some muscle promoters, nucleosomes may be blocking access of MyoD to E boxes. In such cases, MyoD may require the assistance of a resident factor to mark genes for activation and facilitate binding to the appropriate E boxes. Pbx is proposed to play such a role in the context of the myogenin promoter. In liver precursor cells, the transcription factors HNF3 (FoxA) and GATA-4 bind to the albumin gene enhancer in silent chromatin and facilitate opening of an H1-compacted nucleosome array on this enhancer [140]. It is possible that in the context of musclespecific gene regulation, Pbx, and potentially other pioneer factors may possess the intrinsic ability to alter chromatin in a similar manner, or may alternately act to recruit chromatin remodeling factors through protein-protein interactions. Further studies will undoubtedly fine-tune our understanding of muscle-specific gene regulation, but it is unlikely that one model will apply to all muscle-specific genes. In the future, it will be useful to take advantage of improving bioinformatics tools to identify conserved binding sites in the promoters and enhancers of muscle-specific genes and to identify groups of co-regulated genes during development. Emerging themes in the field of myogenic transcriptional regulation are likely to apply broadly to many aspects of development.

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