Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA
e-mail: stapscot{at}fhcrc.org
SUMMARY
The expression of Myod is sufficient to convert a fibroblast to a skeletal muscle cell, and, as such, is a model system in developmental biology for studying how a single initiating event can orchestrate a highly complex and predictable response. Recent findings indicate that Myod functions in an instructive chromatin context and directly regulates genes that are expressed throughout the myogenic program, achieving promoter-specific regulation of its own binding and activity through a feed-forward mechanism. These studies are beginning to merge our understanding of how lineage-specific information is encoded in chromatin with how master regulatory factors drive programs of cell differentiation.
Introduction
Developmental biologists have long recognized the role of cell lineage in
establishing the competence of a cell for terminal differentiation. The
association of specific chromatin modifications with individual cell lineages
for example, the association of DNase hypersensitivity and histone
modifications with the hemoglobin gene in red blood cell nuclei but not in
nuclei from other cells (Litt et al.,
2001; Weintraub and Groudine,
1976
) suggested that developmentally established chromatin
modifications can determine the genes that are available to be expressed in an
individual cell-type. A distinction was made between `housekeeping' genes
genes that are constitutively expressed in all cells and
`luxury' genes, such as the hemoglobin locus, the expression of which is
restricted in certain cell-types or in certain circumstances
(Weintraub, 1972
). A crucial
question in developmental biology since then has been whether such luxury
genes are specifically marked for activation by their inherited chromatin
structure, or whether transcription factors that are unique to a
differentiated cell type can alone induce luxury gene expression and impose
upon cells a chromatin structure, regardless of their lineage-established
chromatin components.
To address this question, it was necessary to identify the factors that
regulate luxury gene expression in a specific cell-type and then to determine
whether they had similar activity in different lineages. It was reasonable to
believe such factors could be identified because several studies had already
indicated that complex programs of cell differentiation might be regulated by
the expression of a very small number of genes, or possibly a single gene, a
so-called `master switch' (Holtzer et al.,
1975a; Holtzer et al.,
1975b
; Weintraub et al.,
1973
). It was in this context that a screen for genes that
regulate skeletal myogenesis led to the identification of Myod1
(Lassar et al., 1986
), a
basic-helix-loop-helix (bHLH) transcription factor that can induce skeletal
muscle differentiation in cells from many different lineages.
The identification of Myod as a master regulatory gene of skeletal muscle differentiation provided the opportunity to assess the roles of tissue-specific transcription factors and chromatin-associated proteins in regulating cell differentiation. It also provided the opportunity to address another important question: how does a single transcription factor execute an entire program of cell differentiation? One reason to be interested in the answer to this question is that it is a specific example of the broader question: how does a single event result in a predictable, complex response? It is likely that understanding the orchestration of skeletal muscle gene expression by Myod will provide insights into the regulation of other complex biological events.
This review describes generally how Myod regulates the program of skeletal
muscle differentiation: the simple story of a transcription factor activating
its target genes [reviews covering other aspects of skeletal muscle
development and Myod function, such as cell cycle regulation, have been
published previously (e.g. Buckingham et
al., 2003; Kitzmann and
Fernandez, 2001
; McKinsey et
al., 2001
; Pownall et al.,
2002
; Puri and Sartorelli,
2000
)]. It also describes how a single factor integrates
information from co-regulators and from chromatin-associated proteins to
achieve promoter-specific binding and promoter-specific transcriptional
activation of target genes to temporally pattern gene expression throughout a
program of cell specification and differentiation. Based on our current
understanding of Myod, it is reasonable to suggest that the regulated activity
of a single factor that broadly interacts with many cellular components might
be a common mechanism for orchestrating a complex response to an initiating
event.
The cloning of Myod, a master switch for skeletal muscle
In 1979, Taylor and Jones demonstrated that treating the mouse fibroblast
cell line 10T1/2 with the demethylating agent 5-azacytidine generated clones
with a skeletal muscle phenotype (Taylor
and Jones, 1979). This finding indicated that DNA demethylation
was sufficient to induce skeletal muscle gene expression in these cells. When
genomic DNA was isolated from these muscle clones and stably transfected into
untreated 10T1/2 cells, myogenic colonies were generated at a frequency that
was consistent with the presence of a single locus that could convert the
fibroblasts into skeletal muscle cells
(Lassar et al., 1986
). The
same cell system was then used to clone the cDNA for the myogenic
determination gene Myod1 (Davis
et al., 1987
), hereafter referred to as Myod. When
expressed in primary fibroblasts or in a wide variety of other cell types,
such as pigment, nerve, fat and liver, Myod can convert these cells to
skeletal muscle (Weintraub et al.,
1989
). These findings provided the first direct evidence that a
single gene can initiate a complex program of differentiation, acting as a
master switch.
It is likely that the azacytidine-mediated demethylation of the
Myod gene results in the conversion of 10T1/2 cells to skeletal
muscle, because the Myod gene is heavily methylated and not expressed
in 10T1/2 cells but becomes relatively demethylated and is expressed following
treatment with 5-azacytidine (Jones et
al., 1990). While the demethylation of the Myod locus is
sufficient to activate its expression in 10T1/2 cells, the Myod gene
is not methylated in primary fibroblasts and is not expressed in these cells.
Heterokaryon formation between primary fibroblasts, which have an unmethylated
Myod gene, and 10T1/2 cells, which contain the trans-acting factors
necessary for the expression of a transfected unmethylated Myod gene,
did not result in the expression of the unmethylated fibroblast Myod
gene (Thayer and Weintraub,
1990
), indicating that expression of the Myod gene is
specifically suppressed in primary fibroblasts. It was subsequently shown that
the homeobox factor Msx1 recruits the linker histone H1B to the Myod
enhancer element to repress its transcription
(Lee et al., 2004
;
Woloshin et al., 1995
).
Therefore, in at least some primary cell types, Myod transcription is actively
suppressed by a combination of Msx1 and linker histones. Interestingly, this
suppression is lost in the generation of many fibroblast cell lines, and
clones that emerge through crisis have a methylated Myod locus that
prevents its expression (Jones et al.,
1990
). This suggests that the unregulated cell divisions that lead
to crisis might release the suppression of differentiation genes, like
Myod, perhaps as a means of limiting growth. Consequently, only
clones with a methylated Myod locus grow through crisis. It is
interesting to speculate that releasing the suppression of Myod, and
perhaps of other differentiation-related genes, might contribute to the
epithelial-to-mesenchymal transition that is associated with the progression
of some cancers (Guarino,
1995
).
Myogenic bHLH transcription factors and skeletal muscle development
The ability of Myod to convert fibroblasts and other cell types into
skeletal muscle strongly indicated that it might have a central role in
myogenesis, and subsequent studies have sought to determine its biological
role in development. The Myod protein contains a bHLH motif that is common to
a large family of transcription factors
(Ledent et al., 2002;
Ledent and Vervoort, 2001
)
(Fig. 1). In addition to Myod,
the highly related bHLH proteins Myf5, Mrf4 and Myogenin (Myog) are also
expressed in skeletal muscle, and each has a crucial role in muscle cell
specification and differentiation
(Buckingham et al., 2003
;
Molkentin and Olson, 1996
;
Perry et al., 2001
;
Pownall et al., 2002
;
Puri and Sartorelli, 2000
), as
described below. Understanding the mechanisms by which the myogenic bHLH
protein family regulates myogenesis is likely to provide insight into the
differentiation of many different cell types, because differentiation in many
different lineages is regulated by specific subfamilies of bHLH proteins. For
example, analogous to the Myod sub-family of bHLH proteins, the Neurod
sub-family of bHLH proteins is largely restricted to neural and neuroendocrine
cells, and regulates neuronal specification and differentiation
(Bertrand et al., 2002
). The
E-protein sub-family of bHLH proteins (Tcf3, Tcf4 and Tcf12) has a crucial
role in lymphocyte differentiation (Engel
and Murre, 2001
; Greenbaum and
Zhuang, 2002
), and its family members also function as heterodimer
partners for many of the tissue-restricted bHLH proteins, such as Myod and
Neurod proteins. [For a general review of HLH transcription factors, see
Massari and Murre (Massari and Murre,
2000
)].
|
During normal development, the expression of the myogenic sub-family of
bHLH proteins is almost entirely restricted to the skeletal muscle lineage,
although Myf5 is also expressed transiently in some cells in the developing
nervous system (Tajbakhsh et al.,
1994). Cells in the pre-somitic mesoderm express both Myf5 and
Mdfi (Kraut et al., 1998
), an
inhibitor of Myf5 also known as I-mfa. It remains unknown whether Myf5 protein
regulates any gene expression in the presomitic mesoderm or whether the
inhibitory Mdfi effectively blocks its activity. The cells of the presomitic
mesoderm that express both genes will ultimately generate the myotome, which
is the region of the dermamyotome in the somite that gives rise to skeletal
muscle [both epaxial (dorsal) and hypaxial (ventral) muscles]. Cells that
express Myf5 and Mdfi will also give rise to sclerotome, the
vertebral-cartilage forming part of the somite, and to other somatically
derived tissues. In the presomitic mesoderm, therefore, the low expression of
Myf5 is not sufficient to commit the cells to an exclusively skeletal muscle
fate. In the dorsal lip of the dermamyotome, however, expression of the
inhibitory Mdfi decreases and the expression of Myf5 protein increases
(Kraut et al., 1998
),
co-incident with the specification of the skeletal muscle lineage.
Early expression of Myf5 is prominent in the epaxial myotome,
where it drives the differentiation of the back, intercostal and abdominal
wall muscles. By contrast, the early expression of Myod is most
prominent in the hypaxial myotome, where it drives the differentiation of the
limb, tongue and diaphragm muscles, and the muscles of branchial arch-derived
tissue (Kablar et al., 1998).
Although Myf5 is not necessary for the expression of Myod, the
combined deletion of Myf5 and Pax3 in mice results in the
absence of Myod expression
(Tajbakhsh et al., 1997
),
suggesting that these two factors are required to initiate Myod
expression; however, as discussed below, the elements regulating Myf5
and Myod transcription are complex and remain poorly understood.
Myod and Myf5: nodal points in skeletal muscle specification
It has been well documented that signaling from the surrounding tissues
regulates the expression of the myogenic bHLH genes in the somite: sonic
hedgehog (Shh) from the notochord and floor-plate, Wnt signaling from the
dorsal neural tube (see Fig.
2), and Bmp4 signaling from the adjacent lateral plate mesoderm
combine to initiate and restrict myogenesis to the muscle-forming region of
the dermamyotome (Cossu and Borello,
1999). It is less clear how these signaling events are integrated
by the regulatory elements of Myf5 or Myod. Multiple
enhancers are spread over hundreds of kilobases in the Myf5 locus,
and each regulates Myf5 expression at particular developmental times
and locations (Buchberger et al.,
2003
; Hadchouel et al.,
2003
). An enhancer necessary for epaxial expression has been shown
to respond to Gli proteins and might account for the role of Shh signaling in
regulating Myf5 expression
(Gustafsson et al., 2002
;
Teboul et al., 2003
). Two
enhancer elements have been identified for Myod: one is necessary for early
myotomal expression, and the other functions slightly later in the myotome and
also during the activation of adult muscle satellite cells in muscle
regeneration (Asakura et al.,
1995
; Goldhamer et al.,
1995
); however, the factors regulating these functions remain
largely unknown.
|
Regulation of Myod binding and activity
Once expressed, how does Myod regulate skeletal muscle cell
differentiation? In one sense, the answer seems fairly simple: Myod is a
transcription factor with binding sites in the regulatory regions of many
genes that are expressed in skeletal muscle. Myod forms heterodimers with the
nearly ubiquitous E-protein sub-family of bHLH proteins through the
interaction of the HLH domains (see Fig.
1) (Lassar et al.,
1991; Murre et al.,
1989
). The basic regions act as sequence-specific DNA-binding
domains that recognize a binding site with the simple core consensus sequence
of CANNTG, termed an E-box, and show additional preferences for internal and
flanking sequences (Blackwell and
Weintraub, 1990
). Myod has a single amino-terminal
acidic-activation domain, as determined by its fusion to the heterologous
DNA-binding domain of the Gal4 protein
(Weintraub et al., 1991b
),
whereas E-proteins have a more complex mix of activation and repression
domains (Markus et al., 2002
)
(see Fig. 1). Therefore, the
simple model of the transcriptional activity of the myogenic bHLH proteins is
that they activate gene transcription by binding to the E-boxes in the
regulatory regions of genes that are expressed in skeletal muscle.
There are several problems with this simple model. First, E-boxes occur frequently in the genome, not just in the regulatory regions of genes expressed in skeletal muscle. Second, the many different sub-families of bHLH proteins recognize the same canonical sequences. For example, the Myod, Neurod and E-protein families can all bind to similar sites: yet Myod makes muscle; Neurod makes neurons; and E-proteins activate genes in B and T cells. Therefore, something must limit the potential of these proteins to promiscuously activate genes. Third, skeletal muscle genes are not all expressed simultaneously. Therefore, temporal specificity and promoter specificity must be superimposed on the simple model of a transcription factor and its binding sites.
Intermolecular interactions appear to be necessary for Myod to activate
gene transcription. Myod does not activate reporter constructs with a single
E-box but robustly activates reporters with paired E-boxes
(Weintraub et al., 1990). This
is at least partly due to the fact that Myod forms a relatively stable complex
with DNA if two E-boxes are present, whereas there is a fast dissociation rate
from a single E-box, indicating that inter-protein interactions stabilize
binding, possibly through induced conformational changes. Binding sites for
other factors, such as Mef2, Sp1, or Pbx and Meis, can functionally substitute
for the second E-box, indicating that cooperative homotypic or heterotypic
interactions with adjacent factors are crucial for establishing a stable and
functional transcriptional complex
(Biesiada et al., 1999
;
Knoepfler et al., 1999
;
Sartorelli et al., 1990
).
Therefore, the presence of certain binding sites paired with an E-box could
confer promoter-specific activity to Myod, or, by extension, to Neurod or the
E-proteins, depending on the availability of the cooperating transcription
factors.
In addition to cooperative binding, co-factor interaction or
sequence-specific DNA/protein interactions might alter the conformation of the
Myod complex to effectively expose activation regions. Myod and the other
myogenic bHLH proteins have a conserved set of amino acids in the basic region
that do not significantly alter the sequence specificity of DNA binding, but
do alter the transcriptional activity of the bound Myod
(Brennan et al., 1991;
Davis et al., 1990
).
Introducing this amino acid motif into the basic region of an E-protein will
convert it into a myogenic protein (Davis
and Weintraub, 1992
). This myogenic `code' in the basic region
might function by interacting with co-factors and an interaction with
Mef2 factors has been demonstrated
(Molkentin et al., 1995
)
or these residues might alter the conformation of the bound protein in
a manner that presents other regions for co-factor interaction
(Bengal et al., 1994
;
Ma et al., 1994
), such as has
been suggested for the promoter specific activity of NF-
B
(Leung et al., 2004
).
Myod and chromatin remodeling
Lineage-centric models of cell specification focus on the role of chromatin
in restricting the response of a cell to transcription factors. Central to
this model is the ability of chromatin to suppress the transcription of genes
that are extraneous to the specific lineage. For example, in the erythrocyte
lineage, hemoglobin expression is associated with an open chromatin structure
and hypersensitive sites, whereas in non-erythroid cells, the chromatin at
this locus adopts a transcriptionally repressive conformation
(Weintraub and Groudine,
1976). The fact that a transiently transfected globin gene is
expressed at low levels in non-erythroid cells, whereas the endogenous gene is
highly repressed, supports a model in which the chromatin context of a
specific gene determines how accessible it is to transcription factors that
may be expressed in many different cell types
(Wold et al., 1979
). Implicit
in this model is the need for factors to establish the lineage-specific
chromatin context. This could occur: (1) in a lineage-dependent manner, by,
for example, the sequential and combinatorial use of factors, such as homeobox
and segmentation genes, that are laid down at sites in the chromatin
throughout a cell lineage; or (2) in a lineage-independent manner, through the
action of a single `pioneer' transcription factor
(Cirillo et al., 2002
) that
can both access genes in a repressive chromatin context and actively remodel
the appropriate loci independent of the prior lineage. The ability of Myod to
convert cells of many different lineages and differentiation states to
skeletal muscle suggests that it has the characteristics of a pioneer
transcription factor; however, as elaborated below, both mechanisms are likely
to function in myogenesis.
The first studies in this area sought to determine whether Myod could gain
access to genes in native chromatin and initiate chromatin remodeling.
Nuclease access studies showed that genes regulated by Myod, such as
Myog, muscle creatine kinase (Ckmm), and the auto-regulated
Myod gene itself, were in an inaccessible chromatin context prior to
the presence of Myod, and that Myod was able to initiate chromatin remodeling
at these loci even in the presence of the protein synthesis inhibitor
cycloheximide (Gerber et al.,
1997). Myod directly binds the histone acetyltransferase (HAT)
p300, and p300 recruits another HAT, the p300/CBP-associated factor (PCAF), to
form a Myod complex with two distinct HAT activities
(Puri et al., 1997a
;
Puri et al., 1997b
;
Sartorelli et al., 1997
;
Sartorelli et al., 1999
).
Based on in vitro transcription studies, the two HATs have distinct functions:
p300 acetylates histones, whereas PCAF acetylates Myod at lysine residues near
its DNA-binding domain; both of these activities are necessary for the full
transcriptional activity of Myod on chromatin-associated templates
(Dilworth et al., 2004
). In
addition to recruiting HATs, Myod recruits the Swi/Snf chromatin-remodeling
complex through an interaction that can be regulated by the p38 MAP kinase
(Simone et al., 2004
).
Inhibition of HAT activity or inhibition of Swi/Snf activity prevents the
ability of Myod to initiate transcription and chromatin remodeling at specific
loci (de la Serna et al.,
2001
; Puri et al.,
1997a
). In these regards, Myod has the characteristics of a
pioneer transcription factor: it can access genes in repressive chromatin and
initiate chromatin remodeling through the recruitment of HATs and the Swi/Snf
complex.
Myod might also have a repressive role at its target genes prior to
initiating chromatin remodeling. Based on chromatin immunoprecipitation (ChIP)
studies, Myod is associated with some promoters, such as those of the
Myog and acetylcholine receptor genes, prior to the onset of
differentiation and expression of these genes
(Liu et al., 2000;
Mal and Harter, 2003
). In
contrast to the differentiating muscle cell, where Myod is associated with
HATs, in the myoblast (the replicating muscle precursor cell), Myod is
associated with histone deacetylases (HDACs) and might actively suppress gene
expression (Fulco et al.,
2003
; Mal and Harter,
2003
; Mal et al.,
2001
).
These findings strongly suggest that Myod acts to negatively regulate the
transcription of some genes in the myoblast and that muscle differentiation is
initiated when Myod switches from its association with repressive factors to
activating factors. The differentiation of Myod-expressing cell
lines, such as the C2C12 myoblast cell line
(Silberstein et al., 1986),
can be induced in culture by removing serum or it can be prevented by adding
growth factors, such as Fgf or Tgfß
(De Angelis et al., 1998
;
Li et al., 1992
), indicating
that mitogen stimulation in vivo might sustain a myoblast state. In addition,
Notch signaling represses both Myod transcription and Myod protein
activity (Kopan et al., 1994
;
Nofziger et al., 1999
), and
probably contributes to regulating differentiation in vivo. It is interesting
that while we have identified several mechanisms that might delay myoblast
differentiation, such as mitogens and Notch signaling, we do not yet have a
good understanding of the events that occur in vivo to overcome these
inhibitory signals and to induce differentiation at a specific time and
place.
A feed-forward circuit as a quantal step
How does a single transcription factor execute an entire program of cell
differentiation? How does a single event result in a predictable and complex
response? Microarray expression studies of cultured C2C12 cells have shown
that expression levels of many RNAs change during skeletal muscle
differentiation (Delgado et al.,
2003; Tomczak et al.,
2004
). To determine how many of these changes are caused by the
expression and activity of Myod, we assessed gene expression changes in
fibroblasts with an inducible Myod protein and observed a similarly large
number of expression changes with
5% of the genes tiled on the array
(Bergstrom et al., 2002
). Many
of the RNAs that showed increased expression in response to Myod were
muscle-specific genes, such as skeletal muscle myosins and actins, but many
were genes expressed in numerous different lineages, such as the Mef2
transcription factors. Surprisingly, some RNAs induced by Myod coded for
proteins that inhibit Myod activity, such as the Id proteins
(Benezra et al., 1990
). This
could partly be due to a non-autonomous inhibitory-surround mechanism. For
example, the Notch ligand Delta is an early target of Myod and its expression
is followed by the expression of the Notch regulated gene Hes1
(Bergstrom et al., 2002
),
indicating that Myod-expressing cells might inhibit muscle
differentiation in their neighbors through the Notch signaling pathway.
Not all genes are simultaneously expressed in response to Myod activation
(Bergstrom et al., 2002). Some
are induced immediately, whereas others are induced over the next two days of
differentiation. In addition, some genes are expressed transiently and some
are directly decreased. Interestingly, the cluster of early expressed genes
contains most of the genes that encode adhesion molecules and extracellular
matrix molecules, including proteases; the intermediate clusters contain most
of the transcription factors; and the latest clusters contain most of the
myofibril and cytoskeletal proteins that are associated with the contractile
function of skeletal muscle. Following the expression of Myod, therefore, the
first sets of genes activated might affect cell migration and positioning,
followed by the activation of a set of transcription factors; only later in
the differentiation program are many of the muscle contractile proteins
expressed.
How is the temporal pattern of gene expression established following the
activation of Myod? It is appealing to consider a simple cascade model because
of the large numbers of transcription factors activated in the intermediate
clusters of Myod-responsive genes. For example, Myod initiates the
transcription of Mef2c, and Mef2c might activate a muscle structural
gene; however, muscle-specific genes have not been shown to be activated by
the expression of Mef2c or any other factor in the absence of Myod or another
myogenic bHLH factor. Also in contradiction of a rigid cascade model of
temporal regulation, ChIP studies have shown that Myod binds directly to the
regulatory elements of genes expressed late in the differentiation program,
just as it binds to the regulatory elements of genes expressed early in the
program (Bergstrom et al.,
2002). Therefore, Myod directly regulates genes throughout the
program of muscle gene expression, and temporal patterning is achieved by a
combination of promoter-specific regulation of Myod binding and activity.
Because Myod initiates the myogenic differentiation program and that
program temporally regulates the activity of Myod, it follows that Myod
programs the regulation of its own activity. It does this, at least in part,
through a feed-forward mechanism (Penn et
al., 2004) (Fig.
3). For example, during the first 24 hours after Myod induction in
our model system of Myod-mediated myogenesis, Myod initiates expression of the
Mef2d gene and activates the p38-signaling pathway. Mef2d and p38
then cooperate with Myod to activate a subset of genes initiated between 24
and 48 hours after induction. The precocious activation of p38 and the
expression of Mef2d permits Myod to activate these normally late
genes within hours of induction, demonstrating that the timing of
Myod-mediated activation of late genes is imposed by the availability of
factors that are activated by Myod at an earlier time-point. In this manner,
Myod is active through the entire program of muscle gene expression, binding
directly to the regulatory elements of genes expressed both early and late in
the program; temporal regulation is achieved by superimposing requirements for
additional Myod-regulated factors at subsets of promoters.
|
This quantal-step model of the evolution of skeletal myogenesis is highly
speculative and other models are evident; for example, Myod might have invaded
a pre-existing cascade of gene expression and modified its outcome. As we
learn more about the skeletal muscle differentiation program, it will be
interesting to consider how new cell types and their regulatory circuits
evolve. For example, Pha4 regulates pharyngeal development in C.
elegans, and, like Myod, establishes a temporal pattern of gene
expression by binding to and activating the promoters of genes that are
expressed throughout the developmental program in a regulated manner
(Ao et al., 2004;
Gaudet and Mango, 2002
;
Gaudet et al., 2004
). Although
it would need to be the subject for a different review, a comparison of
vertebrate and invertebrate skeletal myogenesis, and of skeletal and cardiac
gene regulatory circuits, might help to inform our understanding of the
mechanisms that generate new cell types.
Instructive chromatin and muscle lineage specification
How are Myod and Myf5 capable of accessing the appropriate muscle-specific promoters and specifying the skeletal muscle lineage? As noted above, these factors can recruit histone-modifying and chromatin-remodeling complexes to muscle promoters and reset the cellular chromatin structure and transcriptional program. Rather than acting independently of the pre-existing chromatin, however, recent studies indicate that chromatin-associated complexes instruct these factors about where to bind. As such, chromatin context establishes an instructive environment for the activity of these master regulatory transcription factors.
Several studies led to the characterization of discrete domains in Myod and
Myf5 that are necessary to initiate the myogenic program. When knocked-in to
the Myf5 locus, and in the absence of Myod, Myog did not
efficiently establish the skeletal muscle lineage in mouse embryos
(Wang and Jaenisch, 1997),
indicating that Myod and Myf5 have different intrinsic functions to Myog,
rather than simply different temporal expression patterns. Subsequently, it
was demonstrated that Myod and Myf5 were more efficient than Myog at
initiating the expression of a set of endogenous target genes
(Bergstrom and Tapscott, 2001
).
The ability to efficiently initiate endogenous muscle gene expression mapped
to two domains conserved in Myod and Myf5, a region rich in histidines and
cysteines (H/C domain), which lies immediately N-terminal to the basic region,
and a potential amphipathic alpha-helix in the C-terminal region (Helix 3 see
Fig. 1). The mutation of the
H/C and Helix 3 domains in Myod prevents the initiation of chromatin
remodeling at specific target promoters, indicating that they are required
prior to the recruitment of an active Swi/Snf complex. The H/C and Helix 3
domains are also conserved in Mrf4, consistent with the recent demonstration
that it can also specify the skeletal muscle lineage during embryogenesis
(Kassar-Duchossoy et al.,
2004
).
|
Pbx is bound to the Myog promoter in both muscle and non-muscle
cells, and it is possible that the interaction between Myod and the Pbx
complex is necessary for Myod to initially locate the Myog gene
within condensed chromatin prior to differentiation. The Myog
promoter contains a conserved consensus E-box, 100 bp promoter proximal to the
Pbx site, which is necessary for full transcription
(Berkes et al., 2004;
Cheng et al., 1993
). However,
despite the presence of this intact consensus E-box, ChIP studies show that
the Pbx-interacting domains of Myod are necessary to stably recruit Myod to
the Myog promoter (Berkes et al.,
2004
). It appears, therefore, that Myod needs to interact with
Pbx/Meis and the adjacent non-canonical E-boxes before it can form a stable
binding complex at the consensus E-box.
Indeed, Myod targets chromatin-remodeling complexes to the Myog
promoter prior to forming a stable DNA-bound complex
(de La Serna et al., 2005). A
Myod-dependent histone acetylation is the initial event at the Myog
promoter, followed by Swi/Snf recruitment, and then binding of Myod and Mef2
factors. Therefore, an attractive and consistent model is that the canonical
E-box is `hidden' from Myod and other bHLH factors by chromatin in non-muscle
cells and that Myod is initially recruited to this locus through an
interaction with Pbx (Fig. 5).
As noted above, the HATs p300 and PCAF form a complex with Myod, and tethering
Myod to the Myog promoter results in local histone acetylation. The
acetylated histones could then stabilize the binding of the Swi/Snf complex,
which is recruited by Myod in a p38-dependent complex. Remodeling of the locus
would expose the canonical E-boxes and binding sites to other factors, such as
the Mef2 and Six proteins, leading to the formation of a stable multi-protein
regulatory complex. Although this complex might be similar to the enhancesome
described at the IFN-ß promoter
(Agalioti et al., 2000
), the
formation of the IFN-ß enhancesome occurs as a first step on exposed DNA
with subsequent chromatin remodeling, whereas the emerging model at the
Myog locus indicates that transcription factor-directed chromatin
remodeling must occur before the cognate binding sites are exposed and a
stable complex forms on the promoter.
The requirement for an interaction between Myod and the resident Pbx
complex to reveal the other binding sites might explain why E-proteins or
Neurod do not bind and activate the Myog promoter in vivo, despite
the fact that they can initiate gene expression at other loci when effecting
their own program of differentiation. For example, the E-boxes in the IgH
enhancer are nearly identical to the E-boxes in the muscle creatine kinase
enhancer, and Myod binds with equally high affinity to both sets of E-boxes in
gel shift assays (Kadesch et al.,
1986). In addition, Myod can bind and activate the expression of
reporter constructs driven by the IgH E-boxes in transient transfection
assays. By contrast, despite the ability of Myod to recruit chromatin
modifying complexes and initiate chromatin remodeling at many loci, it does
not bind to the IgH E-boxes in vivo and does not initiate IgH expression
(Bergstrom et al., 2002
).
Therefore, chromatin-mediated repression can partly explain the paradox of
lineage-specific gene activation among a family of factors that bind similar
DNA sequences.
|
Conclusion: wiring the circuitry of a master switch
Myod and the myogenic bHLH proteins are master regulatory genes of skeletal muscle differentiation: they are necessary for skeletal muscle specification, are sufficient to establish the myogenic program, and they directly regulate gene expression throughout the differentiation program. Clearly, however, these master regulatory factors do not act in isolation. There is now evidence that an instructive chromatin environment is developmentally established, as evidenced by the emerging role of the Pbx/Meis complex in marking loci for Myod activation. In addition, the binding and activity of Myod is regulated by other factors to achieve a temporal patterning of gene expression through a feed-forward mechanism. It is likely that other master regulatory factors, such as Neurod in neurogenesis and E-proteins in lymphocyte differentiation, regulate and are regulated through similar mechanisms: a simple DNA-binding site that permits a large sampling of the transcriptional potential of the genome, and super-imposed promoter-specific regulation to achieve a coherent pattern of gene expression.
It is interesting to contrast the role of Myod in muscle cell
differentiation with that of Pax6 in eye development, both of which have been
termed master regulatory genes (Pichaud
and Desplan, 2002; Weintraub
et al., 1991a
). Myod regulates the differentiation of a single
cell type, whereas Pax6 is essential for the development of a complex organ
comprising multiple different specialized cell types. Within the context
established by Pax6 and by the other homeobox factors of the retinal
determination gene network (RDGN) (Silver
and Rebay, 2005
), transcription factors that regulate cell
differentiation in many regions of the body generate the specialized neurons,
glia and melanocytes that compose the retina. Relative to our current
understanding of the role of Pbx in creating an instructive environment for
Myod, it is attractive to think that Pax6 and other factors of the RDGN might
establish a chromatin context that modulates the specific activities of the
bHLH and other transcription factors to generate the distinct cell types of
the eye, similar to the role of Pax and Hox proteins in regulating bHLH
activity proposed by Westerman et al.
(Westerman et al., 2003
).
To extend this speculation, perhaps Pax3 and Pax7 also have instructive
roles in Myod-mediated myogenesis. Forced expression of Pax3 in mouse
fibroblasts does not activate muscle gene expression, but expressing the
PAX3-FKHR fusion protein associated with alveolar rhabdomyosarcomas, a fusion
protein that adds FKHR (Forkhead) regulatory domains to the PAX3 DNA-binding
domain, activates a large number of skeletal muscle genes, including Myod,
Myog, and muscle structural genes
(Khan et al., 1999). Perhaps
Pax3 and Pax7 reside at the regulatory regions of subsets of genes expressed
in skeletal muscle but do not directly regulate transcription in their native
state, similar to the role we are postulating for the Pbx complex at the
Myog promoter. If this is the case, it might be necessary to remove
or replace these factors prior to differentiation, because Pax3 and
Pax7 expression ceases at the time of muscle differentiation and
forced expression actually inhibits muscle differentiation. If, in the future,
we learn that the Pax genes mark regions for a subsequent set of homeobox
genes, such as Pbx or Meis, and, in turn, that these instruct Myod or other
`master regulatory factors', then we will have melded the concepts of
lineage-established chromatin-encoded potential with master regulatory
factor-driven programs of cell differentiation.
ACKNOWLEDGMENTS
I am grateful to Robert Davis and Andrew Lassar for revealing the secrets of Myod; to Howard Holtzer, Harold Weintraub and Wolfram Hortz for their wisdom; and to Mark Groudine and Phil Soriano for their insight (and helpful comments on this manuscript).
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