(Received for publication, December 26, 1995)
From the
The MyoD family of transcription factors regulates muscle-specific gene expression in vertebrates. In the adult rat, MyoD mRNA accumulates predominately in fast-twitch muscle, in particular type IIb and/or IIx fibers, whereas Myogenin mRNA is restricted to slow-twitch type I muscle fibers. Transgenic mice expressing the avian v-ski oncogene from the murine sarcoma virus (MSV) promoter-enhancer display preferential hypertrophy of type IIb fast-twitch muscle apparently because of the restricted expression of the transgene. We tested the hypothesis that preferential interactions of MyoD, as a heterodimer with E12, with the MSV enhancer, which has six E-box targets for MyoD family proteins, could contribute to v-ski gene expression in IIb muscle fibers. A series of quantitative binding studies was performed using an electrophoretic mobility shift assay to test MyoD-E12 versus Myogenin-E12 binding to the MSV enhancer. Our results indicate that MyoD-E12 binds the MSV enhancer with higher affinity and higher cooperativity than Myogenin-E12. Interestingly, MyoD-E12 bound all of the individual E-boxes tested with positive cooperativity indicating DNA-mediated dimerization of the protein subunits.
Activation of muscle-specific gene expression in vertebrates is
controlled by the MyoD family of transcription factors, MyoD, Myogenin,
MRF4, and Myf-5, which are exclusively expressed in skeletal muscle (1, 2, 3, 4, 5) . Ectopic
expression of any one member of this family in non-muscle cells, such
as 10T1/2 or NIH3T3 fibroblasts, results in phenotypic conversion of
that cell to muscle, indicating the trans-dominant nature of
these proteins(3, 6) . The four family members share
80% homology in a 60-amino acid region known as the basic
helix-loop-helix (bHLH) ()domain (7) . The basic
region is responsible for DNA binding activity (8) and is
involved in transactivation of regulated genes, whereas the
helix-loop-helix motif constitutes an interface for dimerization with
other bHLH proteins(9, 10, 11) . MyoD family
proteins are not thought to form efficient homodimers; rather they
readily heterodimerize with other HLH proteins, including the E12 and
E47 products of the E2A gene (12) . Heterodimers are capable of
activating transcription of muscle-specific genes as a result of
binding to the E-box consensus sequence (CANNTG) in muscle-specific
enhancers, presumably facilitating the formation of
transcription-competent RNA polymerase-promoter complexes. The roles of
these transcription factors in myogenesis has been reviewed
recently(13) .
Distinctions between these seemingly redundant transcription factors have been elucidated by developmental expression profiles and null mutant studies from which a general model has been proposed(14) . Whereas myoD or myf-5 null mice have fairly normal muscle development, the double mutant is lethal, as are myogenin null mice(15, 16, 17, 18, 19) . There is a temporal pattern of expression for the MyoD family members in the mouse: Myf-5 is expressed first, followed shortly after by Myogenin, MyoD, and MRF4. In the neonate, all four proteins are expressed at a time when the fetal isoforms of myosin heavy chain (MyHC), rather than the adult isoforms predominate. Following birth and maturation of innervation, Myf-5 expression is reduced to an undetectable level, whereas MRF4 expression remains relatively high. Interestingly, both MyoD and Myogenin expression is also reduced, however, to different extents in different fiber types. MyoD expression predominates over that of Myogenin in fast-twitch type IIb/IIx muscle fibers, whereas Myogenin is preferentially expressed in slow-twitch type I fibers(20, 21) . During this time, the fetal isoform of MyHC is replaced by the adult isoforms. The fiber type-specific expression of MyoD and Myogenin is responsive to hormonal and neural signals. Denervation of either fiber type results in increased expression of both MyoD and Myogenin, as if the cells are reverting to the fetal form; adult MyHC is reduced, and fetal MyHC is reexpressed. Cross-reinnervation of a slow muscle (soleus) with a fast motor neuron or thyroid hormone treatment, both of which induce transformation to fast-twitch fiber types, increased the level of MyoD expression and lowered Myogenin expression in the affected myofibers (20) . These results suggest that the bHLH factors regulate different sets of genes in fetal and adult muscle. In the adult, they may be involved in controlling fiber type-specific gene expression in response to external signals.
The viral ski gene (v-ski) encodes a nuclear oncoprotein capable of causing morphological transformation, allowing cell growth in soft agar and inducing myogenesis in cultured avian cells(22, 23, 24) . The avian c-ski proto-oncogene encodes a nuclear protein of unknown function which can bind DNA in the presence of other nuclear proteins(25) . However, when expressed at high levels, similar to that of v-ski during infection, c-ski acquires the oncogenic and myogenic activities of v-ski(26) . This result led Colmenares et al. (26) to conclude that differences in the level of expression between v-ski and c-ski rather than differences in the encoded proteins per se was sufficient to account for the disparate characteristics of the two evolutionarily related proteins.
c-ski expression driven by the promoter-enhancer region of the murine sarcoma virus (MSV) long terminal repeat has unexpected effects in transgenic mice. In three independent lines of mice, hypertrophy of skeletal muscle was observed. One of these lines(8566) was studied in detail, and the hypertrophy of muscle was found to be the result of specific hypertrophy of type IIb fibers, the fastest of the fast-twitch fibers(27) . c-ski expression and the hypertrophic phenotype become elevated at approximately 12 days postpartum, the time at which innervation matures, and the adult isoforms of contractile proteins accumulate. c-ski mRNA accumulation in MSV-ski transgenic mice was reduced in denervated hindlimb muscle or in response to chemically induced hypothyroidism(27) .
The obvious correlation between the expression patterns of the endogenous myoD gene and the MSV-v-ski transgene suggests the possibility that the MSV promoter-enhancer may be responding to MyoD activity specifically in adult IIb muscle fibers. In this case, the MSV enhancer would be a useful tool, not only in studies of differences in MyoD and Myogenin activity, but also for analyses of changes in MyoD activity during development. The idea that MyoD in type IIb fibers may regulate the MSV enhancer is supported by the finding that the bHLH transcription factor E47 can bind and transactivate the murine leukemia virus promoter-enhancer in vivo(28) . The murine leukemia virus and MSV enhancers, as well as those from all type C retroviruses, are highly homologous(29) . Based on these facts, we postulated that the tissue-specific expression of v-ski in the transgenic mice may result from the action of muscle-specific regulators available in fast fibers but absent in slow fibers, of which MyoD is the most likely candidate. As a first test of this hypothesis, we asked if there were intrinsic differences in the ability of MyoD (fast-specific) and Myogenin (slow-specific) to bind the MSV enhancer. A quantitative, in vitro protein-DNA binding analysis of the MSV enhancer region with MyoD-E12 and Myogenin-E12 heterodimers was performed. Our results indicate that MyoD-E12 has higher affinity for the MSV enhancer and that it binds with higher cooperativity than Myogenin-E12. Taken together our results indicate that MyoD is likely to contribute to the fast fiber-specific expression of c-ski in transgenic mice.
Figure 1: Sequence of the MSV enhancer region and DNA fragments used for EMSA. Panel A, the sequence of the MSV enhancer with the six E-boxes enclosed. Binding sites for other transcription factors are shown below the horizontal bars and labeled. MCREF-1, LVa; GR, glucocorticoid receptor; NF-1, nuclear factor 1; CAT, CCAAT box-binding protein. Panel B, the DNA fragments tested in EMSA.
In this function, A1 is the initial y axis
level, A2 is the final y axis level, x is the center point of the curve, and p is the rate of
increase or slope of the curve. Our analysis of theoretical binding
curves for single and multiple protein/DNA interactions showed that the
variable p exponent existing in the sigmoidal fitting function
can be used as a relative measure of binding cooperativity. A p value of 1.0 indicates no cooperativity; p > 1 or p <1 indicates positive or negative cooperativity,
respectively; and the magnitude of p is indicative of the
degree of cooperativity. It is important to note that the p value is a relative value describing the slope of the fitted curve
and is not directly related to the Hill coefficient.
For the
purposes of this work, we define the binding constant (K) as the concentration of protein required to
bind 50% of the total concentration of DNA. We use the term K
rather than K
because we
are considering multiple equilibria where either MyoD or Myogenin is
interacting with another protein, E12, and binding to multiple,
nonequivalent target sites. Considering a single binding site, there
are at least two formal types of equilibrium which could be occurring.
The MyoD family of proteins is commonly thought to bind as dimers, in
which case, there is a monomer-dimer equilibrium, for example MyoX
(MyoD or Myogenin) dimerizing with E12, described as follows.
where
There is also a protein-DNA equilibrium, for example MyoX-E12 interacting with DNA, described as follows.
where
Since there are no data currently to describe the protein/protein interactions, we are unable to assign accurate values to the dissociation constant for the protein/DNA interaction.
Alternatively, the proteins may bind the target DNA successively as monomers, as described.
where
In this case, the units of K would be M
and cannot be derived directly from our data.
Figure 2:
EMSA
for interaction of MyoD-E12 and Myogenin-E12 with 5` deletions of the
E23456 DNA fragment. MyoD-E12 or Myogenin-E12 and P-labeled DNA fragments (10 pM) were equilibrated
and electrophoresed through native acrylamide gels as described under
``Materials and Methods.'' Panel A, representative
autoradiogram of MyoD-E12 and the E23456 DNA fragment. Panel
B, representative autoradiogram of Myogenin-E12 and the E3456 DNA
fragment. Panels C and D, the amount of unbound DNA
in each lane was quantitated by densitometry and used to calculate the
concentration of bound DNA. The percentage of bound DNA is plotted versus the concentration of protein (calculated as the
heterodimer). Arrows indicate resolved protein-DNA complexes. Panel C, MyoD-E12; panel D, Myogenin-E12.
,
E23456;
, E3456;
, E456;
, E56;
,
E6.
The apparent binding constants for the
parent E23456 fragment in this set were 0.2 nM and 0.3 nM for MyoD-E12 and Myogenin-E12, respectively. The K values for MyoD-E12 increased 16-fold from 0.2
nM for the E23456 fragment to 3.2 nM for the E6
fragment (Table 1). There were only very slight changes in K
when E3 or E5 was deleted from the fragments,
indicating that these E-boxes do not contribute significantly to
binding affinity by MyoD-E12. The changes in the p value in Table 1for MyoD-E12 do not correlate with changes in affinity.
Although removal of E2 from the target DNA increases the K
4-fold, there is no significant change in
cooperativity. However, deletions of E3 and E4 reduce the positive
cooperativity of binding.
The 15-fold increase in K for Myogenin-E12, as a result of deleting all but E2 from the
parent E23456 fragment, is similar to that of MyoD-E12, and the
absolute values for K
are similar (Table 1). However, the large increase in K
(10-fold) caused by deleting E2 indicates that this E-box is
perhaps the most important for Myogenin-E12 binding. The presence of E4
in the fragment does not enhance Myogenin-E12 binding as it did for
MyoD-E12. The p values determined from the curves in Fig. 2D indicate that there is positive cooperativity
in binding for all DNA fragments tested except E6, but less than that
observed for MyoD-E12 binding to the same fragments.
Figure 3:
EMSA for
interaction of MyoD-E12 and Myogenin-E12 with 3` deletions of the
E23456 DNA fragment. MyoD-E12 or Myogenin-E12 and P-labeled DNA fragments (10 pM) were equilibrated
and electrophoresed through native acrylamide gels as described under
``Materials and Methods.'' Panel A, representative
autoradiogram of MyoD-E12 and the E234 DNA fragment. Panel B,
representative autoradiogram of Myogenin-E12 and the E2345 DNA
fragment. Panels C and D, the amount of unbound DNA
in each lane was quantitated by densitometry and used to calculate the
concentration of bound DNA. The percentage of bound DNA is plotted versus the concentration of protein (calculated as the
heterodimer). Arrows indicate resolved protein-DNA complexes. Panel C, MyoD-E12; panel D, Myogenin-E12.
,
E23456;
, E2345;
, E234;
, E23;
,
E2.
Deletion of E6 from the E23456
fragment reduced the affinity of Myogenin-E12 binding very much like it
did MyoD-E12 binding with an increase in K of
approximately 12-fold but without the concomitant reduction in
cooperativity. There was a slight increase in affinity, approximately
2-fold, when E5 was removed, leaving E234. Removal of E4 had
devastating effects on binding: the E23 and E2 fragments were bound by
Myogenin-E12 with K
values of 26 and 23
nM, respectively, and all positive cooperativity of binding
was lost.
Figure 4:
EMSA for
interaction of MyoD-E12 and Myogenin-E12 with 3` deletions of the E123
DNA fragment. MyoD-E12 or Myogenin-E12 and P-labeled DNA
fragments (10 pM) were equilibrated and electrophoresed
through native acrylamide gels as described under ``Materials and
Methods.'' Panel A, representative autoradiogram of
MyoD-E12 and the E123 DNA fragment. Panel B, representative
autoradiogram of Myogenin-E12 and the E123 DNA fragment. Panels C and D, the amount of unbound DNA in each lane was
quantitated by densitometry and used to calculate the concentration of
bound DNA. The percentage of bound DNA is plotted versus the
concentration of protein (calculated as the heterodimer). Arrows indicate resolved protein-DNA complexes. Panel C,
MyoD-E12; panel D, Myogenin-E12.
, E123;
, E12;
, E1.
Figure 5:
EMSA for interaction of MyoD-E12 and
Myogenin-E12 with internal E-box mutations of the E23456 DNA fragment.
MyoD-E12 or Myogenin-E12 and P-labeled DNA fragments (10
pM) were equilibrated and electrophoresed through native
acrylamide gels as described under ``Materials and Methods.'' Panel A, representative autoradiogram of MyoD-E12 and the E13
DNA fragment. Panel B, representative autoradiogram of
Myogenin-E12 and the E13 DNA fragment. Panels C and D, the amount of unbound DNA in each lane was quantitated by
densitometry and used to calculate the concentration of bound DNA. The
percentage of bound DNA is plotted versus the concentration of
protein (calculated as the heterodimer). Arrows indicate
resolved protein-DNA complexes. Panel C, MyoD-E12; panel
D, Myogenin-E12.
, E13;
, E2456;
, E235;
, E46.
We tested the hypothesis that MSV-v-ski transgene expression in fast-twitch muscle fibers was responsive to MyoD activity by examining the DNA binding characteristics of MyoD-E12 and Myogenin-E12 (slow fiber-specific) for the MSV enhancer in vitro. The results reported here are our initial analyses of this system and indicate complex interactions between these proteins bound to the six E-boxes in terms of affinity and cooperativity. Further analyses will be required to understand fully the molecular mechanisms involved in regulating expression of the MSV enhancer-promoter. Nevertheless, our results clearly indicate that MyoD-E12 bound nearly all of the DNA fragments tested with higher affinity and higher cooperativity than Myogenin-E12. Thus, we conclude that fiber type IIb-specific expression of MSV-ski in transgenic mice likely results in part from activation by MyoD, which preferentially accumulates in these fibers.
MyoD-E12 binding to all the DNA
fragments containing single E-boxes (E1, E2, or E6) showed positive
cooperativity, whereas only E1 was bound by Myogenin-E12 cooperatively (Table 1Table 2Table 3). In addition, MyoD-E12 bound
all and Myogenin-E12 bound most of the DNA fragments containing
multiple E-boxes with cooperativity. Thus, there are multiple
contributions of cooperativity to DNA binding for these transcription
factors. These results are strikingly similar to the interactions of
repressor with O
1, O
2, and
O
3(31) .
repressor dimerization is enhanced
by DNA binding, thus there is cooperativity observed in binding a
single operator. Repressor also shows cooperativity in binding tandemly
arranged target DNAs, via protein/protein interactions between
repressor bound to O
1 and O
2. Binding to
O
3 is not cooperative, however, because repressor at
O
2 interacting with repressor at O
1 cannot
interact with repressor at O
3. However, mutations in
O
1, which disallow repressor binding, promote repressor
binding to O
2 and O
3 with cooperativity. Such
high levels of cooperativity dramatically increase the regulatory
response due to small changes in protein concentration or activation (31) .
Cooperativity of dimeric protein interactions with a
single DNA target site can arise from two distinct mechanisms. The
stepwise binding of monomers can show cooperativity by increasing the
affinity of binding for the second monomer in the presence of the first
bound monomer. For example, this is what is observed for the
glucocorticoid receptor DNA binding domain. ()Alternatively,
cooperativity can also arise for binding of dimeric proteins to a
single binding site if protein dimerization is significantly enhanced
by DNA binding. It should also be noted that these two mechanisms are
not mutually exclusive; both can be occurring. Although our results
show cooperativity in binding a single site, they do not help
distinguish between whether these proteins bind DNA as dimers, stepwise
as monomers, or both. We are currently conducting experiments to
clarify the mode of binding utilized by MyoD-E12.
Studies on DNA recognition by bHLH muscle transcription factors showed that the CANNTG E-box sequence establishes a minimal DNA binding site for these proteins. Consensus DNA recognition sequences for MyoD-E12 heterodimer (32) and Myogenin homodimer (and heterodimer with E12 as proposed in (33) ) derived in affinity selection experiments show a high level of similarity. The MSV enhancer contains six E-box sequences. Four of the E-boxes (E2, E4, E5, E6) have the sequence CAGATG, which is different from the consensus sequences determined in selection experiments for either MyoD or Myogenin, but is present in enhancers of muscle-specific promoters (34, 35, 36) . Two of the E-boxes (E1, E3) have the sequence CAGCTG, matching the consensus binding site from selection experiments with Myogenin. The crystal structure of the MyoD bHLH domain-DNA complex (8) revealed protein contacts with the bases of the primary E-box determinants, CA and TG, and some contacts to 5`- and 3`-flanking residues in the double-stranded TCAACAGCTGTTGA DNA, indicating that specific bases outside an E-box may be involved in recognition and binding. It has also been postulated that the central two bases of the E-box motif could contribute to distinct MyoD and Myogenin-DNA binding preferences. Binding site selection experiments showed that Myogenin has a preference for a symmetrical binding site, whereas MyoD binding sequences showed a nonpalindromic distribution of bases in central NN bases of an E-box. In general, our results show only slight differences in affinity for both proteins in combination with E12 binding to single E-box containing DNA fragments (except for E2) and that binding occurs with low to moderate affinity. Thus, MyoD and Myogenin prefer similar DNA targets and bind them with similar affinity.
On the other hand, numerous in vivo experiments have shown that MyoD and Myogenin have distinct functions in the regulation of muscle-specific gene expression. One proposed solution to this apparent contradiction is that heterodimerization, rather than homodimerization, with other bHLH proteins such as ubiquitous E12 and E47 or Id, leads to MyoD- or Myogenin-specific gene activation or inactivation. It has also been shown that other regulatory DNA elements in combination with E-box sequences can differentially contribute to muscle-specific gene activation(37) . These regulatory models, although attractive and well documented, share one weakness: namely, E-box sequences are widespread in genomes (average of one E-box in 256 bases) and are present in the promoters of genes not specifically expressed in muscle. Nevertheless, it has been shown that under certain experimental conditions a single E-box motif can be responsible for Myogenin-dependent gene activation(38) . In our studies, we showed that MyoD-E12 and Myogenin-E12 DNA binding affinity to targets containing multiple E-box sequences is much higher than interactions of these proteins with single DNA fragments containing a single E-box. This indicates that multiple E-box motifs, when present in promoter regions, could contribute to high affinity DNA binding for these proteins and simultaneously provide an additional level to MyoD and Myogenin DNA recognition specificity. Cooperative binding of MyoD-E47 to DNA targets containing multiple E-box motifs (39) and E-box cooperation in the promoter of the rat acetylcholine receptor subunit gene(40, 41) enhance transcriptional activity. We demonstrated that mutations of certain E-box sequences within MSV enhancer significantly reduce affinity of MyoD-E12 and Myogenin-E12 in cooperative DNA binding presumably through lack of communication between proteins. Conversely, the same situation could arise in vivo when two or more transcriptionally active proteins compete for overlapping DNA recognition sites. This competition could result in a delicate balance of repression and activation. Such a situation can be easily predicted in the case of MSV enhancer, where E2 and E4 constitute internal parts of the glucocorticoid response element. Binding of the glucocorticoid receptor in response to steroid hormone treatment could inhibit MyoD-E12 or Myogenin-E12 binding. In fact, clinical application of glucocorticoids in cancer therapy leads to repression of muscle-specific gene expression and a loss of muscle mass(42) . There are similar sequence relationships in other muscle-specific promoters where, in addition to glucocorticoid response element/E-box overlapping motifs, other transcription factor binding sites (e.g. LVa, Ets-1) consistently share the same stretch of bases with E-box sequences.
We have reported the results from an initial probe into the interactions of transcription factors with the MSV enhancer. It is clear from our results that complex protein/protein interactions and protein/DNA interactions are likely to contribute to enhancer activity. Specifically, cooperativity of binding plays a much larger role than previously expected. Currently, we are pursuing experiments that will further address the mechanisms contributing to cooperative binding, the biological relevance of MyoD-E12 and Myogenin-E12 DNA binding within MSV enhancer, and for possible modulation of MSV enhancer activity through glucocorticoid receptor/MyoD, Myogenin competition for their DNA binding sites.