From the Department of Molecular Oncology, Tokyo Medical and Dental University, Graduate School of Medicine and Dentistry, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Received for publication, December 2, 2002, and in revised form, February 10, 2003
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ABSTRACT |
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An EID-1 (E1A-like
inhibitor of differentiation-1)
inhibits differentiation by blocking the histone acetyltransferase
activity of p300. Here we report a novel inhibitor of differentiation
exhibiting homology to EID-1, termed EID-2 (EID-1-like
inhibitor of differentiation-2). EID-2 inhibited MyoD-dependent transcription and muscle
differentiation. Unlike EID-1, EID-2 did not block p300 activity.
Interestingly, EID-2 associated with class I histone deacetylases
(HDACs). The N-terminal portion of EID-2 was required for the binding
to HDACs. This region was also involved in the transcriptional
repression and nuclear localization, suggesting the importance of the
involvement of HDACs in the EID-2 function. These results indicate a
new family of differentiation inhibitors, although there are several
differences in the biochemical mechanisms between EID-2 and EID-1.
The terminal differentiation program is regulated both positively
and negatively. Among various tissues, skeletal muscle is one of the
most well studied in terms of differentiation regulation (1, 2).
Several fate-determining transcription factors regulate the expression
of tissue-specific proteins. As for skeletal muscle differentiation,
ubiquitously expressed basic helix-loop-helix (bHLH)1 transcription factors
such as E proteins heterodimerize with the MyoD family of myogenic bHLH
transcription factors, consisting of MyoD, Myf5, myogenin, and MRF4
(2). These heterodimers bind to the canonical E-box (CANNTG) sequences
of the promoter regions of muscle-specific genes and up-regulate
transcription. Other factors that dimerize with MyoD family proteins
are non-bHLH MCM1, agamous, deficiens serum response partner
(MADS)-box proteins, myocyte enhancer factors (MEFs). This group
consists of MEF2A, -B, -C, and -D (1-3). These tissue-specific
transcription factors recruit coactivators such as
p300/cAMP-response element-binding protein (CBP)-binding protein
and P300/CBP-associated factor (PCAF) and then activate
transcription (2).
In addition to the above-mentioned positive regulators, several
negative regulators for muscle differentiation have been found to date
(2). The Id family consists of four members, namely, Id1, -2, -3, and
-4 (4). Id proteins are HLH proteins that lack DNA-binding
domains. Therefore, they can heterodimerize with bHLH proteins but are
unable to bind to DNA (5); hence Id proteins act as dominant negative
regulators of bHLH proteins.
Histone deacetylases (HDACs) are known to maintain core histones in a
hypoacetylated state, resulting in transcriptional repression. HDACs
comprise three classes, namely RPD3-like HDACs (class I), HDA1-like
HDACs (class II), and newly identified SIR2-like HDACs (class III) (6).
Both class I and class II HDACs exhibit histone deacetylase activities
at the C-terminal portions, but class I HDACs lack an N-terminal
extension. Class I HDACs bind to MyoD and repress transcription (7).
Class II HDACs bind to MEF2 via its N-terminal MEF2-binding domain and
repress transcription (8-11). Class II HDACs are localized to the
nuclei of myoblasts and are exported to the cytoplasm with
differentiation signals (12, 13).
Recently, we and others (14, 15) identified a novel negative regulator
of differentiation, termed EID-1 (E1A-like
inhibitor of differentiation-1), as
a pRB- and p300-binding protein (14, 15). EID-1 inhibits
myogenic differentiation by blocking the histone acetyltransferase
(HAT) activity of p300/cAMP-response element-binding protein-binding
protein (14, 15). This molecule has also been reported as RBP21, which
interacts with pRB (16). EID-1 exhibits no homology to known proteins
including bHLH factors and HDACs. Functionally, EID-1 exhibits
similarity with adenovirus E1A or twist in terms of
inhibition of HAT activity (14, 15, 17).
In this study, we identified a new EID-1 family member, termed EID-2
(EID-1-like inhibitor of
differentiation-2). EID-2 was mainly expressed
in heart, skeletal muscle, kidney, and liver. EID-2 inhibited
MyoD-dependent transcription and blocked muscle differentiation in cultured cells like EID-1. However, EID-2 neither bound to p300 nor inhibited p300-dependent transcription.
Interestingly, EID-2 associated with class I HDACs. This property was
correlated with the ability to repress transcription and its nuclear
localization. These results indicate that EID-2 and EID-1 exhibit
homology and a similar phenotype but have distinct mechanisms in terms
of inhibition of differentiation.
Cell Culture and Transfection--
U-2OS osteosarcoma cells and
10T1/2 murine fibroblasts were grown in Dulbecco's modified
Eagle's medium (Sigma) supplemented with 10% heat-inactivated fetal
bovine serum (FBS), 100 units/ml of penicillin, and 100 µg/ml of
streptomycin (PSG; Invitrogen). C2C12 murine myoblasts were
grown in Dulbecco's modified Eagle's medium supplemented with 20%
FBS and PSG. To induce differentiation, C2C12 cells were grown in
Dulbecco's modified Eagle's medium containing 2% horse serum for 3 days once they had become confluent. Transfection was performed with
TransIT-LT1 (Mirus) according to the manufacturer's instructions.
Plasmids--
pCMV, pcDNA3, pcDNA3-T7-EID-1, pSG5,
pSG5-TetR, pSG5-TetR-EID-1, pMCK-luciferase (15), pUHC13.3 (18),
pCMV-MyoD (19), 3xGal4-luciferase (20), pCMV-Gal4-p300 (21),
pcDNA-FLAG- HDAC1, and pME18S-FLAG-HDAC2 (22) were described
previously. pRL-SV40-luciferase and pGEM-T Easy were purchased from
Promega. EID-2 cDNA fragments were generated by the PCR
reaction using a fetal brain cDNA library (Clontech) as a template. The 5'-primer
(CTATTCGATGATGAAGATAC) was designed as a sense oligonucleotide of the
pGAD10 vector, and the 3'-primer (AAGTAGTGTCACCACATAAC) was designed as
an antisense oligonucleotide whose sequence was obtained from the EST
data base of a putative EID-1-related gene. The PCR product
was purified and subcloned into the pGEM-T Easy vector (Promega) and
sequenced on both strands. The EID-2 cDNA sequence was
deduced by comparing the DNA sequences of multiple overlapping clones.
EID-2 cDNAs encoding the wild-type EID-2, or mutants
thereof, were obtained by PCR using oligonucleotides that introduced a
5'-BamHI site and a 3'-EcoRI site. To obtain
internal deletion mutants, a two-step PCR strategy was used, as
described previously (23). The PCR products were restricted with
BamHI and EcoRI and then subcloned into
pcDNA3-T7, pSG5-TetR (15), and DsRed2
(Clontech), which had been linearized with these
two enzymes or BglII and EcoRI in case of DsRed2.
All of the PCR products were confirmed by DNA sequencing.
Oligo-capping Method--
To determine the transcriptional start
sites for the human EID-2 gene, we used the oligo-capping
method described previously (24). In brief, mRNA was purified from
HepG2 human hepatocellular carcinoma cell lines followed by exchanging
of the cap structure with the adaptor oligonucleotide and reverse
transcription. The cDNA was amplified by nested PCR using a sense
primer of the adaptor sequence and an antisense primer of
EID-2 open reading frame. The first primer set was as
follows: 1st sense adaptor oligo, ATGAGCATCGAGTCGGCCTTG; 1st antisense
EID-2-F, TTCCCGTGTCTGGACCTGGG; 2nd sense adaptor oligo,
AGCATCGAGTCGGCCTTGTTG; 2nd antisense EID-2-G,
TGCCCTGGCCGCCGCCATCC. The PCR fragment was ligated into the pGEM-T Easy
vector and then sequenced on both strands.
Northern Blot Analysis--
Northern blotting was performed with
a multiple tissue northern blot (Clontech) and
detected with a digoxigenin-labeling system (Roche Molecular
Biochemicals) according to the manufacturer's instructions. The EID-2 DNA probe was generated with the PCR
DIG labeling mix (Roche Molecular Biochemicals) using a
sense primer, GGTGCCGGCGGCCAGGGCAG, and an antisense primer,
TTCCCGTGTCTGGACCTGGG. The PCR product was flanked by nucleotides 162 and 392 of EID-2.
Luciferase Assay--
For the TetR-fusion protein
transactivation assay, 50% confluent U-2OS cells were transiently
transfected in 24-well plates in duplicate with 25 ng of pRL-SV40, 50 ng of pUHC-13-3 reporter plasmid, and increasing amounts of
pSG5-TetR-EID-1 or pSG5-TetR-EID-2. Sufficient parental pSG5 was added
so that each reaction mix contained the same amount of pSG5 backbone.
Forty-eight h after transfection, the cells were lysed. Firefly
luciferase activity and Renilla luciferase activity in the
cell extracts were determined by the dual-luciferase reporter assay
(Promega) according to the manufacturer's instructions.
For Gal4-p300 transactivation experiments, 50% confluent U-2OS cells
were transiently transfected in 24-well plates in duplicate with
25 ng of pRL-SV40, 50 ng of pGal4-luciferase reporter plasmid, 50 ng of pGal4-p300, and increasing amounts of pcDNA3-T7-EID-1 or
pcDNA3-T7-EID-2. Sufficient parental pcDNA3 was added so that each reaction mix contained the same amount of pcDNA3 backbone. Cell extracts were prepared 48 h after transfection.
For MyoD transactivation experiments, 50% confluent 10T1/2 cells were
transiently transfected in 24-well plates, in duplicate, with 25 ng of
pRL-SV40, 50 ng of pCMV-MyoD, 50 ng of pMCK-luciferase reporter, and
increasing amounts of pcDNA3-T7-EID-2. Sufficient parental
pcDNA3 was added so that each reaction mix contained the same
amount of pcDNA3 backbone. Cell extracts were prepared 48 h
after transfection. Trichostatin A (TSA; Sigma) was added to the
medium 24 h after transfection at the indicated concentration.
Immunoprecipitation and Immunoblot Analyses--
Cells were
lysed in EBC buffer as described previously (25). Protein
concentrations were determined by the Bradford method (Bio-Rad).
Immunoprecipitation assays of extracts prepared from transfected cells
contained 2 mg of cell extract and 1 µg of anti-T7 (Novagen) antibody
or 1 µg of anti-FLAG (M2; Sigma) antibody, in a final volume of 0.5 ml. Following 1 h of incubation at 4 °C with rocking, the
Sepharose was washed five times with NETN. Bound proteins were
eluted by boiling in SDS-containing sample buffer, resolved by
SDS-polyacrylamide gel electrophoresis and then transferred to a
nitrocellulose filter.
Nitrocellulose filters were blocked in 5% powdered milk in
Tris-buffered saline containing Tween 20 for 1 h at room
temperature prior to incubation with the primary antibody.
Anti-troponin T (JLT-12; Sigma) was used at 1:200 (v/v), anti-tubulin
(B-5-1-2; Sigma) at 1:2000 (v/v), and anti-T7 (Novagen) at a
concentration of 0.2 µg/ml. Following four washes with Tris-buffered
saline containing Tween 20, the bound antibody was detected with
alkaline phosphatase-conjugated secondary antibodies and with
Immun-Star (Bio-Rad) according to the manufacturer's instructions.
Flow Cytometry--
Flow cytometry was performed essentially as
described (26). C2C12 cells stably transfected with pcDNA3 or
pcDNA3-T7-EID-2 were harvested when they were 50 or 100% confluent
or grown in differentiation medium for 3 days, respectively. Samples
were analyzed with a FACSCalibur (BD Biosciences), and the data were analyzed with ModFit LT (BD Biosciences).
Identification and Cloning of EID-2--
According to EST
sequences from a data base, it is suggested that an
EID-1-related gene exists (National Center for Biotechnology Information BLAST Searches, accession numbers AA203157 and AA447078). To obtain a cDNA of the putative
EID-1-related gene, we performed PCR using a human fetal
brain cDNA library (Clontech) as a template.
The 5'-primer was designed as part of the sequence of the pGAD10 vector
upstream of the multi-cloning site, and the 3'-primer was designed as a
sequence corresponding to the putative end of the open reading frame of
the EID-1-related gene obtained from the data base. The PCR
product generated gave a relatively broad single band and was purified
from the gel. The purified PCR product was ligated into the pGEM-T Easy
vector (Promega) and then Escherichia coli was
transformed with this construct. Six independent clones were obtained,
which contained overlapping fragments of the same cDNA, hereafter
called EID-2. All of them contain ATG in-frame with the stop
codon. The deduced polypeptides consist of 236 amino acids, and
homology to EID-1 was observed in both the N and C termini (Fig.
1A). Neither of the six clones had an in-frame stop codon upstream of ATG. Then we used the
oligo-capping method (24) to determine the transcriptional start site,
as described under "Experimental Procedures." We obtained a
cDNA containing 51 additional nucleotides upstream of ATG of
EID-2 (data not shown). There was no additional ATG in this
portion, supporting that the EID-2 sequence contains the
full-length open reading frame.
The pRB-binding motif (LXCXE, where X
equals any amino acid residue), which exists in the C-terminal of
EID-1, is replaced by LXCXK in EID-2 (Fig. 1,
A and B), suggesting that EID-2 may not bind to
pRB via this motif. EID-2 had an alanine-rich region in the middle
instead of the acidic regions observed in EID-1 (Fig. 1B)
(15). We then performed Northern blot analysis to determine the tissue
distribution of EID-2 mRNA. An ~5.0-kb transcript was
detected abundantly in heart, skeletal muscle, kidney, and liver (Fig.
1C, lanes 2, 3, 7, and
8). The EID-2 gene has been mapped to chromosome
19q as an UniGene Cluster Hs.18949 (NCBI).
EID-2 Has a Potential Transrepression Domain--
We showed
previously (14, 15) that EID-1 had a potential transactivation domain
because of its p300-binding property. To determine whether EID-2 has a
similar property to EID-1 in terms of activation of transcription as a
fusion protein with a heterologous DNA-binding domain, EID-2 was fused
to the DNA-binding domain of the TetR, followed by scoring the
ability to activate or repress transcription from the luciferase
reporter plasmid. Surprisingly, although TetR-EID-1 caused an increase
in reporter activity (Fig. 2A,
left panel) (15), TetR-EID-2 caused a modest but
reproducible decrease in reporter activity (Fig. 2A,
right panel). In agreement with this result, EID-2 did not
bind to p300 in vivo, as determined by means of a mammalian
two-hybrid assay (data not shown).
Previous reports showed that EID-1 blocked the HAT activity of p300,
which caused inhibition of p300-mediated transcription (14, 15). To
determine whether EID-2 can block p300-mediated transcription like
EID-1, U-2OS cells were transfected with a reporter plasmid containing
Gal4-DNA-binding sites and a plasmid encoding Gal4 fused to the
full-length p300 in the presence of a plasmid encoding EID-1 or EID-2.
EID-1 inhibited transactivation by p300, whereas EID-2 had no effect on
p300-mediated transcription (Fig. 2B). These results suggest
that EID-1 and EID-2 have distinct mechanisms as to transcriptional repression.
The N-terminal Portion of EID-2 Is Involved in the Inhibition of
MyoD-mediated Transactivation--
EID-1 inhibits transcription by
certain fate-determining proteins such as MyoD (14, 15). To determine
whether EID-2 inhibits the activities of such proteins, 10T1/2 murine
fibroblasts were transiently transfected with a plasmid encoding EID-1
or EID-2, along with a MyoD expression plasmid and a reporter plasmid
containing the MCK promoter. This promoter is activated by MyoD during
myogenic differentiation. As determined by a luciferase assay, both
EID-1 and EID-2 inhibited the MyoD-dependent
transactivation (Fig. 3B).
We tried to determine which region is responsible for the
transrepression activity. As shown in Fig. 1, EID-2 and EID-1 exhibit similarities in three distinct portions, namely, one N-terminal and two
C-terminal regions. To this end we produced various truncation mutants
lacking either the N terminus or C terminus, as well as an internal
deletion mutant lacking the central alanine-rich region (Fig.
3A). All of the EID-2 mutant proteins in these studies were produced at comparable levels, as determined by immunoblot analyses (data not shown). The wild-type EID-2 inhibited
MyoD-dependent transcription, and the various C-terminal
truncation mutants and the internal deletion mutant still retained the
same inhibitory property. In contrast, all of the N-terminal deletion
mutants in this assay showed impaired transrepression ability (Fig.
3B). These results indicated that the N-terminal portion of
EID-2 is involved in the inhibitory function as to
MyoD-dependent transcription.
EID-2 Inhibits Muscle Differentiation of Cultured Cells--
As
EID-1 inhibits muscle differentiation of cultured cells, C2C12
myoblasts were stably transfected with EID-2 DNA so as to produce the wild-type EID-2 protein in the next set of experiments. Totally, five clones with lower expression and three clones with higher
expression were obtained. The protein levels of ectopically produced
EID-2 in representative clones (clones 8 and 15 for lower and higher
expression, respectively) were determined by immunoblotting (Fig.
4A). It is noteworthy that the
EID-2 protein levels were not changing during the course of
differentiation. In contrast, the EID-1 protein level decreased with
differentiation signal because of protein degradation via the
ubiquitin-proteasome pathway (15).
Both the low and high expressing clones were analyzed for myotube
formation (Fig. 4B). There were almost no microscopic
difference between the clones in both sparse and confluent cultures in
growth medium (GM) (Fig. 4B, a, b,
d, e, g, and h). However,
the clones transfected with an empty vector formed myotubes (Fig.
4B, c) upon a shift to differentiation medium
(DM) and expressed markers of muscle differentiation (Fig.
4A, lane 2), whereas the clones producing lower
level of wild-type EID-2 did not (Fig. 4, A, lane 4 and B, f). The higher expression clones
exhibited marked cell death upon a shift to DM (Fig. 4, B,
i and C, l). These results indicated
that EID-2 had a similar phenotype to EID-1, that is, inhibition of
muscle differentiation, when it was ectopically expressed in myoblasts,
even though EID-2 did not have the ability to inhibit p300-mediated
transactivation (Fig. 2B).
EID-2 Does Not Inhibit Cell Cycle Arrest with Differentiation
Signal--
Terminal differentiation and cell cycle arrest are closely
related. Therefore, we determined the cell cycle profiles of C2C12 myoblasts expressing different levels of EID-2. Both in sparse and
confluent cultures in GM, there was almost no difference between the
clones with empty vector and EID-2-expressing cells in terms of the
cell cycle profiles (Fig. 4C, a, b,
e, f, i, and j). After incubation in DM for 3 days, the G1/G0
population of each clone exhibited no difference (Fig. 4C,
c, g, and k). Note that only a few
cells of both the vector transfectant and the lower expresser of EID-2
(clone 8) exhibited cell death (Fig. 4C, c,
d, g, and h); however, for the higher
expresser of EID-2 (clone 15), the number of dead cells increased
dramatically (Fig. 4C, k and l).
N-terminal Portion of EID-2 Is Required for Nuclear
Localization--
As transcriptional regulation occurs mainly in the
nucleus, we next asked whether EID-2 was localized in the nucleus or
the cytoplasm. We transfected U-2OS cells with plasmids encoding red fluorescent protein (DsRed2) or fusion proteins of EID-2 and its mutants (Fig. 5). DsRed2 itself was
localized both in the nucleus and the cytoplasm (Fig. 5, a).
The fusion protein of wild-type EID-2 to DsRed2 was localized
exclusively in the nucleus; however, the fusion protein of the
N-terminal deletion mutant, EID-2 (33-236), was localized exclusively
in the cytoplasm (Fig. 5, b and c). On the other
hand, the fusion proteins of C-terminal deletion mutants still remained
in the nucleus (data not shown). Therefore, the N terminus of EID-2 was
required for nuclear localization.
EID-2 Binds to Class I HDACs in Vivo--
Previous reports
described that HDACs are involved in muscle differentiation (7-11).
The data that EID-2 had a potential transrepression domain prompted us
to examine a possible interaction between EID-2 and HDACs. To determine
whether EID-2 can bind to class I HDACs, U-2OS cells were cotransfected
with plasmids encoding T7 epitope-tagged EID-2, or mutants thereof,
along with a plasmid encoding FLAG epitope-tagged HDAC1 or HDAC2. HDAC
binding to EID-2 was scored by means of anti-T7 immunoblot analysis
of anti-FLAG immunoprecipitates (Fig.
6A). The wild-type and
C-terminal deletion mutants of EID-2 interacted with both HDAC1 and
HDAC2 in vivo, whereas the N-terminal deletion mutants of
EID-2 (33-236, 47-236, 101-236) did not (Fig. 5A) (data
not shown). Next we examined whether the transcriptional repression by
EID-2 was recovered by treatment with an HDAC inhibitor, TSA. As shown
in Fig. 6B, TSA treatment recovered the EID-2-mediated transcriptional repression in a dose-dependent manner. This
result supported that HDAC activity may be involved in the
transrepression activity of EID-2.
In this study, we identified a novel inhibitor of differentiation,
EID-2, which exhibited homology to EID-1. EID-1 and EID-2 showed no
homology to any other known proteins. Neither of them exhibited the
characteristic structures of the known negative regulators of muscle
differentiation. Thus EID-1 and EID-2 comprise a novel family of
inhibitors of differentiation with distinct functions from those of
known negative regulators of differentiation.
In terms of subcellular localization of proteins, the wild-type EID-2
was localized exclusively in the nucleus, whereas the EID-2 mutant
lacking N-terminal 32 amino acids was localized in the cytoplasm.
Because the cytoplasmic localization of the mutant protein was not
affected by the treatment with leptomycin B, an inhibitor of nuclear
export,2 we speculated that
the mutant protein was not able to enter the nucleus, suggesting
the existence of the nuclear localization signal in the N-terminal
portion of EID-2. In contrast, wild-type EID-1 was localized mainly in
the cytoplasm (27).2 However, when the cells were treated
with leptomycin B, EID-1 was found in the nucleus. These data suggested
that EID-1 was originally localized in the nucleus and exported rapidly
to the cytoplasm (27).2 Interestingly, the EID-1 mutant
lacking C-terminal 30 amino acids was localized in the nucleus even
without leptomycin B treatment.2 These data indicated the
difference between EID-2 and EID-1 as to the subcellular localization.
A fusion protein comprising EID-2 and a heterologous DNA-binding domain
exhibited transcriptional repression instead of the activation in the
case of EID-1, which reflects its p300-binding ability (15). EID-1
inhibited transcription caused by fate-determining transcription
factors such as MyoD by blocking the HAT activity of p300. This
inhibitory effect on HAT was mediated by both the C-terminal
p300-binding domain and the middle acidic clusters of EID-1 (15). EID-2
also inhibited MyoD-dependent transactivation. However,
EID-2 neither bound to p300 nor blocked p300-dependent transcription. Deletion analyses showed that the N-terminal region of
EID-2 was involved in the inhibition of MyoD-mediated transcription. Interestingly, both the N-terminal portion of EID-1 and the C-terminal region of EID-2 were dispensable for the inhibitory function as to
transcriptional repression. Thus the mechanisms of transrepression and
differentiation inhibition of EID-2 may be distinct from those of
EID-1.
EID-2 interacted with HDACs, which have an opposite effect on
transcription to p300. The N-terminal region of EID-2 was necessary for
this interaction. EID-1 interacted with p300 via its C-terminal region
but not with HDACs, and EID-2 interacted with HDACs via its N-terminal
region but not with p300, despite these two putative family proteins
exhibited homology in both the N and C termini. The data that the
transrepression activity of EID-2 was alleviated by TSA treatment
supported the physiological importance of the interaction between EID-2
and HDACs.
Like EID-1, EID-2 blocked myogenic differentiation of cultured cells
when stably introduced into murine C2C12 myoblasts (Fig. 4). A previous
report (26) indicated that cell cycle arrest and differentiation could
be separated, although they are closely linked. Our results support
this observation and suggest that EID-2 mainly causes inhibition of
differentiation but not cell cycle progression, which was also observed
in the case for EID-1 (15). In addition to inhibiting differentiation,
EID-2 induced cell death when the protein level was high (Fig. 4). It
remains to be clarified whether this phenomenon is physiologically relevant.
As for the mechanism of the inhibition of differentiation by EID-2, the
N-terminal 32 amino acids were important not only for the nuclear
localization and the transcriptional repression but also HDAC binding.
Because HDACs are the inhibitors of muscle differentiation (7), nuclear
EID-2 may be associated with HDACs, and the resultant complex may
inhibit transcription involved in muscle differentiation. Further
biochemical and biological studies will elucidate the link between the
role of EID-2 in muscle differentiation and the functions of HDACs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequence of EID-2.
A, amino acid sequence of EID-2 and alignment with EID-1.
Homologous regions to EID-1 are underlined. An
asterisk indicates the alteration of the pocket
protein-binding motif. B, schematic comparison of EID-2 and
EID-1. C, Northern blot of mRNAs from the indicated
tissues with a digoxigenin-labeled EID-2 (upper
panel) or -actin (lower panel) cDNA
probe, respectively.
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Fig. 2.
EID-2 contains a potential transrepression
domain. A, U-2OS cells were transiently transfected
with a pRL-SV40 control reporter plasmid, a firefly luciferase reporter
plasmid containing TetR-binding sites, and plasmids encoding the
indicated TetR-EID-1 or TetR-EID-2 fusion proteins or TetR alone. Cell
extracts were prepared, and firefly luciferase activity, corrected for
Renilla luciferase activity, was expressed as -fold
activation relative to cells producing TetR alone. B, U-2OS
cells were transfected with a pRL-SV40 control reporter plasmid, a
firefly luciferase reporter plasmid containing Gal4 DNA-binding sites,
a plasmid encoding Gal4-p300, and plasmids encoding the indicated EID-1
or EID-2 proteins.
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Fig. 3.
N-terminal region of EID-2 is involved in
repression of MyoD-dependent transcription.
A, schematic representation of the EID-2 mutants used in
this study. B, 10T1/2 murine fibroblasts were transfected
with a pRL-SV40 control reporter plasmid, a luciferase reporter plasmid
containing the MCK promoter, a plasmid encoding MyoD, and plasmids
encoding the indicated T7-EID-1 or T7-EID-2 proteins. *,
p < 0.05.
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Fig. 4.
EID-2 inhibits muscle differentiation.
A, stable C2C12 clones producing T7-EID-2, as well as an
empty vector transfectant, were grown under conditions that are (2%
horse serum) or are not (20% FBS) permissive for differentiation. Cell
extracts were prepared and immunoblotted for the indicated proteins.
B, microscopic appearance of C2C12 myoblasts expressing a
low level of EID-2 protein (d, e, and
f), a high level of EID-2 protein (g,
h, and i), and the empty vector (a,
b, and c). Cells were induced to differentiate in
DM for 3 days (c, f, and i) or
maintained in GM at 50% (a, d, and g)
or 100% (b, e, and h) confluency.
C, stable C2C12 clones producing T7-EID-2, as well as an
empty vector transfectant, were grown under conditions that are (2%
horse serum) or are not (20% FBS) permissive for differentiation. Cell
cycle profiles were determined by FACS analyses. The x-axis
in d, h, and l is a log scale to
highlight the sub-G1 population.
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Fig. 5.
Subcellular localization of EID-2.
U-2OS cells were transfected with DsRed2 (a),
DsRed2-EID-2 (b), and DsRed2-EID2 (33-236) (c).
Subcellular localization of the proteins was observed under a
fluorescent microscope at 48 h after transfection.
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Fig. 6.
EID-2 binds to class I HDACs in
vivo. A, U-2OS cells were transfected so as
to produce FLAG-HDAC2 alone (lane 1), T7-EID-2 alone
(lane 2), or FLAG-HDAC2 with the indicated EID-2 proteins
(lanes 3-6). Cell extracts were prepared and immunoblotted
with an anti-FLAG (top panel) or anti-T7 (middle
and lower panels) antibody. In parallel, an aliquot of each
extract was immunoprecipitated with anti-FLAG (lower panel)
antibody. WCE, whole cell extract. B, 10T1/2
murine fibroblasts were transfected with a pRL-SV40 control reporter
plasmid, a luciferase reporter plasmid containing the MCK promoter, a
plasmid encoding MyoD, and a plasmid encoding the EID-2 protein treated
with or without TSA (50, 100, and 200 ng/ml).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Bill Kaelin, Bill Sellers, Shoumo Bhattacharya, Steve Grossman, and Takayuki Yamada for providing the plasmids. We also thank Sumio Sugano and Yutaka Suzuki for providing the cDNAs and Hidenori Ichijo for helpful suggestion.
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
81-3-5803-5183; Fax: 81-3-5803-0125; E-mail:
miyake.monc@tmd.ac.jp.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M212212200
2 S. Miyake and Y. Yuasa, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: bHLH, basic helix-loop-helix; MEF, myocyte enhancer factor; HDAC, histone deacetylase; HAT, histone acetyltransferase; TSA, trichostatin A; GM, growth medium; DM, differentiation media; FBS, fetal bovine serum; CMV, cytomegalovirus; MCK, muscle creatine kinase; TetR, tetracycline repressor; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein.
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