From the Department of Molecular Biology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148
Received for publication, August 14, 2000
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ABSTRACT |
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The class II histone deacetylases (HDACs) 4, 5, and 7 share a common structural organization, with a carboxyl-terminal
catalytic domain and an amino-terminal extension that mediates
interactions with members of the myocyte enhancer factor-2 (MEF2)
family of transcription factors. Association of these HDACs with
MEF2 factors represses transcription of MEF2 target genes.
MEF2-interacting transcription repressor (MITR) shares
homology with the amino-terminal extensions of class II HDACs and also
acts as a transcriptional repressor, but lacks a histone deacetylase
catalytic domain. This suggests that MITR represses transcription by
recruiting other corepressors. We show that the amino-terminal regions
of MITR and class II HDACs interact with the transcriptional
corepressor, COOH-terminal-binding protein (CtBP), through a
CtBP-binding motif (P-X-D-L-R) conserved in MITR and HDACs
4, 5, and 7. Mutation of this sequence in MITR abolishes interaction
with CtBP and impairs, but does not eliminate, the ability of MITR to
inhibit MEF2-dependent transcription. The residual
repressive activity of MITR mutants that fail to bind CtBP can be
attributed to association with other HDAC family members. These
findings reveal CtBP-dependent and -independent mechanisms
for transcriptional repression by MITR and show that MITR
represses MEF2 activity through recruitment of multicomponent
corepressor complexes that include CtBP and HDACs.
Regulation of chromatin structure is a central mechanism for the
control of gene expression and modulation of the acetylation state of
nucleosomal histones has been revealed as a dynamic mechanism for
chromatin remodeling (reviewed in Ref. 1). Acetylation of the
amino-terminal tails of core histones by histone
acetyltransferases results in chromatin relaxation and
transcriptional activation, whereas deacetylation of histones by
histone deacetylases (HDACs)1
is associated with transcriptional repression. Recruitment of histone
acetyltransferases and HDACs by DNA-bound transcription factors results
in the formation of multiprotein transcription regulatory
complexes that confer cell type specificity and
signal-dependent regulation to arrays of subordinate genes.
There is also evidence that direct acetylation/deacetylation of certain
transcription factors provides a mechanism for reversible regulation of transcription.
HDACs can be divided into two classes, I and II, on the basis of size,
sequence homology, and formation of distinct complexes. Class I
includes HDACs 1, 2, and 3, which are expressed ubiquitously (2-5).
Class II includes HDACs 4, 5, 6, and 7, which contain a conserved
domain of several hundred amino acids extending amino-terminal from the
carboxyl-terminal catalytic domain (6-10). Class II HDACs are
tissue-restricted, with especially high levels of expression in heart,
skeletal muscle, and brain. MEF2-interacting transcription repressor
(MITR) (11), also called HDAC-related protein (HDRP) (12), shares
homology with the amino-terminal extension of class II HDACs, but lacks
a carboxyl-terminal catalytic domain.
Class II HDACs and MITR/HDRP have been shown to interact with members
of the MEF2 family of MADS (MCM1, Agamous, Deficiens, serum response
factor) box transcription factors, resulting in transcriptional
repression (7, 8, 11, 13, 14). MEF2 factors play central roles in the
control of muscle differentiation and have been implicated in growth
factor signaling and apoptotic pathways (reviewed in Ref. 15). Thus,
negative regulation of MEF2 activity by association with HDACs may
inhibit growth and/or differentiation, depending on cell type and the
presence of other cofactors for MEF2 and HDACs.
While HDACs are presumed to inhibit transcription as a consequence of
their deacetylase activity, the mechanism whereby MITR represses
transcription is unclear. MITR has been shown to interact with HDAC1,
which could account for its ability to repress
MEF2-dependent transcription (11). Consistent with this
conclusion, the HDAC inhibitor trichostatin (TSA) has been reported to
partially attenuate transcriptional repression by MITR (11). However,
another study reported that TSA had no effect on transcriptional
repression by MITR, concluding that its mechanism for repression did
not involve other HDACs (12).
Here we report that HDACs 4 and 5 and MITR associate with the
transcriptional corepressor COOH-terminal binding protein (CtBP) via a
CtBP-binding motif (P-X-D-L-R) conserved in HDACs 4, 5, and
7 and MITR. Mutation of this sequence abolishes association of CtBP
with MITR and substantially diminishes, but does not eliminate, the
transcriptional repression of basal as well as
MEF2-dependent promoters by MITR. These results demonstrate
that MITR and class II HDACs act as a bridge linking the CtBP
corepressor to target transcription factors, such as MEF2, thereby
establishing a multiprotein corepressor complex directed at specific
downstream genes.
Yeast Two-hybrid Screen--
A mouse 17-day embryo MATCHMAKER
cDNA library (CLONTECH) was screened with
GAL4-HDAC4 bait in the yeast two-hybrid system, as described previously
(16). The bait contained amino acids 1-640 of human HDAC4 fused to the
GAL4 DNA binding domain. Positive clones were subjected to specificity
tests using the GAL4 DNA binding domain alone as bait. Those clones
that were specific for interaction with GAL4-HDAC4 bait were sequenced.
Cloning of Mouse MITR--
A mouse expressed sequence tag
encoding MITR (GenBankTM accession number AV118321),
identified by searching the data base, was used to screen a mouse
embryonic day 10.5 cDNA library (Stratagene) for cDNAs
encompassing the complete open reading frame. The deduced open reading
frame of mouse MITR is 586 amino acids and shows 94 and 66% identity
to human and Xenopus MITR proteins, respectively (11, 12).
The nucleotide sequence of mouse MITR has been deposited in the data
base (GenBankTM accession number AF324492).
Cell Culture, Plasmids, and Transfections--
10T1/2, COS, and
293T cells were maintained in Dulbecco's modified Eagle's
medium containing 10% fetal bovine serum, 2 mM L-glutamine, and penicillin-streptomycin. Transfections
were performed using the lipid-based reagent Fugene 6 (Roche Molecular
Biochemicals) and cells growing at a density of 5-10 × 105 cells/35-mm dish. Epitope-tagged derivatives of CtBP,
MITR, HDAC4, and HDAC5, containing amino-terminal FLAG or Myc
tags, were generated using the pcDNA3.1 expression vector
(Invitrogen). Expression plasmids for carboxyl-terminally FLAG-tagged
HDAC1 (pBJ) and HDAC3 (pcDNA6) were kindly provided by Dr. Stuart
Schreiber (Harvard). For GAL4-dependent reporter assays,
10T1/2 cells were cotransfected with a luciferase reporter plasmid
under control of four GAL4 DNA binding sites and the thymidine kinase
(tk) promoter, pMH100-tk-luc (17), pM1-based expression vectors
encoding MITR or MITR(DL-AS) fused to the GAL4 DNA binding domain, and
a CMV-lacZ (Invitrogen) plasmid to normalize for variable transfection
efficiency. For MEF2-dependent transcription assays, 10T1/2
cells were transfected with a luciferase reporter plasmid under control
of three copies of a consensus MEF2 binding site, 3×MEF-luc, a
pcDNA1-based expression plasmid (Invitrogen) for MEF2C,
pcDNA3.1-based expression vectors encoding either MITR or
MITR(DL-AS), and the CMV-lacZ plasmid. Cells were harvested 48 h
after transfection, and luciferase and GST Interaction Assays--
CtBP protein was translated in
vitro using the TNT kit (Promega) and a pcDNA3.1-based
expression plasmid encoding full-length mouse CtBP1. GST-HDAC and
GST-MITR fusion proteins were expressed in the BL21(DE3) strain of
Escherichia coli using the pGEX-KG vector (Amersham
Pharmacia Biotech). Ten microliters of in vitro translated, [35S]methionine-labeled CtBP (~10,000 cpm)
were incubated with GST-HDAC4 and GST-MITR proteins bound to
glutathione-agarose beads (Sigma) in GST binding buffer (20 mM Tris, pH 7.3, 150 mM NaCl, 0.5% Nonidet P-40, and protease inhibitors (Complete; Roche Molecular
Biochemicals)) for 1 h at 4 °C. Beads were collected by
centrifugation and washed five times in GST binding buffer. Bound
proteins were resolved by SDS-PAGE and analyzed by autoradiography.
Coimmunoprecipitation Assays--
For coimmunoprecipitation
experiments, transiently transfected COS or 293T cells were harvested
48 h following transfection in phosphate-buffered saline
containing 0.5% Triton X-100, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, and protease inhibitors. After
brief sonication and removal of cellular debris by centrifugation, FLAG-tagged proteins were immunoprecipitated from cell lysates using
anti-FLAG affinity resin (Sigma) and washed five times with lysis
buffer. Alternatively, Myc-tagged proteins were immunoprecipitated using polyclonal anti-Myc antibodies (Santa Cruz; A-14) and protein A-Sepharose beads (Zymed Laboratories Inc.).
Precipitated proteins were separated by SDS-PAGE, transferred to
polyvinylidene fluoride membranes, and immunoblotted with either
a monoclonal anti-Myc antibody (Santa Cruz; 9E10) or a monoclonal
anti-FLAG antibody (Sigma; M2). Proteins were visualized with a
chemiluminescence system (Santa Cruz).
Interaction between HDAC4 and CtBP in the Yeast Two-hybrid
System--
HDACs 4, 5, and 7 are bipartite, with an amino-terminal
region that contains a MEF2-binding motif, and a carboxyl-terminal catalytic region (Fig. 1A). In
previous studies, we (13) and others (7, 8, 11, 14) showed that
association of HDACs 4, 5, and 7 with MEF2 is mediated by a conserved
18-amino acid motif near their amino-termini. This motif is also
conserved in MITR/HDRP, which lacks a catalytic domain.
To further investigate the functions of the amino-terminal extension of
class II HDACs and MITR, we performed yeast two-hybrid screens using
amino acids 1-640 of HDAC4 fused to the GAL4 DNA binding domain as
bait (Fig. 1A). Three strong positives obtained from a mouse
17-day embryo cDNA library corresponded to CtBP1, a widely
expressed transcriptional corepressor (18, 19). Two independent
cDNA clones encoded the entire 441-amino acid CtBP1 protein; the
other encoded amino acids 25-441. These CtBP-GAL4 activation
domain fusion proteins interacted specifically with the HDAC4 bait and
not with the GAL4 DNA binding domain alone (see "Experimental
Procedures").
Because CtBP has been shown to interact with the consensus motif
P-X-D-L-S/R (20, 21), we scanned the 640-amino acid region of HDAC4 used as bait and identified a potential CtBP-binding motif
between residues 48 and 52 (Fig. 1B). Similar sequences were
also found in HDACs 5 and 7 and MITR (Fig. 1B). The amino acid sequences surrounding this potential CtBP-binding motif were not
conserved in the different HDACs or MITR.
Association of CtBP with MITR and HDACs in Vivo and in
Vitro--
The possible association of CtBP with MITR and HDACs was
tested further by coimmunoprecipitation assays from transfected cells. As shown in Fig. 2A, HDACs 4 and 5, as well as MITR, were coimmunoprecipitated with CtBP.
To further test the interaction of CtBP with HDACs and MITR, we
performed binding assays with GST fusion proteins. As shown in Fig.
2B, a GST fusion protein containing amino acids 1-60 of HDAC4 (GST-HDAC4-(1-60)), including the CtBP-binding motif, interacted with [35S]methionine-labeled CtBP translated in
vitro, whereas amino acids 49-223 of HDAC4 (GST-HDAC4-(49-223)),
which contains a truncated CtBP-binding motif, did not interact with
CtBP. GST-MITR and GST-HDAC5 also interacted with CtBP (Fig.
2B and data not shown). Residues 1-201 of MITR
(GST-MITR-(1-201)) contained the CtBP-binding region, whereas residues
131-586 (GST-MITR-(131-586)) did not bind CtBP (Fig. 2B).
A further amino-terminal deletion mutant containing only the first 45 amino acids (GST-MITR-(1-45)) also interacted with CtBP.
To determine specifically whether the P-X-D-L-R motif
(residues 23-27) of MITR was responsible for all CtBP-binding
activity, we mutated residues DL to AS, which has been shown previously to abolish CtBP binding to the E1a oncoprotein (20). This mutant, GST-MITR-(1-201DL-AS) showed no CtBP binding activity (Fig.
2B). GST alone also showed no interaction with MITR or
HDAC4, further establishing the specificity of the observed
interactions. The results of the GST pulldown experiments are
summarized in Fig. 2C.
The regions of MITR and HDAC5 responsible for recruitment of CtBP were
also assayed by coimmunoprecipitation assays in transfected 293T cells.
As observed in the GST binding assays, the interaction of MITR with
CtBP required the CtBP-binding motif between residues 23 and 27 and was
abolished by the DL-AS mutation of this motif (Fig. 2D). In
contrast, mutation of the corresponding region of HDAC5 (mutant
HDAC5-(EL-AS)) did not prevent its association of CtBP (Fig.
2E). This result suggested that HDAC5 contained additional CtBP-binding motifs. Indeed, a deletion mutant containing residues 121-664, which lacked the CtBP consensus binding motif at the analogous position to that in MITR, also bound CtBP (Fig.
2E), indicating the presence of an additional CtBP-binding
motif in this region of the protein. We conclude that CtBP is
specifically recruited to MITR through interaction with the
P-X-D-L-R motif, which is also conserved in the
amino-terminal region of class II HDACs. However, additional regions of
HDAC5 can also recruit CtBP.
Association of CtBP Enhances Transcriptional Repression by
MITR--
To investigate the potential significance of CtBP binding to
MITR, we compared the repressive activity of wild-type MITR and MITR
mutant DL-AS, which failed to bind CtBP. As shown in Fig. 3A, wild-type MITR fused to
the GAL4 DNA binding domain potently repressed transcription of a
GAL4-dependent luciferase reporter linked to the highly
active thymidine kinase promoter. GAL4-MITR(DL-AS) also repressed this
reporter, but the level of repression was reduced by at least 50% over
a 30-fold range of expression plasmid. Wild-type and mutant forms of
MITR were expressed at comparable levels in transfected cells (Fig.
2D).
Because MITR has been shown to repress MEF2-dependent
transcription (11), we compared the abilities of wild-type MITR and MITR(DL-AS) to repress activation of a luciferase reporter linked to
three tandem copies of the MEF2 consensus sequence. Consistent with the
results obtained with GAL4-MITR fusions, the MITR(DL-AS) mutant was
compromised in its ability to repress transcription, although it
clearly retained repressive activity (Fig. 3B).
Association of MITR with Class I and Class II HDACs--
The
ability of MITR(DL-AS) to repress transcription in the absence of CtBP
binding suggests the existence of redundant, CtBP-independent mechanisms for MITR-mediated repression. Consistent with this, MITR has
been shown to interact with HDAC1 (11). Coimmunoprecipitation experiments were performed to determine whether MITR(DL-AS) retained the capacity to bind HDAC1. As shown in Fig.
4A, both wild-type MITR and
MITR(DL-AS) efficiently associated with HDAC1 in transfected cells.
Furthermore, both proteins interacted with another class I HDAC, HDAC3,
as well as the class II HDACs 4 and 5. Homodimeric MITR complexes were
also detected in this assay. These results demonstrate that MITR
possesses the capacity to associate with multiple HDACs, independent of
CtBP binding, and suggest the existence of CtBP-dependent
and -independent mechanisms for transcriptional repression by MITR.
The results of this study demonstrate that MITR and class II HDACs
share homology in an amino-terminal CtBP-binding motif that recruits
CtBP to enhance transcriptional repression. Mutations in this
CtBP-binding motif that disrupt CtBP binding diminish the ability of
MITR to repress transcription, but such mutants retain substantial
repressive activity. Thus, we conclude that recruitment of CtBP by MITR
contributes to transcriptional repression. However, maximal inhibition
of transcription by MITR, as well as class II HDACs, likely involves
the cooperative action of CtBP and deacetylase activity provided in
trans by associated HDACs. Indeed, we show that MITR is able
to physically associate with several different class I and II HDACs.
CtBP1, first identified as a cellular protein that bound to the
carboxyl-terminal region of the E1a oncoprotein (18, 19), acts as a
transcriptional corepressor through association with the consensus
sequence P-X-D-L-S/R, found in a variety of transcription factors (20-28). CtBP1 and the closely related CtBP2 are expressed in
a wide range of tissues. The precise mechanism for transcriptional repression by CtBP is unclear. Other corepressors, such as NcoR and SMRT, repress transcription by recruiting HDACs (9, 10). CtBP1 has
also been shown to interact with HDAC1 (27), and TSA has been shown to
partially relieve repression by CtBP. However, other studies have found
that promoters repressed by CtBP are insensitive to TSA (29),
suggesting the existence of HDAC-independent mechanisms for repression
by CtBP.
The results of the present study provide the first demonstration of a
direct interaction between CtBP and class II HDACs. Class II HDACs can
physically associate with class I HDACs, and both types of HDACs can
interact with CtBP. Thus, it is clear that each of these proteins has
the capacity to serve as a bridge to the other, with the potential to
generate repression complexes that are linked to DNA-bound
transcription factors such as MEF2.
In contrast to the ubiquitous expression of CtBP, MITR and class II
HDACs are expressed predominantly in heart, skeletal muscle, and brain
(6, 30, 31), the same tissues in which MEF2 expression is highest (32,
33). While CtBP played only a partial role in MITR-mediated
transcriptional repression in our assays, in other cell backgrounds,
its contribution to the repressive activity of MITR could be greater,
if for example other trans-acting HDACs were expressed at
lower levels or were less active due to intracellular signals (13).
It is becoming increasingly clear that HDACs associate with a complex
array of cell-specific and ubiquitous transcriptional activators and
repressors to control gene expression of large sets of downstream
genes. In this regard, a role for these multiprotein complexes in the
regulation of cell differentiation is strongly supported by our recent
demonstration that class II HDACs, including HDAC4 and HDAC5, potently
repress the transcriptional program required for skeletal muscle
development (34, 35). This inhibitory action of HDAC is absolutely
dependent on its association with MEF2 and can be overcome by
calcium/calmodulin-dependent protein kinase signaling,
which disrupts MEF2·HDAC complexes (13, 34). Understanding in
molecular terms how HDAC·CtBP-containing complexes regulate
divergent gene programs will require identification of the many
components of these transcriptional complexes and elucidation of the
mechanisms by which they are regulated by cell identity and signaling systems.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activities
were measured under conditions of linearity with respect to time and
extract concentration.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic diagrams of class II HDACs and
MITR. A, schematic diagrams of MITR and class II HDACs.
B, amino acid sequences of human (h) and mouse
(m) HDACs and MITR with the CtBP-binding motif shown in
black.
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Fig. 2.
Association of class II HDACs and MITR with
CtBP in vivo and in vitro.
A, 293T cells were cotransfected with expression vectors
encoding FLAG-tagged CtBP and the indicated Myc-tagged derivatives of
HDAC4, HDAC5, or MITR (1 µg each). Myc-tagged proteins were
immunoprecipitated from cell lysates with a polyclonal anti-Myc
antibody, and coimmunoprecipitating FLAG-tagged proteins were detected
by immunoblotting with a monoclonal anti-FLAG antibody (upper
panel). The membrane was reprobed with an anti-Myc monoclonal
antibody to reveal total immunoprecipitated HDAC or MITR protein
(bottom panel). Mock-transfected cells were included as a
negative control. B, GST-HDAC and GST-MITR fusion proteins
were conjugated to glutathione-agarose beads and incubated with
[35S]methionine-labeled CtBP, as described under
"Experimental Procedures." After washing and recovery of the beads,
associated proteins were resolved by SDS-PAGE and analyzed by
autoradiography. In lane 1, 10% of the input
[35S]methionine-labeled CtBP was applied directly to the
gel. The positions of molecular weight markers are indicated to the
left. C, schematic representations of MITR
proteins and summary of their interactions with CtBP. D,
cells were cotransfected with expression vectors encoding FLAG-tagged
CtBP and the indicated Myc-tagged derivatives of MITR (1 µg each).
Myc-tagged proteins were immunoprecipitated from cell lysates with a
polyclonal anti-Myc antibody, and coimmunoprecipitating FLAG-tagged
proteins were detected by immunoblotting with a monoclonal anti-FLAG
antibody (upper panel). The membrane was reprobed with an
anti-Myc monoclonal antibody to reveal total immunoprecipitated MITR
protein (bottom panel). Mock-transfected cells were
included as a negative control. E, 293T cells were transfected with expression vectors (1 µg each) for the
indicated HDAC5 constructs (left-hand panels, FLAG-tagged;
right-hand panels, Myc-tagged) and CtBP (left-hand
panels, Myc-tagged; right-hand panels, FLAG-tagged).
HDAC5 was immunoprecipitated from cell lysates, and
coimmunoprecipitating CtBP was detected by immunoblotting (upper
panels). The membranes were reprobed with the appropriate
antibodies to reveal total immunoprecipitated HDAC5 (lower
panel).
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Fig. 3.
The CtBP-binding motif in MITR is required
for maximal MITR-mediated transcriptional repression.
A, 10T1/2 fibroblasts were transiently cotransfected with a
GAL4-dependent luciferase reporter plasmid, pMH100-tk-luc
(0.4 µg), expression vectors (10-300 ng) for either GAL4-MITR or
GAL4-MITR(DL-AS), and a CMV-lacZ control plasmid (0.1 µg).
Forty-eight hours later, cells were harvested and luciferase, and
-galactosidase activities were measured under conditions of
linearity with respect to time and extract concentration. The
luciferase values were normalized to
-galactosidase activity
to control for differences in transfection efficiency. B,
10T1/2 fibroblasts were transiently cotransfected with a luciferase
reporter plasmid driven by three copies of a MEF2 binding site,
3×MEF2-luciferase (0.3 µg), and expression vectors for MEF2C
(0.3 µg), and either MITR (0.1 µg) or MITR(DL-AS) (0.1 µg).
Luciferase activity in cell extracts was measured 48 h
post-transfection as described above. For A and
B, luciferase activity is depicted as -fold repression of
the reporter gene in cells expressing GAL4-MITR or GAL4-MITR(DL-AS)
relative to activity in cells lacking ectopic MITR. Values represent
the mean ± S.D. from at least two separate experiments.
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Fig. 4.
Association of MITR with class I and class II
HDACs. 293T cells were transiently cotransfected with expression
vectors encoding Myc-tagged MITR (0.75 µg) or MITR(DL-AS) (0.75 µg)
and FLAG-tagged versions of the indicated HDACs (0.75 µg).
Alternatively, cells were transfected with the Myc-MITR expression
vector (0.75 µg) in the absence of an HDAC expression plasmid
(Mock). Cells were harvested 48 h post-transfection,
and FLAG-tagged proteins were immunoprecipitated from cell lysates
using anti-FLAG affinity resin. Precipitated proteins were resolved by
SDS-PAGE and transferred to polyvinylidene difluoride membranes.
Membranes were immunoblotted with anti-Myc antibodies to reveal
MITR-HDAC interactions (upper panels) and
subsequently with anti-FLAG antibodies to control for HDAC expression
levels (bottom panels). The positions of molecular weight
markers are indicated to the left of each image.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to A. Tizenor for graphics and J. Page for editorial assistance.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Heath, The Robert A. Welch Foundation, and The D. W. Reynolds Foundation (to E. N. O).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF324492.
Supported as a Pfizer fellow of The Life Sciences Research Foundation.
§ To whom correspondence should be addressed. Tel.: 214-648-1187; Fax: 214-648-1196; E-mail:eolson@hamon.swmed.edu.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M007364200
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ABBREVIATIONS |
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The abbreviations used are: HDAC, histone deacetylase; CtBP, COOH-terminal-binding protein; HDRP, HDAC-related protein; MEF2, myocyte enhancer factor-2; MITR, myocyte enhancer factor-2-interacting transcription repressor; TSA, trichostatin; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.
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