From the University of Queensland, Institute for
Molecular Bioscience, Centre for Molecular and Cellular Biology,
Ritchie Research Laboratories, B402A, St. Lucia 4072, Queensland,
Australia, ¶ Harvard Medical School, Department of Biological
Chemistry and Molecular Pharmacology, Boston, Massachuttes 02115, and
the
Salk Institute, Howard Hughes Medical Institute, Gene
Expression Laboratory, San Diego, California 92186-5800
Received for publication, February 16, 2001, and in revised form, March 2, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The overlapping expression profile of MEF2 and
the class-II histone deacetylase, HDAC7, led us to investigate the
functional interaction and relationship between these regulatory
proteins. HDAC7 expression inhibits the activity of MEF2 (-A, -C, and
-D), and in contrast MyoD and Myogenin activities are not affected. Glutathione S-transferase pulldown and
immunoprecipitation demonstrate that the repression mechanism involves
direct interactions between MEF2 proteins and HDAC7 and is associated
with the ability of MEF2 to interact with the N-terminal 121 amino
acids of HDAC7 that encode repression domain 1. The MADS domain of MEF2
mediates the direct interaction of MEF2 with HDAC7. MEF2 inhibition by HDAC7 is dependent on the N-terminal repression domain and surprisingly does not involve the C-terminal deacetylase domain. HDAC7 interacts with CtBP and other class-I and -II HDACs suggesting that silencing of
MEF2 activity involves corepressor recruitment. Furthermore, we show
that induction of muscle differentiation by serum withdrawal leads to
the translocation of HDAC7 from the nucleus into the cytoplasm. This
work demonstrates that HDAC7 regulates the function of MEF2 proteins
and suggests that this class-II HDAC regulates this important
transcriptional (and pathophysiological) target in heart and muscle
tissue. The nucleocytoplasmic trafficking of HDAC7 and other class-II
HDACs during myogenesis provides an ideal mechanism for the regulation
of HDAC targets during mammalian development and differentiation.
Skeletal muscle has become a model for understanding many
fundamental principles of development. Differentiation of precursor cells into skeletal muscle cells involves two events, determination into myoblasts and the formation of postmitotic, multinucleated myotubes with contractile phenotype. These processes are under control
of members of the MyoD family of
basic-helix-loop-helix (bHLH)1 transcription factors
(MyoD, Myf5, Myogenin, and MRF4). These proteins can inhibit cell
proliferation, regulate a cascade of muscle-specific gene expression,
auto- and cross-regulate their own and each other's expression, and
induce muscle differentiation in nonmuscle cells (1-3). Myogenic bHLH
proteins activate transcription of muscle-specific genes by forming
heterodimers with other, ubiquitously expressed bHLH proteins known as
E2A proteins (alternatively spliced products of the E2A
gene) (4-7). These heterodimers bind to the E box motif (CANNTG),
which functions as the cognate binding site in the regulatory regions
of most muscle genes (1, 3, 8). MyoD and Myf5 are required for
determination of precursor cells into myoblasts (9), whereas Myogenin
is specifically required for differentiation (10, 11). Therefore
myoD and myf5 are expressed in proliferating
myoblasts and are markers for commitment, whereas the expression of
myogenin is a marker of terminal-differentiation.
Even though members of the MyoD family are the key regulators of muscle
differentiation, the activation of muscle-specific genes is dependent
on the association with members of the MEF2 (myocyte
enhancer factor 2) (12) family of transcription
factors (8). MEF2 proteins cooperatively increase the activity of
myogenic bHLH transcription factors (13). In vertebrates the MEF2
family is encoded by four independent genes, mef2a,
mef2b, mef2c, and mef2d (13). MEF2 factors belong to the
MADS-(MCM1-agamous
deficiens-serum response factor) box family and
share a highly conserved 86-amino acid-region that encodes the MADS and
MEF2 domains, which mediate DNA binding and dimerization, respectively
(2). Loss-of-function mutations in the single Drosophila
mef2 gene prevent myoblast differentiation (14, 15),
and dominant-negative MEF2 mutants inhibit myoblast differentiation in
cell culture (16) demonstrating a critical role of MEF2 proteins in
terminal muscle differentiation. MEF2-proteins are expressed in a wide
range of tissues, whereas MEF2C is restricted mainly to skeletal
muscle, brain, and spleen. However, MEF2C DNA-binding activity is
highly enriched in muscle and neural tissue indicating a critical role
in muscle differentiation.
Positive and negative regulation of eukaryotic transcription has been
shown to be mediated in part by two opposing enzymatic activities,
histone acetylases and histone
deacetylases (HDACs) (17, 18). Whereas histone
acetylases are associated with transcriptional activation,
deacetylation of histone leads to a compact chromatin structure to
which the accessibility of transcriptional activators is impaired, and
thereby transcription is repressed.
Recently, we and others have shown, that the transcriptional activity
of MEF2C is modulated by cofactor recruitment. MEF2C recruits chromatin
remodeling factors such as the histone acetylases p300/CBP and
P/CAF (19-22) to activate gene expression. This recruitment is
probably mediated by the coactivator GRIP1, which has been shown to
directly interact with MEF2C and to be necessary for MEF2C-dependent gene expression and skeletal muscle
differentiation (23). Former studies revealed that GRIP1-mediated
activation is due to interaction with p300/CBP and P/CAF (24, 25).
Interestingly MEF2 proteins also recruit HDACs, which leads to a
repression of MEF2-dependent gene expression and prevents
myogenesis (26-35). Two families of HDACs, referred to as class-I and
class-II HDACs, have been identified in mammals. Class-I HDACs (HDAC1,
-2, and -3) are related to the yeast transcriptional regulator RPD3
(36-39), are found in large corepressor complexes (40), and are
recruited to MEF2 proteins via corepressors such as MITR, Cabin1, and
CtBP (26, 27, 33, 34). Class-II HDACs (HDAC4, -5, -6, and -7) are
related to another yeast transcription factor, Hda1, which was found in
a separate complex from RPD3 (41). HDAC4 and -5 have been reported to
interact directly with MEF2C but also get recruited by corepressors
(27-29). This interaction is disrupted by the
calcium/calmodulin-dependent kinase CaMK (30, 32, 33, 35),
reviewed in Ref. 42.
In this study we report that the recently identified HDAC7 (43), like
other class-II HDACs, represses MEF2-dependent
transcription via physical interaction with the MADS domain of MEF2.
This repression is dependent on the N-terminal repression domain of
HDAC7 and functions independently of the C-terminal deacetylase domain. Furthermore we provide evidence that this functions via the interaction with other corepressors, such as CtBP and class-I and -II HDACs. Finally we demonstrate that the hdac7 mRNA is
down-regulated but constitutively expressed during differentiation of
muscle cells and that the HDAC7 protein shuttles from the nucleus into
the cytoplasm during this process thereby preventing the formation of a
MEF2/HDAC7-repressive complex.
Cell Culture and Transient Transfections--
Pluripotent
C310T1/2 cells were cultured for 24 h in DMEM supplemented with
10% FCS in 6% CO2 before transfection. Cells grown in
12-well plates to 60-70% confluence were transiently transfected using a DOTAP/DOSPER (3:1) liposome (Roche Molecular Biochemicals) mixture in HEBS (42 mM HEPES, 275 mM NaCl, 10 mM KCl, 0.4 mM Na2HPO4, 11 mM Dextrose, pH 7.1), with 2 µg of total DNA. For
elimination of Gal-MEF2C, -A, or -D-mediated transactivation 1 µg of
G5-E1b-Luc reporter, 0.3 µg of the respective MEF2 cDNA in the
pSV40gal expression vector, and 0.6 µg of HDAC4, -5, or -7 in the
pSG5 expression vector/well were used. Empty expression vectors
served as controls. The same conditions applied for the experiments
carried out with Gal-MyoD and Gal-Myogenin.
For elimination of MEF2C-mediated transactivation on MEF2-responsive
elements, 1 µg of pGL3-MEF2[×3]-E1b-Luc reporter (or as control, the pGL3-E1b-Luc plasmid without MEF2-binding sites), 0.5 µg of pSG5-MEF2C, and 0.5 µg of pSG5-HDAC4, -5, or -7 were used. Empty SG5 expression plasmid was used as control. Medium was replaced 24 h after transfection, and cells were
further grown for 25-48 h before harvesting and assayed for
Luciferase activity using the Luclite kit (Packard Instrument
Co.) according to the manufacturer's protocol.
Mouse myogenic C2C12 cells were cultured in growth medium (DMEM
supplemented with 20% FCS) in 6% CO2. For differentiation assays, cells were grown confluent, and the media was changed into
differentiation medium (DMEM supplemented with 2% horse serum), and
cells were harvested at the time points indicated. For visualizing intracellular localization of HDAC7, 5 µg of pCMX-HDAC7-YFP were used
to transiently transfect proliferating C2C12 myoblasts held in growth
medium or C2C12 cells that have been grown in differentiation medium
for 48 h. The appropriate medium was replaced after 24 h and
cells were incubated for an additional 24 h. YFP-HDAC7 was visualized using standard fluorescence procedures after fixation in 4%
paraformaldehyde in phosphate-buffered solution.
Immunoprecipitation--
Cos-7 cells were transfected with
expression constructs encoding either full-length FLAG-tagged MEF2C
and HA-tagged HDAC4, -5, or -7 as indicated using the FuGENE 6 (Roche Molecular Biochemicals) liposome reagent. 48 h after
transfection cells were lysed in phosphate-buffered solution containing
0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, and
protease inhibitors (complete by Roche Molecular Biochemicals) and
subjected to brief sonication, and the resultant cellular debris was
pelleted by centrifugation. Lysates were then incubated with a mouse
monoclonal anti-FLAG M2 antibody (Sigma) for 2 h at 4° C and
precipitated by incubation for a further 1 h with protein A/G
affinity resin (Pierce). Precipitated proteins were resolved by
SDS-polyacrylamide gel electrophoresis and subsequently immunoblotted
with a rabbit polyclonal anti-HA antibody (Bab. Co.)
RNA Extraction, Northern Hybridization, and Probe
Preparation--
Total RNA was extracted by guanidinium
thiocyanate-phenol-chloroform method. Northern blots, random priming,
and hybridization were performed as described previously (44, 45). The
1.8-kilobase pair HindIII fragment of pCMX-m HDAC7-HA
(43) was used as a probe for HDAC7. As a probe for HDAC5 the
KpnI/SmaI 1.7-kilobase pair fragment of
pCMX-mHDAC5-HA was used. Other cDNA probes have been described
previously (23).
cDNA probes were radioactively labeled by random priming. DNA
fragments (200 ng) were boiled with 20 ng of random hexamers (pdN6;
Amersham Pharmacia Biotech) and then incubated over night with EcoPol
buffer (New England Biolabs), 200 µm dGTP/dTTP, 10 µl
[32P]dATP/[32P]dCTP (Bresatec), and 5-10
units of Klenow polymerase (New England Biolabs). Probes were purified
using NICK columns (Amersham Pharmacia Biotech) according to the
manufacturer's protocol.
GST Pulldowns--
GST and GST fusion proteins were expressed in
Escherichia coli (BL21) and purified using
glutathione-agarose affinity chromatography as described previously
(45, 46). Amounts and integrity of GST fusion proteins were checked by
SDS-polyacrylamide gel electrophoresis and Coomassie staining. The
TnT-coupled transcription/translation kit (Promega) was used to
produce [35S]methionine-labeled proteins. Pulldowns were
carried out as described previously (23).
HDAC7 Represses MEF2-mediated Transactivation: MyoD and Myogenin
Activity Is Refractory to Class-II HDAC-mediated Inhibition--
The
class-II HDAC4 and -5 interact with MEF2 and suppress its ability to
activate transcription and cell specific differentiation (26, 27, 29,
31). Therefore we tested if the recently discovered HDAC7 (43), which
is expressed in a cell-specific manner and colocalizes with other
class-I and -II HDACs and corepressors in distinct nuclear
compartments, regulates MEF2-mediated transcription.
MEF2C is highly expressed in developing skeletal, cardiac, and smooth
muscle cells. Weak expression is detected in endothelial cells and
surrounding mesenchyme during embryo development (47-49). Because
HDAC7 and MEF2C are expressed in a similar cell-specific manner, we
initially tested the effect of HDAC7 expression on MEF2C activity.
MEF2C can activate transcription directly and/or by protein-protein
interaction with other DNA bound factors, hence we examined the effect
of HDAC7 (relative to HDAC4 and -5) expression on MEF2C-mediated transactivation in the GAL4 hybrid system (Fig.
1A). In these assays the
activity of MEF2C is independent of binding to its cognate binding
motif, the A/T-rich MEF2 site. If HDAC7 inhibits the transcriptional
activity, then the potential of the GAL4-MEF2C fusion to trans-activate
gene expression should be significantly reduced in this assay.
C3H10T1/2 pluripotent cells were cotransfected with GAL-MEF2C and the
G5E1b-LUC reporter in the presence and absence of the class-II HDAC4,
-5, and -7. G5E1b-LUC contains five copies of the GAL4 binding site
placed upstream of a minimal E1b promoter. Transfection of Gal-MEF2C
alone induced transcription ~10-fold relative to the GAL4 DNA binding
domain. This level of activity was significantly repressed by
the addition of HDAC4 and -7, whereas no significant effect is seen on
the basal activity of the GAL4-expression plasmid. Surprisingly, HDAC5
poorly repressed MEF2C activity at the amounts transfected in this assay.
The MEF2 family is composed of a group of proteins encoded by four
vertebrate genes mef2a, mef2b, mef2c
and mef2d (50). All of these factors bind DNA,
recruit cofactors, and trans-activate gene expression in a similar
manner. Hence we analyzed the effect of HDAC4, -5, and -7 expression on
the transcriptional activation mediated by MEF2A and MEF2D (Fig.
1B). Similarly to the results obtained with MEF2C, we
observed that HDAC4 and -7 dramatically reduced the activity of MEF2A
and MEF2D. Again, HDAC5 weakly repressed MEF2A and MEF2D (Fig.
1B). Thus class-II HDACs, including HDAC7, repress
MEF2-dependent gene expression.
The cell culture experiments presented above suggest that HDAC7
inhibits MEF2 activity. Therefore, we examined whether HDAC7 repressed
MEF2C-dependent activation of a luciferase reporter with 3 tandem copies of the MEF2 cognate binding sites upstream of a basal E1b
promoter (Fig. 1C). These experiments clearly demonstrate again that HDAC7 represses MEF2C-mediated transactivation of a MEF2-dependent reporter. The specificity of this repression
was supported by the inability of class-II HDACs alone to repress the
MEF2-dependent reporter
(MEF2[×3]-E1b-LUC) and the failure of MEF2C
and class-II HDACs to regulate the expression of the vehicle,
i.e. the basal E1b promoter linked to luciferase (Fig.
1C).
MEF2 proteins belong to the MADS box family of transcription factors
that cooperate with the myogenic bHLH proteins, MyoD and Myogenin, in
the activation of the contractile protein gene expression and function
within a regulatory network that establishes the differentiated
phenotype. Therefore, we investigated whether HDAC7 regulates the
transactivation of gene expression mediated by myogenic bHLH proteins.
C310T1/2 cells were transfected with Myogenin or MyoD fused to the
DNA-binding domain of GAL4 and examined for the effect of class-II HDAC
expression on the activity of these transcriptional activators. We
observed that neither HDAC4, -5, or -7 had any significant effect on
the activity of these bHLH factors (Fig. 1D).
This data suggest that HDAC7 specifically represses the activity of the
MADS box proteins, MEF2A, -C, and -D, but not that of the bHLH factors,
MyoD and Myogenin. HDAC7 functions as a negative regulator of
myogenesis by inhibiting MEF2-dependent gene expression and
transactivation dependent on the cooperative function between MEF2 and
the MyoD family of basic helix-loop-helix transcription factors. This
correlates with the observations that have been made with HDAC4 and -5 (26, 27, 29, 31).
HDAC7 Interacts with MEF2C: the N-terminal 121 Amino Acids of HDAC7
Mediate the Interaction--
The class-II HDACs have been reported to
directly interact with the N terminus of MEF2 in vivo
and in vitro (26, 27, 29, 31). Therefore we tested if HDAC7
interacts with MEF2C in vivo. COS-7 cells were transfected
with FLAG-tagged MEF2C (amino acids 1-117), HA-tagged HDAC7, and the
other class-II HDACs (HDAC4 and -5). Coimmunoprecipitation with the
FLAG-antibody demonstrates that ectopic MEF2C interacts with HA-HDAC7,
as well as with HDAC4 and -5 in vivo (Fig.
2A).
The regulation of MEF2-dependent transcription by the
class-II HDACs and the demonstration of interaction between these
factors in the immunoprecipitation assay strongly suggests that these proteins interact by a direct mechanism. However, it does not completely eliminate the possibility of an indirect mechanism in which
additional factor(s) mediate the interaction, although we postulated
that HDAC7 represses MEF2C activity/function by direct interaction. We
tested this hypothesis using a biochemical approach, the in
vitro GST pulldown assay to confirm the direct interaction between
MEF2C and HDAC7.
Glutathione-agarose-immobilized GST-MEF2C was tested for direct
interaction with in vitro 35S-radiolabeled
native HDAC7 (Fig. 2B). Native HDAC7 showed a very strong
direct interaction with GST-MEF2C relative to GST alone, suggesting a
direct protein/protein interaction between these two
transcription factors as it has been shown for HDAC4 and -5 (29,
31).
To delimit the minimal region of HDAC7 required to mediate the
interaction with MEF2C we incubated a number of 35S-labeled
C-terminal unidirectional deletions of HDAC7 with GST-MEF2C. We
observed that the N-terminal fragment of HDAC7 encompassing amino acids
1-121 is sufficient to interact with MEF2C (Fig. 2B). This
fragment encodes repression domain 1 of HDAC7 and contains a highly
conserved motif of 17 amino acids (Fig. 2C), which has been
shown to be necessary for the interaction of HDAC4 and -5 with the MEF2
proteins (29).
The MADS Box Domain of MEF2C Mediates the Physical Association with
HDAC7--
We used the in vitro GST pulldown assay to
confirm the direct interaction between MEF2C and GST-HDAC7 and to
identify the domain in MEF2C that mediates the interaction with this
class-II HDAC.
Glutathione-agarose-immobilized GST-HDAC7 was tested for direct
interaction with in vitro 35S-radiolabeled
native MEF2C, MEF2C-(58-465), MEF2C-(90-465), MEF2C-(1-223) and, MEF2C-(1-177). Native MEF2C showed a very strong interaction with
GST-HDAC7 relative to GST alone. In contrast, MEF2C-(58-465) and MEF2C-(90-465) that, respectively, lacked the MADS box and MADS box/MEF2 domains failed to interact with HDAC7 (Fig.
3). This indicated that the MADS box is
crucial for HDAC7 interaction as it has been shown for the interaction
with HDAC4 and -5 (26, 27, 29, 31). In support of this result the
C-terminal deletions (MEF2C-(1-223) and, MEF2C-(1-177)) that
contained the MADS box efficiently interacted with HDAC7.
Repression of MEF2C-mediated Transcription by HDAC7 Is Mediated by
the N-terminal Repression Domain: the Deacetylase Domain Is Not
Required--
HDACs have been shown to repress transcription at least
partially via deacetylation of histones but also to harbor other
repression domains (43). Therefore we were interested to examine
whether the down-regulation of MEF2-mediated transactivation via HDAC7 requires the enzymatic deacetylation activity. Two deletion mutants of
HDAC7 that still interact with MEF2C but lack the HDAC domain (amino
acids 1-372 and 1-121; Fig. 2) and a point mutation in the HDAC
domain that interferes with the ability to deacetylase histones
(H657A, Ref. 51) were tested for their influence on MEF2C-mediated transactivation. Interestingly, these mutants were also
able to inhibit MEF2C-mediated transactivation and did not interfere
with the full-length HDAC7 (Fig.
4A). No significant effect of
HDAC7 mutants or the full-length construct was observed on the basal
activity when no MEF2C was transfected (Fig. 4B).
Because HDAC7 mutants and deletions lacking deacetylase function are
able to repress MEF2C-mediated transactivation we wanted to gain an
insight in how RD1 and RD2 of HDAC7 mediate repression. We therefore
tested if HDAC7 interacts with other class-I and -II HDACs and
corepressors as it has been shown for HDAC -4 and -5. GST pulldown
experiments using bacterial expressed GST-HDAC7 and in
vitro-translated 35S-labeled class-I and -II HDACs or
the corepressor CtBP demonstrate that HDAC7 interacts with all tested
HDACs (-1, -2, -4, -5, and -7) as well as with the corepressor CtBP
(Fig. 5). This observation provides
evidence that HDAC7-mediated repression of MEF2 activity requires the
recruitment of corepressors and other class-I and -II HDACs.
This is consistent with the identification of a distinct
matrix-associated nuclear structure that contains corepressors (SMRT, members of the Sin 3 and NuRD complexes), class-I and -II HDACs (43,
51). Furthermore the interaction of CtBP with HDAC7 correlates with
several observations that demonstrate that HDAC4, MITR, and Cabin-1
constitute a family of calcium-sensitive transcriptional repressors of
MEF2 and recruit CtBP via the PXDLR motif conserved in the N-terminal
region of HDAC4, -5, and -7 (32-34, 52).
HDAC7 Is Expressed during Skeletal Muscle Differentiation and Is
Regulated via Nucleo-Cytoplasmic Trafficking--
To gain insight into
how class-II HDACs regulate MEF2C-dependent gene activation
during muscle differentiation, we examined the expression pattern of
the mRNAs encoding HDAC5 and -7 during the conversion of mouse
myoblast C2C12 cells into terminal differentiated myotubes.
Proliferating C2C12 myoblasts were induced to biochemically and
morphologically differentiate into postmitotic, multinucleated myotubes
by serum withdrawal in culture over a period of 4-120 h. Total RNA was
isolated from proliferating myoblasts, confluent myoblasts, and
postmitotic myotubes after 4, 8, 24, 48, 72, and 120 h of serum
withdrawal and examined by Northern blot analysis. The mRNAs for
HDAC7 and -5 are expressed in proliferating myoblasts and suppressed as
the cells exit the cell cycle and fuse into terminal differentiated
myoblasts that have acquired a muscle-specific phenotype (Fig.
6A). The repression of the
cyclinD1 mRNA and the induction of the mRNAs
encoding myogenin and p21 relative to
GAPDH confirms that these cells were exiting from the cell
cycle and activating the differentiation program. However, expression
of the class-II HDACs is still observed in postmitotic cells.
If the HDAC7 protein acts as a transcriptional repressor of
MEF2-mediated gene activation it should be found in the nucleus when
MEF2/differentiation-dependant genes are inactive. During myogenesis,
when these genes are activated, HDAC7 should be unable to suppress the
function of MEF2 in the context of HDAC7 expression in postmitotic
differentiated cells. The HDAC7 function as a repressor of MEF2
activity must be overcome by another mechanism such as cellular
trafficking, localization, and/or proteolytic degradation. Observations
reported by others have shown that other class-II HDACs dissociate from
MEF2 proteins and shuttle into the cytoplasm in differentiation
involving calcium/calmodulin and 14-3-3-dependent phosphorylation (27, 29, 32, 35, 42, 52). To address this question we
transfected C2C12 myoblasts with a construct encoding a fusion protein
of HDAC7 and the yellow fluorescent protein (pCMX-HDAC7-YFP) and
examined its sub-cellular localization in C2C12 cells cultured in
growth medium (DMEM supplemented with 20% FCS) or differentiation
medium (DMEM supplemented with 2% horse serum), which leads to
withdrawal from the cell cycle and the induction of the differentiation
program. As seen in Fig. 6B, HDAC7-YFP is predominantly
localized in the nucleus when cells were held in growth medium. After
48 h of serum withdrawal (i.e. culturing in 2% horse
serum), the HDAC7 protein is localized within the cytoplasm, where it
cannot form an inhibitory complex with MEF2 factors. This observation
provides a plausible mechanism for the regulation of MEF2 function
during muscle differentiation: serum withdrawal leads to
nucleo-cytoplasmic trafficking of HDAC7 and therefore allows the
differentiation process to proceed.
The class-II HDACs are very homologous to each other and show many
similar features in the context of transcriptional regulation; however,
they are different proteins with (i) unique amino acid sequences and
(ii) spatio-temporal-specific expression patterns. Currently, it still
remains unclear what their specific roles in vivo are and
whether functional redundancy occurs in the class-II HDACs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 1.
HDAC4, -5, and -7 down-regulate
transactivation mediated by MEF2-proteins but not that of myogenic bHLH
proteins. A, pluripotent C310T1/2 cells were
transfected with G5-E1b-Luc as a reporter, pSV40-gal-MEF2C, pSG5-HDAC4,
-5, and -7 or the empty vectors. B, C310T1/2 cells were
transfected with the same reporter but pSV40-gal-MEF2A or -D and
pSG5-HDAC4, -5, and -7. Fold activation is expressed relative to
Luciferase activity obtained after cotransfection of G5-E1b-Luc,
pSV40-gal0, and empty pSG5 expression vector alone. C,
pluripotent C310T1/2 cells were transfected with either
GL3-MEF2[×3]-E1b-Luc or the GL3-E1b-Luc
plasmid with no MEF2 binding sites as reporter, pSG5-MEF2C, pSG5-HDAC4,
-5, and -7 or the empty pSG5 expression vector. D,
pluripotent C310T1/2 cells were transfected with G5-E1b-Luc as a
reporter, pSV40gal-Myogenin or -MyoD, pSG5-HDAC4, -5, and -7 or the
empty vectors. Fold activation is expressed relative to Luciferase
activity obtained after cotransfection of G5-E1b-Luc, pSV40-gal0, and
empty pSG5 expression vector alone.
View larger version (37K):
[in a new window]
Fig. 2.
HDAC4, -5, and -7 interact with MEF2C
in vivo and in vitro.
A, COS-7 cells were transfected with HA-tagged HDAC4, -5, and -7 and FLAG-tagged MEF2C (amino acids 1-117) or the empty
expression vectors as control. Immunoprecipitation with a FLAG antibody
followed by Western blot analysis with a HA antibody show that class-II
HDACs interact with MEF2C in vivo. B,
glutathione-agarose-immobilized, bacterial expressed GST and GST-MEF2C
proteins were incubated with either 35S-radiolabeled
full-length HDAC7 or fragments of HDAC7 encompassing the indicated
amino acid positions. The input lanes represent ~20% of
total radiolabeled HDAC7 protein. C, schematic
representation showing the HDAC7 regulatory domains and the deletion
fragments used. R1, R2, repression domains 1 and 2.
View larger version (47K):
[in a new window]
Fig. 3.
The MADS box of MEF2C is necessary for
interaction with HDAC7. A, schematic representation
showing the functional MEF2C domains and the deletion fragments used.
B, glutathione-agarose-immobilized, bacterial expressed GST
and GST-HDAC7 proteins were incubated with either
35S-radiolabeled full-length MEF2C or fragments of MEF2C
encompassing the indicated amino acid positions. The input
lanes represent ~10% of total radiolabeled MEF2C protein.
View larger version (13K):
[in a new window]
Fig. 4.
The deacetylase domain of HDAC7 is not
required for repression of MEF2C-mediated transactivation.
A, pluripotent C310T1/2 cells were transfected with
G5-E1b-Luc as a reporter, pSV40-gal-MEF2C, pSG5-HDAC7, and the
indicated mutants of HDAC7 or the empty vectors. Fold activation is
expressed relative to Luciferase activity obtained after cotransfection
of G5-E1b-Luc, pSV40-gal0, and empty pSG5 expression vector alone.
B, the same experiment was performed but using pSV40-gal0
instead of gal-MEF2C to demonstrate that the observed repression is
MEF2C-dependent.
View larger version (31K):
[in a new window]
Fig. 5.
HDAC7 interacts with other corepressors.
Glutathione-agarose-immobilized, bacterial expressed GST and GST-HDAC7
proteins were incubated with either 35S-radiolabeled CtBP
or HDAC7 encompassing the indicated amino acid positions. The
input lanes represent ~10% of total radiolabeled
protein.
View larger version (65K):
[in a new window]
Fig. 6.
HDAC7 is constitutively expressed and
shuttles into the cytoplasm during myoblast differentiation.
A, total RNA was isolated from proliferating C2C12 myoblasts
(PMB), confluent myoblasts (CMB) cultured in
growth medium (DMEM containing 20% FCS), and developing myotubes 4, 8, 24, 72, and 120 h after serum exchange into differentiation medium
(DMEM containing 2% adult horse serum). After blotting, RNA was probed
with 32P-radiolabeled cDNAs encoding
glyceraldehyde-3-phosphate dehydrogenase, HDAC5 and -7, Myogenin, MyoD, p21, and cyclinD1. Induction of Myogenin and p21 as
well as down-regulation of cyclinD1 confirm that these cells have
undergone terminal differentiation into myotubes. B, C2C12
myoblasts were transfected with HDAC7 fused to the yellow fluorescent
protein. Cells kept in growth medium show an exclusive nuclear staining
of HDAC7-YFP. Cells 72 h after serum withdrawal into
differentiation medium show a cytoplasmatic localization of
HDAC7-YFP.
![]() |
FOOTNOTES |
---|
* This work was supported by the National Health and Medical Research Council (NHMRC) of Australia. The Institute for Molecular Bioscience is part of the Special Research Center for Functional and Applied Genomics that is supported by Australia Research Council (ARC).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.
§ Supported by a Postdoctoral Fellowship from the Hochschulsonderprogramm III through the Deutscher Akademischer Austauschdienst (DAAD), Bonn, Germany).
** Principal Research Fellow of the NHMRC. To whom correspondence should be addressed: Univ. of Queensland, Inst. for Molecular Bioscience, Ritchie Research Lab., B402A, St. Lucia 4072, Queensland, Australia. Tel.: 61-7-3365-4492; Fax: 61-7-3365-4388 or 61-7 3279-0640; E-mail: G.Muscat@imb.uq.edu.au.
Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M101508200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: bHLH, basic helix-loop-helix; HDAC, histone deacetylase; DMDM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; HA, hemagglutinin; GST, glutathione S-transferase; MEF2, myocyte enhancer factor 2; MADS, MCM1-agamous deficiens-serum response factor; CtBP, C-terminal binding protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Ludolph, D. C.,
and Konieczny, S. F.
(1995)
FASEB J.
9,
1595-1604 |
2. | Molkentin, J. D., and Olson, E. N. (1996) Curr. Opin. Genet. Dev. 6, 445-453[CrossRef][Medline] [Order article via Infotrieve] |
3. | Yun, K., and Wold, B. (1996) Curr. Opin. Cell Biol. 8, 877-889[CrossRef][Medline] [Order article via Infotrieve] |
4. | Henthorn, P., Kiledjian, M., and Kadesch, T. (1990) Science 247, 467-470[Medline] [Order article via Infotrieve] |
5. | Murre, C., Voronova, A., and Baltimore, D. (1991) Mol. Cell. Biol. 11, 1156-1160[Medline] [Order article via Infotrieve] |
6. | Sun, X. H., and Baltimore, D. (1991) Cell 64, 459-470[Medline] [Order article via Infotrieve] |
7. | Kadesch, T. (1993) Cell Growth Differ. 4, 49-55[Medline] [Order article via Infotrieve] |
8. | Molkentin, J. D., Black, B. L., Martin, J. F., and Olson, E. N. (1995) Cell 83, 1125-1136[Medline] [Order article via Infotrieve] |
9. | Rudnicki, M. A., Schnegelsberg, P. N., Stead, R. H., Braun, T., Arnold, H. H., and Jaenisch, R. (1993) Cell 75, 1351-1359[Medline] [Order article via Infotrieve] |
10. | Hasty, P., Bradley, A., Morris, J. H., Edmondson, D. G., Venuti, J. M., Olson, E. N., and Klein, W. H. (1993) Nature 364, 501-506[CrossRef][Medline] [Order article via Infotrieve] |
11. | Olson, E. N., Arnold, H. H., Rigby, P. W., and Wold, B. J. (1996) Cell 85, 1-4[Medline] [Order article via Infotrieve] |
12. | Gossett, L. A., Kelvin, D. J., Sternberg, E. A., and Olson, E. N. (1989) Mol. Cell. Biol. 9, 5022-5033[Medline] [Order article via Infotrieve] |
13. |
Black, B. L.,
Molkentin, J. D.,
and Olson, E. N.
(1998)
Mol. Cell. Biol.
18,
69-77 |
14. | Bour, B. A., O'Brien, M. A., Lockwood, W. L., Goldstein, E. S., Bodmer, R., Taghert, P. H., Abmayr, S. M., and Nguyen, H. T. (1995) Genes Dev. 9, 730-741[Abstract] |
15. | Lilly, B., Zhao, B., Ranganayakulu, G., Paterson, B. M., Schulz, R. A., and Olson, E. N. (1995) Science 267, 688-693[Medline] [Order article via Infotrieve] |
16. |
Ornatsky, O. I.,
Andreucci, J. J.,
and McDermott, J. C.
(1997)
J. Biol. Chem.
272,
33271-33278 |
17. | Kuo, M. H., and Allis, C. D. (1998) Bioessays 20, 615-626[CrossRef][Medline] [Order article via Infotrieve] |
18. | Xu, L., Glass, C. K., and Rosenfeld, M. G. (1999) Curr. Opin. Genet. Dev. 9, 140-147[CrossRef][Medline] [Order article via Infotrieve] |
19. | Eckner, R., Yao, T. P., Oldread, E., and Livingston, D. M. (1996) Genes Dev. 10, 2478-2490[Abstract] |
20. | Puri, P. L., Sartorelli, V., Yang, X. J., Hamamori, Y., Ogryzko, V. V., Howard, B. H., Kedes, L., Wang, J. Y., Graessmann, A., Nakatani, Y., and Levrero, M. (1997) Mol. Cell 1, 35-45[Medline] [Order article via Infotrieve] |
21. |
Puri, P. L.,
Avantaggiati, M. L.,
Balsano, C.,
Sang, N.,
Graessmann, A.,
Giordano, A.,
and Levrero, M.
(1997)
EMBO J.
16,
369-383 |
22. | Sartorelli, V., Huang, J., Hamamori, Y., and Kedes, L. (1997) Mol. Cell. Biol. 17, 1010-1026[Abstract] |
23. |
Chen, S. L.,
Dowhan, D. H.,
Hosking, B. M.,
and Muscat, G. E.
(2000)
Genes Dev.
14,
1209-1228 |
24. | Onate, S. A., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1995) Science 270, 1354-1357[Abstract] |
25. |
McKenna, N. J.,
Lanz, R. B.,
and O'Malley, B. W.
(1999)
Endocr. Rev.
20,
321-344 |
26. |
Sparrow, D. B.,
Miska, E. A.,
Langley, E.,
Reynaud-Deonauth, S.,
Kotecha, S.,
Towers, N.,
Spohr, G.,
Kouzarides, T.,
and Mohun, T. J.
(1999)
EMBO J.
18,
5085-5098 |
27. |
Miska, E. A.,
Karlsson, C.,
Langley, E.,
Nielsen, S. J.,
Pines, J.,
and Kouzarides, T.
(1999)
EMBO J.
18,
5099-5107 |
28. |
Wang, A. H.,
Bertos, N. R.,
Vezmar, M.,
Pelletier, N.,
Crosato, M.,
Heng, H. H.,
Th'ng, J.,
Han, J.,
and Yang, X. J.
(1999)
Mol. Cell. Biol.
19,
7816-7827 |
29. | Lu, J., McKinsey, T. A., Zhang, C. L., and Olson, E. N. (2000) Mol. Cell 6, 233-244[Medline] [Order article via Infotrieve] |
30. |
Lu, J.,
McKinsey, T. A.,
Nicol, R. L.,
and Olson, E. N.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4070-4075 |
31. |
Lemercier, C.,
Verdel, A.,
Galloo, B.,
Curtet, S.,
Brocard, M. P.,
and Khochbin, S.
(2000)
J. Biol. Chem.
275,
15594-15599 |
32. |
Youn, H. D.,
Grozinger, C. M.,
and Liu, J. O.
(2000)
J. Biol. Chem.
275,
22563-22567 |
33. | Youn, H. D., and Liu, J. O. (2000) Immunity 13, 85-94[Medline] [Order article via Infotrieve] |
34. |
Zhang, C. L.,
McKinsey, T. A.,
Lu, Jr.,
and Olson, E. N.
(2001)
J. Biol. Chem.
276,
35-39 |
35. | McKinsey, T. A., Zhang, C. L., Lu, J., and Olson, E. N. (2000) Nature 408, 106-111[CrossRef][Medline] [Order article via Infotrieve] |
36. | Taunton, J., Hassig, C. A., and Schreiber, S. L. (1996) Science 272, 408-411[Abstract] |
37. |
Yang, W. M.,
Inouye, C.,
Zeng, Y.,
Bearss, D.,
and Seto, E.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12845-12850 |
38. | Dangond, F., Hafler, D. A., Tong, J. K., Randall, J., Kojima, R., Utku, N., and Gullans, S. R. (1998) Biochem. Biophys. Res. Commun. 242, 648-652[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Emiliani, S.,
Fischle, W.,
Van Lint, C.,
Al-Abed, Y.,
and Verdin, E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2795-2800 |
40. |
Burke, L. J.,
and Baniahmad, A.
(2000)
FASEB J.
14,
1876-1888 |
41. |
Rundlett, S. E.,
Carmen, A. A.,
Kobayashi, R.,
Bavykin, S.,
Turner, B. M.,
and Grunstein, M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14503-14508 |
42. | Muscat, G. E., and Dressel, U. (2000) Nat. Med. 6, 1216-1217[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Kao, H. Y.,
Downes, M.,
Ordentlich, P.,
and Evans, R. M.
(2000)
Genes Dev.
14,
55-66 |
44. |
Burke, L.,
Downes, M.,
Carozzi, A.,
Giguere, V.,
and Muscat, G. E.
(1996)
Nucleic Acids Res.
24,
3481-3489 |
45. | Downes, M., Carozzi, A. J., and Muscat, G. E. (1995) Mol. Endocrinol. 9, 1666-1678[Abstract] |
46. | Muscat, G. E., Rea, S., and Downes, M. (1995) Nucleic Acids Res. 23, 1311-1318[Abstract] |
47. | Black, B. L., and Olson, E. N. (1998) Annu. Rev. Cell Dev. Biol. 14, 167-196[CrossRef][Medline] [Order article via Infotrieve] |
48. |
Edmondson, D. G.,
Lyons, G. E.,
Martin, J. F.,
and Olson, E. N.
(1994)
Development
120,
1251-1263 |
49. |
Lin, Q.,
Lu, J.,
Yanagisawa, H.,
Webb, R.,
Lyons, G. E.,
Richardson, J. A.,
and Olson, E. N.
(1998)
Development
125,
4565-4574 |
50. | Subramanian, S. V., and Nadal-Ginard, B. (1996) Mech. Dev. 57, 103-112[CrossRef][Medline] [Order article via Infotrieve] |
51. |
Downes, M.,
Ordentlich, P.,
Kao, H. Y.,
Alvarez, J. G.,
and Evans, R. M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
10330-10335 |
52. |
Grozinger, C. M.,
and Schreiber, S. L.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
7835-7840 |