Báo cáo Y học: When the embryonic genome flexes its muscles Chromatin and myogenic transcription regulation ppt

MINIREVIEW When the embryonic genome flexes its muscles Chromatin and myogenic transcription regulation Ralph A. W. Rupp 1 , Nishant Singhal 1 and Gert Jan C. Veenstra 2 1 Adolf-Butenandt-Institut, Department of Molecular Biology, Mu ¨ nchen, Germany; 2 Department of Molecular Biology, Nijmegen Centre for Molecular Life Sciences, University of Nijmegen, the Netherlands During the development of multicellular organisms, both transient and stable gene expression patterns have to be established in a precisely orchestrated sequence. Evidence from diverse model organisms indicates that this epigenetic program involves not only transcription factors, but also the local structure, composition, and modification of chromatin, which define and maintain the accessibility and transcrip- tional competence of the nucleosomal DNA template. A paradigm for the interdependence of development and chromatin is constituted by the mechanisms controlling the specification and differentiation of the skeletal muscle cell lineage in vertebrates, which is the topic of this review. Keywords: skeletal myogenesis; MyoD family; chromatin remodelling; histone-acetyltransferase; histone H1. INTRODUCTION Following cells through development as they become specified and differentiate into a specific cell type provides a framework, in which developmental and chromatin aspects of gene regulation naturally meet. The determin- ation of cell fate involves invariably induction-dependent changes in gene expression patterns. For the acquisition of skeletal muscle cell identity, this involves the transcriptional activation of the MyoD and Myf5 genes, the two early expressed members of the myogenic bHLH transcription factor family in vertebrates (reviewed in [1]; see Fig. 1). Not surprisingly, many of the Ôusual suspectsÕ of embryonic patterning signals, i.e. SHH, FGFs, TGF-bs, and Wnts, have been implicated as potential activators and repressors, although a direct link between any of these pathways and the myogenic genes has yet to be established. A question of general importance for embryonic induction arises from the pleiotropic nature of these signals: how do they specify the stage- and tissue-specific expression patterns of the myo- genic genes, given their functional cooperation at many different times and places in the embryo? As discussed here, insights from several vertebrate species point to epigentic marks and chromatin as a pivotal regulator of transcrip- tional competence. This is evident from reports showing that regulatory elements of the myf5 and myoD genes from several vertebrate species require a chromosomal context for proper function [2–4] (A. Oho, L. Xiao & R. Rupp, unpublished observation), but this theme extends also beyond the induction of these two master regulatory genes. Once expressed, MyoD and Myf5 protein will drive myoblasts toward cell cycle exit and promote terminal differentiation by activating downstream target genes such as p21, myogenin and members of the Mef-2 protein family (Fig. 1). How fast and efficient this goal is achieved depends on extracellular signals, which control the activity of the myogenic proteins through covalent modifications and a multitude of protein–protein interactions (reviewed in [1,6,7]). Among the interaction partners, particularly MyoD, an increasing number of chromatin modifying and remodelling enzymes are found, which can both restrict and potentiate the activities of the muscle regulatory proteins. In the following, we will discuss regulatory functions of chromatin structure and modifications in prospective muscle cells, as these progress through their normal development. MYOGENESIS AND DNA METHYLATION The most abundant modification of vertebrate genomes is methylation of cytosine at CG dinucleotides. DNA methy- lation is dynamically regulated during embryonic develop- ment, and plays a role in the stable repression of gene expression through nucleosomal histone deacetylation, as in the examples of X-chromosome inactivation, silencing of transposable elements and genomic imprinting in mammals (reviewed by [8,9]). Vertebrate genomes contain at least five genes (MeCP2, MBD1–4) with a highly related methyl-CpG binding domain (MBD). All MBD proteins, except MBD4, have been found associated with histone deacetylases (reviewed in [10,11]), providing a mechanistic link between DNA methylation, chromatin and repression. A potential link between methylation and myogenesis was provided early on by the observation that 5-azacytidine treatment (which inhibits CpG methylation) converts 10T1/ 2 mouse embryonic fibroblasts at high frequency to muscle Correspondence to R. A. W. Rupp, Adolf-Butenandt-Institut, Department of Molecular Biology, Schillerstr. 44, D-80336 Mu ¨ nchen, Germany. Fax: + 49 89 5996440, Tel.: + 49 89 5996438, E-mail: ralph.rupp@med.uni-muenchen.de Abbreviations: HAT, histone acetyltransferases; HDAC, histone deacetylase. Dedication: This Minireview Series is dedicated to Dr Alan Wolffe, deceased 26 May 2001. (Received 15 November 2001, revised 19 February 2002, accepted 18 March 2002) Eur. J. Biochem. 269, 2294–2299 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02885.x [12]. This observation led to the cloning of MyoD [13]. Whether transcription of this master control gene is normally regulated by DNA-methylation has remained controversial, because the CpG island of the MyoD promoter is constitutively unmethylated in vivo [14]. How- ever, this possibility has been revived by the identification of a conserved distal enhancer element, which mediates the primary induction of the human and mouse myoD genes in development [3,15], and by the subsequent finding that this enhancer undergoes a regulated demethylation in some somitic cells, before the myoD gene is activated [16]. Through this transition, the methylation status of the distal enhancer becomes tightly correlated with myoD transcrip- tion, i.e. completely unmethylated in myogenic cells but significantly methylated in nonmuscle cells and tissues. Reporter genes, in which the CpG dinucleotides of this enhancer were mutated, expressed normally in transgenic mice, indicating that methylation at the distal enhancer is not required to prevent precocious or ectopic MyoD transcription in the mouse [16]. Instead, the authors proposed that the regulated demethylation could imprint the distal enhancer for subsequent induction by growth factor signals. Linker scanner mutations have identified functionally important subelements within the distal MyoD enhancer [17]. Once the corresponding trans-acting factors are known, it can be tested, whether their functions depend on the methylation (and acetylation?) status of the enhancer element. In Xenopus, the coding region of the myoD gene is constitutively hypomethylated during larval development, a feature that myoD shares with a comparatively small fraction of the otherwise constitutively hypermethylated frog genome [18]. This hypomethylated state of the XmyoD gene may contribute to a short period of ubiquitous MyoD expression at the midblastula transition, which precedes the local stabilization and up-regulation of myoD transcription in prospective myoblasts at the beginning of gastrulation [19]. The subsequent silencing of this ubiquitous myoD transcription in nonmuscle cells occurs without de novo methylation [18]. Whether the DNA methylation patterns of critical regulatory elements of frog myoD undergo developmental changes, as in the mouse, remains to be seen. H1 linker histones and myogenic competence Linker histones and a number of other proteins bind to internucleosomal or linker DNA. The high mobility group proteins HMG1 and HMG2, for example, share a similar structural role in binding to linker DNA with several variants of linker histones [20], but differ in their affinity for nucleosomal DNA and their ability to repress transcription of the genes with which they associate [21]. One of the linker histone variants, referred to as H1oo or B4, is expressed specifically in the oocyte and early embryo [22,23], and has been shown in Xenopus to be required for myogenic competence during early embryogenesis [24]. Xenopus MyoD is expressed at low levels at the late blastula stage, which requires developmentally regulated translation of maternal TBP RNA [25], but may require other regulatory events as well. Subsequently, MyoD and Myf5 expression are induced by secreted growth factors of the TGF-b, Wnt- and FGF-families [26] in prospective muscle, when mesoderm forms at the onset of gastrulation. This in contrast to mammals, in which early myogenic gene expression is coupled to somitogenesis. The timing of MyoD induction in Xenopus embryonic explant assays is largely independent from the timepoint of inducer application [26]. An independent mechanism ensures that MyoD transcrip- tion can only be induced until the midgastrula stage, although signalling pathways, for example that of activin (a TGF-b family member and potent muscle inducer in Xenopus), remain functional [24]. The loss of MyoD-indu- cibility coincides precisely with the disappearance of both muscle-forming competence and general mesodermal com- petence. Together, these observations describe a window of opportunity to induce MyoD transcription of less than two hours, which is a reflection of chromosomal epigenetic state of the MyoD locus [26]. Indeed, the ability to induce MyoD is under the control of linker histone proteins, which act as transcriptional inhibitors of MyoD induction [24]. The chromatin of early frog embryos undergoes major transitions in chromatin structure from cleavage to neurula stages, including a gradual replacement of a maternal linker histone variant (known as B4 or H1M) by somatic H1 protein subtypes after the midblastula transition [27]. Delaying the accumulation of somatic H1 protein by antisense methodology prolonged the period of myoD induciblility and muscle-forming competence, while overex- pression of somatic (but not the maternal) H1 variants had the opposite effect. Thus, accumulation of somatic H1 protein is rate-limiting for the timepoint, at which embry- onic cells loose competence to enter the myogenic program. In vivo, H1A was shown both to control the number of Myf5 MRF 4/ Myogenin MEF-2 Muscle Structural Genes Induction Determination Differentiation ? ? Transcriptional Competence (H1-Silencing De-Methylation) Chromatin Remodeling (BRM/BRG-1) SHH TGF-ß Wnt/ß-Catenin G0 Arrest Acetylation/ Deacetylation (p300/ P/CAF HDAC-1/N-Co-R) Chromatin Remodeling (BRM/BRG-1) Mesodermal Precursor Cell Myozyte MyotubeMyoblast ? MyoD Fig. 1. Simplified diagram of the skeletal muscle differentiation program in vertebrates. Solid arrows symbolize progression through development or genetic epistasis, respectively. Dashed arrows define epigenetic inputs; the question marks indicate either unclear mech- anisms (myoD/myf5 inductions) or potential contribution (b-catenin/Brm interaction) for the transcriptional induction of myoD and myf5 genes. Ó FEBS 2002 Muscle chromatin (Eur. J. Biochem. 269) 2295 MyoD-positive cells, and to prevent ectopic induction of MyoD transcription in the neural plate, apparently uncoupling some redundancy between mesodermal and neural induction processes [24]. In addition, somatic H1A represses the oocyte-specific 5S RNA genes from the late blastula stage on, and prevents precocious activation of the H1° gene, an H1 variant found in differentiated somatic cells [28,29]. Mounting evidence from diverse organisms describes H1 linker histones as gene-specific regulators of transcription [24,30], but many aspects of its function are still unknown. Overexpression of H1 subtypes can have selective effects on gene expression, due to differences in the central globular domain [31], and phosphorylation of H1 is implicated as a necessary step in the hormone dependent activation of the MMTV-LTR promoter by the glucocorticoid receptor [32]. So far, all H1 functions described in Xenopus require only the globular DNA-binding domain of H1, but not the charged N- and C-terminal tails, through which H1 affects higher-order chromatin structure [33]. Thus, the mechanism whereby histone H1 restricts induction of myoD, is through stabilization of nucleosome cores [30] and, possibly, inter- ference with nucleosome remodelling by SWI/SNF com- plexes [34]. The target of histone H1 in frog myogenesis could either be the MyoD locus itself, or the locus of an upstream regulator. Identification of the transcription factors inducing MyoD transcription will allow distinction between these possibilities. ACETYLATION AND MYOGENIC REGULATORS Histone acetyltransferases (HATs) such as p300/CBP, P/CAF and GCN5/2 are integrators for transcriptional regulation, which play important roles in development [35]. Likewise, histone deacetylases (HDACs) [36] also play important biological roles (reviewed in [37]). Both enzyme families have been shown to regulate myogenic protein activities, in particular that of MyoD (see Fig. 2). A first link between HATs and myogenesis was estab- lished by the finding that p300/CBP and P/CAF function as coactivators of MyoD [38,39]. This involves direct acetyla- tion of MyoD protein through both P/CAF [40] and p300 [41] at conserved lysine residues near the basic region of MyoD. This modification increases both MyoD’s DNA binding activity [42] and its affinity for p300/CBP [43]. Preventing acetylation of MyoD by mutating these lysines impaired the ability of MyoD to transactivate target genes and induce myogenic conversion [42], implicating MyoD acetylation as an important differentiation promoting event. However, the situation is more complicated because MyoD acetylation and association with p300 and P/CAF has been observed only in postmitotic, differentiating cells. In prolif- erating cells, MyoD is associated with the histone deacety- lase HDAC-1, either by direct interaction or by recruitment through the nuclear receptor corepressor N-CoR [44,45]. HDAC-1 can deacetylate MyoD protein in vitro,andits enzymatic activity is required to silence endogenous MyoD target genes, such as p21, in proliferating cells. These in vitro studies suggest that the transcriptional activity of MyoD is temporally controlled during myogenesis through interac- tions with HDAC-1/N-CoR or p300/PCAF. The equilib- rium between these complexes appears to be shifted by phosphorylation of MyoD protein through cdk1 and cdk2 [7], with HATs associating with underphosphorylated MyoD protein [44,46]. Unfortunately, not enough is known about the temporal correlation between Myf5/MyoD- mediated gene activation, cell cycle exit, and muscle differentiation to know, whether a quantitative switch from HDAC- to HAT-containing myogenic protein complexes occurs in vivo, or whether both complexes coexist in a dynamic equilibrium controlled by extracellular signals and cell cycle regulators [7]. Because the binding-domains for p300/CBP and HDAC-1/N-CoR do not overlap on MyoD (see Fig. 2), it is formally possible that both enzyme classes could be associated with MyoD simultaneously. In fact, this could contribute to the observed block of MyoD protein function by the HDAC-inhibitor TSA in early embryonic frog cells [47]. This issue relates also to the question, whether Myf5/MyoD corepressor complexes function as repressors during the establishment of muscle cell identity, which is an issue of controversy in the field [48,49]. A regulated transition between HAT- and HDAC- interactions has been reported for MEF-2 proteins. Mem- bers of this protein family lack myogenic activity, but are essential potentiators of myogenic bHLH protein activities during muscle differentiation [50]. Specifically, class II HDACs 4 and 5 bind and inhibit the transactivation activity of MEF-2 [51,52]. Interestingly, the MEF-2/HDAC complex is sensitive to calcium-calmodulin-dependent pro- tein kinsase (CamK) activity, which mediates the muscle differentiation promoting effect of Insulin-like growth factor 1 [53]. Increased CamK activity leads to phosphory- lation of HDAC5 on two serine residues, which induces binding of the chaperone protein 14-3-3, activation of the HDAC5 nuclear export signal, and consequent export of the transcriptional repressor to the cytoplam [54]. HDAC- free nuclear MEF-2 proteins interact instead with GRIP-1, 3181 60 162 Transactivation Domain P 300 - P/CAF Acetylated residues 124 P300/CBP interaction domain Activation of silent genes Chromatin remodelling K K K 99 101 102 218 26963 99 102 Helix IIIHelix Loop HelixBasicCys/His DNA Binding & Dimerization Domain Activation of silent genes Chromatin remodelling HDAC-1/NCo-R interaction domain Fig. 2. MyoD’s toolbox for changing chromatin structure. Functional subdomains are indicated as segments, with numbers referring to amino-acid residues of mouse MyoD protein. Only interactions between MyoD and chromatin modifying enzymes are shown. For further information about myoD protein partners refer to the references [1,6,7]. 2296 R. A. W. Rupp et al. (Eur. J. Biochem. 269) Ó FEBS 2002 a transcriptional coactivator with intrinsic HAT-activity, which is required for MEF-2C dependent gene expression and skeletal muscle differentiation [55]. These studies establish a mechanistic model for how extracellular cues control progression of muscle differentiation. There may be many targets for the enzymatic activities of the various HAT- and HDAC-complexes with myogenic regulators, but nucleosomal histones are certainly among them. For instance, histone octamers at the XMyoD promoter are hyperacteylated in frog oocytes by MyoD/ P300/PCAF-dependent transactivation (S. Ait-Si-Ali, A. Wolffe and R. Rupp, unpublished results), and the nucleosomes at the regulatory sites of the myogenin and MCK genes, which are bound by MyoD and MEF-2 family members, shift from a hypo- to a hyper-acteylated state during muscle differentiation [51]. Thus, either directly or indirectly, the core myogenic regulators induce modifica- tions of the local chromatin infrastructure through recruit- ment of HAT- and possibly HDAC-enzymes in the course of myogenesis. CHROMATIN REMODELING IN MUSCLE DIFFERENTIATION Chromatin structure and nucleosomal positions are subject to dynamic regulation in the cell nucleus. This nucleosome remodeling can either facilitate transcription or repress it, depending on where the nucleosomes are moved relative to important regulatory regions of specific genes. A key role in shuffling nucleosomes is played by nucleosomal ATPases of the SWI/SNF superfamily (reviewed in [56]). By regulating chromatin structure, nucleosomal ATPases play important roles in differentiation and development. Here we will briefly review the contribution of these enzymes to myo- genesis. In vivo, Myf5 and MyoD have to initiate gene transcrip- tion at many previously silent loci. MyoD utilizes two domains (see Fig. 2) to remodel the repressive chromatin structure over regulatory regions of target genes such as myogenin or muscle creatine kinase [57]. Repressors of muscle differentiation, such as TGF-b, bFGF and sodium butyrate, inhibit MyoD-mediated transactivation only before, but not after, chromatin remodelling. The ability to remodel chromatin is therefore a pivotal aspect of MyoD function, which may be regulated in vivo by extracellular signals. Interestingly, myogenin was significantly less efficient than MyoD or Myf5 in initiating endogenous muscle gene expression. Domain-swap experiments between MyoD and myogenin, both members of the myogenic bHLH family of transcription factors, revealed that sequence divergence in a C-terminal domain (i.e. Ôhelix IIIÕ; Fig. 2) is responsible for this difference [58]. Helix III of MyoD contributes to chromatin remodelling, while in myogenin this domains acts as a general transcriptional activation domain. The func- tional specialization of helix III sequences provides a molecular basis for the apparent distinction between specification (MyoD/Myf5) and differentiation (myogenin) functions within the myogenic bHLH gene family [58]. Recently, dominant-negative variants of human Brahma (Brm) or Brahma-related gene-1 (BRG-1) proteins, related ATPase subunits of two mammalian SWI/SNF complexes, were shown to block MyoD-mediated chromatin-remodel- ling, transactivation and muscle differentiation in cell lines [59]. Interestingly, while activation of all muscle-specific regulatory or structural genes was inhibited, the ability of MyoD to cause cell-cycle arrest through activation of cdk inhibitor p21, cyclin D3 and the retinoblastoma protein was unaffected by the dominant-negative variants [60]. This may reflect differences in chromatin structure of cell cycle regulatory genes vs. muscle differentiation genes, or indicate different activation requirements by MyoD protein. Consistent with the latter possibility, the basic region of MyoD, which is required for the activation of muscle specific genes, is dispensable for MyoD-mediated cell cycle arrest [61]. Together, the data suggest that MyoD (and probably Myf5) recruit mammalian SWI/SNF complexes to initiate target gene transcription. However, formal proof of a physical interaction between MyoD and Brm/Brg-1 containing chromatin remodelling complexes is still missing, as well as formal genetic evidence for an involvement of hSWI/SNF complexes in myogenesis. Indeed, the latter may not be easy to obtain, given that gene knock-outs for several components of Brm/BRG-1 complexes, including BRG-1 [62], SNF5/INI1 [63–65], and Srg3 [66], result in early embryonic lethality. MyoD induced chromatin remodelling is not restricted to promoter regions, but has been observed at several kb distance from the promoter and in the absence of tran- scription. Thus, it could provide a priming function for the activation of late muscle genes by factors like myogenin, which cannot efficiently remodel chromatin [57]. Precedence for a preconfiguration of chromatin structure, which occurs before transcriptional activation, has been reported for example for the lysozyme gene in myeloid cells [67], and for the b-globin gene in erythrocytes [68]. Priming by SWI/SNF complexes may even apply to the myogenic determination genes themselves. It was recently shown that b-catenin interacts directly with BRG-1 to promote target gene activation in response to Wnt ligands [69], one of the presumed inducers of Myf5 and MyoD transcription [1]. b-Catenin binds to Tcf transcription factors, which can also serve as docking sites for corepressors [70]. Indeed, a distal Tcf binding site mediates repression of Myf5 in nonmuscle mesoderm in Xenopus, i.e. in regions devoid of Wnt signaling [5]. Although the mechanism of repression is not known in this case, evidence from Drosophila indicates that some Wingless target genes are silenced in the absence of the Wg ligand through a specialized Brm chromatin remodel- ling complex, characterized by the conserved Osa protein [71]. It remains to be seen whether vertebrates contain an equivalent remodelling complex to repress Wnt target genes such as Xmyf5, or whether this involves the groucho/ HDAC corepressor complexes, which interact with Tcf proteins in the absence of Wnt signalling [70]. CONCLUSIONS We have limited this review to interactions between the core network of myogenic regulatory proteins and chromatin modifying enzymes. Correlating the genetic blueprint of skeletal myogenesis with these epigentic regulatory inputs reveals that embryonic cells need to modify the chromatin structure in many ways in order to become muscle (Fig. 1). Additional, pivotal control levels, such as the interplay between the myogenic bHLH regulators and the cell cycle Ó FEBS 2002 Muscle chromatin (Eur. J. Biochem. 269) 2297 machinery, have not been elaborated here. As exemplified by the retinoblastoma protein, whose antiproliferative activity depends at least in part on SWI/SNF and HDAC protein functions [7], chromatin would again emerge as one important interface of regulation. Given the rapid advances of the field in recent years, it can be expected that the tuning of the chromatin fibre will soon be recognized as a general and essential aspect of developmental gene regulation. ACKNOWLEDGEMENTS This review is dedicated to the late Dr Alan P. Wolffe, whose scientific passion, insight and vision constituted an important motor for the chromatin field. REFERENCES 1. Buckingham, M. (2001) Skeletal muscle formation in vertebrates. Curr. Opin. Genet. Develop. 11, 440–448. 2. Tapscott, S.J., Lassar, A.B. & Weintraub, H. 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Veenstra 2 1 Adolf-Butenandt-Institut,. for example for the lysozyme gene in myeloid cells [67], and for the b-globin gene in erythrocytes [68]. Priming by SWI/SNF complexes may even apply to the myogenic determination genes themselves of MyoD. This modification increases both MyoD’s DNA binding activity [42] and its affinity for p300/CBP [43]. Preventing acetylation of MyoD by mutating these lysines impaired the ability of MyoD