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INTRODUCTION |
Basic helix-loop-helix
(bHLH)1 transcription factors
have been demonstrated to play critical roles in cell fate
determination including differentiation in a variety of tissues of both
vertebrates and invertebrates (1, 2). The bHLH proteins form homodimers or heterodimers through the helix-loop-helix domains and enable the
basic regions to form a bipartite DNA-binding motif that recognizes the
so-called E-box sequences, CANNTG (3). Typically, tissue-specific bHLH
factors, such as MyoD and BETA2/NeuroD, dimerize with ubiquitously expressed bHLH factors, such as E2A gene products, and
promote cell fate determination to differentiate into specific lineages (3, 4). The E2A gene encodes two alternatively spliced
products, E12 and E47, which differ in their bHLH domains and hence
their DNA-binding properties (5, 6).
Epicardin (7)/capsulin (8, 9)/Pod-1 (10) is a bHLH transcription factor
expressed in brachial muscle precursors and mesenchymal cells at
sites of epithelial-mesenchymal interactions in the kidney, lung,
intestine, pancreas, spleen, developing respiratory, gastrointestinal,
urogenital, and cardiovascular systems (7, 8, 10). The phenotypic
analysis of homozygous epicardin/capsulin/Pod-1 mouse
mutants reveals a critical role for epicardin/capsulin/Pod-1 in the
formation of spleen (11), lung, and kidney (12).
Epicardin/capsulin/Pod-1 binds the E box consensus sequence as a
heterodimer with the ubiquitous bHLH protein E12 (8, 9). Although
epicardin/capsulin/Pod-1 seems to be involved in organogenesis in
vivo, little is known concerning the functions of
epicardin/capsulin/Pod-1 in controlling tissue-specific gene expression
and differentiation.
bHLH factors are involved in at least two distinct steps, cell cycle
arrest and tissue-specific gene expression. The E2A proteins, another
bHLH factor Twist, and the dominant negative-type helix-loop-helix proteins Ids regulate expression of the gene for an inhibitor of
cyclin-dependent kinases (Cdks) p21(WAF1/Cip1) (13, 14), which is induced early during the differentiation program in myogenesis (15, 16). Previous studies (17, 18) using cultured cells have shown
that MyoD promotes cell cycle arrest through induction of p21 in
differentiating myoblasts. In addition, the combination of p21 and a
related molecule p57 has been shown to be essential for muscle
differentiation during embryonic development in mice (19). bHLH factors
also regulate expression of fibroblast growth factor receptor 3 (FGFR3) and muscle creatine kinase (MCK) genes (14, 20), which are responsible for differentiation of osteoblasts and
myoblasts, respectively (21, 22).
To investigate the implication of epicardin/capsulin/Pod-1 in
differentiation, we examined how epicardin/capsulin/Pod-1 is involved
in the transcriptional regulation of cell cycle arrest and
lineage-specific gene expression. Ectopic expression of
epicardin/capsulin/Pod-1 inhibited E12- and MyoD-induced
transactivation of p21. This is also the case in the
regulation of MCK expression, demonstrating that common
transcriptional regulation controls cell cycle arrest and
differentiation of myoblasts. Furthermore, introduction of epicardin/capsulin/Pod-1 hampered terminal differentiation of C2C12
myoblasts. These functional characteristics indicate that epicardin/capsulin/Pod-1 is a negative transcriptional regulator of
differentiation-related genes similar to Twist.
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EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Screening--
All plasmids and strains for
two-hybrid analysis were obtained from the Matchmaker System-3 kit
(Clontech). The bHLH domain of E12 (amino acids
510-654) was cloned in-frame with the GAL4 DNA-binding domain (DBD) in
pGBKT7 (Clontech), yielding pGBKT7-E12 (bHLH). An
MG63 cDNA library in the pGADT7 prey plasmid was co-transfected with the pGBKT7-E12 (bHLH) bait plasmid into AH109 yeast cells. Yeast
two-hybrid screening was performed as described by the manufacturer's protocol (Clontech Matchmaker Two-hybrid Protocol).
For conditional growth assays, yeast cells transfected with the
expression plasmids for pGAL4DBD-fusion constructs and
pGAL4AD-epicardin were grown on the appropriate selective medium at
30 °C for 5 days.
Mammalian Two-hybrid Assay--
All plasmids and strains for
mammalian two-hybrid assays were obtained from Promega. Subconfluent
cultures of C3H10T1/2 cells (2 × 105 cells/dish of a
60-mm diameter) were transfected with 2 µg of pG5luc reporter, pGAL
chimeras, and pVP16 chimeras together with pCMV-
-Gal by the
calcium-phosphate method. After incubation overnight with the DNA
precipitate, cells were then cultured in Dulbecco's modified Eagle's
medium (DMEM) containing 2% horse serum for a further 48 h for
the luciferase assay. Luciferase and
-galactosidase assays were done.
Cell Culture--
The human osteosarcoma osteoblast-like cell
line MG63, which is negative for p53 (23), C3H10T1/2 fibroblasts (Riken
Cell Bank), and myoblast cell line C2C12 (Riken Cell Bank) were
cultured in DMEM with 10% fetal bovine serum (FBS) at 37 °C under a
humidified atmosphere of 5% CO2.
Plasmid Construction--
E12 and E47 cDNAs were kindly
given by C. Murre (University of California, San Diego). Expression
vectors pCMV-E12, pCMV-E47, pCMV-Twist (14), pCMV-Id1 and pCMV-Id2 (24)
were used. Reporter plasmids p21-luc (25) and pMCK-luc were
generous gifts from B. Vogelstein (The Johns Hopkins University), and
S. D. Hauschka and J. Buskin (University of Washington),
respectively. The expression vectors, pCMV-MyoD (26) and pRSV-CBP, were
generous gifts of S. J. Tapscott (Fred Hutchinson Cancer Research
Center) and T. Nakajima (St. Marianna University), respectively.
The epicardin/capsulin/Pod-1 cDNA fragment was recovered from the
pGADT7 prey plasmids by digestion with EcoRI and subcloned into the EcoRI site of pcDNA3 (Invitrogen), generating
pCMV-epicardin. Epicardin/capsulin/Pod-1 cDNA was fused to pEGFP-C2
(Clontech) for expression of the green fluorescent
protein (GFP), generating the vector pGFP::epicardin.
Epicardin/capsulin/Pod-1 cDNA was fused to pCMV-Myc
(Clontech) or pCMV-HA
(Clontech) for expression of the Myc- or HA-tagged
epicardin/capsulin/Pod-1, generating the vector pMyc-epicardin or
pHA-epicardin, respectively.
Transient Transfection and Luciferase Assay--
Subconfluent
cultures of MG63 cells, C3H10T1/2 cells, or C2C12 myoblasts (2 × 105 cells/dish of a 60-mm diameter) were transfected with a
total of 10 µg of expression and reporter plasmids by the
calcium-phosphate method together with the
-galactosidase expression
vector, pCMV-
-Gal, which was used as an internal control to monitor
the transfection efficiency. After incubation overnight with the DNA
precipitate, cells were then cultured in DMEM containing 2 or 10% FBS
or 2% horse serum for a further 48 h for the luciferase assay and
10% FBS for a further 24 or 72 h for immunostaining. Luciferase
activity was assayed using the luciferase assay system (Promega) and
normalized to
-galactosidase activity, which was determined by the
method of Rose and Botstein (27). All assays were performed at least three times in duplicate, and representative data are presented. The
results are the mean of different experiments ± S.E.
Immunofluorescence Stains--
Cells were fixed in
phosphate-buffered saline (PBS) with 3.7% formaldehyde, permeabilized
with Triton X-100 (0.1%) in PBS, and then treated with goat or horse
serum to block nonspecific binding sites. Polyclonal rabbit antibodies
against E2A (sc-349, Santa Cruz Biotechnology) and a mouse monoclonal
antibody to human p21 (sc-817, Santa Cruz Biotechnology) were used.
After permeabilization, cells were incubated for 1 h at room
temperature with the above-mentioned primary antibodies at the
respective dilutions of 1:100 to 1:250. Immune complexes containing E2A
and p21 were detected with rhodamine-conjugated anti-rabbit IgG (Santa
Cruz Biotechnology) and fluorescein isothiocyanate-conjugated anti-mouse IgG (Chemicon), respectively. DNA in the nuclei was detected
with 4',6'-diamidino-2-phenylindole. In the quantitative analysis, a
minimum of 150 positive cells was examined in each transfection under
an Olympus fluorescence microscope (BX-FLA) or a confocal
laser-scanning microscope (LSM510; Carl Zeiss).
Differentiation of C2C12 Myoblasts--
The myoblast
differentiation assays were performed as described previously (28). In
brief, C2C12 myoblasts were transiently transfected in DMEM containing
10% FBS (growth medium) with 2.5 µg of pCMV-
-Gal together with or
without 2.5 µg of pCMV-epicardin per 30-mm well. The total amount of
DNA added to C2C12 cells was adjusted to 2.5 µg by addition of empty
pCMV vector. After culture in DMEM containing 2% horse serum
(differentiation medium) for 96 h, cells were fixed,
permeabilized, and stained with anti-troponin T (TnT) antibody (Sigma).
Differentiation was evaluated by counting the number of TnT-positive
cells relative to that of
-galactosidase-positive cells.
Small Interfering RNA (siRNA)--
The coding strand sequence of
the siRNA for epicardin/capsulin/Pod-1 was
TTAAGGCCTTCTCCAGACTCAAG. siRNA duplexes were prepared by
annealing two pairs of 21 oligonucleotides synthesized by
Qiagen. As a control, non-silencing siRNA was purchased from Dharmacon. Twenty four hours before transfection, mammalian cells were trypsinized and transferred to 6-well plates (5 × 105
cells/well). Reporter plasmids and expression plasmids were transfected by the calcium-phosphate method. Cells were incubated for 5 h and
then transfected with 0.5 µg of siRNA duplexes using TransMessenger Transfection Reagent (Qiagen) as described by the manufacturer for
adherent cell lines. Forty eight hours later, cell lysates were
prepared and subjected to Western blotting and luciferase assay, respectively.
Immunoblot Analysis--
Total cell lysates were fractionated by
SDS-PAGE in a 12% gel according to standard protocols, and proteins
were transferred to nitrocellulose filters. Polyclonal antibodies
against
-galactosidase (Zymed Laboratories Inc.)
and mouse monoclonal antibodies to Myc epitope
(Clontech), p21 (sc-817; Santa Cruz Biotechnology),
MHC (MY-32; Sigma), and
-tubulin (DM1A; Sigma) were used at the
respective dilutions of 1:400 to 1:1000 in blocking buffer (1% bovine
serum albumin and 0.1% Tween-20 in PBS) at room temperature for 2 h, and immune complexes were detected by the ECL method as described by
the manufacturer (Amersham Biosciences).
Chromatin Immunoprecipitation (ChIP) Assays--
ChIP assays
were done as described previously by Shang et al. (29).
Approximately 1 × 107 C2C12 cells were transfected
with 5 µg of pMCK-luc and 15 µg of pHA-epicardin by the
calcium-phosphate method and cultured in DMEM containing 2% horse
serum for 48 h. After washing twice with PBS, cells were
cross-linked with 1% formaldehyde at room temperature for 10 min,
rinsed with ice-cold PBS twice, and collected into ice-cold PBS. Cells
were then resuspended in 0.3 ml of lysis buffer (1% SDS, 5 mM EDTA, 50 mM Tris-HCl, pH 8.1) with protease inhibitor mixture (Roche Molecular Biochemicals) and sonicated three
times for 15 s each (Tomy Ultrasonic disrupter, model UD-201) followed by centrifugation for 10 min. Supernatants were collected and
diluted in a dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1) with
protease inhibitor mixture followed by immunoclearing with 2 µg of
sheared salmon sperm DNA and protein G-Sepharose (45 µl of 50%
slurry in 10 mM Tris-HCl, pH 8.1, 1 mM EDTA)
for 2 h at 4 °C. Immunoprecipitation was performed overnight at
4 °C with anti-HA antibody (3F10; Roche Molecular Biochemicals) or
anti-Myc antibody (Clontech). After
immunoprecipitation, 45 µl of protein G-Sepharose and 2 µg of
salmon sperm DNA were added, and incubation was continued for another
1 h. Precipitates were washed sequentially for 5 min each in TSE I
(0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM
Tris-HCl, pH 8.1, 150 mM NaCl), TSE II (0.1% SDS, 1%
Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH
8.1, 500 mM NaCl), and buffer III (0.25 M LiCl,
1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), further washed three times with TE
buffer, and extracted with 1% SDS, 0.1 M
NaHCO3. Elutes were heated at 65 °C for 6 h to
reverse the formaldehyde cross-linking. DNA fragments were purified
with a QIAquick Spin Kit (Qiagen, CA). A portion (4%) of purified DNA extraction was subjected to 30 cycles of PCR with primers specific for
either the MCK enhancer ((+), 5'-GACACCCGAGATGCCTGGTT-3'; (
),
5'-GATCCACCAGGGACAGGGTT-3'). As a control for DNA content, PCRs were
performed with samples without immunoprecipitation. A portion (20%) of
PCR product of each reaction mixture was resolved through a 5% native
acrylamide gel.
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RESULTS |
Association of Epicardin/Capsulin/Pod-1 with
bHLH of E12--
We employed the yeast two-hybrid system to search for
proteins that bind to E12, using the E12-bHLH region fused to the GAL4 DNA-binding domain as bait. In a screen of 2 × 106
human MG63 cell library clones, 35 clones specifically interacted with
E12-bHLH. DNA sequence analysis of these clones revealed that half of
them encoded epicardin/capsulin/Pod-1 fused in-frame to the
transcriptional activation domain in the prey plasmid. We confirmed
interaction of intact epicardin/capsulin/Pod-1 with either full-length
E12 or the bHLH domain of E12 (E12-bHLH) in a yeast two-hybrid assay
(Fig. 1, A and B).
The result is consistent with the observation that
epicardin/capsulin/Pod-1 heterodimerizes with the ubiquitous bHLH
protein E12 (8). Epicardin/capsulin/Pod-1 did not bind to the Twist
bHLH domain (Fig. 1B), indicating that the association of
epicardin/capsulin/Pod-1 with E12 was specific.

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Fig. 1.
Association of epicardin/capsulin/Pod-1 with
E12. A, schematic representation of wild-type and mutant
proteins of E12 and Twist. B, interaction of
epicardin/capsulin/Pod-1 with the wild-type E12 protein in yeast. A
plasmid encoding full-length epicardin/capsulin/Pod-1 fused to the GAL4
activating domain (AD) was cotransfected into yeast AH109
cells with plasmids encoding the indicated forms of E12 or Twist fused
to the GAL4 DNA-binding domain (DBD). Transformants were
streaked onto a control plate lacking leucine and tryptophan
(SD-Leu-Trp) and an indicator plate lacking leucine, tryptophan,
histidine, and adenine (SD-Leu-Trp-His-Ade). C, diagrammatic
representation of the mammalian two-hybrid assay. Constructs for
chimeric proteins of GAL4-DBD and epicardin/capsulin/Pod-1 bHLH and of
VP16 and E12 were generated and named pGAL-epi (bHLH) and pVP16-E12,
respectively. D, interaction of epicardin/capsulin/Pod-1
with E12. MG63 cells were transfected with combinations of expression
plasmids and control plasmids along with the reporter plasmid pG5-luc,
and luciferase activity was determined. Fold activation is expressed as
a ratio of luciferase activity relative to that obtained with the GAL4
DBD and VP16 cassettes, which is arbitrarily set at 1.
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Similar observations were made in mammalian cells. The pG5luc vector
contains five tandem copies of the GAL4-binding site upstream of a
minimal TATA box linked to the firefly luciferase gene (Fig.
1C). Cotransfection of this reporter plasmid with the expression vectors for the fusion proteins of GAL4DBD-epicartdin bHLH
and VP16-E12 effectively induced profound activation of the promoter,
whereas introduction of each of GAL4DBD-epicardin bHLH and VP16-E12
showed slight and little, if any, activation, respectively (Fig.
1D). These results clearly indicate that E12 physically interacts with the bHLH domain of epicardin/capsulin/Pod-1.
This conclusion was further supported by colocalization of
epicardin/capsulin/Pod-1 and E12 in the nuclei. GFP alone was in both
the cytoplasm and nuclei in MG63 cells (Fig.
2b), whereas the
epicardin/capsulin/Pod-1-GFP fusion protein from
pGFP::epicardin were found in the nuclei (Fig.
2e).

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Fig. 2.
Co-localization of
epicardin/capsulin/Pod-1 with E12 in the nucleus. MG63 cells were
transfected with pGFP (a-c) or pGFP::epicardin
(d-f) together with expression vector for E12 (pCMV-E12).
The GFP signal (green; b and e) was
subsequently visualized by confocal microscopy. Immunofluorescence
associated with E12 expression was detected by immunostaining with the
antibody specific to E12 (rhodamine; a and d).
c and f show images merged with a and
b, d and e, respectively. Scale
bars, 20 µm.
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Transcriptional Inhibition of the p21 Gene by
Epicardin/Capsulin/Pod-1--
Expression of p21
has been shown to parallel the irreversible withdrawal from the cell
cycle, following the induction of MyoD in myogenic differentiation (17,
18, 30). We have shown previously (14) that there is a close link
between p21 induction and cell cycle arrest in MG63 cells. The MyoD/E12
heterodimer has been shown to bind to specific E-box DNA sequences and
activate muscle-specific genes (5, 20, 31). To gain insight into the
biological significance of the interaction of epicardin/capsulin/Pod-1 with E12, we tested the role of epicardin/capsulin/Pod-1 in
E2A-dependent activation of p21 transcription with MG63
cells, in which we have previously shown the inhibitory effects of
Twist on E2A-dependent p21 promoter activation (14). MG63
cells were cotransfected with p21-luc, an E2A expression plasmid
(pCMV-E12 or pCMV-E47) and/or pCMV-epicardin and luciferase activities
were determined after 48 h of culture. Concentrations of the
epicardin/capsulin/Pod-1 plasmid were equivalent to those for Twist, at
which muscle differentiation is inhibited (20, 32). When p21-luc was
transfected along with pCMV-epicardin, a major reduction in luciferase
activities was observed in either E12 or E47 expressing cells over a
range of pCMV-epicardin concentrations used, similar to Twist (Fig. 3B). The magnitude of
inhibition by epicardin/capsulin/Pod-1 was equivalent to those induced
by Twist and Id1 except for the case of Id1 in E47-driven transcription
(Fig. 3A). The inhibition of the E2A-dependent
p21 promoter activation by epicardin/capsulin/Pod-1 was further
enhanced by the addition of Twist or Id1 (Fig. 3A), suggesting that epicardin/capsulin/Pod-1, Twist, and Id1 inhibit p21
expression in a cooperative manner. These results demonstrate that the
epicardin/capsulin/Pod-1 functions as a negative transcriptional regulator in the E2A-dependent p21 promoter activation in
MG63 cells, like Twist and Id1.

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Fig. 3.
Effects of epicardin/capsulin/Pod-1 on
E2A-dependent transactivation of p21. A,
inhibition of E12- and E47-dependent transactivation of p21
by epicardin/capsulin/Pod-1. MG63 cells were cotransfected with 2.5 µg of p21-luc in combination with pCMV-E12, pCMV-E47, pCMV-epicardin,
pCMV-Twist, and pCMV-Id1. The cells were harvested 48 h after
transfection and then assayed for reporter gene expression. Empty
vector pcDNA3 was included to adjust DNA amounts. Activation
mediated by pcDNA3 is arbitrarily set at 1. B,
inhibition of E2A-dependent transactivation of p21 by
epicardin/capsulin/Pod-1 in a dose-dependent manner. MG63
cells were cotransfected with 2.5 µg of p21-luc in combination with
pCMV-E12 and pCMV-E47 and with indicated amounts of pCMV-epicardin or
pCMV-Twist. Empty vector pcDNA3 was included to adjust DNA amounts.
C, inhibition of E12- and CBP-dependent p21
transcription by epicardin/capsulin/Pod-1. MG63 cells were
cotransfected with 1.0 µg of p21-luc and 0.75 µg of either
pcDNA3 or pCMV-E12. The cells were cultured in DMEM containing 2%
FBS for 48 h and collected for the luciferase assay. D,
inhibition of E12- and MyoD-dependent transactivation of
p21 by epicardin/capsulin/Pod-1. C2C12 myoblasts were cotransfected
with 2.5 µg of p21-luc in combination with pCMV-E12, pCMV-MyoD, and
pCMV-epicardin. The cells were cultured in DMEM containing 10% FBS for
48 h and collected for luciferase assay. Activation mediated by
pcDNA3 is arbitrarily set at 1. E, reduced
epicardin/capsulin/Pod-1 in MG63 cells transfected with
epicardin/capsulin/Pod-1 siRNA. Five hours prior to siRNA transfection,
cells were cotransfected with pCMV-Myc-epicardin and the
-galactosidase expression vector, pCMV- -Gal, which was used as an
internal control to monitor the transfection efficiency. Cells were
harvested 48 h following siRNA transfection, and cell lysates were
generated. Samples were immunoblotted with antibodies to the Myc
epitope, -galactosidase, and -tubulin. F, abolishment
of epicardin/capsulin/Pod-1-mediated E12 inhibition by
epicardin/capsulin/Pod-1 siRNA. Five hours prior to siRNA transfection,
MG63 cells were cotransfected with 1.0 µg of p21-luc in combination
with 1.0 µg of pCMV-E12 and pMyc-epicardin. The cells were harvested
48 h later, and luciferase activities were determined. Empty
vector pCMV-Myc was included to adjust DNA amounts.
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We further tested if epicardin/capsulin/Pod-1 protein could inhibit the
expression of the endogenous p21 gene. MG63 cells were transfected with
expression plasmids for E12 with or without epicardin/capsulin/Pod-1
and then examined for p21 expression by immunostaining. Upon
introduction of E12, endogenous p21 expression was induced in cells
expressing high levels of E12 even when cultured in high serum (Fig.
4A) as reported previously
(14). Overexpression of epicardin/capsulin/Pod-1 abolished the
endogenous p21 expression induced by E12 (Fig. 4B and Table
I), confirming the antagonistic function
of epicardin/capsulin/Pod-1 in E12-induced gene expression.

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Fig. 4.
Inhibition of E12-dependent
endogenous p21 induction by epicardin/capsulin/Pod-1. MG63 cells
were transfected with pCMV-E12 and either pcDNA3 (an empty vector)
(A) or pCMV-epicardin (B). The cells were
maintained in DMEM containing 10% FBS for 72 h. The cells were
fixed and stained for E12 (rhodamine, a), p21 (fluorescein,
b), and DNA (4',6'-diamidino-2-phenylindole
(DAPI), c). Arrows indicate cells
expressing E12. Scale bar, 20 µm.
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Table I
Effects of epicardin/capsulin/Pod-1 on
E12-dependent p21 expression
MG63 cells were transiently cotransfected with pCMV-E12 and
pCMV-epicardin/capsulin/Pod-1. The cells were maintained in DMEM
containing 10% FBS. The expression of p21 was monitored by
immunostaining of E12 and p21 (Fig. 4). At least 150 cells were counted
in each category, and results are representative of multiple
experiments performed.
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The coactivator p300/cyclic AMP-responsive element binding factor
CREB-binding protein (CBP) harboring histone acetyltransferase activity
has been shown to interact with E12 (33). Histone acetyltransferase activity is important for decondensing the chromatin (34) and changing the accessibility of the transcription machinery (35). To
analyze the functional implications of the interactions between E12-CBP
complex and epicardin/capsulin/Pod-1, we studied the effects of
epicardin/capsulin/Pod-1 on E12-CBP-dependent
transcriptional activation of the p21 gene.
Epicardin/capsulin/Pod-1-induced inhibition of p21 promoter activation
in response to E12 was partly overcome by the addition of CBP in a
dose-dependent manner (Fig. 3C). Taken together,
these findings support the view that epicardin/capsulin/Pod-1 suppresses transcriptional activity conferred by a combination of E12
and CBP.
To confirm involvement of epicardin/capsulin/Pod-1 in regulation of p21
gene expression, the effect of loss of function of epicardin/capsulin/Pod-1 was examined using siRNA specific to epicardin/capsulin/Pod-1. Transfection of MG63 cells with
epicardin/capsulin/Pod-1 siRNA along with the epicardin/capsulin/Pod-1
expression vector relieved epicardin/capsulin/Pod-1-mediated
suppression of E12-dependent activation of the p21 promoter
(Fig. 3F), in agreement with reduction of the expression of
epicardin/capsulin/Pod-1 at the protein level (Fig. 3E).
Introduction of epicardin/capsulin/Pod-1 siRNA alone induced little, if
any, change in p21 expression (Fig. 3F).
Transcriptional Inhibition of the MCK Gene by
Epicardin/Capsulin/Pod-1--
Expression of
epicardin/capsulin/Pod-1 and
MyoR has been shown to overlap in head muscle precursors,
and the lack of an appreciable change in phenotype of the head
musculature in
epicardin/capsulin/Pod-1 mutant mice
(36) raises the possibility of functional redundancy between
epicardin/capsulin/Pod-1 and MyoR. We thus investigated the
transcriptional regulatory mechanism that involves
epicardin/capsulin/Pod-1 in myofibroblast differentiation beyond the
cell cycle arrest. Terminal differentiation of skeletal myoblasts is
accompanied by induction of a series of tissue-specific genes including
the MCK gene. MyoD and E12 have been shown to efficiently
transactivate the MCK gene in a cooperative manner (20, 37).
The effects of epicardin/capsulin/Pod-1 on expression of the
MCK gene were examined. 10T1/2 cells were cotransfected with
a luciferase reporter construct pMCK-luc containing the 1.3-kb promoter
sequence of the MCK gene, along with pCMV-E12, pCMV-MyoD,
and pCMV-epicardin. The epicardin/capsulin/Pod-1 expression plasmid was
added at concentrations over a range equivalent to those for Id1 and
Id2 which showed inhibition of muscle differentiation through
suppression of production of molecules, such as MCK (20, 38, 39) and
p204 (40). Epicardin/capsulin/Pod-1 reduced luciferase activity
driven by the MCK promoter in response to E12 and MyoD
in a dose-dependent manner (Fig.
5, A and B).
Similar results were obtained in the regulation of p21 and
MCK expression in C2C12 myoblasts (Figs. 3D and
5C). These results demonstrate that the MCK gene
is negatively regulated by epicardin/capsulin/Pod-1, similar to the p21
gene. Regulation of the MCK promoter was also examined with
epicardin/capsulin/Pod-1 siRNA. The addition of siRNA partly prevented
epicardin/capsulin/Pod-1-mediated suppression of the MCK
promoter (Fig. 5D), similar to the effect of siRNA on the
p21 promoter (Fig. 3F).

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Fig. 5.
Inhibition of MCK transcription by
epicardin/capsulin/Pod-1. A, inhibition of E12- and
MyoD-dependent transactivation of MCK by
epicardin/capsulin/Pod-1. 10T1/2 cells were cotransfected with 2.5 µg
of pMCK-luc in combination with pCMV-E12, pCMV-MyoD, pCMV-epicardin,
pCMV-Twist, and pCMV-Id1. The cells were harvested 48 h after
transfection and then assayed for reporter gene expression.
B, inhibition of MyoD- and E12-dependent
transactivation of MCK by epicardin/capsulin/Pod-1 in a
dose-dependent manner. 10T1/2 cells were cotransfected with
pMCK-luc, pCMV-E12, and pCMV-MyoD in combination with the indicated
amounts of either pCMV-epicardin, pCMV-Id1, or pCMV-Id2. The cells were
harvested 48 h after transfection and then assayed for reporter
gene expression. Empty vector pcDNA3 was included to adjust DNA
amounts. C, inhibition of MyoD- and
E12-dependent transactivation of MCK by
epicardin/capsulin/Pod-1 in C2C12 myoblasts. C2C12 myoblasts were
cotransfected with 2.5 µg of pMCK-luc in combinations with pCMV-E12,
pCMV-MyoD, and pCMV-epicardin. The cells were cultured in DMEM
containing 10% FBS for 48 h and collected for the luciferase
assay. Activation mediated by pcDNA3 is arbitrarily set at 1. D, abolishment of epicardin/capsulin/Pod-1-mediated E12
inhibition by epicardin/capsulin/Pod-1 siRNA. Five hours prior to siRNA
transfection, C3H10T1/2 cells were cotransfected with 1.0 µg of
pMCK-luc in combination with 1.0 µg of pCMV-E12 and
pCMV-Myc-epicardin. Empty vector pCMV-Myc was included to adjust DNA
amounts. E, physical association of epicardin/capsulin/Pod-1
to the MCK promoter. Chromatin immunoprecipitated from C2C12
cells transfected with pHA-epicardin and pMCK-luc was subjected to PCR
with primers specific to the MCK promoter. A portion (10%)
of total amounts of chromatin prior to immunoprecipitation was also
subjected to PCR (Input). Anti-Myc antibody was used as an
irrelevant control antibody (Mock).
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Binding of Epicardin/Capsulin/Pod-1 to the
MCK Promoter in Vivo--
Because epicardin/capsulin/Pod-1 was
initially found as a molecule physically interacting with E12 (Fig.
1B), integration of epicardin/capsulin/Pod-1 into a complex
with the MCK promoter would be expected. To address this
view, we further investigated the recruitment of
epicardin/capsulin/Pod-1 to the MyoD-dependent MCK enhancer
by the ChIP assay in vivo. Soluble chromatin from C2C12
transfected with pHA-epicardin and pMCK-luc was immunoprecipitated with
an antibody specific to the HA epitope, and was examined for the
presence of the MyoD-binding site by PCR. PCR produced only one band
with a molecular size of 162 bp (Fig. 5E), and DNA sequencing confirmed that the product contained the MyoD-binding sequence in the MCK promoter. The soluble chromatin treated
with an antibody specific to the Myc epitope did not produce any PCR products (Fig. 5E). These results demonstrate that
epicardin/capsulin/Pod-1 binds to the MCK promoter in C2C12 cells.
Inhibition of Muscle Gene Expression by
Epicardin/Capsulin/Pod-1--
The results in
this study convinced us that the induced expression of
epicardin/capsulin/Pod-1 could interfere with the activation of
endogenous muscle-specific genes. To examine this possibility, C2C12
myoblasts capable of differentiation to myotubes were transfected with
pCMV-epicardin along with pCMV-
-Gal and then cultured in the
differentiation medium. Induction of epicardin/capsulin/Pod-1 greatly
inhibited expression of TnT, a marker of differentiated muscle cells.
Immunostaining analysis exhibited that epicardin/capsulin/Pod-1 expression decreased a percentage of TnT-positive cells to 26.6% among
cells expressing
-galactosidase, whereas nearly all cells expressing
-galactosidase were positive for TnT in a mock empty vector
transfection experiment (Fig. 6). In all
experiments,
-galactosidase-negative cells differentiated to muscle
with expression of TnT to a similar extent. Thus, the results indicate
that the terminal differentiation to myotubes of C2C12 cells is greatly
suppressed by epicardin/capsulin/Pod-1 presumably via its inhibitory
function on the expression of genes specific to muscle cells.

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Fig. 6.
Effect of epicardin overexpression on
muscle-specific gene expression. C2C12 cells were transfected in
the growth medium with pCMV empty vector or pCMV-epicardin together
with the -galactosidase ( -gal) expression vector and
then induced to differentiate in the differentiation medium for 96 h. After fixation in paraformaldehyde, productively transfected cells
were visualized by the expression of cotransfected -galactosidase
(green). Myogenic differentiation was scored by determining
expression of TnT (red) in -galactosidase-positive cells.
Scale bars, 20 µm. Vector,
pcDNA3-transfected cells; Epicardin,
pCMV-epicardin-transfected cells.
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DISCUSSION |
In this study we found that epicardin/capsulin/Pod-1 acts as a
transcriptional suppressor of E2A-induced gene expression. The
inhibitory effect was seen on the expression of p21 and MCK, which are
activated by E2A proteins and by the combination of E12 and MyoD,
respectively. Physical interaction of epicardin/capsulin/Pod-1 with the
bHLH domain of E12 was shown by the yeast and mammalian two-hybrid
methods in addition to the demonstration of colocalization of
epicardin/capsulin/Pod-1 and E12 in the nuclei. Moreover, ectopic expression of epicardin/capsulin/Pod-1 blocked the terminal
differentiation of C2C12 myoblasts. Collectively,
epicardin/capsulin/Pod-1 is involved in the inhibition of
differentiation through the regulation of the expression of genes
related to cell cycle arrest and of tissue-specific differentiation.
Little is known about the transcriptional suppression which regulates
early processes of organogenesis. Our results provide a hypothesis that
epicardin/capsulin/Pod-1 normally suppresses differentiation through
inhibition of cell cycle arrest by regulating p21 expression,
presumably resulting in precursor cells remaining in an
undifferentiated state. This in turn leads to the expansion of
precursor cells for the generation of the organ.
Epicardin/capsulin/Pod-1 is expressed in brachial muscle
precursors and mesenchymal cells at sites of epithelial-mesenchymal
interactions in various organs (8, 10, 36). In this study, we showed
that myoblast differentiation was inhibited by
epicardin/capsulin/Pod-1-mediated suppression of p21 expression.
Regulation of expression of the p21 gene is presumably involved in the
differentiation of other cell lineages. Indeed, we have previously
reported that bHLH proteins regulate p21 expression and osteoblast
differentiation (14). Because E12 has been shown to activate p16 and
p15 (41) besides p21, epicardin/capsulin/Pod-1 may participate in the
regulation of other Cdk inhibitors, and such a regulation may be
essential for differentiation. We observed that, in 10T1/2 cells,
introduction of p21 alone could not rescue
epicardin/capsulin/Pod-1-mediated inhibition of MCK promoter
activity induced by E12 and MyoD (data not shown). Our observation is
compatible with a previous report (42) that ectopic expression of
either p21 or p16 partially reversed the inhibitory effect of the
growth medium on the MCK promoter and that coexpression of
p21 and p16 resulted in the reverse of the suppressed MCK
promoter activity. Previous studies demonstrated that the Cdk
inhibitors are implicated in myogenesis; p21 and p16 promote
differentiation (42), p21, p27, p57, and p18 are all up-regulated
coincident with terminal growth arrest (17-19, 30), and the expression
of p21 and p57 is essential for muscle differentiation during mice
embryonic development (19). Simultaneously, tissue-specific gene
expression is inhibited by epicardin/capsulin/Pod-1, resulting in
suppression of the organ development. A set of lineage
differentiation-specific genes obviously differs from tissue to tissue
and seems to be commonly regulated by transcriptional signals from
tissue-specific master molecules, such as MCK for myoblasts and FGFR3
for osteoblasts. In addition, epicardin/capsulin/Pod-1 has been
reported to suppress expression of Ad4BP/SF-1, a zinc finger
transcription factor, which plays important roles in gonadogenesis
(43).
Epicardin/capsulin/Pod-1-meidiated suppression of transcription may be
selective within genes that are regulated by E2A. The expression of
epicardin/capsulin/Pod-1 seems to be more restricted than that of E2A
in terms of location and timing during differentiation (7, 8). This may
be a mechanism underlying the selective suppression by
epicardin/capsulin/Pod-1. Related molecules have been shown to function
selectively depending on promoters. OUT, structurally related to
epicardin/capsulin/Pod-1, inhibited the induction of
E-box-dependent MCK promoter activation by
MyoD-E12 heterodimers, whereas similar bHLH factors, neural bHLH TAL2, and placental bHLH Mash2 failed to inhibit the MCK expression (44). It
may also be possible that the specificity of action of
epicardin/capsulin/Pod-1 is dependent on the context of DNA elements in
a promoter; interaction of epicardin/capsulin/Pod-1 with another factor
that binds different DNA elements in the same promoter may be
important. Alternatively, there may be an unidentified mechanism by
which epicardin/capsulin/Pod-1 selects an E-box in E2A-targeted genes,
as shown in the case of MyoD, which selectively binds some of the
E-boxes (45).
It is also possible that epicardin/capsulin/Pod-1 may inhibit
apoptosis. Some bHLH factors are implicated in inhibition of apoptosis
by negative regulation of transcription. Twist and Dermo-1, which
suppresses transcriptional activity of myogenic bHLH proteins (46),
have been shown to inhibit oncogene-dependent and
p53-dependent cell death (47). This notion may be supported
by the observation that apoptosis was readily detected in the
presumptive splenic forming region of
epicardin/capsulin/Pod-1 mutant
embryos, whereas only random and occasional apoptosis was observed in
the internal organs of wild-type embryos (11).
The importance of negative regulation by bHLH factors has been
emphasized in cell differentiation of Drosophila and mice. Inactivation of twist results in defective dorso-ventral
patterning due to disturbed gastrulation in Drosophila (48)
as well as defects in cranial neural tube closure and mesodermal
derivatives in mice (49). Saethre-Chotzen syndrome
(acrocephalosyndactyly type III; OMIM 101400), characteristic of skull
deformity due to craniosynostosis, is caused by mutations in the gene
encoding Twist (50, 51), which is a transcriptional suppressor for the
p21 and FGFR3 genes in osteoblasts (14).
The asplenic phenotype of
epicardin/capsulin/Pod-1 mutant mice
resembles those of mice lacking Bapx1 (52, 53) and
Hox11 (54, 55), as well as the Wilm's tumor suppressor
gene, WT-1 (56). They are both coexpressed with
epicardin/capsulin/Pod-1 in the spleen during development and function
as essential regulators of spleen organogenesis. These genes may
constitute a genetic cascade that acts in concert to regulate a common
early event in spleen organogenesis. In the developing lung,
epicardin/capsulin/Pod-1 in the mesenchyme has been shown to be
required to activate expression of bone morphogenetic protein-4 (BMP-4)
in the adjacent epithelium; in the absence of BMP-4 expression, the
airway epithelium fails to differentiate (12). This result together
with our findings suggest that epicardin/capsulin/Pod-1 acts as a
transcriptional suppressor and raises the possibility that
epicardin/capsulin/Pod-1 does not directly regulate expression of
BMP-4, rather that it is implicated in regulation upstream of an
unknown transcription factor that suppresses transcription of
BMP-4.
Among bHLH members, epicardin/capsulin/Pod-1 shows a high degree of
homology to a group of bHLH factors that are expressed in tissues of
mesodermal origin. In particular, epicardin/capsulin/Pod-1 is closely
related to MyoR (57) with 94.5% identity in the bHLH region at the
amino acid level, presumably generating a subfamily within the bHLH
factors. MyoR acts in undifferentiated skeletal myoblasts as a potent
transcriptional suppressor that can block myoblast differentiation by
interfering with the activity of MyoD (36, 57). The expression of
epicardin/capsulin/Pod-1 and
MyoR overlaps in head muscle precursors (7, 57), suggesting
functional redundancy between
epicardin/capsulin/Pod-1 and
MyoR. This notion may be supported by the phenotypic
observation made in
epicardin/capsulin/Pod-1 mutant mice,
which have no defect in head musculature (11). Our preliminary
experiments showed that epicardin/capsulin/Pod-1 siRNA did not affect
p21 in MG63 cells and TnT and myosin heavy chain in C2C12 cells,
markers of myoblast differentiation. This may result from functional
redundancy between
epicardin/capsulin/Pod-1 and
TWIST, Id1, or MyoR.
In a broader context, our study demonstrates that
epicardin/capsulin/Pod-1 inhibits cell cycle arrest and tissue-specific gene induction, resulting in regulation of cell differentiation. Identification of other molecules that bind to epicardin/capsulin/Pod-1 and elucidation of the functional significance of the interaction of
those molecules represent important issues for the future.