From the Unité de Biochimie, Département de Biologie Moléculaire, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
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ABSTRACT |
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The murine adult IIB myosin heavy chain (IIB MyHC) gene is expressed only in certain skeletal muscle fibers. Within the proximal promoter are two A + T-rich motifs, mAT1 and mAT2, which greatly enhance muscle-specific transcription; myogenic cells contain proteins that bind to these sequences. MEF-2 binds to both mAT1 and mAT2; a mutation abolishing its binding to mAT1 greatly diminishes the activity of the promoter. Both mAT motifs also form complexes with a protein requiring a target sequence typical of POU domain proteins, which migrate in electrophoretic mobility shift assays to the same position as a complex containing purified Oct-1 and which are supershifted by an antibody specific to Oct-1; this protein is therefore probably Oct-1. Footprinting experiments demonstrate that mAT1 is preferentially occupied by MEF-2 and mAT2 by Oct-1 and that these two proteins appear to bind cooperatively to their respective sites. Although the two mAT motifs have sequences that are very similar, they nonetheless exhibit distinct behaviors and perform differently in the activation of the promoter. The contribution of the IIB MyHC gene to specification of the myogenic phenotype is thus at least in part regulated by MEF-2 and Oct-1.
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INTRODUCTION |
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Skeletal muscle represents an excellent model to examine
tissue-specific controls on transcription, since most of the major muscle structural proteins have several isoforms that often have characteristic spatiotemporal patterns of expression (reviewed in Ref.
1). The mouse adult IIB myosin heavy chain (IIB
MyHC)1 gene, for example, is
expressed only at a mature stage of development and only in the
fast-twitch glycolytic fibers of differentiated skeletal muscle (2-4).
Transcriptional control is thought to be a primary level of regulation
for many of the genes coding for these different isoforms (5-7). Among
the transcription factors important for muscle gene expression are the
myogenic basic helix-loop-helix (bHLH) regulatory factors (myf-5, MyoD,
myogenin, and MRF4), which are able to induce muscle differentiation in
non-myogenic cells (reviewed in Ref. 8). The MEF-2 proteins (summarized
in Ref. 9) are also often required for the transcription of muscle genes. Many genes require interactions among several promoter-bound proteins, both tissue-specific and non-tissue-specific, to be expressed
in a tissue-specific manner. We have previously characterized numerous
potential binding sites for known transcriptional activators within the
5'-flanking region of the IIB MyHC gene (10-12). In particular, two
regions rich in the nucleotides A and T, situated between 140 and
190 bp, enhance the transcriptional activity of those constructions
that contain them. This activation is found only in differentiated
myotubes and not in undifferentiated myoblasts, implying that these
sites contribute to the restriction of the expression of the IIB MyHC
gene to mature muscle tissue.
There are numerous DNA-binding proteins that recognize and bind to sequences that are predominantly composed of A and T nucleotides. Among these are the ubiquitous SRF (13) and the "related-to-SRF" proteins that share with SRF the existence of an amino-terminal MADS domain (13, 14); the nuclear protein MEF-2 belongs to the related-to-SRF family. There are at least four different mef2 genes, each of which may be alternatively spliced to produce several isoforms of the MEF-2 protein, some of which are selectively expressed in muscle and brain (9, 15-17). Proteins containing a homeodomain also generally have at least a short AT-rich motif (often TAAT) as part of their target sequence (reviewed in Ref. 18); among the homeodomain proteins are the POU domain proteins, such as Oct-1 and Pit-1 (19).
The sequences of the two related AT-rich motifs found in the proximal region of the IIB MyHC are conserved among numerous distinct skeletal muscle MyHC genes of several vertebrate species (11), suggesting an important role for these "mAT" (myosin AT-rich) sites in the regulation of this family of genes. This observation led us to examine in detail the interactions among the two mAT sites and the DNA-binding proteins that presumably associate with them in vivo to effect transcriptional activation. In this study we demonstrate that Oct-1, bound to mAT2, and a MEF-2 protein, bound to the more downstream mAT1, are both involved in the regulation of the activity of the promoter. Muscle-specific transcription of this gene may thus be achieved by combinations of widely and narrowly expressed factors, and the interactions among them appear to be important for the activation of the IIB MyHC gene.
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EXPERIMENTAL PROCEDURES |
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Plasmids-- All the deletion constructions of the 5'-flanking sequence of the murine IIB MyHC gene (inserted into the pCAT-Basic vector) used in this study are based on those described by Takeda et al. (12). The constructions mutated in the mAT and palindrome regions were prepared by polymerase chain reaction, and the sequence of the mutations was verified experimentally.
Transfections and CAT Assays-- Murine C2C12 myoblasts (20, 21) were maintained in Dulbecco's modified Eagle's medium with 20% fetal calf serum at 37 °C in an atmosphere of 7% CO2. One day before transfection, approximately 4 × 105 myoblasts were plated; they were transfected with 2 µg of the deletion construction in the presence of the cationic lipid dioctadecylamidoglycyl spermine (22), as described previously (12). The activity of the CAT reporter gene in the cellular extract was measured according to the method of Seed and Sheen (23).
Nuclear Extract Preparation-- To prepare extracts of nuclear proteins from cultured cells, we used a method based on that of Dignam et al. (24). The method used for the preparation of nuclear proteins from hindlimb muscle of rats (1 week to 10 days old) or mice (2 weeks old) was that of Mar and Ordahl (25).
DNA Binding Assays--
Fragments of DNA (100-400 bp) used as
probes for DNase I footprinting assays were isolated from plasmids
containing the promoter region of the IIB MyHC gene (12). The probes
were labeled with [32P]dXTP and the Klenow fragment of
DNA polymerase I and purified on a 3.5-5% polyacrylamide gel.
Oligonucleotides used as probes were gel-purified and labeled with T4
polynucleotide kinase and 1 µM
[-32P]ATP. The sequences of the upper strand of the
double-stranded oligonucleotides used as probes and competitors are
given in Table I.
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RESULTS |
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Nuclear Proteins from Myogenic Cells Bind the mAT1 and mAT2 Elements in Vitro and Are Not All Present at the Same Levels in Different Cell Types-- To identify nuclear proteins that bind specifically to the mAT1 and mAT2 sequence motifs of the IIB MyHC promoter, we performed electrophoretic mobility shift assays (EMSAs) with oligonucleotide probes containing the sequences of these sites (refer to Table I for the sequences of all oligonucleotides used). By using nuclear extracts from myotubes of the murine skeletal muscle C2 cell line, we observed the formation of three main complexes with a probe (entire mAT1) containing the 23-bp mAT1 site (Fig. 1A, lane 3), and two main complexes with a probe (mAT2) containing the 17-bp mAT2 site (Fig. 1A, lane 4). Further experiments suggested that the binding of these proteins is mutually exclusive.
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The Protein That Forms Complex 1 Binds to MEF-2-like Recognition Sequences within the mAT1 and mAT2 Sites-- A sequence with homology to the consensus binding site for the MEF-2 protein ((C/T)T(A/t)(A/T)AAATA(A/G); see Ref. 27) is found within both of the mAT sites (mAT1 = ATTTCTAATTATATCCATTCA; mAT2 = TGTCAAATTATTTATAG). The mobility in an EMSA of the complex 1 formed with an oligonucleotide probe of either the mAT1 or the mAT2 sites was identical to that of a complex formed with an oligonucleotide probe containing the MEF-2-binding site from the mouse mck enhancer (Fig. 2A). In addition, the formation of the complex with the mAT1 or mAT2 probes could be inhibited by an excess of the mck site oligonucleotide, although not by an excess of the mAT1 oligonucleotide mutated to prevent binding of MEF-2 (mutant 2 mAT1; Fig. 2B, lanes 4 versus 3). The protein had approximately the same affinity for the mck MEF-2 and IIB MyHC mAT1 sites but a slightly reduced affinity for the IIB MyHC mAT2 site (Fig. 2C). From EMSAs in which a series of competing oligonucleotides was used, it was evident that the protein that forms complex 1 bound to the CTAATTATAT sequence within the mAT1 site and the CTATAAATAA sequence (lower strand) within the mAT2 site (Figs. 2B and 3A, and additional data), both of which fit the established MEF-2 consensus sequence. Based on its recognition sequence and tissue distribution, the protein that bound to the mAT1 and mAT2 sites to produce the abundant complex 1 therefore appears to be MEF-2.
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The Protein That Forms Complex 2 Requires a Target Sequence Containing a ATAAT, a TAAT(T/A) or a ATGAATA Motif-- Complex 2, whose presence was independent of the state of myogenic differentiation of the cells, migrated immediately ahead of complex 1 in EMSAs; indeed, the two could only be resolved on gels containing an elevated concentration of acrylamide and run for a long period. For the purposes of this study, we designated the protein that formed complex 2 as "2BB2," since in an EMSA with probes of the IIB (2B) MyHC promoter it formed the second band (B2). Based on competition EMSAs, 2BB2 appeared to require a typical homeodomain protein TAAT target sequence (18, 28), ATAAT or TAAT(T/A), in its binding site (Figs. 2B (compare lanes 3 and 4) and 3A and Table II). In addition, the formation of the 2BB2·mAT1 complex was also inhibited by a second group of oligonucleotide competitors whose sequences do not conform to a homeodomain consensus binding sequence but instead have in common an ATGAATA motif (two Pit-1 binding sequences (29), two octamer binding sequences (30, 31), and the heptamer sequence (32)). This motif is homologous to part of the consensus binding sequence for the POU domain transcription factor Pit-1, ATGNATA(T/A) (29, 33).
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2BB2 Is Oct-1 or an Oct-1-related Protein-- Since 2BB2 is widely expressed, and since it has a binding specificity which resembles that of a POU domain factor, we considered that it could be the DNA-binding POU domain protein Oct-1, which is expressed in many types of mammalian cells (19, 37). When an EMSA was performed with nuclear extracts from C2 myoblasts or myotubes and an oligonucleotide probe of the mAT2 site (mAT2 mutant MEF-2, mutated to selectively prevent binding of MEF-2 and thus render the 2BB2 band easier to distinguish), the 2BB2 complex could be supershifted by an antibody that specifically recognizes Oct-1 (Fig. 3B, lanes 1-4). This same complex was formed with a nuclear protein found in CV-1 cells (lanes 5 and 6). In addition, when the same mAT2 probe was used in an EMSA with purified Oct-1 protein, the band produced migrated to the same position as the 2BB2 band (lanes 7 and 8). We believe that the prominent, rapidly migrating complex seen in the Oct-1 lanes was due to a proteolytic degradation product of Oct-1, since it was apparently smaller than Oct-1 yet still reacted with the antibody. Thus 2BB2 is antigenically related to Oct-1, appears to have approximately the same molecular weight as Oct-1, has a binding specificity similar to that of Oct-1, and has the same widespread tissue distribution as Oct-1. We conclude therefore that 2BB2 is either Oct-1 or a very closely related protein.
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The Protein That Forms Complex 3 Binds to a Palindromic Sequence
Situated between the mAT1 and mAT2 Sites--
Complex 3 was formed in
EMSAs with nuclear extracts from differentiated C2 muscle cells and an
oligonucleotide probe of the mAT1 region (entire mAT1, 170/
138)
that extends several nucleotides upstream of the sequence required for
the binding of MEF-2 (
155/
142 bp) (Fig. 1A). Further
experiments demonstrated that the palindromic sequence situated at
154/
165 bp, AGAAATATTTCT, was the target sequence (Fig.
4A); this motif plus 4 bp
flanking on each side was sufficient for binding. The central 8 bp of
the motif were the most important and are also found in the same
position (
171/
155 bp) in the chicken neonatal MyHC gene (gene clone
N125 from Ref. 38) which is also expressed in fast muscles. The
symmetrical structure of this motif and the importance of the spacing
of the half-sites suggest that this palindrome-binding protein (Pal-BP) binds as a dimer.
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The Proteins That Form Complex 4a and Complex 4b Require a Sequence Similar to That of MHox-- The formation of complex 4a and complex 4b in an EMSA with an oligonucleotide probe of the mAT1 region could be totally inhibited by competition with an excess of unlabeled oligonucleotide (MHox control, modified) containing an MHox-binding site (from the mck enhancer (35), but with a point mutation to prevent binding by MEF-2, which was otherwise able also to bind to the site). Partial inhibition was obtained by an oligonucleotide (mutant 1 mAT1) containing the mAT1 site mutated to prevent binding of MEF-2 but not Oct-1 (Fig. 4B). The protein or proteins that formed these complexes therefore probably required a TAAT motif.
These complexes migrated in an EMSA to approximately the same position as does the MHox complex, with respect to the MEF-2 complex (35), and the proteins required target sequences similar to the consensus binding sequence of MHox. However, the amount of the proteins forming complexes 4a and 4b (as detected by EMSAs) declined drastically as myoblasts differentiated into myotubes (Fig. 4C), whereas that of MHox (as inferred from its mRNA detected by Northern blots), in contrast, declines only very slightly, if at all, during this transition (35).mAT1 Is Preferentially Occupied by MEF-2 and mAT2 by Oct-1 in
Differentiated Myogenic Cells--
Proteins present in nuclear
extracts from both myoblasts and myotubes (C2 and quail) produced a
similar footprint over the mAT1-mAT2 region (Fig.
5A, lanes 1 versus 2, Fig.
5B, lanes 2 versus 3 and 13 versus 12; only
footprints obtained with C2 myotube extracts are shown). Moreover,
there was no difference between the patterns of protection over the
mAT1-mAT2 region produced by nuclear extracts from C2 myotubes or from
adult rat muscles,2
indicating that the two sites are occupied at all stages of myogenic differentiation. This footprint was abolished by competition with an
oligonucleotide (entire mAT1) containing the mAT1 site (Fig. 5B,
lanes 4 and 5). The footprint on the coding strand
extended from 187 bp through
141 bp, that is from six nucleotides
upstream of the end of the mAT2 site MEF-2 sequence through four
nucleotides downstream of the end of the mAT1 site MEF-2 sequence. The
same region was also protected on the non-coding strand (data not
shown).
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Transcriptional Activity of the IIB MyHC Promoter Requires MEF-2
Binding to mAT1 and Oct-1 or MEF-2 Binding to mAT2--
We have
previously demonstrated that the presence of the mAT1 site or the mAT1
plus mAT2 sites leads to an increased level of transcriptional
activation when included in reporter gene plasmid constructions used
for transient transfection assays in quail myotubes (12) and C2
myotubes (10). (Note that in order for the IIB MyHC promoter to be
active in C2 myogenic cells, co-transfection with an expression vector
for one of the myogenic bHLH factors is necessary; this system has been
analyzed and validated in our laboratory (10, 39).) The 192-bp
construct (192 bp/+13 bp of the IIB MyHC promoter immediately
upstream of the CAT reporter gene), containing both mAT sites, is the
most active of all the promoter constructs, even including those which
are much larger (e.g. to
2330 bp).
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DISCUSSION |
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Both a Muscle-specific and a Ubiquitous Factor Are Involved in the
Muscle-specific Expression of the IIB MyHC Gene--
Combinations of
tissue-restricted and widely expressed factors are not infrequently
used to mediate muscle-specific expression (e.g. Refs. 35,
40, and 41-46). The muscle-specific expression of the IIB MyHC gene
studied here is at least partially controlled by the factors MEF-2 and
Oct-1 binding to the mAT1 and mAT2 motifs. The MEF-2 isoform that
activates the IIB MyHC promoter is restricted to differentiated muscle
cells (this study and Ref. 12), whereas Oct-1 is present in a wide
variety of tissues (19, 37). These two proteins contain distinct
DNA-binding domains: MEF-2 (a MADS box) and Oct-1 (a POU domain, which
itself contains both a homeodomain and a POU-specific domain). There
are several known MADS domain-homeodomain protein pairs that are
important for transcriptional activation. Unlike the MCM1/MAT2 (47)
and SRF/Phox1 (48) pairs, where the homeodomain partner contributes the
tissue specificity, in the MEF-2/Oct-1 pair on the IIB MyHC promoter it
is rather the MADS domain partner that is specific to muscle.
MEF-2C Activates the IIB MyHC Promoter through mAT1-- The four vertebrate mef2 genes give rise via alternative splicing to numerous MEF-2 proteins (15-17). MEF-2A (9) and MEF-2C (15, 16) are specific to differentiated muscle tissue and to nerve cells. MEF-2D is present at the myoblast stage, and MEF-2A appears almost immediately after myoblasts are transferred to differentiation medium, whereas MEF-2C is expressed only after several days of differentiation. The myotubes used here to prepare the nuclear extracts have spent 3 to 4 days in differentiation medium. The endogenous IIB MyHC gene is expressed at a significant level in C2 cells only after several days in differentiation medium (6). The MEF-2C mRNA levels are extremely low in C2C12 myotubes (39), and overexpression of MEF-2C activates the IIB MyHC promoter. These observations strongly argue in favor of importance of the MEF-2C isoform.
Oct-1 or a Closely Related Protein Binds the mAT2 Site in Vivo-- We have shown that the protein 2BB2 has binding requirements similar to those of a protein containing not only a homeodomain but also a POU-specific domain and that it is bound by an antibody against Oct-1. Moreover, we have shown using chimeric constructs in transfection experiments that the mAT2 element cis-activates the minimal IIB promoter in muscle and non-muscle cells. There is strong reason therefore to believe that the widely expressed POU domain protein Oct-1, or a closely related protein, is bound in vivo to the mAT2 element.
This protein is present in undifferentiated cells, in determined but not yet differentiated myogenic cells, in differentiated myogenic cells, in mature muscle fibers from rodent muscle, and in CV-1 cells. In addition to Oct-1, which is widely expressed among mammalian cells (19, 37), the alternatively spliced isoform Oct-1B has the same wide tissue distribution as Oct-1 (49). There is also evidence for other alternatively spliced isoforms of Oct-1, which exhibit some degree of tissue-restricted expression (50). Oct-1, despite its wide expression pattern, is involved in the cell-specific activation of several genes (51-56). Although we cannot formally attribute a function to the binding of Oct-1 to the mAT2 motif (discussed below), we suggest that Oct-1 could be involved in the specification of the myogenic phenotype, in conjunction with muscle-specific factors.The Homologous mAT1 and mAT2 Motifs Are Functionally Different-- Although our transfection data with a construct containing both mAT sites together support the idea of cooperative binding between the proteins interacting with these two sites, and clearly show that the mAT1 motif is the target of MEF-2, they do not allow us to tell whether Oct-1 or MEF-2 recognizes the mAT2 motif in mature myotubes. However, although in the context of the entire 192-bp of 5'-flanking sequence the respective roles of Oct-1 and MEF-2 were not sufficiently clear, isolation of the elements allowed us to conclude that, while MEF-2 can bind to mAT2, it is unlikely that MEF-2 occupies this site, at least up to the stage of early myotubes. The activities of the chimeric construct in non-myogenic and myogenic cells argue in favor of Oct-1 being bound to this element. Indeed, Oct-1 may normally out-compete MEF-2 for the mAT2 site in vivo. However, when Oct-1 is prevented from binding by the Oct-1 mutation, MEF-2 usurps its place and is actually better able to activate the promoter in the context of cultured myotubes.
Thus the two strikingly similar mAT motifs have distinct and separate binding proteins, MEF-2 on mAT1 and Oct-1 on mAT2. However, although our data with the entire IIB MyHC promoter clearly show that MEF-2 acts as an activator through the mAT1 site, we cannot rigorously determine the role of Oct-1. We suggest that the possibility of modulating the protein binding to the mAT2 site is essential for the tissue and/or temporal specificity of the transcription of the IIB MyHC gene. In non-muscle tissue or myoblasts, where the IIB MyHC gene is not expressed, the mAT sites could be occupied by the MHox-like proteins or by Oct-1; it is unlikely they are occupied by the more precociously expressed isoform MEF-2D, since there was no corresponding complex in EMSAs with myoblast nuclear proteins. In mature skeletal muscle, on the other hand, these sites are probably bound by Oct-1 and MEF-2. Our co-transfection experiments with MEF-2C and myogenin suggest that MEF-2 activates this muscle-specific promoter perhaps by interacting with a myogenic bHLH protein which would provide a functional activation domain, as in the model suggested by Molkentin et al. (57). MEF-2, bound to mAT1, could recruit or help Oct-1 bind to mAT2 (47, 58). Oct-1 itself has been shown to activate transcription by aiding the binding of other factors (54, 59). Oct-1 bound to mAT2 could thus improve the binding, interaction, and/or activation properties of MEF-2 or of other transcription factors bound elsewhere on the promoter, e.g. the CArG box at ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. Shin'ichi Takeda for discussion and assistance. We also thank Dr. Barbara Demeneix for the dioctadecyl amidoglycyl spermine; Dr. R. G. Roeder for the Oct-1 antibody; Dr. P. C. van der Vliet for the Oct-1 protein; Dr. E. Olson for the MEF-2C expression vector; and Prof. François Gros for continuous support.
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FOOTNOTES |
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* This work was supported by the Pasteur Institute, the CNRS, and by grants (to R. G. W.) from INSERM, the French Ministry of Research (to R. G. W., M. M. L., and D. L. N.), the Association Française Contre les Myopathies, and (to T. T. D.) from the Ministère de la Recherche et de la Technologie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Current address:
Molecular Biology and Vectorology Dept., Rhone-Poulenc Rorer-Gencell, 5301 Patrick Henry Dr., Santa Clara, CA 95054. E-mail:
melissa-lakich{at}rp.rorer.com.
§ Current address: Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Rd., La Jolla, CA 92037.
¶ Current address: 9003inc. Canada Corp., 181 Eglinton Ave. East, Suite 305, Toronto, Ontario, Canada M2P 1J4.
Current address: 332, rue Lecourbe, 75015 Paris, France.
1 The abbreviations used are: IIB MyHC, mouse adult IIB myosin heavy chain; EMSA, electrophoretic mobility shift assay; bHLH, basic helix-loop-helix; WT, wild type; mut., mutant; CAT, chloramphenicol acetyltransferase; MADS, MCM1-agamous-deficiens-serum response factor family of DNA-binding proteins.
2 M. Salminen, personal communication.
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REFERENCES |
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