Regulation of Myogenic Terminal Differentiation by the Hairy-related Transcription Factor CHF2*

Jianxin Sun, Caramai N. Kamei, Matthew D. Layne, Mukesh K. Jain, James K. Liao, Mu-En Leedagger, and Michael T. ChinDagger

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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). beta -Galactosidase assays were performed as described (23).

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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.


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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 beta -galactosidase reporter plasmid, pSVbeta 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 beta -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.

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.


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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. alpha -MyoD, antibody to MyoD; alpha -E2A, antibody to E2A proteins.

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.


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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.

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.


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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).

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).


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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

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.

    ACKNOWLEDGEMENT

We are grateful to Sunjay Kaushal for the MyoD expression plasmid pV2C11B.

    FOOTNOTES

* 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.

dagger Deceased April 10, 2000.

Dagger 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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