REVIEW ARTICLE
Differential modulation of cell cycle progression
distinguishes members of the myogenic regulatory factor
family of transcription factors
Kulwant Singh1 and F. Jeffrey Dilworth1,2
1 Sprott Center for Stem Cell Research, Ottawa Hospital Research Institute, ON, Canada
2 Department of Cellular and Molecular Medicine, University of Ottawa, ON, Canada
Keywords
cell cycle; DNA repair; gene expression;
MRF4; Myf5; MyoD; myogenesis; myogenin
(Myog)
Correspondence
F. J. Dilworth, Ottawa Hospital Research
Institute, 501 Smyth Rd, Mailbox 511,
Ottawa, ON, Canada K1H 8L6
Fax: +1 613 739 6294
Tel: +1 613 737 8899 ext 70339
E-mail: [email protected]
(Received 7 January 2013, revised 1
February 2013, accepted 5 February 2013)
doi:10.1111/febs.12188
The muscle-specific basic helix–loop–helix proteins MyoD, Myf5, myogenin
(Myog) and MRF4 constitute the myogenic regulatory factor (MRF) family of transcription factors that drive muscle gene expression during myogenesis. Having evolved from a single ancestral gene, the spatial and
temporal specificity of expression for each family member has been used to
define a hierarchical relationship between the four MRFs. Molecular characterization of two of the MRFs (MyoD and Myog) suggests an important
distinction between these factors, whereby MyoD establishes an open chromatin structure at muscle-specific genes, whereas Myog drives high levels
of transcription of genes within this open chromatin state. Furthermore,
recent data have provided an additional distinction between MRF function
with respect to cell cycle regulation. Indeed, MyoD has been shown to
directly activate genes involved in cell cycle progression, leading to myoblast proliferation. In contrast, Myog has antiproliferative activity through
the activation of genes that shut down the cell proliferation machinery,
leading to cell cycle exit and myoblast differentiation. Although the transcriptional activities of MyoD and Myog synergize to drive muscle differentiation, it is the expression of Myog that sets in motion a gene
expression program that constitutes a ‘point of no return’, leading to cell
cycle exit. In this review, we compare and contrast the current literature
with respect to MRF function, with a particular emphasis on the differential role of MRFs in modulating the cell cycle.
Introduction
Developmental gene expression programs are established through the combined activity of tissuerestricted and ubiquitously expressed transcription factors that allow communication between distal and
proximal regulatory regions of genes [1,2]. According
to current models, tissue-restricted transcription factors help to establish a remodeled chromatin structure
that is permissive to binding of the transcriptional
machinery at specific genes within a cell type [3]. The
paradigm for such a model has been the transcriptional regulatory network that governs vertebrate skeletal myogenesis. Indeed, expression of the skeletal
muscle-specific transcription factor MyoD was long
ago shown to be sufficient to mediate lineage repro-
Abbreviations
bHLH, basic helix–loop–helix; C/H, cysteine/histidine-rich; ChIP, chromatin immunoprecipitation; E, embryonic day; KO, knockout; MAPK,
mitogen-activated protein kinase; MRF, myogenic regulatory factor; Myog, myogenin; pRB, retinoblastoma protein; TAD, transactivation
domain.
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Modulation of cell cycle by MRFs
K. Singh and F. Jeffrey Dilworth
gramming through activation of the muscle gene
expression program in multiple cell types [4]. This seminal finding led to the cloning of three related skeletal
muscle-specific transcription factors [5]. Collectively,
MyoD, Myf5, myogenin (Myog), and MRF4 constitute a family of transcription factors that have been
termed myogenic regulatory factors (MRFs). Characteristic of MRFs, these proteins share a highly conserved variant of the basic helix–loop–helix (bHLH)
domain [6] that confers their myogenic potential [7].
Whereas the expression of MRFs is skeletal musclespecific, their function requires heterodimerization with
a member of the ubiquitously expressed E-protein family of bHLH proteins. Upon dimerization, the MRF–
E-protein heterodimeric complexes are able to bind the
E-box consensus sequence (CANNTG), which is present in the regulatory regions of muscle-specific genes
[8]. Although E-boxes are well represented in the genome, with more than 14 million sites, MRFs bind only
a small fraction of these sequences [9,10]. Furthermore, chromatin immunoprecipitation (ChIP) sequencing analysis has shown that MRFs bind both
overlapping and distinct loci within the genome
[10,11]. This suggests that the binding of MRFs to
their target E-box is limited by chromatin accessibility,
but also through properties intrinsic to the individual
family members.
Phylogenetic analysis indicates that the four vertebrate MRF genes have evolved from a single ancestral
MRF gene as a result of gene duplication events and
subsequent divergent mutations [12]. Indeed, many
invertebrates, such as Caenorhabditis elegans, Drosophila, sea urchins, and acidians, continue to develop their
musculature in the presence of a single MRF gene [13–
15]. However, the more complex musculature of
vertebrates has forced the evolution of four MRFs to
regulate the complex gene expression program in myogenesis. To establish this complex regulation of gene
expression, MRFs have retained high conservation of
their DNA-binding domain (bHLH), while the transactivation domains of each transcription factor have
diverges. This has allowed MyoD, Myf5, Myog and
MRF4 to retain a certain degree of functional overlap
while acquiring the unique properties required for
development of the mammalian musculature. This has
been clearly demonstrated in genetic models, where
knock-in studies have shown that MRFs display a certain degree of functional redundancy, but are not completely interchangeable [16,17]. Although elegant
experiments involving swapping between MyoD and
Myog have provided some insights into the roles of
specific protein domains in mediating differential
transactivation by these transcription factors [18,19],
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most studies examining the mechanisms of transactivation have focused on the activities of individual
MRFs. Thus, we currently lack a good understanding
of the specific functional roles played by individual
MRFs in the processes of muscle development, repair,
and homeostasis. Here, we review the current literature
concerning MRF functions that supports the notion
that MRFs play differential roles in mediating cell
cycle progression.
Understanding the role of MRFs
through mouse genetics
Studies over the last few decades have made significant
advances in demarcating the functions of individual
MRFs in myogenesis. Importantly, these studies have
shown that these muscle-specific transcription factors
have evolved a hierarchical relationship that retains a
certain degree of functional redundancy, while highlighting the fact that each MRF also possess unique
properties that subtly affect the myogenic process.
Mouse genetic studies have played a key role in identifying the hierarchical relationship between MRFs,
whereby MyoD and Myf5 are generally viewed as factors involved in the determination of myogenic cells,
whereas Myog and MRF4 are more closely associated
with terminal differentiation and homeostasis of myofibers [5]. The definition of MyoD and Myf5 as determination factors comes from genetic studies in mice,
where the double knockout (KO) of these two MRFs
resulted in postnatal lethality, owing to a complete
absence of skeletal myoblasts or myofibers [20–22].
The absence of an overt phenotype in mice lacking
either MyoD or Myf5 provides strong evidence for
functional overlap between these two determination
factors [20–22]. However, further exploration of myogenesis in the absence of MyoD or Myf5 has revealed
subtle difference between these two transcription factors. Developmental studies of the single KO mice
showed delayed myogenesis in the epaxial myotome in
the absence of Myf5, whereas myogenesis in the hypaxial myotome was delayed in mice lacking MyoD
[23]. Further evidence for MyoD and Myf5 determining distinct muscle populations in the myotome comes
from studies showing that ablation of Myf5-expressing
cells failed to prevent MyoD-dependent skeletal muscle
differentiation [24,25]. This suggests that MyoD and
Myf5 are responsible for determining distinct myogenic populations in the myotome that have the potential to compensate for each other in development.
Mice that lack Myog continue to specify the muscle
lineage through the formation of myoblasts. However,
these mice show perinatal lethality, because of severe
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K. Singh and F. Jeffrey Dilworth
disruption of myoblast differentiation and muscle fiber
formation, leading to the idea that Myog is a differentiation factor in the myogenic process [26,27]. This
functional distinction between Myog and its related
family members MyoD and Myf5 is further highlighted by studies showing that the Myog/MyoD or
Myog/Myf5 double KO mice specify the muscle lineage but do not form muscle fibers – a phenotype similar to that of the Myog KO mouse [28]. These findings
demonstrate that the function of Myog does not overlap with those of MyoD and Myf5, and that Myog
acts downstream of MyoD and Myf5 in skeletal muscle development as a differentiation factor.
Studies have demonstrated that MRF4 can act as
both a determination and a differentiation factor [29].
Similar to the effect of KO of the determination factors MyoD and Myf5, MRF4 null mice do not show
any overt muscle phenotype [30]. This would suggest
that the determination role of MRF4 is functionally
redundant with MyoD and Myf5, whereas its differentiation role is redundant with that of Myog. Indeed,
MRF4 KO mice did show strong upregulation of
Myog expression, suggesting that MRF4 probably
functions to downregulate Myog expression in the
mature myofiber [30]. Interestingly, MRF4/MyoD KO
mice died at birth, showing a phenotype highly similar
to that of the Myog null mutant mice [31]. In MRF4/
MyoD double KO mice, Myog was expressed, but this
expression was insufficient to support normal myogenesis in vivo. This suggests that MRF4 and MyoD play
a redundant role in mediating skeletal muscle differentiation during development. Taken together, these
genetic studies establish the need for a minimum of
two MRFs to generate functional muscle during development: one to determine the muscle lineage, and a
second to permit terminal differentiation.
Spatial and temporal control of MRF
expression
The four members of the MRF family can act as master regulators of skeletal myogenesis, whereby their
exogenous expression can hijack the inherent gene
expression program of a nonmuscle cell and drive it
towards a myogenic fate [4,32–34]. Therefore, the location, timing and expression levels of the MRFs during
embryonic development are tightly regulated to ensure
the accurate progression of the developmental process.
In the murine model, precursor cells for myogenesis
originate in the segmentally arranged nascent somites
that flank both side of the neural tube and notochord.
As embryonic development progresses (Fig. 1), a
portion of the somites transforms into a transient
Modulation of cell cycle by MRFs
structure called the dermomyotome [35]. Within these
structures, expression of MRFs is first detected at
around embryonic day (E)8.0, as sonic hedgehog signaling from the notochord and floor plate induces epaxial expression of Myf5 in the dorsal lips of the
dermomyotome, committing these cells to become the
epaxial myotome [36,37]. At E10.5, Wnt (from the
dorsal ectoderm) and Bmp4 (from the lateral mesoderm) signaling establishes MyoD expression in the
hypaxial dermomyotome, causing these cells to establish the hypaxial myotome [38,39]. Transcripts for
Myog and MRF4 are first detected at E8.5 and E9.0,
respectively, and their expression is evenly distributed
throughout the myotome [39–42]. Consistent with a
dual role in both determination and differentiation of
the muscle lineage, MRF4 transcripts show biphasic
expression, whereby they are downregulated by E11.5,
but reappear at E16.0 in differentiated muscle fibers
[40]. These MRFs execute the myogenic differentiation
program, via expression of downstream targets, that
results in the development of trunk muscle from the
epaxial myotome, whereas limbs, diaphragm and body
wall muscle develop from the hypaxial myotome.
Whereas in vivo studies have provided important
spatial information about the expression of MRFs, the
hierarchical relationship regulating the expression of
the different family members has been defined through
ex vivo studies using satellite cells. Satellite cells (SCs)
are resident muscle stem cells that are mitotically quiescent in healthy adult muscle and become activated
upon damage to the muscle fiber [43]. Overall, these
studies have shown that the progression of activated
satellite cells towards the myogenic lineage is mainly
controlled by expression of Myf5 and MyoD [44].
Recent work suggests that Myf5 sits at the top of the
hierarchy, as it was shown that the majority of quiescent satellite cells transcribe the Myf5 gene and are
poised to enter the myogenic program. To maintain
these poised cells in the quiescent state, mRNAs
encoding Myf5 remain untranslated, owing to miR-31dependent sequestration of the transcripts in mRNP
granules. Upon satellite cell activation, mRNP granule
dissociation results in the release of sequestered transcripts and rapid translation of the Myf5 mRNAs [45].
Soon afterwards, MyoD expression is initiated through
the activity of Pax3/7, Six1/4, and FoxO3, to permit
expansion of a cell population that is committed to the
myogenic lineage [46,47]. In the proliferating myoblasts, the MyoD gene is transcribed through a
TFIID-dependent mechanism, whereby a moderate
level of expression is ensured through spatial localization near the periphery of the nucleus [48,49]. As myoblasts initiate differentiation towards myotubes, the
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K. Singh and F. Jeffrey Dilworth
Fig. 1. A schematic representation of embryonic and postnatal skeletal myogenesis. During vertebrate myogenesis, somites differentiate
and subdivide to give rise to the dermomyotome (DM) and sclerotome (SCL) in response to signals from the neural tube (NT) and notochord
(N). Cells from the dorsomedial lip (DML) migrate under the DM to form the epaxial myotome, which is the main source of the deep back
musculature. Similarly, cells originating in the ventrolateral lip (VLL) migrate under the DM to generate the hypaxial myotome, which gives
rise to the lateral trunk musculature. A proportion of cells from the VLL undergoing epithelial–mesenchymal transition delaminate and
migrate to the region of presumptive limb muscle development (migrating limb precursors). The paired homeobox gene Pax3 has been
demonstrated to be a key regulator of embryonic skeletal myogenesis, and sufficient to activate the expression of Myf5 and MyoD and
initiate the myogenic program that leads to the development of myoblasts. Although myogenic precursor cells express both Pax3 and Pax7,
Pax7-expressing cells are more prominent in the central region of the DM, and this is the population that gives rise to the major pool of
Pax7+ satellite cells (SC) in the body. SCs are mitotically quiescent in adult muscle, and are located underneath the basal lamina. In
response to muscle injury, SCs are activated and give rise to Myf5 /MyoD cells, which return to the quiescent stage, and a Myf5+/MyoD+
myoblast population. Myoblasts generated from both embryonic and postnatal myogenesis undergo extensive proliferation, which leads to
the generation of Myog+ myocytes in response to appropriate differentiation cues. Finally, these myocytes fuse to form myotubes and
subsequently myofibers, which continue to express the terminal differentiation marker MRF4, structural and metabolic genes such as the
myosin heavy chain (MHC) and the muscle creatinine kinase (MCK) genes.
MyoD gene moves towards the lumen of the nucleus,
where a TAF3/TRF3-dependent transcriptional mechanism results in a high level of MyoD expression
[48,49]. Under these conditions, MyoD induces Myog
expression, which results in downregulation of Myf5
expression [50]. This switch in expression from Myf5
to Myog coincides with cell cycle exit and a commitment to differentiate [2,51]. The combined activity of
MyoD and Myog leads to the expression of the
MRF4 gene and other late muscle differentiation genes
to permit the formation of multinucleated fibers. In
mature muscle fibers, expression of MyoD and Myog
is then downregulated, whereas MRF4 continues to be
expressed at high levels to act as the predominant
MRF in adult muscle [41].
The sequential expression of MRFs during muscle
differentiation, combined with their functional redundancy, would suggest that the timing of MRF expression could be responsible for the differential roles of
family members in muscle formation. Supporting such
a possibility, insertion of the Myog cDNA into the
Myf5 locus in Myf5/MyoD double KO mice allowed
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Myog to rescue the absence of skeletal muscle [16].
The formation of healthy myofibers in these knock-in
mice demonstrated that Myog can act to specify the
muscle lineage in the proper context. However, it is
noted that these mice died at birth, owing to reduced
skeletal muscle formation. Thus, whereas Myog was
able to specify the muscle lineage, it appears that
Myog may not have allowed for the expansion of the
muscle progenitor population to generate sufficient
cells to establish a complete musculature. Therefore,
these findings demonstrate that there are functional
differences inherent to MRF family members beyond
their specific spatial and temporal expression patterns
during development.
Divergence within structural domains
of MRFs
Although all four MRFs are able to induce lineage
reprogramming of fibroblasts towards a skeletal muscle lineage, studies in cultured cells have demonstrated
differential efficiency between MRFs in activating the
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K. Singh and F. Jeffrey Dilworth
Modulation of cell cycle by MRFs
expression of silent muscle genes [18,52]. To explain
this divergent ability to activate gene expression within
chromatin, differences in protein structure have been
explored. Examination of the protein sequences shows
that MRFs vary in size from 224 amino acids (Myog)
to 319 amino acids (MyoD). Structurally, the MRFs
are highly similar, in that they possess a conserved
bHLH domain for DNA binding that is flanked by
less conserved N-terminal and C-terminal domains
that mediate transcriptional activation (Fig. 2). However, the divergent amino acid sequences in the transactivation domains (TADs) of MRFs raises the
possibility that they may be responsible for the inherent differences between family members. This hypothesis was tested by Ishibashi et al. [53], who
interchanged the TADs between MyoD and Myf5.
These experiments demonstrated that the N-terminal
and C-terminal domains of MyoD and Myf5 were
interchangeable for the activation of gene expression
involved in myoblast proliferation. However, in conditions of differentiation, the N-terminal and C-terminal
TADs of MyoD act in a synergistic manner to induce
the expression of muscle differentiation genes, and this
synergy could not be achieved in combination with
either of the Myf5 TADs [53].
Within the TADs of MyoD, two structural domains
have been identified that allow MyoD to activate the
expression of silent genes during myogenesis [18].
These domains are termed the cysteine/histidine-rich
(C/H) domain and helix 3 domain, and they lie within
the N-terminal and C-terminal TADs respectively.
Interestingly, swapping of the C/H and helix 3
domains of MyoD into Myog allows Myog to efficiently activate the expression of silent muscle genes
[18]. Characterization of the C/H and helix 3 domains
showed that this structure is important for targeting
MyoD to E-boxes within repressive chromatin through
an interaction with the homeodomain protein Pbx1
[19]. Once tethered to the chromatin by Pbx1, MyoD
can then recruit the SWI/SNF ATP-dependent chromatin remodeling complex [54] and the p300 histone
acetyltransferase [55,56] to muscle-specific genes for
the establishment of an open chromatin structure.
Importantly, the C/H and helix 3 domains of MyoD
and Myf5 are highly conserved, whereas the domains
diverge in the determination factors Myog and MRF4
[18]. Genome-wide binding analyses have demonstrated that MyoD and Myf5 share a significant proportion of their targets in proliferating myoblasts [10].
This would suggest that a shared ability to remodel
chromatin might be at the heart of the redundant ability of MyoD and Myf5 to efficiently specify the muscle
lineage. Importantly, the C/H and helix 3 domains of
Myog cannot mediate remodeling of chromatin within
the promoter of muscle genes [18,52]. Instead, Myog is
a strong activator of transcription at loci with an open
chromatin structure previously established by MyoD
[57]. That being said, Myog can function in concert
with transcription factors such as Mef2D to recruit
chromatin remodeling machinery at muscle genes to
permit transcriptional activation [58]. This observation
would explain how Myog could act as a specification
factor when expressed from the Myf5 locus in the
MyoD/Myf5 double KO background [16]. Thus,
MyoD and Myf5 possess an activity that allows for
the opening of chromatin that is not shared by Myog.
Domain swapping studies have also revealed functional differences between MyoD and MRF4, whereby
the N-terminal domain of MRF4 can act as either a
transcriptional activator or as a repressor, depending
on the promoter context [59]. Among the target genes
repressed by MRF4 is the cardiac a-actin gene, which
N-terminal TAD
C-terminal
C = conserved
NC = not conserved
p = phosphorylation
Ac = acetylation
Fig. 2. A schematic diagram of the structure of MyoD as a representative of the MRF family. The amino acids encompassed by the
different domains are indicated. Various known post-translational modifications (PTMs) are presented in table below, and their conservation
is determined on the basis of multiple sequence alignment.
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is activated by MyoD expression [59]. Direct competition studies on the cardiac a-actin promoter showed
that the repressive property of MRF4 predominates
over MyoD-mediated transactivation, suggesting that
the relative levels of different MRFs may modulate the
transcriptional output of specific muscle genes [59].
Interestingly, MRF4 activity is modulated by the p38
mitogen-activated protein kinase (MAPK) signaling
pathway during the course of myoblast differentiation
[60]. Phosphorylation of MRF4 by p38 MAPK at
Ser31 and Ser42 within the N-terminal transactivation
domain inhibits its function, permitting activation of
the cardiac a-actin gerne while blocking CKm gene
expression [60]. Thus, the N-terminal TAD of MRF4
cooperates with p38 MAPK to selectively modulate
the expression of the late myogenic transcriptional
program. Taken together, these results suggest that
divergent TADs within the MRF family play a role in
modulating their targeting to specific genomic loci.
MRFs and their transcriptional targets
The binding of several MRFs has been explored in
cultured cell systems with high-throughput technologies such as ChIP microarray and ChIP sequencing
[9,10,57,61]. Among these, genome-wide binding of
MyoD has been the most thoroughly explored. The
consensus among these different studies is that MyoD
binds a large number of genes in muscle cells while
modifying gene expression at only a fraction of its targets [9,10,57,61]. However, an important finding of the
study by Cao et al. was the observation that binding
of MyoD within the genome correlated well with
opening of the chromatin structure through acetylation
of histones [9]. This opening of chromatin has recently
been shown to establish enhancer elements that can
control the expression of both coding and noncoding
RNAs [62]. This would suggest that a major role of
MyoD in the myogenic process is the opening of chromatin at specific loci to establish the muscle-specific
chromatin state. Consistent with this idea, studies in
the Tapscott laboratory demonstrated that MyoDdependent activation of late muscle genes required the
activity of Myog [57]. On the basis of these findings, it
was proposed that the transcriptional activation
domain of Myog drives high-level expression at genes
that have an open chromatin structure established by
MyoD [57]. Nevertheless, MyoD can also directly
modulate the activation of gene expression. During
differentiation, MyoD-dependent activation of immediate-early genes occurs in the absence of Myog [57].
Similarly, MyoD has been shown to directly regulate
the expression of a subset of genes in proliferating
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myoblasts through direct binding to the promoter
[9,10]. Gene ontology analysis of these different target
genes revealed that MyoD displayed enhanced binding
in myotubes at genes involved in muscle development,
whereas genes involved in the regulation of cell cycle
were preferentially bound by MyoD in proliferating
myoblasts [9,61]. Thus, MyoD binding facilitates the
expression of genes that promote proliferation or differentiation of myoblasts, depending on the cellular
environment.
Genome-wide occupancy of Myf5 has been reported
recently in proliferating myoblasts [10]. Comparative
analysis of MyoD and Myf5 binding sites showed a
highly significant overlap of ~ 30% [10]. This result is
consistent with the notion that MyoD and Myf5 share
a role in defining myoblast identity. Unfortunately, the
study did not report the ontology of Myf5 target
genes, and thus it remains to be seen whether Myf5
modulates myoblast proliferation through direct upregulation of genes involved in cell cycle regulation.
Analysis of the genome-wide binding of Myog that
was performed within the ENCODE project has not
yet been published [63]. Thus, our understanding of
Myog binding is shaped mostly by ChIP array studies
performed to identify associations with defined promoter regions [57,61]. These studies revealed that
Myog is bound to 75% of the promoters that are targeted by MyoD in differentiating myotubes [61]. This
finding is consistent with the view that MyoD establishes an open chromatin structure that, in turn, permits binding of Myog to establish a high level of
transcriptional activation [57]. Examination of the
genes co-bound by MyoD and Myog identified genes
involved in muscle development [57,61]. Interestingly,
microarray studies examining the role of Myog in differentiation recently identified genes involved in cell
cycle progression as key transcriptional targets that
are downregulated by Myog during differentiation
[51]. This suggests that Myog is an important modulator of cell cycle exit during differentiation. Thus, in
contrast to MyoD, which promotes proliferation in
growing myoblasts, Myog attenuates the expression of
genes that mediate cell cycle progression.
MRFs as modulators of the cell cycle
Studies from cultured cell systems support the notion
that MyoD and Myog have opposing roles in modulating the cell cycle. In fact, both MyoD and Myf5
have been shown to promote expansion of the muscle
progenitor population [64–66]. In contrast, Myog
appears to possess intrinsic activity that is required to
mediate cell cycle exit [51]. Indeed, it was recently
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K. Singh and F. Jeffrey Dilworth
demonstrated that ectopic expression of Myog in proliferating myoblasts leads to exit from the cell cycle
[51]. This finding provides an explanation for the phenotype observed in the studies where Myog is
expressed from the Myf5 locus in the Myf5/MyoD
double KO mouse [16]. These mice had specified the
muscle lineage and established healthy muscle fibers.
However, the mice died at birth, owing to insufficient
musculature. Thus, expression of Myog from the Myf5
locus is sufficient to determine the muscle lineage,
although it probably caused precocious exit from the
cell cycle, leading to impaired expansion of the progenitor pool, resulting in less muscle. Furthermore, this
finding might explain why stable expression of Myog
in primary myoblasts has never been reported. Taken
together, these findings support the notion that MyoD
and Myf5 promote expansion of muscle progenitor
pools, whereas Myog induces cell cycle exit.
Characterization of satellite cells from MyoD KO
[65] and Myf5 KO [66] mice showed that they both
had proliferation defects. This suggests that the two
determination MRF factors play nonredundant roles
in the cell cycle. Further evidence for this notion come
Modulation of cell cycle by MRFs
from studies showing the distinct and contrasting
expression patterns of MyoD and Myf5 during the different phases of the cell cycle [67]. Myf5 protein levels
peak in G0, decrease during G1, and then rise again at
the end of G1 where they remain stable through mitosis (Fig. 3). In contrast, MyoD has been shown to
block the G1/S transition [68]. Thus, MyoD protein
levels peak in mid-G1, are reduced to their minimum
level on G1/S transition, and are reaugmented from S
to M [67]. These changes in MyoD and Myf5 protein
levels during the cell cycle are modulated through
post-translational modifications that signal their degradation. Although not all of the mechanisms regulating
MRF stability have been established, several studies
have highlighted modifications that lead to degradation of MyoD or Myf5. In particular, the Myf5 protein level is modulated by its phosphorylation and
subsequent degradation at mitosis [69,70]. In the case
of MyoD, degradation at late G1 is mediated by the
ubiquitin proteasome system, which is triggered by cyclin E/CDK2-dependent phosphorylation of MyoD on
Ser200 [71,72]. The transcriptional activity of MRFs
can also be modulated during the cell cycle, as it has
Fig. 3. A schematic overview of crosstalk between MRFs and cell cycle regulation. MyoD and Myf5 show distinct and contrasting
expression patterns during the different phases of the cell cycle. The Myf5 protein level peaks in G0, decreases during G1, and then rise
again at the end of G1 where they remain stable through mitosis. In contrast, the MyoD protein level peaks in mid-G1, decreases to its
minimum level in the G1/S transition, and is reaugmented from S to M. The levels of both proteins during the different phases of the cell
cycle are regulated by phosphorylation-dependent degradation via the 26S proteasome. In proliferating myoblasts, MyoD initiates the
expression of two genes, CDC6 and MCM2, which are primarily involved in making chromatin operational for DNA replication and
progression of cells through S-phase. In response to appropriate differentiation signals, MyoD induces the expression of Myog, and
establishes a transcriptionally permissive chromatin structure at the p21cip, p57kip and pRB genes. Myog and MyoD then synergize to
upregulate the expression of these key regulators of cell cycle exit – p21cip and p57kip repress the activity of Cdks and cyclins, whereas
pRB targets E2F family members, which are major regulators of the expression of Cdks and cyclins. Myog also interfers with cell cycle
progression by upregulating the expression of miR-20a and LATS2. miR-20a is a microRNA that is well characterized for its ability to
downregulate the transcription factors E2F1 and E2F3 through targeting the 3′-UTRs of their mRNAs. LATS2 is a protein kinase that has
been implicated in targeting of the transcriptional repressor complex DREAM to E2F target genes to block cell cycle progression.
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K. Singh and F. Jeffrey Dilworth
been shown that MyoD is phosphorylated at Ser5 and
Ser200 by cyclin B/Cdc2 in mitosis to inhibit its DNAbinding activity and transcriptional activation ability
[73]. Thus, the activities of Myf5 and MyoD are
dynamically regulated throughout the cell cycle via
modulation of transcriptional activity and protein
abundance.
Although distinctions in the expression patterns
have been observed, the relative importance of MyoD
and Myf5 to cell cycle progression has not been established. Genome-wide binding studies have previously
established that MyoD binds to the transcriptional
regulatory region of genes with critical roles in the cell
cycle [9,57,61]. Characterization of specific promoters
in growth-stimulated quiescent myoblasts showed that
MyoD directly activates expression of the CDC6 and
MCM2 genes, which are involved in preparing chromatin for DNA replication, and consequently progression of cell through S-phase [64]. We note that,
whereas MyoD shows greater efficiency in establishing
transcription at two genes that modulate the cell cycle,
Myf5 can also perform this function, showing some
degree of functional redundancy [64]. Future mechanistic studies based on the plethora of genome-wide
data should prove highly informative with respect to
the transcriptional activation of specific genes by Myf5
and MyoD in proliferating myoblasts.
An interesting distinction between MyoD and Myf5
is their ability to modulate cell cycle progression in
response to DNA damage [74]. The cellular response
to DNA damage is to block cell cycle progression at
specific checkpoints, which allows DNA repair to prevent the propagation of genomic mutations in daughter cells [75,76]. In the case of skeletal myogenesis,
MyoD is a genuine target for the differentiation checkpoint. In response to genotoxic stress, the transcriptional activity of MyoD is repressed to prevent
myotube formation in cells that have arrested the cell
cycle because of DNA damage. This transcriptional
inhibition requires phosphorylation of MyoD at Tyr30
by c-Abl tyrosine kinase [77]. Interestingly, the consensus site for Abl-mediated tyrosine phosphorylation is
absent in Myf5, making this protein insensitive to the
DNA damage response. The differential susceptibility
of MyoD and Myf5 to c-Abl-mediated repression is
not surprising, as MyoD levels peak in mid-G1, where
myoblasts exit the cell cycle, whereas Myf5 levels are
minimal at this stage of the cell cycle. Interestingly,
the c-Abl consensus binding site (YDDP) is also
absent in Myog and MRF4, suggesting that this role
in facilitating DNA repair is unique to MyoD [74,77].
For successful execution of the myogenic differentiation program, myoblasts must exit the cell cycle. An
3998
extensive literature has supported a role for MyoD in
the induction of cell cycle exit during terminal differentiation (reviewed in [78]). Indeed, it has been shown
that MyoD / myoblasts fail to exit the cell cycle, as
the transcriptional regulator nuclear factor-jB (NF-jB)
maintains a nuclear localization to activate the transcription of key cell cycle regulators [79]. However,
recent studies examining bromodeoxyuridine staining in
differentiating myoblasts have demonstrated that
MyoD expression in promyogenic conditions is not sufficient for cell cycle exit [51]. Instead, it appears that the
activation of Myog expression by MyoD constitutes a
‘point of no return’, where Myog initiates a gene expression program that commits the differentiating myoblast
to exit the cell cycle [51]. Consistent with the existing literature, Myog-induced cell cycle exit appears to occur
through the activation of CDKN1a (p21cip), as well as
pathways that inhibit the expression of E2F target
genes – cell cycle targets that have previously been
attributed to MyoD [45,78,80–82]. Indeed, exogenous
expression of Myog in proliferating myoblasts leads to
upregulation of p21cip, which triggers cell cycle exit [51].
This appears to be a direct modulation of p21cip expression, as a binding site for Myog has been identified at
the CDKN1a promoter (unpublished observation based
on the ENCODE data from CalTech, K. Singh and F.
J. Dilworth). In the case of E2F target genes, Myog
takes a multipronged approach to ensure efficient silencing of these loci. In a first mechanism, Myog upregulates
transcription of the miR17-92 cluster of microRNAs
[51]. Among these microRNAs, miR-20a has been
shown to target the mRNA of E2F1, E2F2 and E2F3 to
inhibit their translation [83–85]. Therefore Myog can
indirectly downregulate the cellular protein levels of
E2F family members. A second mechanism used by
Myog to inhibit E2F target gene expression is upregulation of Lats2 kinase [51], which has been shown to inhibit transactivation by E2F proteins. Lats2 was identified
in a small hairpin RNA screen of factors that function
with retinoblastoma protein (pRB) to mediate cell cycle
exit [86]. There, it was shown that Lats2, a component
of the Hippo pathway [87], is required to initiate a phosphorylation cascade that leads to pRB-dependent binding of the repressive complex DREAM to E2F target
genes [86]. This finding is particularly interesting, as it
has been shown that downregulation of pRB and Arf
allows Myog-expressing myocytes to re-enter the cell
cycle [88]. Furthermore, MyoD-induced myogenesis
leads to upregulation of pRB to mediate cell cycle exit
[89] through the establishment of repressive histone
methylation at cell cycle genes [90]. Although we have
not examined whether Myog can directly lead to upregulation of pRB and ARF, we note that MyoD and
FEBS Journal 280 (2013) 3991–4003 ª 2013 The Authors Journal compilation ª 2013 FEBS
K. Singh and F. Jeffrey Dilworth
Myog both bind to the promoter and an intronic enhancer of the Rb1 gene (unpublished observation based on
the ENCODE data from Caltech). On the basis of this
observation, we suggest that MyoD may create an open
chromatin structure at the Rb1 gene that facilitates
strong transactivation by Myog. However, this possibility still needs to be formally tested. Nevertheless, it
appears that MyoD and Myog act in a coordinated
manner to ensure cell cycle exit through the activation
of key genes involved in suppressing E2F activity. The
mechanism by which NF-jB activity is inhibited during
cell cycle exit, and whether Myog participates in this
process, remain to be determined.
It is important to note that the mechanisms
described above for Myog-mediated cell cycle exit are
all indirect, acting by controlling the upregulation of
target genes that are directly involved in blocking cell
cycle progression. This distinction is important, as it
explains observations made by the Walsh group
showing that a small percentage of differentiating
myoblasts staining positive for Myog can proceed
through a round of cell cycle to incorporate bromodeoxyuridine [91]. Thus, we propose that expression
of Myog during myogenic differentiation represents a
‘point of no return’, where the MRF initiates a gene
expression program that commits the myoblast to
withdraw from the cell cycle. In this model, cells that
had committed to division prior to achieving sufficient expression of Myog target genes would complete
a final round of replication before permanently exiting the cell cycle.
The molecular characterization of cultured cell systems has thus provided some mechanistic insights into
the differential roles of MRFs in cell cycle progression
as defined through ex vivo cellular studies of myoblasts from KO mice. Combining these findings, we
propose the following generalization: the MRFs that
play a role in determination (MyoD and Myf5) facilitate cell cycle progression, whereas the MRFs that
mediate differentiation (Myog and MRF4) induce cell
cycle exit. Confirmation of this generalization will
require studies to examine the role of MRF4 in the
cell cycle.
Conclusions and perspective
The functional redundancy within the MRF family is
evident from genetic studies in mice showing that the
development of the murine skeletal musculature can be
directed through the activities of as few as two MRFs
– a determination MRF and a differentiation MRF.
Although it has been difficult to unravel the subtle differences in function between MRFs, high-throughput
Modulation of cell cycle by MRFs
technologies that permit us to pursue unique target
genes as well as changes in gene expression and chromatin structure at a global level have begun to provide
novel insights into the importance of the multiple
MRFs in mediating myogenesis. These studies have
identified a role for the determination MRFs in specifying the muscle lineage through the opening of chromatin structure at genes involved in muscle
development, but also in expanding the muscle progenitor population. In addition, they have identified a role
for the differentiation MRF, Myog, in activating highlevel expression of muscle genes that lie in an open
chromatin structure while also activating signaling cascades to ensure cell cycle withdrawal. Further examination of the plethora of information present in the
high-throughput datasets is certain to provide us with
further insights into the subtle difference between the
various MRFs. Unfortunately, our knowledge of
MRF4 function in myogenesis remains poorly characterized, owing to the absence of good cellular models
for study of its function ex vivo. The current development of more sensitive techniques to exploit ChIP
sequencing and RNA sequencing technologies will
hopefully soon allow for the isolation of MRF4expressing cells from the developing mouse, so that we
can explore the distinct functional features of this
MRF, which displays both determination and differentiation characteristics.
Acknowledgements
We would like to thank M. Brand for critically reading the manuscript. Work in the Dilworth laboratory
on MRF function is supported by a grant from the
Canadian Institutes of Health Research (MOP-77778).
F. J. Dilworth is the Canada Research Chair in Epigenetic Regulation of Transcription.
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