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Originally published In Press as doi:10.1074/jbc.M611513200 on January 22, 2007

J. Biol. Chem., Vol. 282, Issue 12, 9017-9028, March 23, 2007
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TBP-interacting Protein 120B (TIP120B)/Cullin-associated and Neddylation-dissociated 2 (CAND2) Inhibits SCF-dependent Ubiquitination of Myogenin and Accelerates Myogenic Differentiation*Formula

Seiji Shiraishi{ddagger}, Chang Zhou{ddagger}, Tsutomu Aoki{ddagger}1, Naruki Sato{ddagger}, Tomoki Chiba§, Keiji Tanaka§, Shosei Yoshida, Yoko Nabeshima, Yo-ichi Nabeshima, and Taka-aki Tamura{ddagger}2

From the {ddagger}Department of Biology, Faculty of Science, Chiba University, 1-33 Yayoicho, Inage-ku, Chiba 263-8522, Japan, §The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan, and Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan

Received for publication, December 15, 2006 , and in revised form, January 22, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite fast protein degradation in muscles, protein concentrations remain constant during differentiation and maintenance of muscle tissues. Myogenin, a basic helix-loop-helix-type myogenic transcription factor, plays a critical role through transcriptional activation in myogenesis as well as muscle maintenance. TBP-interacting protein 120/cullin-associated neddylation-dissociated (TIP120/CAND) is known to bind to cullin and negatively regulate SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase, although its physiological role has not been elucidated. We have identified a muscle-specific isoform of TIP120, named TIP120B/CAND2. In this study, we found that TIP120B is not only induced in association with myogenic differentiation but also actively accelerates the myogenic differentiation of C2C12 cells. Although myogenin is a short lived protein and is degraded by a ubiquitin-proteasome system, TIP120B suppressed its ubiquitination and subsequent degradation of myogenin. TIP120B bound to cullin family proteins, especially Cullin 1 (CUL1), and was associated with SCF complex in cells. It was demonstrated that myogenin was also associated with SCF and that CUL1 small interference RNA treatment inhibited ubiquitination of myogenin and stabilized it. TIP120B was found to break down the SCF-myogenin complex. Consequently suppression of SCF-dependent ubiquitination of myogenin by TIP120B, which leads to stabilization of myogenin, can account for the TIP120B-directed accelerated differentiation of C2C12 cells. TIP120B is proposed to be a novel regulator for myogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transcription factors play major roles in development and differentiation of multicellular organisms through regulation of target genes in temporally and spatially specific ways. To understand the cell fate-determining pathway, it is essential to clarify how the amount, activity, and localization of relating transcription factors are regulated. Myogenesis is governed critically by a set of myogenic regulatory factors (MRFs)3 that belong to the basic helix-loop-helix family of proteins (1). Tissue-specific basic helix-loop-helix proteins form heterodimers with ubiquitous E-proteins such as E47 and bind to an E-box motif (CANNTG) followed by subsequent transcriptional activation of the target gene (2). They include structurally similar MyoD, myogenin, Myf5, and MRF4. Genetic and developmental studies have revealed that MyoD and Myf5, referred to as early genes, have complementary roles and are required for genesis of myoblasts (3). Either gene alone allows normal myogenesis, whereas knock-out of both genes results in failure of normal myoblast generation and subsequent muscle formation (3). On the other hand, myogenin and MRF4, referred to as intermediate genes, participate in the middle stage of myogenesis (i.e. myotube formation from myoblasts). Gene expression of these two factors does not occur in myoblasts but is induced during myotube differentiation and persists in matured muscles (4, 5). Embryos of myogenin knock-out mice exhibit aberrant myotube formation followed by neonatal death (6, 7). Targeting of myogenin and MRF4 genes does not produce the same phenotype although they have some common roles, implying that these two factors have specific roles in myogenesis in addition to a common role (8, 9). Terminal differentiation of muscle, during which muscle-specific proteins such as myosin heavy chain (MHC) and troponinT, referred to as late genes, are induced, is attributed to temporally specific expression of these two factors in myoblasts in addition to MyoD and Myf5.

It is well known that a large number of transcription factors participating in cell dynamics such as cell cycle regulation, checkpoint, apoptosis, stimulus response, and differentiation are degraded rapidly through the ubiquitin-proteasome pathway. In general, spatially and temporally specific transcription factors in addition to stress-stimulated factors are sensitive to proteolytic degradation. Proteasome-dependent rapid degradation of those transcription factors enables cells to respond to precise and quick gene expression and subsequent cell status alteration. Among MRF family proteins, it is known that MyoD is rapidly degraded by the ubiquitin-proteasome system with a half-life of ~60 min (10, 11). In the case of MyoD ubiquitination, UbcH3/CDC34 serves as an E2 ubiquitin conjugation enzyme (12). MyoD is lysine-independently ubiquitinated by a mechanism called "N-end rule" (13). Furthermore the 133rd lysine of MyoD is ubiquitinated by SCFMAFbx complex (14, 15). MAFbx/Atrogin1 is a muscle-specific F-box protein (16). Degradation of MyoD is stimulated by hypoxia and tumor necrosis factor-{alpha} and results in inhibition of myogenic differentiation of C2C12 myoblastic cells (17, 18). A mutant MyoD that has a prolonged half-life exhibits increased activity in transcription stimulation and in vitro myogenesis (12, 19). Taken together, these findings imply that stability of MRFs is involved in myogenesis, although little is known about the mechanism by which the proteolysis of MRFs is regulated in a physiological condition.

TBP-interacting protein 120 (TIP120)/cullin-associated and neddylation-dissociated (CAND) was originally identified as a TATA-binding protein (TBP)-interacting protein by our group (20). The TIP120 family includes TIP120A/CAND1 and TIP120B/CAND2. TIP120A facilitates transcription driven by all classes of RNA polymerases (21, 22). On the other hand, TIP120A has also been identified as a protein that directly binds to cullin family proteins (23–25). Cullins associate with several proteins and work as E3 ubiquitin ligases (26). Cullin 1 (CUL1) forms an SCF complex consisting of Skp1, CUL1 (+Rbx1/Roc1), and F-box protein (27, 28). In the complex, a specific lysine in the C terminus of CUL1 is covalently modified with Nedd8/Rub1, which facilitates association of CUL1 with E2 enzyme and subsequently stimulates the ligation activity (29, 30). TIP120A binds to un-neddylated CUL1 and inhibits the assembly of CUL1-associating adaptor proteins (24, 25). X-ray crystallography of the CUL1-TIP120A complex revealed that the neddylated 720th lysine of CUL1 is a critical TIP120A-binding site (31). This evidence suggests that association of CUL1 with Nedd8 and TIP120A is competitive and that TIP120A negatively regulates the enzyme activity of the SCF complex (32). At the present time, however, we do not know what kind of physiological event TIP120A regulates.

TIP120B/CAND2, which is another member of the TIP120 family of proteins, has 1235 amino acids and 60% identity with TIP120A (supplemental Fig. 1) (33). Unlike TIP120A, however, TIP120B has been identified only in mammals. Moreover TIP120B gene is expressed restrictedly in skeletal muscles and cardiac muscles, whereas TIP120A is expressed ubiquitously (33). In a previous work, we found that TIP120B is induced in association with differentiation of C2C12 cells (34). High expression levels of TIP120B mRNA were detected in limb buds of day 14 mouse embryos where muscle tissues are actively forming. These findings led to a hypothesis that TIP120B is involved in myogenesis. It has been demonstrated that both termini of TIP120A interact with CUL1 (31). We reported previously that both termini of TIP120A are required for transcription stimulation function (22). Moreover structures of these regions of TIP120 proteins are conserved between paralogues (i.e. TIP120A and TIP120B) as well as among orthologues (i.e. among TIP120As of various organisms) (supplemental Fig. 1). Accumulating findings as described above suggest that TIP120B regulates muscle-specific proteins by affecting their ubiquitination.

In this study, we found that TIP120B positively regulates myogenesis of C2C12 cells through suppression of myogenin degradation. Myogenin is ubiquitinated by SCF complex, and binding of TIP120B to CUL1 leads to dissociation of the SCF-myogenin complex and subsequent inhibition of ubiquitin-proteasome-dependent myogenin degradation. Results of this study suggest that TIP120B accelerates the differentiation from myoblasts to myotubes through suppression of ubiquitination of myogenin.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfection—Mouse myoblast C2C12 cells were cultured in growth medium (GM) consisting of Dulbecco's modified Eagle's medium with high glucose (Sigma), 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin under 5% CO2 at 37 °C. To induce myogenic differentiation, 80–100% confluent cells in GM were washed in phosphate-buffered saline (PBS), and the medium was replaced with differentiation medium (DM) consisting of Dulbecco's modified Eagle's medium with high glucose, 2% horse serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The medium was exchanged every day. Mouse fibroblast C3H10T1/2 cells were maintained in Dulbecco's modified Eagle's medium with low glucose (Sigma). These cells can differentiate into myotubes by ectopic expression of MyoD in the DM. For transfection, cells were transfected with the indicated combination of expression plasmids (0.5–2.0 µg/35-mm dish) using Lipofectamine and PLUS reagent (Invitrogen) according to the manufacturer's instructions.

RNA Interference—Small interference RNA (siRNA) oligonucleotides were prepared with a Silencer siRNA Construction kit (Ambion) according to the manufacturer's instructions. Sequences for targeting the mouse TIP120B/CAND2 and CUL1 were 5'-AAGGTGAAGGAGTACCAAGTG and 5'-AATAGACATTGGGTTCGCCGT, respectively. siRNA for glyceraldehyde-3-phosphate dehydrogenase as a negative control was supplied as a reagent in the kit. Two 29-mer oligodeoxynucleotides for TIP120B/CAND2 (sense, 5'-AACACTTGGTACTCCTTCACCCCTGTCTC; and antisense, 5'-AAGGTGAAGGAGTACCAAGTGCCTGTCTC) and for CUL1 (sense, 5'-AAACGGCGAACCCAATGTCTACCTGTCTC; and antisense, 5'-AATAGACATTGGGTTCGCCGTCCTGTCTC) contain corresponding siRNA sequences (underlined) and flanking sequences complementary to the T7 primer. Templates for siRNA were hybridized with the primer sequence and extended with the Klenow enzyme. The extended sense and corresponding antisense siRNA templates were transcribed with T7 RNA polymerase, and transcripts were hybridized to generate double-stranded siRNA according to the manufacturer's instructions. Cells were transfected with 100 nM siRNA for each gene using Lipofectamine and PLUS reagent.

Antibodies and Plasmids—Rabbit polyclonal antibodies against TIP120B (33) and TIP120A (20) and a mouse monoclonal antibody against MHC (MF20) (35) were described previously. Antibodies against FLAG (M2 and M5; Sigma), influenza virus hemagglutinin (HA) (12CA5; Roche Applied Science), CUL1 (Zymed Laboratories Inc.), CUL2, CUL3, Skp1 (Rockland), CUL4, glutathione S-transferase (GST) (Santa Cruz Biotechnology), troponinT, beta-actin (Sigma), glyceraldehyde-3-phosphate dehydrogenase (Ambion), and myogenin (Pharmingen) were commercial products.

pcDNA for N-terminally FLAG-tagged TIP120B/CAND2 (FLAG-TIP120B/TIP120B FL) was described previously (36). TIP120B {Delta}C and TIP120B {Delta}N are derivatives of TIP120B FL that contain TIP120B amino acids from 1 to 1009 and 330 to 1235, respectively. N-terminally HA-tagged TIP120B expression plasmid (HA-TIP120B/HA-TIP120B FL) was inserted into pcDNA (Invitrogen). TIP120B portions in HA-TIP120B {Delta}C and TIP120B {Delta}N are the same as those in TIP120B {Delta}C and TIP120B {Delta}N, respectively. Mouse myogenin cDNA (pEMSV-myogenin) was a gift from Dr. A. Asakura (University of Minnesota). pcDNA for FLAG-tagged CUL1 (FLAG-CUL1) was a gift from Dr. J. B. Yoon (University of Yonsei). To construct plasmids for N-terminally FLAG-, HA-, green fluorescent protein (GFP)-, and GST-tagged proteins, cDNAs were amplified by PCR with appropriate primers or cleaved with restriction enzymes followed by ligation into pcDNA (Invitrogen), pGEX4T-1 (GE Healthcare), or pEGFP-N1 (Clontech) vector. Expression plasmids for MyoD (pcDNA-MyoD) (37) and HA-tagged ubiquitin (pcDNA3.1-HA-Ub) (38) have been described elsewhere.

Immunofluorescence Staining—Cells on a coverslip were washed three times with PBS. Cells were fixed with 4% paraformaldehyde for 10 min, washed three times with PBS, and permeabilized with 0.5% Triton X-100 for 5 min. For blocking, cells were incubated with 1% bovine serum albumin in PBS at 37 °C for 1 h and then incubated with a primary antibody at 37 °C for 1 h. Cells were washed three times with PBS and incubated with fluorescein isothiocyanate isomer or Texas Red-conjugated anti-mouse IgG or anti-rabbit IgG antibody at 37 °C for 1 h. For nucleus staining, cells were washed three times with PBS, incubated with PBS containing diamidino-2-phenylindole (1 µg/ml; Sigma) for 1 min, and mounted on a slide glass.

Preparation of Whole Cell Extracts and Immunoblotting— Preparation of whole cell extracts was described previously (39). Briefly cells were lysed with buffer C (50 mM HEPES-KOH (pH 7.8), 420 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, 0.1% Nonidet P-40, 20% glycerol) supplemented with a protease inhibitor (PI) mixture (1 mM benzamidine HCl, 1 µg/ml pepstatin A, 0.5 mM phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin). Cell lysate was centrifuged at 13,000 rpm for 20 min at 4 °C. The supernatant fraction was collected as a whole cell extract. Protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce). For immunoblotting, 5–30 µg of proteins were separated by SDS-PAGE and transferred onto an Immobilon-P membrane (Millipore). Proteins on the membrane were incubated with a primary antibody as described in figure legends and incubated with horseradish peroxidase-conjugated antibodies against rat IgG (Santa Cruz Biotechnology), mouse IgG, or rabbit IgG (Cell Signaling Technology). The immunological reaction was detected with ECL-Plus immunoblot detection reagent (GE Healthcare).

Immunoprecipitation—For immunoprecipitation, cells were lysed with TNE buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 10% glycerol) containing 1.0% Triton X-100 and the PI mixture. Lysates were passed through a 26-gauge needle 10 times and centrifuged at 13,000 rpm at 4 °C for 20 min. The whole cell extracts were incubated with protein A-Sepharose beads (GE Healthcare) at 4 °C for 1 h and then immunoprecipitated with M2-agarose beads (Sigma) at 4 °C for 4 h. In the experiments for which results are shown in Figs. 4C and 7D, anti-TIP120B antibody, anti-Skp1 antibody, or control rabbit IgG (Santa Cruz Biotechnology) was added and incubated at 4 °C for 2 h. Protein A-Sepharose beads were added and incubated for 2 h at 4 °C. Beads were washed four times with TNE buffer containing 0.5% Triton X-100. Immunocomplexes were eluted with SDS sample buffer by boiling for 5 min, and proteins were detected by immunoblotting with a corresponding antibody.

Determination of Half-life of Myogenin—C2C12 cells (1.5 x 105 cells/well) were plated onto a 6-well plate. The following day, cells were transfected with 1.5 µg of FLAG-TIP120B or 100 nM TIP120B siRNA. Twenty-four hours after transfection, cells were cultured in DM for 24 h. After cells has been treated with cycloheximide (Sigma) at 50 µg/ml for the indicated times and harvested, myogenin in the lysate was detected by immunoblotting.

Purification of Bacterially Expressed Proteins and Pulldown Assay—Mouse myogenin cDNA was subcloned into pGEX4T-1 vector (GE Healthcare). All constructs were introduced into Escherichia coli BL21 (DE3) (Stratagene). Expression of GST-myogenin in E. coli was performed according to the manufacturer's instructions. GST-myogenin was purified by using glutathione-agarose beads (Clontech) according to the manufacturer's instructions. Recombinant proteins were finally dialyzed against an appropriate buffer before use.

For GST pulldown assay, C2C12 cells were lysed with TNE buffer containing 1.0% Triton X-100, and the protein concentration was determined. Whole cell extracts (1.5 mg) were incubated with 170 pmol of GST-myogenin and mixed with glutathione-agarose beads at 4 °C for 4 h. Beads were washed four times with TNE buffer containing 0.1% Triton X-100. Adsorbed proteins were eluted with the SDS sample buffer by boiling for 5 min and were detected by immunoblotting.

In Vivo Ubiquitination Assay—To detect in vivo ubiquitination of myogenin, C2C12 cells (1.0 x 106 cells) were plated onto a 100-mm dish. The next day, cells were transfected with various combinations of plasmids and siRNA as described in figure legends. Twenty-four or 36 h after transfection, cells were treated with 25 µM MG132 (Biomol) for 4 h. Cells were collected, lysed with 500 µl of RIPA buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing 20 mM N-ethylmaleimide and the PI mixture, and subsequently immunoprecipitated with M2-agarose beads at 4 °C for 4 h. Beads were washed four times with the RIPA buffer. To elute proteins from beads, beads were incubated with the RIPA buffer supplemented with FLAG peptide (0.3 mg/ml) at 4 °C for 1 h. After boiling for 5 min in the SDS sampling buffer, ubiquitinated myogenin was detected by immunoblotting.


Figure 1
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  		FIGURE 1.
Induction of TIP120B/CAND2 protein in C2C12 cells during myogenic differentiation. A, amounts of various proteins in C2C12 cells were determined by immunoblotting (IB). Whole cell extracts of undifferentiated (lane 1) and differentiated cells (differentiation period, day 1 to day 6; lanes 2–7) were analyzed for TIP120B/CAND2, TIP120A/CAND1, MHC, and beta-actin. B, detection of TIP120B in differentiated cells by immunostaining using anti ({alpha})-TIP120B antibody. Cells were cultured in GM (Day 0) or in DM for 1 day (Day 1) and 3 days (Day 3). C and D, localization of TIP120B and myogenin at differentiation day 1(C) and localization of MHC on differentiation day 3 (D). DNA was stained with diamidino-2-phenylindole (DAPI). E, localization of TIP120B in differentiated C3H10T1/2 cells. Mouse embryonic fibroblast C3H10T1/2 cells were transfected with MyoD expression plasmid. Twenty-four hours after transfection, cells were cultured in DM for 5 days and immunostained with anti-TIP120B and anti-troponinT antibodies.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Myogenic Differentiation of C2C12 Cells Associated with Expression of TIP120B/CAND2 Gene—We previously demonstrated that TIP120A/CAND1 is expressed ubiquitously in mouse tissues, whereas its paralogue TIP120B/CAND2 is expressed restrictedly in cardiac muscle and skeletal muscle (33, 34). Moreover we found that TIP120B mRNA and protein are induced in C2C12 cells when myoblasts have differentiated into myotubes (34). In this study, we first investigated in detail the induction profile of TIP120B protein by immunostaining and immunoblotting. C2C12 myoblast cells differentiate into myotubes when a growth medium is changed to a serum-limited differentiation medium. Although only a small amount of TIP120B was detected in undifferentiated cells, the differentiation treatment considerably increased TIP120B in cells soon after the medium change, whereas the amount of TIP120A was unchanged regardless of the differentiation status (Fig. 1A). Immunostaining assay provided the same results (Fig. 1B). The differentiated cells having a spindle shape restrictedly expressed TIP120B (Fig. 1B). Muscle-specific MHC was distinctly induced on differentiation day 3.

We analyzed the localization of TIP120B. Consistent with our previous finding using HeLa cells, TIP120A was constitutively expressed and concentrated in the nucleus of C2C12 cells regardless of the differentiation status (data not shown). TIP120B exists both in the nucleus and cytoplasm in differentiating C2C12 cells (Fig. 1, C and D). Moreover it was clearly shown that TIP120B-expressing cells coincided with those containing myogenin in the nucleus (Fig. 1C) and MHC in the cytoplasm (Fig. 1D). TIP120B was detected in myogenin-positive and single nucleus-containing cells as well as multiple nuclei-containing typical myotubes (Fig. 1, C and D). C3H10T1/2 cells that had been transfected with MyoD expression plasmid and had differentiated into myotubes (i.e. troponinT-positive fusion cells) also expressed a high level of TIP120B (Fig. 1E). Accordingly it was demonstrated that TIP120B is a myotube-specific protein and appears from the initial stage of the differentiation process.

TIP120B Is Involved in the Differentiation—The results described above raise the possibility that TIP120B itself is positively involved in in vitro myogenesis. TIP120B was overexpressed in C2C12 cells just before the cells were subjected to the differentiation protocol, and amounts of various proteins were determined 48 h after the differentiation treatment. It was found that the amount of troponinT also increased (~3-fold) depending on the amount of exogenous TIP120B (Fig. 2A). Myogenin also increased slightly but significantly (1.5-fold) (Fig. 2A). Immunostaining also provided consistent data because TIP120B overexpression increased 1.8-fold in troponinT-positive myotubes (Fig. 2B). We constructed N-terminal and C-terminal deletion versions of TP120B and examined their roles in myogenic differentiation by immunoblotting. Both deletions impaired the regulatory activity of TIP120B because overexpression of these mutants did not change the level of troponinT and myogenin in 48-h differentiation treatment.


Figure 2
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  		FIGURE 2.
Acceleration of myogenic differentiation of C2C12 cells by TIP120B. A, amounts of MHC and myogenin in TIP120B-overexpressing cells. Cells were transfected with 1.0 µg of vacant plasmid (lane 1)or0.5 (lane 2) and 1.0 µg(lane 3) of FLAG-TIP120B expression plasmids. Twenty-four hours after transfection, cells were cultured in DM for 48 h, and proteins in whole cell extracts were detected by immunoblotting. Arrowhead, FLAG-specific band. B, enhanced expression of troponinT in TIP120B-overexpressing cells. Cells were transfected with 1.0 µg of vacant plasmid (open column) and TIP120B expression plasmid (solid column) and cultured in DM for 48 h. TroponinT was detected by immunostaining (a), and the proportion of troponinT-expressing cells obtained from three independent experiments is shown (b). C, effect of N- or C-terminal domain of TIP120B on the accelerated myogenic differentiation of C2C12 cells. Cells were transfected with 1.5 µgof vacant plasmid (lane 1), TIP120B FL (FL; lane 2), TIP120B {Delta}C({Delta}C; lane 3), or TIP120B {Delta}N({Delta}N; lane 4) and analyzed for troponinT, myogenin, beta-actin, and exogenous TIP120B proteins (arrowheads)(a). Relative band intensities of myogenin and troponinT obtained from several experiments are shown (b). {alpha}-, anti-; IB, immunoblotting; DAPI, diamidino-2-phenylindole.

 


Figure 3
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  		FIGURE 3.
Delay in myogenic differentiation by TIP120B knockdown. A, effect of TIP120B knockdown on muscle-specific proteins. C2C12 cells were transfected with 100 nM TIP120B siRNA or control siRNA. Twenty-four hours after transfection, cells were cultured in DM for 1, 3, and 5 days, and amounts of various proteins indicated were determined. B and C, cells were transfected with TIP120B siRNA (gray column) or control siRNA (solid column). Twenty-four hours after transfection, cells were cultured in DM for 24 (B) and 48 h (C) and immunostained with antibodies against myogenin (B) and troponinT (C)(a). Open column, mock-transfected cells. Relative proportions of cells expressing myogenin (B) and troponinT (C) are shown (b). {alpha}-, anti-; IB, immunoblotting; DAPI, diamidino-2-phenylindole.

 
The TIP120B-stimulated differentiation was investigated by siRNA-mediated knockdown experiments. Small interference RNA for TIP120B used in this study interfered with TIP120B but not with TIP120A (data not shown). The amounts of myogenin and MHC were specifically decreased by the siRNA 1 and 3 days after transfection, respectively (Fig. 3A, lanes 3 and 6, respectively). TIP120B was not greatly affected by the siRNA on day 5, possibly due to decay of the transfected siRNA, and the knockdown effect on myogenin and MHC became unclear (Fig. 3A, lane 9). Immunostaining revealed that proportions of myogenin-positive cells (Fig. 3B) and troponinT-positive myotubes (Fig. 3C) decreased to 40 and 60% of control siRNA-transfected cells, respectively (Fig. 3). It is noteworthy that only single nucleus-containing cells exhibited troponinT-positive signals when cells were treated with the specific siRNA (Fig. 3C, TIP120B siRNA), suggesting that TIP120B siRNA works not only to decrease the proportion of differentiated cells but also to delay progression of differentiation. In conclusion, TIP120B was shown to accelerate myogenic differentiation in vitro. Moreover TIP120B is thought to work at a relatively early stage in the differentiation of C2C12 cells.

Association of TIP120B with CUL1—TIP120A/CAND1 binds directly to a cullin family protein (23–25). The amino acids of TIP120A required for CUL1 binding have been determined by x-ray crystallography (31), and these critical amino acids are also conserved in TIP120B. Immunoprecipitation and immunoblotting were performed to examine the association of TIP120B with CUL1 using C2C12 cells that express FLAG-TIP120B and HA-CUL1. It was found that anti-FLAG antibody-derived immunoprecipitates contained HA peptide, implying that TIP120B is associated with CUL1 in an overexpression condition (Fig. 4A). The same conclusion was obtained from reciprocal immunoprecipitation and immunoblotting analyses (Fig. 4B). A similar assay performed with specific antibodies against TIP120B and CUL1 clarified association between endogenous TIP120B and CUL1 in differentiated C2C12 cells (Fig. 4C). Localization of these two proteins in undifferentiated C2C12 cells was investigated by immunostaining using tagged proteins. GFP-tagged TIP120B was observed ubiquitously in a cell but was slightly concentrated in the nucleus (Fig. 4D). It is known that CUL1 exists both in the cytoplasm and nucleus. As seen in Fig. 4D, FLAG-CUL1 was observed ubiquitously in a cell and was co-localized with TIP120B (Fig. 4D, merge). It was found that the amount of HA-CUL1 in truncated TIP120B-derived immunoprecipitates was much smaller than that in intact TIP120B-derived immunoprecipitates (Fig. 4E, lanes 6–8). The C terminus-truncated TIP120B seemed to have lost almost all CUL1-associating ability (Fig. 4E, lane 7). Hence it was demonstrated that TIP120B is associated with CUL1 in cells and that both the N terminus and C terminus of TIP120B are required for the association. This concept is consistent with results of Fig. 2C showing that both termini of TIP120B need to accelerate the differentiation. This ability of TIP120B is most likely due to its CUL1-associating capacity.


Figure 4
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  		FIGURE 4.
Binding of TIP120B to CUL1. A and B, association between exogenous TIP120B and CUL1 was examined by immunoprecipitation (IP) and immunoblotting in undifferentiated C2C12 cells. Cells were transfected with HA-CUL1 and FLAG-TIP120B (A) or with HA-TIP120B and FLAG-CUL1 (B). Twenty-four hours after transfection, whole cell extracts (lanes 1 and 2) were immunoprecipitated with anti-FLAG antibody-conjugated beads. Proteins in immunocomplexes were detected by immunoblotting using antibodies against HA and FLAG tags (lanes 3 and 4). C, association between endogenous TIP120B and CUL1 in differentiated C2C12 cells. Whole cell extracts of cells cultured in DM for 3 days (lane 1) were immunoprecipitated with anti-TIP120B antibody (lane 3) or control rabbit IgG (lane 2), and proteins in immunocomplexes were detected by using antibodies against CUL1 and TIP120B. D, co-localization of exogenously expressed TIP120B and CUL1 in C2C12 cells. Cells were transfected with GFP-TIP120B and FLAG-CUL1 expression plasmids. Twenty-four hours after transfection, cells were immunostained with anti-FLAG antibody. GFP-TIP120B- and FLAG-CUL1-expressing cells are indicated by arrowheads. E, domain of TIP120B required for CUL1 interaction. C2C12 cells were transfected with HA-CUL1 and various kinds of FLAG-TIP120B expression plasmids: empty plasmid (lanes 1 and 5), TIP120B FL (lanes 2 and 6), TIP120B {Delta}C(lanes 3 and 7), and TIP120B {Delta}N(lanes 4 and8).Twenty-fourhoursaftertransfection,cellswerelysed,andproteinswereimmunoprecipitatedwithanti-FLAG antibody-conjugated beads (lanes 5–8). Immunocomplexes were analyzed for HA- and FLAG-containing proteins. {alpha}-, anti-; IB, immunoblotting; DAPI, diamidino-2-phenylindole.

 


Figure 5
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  		FIGURE 5.
Stabilization of myogenin by TIP120B. The effects of TIP120B overexpression (A) and knockdown (B) on the half-life of myogenin are shown. Half-life of myogenin in C2C12 cells was determined using cycloheximide (CHX) as described under "Experimental Procedures." A, C2C12 cells were transfected with an empty plasmid (lanes 1–6) or FLAG-TIP120B expression plasmid (lanes 7–12). B, cells were transfected with 100 nM control siRNA (lanes 1–6) or TIP120B siRNA (lanes 7–12). Three kinds of proteins were measured by immunoblotting. Relative amounts of myogenin in control cells (•) and TIP120B-overexpressing cells ({blacksquare}; A) or TIP120B knockdown cells ({blacktriangleup}; B) at various cycloheximide treatment times are plotted. C, effect of truncation of TIP120B on exogenously expressed myogenin. C2C12 cells were transfected with 0.5 µg of FLAG-myogenin expression plasmid and 1.5 µg of HA-TIP120B FL (FL; lane 3), HA-TIP120B {Delta}C({Delta}C; lane 4), or HA-TIP120B {Delta}N({Delta}N; lane 5) expression plasmid as indicated. Proteins in whole cell extracts were detected by immunoblotting. {alpha}-, anti-; IB, immunoblotting.

 
TIP120B Represses Proteolysis of Myogenin—Net amounts of transcription factors in cells are regulated positively and negatively through synthesis and degradation, respectively. Hence proteolysis is critical for regulation of functional gene expression. Myogenesis is governed by multiple MRFs, including MyoD and myogenin (1). The results described above suggest that TIP120B-facilitated myogenesis is involved in the binding to cullins, especially CUL1, which is an essential component of the SCF complex that serves as a ubiquitin ligase (26, 40). Moreover a change in myogenin level was strongly correlated with TIP120B level (Fig. 2). We determined the half-life of myogenin in C2C12 cells using cycloheximide. First it was found that myogenin is extremely fragile in differentiated cells. Its half-life was ~15 min (Fig. 5A, vector) and ~20 min (Fig. 5B, control siRNA). However, the half-life was prolonged from 15 to 60 min by TIP120B overexpression. This result is consistent with results of a knockdown experiment showing that the half-life was shortened from 20 to 10 min in TIP120B siRNA-treated cells (Fig. 5B). The half-life of MyoD (~80 min) was not greatly extended (~120 min) by TIP120B overexpression (data not shown). Moreover overexpression of intact TIP120B in undifferentiated C2C12 cells increased the amount of exogenous myogenin, whereas truncated TIP120B did not have such an effect (Fig. 5C). The results suggest that myogenin is stabilized by TIP120B.

Because CUL1 is an essential component of SCF ubiquitin ligase, myogenin is thought to be hydrolyzed by proteasome. When C2C12 cells under the differentiation condition were treated with a proteasome inhibitor, MG132, the amount of intracellular myogenin was greater than that in normal cells (Fig. 6A, lane 1 versus lane 4). As shown in Fig. 3, TIP120B siRNA specifically decreased the level of myogenin (Fig. 6A, lanes 2 and 3), whereas MG132 treatment resulted in nearly the same level of endogenous myogenin despite TIP120B knockdown (Fig. 6A, lanes 5 and 6). Therefore, myogenin might be degraded by a ubiquitin-proteasome system. To confirm this view, we performed an in vivo ubiquitination assay of myogenin. Immunoprecipitation followed by immunoblotting clearly revealed that myogenin was polyubiquitinated (Fig. 6B, lane 6). Interestingly this ubiquitination was considerably repressed by intact TIP120B but not by its truncation version (Fig. 6B, lanes 7 and 8). Taken together, the results suggested that polyubiquitination of myogenin is suppressed by TIP120B, resulting in stabilization of myogenin and subsequent accelerated myogenic differentiation.

SCF-dependent Ubiquitination of Myogenin and Role of TIP120B—A recent study has shown that MyoD is ubiquitinated by SCF complex (15). We therefore decided to investigate whether myogenin is also ubiquitinated by SCF complex. First a GST pulldown assay was performed using recombinant GST-myogenin and C2C12 cell extract. Each isoform of cullin adsorbed with GST-myogenin on glutathione beads was analyzed using isoform-specific cullin antibodies. It was found that myogenin was associated with CUL1 and CUL2 but not with CUL3 and CUL4 (Fig. 7A, lane 3). Addition of MG132 to cell extracts during the binding reaction resulted in increased amounts of those cullin proteins (Fig. 7A, lane 5), suggesting that CUL1 and CUL2 function as components of ubiquitin ligase for myogenin. The results shown in Fig. 7B further confirmed the association of CUL1 with myogenin (lane 6). However, myogenin could not be detected in FLAG-CUL2-directed immunoprecipitates, possibly due to poor expression of exogenous CUL2 in C2C12 cells (data not shown).


Figure 6
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  		FIGURE 6.
Inhibition of ubiquitin-proteasome pathway-dependent degradation of myogenin by TIP120B. A, effect of proteasome inhibitor MG132 on myogenin in TIP120B knockdown cells. C2C12 cells transfected with 100 nM TIP120B siRNA or control siRNA were cultured in DM for 24 h and treated with (lanes 4–6) or not treated with (lanes 1–3)25 µM MG132 for 2 h. Amounts of myogenin, TIP120B, and beta-actin in cell lysates were determined by immunoblotting. B, decrease in polyubiquitinated myogenin by TIP120B overexpression. Cells were transfected with the indicated combination of expression plasmids for HA-ubiquitin, FLAG-myogenin, TIP120B FL (FL), and TIP120B {Delta}C({Delta}C). Cells were treated with 25 µM MG132 for 4 h, lysed, and immunoprecipitated with anti-FLAG antibody-conjugated beads (lanes 5–8). Proteins in immunoprecipitates were detected using anti-HA (ubiquitin), anti-myogenin, and anti-TIP120B antibodies. The position of polyubiquitinated (poly-ub) myogenin is indicated.{alpha}-, anti-; IB, immunoblotting; IP, immunoprecipitation; ub, ubiquitin.

 
Immunoprecipitation and immunoblotting assays were performed to detect association of myogenin with SCF components such as CUL1 and Skp1. Experiments using exogenous FLAG-myogenin and HA-CUL1 revealed that overexpressed myogenin is associated with overexpressed CUL1 and endogenous Skp1 (Fig. 7C, lane 4). The same result was obtained even when endogenous proteins were analyzed (Fig. 7D, lane 3). It was found that a major band of myogenin- and Skp1-associated CUL1 was larger than that of usual CUL1. This major band is thought to be a neddylated CUL1 (i.e. a potentiated form of CUL1) (Fig. 7D, lane 3, NEDD8-CUL1). Because Skp1 is another component of SCF complex, it is thought that SCF complex binds to myogenin.


Figure 7
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  		FIGURE 7.
Association of myogenin with SCF complex. A, binding of myogenin to various cullin family proteins was analyzed by GST pulldown assay. Whole cell extract of C2C12 cells (lane 1) was mixed with purified GST (lanes 2 and 4) or GST-myogenin (lanes 3 and 5) and then precipitated with glutathione-agarose beads. Adsorbed proteins were analyzed by immunoblotting using subtype-specific antibodies for cullins. In lanes 4 and 5,50 µM MG132 was added to the binding solution. B, interaction between myogenin and CUL1 was analyzed by immunoprecipitation. C2C12 cells were transiently transfected with FLAG-CUL1- and myogenin-expressing plasmids. Twenty-four hours after transfection, cells were treated with 25 µM MG132 for 4 h. Whole cell extracts (lanes 1–3) were immunoprecipitated with anti-FLAG antibody-conjugated beads (lanes 4–6). Proteins in immunocomplexes were detected by immunoblotting with antibodies against HA and FLAG. C, interaction of exogenous myogenin with the SCF complex. C2C12 cells were transfected with FLAG-myogenin and HA-CUL1 expression plasmids. Cells were treated with 25 µM MG132 for 4 h. Whole cell extracts (lanes 1 and 2) were immunoprecipitated with anti-FLAG antibody-conjugated beads (lanes 3 and 4). Proteins in immunocomplexes were detected with antibodies against HA, Skp1, and FLAG. D, interaction of endogenous myogenin with SCF complex in differentiated C2C12 cells. Cells were cultured in DM for 24 h and treated with 25 µM MG132 for 4 h. Cell lysate (lane 1) was immunoprecipitated with anti-Skp1 antibody (lane 3) or control rabbit IgG (lane 2). Proteins in immunocomplexes were detected with antibodies against CUL1, myogenin, and Skp1. {alpha}-, anti-; IB, immunoblotting; IP, immunoprecipitation.

 


Figure 8
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  		FIGURE 8.
Ubiquitination of myogenin by SCF complex. A, C2C12 cells were transfected with FLAG-myogenin expression plasmid together with 100 nM control siRNA (lane 2) or CUL1 siRNA (lane 3). FLAG-myogenin, CUL1, CUL2, CUL3, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-actin in cell lysates were detected by immunoblotting. B, C2C12 cells were transfected with FLAG-myogenin and HA-ubiquitin expression plasmids together with 100 nM control siRNA (lane 2) or CUL1 siRNA (lane 3). Thirty-six hours after transfection, cells were treated with 25 µM MG132 for 4 h. Whole cell extracts were immunoprecipitated with anti-FLAG antibody-conjugated beads. Proteins in immunoprecipitates were detected with antibodies against HA (for ubiquitin), FLAG, CUL1, glyceraldehyde-3-phosphate dehydrogenase, and beta-actin. Positions of polyubiquitinated (poly-ub) myogenin, CUL1, and Nedd8-bound CUL1 are indicated. {alpha}-, anti-; IB, immunoblotting; IP, immunoprecipitation.

 
Although proteasome-dependent degradation and SCF complex association of myogenin were shown by the above described investigations, it remains unclear whether myogenin is ubiquitinated by SCF complex. Because CUL1 is an essential component of SCF complex, CUL1 in C2C12 cells was knocked down, and the status of myogenin was examined. Fig. 8A shows that CUL1 siRNA that specifically interferes with CUL1 (i.e. immunoblotting for CUL1–CUL3) increased the amount of exogenously expressed FLAG-myogenin (Fig. 8A, lane 3) compared with that in the case of control siRNA (Fig. 8A, lane 2). We also analyzed ubiquitination of myogenin in siRNA-introduced cells and found that CUL1 siRNA treatment lowered the ubiquitination degree of myogenin (Fig. 8B, lane 6). CUL1 level was evidently correlated with ubiquitination of myogenin. Taken together, the results suggested that myogenin is ubiquitinated by SCF complex.

Because it was thought that TIP120B affects the function of SCF complex, we examined whether TIP120B interferes with an SCF-myogenin complex. In extracts of cells in which CUL1 and myogenin were overexpressed, CUL1-derived immunocomplexes contained myogenin as described above (Fig. 9A, lane 6). However, co-transfection of TIP120B decreased the amounts of CUL1-associated myogenin dose-dependently (Fig. 9A, lanes 7 and 8). We further investigated the effect of TIP120B on the association of Skp1 in SCF complexes, and we found that the amount of Skp1 decreased depending on the dose of the TIP120B expression plasmid, HA-TIP120B (Fig. 9B, lanes 6–8). These results suggest that CUL1-mediated binding of TIP120B to SCF complex allows dissociation of an Skp1-F-box protein-myogenin moiety from CUL1, resulting in a breakdown of the intact SCF complex. Consequently this mechanism is thought to inhibit ubiquitination of myogenin, which results in extended half-life of myogenin and promotes myogenic differentiation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Involvement of TP120B in Myogenesis—Transcription factors play key roles in development and differentiation. Myogenesis is one of the most typical model systems of cell differentiation. Many MRFs, including MyoD and myogenin, have been identified using an in vitro myogenesis system (41, 42). It has been shown that they bind to an E-box element of a target gene and exhibit transcription activation functions during a particular period of myogenesis. Although an outline of the roles of MRFs in myogenesis has been elucidated, the regulation mechanisms of MRFs in cells are not yet fully understood. We have identified a homologue of TIP120A/CAND1, TIP120B/CAND2, that is expressed restrictedly in mammalian muscle tissues (33, 34). TIP120B has also been detected in myotubes in culture. In the present study, we investigated in detail the expression of TIP120B during myogenic differentiation of C2C12 cells and found that TIP120B appears in association with the differentiation (Fig. 1). TIP120B was detected only in morphologically altered myotubes and was constitutively expressed in differentiated cells (Fig. 1). Hence we concluded that TIP120B is a myogenesis-related protein. In a previous work, we found a high TIP120B expression level in limb buds of day 14 mouse embryos where myogenesis occurs actively (34).

The findings described above indicate the possibility that TIP120B participates in differentiation and maintenance of muscle cells at a translational or post-translational step because TIP120B exists in the cytoplasm (Fig. 1, C–E). We investigated the involvement of TIP120B in C2C12 cell differentiation and found that overexpression of intact TIP120B, but not that of truncated TIP120B, in differentiated cells significantly increased the amounts of troponinT and myogenin as well as the proportion of differentiated cells (Fig. 2). It was also found that TIP120B siRNA decreased the amount of myogenin on day 1 and the amount of MHC on day 3 in the differentiation process (Fig. 3A). Moreover the siRNA decreased the population of both myogenin- and troponinT-positive cells (Fig. 3, B and C). These results indicated that TIP120B is positively involved in myogenic differentiation. Because the siRNA used in this work persisted for only few days in cells, a decrease in myogenin on differentiation day 3 was not obvious, but a decrease in MHC was observed on day 3 (Fig. 3A, lane 3). These phenomena imply that myogenin is responsible for MHC gene expression. Cao et al. (43) revealed through a chromatin immunoprecipitation-on-chip technique that MHC is regulated synergistically by myogenin and MyoD. They suggested that myogenin, categorized as an intermediate gene, is critical for expression of late genes (e.g. MHC and troponinT) in myogenesis. Taken together, the results suggest that TIP120B, which is coexpressed with myogenin and regulates myogenin level, should also be classified as an intermediate myogenesis gene.


Figure 9
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  		FIGURE 9.
Inhibition of association of myogenin and SCF complex by TIP120B. A, C2C12 cells were transiently transfected with the indicated combination of FLAG-CUL1 and increasing amounts of HA-TIP120B expression plasmids (5 and 10 µg in 100-mm dishes for lanes 3/7 and 4/8, respectively). Whole cell extracts (lanes 1–4) were immunoprecipitated with anti-FLAG antibody-conjugated beads (lanes 5–8). Proteins in immunocomplexes were detected by immunoblotting using specific antibodies as indicated. B, C2C12 cells were transiently transfected with myogenin, FLAG-CUL1, and increasing amounts of HA-TIP120B expression plasmids (5 and 10 µg in 100-mm dishes for lanes 3/7 and 4/8, respectively). Twenty-four hours after transfection, cells were treated with 50 µM MG132 for 2 h. Whole cell extracts (lanes 1–4) were immunoprecipitated with anti-FLAG antibody-conjugated beads (lanes 5–8). Proteins in immunocomplexes were detected with specific antibodies as indicated. {alpha}-, anti-; IB, immunoblotting; IP, immunoprecipitation.

 


Figure 10
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  		FIGURE 10.
Model of TIP120B-accelerated myogenic differentiation. In this model, TIP120B suppresses the SCF complex-mediated ubiquitination through binding to CUL1 and subsequent dissociation of the complex. If TIP120B is absent, myogenin is ubiquitinated in an SCF-dependent manner and degraded rapidly by proteasome. Consequently TIP120B-directed suppression of myogenin degradation leads to acceleration of myogenic differentiation. ub, ubiquitin.

 
Association of TIP120B with CUL1—Because TIP120A has been demonstrated to bind to CUL1 (23–25), it can be speculated that TIP120B also binds to CUL1. We examined whether TIP120B is associated with CUL1 and found that TIP120B is associated with CUL1 in cells (Fig. 4, A–C). Because two TIP120B truncation versions showed decreased CUL1-associating abilities (Fig. 4E) in addition to decreased differentiation abilities (Fig. 2C), it is thought that TIP120B-mediated acceleration of myogenesis is attributed to CUL1-TIP120B association. Moreover GFP-TIP120B was found to be co-localized with FLAG-CUL1 in cells (Fig. 4D). In overexpression conditions, TIP120B was able to be associated with CUL2, CUL3, and CUL4A.4 A yeast two-hybrid assay also revealed an association of CUL4B and TIP120B (data not shown). Both termini of TIP120A have been demonstrated to join to the CUL1 molecule (31). It was found that both termini of TIP120B exhibit high similarity with TIP120A (72–77%), whereas its central region showed lower similarity (52%) (supplemental Fig. 1). It is note-worthy that CUL1-interacting TIP120A amino acid residues were completely conserved in TIP120B (data not shown). Taken together, the results suggest that, like TIP120A, TIP120B binds to CUL1.

Rapid Degradation of Myogenin and Its Suppression by TIP120B—The amount of intracellular proteins is regulated by a balanced rate of synthesis and degradation. Intracellular proteins are degraded either by a lysosomal pathway or ubiquitin-proteasome pathway (44, 45). Short lived proteins, inappropriately synthesized proteins, and denatured proteins are degraded by a ubiquitin-proteasome pathway (46). MyoD is unstable in cells, with a half-life of 45–60 min, and is rapidly degraded by proteasome (11). It is also known that MyoD is ubiquitinated by SCFMAFbx complex (15). It was demonstrated that the amount of myogenin is affected by TIP120B, which interacts with CUL1 (Figs. 2 and 3). Because CUL1 is an essential component of SCF, it is likely that myogenin is ubiquitinated SCF-dependently. We found that myogenin is extremely unstable (Fig. 5, A and B) as described previously (47) and stabilized by MG132 (Fig. 6A). Moreover because myogenin is a polyubiquitinated protein (Fig. 6B), it is thought that myogenin is rapidly degraded by proteasome. Results shown in Fig. 5, A and B, imply that degradation of myogenin is divided into two phases: an initial phase (~30 min) and the later phase in which myogenin is degraded rapidly and slowly, respectively. Previous studies have indicated that an E-protein modulates degradation of MyoD, and DNA-associated MyoD is more stable than free MyoD (11, 48). Myogenin in the later phase may exist in a stable form because of E-protein binding and participation in transcription. Interestingly TIP120B stabilized myogenin (Fig. 5C). TIP120B seems to affect the first phase predominantly (Fig. 5, A and B). These results suggest that TIP120B is a positive regulator of myogenin in C2C12 cells. C2C12 cells constitutively express MyoD. Because expression of myogenin is a critical event of the myogenic differentiation of C2C12 cells, TIP120B is thought to accelerate myogenesis through inhibition of myogenin degradation. This concept is consistent with the fact that TIP120B and myogenin are induced synchronously during C2C12 myogenesis.4

SCF Complex as a Myogenin-ubiquitinating Enzyme—We investigated an E3 enzyme of myogenin. Intensive immunoprecipitation and immunoblotting experiments revealed that myogenin is associated with CUL1, which is an essential component of SCF complex (28). Similar experiments revealed that Skp1, a hinge protein between cullin and F-box protein, is also associated with CUL1 and myogenin (Fig. 7). We did not succeed in in vitro ubiquitination for myogenin using FLAG-CUL1-derived immunocomplexes. However, knockdown of intercellular CUL1 resulted in stabilization of myogenin (Fig. 8A). Moreover the degree of ubiquitination of myogenin was lowered by CUL1 knockdown (Fig. 8B). Although we could not identify an F-box protein in the present study, it is thought that myogenin is ubiquitinated by SCF complex because CUL1 is an essential component of SCF.

Because TIP120B stabilizes intracellular myogenin (Figs. 2, 3, 5, and 6A) and associates with CUL1 (Fig. 4), TIP120B is thought to affect SCF-dependent ubiquitination of myogenin. It was also shown that TIP120B inhibits myogenin ubiquitination (Fig. 6B) and dissociates Skp1 from CUL1 (Fig. 9A). Moreover TIP120B dissociates myogenin from the complex or at least from CUL1 (Fig. 9B). Taken together, the results suggest that TIP120B breaks the SCF complex and releases a free myogenin. Consistent results were obtained in the case of TIP120A, although the physiological role of TIP120A has not yet been elucidated. It is known that Skp1 is dissociated from CUL1 if TIP120A is added (23–25).

Role of TIP120B in Myogenesis—MyoD plays a role in the early phase of myogenesis, whereas myogenin plays a role in the intermediate and late phase (43, 49). Myogenin is thus required for both differentiation and maintenance of muscle cells. It is thought that MyoD induces chromatin modification for expression of myogenesis-related genes and that it is sufficient for activation of intermediate genes, including myogenin, but is insufficient for induction of late genes such as MHC and troponinT (43). This concept can account for how myogenin is required to accomplish myogenesis. We demonstrated that myogenin is extraordinary fragile in C2C12 cells (Fig. 5). It is thought that such rapid degradation must be suppressed if myogenin function is expressed stably in differentiated cells. We believe that TP120B is responsible for determination of the level of intracellular myogenin. Thus it is thought that TIP120B is involved in differentiation and maintenance of muscle tissues. We found that myogenin interacts with SCF complex and probably binds directly to an F-box protein (Fig. 7). If TIP120B is present in a cell, TIP120B binds to CUL1, resulting in dissociation of an Skp1-F-box protein-myogenin moiety from an E3 activity-carrying (Nedd8) CUL1-Roc1 moiety (Fig. 9). Finally myogenin is dissociated from the F-box protein, and such free myogenin participates in transcriptional activation of muscle-specific genes. This scheme can account for how TIP120B accelerates myogenic differentiation (Fig. 10).

A recent study has suggested that TIP120A/CAND1 inhibits CUL3-keap1 complex-dependent ubiquitination of Nrf2, although its physiological role is not fully understood (50). In this study, we succeeded in revealing a physiological role of TIP120/CAND protein using TIP120B and myogenin with a myogenic differentiation system. The present study provides a new insight into the regulation system of intracellular muscle proteins during muscle differentiation and maintenance. It is well known that muscle proteins, including myogenic factors, turn over rapidly compared with proteins in other tissues. If degradation dominates in a balance between protein synthesis and degradation, muscle tissues must fall into atrophy. Therefore, the concentration of myogenic factors in matured muscles is critical for muscle maintenance as well as differentiation. TIP120B, which is required for muscle differentiation, may also be involved in muscle maintenance through regulation of myogenin degradation.


   FOOTNOTES
 
* A part of this work was supported by a grant from Nisshinbo Industries Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1. Back

1 Present address: Dept. of Molecular Biology, Princeton University, Princeton, NJ 08544. Back

2 To whom correspondence should be addressed. Tel.: 81-43-290-2823; Fax: 81-43-290-2824; E-mail: ttamura{at}faculty.chiba-u.jp.

3 The abbreviations used are: MRF, myogenic regulatory factor; TBP, TATA-binding protein; TIP, TBP-interacting protein; SCF, Skp1-CUL1-F-box protein; CUL, Cullin; MHC, myosin heavy chain; GST, glutathione S-transferase; PBS, phosphate-buffered saline; siRNA, small interference RNA; PI, proteinase inhibitor; GM, growth medium; DM, differentiation medium; HA, hemagglutinin; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; CAND, cullin-associated neddylation-dissociated; GFP, green fluorescent protein; RIPA, radioimmune precipitation assay. Back

4 S. Shiraishi, C. Zhou, T. Aoki, N. Sato, T. Chiba, K. Tanaka, S. Yoshida, Y. Nabeshima, Y.-i. Nabeshima, and T.-a. Tamura, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Drs. A. Asakura and J. B. Yoon for providing plasmids.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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