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(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) ( 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) 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 [
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].
Volume 270,
Number 7,
Issue of February 17, 1995 pp. 2889-2892
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)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) .
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.
Isolation of MRF4 Upstream Region
To isolate MRF4 genomic clones, a mouse genomic library was screened by
hybridization to the full-length rat MRF4 cDNA(5) .
Hybridizations and purification of phage clones were performed using
standard techniques(32) . PCR was then used to isolate the
390-bp upstream fragment used in these studies using 5` primer
5`-CTACTACTACTAAAGCTTGTGTTAATCCCCAGT TGT-3` and 3` primer
5`-CATCATCATCATTCTAGACTCCTCCTTGGCCTCTGA-3`.Plasmids, Mutagenesis, and Sequence Analysis
The
390-bp mouse MRF4 promoter fragment was isolated using the
primers described above. This fragment was then cloned into the
polylinker of plasmid pCAT-BASIC (Promega, Inc.) to create plasmid
pMRF4.CAT. The E2 mutant MRF4 promoter was cloned by the same
strategy except that the following 5` primer was used to remove the E2
E box from the 5` end of the fragment:
5`-AAAACTGCAGTGAAGTTGCCTGGTTAGCAGG-3`. All other mutants were made by
utilizing the PCR mutagenesis technique of gene splicing by overlap
extension (``gene SOEing'') (33) creating the
following mutant sequences in the wild-type promoter context: E1,
5`-AATTAAATGATATCTGGGTGGCTCC-3`; and MEF2,
5`-TAGCTAGTATATAAGCAGCTGGGTCGA-3`. The MEF2 mutagenic primers mutate
the outer sequences of the MEF2 consensus, while leaving the TATA motif
contained within the larger MEF2 sequence
intact(26, 34) . Each of the MRF4 promoter-CAT
constructs was sequenced from both ends to confirm that the mutations
were in place and that no unintentional mutations were introduced by
the PCR. MEF2 expression plasmids were made by cloning the coding
regions for MEF2A(14) , MEF2C(19) , and
MEF2D(20) , into the polylinker of plasmid pCDNAI/amp
(Invitrogen, Inc.). The expression plasmids encoding
myogenin(2) , MyoD(1) , myf5(4) , and MRF4 (5) all contain the complete coding region cDNAs cloned into
plasmid pEMSVscribe(1) . Plasmid pCDNAIII.CAT (Invitrogen,
Inc.) encodes the CAT gene under control of the cytomegalovirus
immediate-early promoter and was used as a positive control for
expression.Cell Culture and Transfections
C3H10T1/2 (10T1/2)
and C2C12 (C2) cells were maintained essentially as described
previously(2) . Chick primary myoblasts were isolated and grown
overnight as described previously(35) . Differentiation medium
(DM) is Dulbecco's modified Eagle's medium supplemented
with 5 mML-glutamine, 1 mM sodium pyruvate,
100 IU of penicillin, 100 µg/ml streptomycin, and 2% horse serum.
Growth medium (GM) is Dulbecco's modified Eagle's medium
supplemented with 5 mML-glutamine, 1 mM sodium pyruvate, and either 10% fetal calf serum (10T1/2) or 15%
fetal calf serum (C2C12, chick myoblasts).CAT Assays
Transfected cells were harvested, and
extracts were prepared by three freeze-thaw cycles and heat
inactivation as described previously(37) . Cell lysates were
then quantitated for total protein(38) , and an equivalent
amount of cell lysate (normalized for total protein) from each
transfection was assayed for CAT activity as described
previously(37) . 100 µg (10T1/2 and C2C12 transfected
cells) or 20 µg (chick primary myoblasts) of total cellular protein
was assayed for each reaction. Reactions were conducted for 5 h at 37
°C. Conversion to acetylated forms was analyzed by thin-layer
chromatography and quantitated by phosphorimager analysis (Molecular
Dynamics, Inc.).
The Upstream Region of the Mouse MRF4 Gene Contains
Putative Muscle-specific cis-Acting Regulatory Elements
To begin
to define the muscle-specific regulation of MRF4 gene
transcription, we isolated a 390 bp fragment extending from -300
to +90 relative to the primary transcription start site at
+1. Location of the transcription start site is based on complete
sequence homology to the published transcription start site of the rat MRF4 gene (39) . The sequence of this region of the
mouse gene is shown in Fig. 1. We chose to focus on this region
of sequence surrounding the MRF4 transcription start site
because it contains several potential muscle-specific cis-acting elements. Furthermore, sequence analysis of this
390-bp fragment showed that it had a strong overall resemblance to the
proximal region of the myogenin gene promoter(23) .
Two E boxes are present in this region, one at +22 to +27 and
the other at -287 to -282, designated as E1 and E2,
respectively. There is also an A+T-rich element in this region at
-26 to -17, which serves as the primary transcription start
site(39) , as well as meeting the consensus sequence
requirements for MEF2 transcription factor
binding(13, 22) . An additional A+T-rich region,
which resembles a TATA motif, is present at +11 to +19.
The Upstream Region of the MRF4 Gene Mediates
Muscle-specific Expression
Based on its similarity to the myogenin promoter, we predicted that this region of the mouse MRF4 gene would serve as a muscle-specific promoter. To test
this hypothesis and determine if this region could provide
muscle-specific expression, we subcloned this 390 bp of 5`-flanking
sequence upstream of the CAT reporter gene into plasmid
pCATBASIC to create plasmid pMRF4.CAT and we transfected this plasmid
into both muscle and non-muscle cell types (Fig. 2). The results
showed that this MRF4 upstream fragment is incapable of
directing expression of CAT in 10T1/2 fibroblast cells but mediates
significant expression in the C2 muscle cell line and in primary chick
muscle cells. These results indicate that this 390 bp of MRF4 5`-flanking sequence contains promoter and enhancer elements
sufficient to mediate muscle-specific activation, and that this
promoter element is transcriptionally silent in 10T1/2 fibroblasts.
While the activity of the MRF4 promoter was relatively weak in
C2 myotubes, it was clearly muscle-specific. The relatively weak
activity of the promoter most likely reflects the fact that MRF4 is
up-regulated late in the differentiation program and is not expressed
at high levels in C2
myotubes(5, 6, 7, 29, 30) .
It is also possible that while the promoter sequence used here contains
the elements necessary for muscle-specificity, it may not contain all
of the enhancer elements required for high level expression in these
cell types.
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.
Muscle-specific Transcription Factors trans-Activate the
MRF4 Promoter
Since the MRF4 promoter contains
potential myogenic bHLH and MEF2 binding sites (Fig. 1) and was
capable of directing muscle-specific transcription (Fig. 2), we
wanted to determine if these two classes of transcription factors were
involved in activation of the MRF4 promoter. We cotransfected
pMRF4.CAT into 10T1/2 fibroblasts along with activator plasmids
encoding one of the four myogenic bHLH factors or MEF2A, MEF2C, or
MEF2D. As shown in Fig. 3, myogenin, myf5, and MyoD all mediate
a strong trans-activation (about 20-fold) of the MRF4 promoter. In contrast, MRF4 was unable to activate its own
promoter in this assay. All of the MEF2 factors tested were able to
reproducibly activate the MRF4 promoter from 4-10-fold.
These trans-activations were specific for the MRF4 promoter and were not the result of a general transcriptional
activation, since neither the bHLH nor the MEF2 activators affected the
level of CAT expression when CAT was under the control of a
constitutively active viral promoter (data not shown). These results
indicate that the MRF4 promoter can be trans-activated by muscle-specific transcription factors and
suggest that the putative E boxes and MEF2 sites in the promoter may be
involved in this activation.
Myogenin trans-Activation of the MRF4 Promoter is E
Box-dependent
In order to determine whether the two E boxes and
the single MEF2 site in the MRF4 promoter were involved in trans-activation of the promoter by myogenic bHLH and MEF2
proteins, we mutated each of these elements singly and in combination
to produce each possible double and triple mutant. The wild-type 390-bp MRF4 promoter and each of the seven mutant promoter-CAT
constructs were then cotransfected into 10T1/2 cells along with a
myogenin expression plasmid or with a control plasmid expressing the
neomycin resistance (neo) gene. Following transfection, the
cells were harvested and assayed for CAT activity. The results in Fig. 4A show that at least one E box is required for trans-activation of the MRF4 promoter by myogenin.
Mutation of either one of the E boxes (E1(-) or E2(-)) had
no effect on promoter activity, suggesting that only one E box is
necessary and sufficient for full activation of the promoter by
myogenin. These data also show that the MEF2 site in the MRF4 promoter is not required for indirect activation by myogenic bHLH
proteins, since the MEF2 mutations had no effect on trans-activation by myogenin. The activity of the promoter was
greatly reduced only when both E boxes were mutated. These results
suggest that in 10T1/2 cells, trans-activation of the MRF4 promoter by myogenin, MyoD, and myf5 is direct and requires at
least one intact E box motif. The direct nature of this interaction is
further supported by gel shift analyses, which showed that both E boxes
specifically bound myogenin-E12 heterodimers (data not shown).
)
We appreciate the sequence analysis provided by Mike
Chase and the help with chicken cell culture provided by Janet Mar and
Mei Zhang. We also thank Janet Mar for critical review of the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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B. Datta, W. Min, S. Burma, and P. Lengyel Increase in p202 Expression during Skeletal Muscle Differentiation: Inhibition of MyoD Protein Expression and Activity by p202 Mol. Cell. Biol., February 1, 1998; 18(2): 1074 - 1083. [Abstract] [Full Text]
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B. L. Black, J. D. Molkentin, and E. N. Olson Multiple Roles for the MyoD Basic Region in Transmission of Transcriptional Activation Signals and Interaction with MEF2 Mol. Cell. Biol., January 1, 1998; 18(1): 69 - 77. [Abstract] [Full Text]
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A N Gerber, T R Klesert, D A Bergstrom, and S J Tapscott Two domains of MyoD mediate transcriptional activation of genes in repressive chromatin: a mechanism for lineage determination in myogenesis. Genes & Dev., February 15, 1997; 11(4): 436 - 450. [Abstract] [PDF]
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Y Wang and R Jaenisch Myogenin can substitute for Myf5 in promoting myogenesis but less efficiently Development, January 7, 1997; 124(13): 2507 - 2513. [Abstract] [PDF]
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B. L. Ziober and R. H. Kramer Identification and Characterization of the Cell Type-specific and Developmentally Regulated alpha 7 Integrin Gene Promoter J. Biol. Chem., September 13, 1996; 271(37): 22915 - 22922. [Abstract] [Full Text] [PDF]
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W Zhang, R R Behringer, and E N Olson Inactivation of the myogenic bHLH gene MRF4 results in up-regulation of myogenin and rib anomalies. Genes & Dev., June 1, 1995; 9(11): 1388 - 1399. [Abstract] [PDF]
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F. B. Berry, Y. Miura, K. Mihara, P. Kaspar, N. Sakata, T. Hashimoto-Tamaoki, and T. Tamaoki Positive and Negative Regulation of Myogenic Differentiation of C2C12 Cells by Isoforms of the Multiple Homeodomain Zinc Finger Transcription Factor ATBF1 J. Biol. Chem., June 29, 2001; 276(27): 25057 - 25065. [Abstract] [Full Text] [PDF]
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