(Received for publication, November 18, 1994; and in revised form, December 13, 1994)
From the
MRF4 is a member of the basic helix-loop-helix (bHLH) family of muscle-specific transcription factors, which also includes MyoD, myogenin, and myf5. The myocyte enhancer binding factor 2 (MEF2) proteins also serve as important muscle-specific transcription factors. In addition to activating the expression of many muscle-specific structural genes, various members of these two classes of proteins activate their own expression and the expression of each other in a complex transcriptional network that results in the establishment and maintenance of the muscle phenotype. To begin to determine how the expression of MRF4 is regulated by other muscle-specific transcription factors, we have isolated a region of the MRF4 gene that confers muscle-specific expression and have analyzed this promoter region for cis-acting elements involved in trans-activation by the myogenic bHLH and MEF2 transcription factors. Here, we show that in 10T1/2 fibroblasts the MRF4 promoter is trans-activated by myogenin, MyoD, myf5, and by the MEF2 factors, but that MRF4 does not activate expression of its own promoter. Myogenin activated the MRF4 promoter directly by an E box-dependent mechanism, while MEF2 factors activated the promoter through an indirect pathway. The E box-dependent regulation of the MRF4 promoter is in contrast to the regulation of the myogenin and MyoD promoters and may represent a mechanism for the differential expression of these factors during myogenesis.
During skeletal muscle development, a wide array of
muscle-specific genes are expressed in an ordered pattern, which
results in the myogenic phenotype. One set of transcription factors
that is involved in the regulation of the muscle transcriptional
network is the myogenic basic helix-loop-helix (bHLH) ()family. This family of transcription factors includes
MyoD(1) , myogenin(2, 3) , myf5(4) ,
and MRF4(5, 6, 7) . These factors induce
transcription by binding as heterodimers with ubiquitously expressed
E-proteins to the E box consensus sequence (CANNTG), which is found in
the control regions of numerous muscle-specific genes(8) . When
expressed in a variety of non-muscle cells, each of these four factors
is capable of inducing myogenic conversion and
differentiation(1, 2, 3, 5, 7) .
In addition to activating the expression of muscle-specific structural
genes, in many cell types, these myogenic bHLH factors have been shown
to activate their own and each other's
expression(9, 10, 11) .
The MEF2 family of
MADS box transcription factors has also been shown to play a role in
the activation of muscle-specific gene transcription (12) .
MEF2 factors are the products of four separate genes, mef2a, mef2b, mef2c, and mef2d(13, 14, 15, 16, 17, 18, 19, 20) ,
and they activate transcription by binding to the consensus MEF2 site
sequence, CTA(A/T)TA(G/A), as homo- and
heterodimers(13, 20, 21, 22) .
Recently, the MEF2 binding site has been shown to be required for
expression of several of the myogenic bHLH
factors(18, 23, 24, 25, 26) ;
likewise, members of the myogenic bHLH family can activate the
expression of MEF2 factors(19, 22, 27) .
Thus, the MEF2 factors and the myogenic bHLH proteins appear to
function in a complex network by auto- and cross-activating their own
and each others expression to establish and maintain the expression of
muscle genes that give rise to the myogenic phenotype.
During mouse development, each of the myogenic bHLH factors is expressed in a precise temporal and spatial pattern to give rise to muscle(28) . In the developing myotome, for example, myf5 is the first of the bHLH factors to be expressed, followed shortly thereafter by the expression of myogenin(28) . MRF4 is expressed in a biphasic pattern in the somite and is the last of the myogenic bHLH factors to be expressed in the developing muscle of the limb(29, 30) . It is the most highly expressed of the bHLH factors at birth and is the only myogenic bHLH factor to be expressed at high levels in adult muscle(5, 7, 29, 30) . Based on these observations, it has been proposed that MRF4 primarily functions downstream of the other myogenic bHLH factors(7, 11, 29, 31, 42) . In this regard, MRF4 has been postulated to be involved in myofiber formation (11, 29, 31) and in the maintenance of the muscle phenotype(7, 29) .
Based on these hypotheses, which suggest that MRF4 plays a downstream role in myogenesis, we wanted to determine if MRF4 expression is directly activated by other muscle-specific transcription factors. To begin to define the mechanisms involved in the activation of MRF4 expression, we have isolated a region of the MRF4 promoter that confers muscle-specific expression and have analyzed this promoter region for muscle-specific sequence elements involved in activation by the myogenic bHLH and MEF2 transcription factors. Here, we show that the MRF4 promoter is trans-activated directly by the myogenic bHLH factor myogenin via an E box-dependent mechanism and is activated by MEF2 proteins via an indirect pathway.
All transfections were performed by calcium phosphate precipitation for 12 h as described previously(36) . In each transfection, 20 µg of plasmid DNA was transfected. Chick primary myoblasts and late C2C12 myotubes were transfected in DM. 10T1/2 cells and early C2C12 myotubes were transfected in GM. Following transfection, early C2C12 myotubes and 10T1/2 cells were grown for 12 h in GM followed by 48 h in DM prior to harvesting, and chick primary myoblasts were maintained in DM supplemented with chick embryo extract (35) and without antibiotics for 48 h prior to harvesting. Late C2C12 myotubes were allowed to differentiate for 5 days in DM, transfected in DM for 12 h, then maintained for 48 h in DM prior to harvesting.
Figure 1: Nucleotide sequence of the upstream region of the mouse MRF4 gene. Figure shows nucleotide sequence of the 390-bp fragment of the MRF4 gene used in this study. The E boxes (E1 and E2) and MEF2/TATA element are underlined. Numbers are relative to the transcriptional start site at +1(39) .
Figure 2:
Muscle-specific activity of the 390 bp MRF4 fragment. 10T1/2 cells, early and late C2C12 myotubes,
and day 12 chicken primary myoblasts were transfected with equal
amounts of plasmid pMRF4.CAT (MRF4), pCATBASIC (BASIC), or pCDNAIII.CAT (CMV) or were untransfected (UNTR). Transfection of each cell type was performed as
described under ``Materials and Methods.'' Reactions were
analyzed by thin-layer chromatography, an autoradiograph of which is
shown. The MRF4 promoter-CAT construct exhibited 1.0-, 2.9-,
3.8-, and 5.2-fold activation over the activity of the pCATBASIC vector
in 10T1/2, early C2C12, late C2C12, and chick myoblasts, respectively.
CAT activity of cell extracts was determined by the percent conversion
of [C]chloramphenicol (Cm) to
acetylated forms (AcCm). Percent conversion of each reaction
is shown at the top of the figure. All four cell types were similarly
transfected as the activation of the positive control plasmid
pCDNAIII.CAT was approximately equivalent in each. Quantitation was by
phosphorimager analysis (Molecular Dynamics, Inc.). Comparable results
were obtained in three separate sets of
experiments.
Figure 3: trans-Activation of the MRF4 promoter by muscle-specific transcription factors. Expression plasmids encoding myogenin, myf5, MyoD, MRF4, MEF2A, MEF2C, or MEF2D were cotransfected into 10T1/2 cells along with the MRF4 promoter reporter plasmid, pMRF4.CAT. In each case 10 µg of reporter and 10 µg of activator were cotransfected. The data are expressed as the -fold activation of the activator cotransfection over a control cotransfection in which the activator plasmid encoded the neo gene rather than one of the muscle-specific transcription factors. CAT activity of cell extracts was determined by thin-layer chromatography and was quantitated by phosphorimager analysis (Molecular Dynamics, Inc.). The data are the average of three independent experiments. Error bars indicate the standard error of the mean for the three experiments.
Figure 4: Effect of MRF4 promoter mutations on trans-activation by myogenin and MEF2A. Myogenin (A) or MEF2A (B) expression plasmid was cotransfected into 10T1/2 cells with either the wild-type (wt) or any of seven mutants of the MRF4 promoter-CAT plasmid pMRF4.CAT. The data are expressed as fold activation of the myogenin (A) or MEF2A (B) cotransfection over a neo-only activator cotransfection using the same MRF4 reporter construct. In each case 10 µg of reporter and 10 µg of activator were cotransfected. CAT activity of cell extracts was determined by thin-layer chromatography and was quantitated by phosphorimager analysis (Molecular Dynamics, Inc.). The results presented are from a representative cotransfection analysis. The same results were obtained from two independent transfections and analyses. Similar results to those in A were also obtained with myf5 and MyoD activators. Similar results to those in B were also obtained with MEF2C and MEF2D activators. MRF4 promoter mutants: wt, wild-type; E1(-), mutant E1 E box; E2(-), deleted E2 E box; E1/E2(-), mutant E1 E box and deleted E2 E box; M2(-), mutant MEF2 site (TATA site remains intact); M2/E1(-), mutant MEF2 site and mutant E1 E box; M2/E2(-), mutant MEF2 site and deleted E2 E box; M2/E1/E2(-), mutant MEF2 site, mutant E1 E box, and deleted E2 E box.
We also tested the MRF4 promoter mutants for the ability to be trans-activated by MEF2 factors. Whereas the MEF2 factors were able to activate the MRF4 promoter in 10T1/2 cells (Fig. 3), this activation was through an indirect pathway since all of the mutant reporters were as active as the wild-type construct (Fig. 4B). This result is also supported by gel shift data, which showed that this consensus MEF2 binding sequence in the promoter is bound only very weakly by in vitro translated MEF2 proteins (data not shown). The indirect nature of the MEF2 trans-activation seen in Fig. 4B suggests that in vivo the MEF2 consensus sequence in the MRF4 promoter serves as the TATA box as has been previously demonstrated (39) but that it does not function as a MEF2 site.
The data presented in this study provide evidence for direct and indirect trans-activation of the MRF4 promoter by muscle-specific transcription factors. We have shown that a small region of the MRF4 gene surrounding the transcription start site directs muscle-specific expression. Myogenin, MyoD, and myf5 strongly trans-activate the MRF4 promoter while MRF4 is incapable of efficiently activating the expression of its own promoter. This activation by other members of the bHLH family is direct and requires at least one intact E box motif. Likewise, members of the MEF2 family of transcription factors are able to trans-activate the MRF4 promoter; however, this activation is weak and appears to function via an indirect mechanism that does not require an intact MEF2 site or E box sequence elements in the promoter. The indirect nature of the MEF2 trans-activation of the MRF4 promoter probably occurs through protein-protein interactions with the basal transcription machinery. Such an interaction would imply that the sequence of the TATA element in the MRF4 promoter imparts a degree of muscle specificity since MEF2 activates the expression of the MRF4 promoter but not the CMV promoter in these cotransfection analyses. This type of functional heterogeneity of TATA elements resulting in muscle-specific activation has been proposed previously(40) . The low level of activation seen using the E1/E2(-) mutants in the myogenin cotransfection (Fig. 4A) may be a result of myogenin activation of endogenous MEF2 factors, which could then indirectly regulate the MRF4 promoter, as in Fig. 4B. Depending on which muscle-specific transcription factors are present, both the direct and indirect pathways described in this study may function in muscle cells in vivo. It is also possible that the regulatory pathways that govern MRF4 expression in muscle cells may vary from these as a result of a more complex set of muscle-specific trans-acting factors present at times during muscle development. It is also likely that additional complexity in MRF4 regulation exists in additional enhancer sequences since 6.5 kb of MRF4 upstream sequence linked to lacZ in transgenic mice recapitulated only part of the pattern of endogenous MRF4 expression(41) .
In spite of the similarity of the MRF4 promoter to the proximal myogenin promoter, the two promoters are regulated quite differently. The myogenin promoter is trans-activated by myogenic bHLH factors via an indirect pathway dependent upon the MEF2 site in the promoter(23) , while the MRF4 promoter is regulated directly by the other bHLH factors. The regulation of the MRF4 promoter is also in contrast to the regulation of the Xenopus and chicken MyoD promoters, which are regulated independently of their E box motifs(26, 40) . Thus, while all of the myogenic bHLH factors are able to mediate myogenesis, it is becoming clear that they are regulated by different mechanisms. It is these different mechanisms that are likely to account for proper expression of each of these bHLH factors during myogenesis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U18131[GenBank].