(Received for publication, September 11, 1995; and in revised form, December 28, 1995)
From the
Terminal differentiation of muscle cells results in opposite effects on gene promoters: muscle-specific promoters, which are repressed during active proliferation of myoblasts, are turned on, whereas at least some proliferation-associated promoters, such as c-fos, which are active during cell division, are turned off. MyoD and myogenin, transcription factors from the basic-helix-loop-helix (bHLH) family, are involved in both processes, up-regulating muscle genes and down-regulating c-fos. On the other hand, the serum response factor (SRF) is involved in the activation of muscle-specific genes, such as c-fos, as well as in the up-regulation of a subset of genes that are responsive to mitogens. Upon terminal differentiation, the activity of these various transcription factors could be modulated by the formation of distinct protein-protein complexes. Here, we have investigated the hypothesis that the function of SRF and/or MyoD and myogenin could be modulated by a physical association between these transcription factors.
We show that myogenin from differentiating myoblasts specifically binds to SRF. In vitro analysis, using the glutathione S-transferase pull-down assay, indicates that SRF-myogenin interactions occur only with myogenin-E12 heterodimers and not with isolated myogenin. A physical interaction between myogenin, E12, and SRF could also be demonstrated in vivo using a triple-hybrid approach in yeast.
Glutathione S-transferase pull-down analysis of various mutants of the proteins demonstrated that the bHLH domain of myogenin and that of E12 were necessary and sufficient for the interaction to be observed. Specific binding to SRF was also seen with MyoD. In contrast, Id, a natural inhibitor of myogenic bHLH proteins, did not bind SRF in any of the situations tested. These data suggest that SRF, on one hand, and myogenic bHLH, on the other, could modulate each other's activity through the formation of a heterotrimeric complex.
Proliferation and cell differentiation are mutually exclusive
processes, as best exemplified in muscle precursor cells. Proliferation
inhibition is a crucial step that precedes muscle-specific gene
expression and cell fusion into myotubes in the process of muscle cell
terminal differentiation(1) . Indeed, terminal differentiation
of myoblastic cell lines in vitro is triggered by the
accumulation of the precursor cells (myoblasts) in a G state(1) . This step is a prerequisite, and a number of
mitogens(2, 3, 4, 5) or oncogenes (6, 7, 8, 9, 10) inhibit
terminal differentiation.
Two distinct families of transcription
factors, MEF-1 and MEF-2, ()are instrumental to the muscle
cell differentiation process. The MEF-1 family includes the myogenic
bHLH proteins MyoD(11, 12, 13) ,
myogenin(14, 15) , Myf5 (16) and
MRF4/herculin/Myf6(17) . These proteins are all able to elicit in vitro a muscle determination program in a number of
nonmuscle cell types(18, 19) . These muscle-restricted
proteins share a domain of homology, the bHLH, which is also common to
ubiquitous transcription factors such as the products of the E2A gene,
E12 and E47(20, 21) , with which myogenic factors form
heterodimeric complexes. Heterodimers between myogenic bHLH and E12 or
E47 (22, 23, 24, 25, 26) bind
to upstream regulatory sequences of the form CANNTG (E boxes) (27) in muscle-specific gene promoters. MyoD, myogenin, and
Myf5 are all able to transactivate these promoters
efficiently(28) , a function that involves a common motif in
the basic domain(29) .
MEF-2 transcription factors are members of the MADS box protein family. MADS box proteins are widely expressed, from plants to man and including yeast(30) . MADS box proteins exert a wide range of functions from development in plant cells to muscle differentiation or growth factor responses in mammalian cells. In yeast, the MADS box protein MCM1 is involved in the mating-type phenotype(31) . A highly homologous protein, the serum response factor (SRF), is instrumental to immediate early gene mitogenic responses in mammals(32, 33) . The sequences that are recognized by the two proteins are homologous(34) . The two proteins function in a similar manner: although both proteins are able to transactivate transcription(35, 36) , they mainly function by recruiting ternary complex-forming factors through a protein motif located in the MADS box(37, 38) .
The
MEF-2 subset of MADS box proteins includes four members, MEF-2A,
MEF-2B, MEF-2C, and MEF-2D, some of which are ubiquitous and some
restricted to differentiated muscle cells. These proteins are related
to SRF and are often referred to as rSRF proteins(39) . They
bind to and transactivate CTA(A/T)TAG DNA elements, a
consensus sequence that is homologous, but not identical, to the CArG
boxes (CC(A/T)
GG), which are recognized by SRF. In fact,
SRF and rSRF(MEF-2) cannot heterodimerize with each other(39) ,
and thus, they belong to distinct subsets of MADS box proteins.
The factors that control the balance between proliferation and differentiation in muscle are not fully understood at present. However, myogenic factors of the bHLH family seem to be involved in this delicate control. In particular, MyoD is both a target for mitogens that are inhibiting differentiation (2, 7, 10) and a negative regulator for cell proliferation in vitro(40, 41) . Part of the mechanism by which MyoD blocks cell proliferation in vitro seems to be the repression of proliferation-associated genes, and indeed, most of the proliferation-associated genes are silent in differentiated muscle cells(42) . In particular, immediate early genes such as c-fos are repressed on terminal differentiation(42, 43) . In fact, the transcription factor AP1, which is formed by heterodimers between members of the Fos-related proteins and members of the Jun-related factors, is a prominent target for MyoD: MyoD interferes with AP1 function by forming specific complexes with Jun proteins(44) . In addition, MyoD (43) and myogenin (45) both act as repressors for the c-fos promoter through the inhibition of its main element, the serum response element (SRE)(46) . The SRE includes a CArG box, the binding site for SRF, which is repressed on differentiation.
Paradoxically, CArG boxes are involved in the up-regulation of some
muscle-specific genes such as cardiac
-actin(22, 47, 48, 49) ,
skeletal
-actin(50, 51) , myosin light chain
1A(52) , dystrophin(53) , and muscle creatine
kinase(54) . SRF itself has been shown to be involved in muscle
gene up-regulation(51, 55, 56) . Furthermore,
SRF seems to be indispensable for muscle cell terminal
differentiation(57) . Indeed, CArG boxes of muscle genes can be
replaced by the c-fos SRF-binding site without any loss of
function(58) , strongly suggesting that the same protein is
involved in the activation of both genes. Thus, SRF is instrumental to
the activation of both mitogen-responsive genes, such as c-fos or
-actin, which are down-regulated on muscle cell terminal
differentiation(32, 33, 46) , and
muscle-specific genes.
The mechanism by which SRF is converted into a muscle-specific transactivator is largely unknown. In muscle-specific promoters, transactivation by SRF could use a slightly different mechanism than that used in the c-fos promoter: the affinity of SRF for muscle CArG boxes is lower than its affinity for c-fos SRE (58, 59) . Furthermore, whereas one SRF-binding site is sufficient for mitogen responses, several sites are necessary to observe muscle-specific expression(51, 60) . Therefore, it seems likely that with regard to muscle genes, SRF needs to cooperate with other factors for optimal transactivation. A tempting hypothesis would thus be that, in order to be active on muscle promoters, SRF needs to interact physically with other factors.
In this paper, we have tested the hypothesis that SRF interacts with the myogenic bHLH proteins. We demonstrate that a heterotrimeric complex forms between bHLH proteins and SRF both in vitro and in cultured cells. The formation of this complex could be one of the means by which SRF activity is deviated from proliferative to differentiating genes upon muscle cell terminal differentiation.
Plasmids pGAD424 and pGBT9 were
a kind gift of Dr. S. Fields(63) . Plasmid pRS313 has been
described by Sikorski and Hieter(64) . Plasmids pGAD-E12 and
pGAD-myogenin were constructed by inserting the corresponding complete
coding sequence, obtained by polymerase chain reaction using the
above-described primers for myogenin or the primers described in (43) for E12, into the EcoRI site of pGAD424. Plasmid
pGB-SRF was constructed by inserting the complete coding sequence of
SRF, obtained by polymerase chain reaction using the above-described
primers and subcloned into a Bluescribe vector,
between the EcoRI and SalI sites of pGBT9. pRS-E12
and pRS-myogenin were constructed by subcloning an SphI-SphI insert from the corresponding pGAD
construct into the SmaI site of pRS313. These constructs were
controlled by partial sequence (which did not reveal any mutations),
and results obtained with these constructs were confirmed using two
independent clones.
pEMSV-MyoD, pEMSV-MyoD-bH, pEMSV-MyoD-bE12,
and pEMSVMyoD-bT4 were kind gifts of Dr. H. Weintraub(27) .
pEMSV-myogenin, pEMSV-
N-myogenin, pEMSV-
C-myogenin, and
pEMSV-
N/
C-myogenin were kind gifts of Dr. E.
Olson(65) .
Figure 1:
In vitro and in vivo interactions between myogenin and SRF. A, GST pull-down
assay. E12 (lane 1) and myogenin (lane 2) were in
vitro translated alone or in combination (lane 3), as
indicated (Input: one-tenth of the amount used in the GST
pull-down assay). Translation products were incubated with beads
covered with GST (G; lanes 5, 7, and 9) or GST-SRF (S; lanes 4, 6, and 8). Bound products were analyzed by SDS-polyacrylamide gel
electrophoresis. The positions of E12 and myogenin translation products
are shown. B, triple-hybrid assay. Indicator yeast cells were
transfected with expression vectors for fusion proteins between the
GAL4-activating domain (pGAD) or the DNA-binding domain (pGB) and a
third construct, pRS, resulting in the expression of GAL4 AD-E12 and/or
GAL4 AD-myogenin (Myog) and GAL4 GB-SRF, as indicated.
Transfected yeast cells were analyzed for -galactosidase
expression by a colorimetric assay (each streak represents an
independent clone) or by a quantitative chemiluminescent assay (RLUs, arbitrary light units), as indicated. C,
confluent C2C12 mouse myoblastic cells were kept in high serum (lanes 3, 6, and 7) or induced to
differentiate for 24 h (lanes 4, 8, and 9)
or 48 h (lanes 5, 10, and 11). Nuclear
proteins were analyzed by Western blotting using anti-myogenin
antibodies, either directly (lanes 3-5) or after
absorption onto GST-covered beads (G; lanes 7, 9, and 11) or GST-SRF-covered beads (S; lanes 6, 8, and 10). Lanes 1 and 2 show a direct analysis by Western blotting of in vitro translated myogenin. NP, nonprogramed lysate; myog, myogenin.
These
results were confirmed in vivo using a double-hybrid approach
in yeast(63) . Indicator yeast cells (permanently transfected
with a GAL1-lacZ reporter construct) were transfected with expression
vectors encoding fusion proteins between the GAL4 DNA-binding domain
(pGB series) or transactivating domain (pGAD series) and SRF, myogenin,
or E12 in all combinations. These experiments showed that expression of
fusion products between the GAL4 DNA-binding domain and E12 or myogenin
resulted in a high background, indicating that the transactivation
domains of these proteins are active in yeast (data not shown).
Therefore, these constructs could not be used in subsequent assays.
When yeast cells were transfected with pGB-SRF and pGAD-myogenin or
pGAD-E12, no -galactosidase could be detected, suggesting that a
heterodimer does not form between SRF and E12 or myogenin, as expected
from the results of GST pull-down assays. We next introduced, into the
same yeast indicator cells, three expression vectors bearing three
distinct selectable markers and encoding a fusion protein between the
GAL4 DNA-binding domain and SRF and fusion proteins between the GAL4
activation domain and myogenin or E12, respectively. Yeast that had
received the three expression vectors expressed significant amounts of
-galactosidase (Fig. 1B), indicating that a
heterotrimer had formed in the cells, resulting in the activation of
the GAL4-responsive promoter. In contrast, galactosidase activity was
hardly detectable in yeast transfected with SRF and E12 or with SRF and
myogenin (lanes 1 and 2), confirming that complex
formation between SRF and isolated E12 or myogenin was not very
efficient. All other combinations, including pGAD-myogenin (or
pGAD-E12), pGB-SRF, and pRS as a backbone vector, resulted in
background or low expression of
-galactosidase (data not shown).
Taken together, these data indicate that heterodimers of myogenin and
E12 physically interact with SRF in vitro and in
vivo.
A complex between SRF and myogenin was also detected when nuclear extracts from differentiating myoblasts were used (Fig. 1C). C2C12 cells were or were not induced to differentiate in low serum. Nuclear proteins were extracted at different time points and incubated with GST- or GST-SRF-covered agarose beads. Bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blotting using an anti-myogenin monoclonal antibody. These experiments demonstrated that cellular myogenin, amounts of which increased during differentiation, was specifically retained on SRF-coated beads in differentiating myoblasts. Taken together, these data demonstrate that myogenin, in its physiological heterodimeric form, binds to SRF.
Figure 2:
The bHLH domains of E12 and myogenin are
necessary and sufficient for specific SRF recognition. A,
deletion mutants used. Solid boxes, basic domain; hatched
boxes, HLH domain. B, GST pull-down analysis of wild-type
myogenin and E12 (lanes 1 and 2) or deletion mutants
of myogenin (lanes 3-8) or E12 (lanes
9-12). The positions of the various mutants are indicated by arrows, and the corresponding inputs are shown in C. D, GST pull-down analysis of wild-type myogenin (lanes
1, 4, 5, and 10) or deletion mutants
that have lost the b and H1 region (bH1; lanes 2, 6, 7, and 11) or the entire bHLH domain
(
H; lanes 3, 8, 9, and 12).
Translation products were analyzed on beads coated with GST (G; lanes 5, 7, and 9), GST-SRF (S; lanes 4, 6, and 8), or
GST-E12/myogenin (lanes
10-12).
On the other hand, experiments performed with deletion mutants of SRF indicated that, similarly, core SRF (a minimal domain of SRF that includes the DNA-binding and dimerization domains) was sufficient to observe a specific interaction with myogenin (Fig. 3). No interaction could be detected with the N- or C-terminal moiety of SRF.
Figure 3:
Core SRF is necessary and sufficient to
interact specifically with bHLH proteins. A, deletion mutants
of SRF used. The hatched boxes indicate the location of the
MADS box. B, GST-pull down analysis of wild-type myogenin and
E12 on beads coated with GST (lane 2), GST-wild-type SRF (lane 1), or mutants that have lost amino acids 11-244 (C-SRF; lane 3), 142-508 (N-SRF; lane 4), or 11-142 and 244-508 (N
C-SRF; lane 5). Translation products were
also analyzed on beads coated with core SRF as an internal control for
the various beads (MADS-SRF; lanes
6-10).
Taken together, these results indicate
that the DNA-binding domains of SRF, E12, and myogenin are both
necessary and sufficient to observe the interaction, raising the
possibility that the interaction occurs through nonspecific binding of
these proteins to contaminating DNA during the GST pull-down assay.
However, the interaction among the three proteins was still observed
when contaminating DNA was degraded using nuclease or when the GST
pull-down assay was performed in the presence of ethidium bromide,
which prevents DNA-protein interaction (data not shown). The results of
these control experiments rule out the possibility that the
SRFbHLH complex could be due to artifactual nonspecific binding
to DNA.
Figure 4: Analysis of various bHLH proteins. A, MyoD mutants used in the assay. B, GST-pull down analysis of wild-type MyoD (lanes 1, 3, and 4) or a mutant that has lost the N-terminal part of the bHLH domain (lanes 2, 5, and 6) on GST-coated (G; lanes 4 and 6) or GST-SRF-coated (S; lanes 3 and 5) beads. Lanes 1 and 2 show the input. C, GST pull-down analysis of Id on GST (lane 3), GST-SRF (lane 2), or GST-E12/myogenin (lane 4). Lane 1 shows the input. D, GST pull-down analysis of wild-type MyoD (lanes 1, 2, 7, and 8), a deletion mutant that has lost the N-terminal part of the bHLH domain (lanes 3 and 4), or mutants in which the b domain has been replaced by that of E12 (lanes 5 and 6) or T4 achaete scute (lanes 9 and 10).
However, the ability to physically interact with SRF was not observed for all members of the bHLH family. In particular, Id, a functional antagonist of MyoD or myogenin, did not bind to SRF, either alone or in combination with E12 (Fig. 4C). We have also tested several mutants of MyoD or myogenin (Fig. 4D). MyoD-bE12 (a mutant of MyoD in which the basic domain has been replaced by the E12 basic domain) and MyoD-bT4 (in which the basic domain has been replaced by that of the T4 achaete scute Drosophila protein) both bind to DNA efficiently(27) , but do not transactivate muscle genes. Whereas MyoD-bE12 retains a high affinity for SRF, MyoD-bT4 has lost SRF recognition.
Terminal differentiation of myoblasts is accompanied by
drastic changes in the pattern of gene expression: muscle-specific
genes are up-regulated, while mitogen-responsive promoters are
repressed. Paradoxically, some enhancer elements are involved in the
activation of both mitogen-responsive and differentiation-associated
genes. In particular, CArG boxes or related elements are active in a
number of muscle promoters(51, 55, 60, 72, 73, 74, 75, 76) as
well as in immediate early genes such as c-fos or cytoskeletal
actin(33, 77) . Furthermore, the CArG box-binding
protein SRF, a prominent factor in the immediate early response to
mitogens, seems also to be indispensable for muscle cell terminal
differentiation(57) . Thus, SRF is involved in the
up-regulation of both mitogen-responsive genes (such as c-fos)
and differentiation-associated genes (such as those encoding
muscle-specific proteins), although these two sets of genes are
regulated in an opposite manner. A tempting hypothesis to explain this
apparent paradox is that SRF activity is regulated through the
formation of distinct protein complexes. In particular, SRF activity,
during muscle terminal differentiation, could be modulated by a
physical interaction with factors involved in this process. We have
thus tested the hypothesis that such a physical interaction could occur
between the myogenic bHLH proteins and SRF and found that complexes
could indeed be detected between MyoD or myogenin using various assays in vitro or in vivo, including GST pull-down and
hybrid assays in yeast. Interestingly, this interaction requires that
myogenic bHLH be in a heterodimeric form: no complex could be detected
between myogenin or MyoD and SRF in the absence of E12. Accordingly,
complex formation required the integrity of the bHLH domain on both E12
and MyoD or myogenin. A likely interpretation of these results is that
heterodimeric formation between E12 and MyoD or myogenin induces a
conformational change in the proteins that unmasks a site of
interaction with SRF. The bHLH domain is also sufficient for the
interaction to occur, suggesting that this conformational change occurs
in the bHLH domain, which is not unexpected. Interestingly, core SRF is
also necessary and sufficient for complex formation with bHLH. This
suggests that core SRF is involved in a wide variety of functions,
including dimerization and DNA binding(32) , interaction with
p62(78) , transcriptional activation in response
to some signal transduction pathways(36) , and interaction with
members of distinct transcription factor families (this study).
Furthermore, this interaction might also be observed with other members
of the MADS box family that share large domains of homology with core
SRF. Indeed, a similar physical interaction has been demonstrated for
MEF-2(79) , although for MEF-2, the formation of the complex
did not require the presence of E12.
From a biochemical point of view, we do not know if the interaction between SRF and the heterodimeric bHLH proteins involves amino acids from the three proteins or if only two of the proteins are physically involved. It should be noted, however, that some mutations in MyoD the bHLH domain result in inactivation of SRF recognition, suggesting that MyoD and myogenin are directly involved in SRF binding. Our experiments have been performed in various systems, from reticulocytes to yeast, and thus, it is likely that the interaction among the three proteins is direct. However, we cannot rule out the possibility that a fourth protein, which would be ubiquitous, is also involved.
From a
functional point of view, it is not clear which activity, that of SRF
or that of myogenic bHLH, is modulated by the formation of the
trimolecular complex. It is, however, clear that formation of this
trimeric complex is associated with the terminal step of muscle
differentiation since 1) it is detected only with cells that have
entered the differentiation process; 2) it requires heterodimerization
of MyoD or myogenin with E12, an event that occurs only at this step of
the process, even if MyoD pre-exists in myoblasts; and 3) Id-E12
complexes, which are present in proliferating myoblasts, do not bind to
SRF. The formation of the complex on terminal differentiation might
turn SRF, a ubiquitous protein that is a key participant in immediate
early responses, into a muscle-specific transactivator. In this regard,
this interaction could explain several puzzling observations. First,
SRF has a low affinity for muscle gene CArG
boxes(51, 58, 59) , although it seems
instrumental to the up-regulation of these genes. By a cooperative
binding with heterodimers of myogenic bHLH proteins, SRFDNA
complexes could gain enough stability for SRF to be active. Second,
although SRF is present and active on mitogen-responsive genes in
proliferating myoblasts, muscle-specific genes remain silent until
terminal differentiation. This could easily be explained by the fact
that transactivation of muscle genes by SRF requires the formation of a
complex with myogenic bHLH proteins.
However, we cannot rule out the possibility that the formation of this complex also results in MyoD/myogenin modulation by SRF. In fact, for some muscle promoters, the transactivation of E boxes by MyoD or myogenin requires that an intact CArG box also be present on the promoter(52) . This suggests some cooperation between myogenic bHLH and proteins binding to the CArG box. Clearly, however, the interaction with SRF is not sufficient for myogenic activity of bHLH proteins: MyoD-bE12, a MyoD mutant that has lost myogenic activity(27) , is still able to recognize SRF in a specific manner; thus, some functionality of the basic domain distinct from SRF recognition is required for myogenic activity.
It is thus possible that the formation of the
SRFbHLH complex results in the modulation of each of the partners
of the complex, SRF and myogenic bHLH proteins. Various functions could
be regulated in this manner. For example, the formation of this complex
could be a mechanism used by myogenic bHLH proteins to repress SRF from
transactivating mitogen-responsive genes such as
c-fos(43) . Myogenic bHLH proteins repress c-fos SRE through binding to an E box overlapping the CArG box in the
DNA element. The formation of a multiprotein complex on the element
could explain the observed inhibition. Preliminary data, using an
improved electrophoretic mobility shift assay(80) , suggested
that such a multiprotein complex (including myogenin, E12, and SRF)
could assemble on c-fos SRE, although this complex seems to be
unstable (data not shown). We are currently analyzing this complex in
more detail.
An alternative hypothesis would be that the interaction between SRF and myogenic bHLH increases the affinity of SRF for noncanonical CArG boxes or the affinity of myogenic bHLH for E boxes that are distant from the consensus sequence. We are currently running experiments to test this hypothesis. It is also possible that this interaction is involved in a step that follows the binding to DNA, such as transcriptional transactivation. It is important to note that the cooperation between CArG boxes and E boxes in muscle genes is mainly observed in cardiac muscle cells. It is thus possible that a similar interaction involves members of both families of protein that are specifically expressed in cardiac muscle.
Taken together, our results indicate that myogenic bHLH proteins are able to form a heterotrimeric complex with SRF both in vitro and in vivo. The existence of this complex could help explain how SRF, which is a ubiquitous factor involved in immediate early responses, is converted into a muscle-specific transactivator on muscle cell differentiation.