From the Cardiovascular Division, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, February 6, 2001, and in revised form, February 21, 2001
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ABSTRACT |
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We have recently cloned a novel basic
helix-loop-helix factor, CHF2, that functions as a transcriptional
repressor. To address its role in the regulation of myogenic terminal
differentiation, we analyzed its expression pattern during C2C12 mouse
myotube formation. In undifferentiated myoblasts, CHF2 is expressed at high levels. After induction of myotube formation in low serum, CHF2
expression is barely detectable at 3 days after induction. Myogenin
expression, in contrast, peaks at 3 days. In transiently transfected
10T1/2 embryonic fibroblasts, CHF2 inhibited MyoD-dependent activation of the myogenin promoter in a dose-dependent
fashion. Electrophoretic mobility shift analysis indicated that CHF2
inhibits the binding of the MyoD·E47 heterodimer to the E-box
binding site. CHF2 also inhibited myogenic conversion of 10T1/2 cells
by MyoD, as measured by skeletal myosin heavy chain protein expression. Coimmunoprecipitation analysis indicated that CHF2 forms a protein complex with MyoD. Mutational analysis of CHF2 indicated that the
repression activity for both transcription and myogenic conversion mapped to a hydrophobic carboxyl-terminal region and did not require either the basic helix-loop-helix or YRPW motifs. Our data indicate that CHF2 functions as a transcriptional repressor of myogenesis by
formation of an inactive heterodimeric complex with MyoD and likely
plays an important role in muscle development.
The hairy family of transcriptional repressor
bHLH1 proteins has been
implicated in controlling important developmental processes, including
neurogenesis and somitogenesis (reviewed in Ref. 1). These proteins are
thought to act as downstream effectors of Notch signaling,
during embryo pattern formation and lateral inhibition of neurogenic
and myogenic precursors. In the developing chick and zebrafish, hairy
homologues are expressed in dynamic oscillating patterns that
correlate with the generation of each somite (2, 3).
We have recently described two novel hairy-related bHLH proteins, CHF1
and CHF2, that function as transcriptional repressors and possibly
regulate terminal differentiation (4). Others have independently cloned
the same genes (referred to as Hey2, Hrt2, and gridlock for CHF1; Hey1,
Hrt1, and Hesr-1 for CHF2) and have described a transient expression
pattern in the developing somites and limb buds, suggesting a potential
role in skeletal muscle development (5-8). We have found that CHF2 is
expressed at high levels in undifferentiated myoblasts. Accordingly, we have examined the role of CHF2 in vitro with established
models of skeletal muscle differentiation.
Skeletal muscle ontogeny has been extensively studied. The bHLH muscle
regulatory factors MyoD, myogenin, Myf-5, and MRF4 are expressed
hierarchically during mouse embryo development and serve as nodal
regulators for myogenesis by turning on the transcription of a cascade
of skeletal muscle specific genes (reviewed in Ref. 9). Myogenesis is
also negatively regulated by other helix-loop-helix proteins that
function as transcriptional repressors, including Id (10), MyoR (11),
and I-mf (12). To date, little is known about how members of the hairy
family of transcriptional repressors act within the known hierarchy of
skeletal muscle regulatory factors.
The domain structure of hairy family members has been inferred from
structure-function studies. Most members of the hairy family of
transcriptional repressors have three functional domains. They contain
a distinctive bHLH domain, an Orange domain, and a WRPW motif at or
adjacent to the carboxyl terminus (reviewed in Ref. 1). The bHLH region
is distinct by virtue of a proline in the basic region that is thought
to affect the DNA binding specificity. Most of the hairy proteins bind
to class B E-boxes or N-boxes (13-15). The Orange domain mediates
interaction with specific transcriptional activators and is essential
for full transcriptional repression (16). The WRPW motif is critical for recruitment of members of the groucho family of WD40 repeat proteins, which act as transcriptional corepressors (17).
CHF1 and CHF2 are members of a novel subclass of hairy-related genes
that diverge from other hairy proteins by the substitution of glycine
for proline in the basic region and by the substitution of YRPW for
WRPW near the carboxyl terminus (4-7). To date, very little is known
about how CHF1, CHF2, and their relatives function in vivo,
although they are known to function in some contexts as transcriptional
repressors (4). Because of their relationship to other hairy proteins
and their expression pattern in the developing somites and limb buds,
we investigated the role of CHF1 and CHF2 in skeletal muscle
differentiation. We found that CHF1 is not expressed in proliferating
myoblasts, whereas CHF2 expression is high in myoblasts and is
dramatically reduced after induction of terminal differentiation, as
myogenin expression rises. We show that CHF1 and CHF2 act as repressors
of the myogenin promoter, most likely by preventing the binding of the
MyoD·E47 heterodimer to the E-box and by forming an inactive
heterodimer with MyoD. CHF1 and CHF2 also repress myogenic conversion
of 10T1/2 embryonic fibroblasts by MyoD, as measured by induction of
skeletal myosin heavy chain protein expression. Mutational analysis of
CHF2 demonstrated that the bHLH and the YRPW motifs are completely
dispensable for repression of both myogenin transcription and myogenic
conversion, suggesting that a previously undescribed alanine-rich
region is required. Our results suggest that CHF1 and CHF2 may regulate the expression of myogenin in the developing somites and limb buds and
thereby control the timing of myogenesis in vivo.
Construction of Plasmids--
The human CHF1 cDNA
expression construct, the human arylhydrocarbon receptor nuclear
translocator (ARNT) expression construct, and the Myc-tagged MyoD
expression construct pCS26MTMyoD have been described previously (4).
The myogenin core promoter/luciferase construct was created by
polymerase chain reaction (PCR) amplification of the 184-base pair
myogenin core promoter (18) from the template p184CAT with the primers
5'-CTCGAGCCTGCAGGGTGGGGTGGGGG-3' and 5'-AAGCTTCCCCCAAGCTCCCGCAGCCC-3'.
The PCR product was cloned directly into pCRII (Invitrogen, Carlsbad,
CA). This insert was subsequently excised with the restriction enzymes
XhoI and HindIII, and then cloned into the
XhoI and HindIII sites of the luciferase reporter plasmid pGL2basic (Promega, Madison, WI). The hCHF2 expression plasmid
pcDNA-hCHF2 was constructed by taking the full-length 2.2-kb
EcoRI/XhoI cDNA and cloning it into the
EcoRI and XhoI sites of pcDNA3 (Invitrogen).
The MyoD construct used as a template for in vitro
transcription and translation was made by excision of the MyoD cDNA
from pV2C11B (obtained from Sunjay Kaushal) with EcoRI and
cloning into the EcoRI site of pcDNA3. The human E47 expression construct was cloned by PCR amplification with
Pfu polymerase of the E47 cDNA (19) with the primers
5'-CGGGATCCAGGAGAATGAACCAGCCG-3' and 5' -CGGAATTCGGAGGCATACCTTTCACA-3',
followed by digestion with BamHI and EcoRI. The
fragment was then ligated into the BamHI and
EcoRI sites of pcDNA3.
The point mutant of hCHF2 and the deletion mutants of hCHF2 were
generated by oligonucleotide-directed mutagenesis of pcDNA-hCHF2 with the QuikChange kit according to the manufacturer's instructions (Stratagene, La Jolla, CA). To introduce stop signals at codons 112, 172, 232, and 292 in pcDNA-hCHF2, the primer pairs
5'-GCAGGAGGGAAAGGTTAATTTGACGCGCACGCCCTTGC-3' and
5'-GCAAGGGCGTGCGCGTCAAATTAACCTTTCCCTCCTGC-3',
5'-GAAGCCGCGAGCGGCGCCTAGGCGGGCCTCGGACACATTC-3' and
5'-GAATGTGTCCGAGGCCCGCCTAGGCGCCGCTCGCGGCTTC-3',
5'-GAGGCGCCTGCTTTCGAGCGTAACCTAGCGGCAGCCTCGGACCG-3' and
5'-CGGTCCGAGGCTGCCGCTAGGTTACGCTCGCAAAGCAGGCGCCTC-3',
5'-CAGGCTGCTGCAAACCTTGGCTAGCCCTATAGACCTTGG-3'and 5'-CCAAGGTCTATAGGGCTAGCCAAGGTTTGCAGCAGCCTG-3', were used, respectively. To generate the point mutation glycine to proline at codon 55, the
primer pair 5'-GCCAGAAAAAGACGGAGACCAATAATTGAGAAGCGCCGACGAGAC-3' and
5'GTCTCGTCGGCGCTTCTCAATTATTGGTCTCCGTCTTTTTCTGGC-3' was used. All
mutations were confirmed by DNA sequencing. To generate the amino-terminal deletion mutant pFLAG-CHF2(76-304), a 1.8-kb
KpnI-XbaI hCHF2 cDNA fragment was excised
from pcDNA-hCHF2 and cloned into the KpnI and
XbaI sites of pFLAGCMV2 (Sigma).
For coimmunoprecipitation experiments, the FLAG-tagged CHF2 construct
was generated by excision of the 2.1-kb AvaI fragment from
pcDNA-hCHF2 and treatment with the Klenow fragment of E. coli DNA polymerase I to generate blunt ends, followed by ligation to the Klenow-treated EcoRI site of pFLAGCMV2. This
construct encodes amino acids 7-304 downstream of the FLAG peptide.
RNA Preparation and Northern Blot Analysis--
Total RNA
was prepared from cultured C2C12 myoblasts and myotubes as described
(20). Northern blot analysis was performed as described (20). The probe
for mCHF2 consisted of a 2.2-kb full-length mouse EcoRI
cDNA fragment (4), while the probe for mouse myogenin has been
described previously (21).
Cell Culture, Transient Transfection, Luciferase, and
Electrophoretic Mobility Shift Assays (EMSAs)--
EMSAs with
in vitro transcribed and translated hCHF2, hCHF2, MyoD, and
E47 binding to the E-box were performed as described (4). In
vitro transcribed and translated hCHF1, hCHF2, MyoD, and E47 were
generated using the plasmids pcDNA-hCHF1, pcDNA-hCHF2, pcDNA-MyoD, and pcDNA-E47, respectively (see above). All
in vitro transcription and translation reactions were
verified by SDS-PAGE. The E-box oligonucleotides
5'-AGCTTCCAACACCTGCTGCAAGCT-3' and 5'-AGCTTGCAGCAGGTGTTGGAAGCT-3' and
the mutant E box oligonucleotides 5'-AGCTTCCAAGACCTGCTGCAAGCT-3' and
5'-AGCTTGCAGCAGGTCTTGGAAGCT-3' (for nonspecific competition) were
annealed and labeled as described previously (23). Supershift
antibodies for MyoD and E2A were obtained commercially (Santa Cruz
Biotechnology, Santa Cruz, CA) and used according to the
manufacturer's instructions.
Immunocytochemistry--
Glass coverslips were immersed in
ethanol, flame-sterilized, and placed in six-well trays. 10T1/2 cells
were plated and transfected as described above. After 24 h, the
culture medium was changed to Dulbecco's modified Eagle's medium
supplemented with 2% horse serum. After an additional 48 h, the
cells were washed in phosphate-buffered saline (PBS), fixed in 4%
paraformaldehyde/PBS for 15 min at room temperature, and permeabilized
with PBS, 0.1% Triton X-100 for 5 min at room temperature. The
cells were then incubated in PBS, 0.1% Triton X-100, 3% BSA for 20 min to decrease nonspecific binding. The cells were incubated next with
a mouse monoclonal antibody directed against skeletal myosin heavy
chain (MY-32, Sigma), at 1:1000 dilution in PBS, 0.1% Triton X-100,
3% BSA, for 1 h at room temperature. The cells were next washed
five times in PBS, 0.1% Triton X-100 and incubated with the
fluorescent secondary antibody ALEXA 546 anti-mouse (Molecular Probes,
Eugene, OR) at 1:200 dilution in PBS, 0.1% Triton X-100, 3% BSA for
1 h in the dark at room temperature. The cells were then washed
twice with PBS, 0.1% Triton X-100, stained with Hoechst 33258, and
washed an additional two times. The coverslips were then mounted on
glass slides with antifade solution (Molecular Probes). Myosin heavy chain-positive cells were detected with fluorescence microscopy, and
the number of positive cells was quantified by counting the number in
six consecutive 100× fields.
Coimmunoprecipitation--
COS7 cells were cotransfected with
either pFLAG-CHF2 and pCS26MTMyoD or pFLAG-CHF2 and pCS26MT (empty
vector) with FuGENE 6 as described above. After 48 h, cells were
lysed in ice-cold radioimmunoprecipitation assay buffer (20 mM sodium phosphate, pH 7.5, 140 mM NaCl, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate)
with protease and phosphatase inhibitors (4 mM sodium
fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml
phenylmethylsulfonyl fluoride, 0.1 mM sodium orthovanadate, and 10 µg/ml glycerol phosphate). Lysates were cleared by
centrifugation at 15,000 × g for 15-30 min. 2 µg of
9E10 anti-Myc monoclonal antibody was added to 1 ml of clarified lysate
containing 500 µg of total cellular protein and incubated overnight
at 4 °C with rotation. 25 µl of Protein G Plus-Agarose (Santa
Cruz) was added and incubated for 6 h at 4 °C. The
immunoprecipitates were collected by centrifugation followed by four
washes in radioimmunoprecipitation assay buffer. The pellets were then
resuspended in an equal volume of 1× SDS-PAGE sample buffer, boiled 5 min, and analyzed by SDS-PAGE. The separated proteins were then
electrophoretically transferred to nitrocellulose and subjected to
Western blotting with the M2 anti-FLAG antibody (Sigma) or the 9E10
anti-Myc antibody. Bands were visualized by incubation with horseradish
peroxidase-conjugated anti-mouse antibody (1:4000), followed by
enhanced chemiluminescence according to the manufacturer's protocol
(PerkinElmer Life Sciences).
Statistical Methods--
Groups were compared by one-way
analysis of variance with statistical significance set at a
p value of < 0.05. For reporter gene assays, all
groups involving cotransfection of CHF1, CHF2, or its derivatives were
compared with the MyoD-stimulated activity of the myogenin promoter in
the absence of CHF1 or CHF2. For myogenic conversion assays, a similar
comparison was performed.
Regulation of CHF2 Expression in C2C12 Cells--
To determine
whether CHF2 is regulated during myoblast to myotube differentiation,
we used the well characterized C2C12 myoblast in vitro
differentiation system (22). Under conditions of low serum and high
cell density, C2C12 myoblasts spontaneously fuse, show a dramatic
increase in myogenin expression, and express a variety of skeletal
muscle contractile proteins such as myosin heavy chain. Given the
potential role of CHF2 in muscle differentiation, we examined the time
course of CHF2 expression in differentiating muscle cells. As shown in
Fig. 1, CHF2 mRNA is easily
detectable in proliferating myoblasts. After induction of
differentiation, CHF2 expression decreases to nearly undetectable
levels by 3 days. Myogenin expression, in contrast, is markedly
increased, peaking 3 days after differentiation was induced. These
findings suggest that decreased expression of CHF2 may be required for
full induction of myogenin expression. We also examined the expression
of CHF1, but no signal was seen in either proliferating myoblasts or
differentiated myotubes (data not shown).
Regulation of Myogenin Promoter Activity by CHF1 and CHF2--
To
test the hypothesis that CHF2 negatively regulates the myogenin
promoter, we performed assays of myogenin promoter activity using a
luciferase reporter after transient transfection of various regulators
into 10T1/2 cells. We also tested the effect of CHF1, because of its
transient expression in somites and to address functional redundancy
with CHF2. As shown in Fig.
2A, cotransfection of MyoD
with the myogenin promoter led to an 8.5-fold induction of reporter
gene activity. When CHF1 was cotransfected with MyoD, this MyoD-induced
increase in promoter activity was reduced by 65%. When CHF2 was
cotransfected with MyoD, the MyoD-induced increase in promoter activity
was almost completely abolished. As a control, cotransfection of an
expression plasmid for the bHLH-Period ARNT-SIM domain protein
ARNT had no significant effect on myogenin promoter activity. These
findings suggest that CHF1 and CHF2 function as negative regulators of
myogenin expression. To verify that the effect on myogenin promoter
activity is dose-dependent, we transfected increasing doses
of CHF1 or CHF2, keeping total DNA constant by the addition of vector
DNA. As shown in Fig. 2B, the repression of myogenin
promoter activity by both CHF1 and CHF2 is
dose-dependent.
CHF1 and CHF2 Prevent Binding of MyoD·E47 to the E-box Binding
Site--
Binding of bHLH proteins to consensus E-box targets induces
myogenin promoter activity (18). In order to determine the mechanism by
which CHF1 and CHF2 inhibit myogenin promoter activation, we performed
EMSAs to determine the effect of CHF1 and CHF2 on the binding of the
MyoD·E47 heterodimer to the E-box binding site, as shown in Fig.
3. The control lanes demonstrate that no
binding activity is present in the reticulocyte lysate alone
(lane 1) and that MyoD does not bind as a
homodimer (lane 2), but that E47 does bind as a
homodimer (lane 3). MyoD and E47 together form a
heterodimer, as verified with supershift antibodies to MyoD and E47,
that prevent the complexes from entering the gel (lanes 4-6). When cotranslated CHF1 or CHF2 is present, the
MyoD·E47 complex is virtually abolished, and no new bands appear,
which suggests that these proteins prevent formation of a DNA-binding complex (lanes 7 and 8). In contrast,
cotranslation of the ARNT bHLH-Period ARNT-SIM domain protein
has no effect on the MyoD·E47 complex (lane 9).
To verify that this band represents a complex that binds specifically
to the E-box, unlabeled E-box oligonucleotides (specific competitor)
were also added to the reaction at a 20-fold molar excess and inhibited
formation of the labeled complex. In contrast, a nonspecific competitor
oligonucleotide containing a mutated E-box did not inhibit formation of
the observed complex (lane 10). These findings
suggest that the mechanism by which CHF1 and CHF2 repress
MyoD-dependent activation of the myogenin promoter is
inhibition of MyoD·E47 binding to the E-box.
CHF1 and CHF2 Inhibit Myogenic Conversion of 10T1/2 Cells--
To
verify that the repression of the myogenin promoter seen with reporter
gene assays is biologically relevant to myogenesis, we overexpressed
CHF1 and CHF2 in C2C12 myoblasts using adenovirus-mediated transfection. We infected myoblasts at a multiplicity of infection of
10 or 50 just prior to induction of differentiation. Unfortunately, the
control vector expressing only green fluorescent protein was a potent
inhibitor of myogenic terminal differentiation, as no cells that were
both myosin heavy chain-positive and green fluorescent protein-positive
could be identified (data not shown). We subsequently performed
transient transfection of 10T1/2 cells with a MyoD expression plasmid
and tested the effect of cotransfection of CHF1 and CHF2. As shown in
Fig. 4A, MyoD transfection
results in a significant number of myosin heavy chain-positive cells.
When CHF1 or CHF2 was cotransfected, virtually no myosin heavy
chain-positive cells are seen. Representative microscope fields for
each transfection are shown in Fig. 4B. These results
indicate that CHF1 and CHF2 can inhibit myogenesis, and are likely to
play an important role in skeletal muscle differentiation.
CHF2 Forms a Heterodimer with MyoD--
In order to elucidate
further the mechanisms by which CHF2 inhibits myogenesis,
coimmunoprecipitation experiments were performed. One potential
mechanism is CHF binding and sequestering MyoD·E2A. Another potential
mechanism would be by forming an inactive heterodimer with either MyoD
or E2A gene products (E12 or E47). Our previous study indicated that
CHF1 and CHF2 did not bind to E12 in a yeast two-hybrid assay (4).
Accordingly, we attempted to coimmunoprecipitate MyoD and CHF2, to test
the hypothesis that repression is mediated by inactive heterodimer
formation. As shown in Fig. 5,
immunoprecipitation of Myc-tagged MyoD leads to coimmunoprecipitation
of FLAG-tagged CHF2 when both proteins are cotransfected. As a control,
the anti-Myc antibody did not immunoprecipitate FLAG-CHF2 in the
absence of Myc-MyoD. These findings suggest that the mechanism of
repression involves formation of an inactive heterodimer with MyoD.
Mutation of CHF2 Abolishes Transcriptional Repression and
Repression of Myogenic Terminal Differentiation--
In order to
identify portions of the molecule that are critical for repression of
myogenin promoter activity and myogenic terminal differentiation, we
generated a series of mutations in CHF2. These mutations specifically
target the bHLH domain and the YRPW motif. In transient transfection
analysis, full-length CHF2 repressed MyoD-dependent
transcription, as shown in Fig. 6.
Deletion of the COOH-terminal 13 amino acids, including the YRPW motif,
had no effect on transcriptional repression. Further deletion of the
COOH-terminal 73 amino acids led to a sharp decrease in repressor
activity. Surprisingly, deletion of the amino-terminal 75 amino acids,
containing a portion of the bHLH domain, also had no effect on
transcriptional repression. Mutation of the glycine at position 55 to a
proline residue in CHF2 (Pro55) also did not significantly
affect the ability of the protein to repress transcription, indicating
that an intact bHLH domain is dispensable for transcriptional
repression. To test whether these mutations also affect myogenic
terminal differentiation, we transfected 10T1/2 cells, as shown in Fig.
7. As expected, mutations that affect
transcriptional repression also affect myogenic conversion. In summary,
our findings indicate that neither the YRPW motif nor the bHLH are
absolutely required for transcriptional repression or repression of
myogenic terminal differentiation, and a critical domain is located
between amino acids 76 and 291. Analysis of the deleted region between
amino acids 231 and 291 reveals no known structural motifs, however
this region is striking for its preponderance of alanine residues,
which predicts a markedly hydrophobic domain by Kyte-Doolittle
calculations (data not shown).
We have studied the functional role of the hairy-related bHLH
transcription factor CHF2 in myogenic terminal differentiation. Others
have reported previously that CHF2 and its relative CHF1 are expressed
during somitogenesis and in the limb buds, suggesting a potential role
in regulation of myogenesis (5-7). Our data indicate that CHF2
represses myogenic differentiation in vitro, suggesting that
it may serve a similar role during embryonic skeletal muscle development.
CHF1, CHF2, and their relatives form a novel subclass of hairy-related
bHLH proteins (4-8). Our work provides a systematic analysis of
structure and function, assesses the contribution of each domain to
transcriptional repression, and highlights functional divergence from
other hairy-related proteins. Most hairy family members have an
invariant proline within the basic region, whereas CHF1 and its
relatives have glycine in place of proline. Our mutational analysis was
designed to assess the importance of this glycine residue, by
substituting proline for glycine to make CHF2 more like other hairy
family proteins. As shown in Fig. 5, the mutation of glycine to proline
does not significantly affect the ability of the protein to function as
a transcriptional repressor, raising questions about the importance
of this residue.
In Drosophila hairy, the Orange domain, in conjunction with
the bHLH domain, is thought to mediate repression of specific transcriptional activators, such as Scute (16). Our mutational analysis
tested whether the CHF2 Orange domain plays a similar role in
conjunction with the bHLH domain to regulate myogenesis. As shown in
Fig. 5, the combination of the bHLH domain and the Orange was not
sufficient to mediate repression of myogenin promoter activity. These
findings indicate that the function of the Orange domain in CHF2 may
not parallel the function of the homologous domain in hairy.
In most hairy family members, the WRPW motif is critical for
interaction with members of the groucho family of transcriptional co-repressors (17). In CHF2 and its relatives, the WRPW motif is
replaced by the related sequence YRPW. Our data indicate that this
motif is dispensable for transcriptional repression, which suggests
that an interaction with groucho-related proteins is not required for
repression of myogenesis. Our mutational analysis represents the first
systematic assessment of each domain identified by protein homology,
and confirms that the structural divergence of CHF2 and its relatives
also leads to functional divergence.
At present, it is unclear whether CHF1 and CHF2 are functionally
distinct, since they behave similarly in vitro (Figs. 2-4
and data not shown). This functional overlap is surprising, given their
very different expression patterns. Targeted disruption of each gene in
mice will determine their contribution to muscle development and shed
insight into their functional divergence. Distinction of function
in vivo will further be enhanced by knock-in of each
cDNA into the locus of the other. These studies are ongoing.
The myogenic bHLH factors are among the most powerful tissue-restricted
transcription factors, by virtue of their ability to activate a
muscle-specific transcriptional program in a variety of cell types. A
number of myogenic repressors have previously been identified (10-12).
Negative regulators of the myogenic program are hypothesized to play
important roles in preventing the expression of a myogenic program in
inappropriate embryonic tissues or at inappropriate times during
development. The mechanisms by which they repress the activity of
myogenic bHLH factors include formation of heterodimers that do not
bind DNA (10, 12), by masking their nuclear localization signal (12),
and by formation of heterodimers that bind DNA but are
transcriptionally inactive (11). Of the hairy family members studied,
HES-1 has been shown to inhibit in vitro myogenesis by
inhibition of DNA binding (13). Our data indicate that CHF2 functions
similarly to HES-1 by inhibiting binding of the MyoD·E2A heterodimer
to DNA. Furthermore, we show that CHF2 forms a heterodimer with MyoD
that does not bind to the E-box, in a manner analogous to the Id
protein (10). It remains possible that the CHF2·MyoD complex may bind
to an as yet undetermined DNA sequence, and this hypothesis is under
active investigation.
The regulation of myogenic bHLH protein function and myogenic
differentiation in general is highly complex. The MADS box protein MEF2C has been shown to activate the myogenin promoter and also interacts directly with myogenic bHLH proteins to synergistically activate muscle-specific genes (18, 24). The bHLH factor Twist has been
shown to function as a negative regulator of myogenesis through
interactions with both myogenic bHLH proteins and MEF2C (25). In
addition, activation of the myogenic program is enhanced by the
transcriptional coactivator p300 (26). The function of CHF2 within this
regulatory network is currently under study.
Our previous work describing the cloning of CHF1 and CHF2 demonstrated
that CHF1 functions as a transcriptional repressor and may control the
timing of terminal differentiation in ventricular cardiac myocytes (4),
by virtue of its expression in the developing but not mature ventricle.
Our current work indicates that CHF2 is likely to play an important
role in controlling the timing of terminal differentiation of skeletal
muscle. Interestingly, CHF1 expression persists in adult aortic smooth
muscle cells (4), which are neither striated nor terminally
differentiated. It is tempting to speculate that CHF1 and CHF2 may
function in general as molecular regulators of terminal differentiation
in muscle tissue, where persistent expression leads to a proliferative, non-terminally differentiated phenotype. This hypothesis is subject to
ongoing investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Galactosidase Assays--
C2C12 myoblasts were cultured in growth
medium and induced to differentiate under low serum conditions as
described (22). 10T1/2 cells were cultured in basal Eagle's medium
containing 10% fetal bovine serum. Cells were plated onto six-well
trays 1 day prior to transfection and were transfected at 70%
confluence with the indicated plasmids mixed with FuGENE 6 transfection
reagent according to the manufacturer's protocol (Roche Molecular
Biochemicals). Total DNA transfected per well was kept constant at 2 µg by adding vector DNA as needed. After 48 h, cells were
harvested with passive lysis buffer and luciferase assays were
performed according to the manufacturer's instructions (Promega).
-Galactosidase assays were performed as described (23).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
CHF2 is down-regulated during myoblast to
myotube differentiation. C2C12 myoblasts were cultured and induced
to differentiate into myotubes as described under "Experimental
Procedures." Total RNA (10 µg) from each time point was
fractionated on MOPS-formaldehyde 1.3% agarose gels and transferred to
nitrocellulose. The blot was probed with a 32P-labeled
2.2-kb cDNA fragment containing the entire coding region of mouse
CHF2 and a mouse cDNA for mouse myogenin. To control for loading,
the blot was also probed with an oligonucleotide complementary to 28 S
ribosomal RNA.
View larger version (20K):
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Fig. 2.
CHF1 and CHF2 negatively regulate the
myogenin promoter. A, 10T1/2 cells were cotransfected
with the 184-base pair myogenin promoter/luciferase reporter plasmid
(0.3 µg) with MyoD (0.3 µg) as indicated. CHF1, CHF2, and ARNT
expression plasmids (0.9 µg) were cotransfected as shown. Total DNA
was kept constant by the addition of the appropriate amounts of
pcDNA3. For all transfections, an SV40 promoter-driven
-galactosidase reporter plasmid, pSV
gal (0.25 µg; Promega), was
included to correct for differences in transfection efficiency. All
transfections were performed in triplicate at least three times.
Transfection efficiencies as measured by
-galactosidase activity
were similar in all experiments. Myogenin promoter activity is
presented in relative units normalized to basal activity of the
myogenin promoter (mean ± S.E.). Asterisk (*) denotes
statistical significance when compared with cells transfected with MyoD
alone. B, repression of the myogenin promoter by CHF1 and
CHF2 is dose-dependent. 10T1/2 cells were transfected with
the myogenin promoter and MyoD expression plasmids along with varying
amounts of pcDNA-CHF1 or pcDNA-CHF2 (0, 0.15, 0.3, 0.45, 0.6, and 0.9 µg). Total DNA was kept constant by the addition of
appropriate amounts of pcDNA3 vector.
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Fig. 3.
CHF1 and CHF2 inhibit binding of the
MyoD·E47 heterodimer to the E-box binding site. The indicated
proteins were expressed by in vitro transcription and
translation and incubated with radiolabeled E-box oligonucleotides as
described under "Experimental Procedures." Protein-DNA complexes
were separated on 4% low ionic strength, nondenaturing gels, dried,
and exposed to film. SC, specific competitor
oligonucleotide; NSC, nonspecific competitor
oligonucleotide. -MyoD, antibody to MyoD;
-E2A, antibody to E2A proteins.
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Fig. 4.
CHF1 and CHF2 inhibit myogenic conversion of
10T1/2 cells. 10T1/2 cells were cotransfected with MyoD
(0.5 µg) and pcDNA, pcDNA-CHF1, or pcDNA-CHF2 (1.5 µg).
Immunostaining and nuclear counterstaining with Hoechst 33258 was
performed as described under "Experimental Procedures."
A, total number of myosin heavy chain-positive cells after
transfection as counted in six 100× fields. Each transfection was
repeated at least three times, and the total number is presented as the
mean ± S.E. B, representative microscope fields after
myosin heavy chain (MHC) staining. Original magnification,
×400. Asterisk (*) denotes statistical significance when
compared with cells transfected with MyoD alone.
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Fig. 5.
CHF2 coimmunoprecipitates with MyoD.
COS-7 cells were cotransfected with FLAG-CHF2 and either Myc-MyoD or
Myc tag expression vector alone, as described under "Experimental
Procedures." Lysates were prepared and immunoprecipitations
(IP) were performed and analyzed as described under
"Experimental Procedures." Total cell lysates were analyzed before
coimmunoprecipitation to verify expression of FLAG-CHF2 and Myc-MyoD
(lanes 1 and 2). After
coimmunoprecipitation with antibody to the Myc tag, FLAG-CHF2 was found
only in the presence of Myc-MyoD (lanes 3 and
4).
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Fig. 6.
Identification of CHF2 domains required for
transcriptional repression. Mutations were generated as described
under "Experimental Procedures." Transient transfections were
performed as described under "Experimental Procedures" and in the
legend to Fig. 2. 10T1/2 cells were transfected with 0.3 µg of
reporter, 0.3 µg of MyoD as indicated, and 0.9 µg of CHF2 wild type
and mutant expression plasmids as indicated. All transfections were
performed in triplicate at least three times. Relative myogenin
promoter activity is presented in arbitrary units (mean ± S.E.).
Asterisk (*) denotes statistical significance when compared
with cells transfected with MyoD alone.
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Fig. 7.
Identification of CHF2 domains required for
repression of myogenic terminal differentiation. Mutations were
generated as described under "Experimental Procedures." Transient
transfections and immunostaining for skeletal myosin heavy chain
(MHC) were performed as described under "Experimental
Procedures" and in the legend to Fig. 4. Asterisk (*)
denotes statistical significance when compared with cells transfected
with MyoD alone.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We are grateful to Sunjay Kaushal for the MyoD expression plasmid pV2C11B.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL 03745 (to M. T. C.), HL 57664 (to M.-E. L.), HL 03747 (to M. K. J.), HL 10113 (to M. D. L.), and HL 48743 (to J. K. L.).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: Vascular
Medicine Unit, Brigham and Women's Hospital, 221 Longwood Ave.,
Boston, MA 02115. Tel.: 617-732-7604; Fax: 617-264-5253; E-mail:
mchin@rics.bwh.harvard.edu.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M101163200
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ABBREVIATIONS |
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The abbreviations used are: bHLH, basic helix-loop-helix; CHF1, cardiovascular basic helix-loop-helix factor 1; CHF2, cardiovascular basic helix-loop-helix factor 2; PCR, polymerase chain reaction; kb, kilobase pair(s); EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BSA, bovine serum albumin; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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