From the Department of Molecular and Cellular
Pharmacology, University of Miami, Miami, Florida 33101 and the
Departments of § Molecular Genetics and ¶ Medical
Cardiology, Glasgow Royal Infirmary, University of Glasgow,
Glasgow G11 6 NU, United Kingdom
Received for publication, May 29, 2000, and in revised form, October 16, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The transcriptional integrator p300
regulates gene expression by interaction with sequence-specific
DNA-binding proteins and local remodeling of chromatin. p300 is
required for cardiac-specific gene transcription, but the molecular
basis of this requirement is unknown. Here we report that the MADS
(MCM-1, agamous, deficiens, serum response factor) box transcription
factor myocyte enhancer factor-2D (MEF-2D) acts as the principal
conduit for cardiac transcriptional activation by p300. p300 activation
of the native 2130-base pair human skeletal Transcriptional coactivators, or integrators, are members of a
class of transcription factors that bring about tissue- and stimulus-specific changes in gene expression by coordinating groups of
signal responsive proteins in the cell. Coactivators do not bind
independently to DNA, but are thought to stabilize the formation of
specific transcription factor-DNA complexes and to cause the local
unwinding of chromatin directly or by recruitment of histone-remodeling enzymes (1, 2). Specific coactivator families have been identified for
regulation of steroid hormone-responsive genes (SRC-1, SRC-3) (3-5)
and fatty acid regulatory proteins (PGC-1) (6, 7). Gene targeting
experiments have confirmed that the activities of these coactivators
in vivo are highly tissue-restricted, although the molecular
basis of this specificity is not well understood (4, 6, 8-10).
An important subgroup of coactivators is represented by the
closely related transcriptional integrators p300 and cAMP-responsive element binding protein-binding protein
(CBP).1 These large
proteins share extensive sequence homology and many structural features
and appear to have arisen as part of an ancestral gene duplication
(11-13). The cellular levels and activities of p300 and CBP are
tightly regulated through steroid, adrenergic, and growth factor
signals as well as during the cell cycle (14). Critical roles for p300
and CBP have been identified in growth, differentiation, apoptosis, and
tissue-specific gene expression, reflecting their interaction with
multiple cellular regulatory proteins (13). A particular requirement
for p300 is observed in the heart. Mice deficient in p300 have an
embryonic lethal phenotype characterized by failure of cardiac myocyte
proliferation and muscle-specific gene expression (10). In the
postnatal heart, adenovirus E1A selectively inhibits cardiac
muscle-specific gene expression by binding to p300 and/or related
proteins (15-17). Although p300 is also required for skeletal muscle
gene transcription by the tissue-specific basic helix-loop helix
protein MyoD, the heart lacks any equivalent to this transcription
factor (18). The molecular partners and pathways of
p300-mediated transcriptional regulation in the heart are not known.
Expression of the human skeletal Materials--
Expression vectors encoding MEF-2A-D were
generously provided by Dr. Eric Olson. A p300 expression vector
(pCMVp300 Plasmid Construction--
The distal hSA promoter sequence was
determined by automated DNA sequencing using an ABI 377 DNA sequencer
in the Biochemistry Core Facility, University of California, San
Francisco, CA. The resulting information was used to construct skeletal
actin promoter-luciferase chimeras. A 2335-bp HindIII
genomic fragment containing the hSA promoter sequence, comprising 2130 bp 5' and 203 bp 3' to the start of transcription, including all of
exon 1, was cloned in sense orientation into the HindIII
site of pGL-2Basic (Promega). Truncations and point mutations were
generated by cloning and/or PCR-mediated mutagenesis as described (32).
Fig. 1 shows the structure of each construct used in this paper and the
specific point mutations introduced. Primers used for introduction of
point mutations are shown in Table I.
Mutated bases are shown in bold letters. Numbering reflects the
position of hSA gene sequences relative to the transcription start
site. Our numbering reflects a systematic difference of +16 bp with the
sequence previously published by Muscat et al. (33) as
nucleotides
Internal and 5' deletions of the distal promoter were generated by
digestion of the hSA at unique restriction sites at
Introduction of point mutations to In Vivo Gene Transfer to Rat Myocardium--
Male Harlan
Sprague-Dawley rats (250-400 g) were premedicated with a mixture of
10-20 mg/kg fluanisone, 0.315-0.630 mg/kg fentanyl citrate (Hypnorm,
Jansen Pharmaceuticals), and 0.5-1.0 mg/kg midazolam (Hypnovel, Roche
Pharmaceuticals) given intraperitoneally. Animals were ventilated
(0.04-0.06 liters/min/kg) on a small animal respirator with 0.5-1.0
cmH2O of positive end-expiratory pressure and maintained
under anesthesia with a mixture of nitrous oxide and oxygen in a 1:1
ratio plus 0.5-1% halothane. The chest was opened by a left
thoracotomy and the pericardium removed for DNA injection. DNA was
directly injected into the apex of the left ventricle, using 100 µl/injection in a Hamilton syringe (25 µg of internal control
plasmid and 50 µg of hSA-luciferase plasmid suspended in
phosphate-buffered saline). Postoperative analgesia was administered
for at least the next 24 h with 0.2 mg/kg intramuscular buprenorphine (Vetergesic, Reckitt & Coleman). Seven days after surgery, animals were sacrificed with a lethal dose of pentobarbital sodium and the heart excised for assays of reporter gene expression. Enzyme determinations in tissue extracts were performed as described previously (35). pRSV-luciferase and pCAT3PV (SV40 promoter linked to
CAT) were used as internal controls and were obtained from Promega
Biotech (Madison, WI). All procedures were performed under license in
accordance with National Institutes of Health guidelines or in
accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986.
Cell Culture and Transfection--
HeLa cells were grown in MEM
with Eagle's salts, penicillin, streptomycin, and 10% fetal bovine
serum. Neonatal rat myocardial cells were isolated as described
previously (19) by gentle trypsinization and mechanical dissociation
over a period of 4-5 h, and plated at a density of 3.5-4 × 106 cells/60-mm culture dish. Cultures were enriched for
myocardial cells by preplating for 30-60 min to deplete the population
of nonmyocardial cells. Prior to and during transfection, cells were maintained in MEM with Eagle's salts, penicillin, streptomycin, and
5% fetal bovine serum (MEM-FBS). Following transfection, cells were
incubated in a serum-free medium consisting of MEM supplemented with
insulin, transferrin, vitamin B12, penicillin, and
streptomycin (MEM-TIB). All cells were maintained in 5%
CO2 atmosphere at 37 °C and transfected as described below.
Cardiac myocytes were transfected with reporter plasmids and other
constructs on the day following plating, using an adaptation of the
calcium phosphate method (36). Equal numbers of myocytes were
cotransfected with 10 µg of reporter plasmid and either 5 µg of
p300 expression vector, 5 µg of MEF-2 expression plasmid, or an equal
amount of blank CMV expression vector. 24 h after transfection,
cells were washed twice with MEM-TIB and maintained in that medium for
an additional 48 h. Cells were then washed twice with
phosphate-buffered saline, pH 7.4, and collected in 1× reporter lysis
buffer (Promega).
HeLa and C2C12 cells were transfected at a confluence of 50-70%, also
using the calcium phosphate method. Cells were maintained prior and
during transfection in MEM (HeLa) or DMEM (C2C12) supplemented with
10% fetal bovine serum, penicillin, and streptomycin. On the day after
transfection, cells were rinsed twice with fresh media and incubated
for 24-48 h before harvesting as described above. A commercially
available kit was used to measure luciferase activity in cell lysates
(Promega).
Gel Mobility Shift Assays--
For preparation of nuclear
extracts, cardiac myocytes were plated in 10-cm dishes and cultured in
MEM-FBS for 2 days, then switched to MEM-TIB for 3-5 days. Nuclear
extracts were prepared essentially as described previously (37), except
that the final extract was desalted on a 1-2-ml Sephadex G-25
chromatography mini-column instead of by dialysis. Protein
concentrations were determined using the Bio-Rad protein assay kit.
Aliquots of nuclear extract were frozen and stored at
Double-stranded oligonucleotide gel shift probes containing the
wild-type and mutant Immunoprecipitation--
50 µg of nuclear extract from cardiac
myocytes (prepared as described above) was mixed in equal amounts with
2× immunoprecipitation buffer (2% Triton X-100, 300 mM
NaCl, 20 mM Tris, pH 7.4, 2 mM EDTA, 2 mM EGTA, pH 8.0, 0.4 mM sodium orthovanadate,
0.4 mM phenylmethylsulfonyl fluoride, 1.0% Nonidet P-40).
Subsequently, 1-5 µg of either MEF-2 or p300 antibody was added.
Mixtures were incubated for 1 h at 4 °C. with constant
agitation. Protein A-agarose (20 µl; Santa Cruz Biotechnology) was
then added to the protein-antibody mixture, and tubes were incubated
for an additional 30 min at 4 °C. At the end of the incubation,
agarose beads were spun down, and the supernatant was reserved to
quantitate unbound proteins. Beads were washed three times with 1×
immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl,
10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM sodium orthovanadate, 0.2 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40),
resuspended in 30 ml of 2× electrophoresis sample buffer (250 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol
blue, 2% Coprecipitation of MEF-2/p300 Complexes Using a Biotinylated
Probe--
A biotinylated oligonucleotide containing the antisense ATr
motif (5'-biotin-TTACCAGAGCCTGCTGCAGGTTCTATTTATATCA-3') was annealed to
the complementary ATr (R) sense oligonucleotide (see Fig.
7A) at a final concentration of 10 µM and
linked to Dynabeads M-280 streptavidin (Dynal Inc., Lake Success, NY)
as described previously (39). For coprecipitation of MEF-2 and p300,
the beads were initially washed three times with phosphate-buffered
saline, pH 7.4, containing 0.1% bovine serum albumin, and twice with a
buffer containing 1 M NaCl, 10 mM Tris, 1 mM EDTA (TE-NaCl, pH 7.5). The beads were then mixed with
10 pmol of double-stranded biotinylated probe and incubated 20 min at
room temperature in TE-NaCl. Sequential washes with TE-NaCl and 1×
binding buffer (10 mM Tris, pH 7.5, 50 mM KCl,
1 mM MgCl2, 1 mM EDTA, 5.5 mM dithiothreitol, 5% glycerol, 0.3% Nonidet P-40) were
used to remove excess unbound oligonucleotide and to equilibrate the
beads with the binding buffer. Cardiac myocyte nuclear extract (50 µg) was mixed with 1× binding buffer for 5 min at room temperature
and incubated with the probe-linked beads for 20 min. Unbound proteins
were washed from the beads three times with 1× buffer containing 0.5 µg/ml poly(dI-dC), and aliquots of each wash were retained for
analysis. Finally, proteins specifically bound to the beads were eluted
with 1× binding buffer containing 1 M NaCl.
All fractions were resolved by SDS-PAGE (6% for p300 and 12% for
MEF-2) and analyzed by Western blot using antibodies to p300 and MEF-2
as described above.
MEF-2, SRF, and TEF-1 Binding Sites in the hSA Promoter Are
Required for Maximal Cardiac Expression in Vivo--
p300/CBP does not
bind DNA directly, but is recruited to specific gene control regions by
transcription factors that bind to these sites. Thus, we looked for
binding sites for known DNA-binding transcription factors in the hSA
promoter that could represent partners or mediators of
p300-dependent transactivation. The proximal human skeletal
actin promoter contains consensus binding sites for SRF, TEF-1, and
Sp-1, similar to the chick, mouse, and rat skeletal actin promoters
(Refs. 41, 35, and 56, respectively; see Fig.
1A), and additional regulatory
elements have been localized to the distal promoter (5' to
To test the functional relevance of these elements, the hSA promoter
was subjected to serial 5' truncations and point mutagenesis (Fig. 1,
B and C). The resulting constructs were assayed
for activity in adult myocardium by direct injection as described under
"Experimental Procedures" (Fig. 2).
These studies showed that the ATr site at A Distal AT-rich Site Is the Major p300-responsive Element--
We
next looked for specific DNA sequences in the hSA promoter that mediate
its activation by p300. Although the full-length hSA promoter
(p2130luc1) has constitutively high basal activity in cardiac myocytes,
comparable to that of The ATr Element Is a MEF-2 Binding Site--
The ATr site is
assumed to be a MEF-2 binding site based on its homology to similar
sites in other muscle-specific promoters (42). To identify cardiac
nuclear proteins binding to the p300-responsive ATr element, we
synthesized oligonucleotides containing both the wild type and mutant
sequences used for functional assays above (Table
II). Electrophoretic mobility shift
assays (EMSAs) were performed with nuclear extracts from cardiac
myocytes and from endothelial cells. Cardiac nuclear proteins
interacting with the ATr element formed at least three
sequence-specific bands (labeled 1-3 in Fig.
4A). The upper two bands
required the core ATr sequence, and did not form on a mutant ATr site
(Fig. 4A, lane 6), or on the GATA-like
sequence at
We next confirmed that MEF-2 proteins bind to the ATr site. The same
three nucleoprotein complexes identified in Fig. 4A were again seen forming on the ATr site, as well as on a synthetic MEF-2
site (Fig. 4B). The synthetic MEF-2 oligonucleotide also competed effectively for the three complexes binding to ATr. In both
cases, bands 1 and 2 were specifically and quantitatively supershifted
by a MEF-2 antibody that recognizes MEF-2 subtypes A, C, and D (Fig.
4B, lanes 5 and 10),
whereas a control MyoD antibody did not (data not shown). Thus, MEF-2
is present in two of three tissue-specific complexes bound to the ATr site.
The ATr site has significant homology to a GATA-4 binding site from the
B-type natriuretic peptide promoter that is required for its
cardiac-specific activation (43). To determine whether GATA-4 also
bound to the ATr site, we synthesized shorter oligomers representing
sequences centered at
Taken together, these data show that the distal hSA cardiac-specific
element is occupied by at least three muscle-specific protein
complexes: two containing MEF-2, and one with GATA-4. Moreover, MEF-2
and GATA-4 complexes form on distinct but overlapping sites within the
ATr motif.
p300 Binds Specifically to the Skeletal Actin MEF-2 Binding
Site--
We next looked for evidence that p300 interacts directly
with the MEF-2 DNA-binding complexes. The ATr and Atr-Left
oligonucleotides shown in Fig. 5A were labeled and allowed
to interact with cardiac nuclear proteins in the presence or absence of
specific antibodies. As in Figs. 4B and 5B, the
polyspecific MEF-2 antibody completely supershifted bands 1 and 2 (Fig.
6, lane 8). A
second, MEF-2D-specific antibody reacted only with band 1 (Fig. 6,
lane 7.) Band 3 on both oligonucleotides was
supershifted by a GATA-4 antibody (Fig. 6, lanes
3 and 10). These results show that the more
rapidly migrating complex is likely to contain MEF-2A, MEF-2C, or both,
whereas the slower moving complex (band 1) probably contains only the MEF-2D isoform.
Band 1, containing MEF-2D, was also the only complex that could be
shown to contain p300. A polyclonal antibody directed against the
NH2 terminus of p300 reacted very specifically with the
MEF-2D-ATr complex (Fig. 6, lane 9); bands 2 and
3 were not affected. None of the protein-DNA complexes on the Atr-Left
oligomer reacted with the p300 antibody. These data show that p300 is
specifically present in a complex with MEF-2D on the ATr, and not with
GATA-4 or other MEF-2 species. Morever, 3'-flanking sequences of the ATr are required for formation of this complex.
A MEF-2 site in the cardiac Endogenous Cardiac MEF-2 and p300 Interact on the ATr
Element--
We next asked whether MEF-2 and p300 bound to each other
directly or via contact with DNA. Initially, we attempted to
coimmunoprecipitate MEF-2 and p300 from cardiac nuclear extracts, using
antibodies against both p300 and MEF-2. The polyclonal MEF-2 antibody
was able to quantitatively immunoprecipitate at least two MEF-2 species (Fig. 8A, lane
4). However, we detected no p300 protein in the MEF-2
immunoprecipitates using either monoclonal or polyclonal p300
antibodies (Fig. 8A, lane 9).
Conversely, the anti-p300 monoclonal antibody (NM-11) successfully
immunoprecipitated p300 from cardiac nuclear extracts (Fig.
8A, lane 10), but did not
coprecipitate detectable MEF-2 species (data not shown). This may be
attributable to inaccessibility of the interaction domains in the
presence of antibody or to the lack of high affinity interaction
between the two proteins in the absence of DNA.
To determine whether MEF-2 and p300 interacted through DNA binding, we
performed DNA "pull-down" assays using Dynal beads linked to a
double-stranded oligonucleotide containing the hSA MEF-2 site (ATr) as
described previously (39). Cardiac nuclear proteins binding
specifically to this oligonucleotide were eluted from the beads and
characterized by Western analysis with MEF-2 and p300 antibodies. Two
MEF-2 proteins eluted from the hSA ATr site (Fig. 8B,
upper panel, lane 4).
Western analysis of the same blot using a MEF-2D-specific antibody
confirmed that the upper band contains MEF-2D (data not shown).
Importantly, a band corresponding to p300 was present in the same
eluate (Fig. 8B, lower panel, lane 4). A second protein of smaller size (about
140 kDa) was also detected by the NM-11 antibody and may represent a
degradation product. A biotinylated mutant ATr did not bind to either
MEF-2 or p300 (data not shown). These data show that endogenous cardiac MEF-2 and p300 form a specific complex with the ATr element, and they
support the results of the gel mobility retardation assays shown above.
MEF-2, but Not GATA-4, Is Synergistic with p300--
To address
the functional significance of the MEF-2-p300 interaction, we asked
whether the two proteins could activate cardiac transcription
synergistically. We measured the activity of the hSA wild-type
promoter, and the same promoter containing a point mutation in the ATr
motif (p2130luc1 and mATr, Fig. 1, B and C) in
the presence and absence of p300 and one of the four MEF-2 species.
Parallel experiments were performed in cardiac myocytes and HeLa cells
(Fig. 9A). Alone, none of the
individual MEF-2 subtypes significantly activated either the wild type
or mutant hSA promoters in cardiac myocytes, although a small amount of activation was seen in HeLa cells (Fig. 9A, white
bars). In cardiac myocytes, activation of the wild-type
promoter by MEF-2A, -B, or -C did not significantly increase in the
presence of p300 (Fig. 9A, blue bars).
However, the combination of MEF-2D and p300 significantly activated hSA
expression, compared with either blank vector or p300 alone
(p < 0.01, Fig. 9A). Because it is not
possible to establish the relative expression of the different MEF-2
species from these vectors, it may be that the divergent behavior of
MEF-2C and MEF-2D is due to quantitative differences in MEF-2 delivery or expression. However, all vectors contained the same CMV promoter and
were expressed at measurable levels in a HeLa cell background, suggesting that transfer and expression were not qualitatively defective for the MEF-2C vector (data not shown).
In HeLa cells, the combination of p300 and MEF-2D failed to activate
hSA transcription above 10% of its activity in cardiac myocytes, and
this activation was largely independent of the ATr site, suggesting
that additional cell type-specific factors are required for maximal
expression of this promoter. As expected, neither p300 nor MEF-2
species activated an hSA mutant lacking the ATr site (mATr) in cardiac
myocytes. These results show that p300 and MEF-2D are synergistic for
cardiac transcription of the hSA promoter and that this synergy is
exerted through the ATr site at
The skeletal actin gene has not previously been shown to be a target
for activation by GATA-4. However, as shown above, GATA-4 binds to the
hSA ATr site, flanking the MEF-2 binding sequences. Thus, GATA-4 might
also transactivate the hSA promoter, or participate in activation of
hSA by p300 through the ATr site. To investigate these possibilities, a
GATA-4 expression vector was cotransfected with the hSA wild type and
ATr site mutants. The ATr mutant was expressed at ~50% of wild type
promoter levels, as reported above. Coexpression of GATA-4 did not
activate basal expression of the wild type promoter at any
concentration (Fig. 9 and data not shown), and significantly reduced
its activation by p300 in the presence of an intact ATr (Fig. 9,
light bars). Together with the observed lack of
physical interaction in vivo, these observations suggest that GATA-4 is not directly involved in p300-mediated activation of
skeletal actin transcription.
Studies described here identify the myogenic transcription factor
MEF-2D as a specific physical and functional target of p300 in the
heart. Activation of the native 2180-bp human skeletal actin promoter
by p300 required a single short DNA sequence in the distal promoter, a
hybrid binding site for MEF-2 and GATA-4. Full myocardial expression of
the skeletal actin promoter required this same element, both in
vitro and in vivo. p300 bound exclusively to this
element, as part of a complex with MEF-2D, and could not be identified
in DNA complexes with GATA-4 or in a structurally related DNA-SRF
complex. Furthermore, p300 and MEF-2D displayed cooperative
transcriptional activation of the hSA promoter. This functional synergy
was not observed in HeLa cells, suggesting a requirement for additional
cell type-specific factors. Taken together, these findings identify
MEF-2D as a dominant partner for p300 in the myocardium and place p300
in the regulatory hierarchy of the cardiac phenotype (10, 15-17).
Our data show that p300 targeting of MEF-2D takes priority over its
potential interactions with several other proteins that bind to the
2130-bp hSA promoter, including GATA-4 and SRF. This observation is
remarkable, since other studies have shown that p300 can bind to both
SRF and members of the GATA family and can activate transcription
through their cognate binding sites (47, 48, 49). In contrast, we did
not observe physical or functional interaction between p300 and GATA-4
on the hSA promoter, nor did we detect p300 at GATA-4 binding sites in
the B-type natriuretic peptide, The MEF-2 family of transcription factors belongs to the MADS group of
DNA-binding proteins, characterized by their homology within an
amino-terminal domain ("MADS box"). Although MEF-2 is widely
expressed, its role in cardiac-specific transcription been well
documented. MEF-2C is required for normal cardiac morphogenesis, and
most if not all cardiac genes possess functionally important MEF-2
binding sites (42). MEF-2 species form DNA-binding homo- and
heterodimers and also bind to myogenic helix-loop-helix proteins as
coregulators. In skeletal muscle, MEF-2 proteins collaborate with MyoD
and p300 to promote myogenesis and muscle-specific transcription and
are thought to act in a positive autoregulatory loop that maintains
myogenic differentiation (18, 40, 52, 53). Other partners for MEF-2
proteins in cardiac transcriptional activation remain to be identified.
Our data suggest that the MEF-2D isoform may be targeted by p300 in
preference to other MEF species. We were able to demonstrate cooperative transcriptional activation between p300 and MEF-2D, but not
with the other MEF-2 isoforms, and p300 was localized to the DNA
complex containing MEF-2D. Our data do not exclude limited functional
interaction between p300 and other MEF-2 species at this promoter, but
it is also possible that the small positive interaction between the
other MEF species and p300 is mediated by heterodimerization with
endogenous MEF-2D. This finding is important because, to date, there
are few examples of functional differences between the four known MEF-2
species. All four mammalian MEF-2 species are expressed in the heart
(reviewed in Ref. 42), and all subtypes recognize the same AT-rich
consensus sequence (YTA(A/T)4TAR). Differential recruitment
of essential coactivator proteins may confer distinct transcriptional
activation properties on MEF-2D.
The observed lack of coactivation between MEF2C and p300 at this
promoter was unexpected. Several previous findings suggest that p300
and MEF-2C may act cooperatively in the cardiovascular system;
p300-deficient and MEF-2C-deficient mice have overlapping defects in
vascularization, and both have defects in cardiac development, although
these are dissimilar (54). MEF2C and D have closely parallel expression
patterns throughout cardiac development, and MEF2C expression actually
precedes that of MEF2D in the cardiac mesoderm (42). Furthermore,
cotranslated p300 and MEF2C have been shown to interact in
vitro (18). However, the physical and functional data presented
here suggest that MEF-2-p300 interactions may be subject to
modification by cell type-specific or promoter-specific factors. One
possibility is that MEF2C-p300 interactions play critical roles in
mesodermal patterning and in vasculogenesis, whereas MEF2D-p300 may be
more important for tissue-specific gene expression in the
differentiated cardiac myocyte. Further studies of MEF2D in
gene-targeted mice will help to clarify its specific roles.
The muscle-specific actions of MEF-2 cannot be explained by
tissue-restricted expression, or by differences between MEF-2 binding
sites in muscle-specific and ubiquitously expressed genes (55).
Instead, MEF-2 activity appears to be subject to significant post-transcriptional control by phosphorylation, and by the recruitment of additional factors (40, 56, 59). In support of this hypothesis, it
has been suggested that MEF-2-dependent transcription is
silenced in nonmuscle cell types by recruitment of HDAC-4, a histone
deacetylase (31). We propose that the converse is also true; activation of MEF-2-dependent genes in cardiac muscle requires
recruitment of histone acetyltransferase via p300. These findings point
to a unique role for MEF-2D in channeling both the activation and silencing signals of chromatin-remodeling enzymes to cardiac-specific promoters.
Our studies provide the first direct evidence that MEF-2 and p300
interact to regulate cardiac-specific transcription. Although MEF-2
proteins and p300 have been shown to interact in vitro (52, 18), and at artificial MEF-2-dependent promoters in
nonmuscle cells (18), the significance of these findings in
tissue-specific transcription has been unclear. In skeletal muscle, the
MEF-2-p300 interaction may serve to stabilize critical MyoD-p300
complexes on adjacent E boxes (52). However, our data indicate that the MEF-2-p300 complex can activate the tissue-specific transcription of
promoters that lack essential E-boxes, and can do so in the apparent
absence of basic helix-loop-helix proteins analogous to MyoD or
NeuroD/Beta2 (60, 61). It seems likely that additional, unidentified
cell type-specific factors cooperate with MEF-2 and p300 in the cardiac myocyte.
-actin promoter required
a single hybrid MEF-2/GATA-4 DNA motif centered at
1256 base pairs.
Maximal expression of the promoter in cultured myocytes and in
vivo correlated with binding of both MEF-2 and p300, but not
GATA-4, to this AT-rich motif. p300 and MEF-2 were coprecipitated from
cardiac nuclear extracts by an oligomer containing this element. p300
was found exclusively in a complex with MEF-2D at this and related
sites in other cardiac-restricted promoters. MEF-2D, but not other
MEFs, significantly potentiated cardiac-specific transcription by p300. No physical or functional interaction was observed between p300 and
other factors implicated in skeletal actin transcription, including
GATA-4, TEF-1, or SRF. These results show that, in the intact cell,
p300 interactions with its protein targets are highly selective and
that MEF-2D is the preferred channel for p300-mediated transcriptional
control in the heart.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin gene is tightly restricted
to striated muscle. In the myocardium, skeletal actin is one of a group
of "fetal" genes up-regulated in response to hypertrophic stresses
such as pressure overload in vivo, and during the response
to adrenergic stimulation, growth factors, and other neurohormonal
effectors in cell culture models (19-22). Although skeletal actin is
the predominant sarcomeric actin isoform in the human heart, it is
further up-regulated during hypertrophy (23-25) and is considered an
important marker of hypertrophy in the rat (20, 26-29). Regulation of
skeletal actin expression in the heart involves both extracellular
signal-responsive and cardiac-specific transcription factors; the
latter have not yet been identified. The
2130 bp human skeletal actin
promoter has two major transcriptional activation domains, proximal
(
153 to
87) and distal (
2130 to
710), which are required for
maximal tissue-specific expression in both skeletal and cardiac
myocytes (19). The proximal promoter contains functional binding sites for Sp-1, SRF, and TEF-1 (30). Here, we report the sequence of the
distal human skeletal actin (hSA) promoter, and demonstrate that only
one DNA motif within the entire 2130-bp transcriptional unit is capable
of transmitting the p300 activation signal. This motif, centered at
1256, binds the MADS (MCM-1, agamous, deficiens, serum response
factor) box transcription factor MEF-2 as well as GATA-4, and is
required for maximal expression in both neonatal and adult rat cardiac
myocytes. The motif also binds endogenous cardiac nuclear p300, and
coordinates synergistic activation of the hSA promoter by p300 and
MEF-2D. Remarkably, p300-dependent activation did not
involve interaction with GATA-4, SRF, or TEF-1, indicating that p300
selectively targets MEF-2 in the context of a native promoter. Based on
these findings and on recent data showing interactions between
MEF-2 and HDAC-4 (31), we propose that MEF-2 governs expression of the
cardiac phenotype by acting as a primary channel for chromatin
remodeling on cardiac-specific promoters.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) was the kind gift of Dr. R. Eckner. Polyclonal antibodies
against GATA-4, MEF-2, and p300 were obtained from Santa Cruz
Biotechnology. The monoclonal antibody against p300 (NM-11) was
supplied by PharMingen, and restriction enzymes were from New England
Biolabs. All other molecular biology reagents were purchased from Sigma
except as indicated, and were of the highest grade available.
1282 through
1177; the corresponding numbers in our
sequence are
1298 through
1193.
Oligonucleotides used for point mutagenesis
1787,
1656,
1298, and
1243, and internal religation or ligation to compatible
sites in the polylinker (Fig. 1B). The p1787luc1 plasmid was
created by digestion at the hSA SpeI site and the polylinker NheI site, and re-ligating. p1656 was generated by digestion
at the hSA NdeI site and religation of the proximal end to
the polylinker BglII site. Digestion with NdeI
and partial digestion with XbaI removed a small fragment of
358 bp, resulting in the dNX plasmid. The dNP plasmid was created by
double digestion with NdeI and PstI, gel
purification of the vector fragment, and religation.
GATA and ATr sites was performed
as described (34) with slight modifications. Briefly, two adjacent
primers were designed on opposite DNA strands with the mutation encoded
at the 5' end of one primer. Both primers were phosphorylated before
PCR. PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) was used to
increase fidelity of DNA replication and create blunt-ended PCR
products. The amplified plasmid with the introduced mutation was
isolated from agarose, religated and transformed to bacteria. Mutations
in the proximal promoter were made by directional cloning of
double-stranded 52-bp oligomers containing mutations in CArG I (m8,
m9), a TEF-1 site (mTEF), or sequences 5' to the CArG box (m6), flanked
by XbaIII and XhoI sites at the 5' and 3' ends,
respectively. The parental pluc1 vector was linearized with
XhoI and subjected to partial digestion with
XbaIII; each oligonucleotide was then ligated into this
vector to generate m6, m8, m9, and mTEF constructs. All clones were
screened by restriction analysis and sequenced to confirm the presence of each mutation.
80 °C.
GATA and ATr motifs were synthesized as shown
in Table II. Gel-purified oligonucleotide pairs were annealed and
end-labeled with [32P]ATP using T4 polynucleotide kinase
(New England Biolabs) and [
-32P]ATP (PerkinElmer Life
Sciences). Gel mobility shift assays were performed as described
previously (38) (69). In brief, equal amounts of radioactive probe
(1.5-2.5 × 104 cpm) were added to binding reactions
that contained 6 µg of nuclear extract protein in 20 µl of a buffer
containing 4 mM Tris (pH 7.8), 12 mM HEPES (pH
7.9), 60 mM KCl, 30 mM NaCl, 0.1 mM
EDTA, 1 µg/ml poly(dI-dC) (Amersham Pharmacia Biotech). Reactions
were incubated for 20 min at 22 °C and then separated on a
nondenaturing 5% polyacrylamide gel at 4 °C. Where indicated,
antibodies (2-4 µg/reaction) were incubated with the binding
reactions for 30 min at 22 °C before addition of the probe. For
determination of sequence-specific binding, a 100-fold molar excess of
unlabeled oligonucleotides was added immediately before the probe. No
DNA-antibody interaction was observed in the absence of nuclear protein
(data not shown).
-mercaptoethanol) and then boiled for 5 min. Aliquots of
the original nuclear extract, unbound proteins, and wash fractions were
also mixed with 2× electrophoresis sample buffer and boiled. All
protein samples were resolved on 6% SDS-polyacrylamide gels (for p300)
or 8% SDS-polyacrylamide (for detection of MEF-2 proteins). Gels were
transferred overnight to nitrocellulose membranes using a Transblot
electrophoresis transfer cell (Bio-Rad). Membranes were probed with
polyclonal MEF-2 antibody (SC-313, Santa Cruz Biotechnology),
polyclonal p300 (N-15) (sc-584, Santa Cruz Biotechnology), and
monoclonal p300 NM11 (14991A, PharMingen). Antigen-antibody complexes
were visualized by enhanced chemiluminescence (Pierce).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
710) (19).
To characterize these elements, we sequenced the distal human skeletal
actin promoter from
710 to
2130 (data not shown). Within this
sequence are two DNA elements resembling AP-1 binding sites (centered
at
1494 and
1300, Fig. 1A), as well as six variant
E-boxes (CANNTG; data not shown). A previously described AT-rich motif
was found centered at
1256 (33) (Fig. 1, A and
C). This AT-rich site differs by one nucleotide from the
consensus binding site for MEF-2 (CTA(A/T)4TAG; Ref. 40).
We also searched for GATA binding sites by BLAST screening of the hSA
promoter with the GATA binding site from the brain natriuretic peptide
promoter (CTGATAAATCAGAGATAACC). This search revealed two potential
GATA binding sites, one of which overlapped with the MEF-2 consensus
sequence. For convenience, the compound MEF-2/GATA binding motif was
called "ATr." An additional putative GATA site was centered at
1798 (Fig. 1, A and C) and designated
GATA.
View larger version (20K):
[in a new window]
Fig. 1.
Diagram of the hSA promoter and mutants.
A, schematic diagram of the hSA promoter, showing location
of major restriction sites and DNA motifs resembling binding sites for
AP-1 (white boxes), GATA-4 ( GATA),
GATA-4/MEF-2 (ATr), serum response factor (CArG),
and TEF-1 as indicated. The complete sequence has been published in
GenBankTM (accession no. AF288779). B, deletion and point
mutants of hSA promoter constructs. Arrow indicates
transcriptional start site. Shaded box, exon 1;
open box, start of luciferase sequence in
pGL2Basic. X indicates site of point mutations illustrated
in C. C, sequence of point mutations introduced
in designated hSA promoter constructs. The targeted DNA binding motifs
are boxed. Specific mutated bases are shown below
the corresponding wild type nucleotides (in bold). Details
of plasmid construction are included under "Experimental
Procedures."
1256 (mATr; Fig. 2), as
well as the TEF-1 and SRF binding sites in the proximal promoter
(mutants mTEF and m9, respectively; Fig. 2), were required for maximal
expression of the human skeletal actin promoter in the adult rat heart.
Deletion of sequences upstream of
1298 did not reduce, and in fact
enhanced expression, indicating that elements in this region (including
GATA) do not confer tissue-specific activation in the heart. In
contrast, point mutagenesis of ATr reduced hSA promoter activity by
about 50% (Fig. 2), suggesting that this element forms the core of the
previously reported distal tissue-specific element (19).
View larger version (13K):
[in a new window]
Fig. 2.
Expression of hSA promoter in adult rat
myocardium requires proximal and distal enhancer motifs. DNA was
delivered by injection into rat myocardium as described under
"Experimental Procedures," and luciferase activity was measured and
normalized after 1 week. p2130, p2130luc1; p1656,
p1656luc1; p1298, p1298luc1; p87, p87luc1 (please
refer to Fig. 1B). This graph summarizes data from a minimum
of 4 different animals and at least two different plasmid preparations
per construct.
actin (19), coexpression of p300 further
activated it by more than 3-fold (Fig.
3). Similar to our findings in
vivo, two different point mutations of the proximal SRE (CArG I)
and mutagenesis of the TEF-1 binding site each reduced basal hSA
promoter activity in cardiac myocytes by > 60% (mutants m8, m9,
and mTEF; Fig. 3). However, these sites were not required for
transactivation by p300 (Fig. 3). Mutation of the Sp-1 site in the
proximal promoter also reduced basal activity, but did not affect p300
transactivation (data not shown). Minimal expression of the basal
promoter (truncated at
87) was increased in the presence of p300,
possibly reflecting interaction of p300 with TATAA binding factors
(41). In contrast, point mutation of the AT-rich motif centered at
1256 (ATr) not only reduced basal hSA promoter activity, but also
abrogated transactivation by p300 (Fig. 3, mutants mATr and mATr
GA).
Although in some experiments mutation of the
GATA site at
1798
appeared to further reduce p300 transactivation, this effect was not
reproducible and did not achieve statistical significance
(p > 0.08). Deletion or mutation of other distal sites
had no significant on p300 transactivation. These findings indicated
that transcriptional activation by p300 required a single
tissue-specific AT-rich enhancer element in the hSA promoter.
View larger version (29K):
[in a new window]
Fig. 3.
Identification of a single p300-responsive
element in the hSA promoter. In vitro activity of the
2130-bp hSA promoter construct and mutants in the presence and absence
of cotransfected p300. Transfections were performed in neonatal rat
cardiac myocyte cultures as detailed under "Experimental
Procedures," and luciferase activity was measured 40-48 h later.
Light bars, hSA-luciferase construct alone;
dark bars, hSA luciferase construct + pCMVp300 . For all constructs, p
0.05 for
comparison between
p300 and +p300, except mATr (p = 0.3705) and mATr
GA (p = 1.0) (asterisks).
These data summarize a minimum of eight different transfection
experiments and three separate plasmid preparations per
construct.
1798,
GATA (Fig. 4A, lane
2). The third nucleoprotein complex formed specifically on
both ATr and mATr, suggesting that it interacts with sequences flanking
the core ATATA sequence (Fig. 4A, lanes
6 and 12). All three major nucleoprotein
complexes were muscle-restricted, as they were absent in endothelial
cell nuclear extracts (Fig. 4A, lanes
13 and 14).
Oligonucleotides used in EMSA assays
View larger version (52K):
[in a new window]
Fig. 4.
Muscle-specific proteins bind to the
p300-responsive element. A, cardiomyocyte and
endothelial cell nuclear protein interactions with hSA promoter.
Oligonucleotides corresponding to GATA and ATr motifs (see Fig.
1C and Table I) were synthesized and used to define
nucleoprotein interactions with these sites. A mutant ATr motif was
also used as a probe (mATr). The ATr motif forms three
specific protein complexes with cardiac myocyte nuclear proteins
(Cardiomyocytes; arrows labeled 1,
2, and 3). None of these bands are seen when
endothelial cell nuclear extract is used.
, labeled probe only;
NE, probe + nuclear extract; S, 100× unlabeled
self probe competitor; m
GA, unlabeled mutant
GATA oligonucleotide competitor; ATr, unlabeled ATr probe
competitor; mATr, unlabeled mutant ATr competitor.
B, MEF-2 binds to the hSA p300-responsive element. The
p300-responsive element (ATr) was labeled and used as a probe in EMSAs
as in Fig. 4A. For comparison, a commercially available
synthetic MEF-2 oligonucleotide (MEF-2) was also used as a
probe. Cardiac nuclear proteins formed three sequence-specific bands
with both of these probes (labeled 1,
2, and 3 as in Fig. 4A). A MEF-2
antibody supershifted bands 1 and 2 on both oligonucleotides; three
novel supershifted bands are seen (black
arrowheads).
, probe alone; NE, probe + nuclear
extract; S, 100× cold self competitor; MEF,
unlabeled synthetic MEF-2 oligonucleotide; ATr, unlabeled
ATr oligonucleotide; Ab, antibody against MEF-2.
1260 ("left"),
1252 ("right"), and
1256 ("center") (Fig.
5A). All three previously
detected complexes formed on each of these shorter oligonucleotides
(Fig. 5B), but with varying efficiency. Complexes 1 and 2 preferentially interacted with the right side of ATr (Fig.
5B, lane 9), whereas complex 3 preferentially bound to the left (Fig. 5B, lane
5). For all three oligonucleotides, only complexes 1 and 2 were supershifted by the MEF-2 antibody (Fig. 5B,
lanes 3, 7, and 11). Thus,
complex 3 binds a site adjacent to and overlapping that of complexes 1 and 2, and does not contain MEF-2. In contrast, a polyclonal
anti-GATA-4 antibody selectively depleted ATr complex 3, suggesting
that it contained GATA-4 (Fig. 5C, lane
4). This depletion was accompanied by appearance or
enhancement of a band comigrating with complex 1 (lane
4). When a synthetic GATA binding site was used as the probe
(Fig. 5C, lanes 5-9), we observed a
single cardiac nucleoprotein complex that comigrated with ATr complex 3 and was effectively competed by an ATr oligomer (Fig. 5C,
lane 8). Incubation with the GATA-4 antibody
generated a supershifted complex that migrated roughly in tandem with
complex 1 on the ATr site (Fig. 5C, compare lanes
9 and 4). Thus, the enhanced binding in
lane 4 appears to be identical with a
supershifted GATA-DNA complex, although we cannot formally exclude the
possibility that this represents enhanced binding of MEF-2. In either
case, it is clear that the most rapidly migrating nucleoprotein complex
on the ATr sequence contains GATA-4.
View larger version (35K):
[in a new window]
Fig. 5.
Overlapping binding sites for MEF-2 and
GATA-4 on the p300-responsive element. A, sequences of
original and truncated ATr oligonucleotides. Sequences are aligned with
a GATA consensus binding site to show the relative position of the GATA
(bold) and MEF-2 consensus sequences (boxed). The
position of nucleotide 1256 in the hSA promoter sequence is shown.
B, bands 1, 2, and 3 exhibit differential affinity for
truncated ATr sites. The oligonucleotides shown in A were
labeled and used as probes in EMSAs with cardiac myocyte nuclear
extracts as in Fig. 4. A MEF-2-specific antibody was used to confirm
the presence of MEF-2 in complexes 1 and 2 (bracket),
forming at least two supershifted bands (white
arrowheads). Note that complex 3 (arrow) is
enhanced on the "left" truncated oligomer (Left) and
absent or diminished from the "right" oligomer (Right).
As in Fig. 4, complex 3 is not supershifted by the MEF-2 antibody.
,
probe alone; NE, probe + nuclear extract; S,
100× cold self competitor; ab, probe, nuclear extract, and
MEF-2-specific antibody. C, GATA-4 binds to the hSA
p300-responsive element. EMSA of cardiac nuclear extracts was performed
as above using the ATr (Left) oligonucleotide and a
commercially available oligonucleotide containing two GATA binding
sites (Santa Cruz Biotechnology). A band similar to complex 3 appears
on the GATA consensus probe and is effectively competed by ATr
(black arrowheads; bar labeled
3). Both complexes 3 are supershifted by a GATA-4-specific
antibody (white arrowheads).
, probe alone;
NE, probe + nuclear extract; S, 100× cold self
competitor; ATr, 100× cold ATr oligonucleotide competitor;
ab, GATA-4-specific antibody.
View larger version (117K):
[in a new window]
Fig. 6.
Both MEF-2 and p300 contact the
p300-responsive element. EMSAs were performed as above, using the
original ATr (ATr (Full)) and ATr (ATr
(Left)) motifs as probes (please refer to Fig. 5A). A
polyspecific antibody against MEF-2 isoforms (MEF2) and
antibodies specific for MEF-2D (MEF2D), p300
(p300), and GATA-4 (GATA) were used to identify
these proteins in DNA-protein complexes, as indicated. The three major
protein-DNA complexes are numbered (1,
2, and 3). ATr (Full)
panels, as in Figs. 4B and 5B, the MEF-2
antibody generated three supershifted bands (black
arrows) and depleted bands 1 and 2 (lane
8). In contrast, the MEF-2D-specific antibody supershifted
only band 1 (lane 7, black
arrowhead). The p300 antibody also selectively depleted band
1 and generated at least 1 supershifted band (white
arrowhead). Band 3 (white arrow) is
supershifted exclusively by a GATA-4 antibody. ATr (Left)
panels, the left Atr motif was examined for interaction with p300 since
this oligomer maximizes GATA-4 binding. As with the full-length ATr,
band 3 is supershifted by a GATA-4 antibody, but not by a p300
antibody. None of the complexes formed on the ATr (left) motif appear
to interact with p300. NE, nuclear extract.
-myosin heavy chain (
-MHC) promoter
is required for maximal activity in cardiac myocytes (44-46). We were
interested in determining whether p300 could be identified at these
sites, or at a binding site for the related MADS protein, SRF, in the
proximal hSA promoter (Fig. 1A). Oligonucleotide sequences used for these studies are given in Table II. Fig.
7 shows that the AT-rich motifs from the
-MHC and M-creatine kinase (M-CK) form complexes similar
to hSA complexes 1, 2, and 3 (Fig. 7, lanes 2,
7, and 12). In each case, complexes 1 and 2 were
supershifted by a MEF-2 antibody (lanes 4,
9, and 14). Furthermore, each complex 1 was
supershifted by the p300 antibody (lanes 5,
10, and 15). No other bands were visibly
affected. Neither of two distinct sequence-specific complexes formed on
the hSA SRE was supershifted by the MEF-2 or p300 antibodies. These
results suggest that p300 interacts differentially with these two MADS
proteins and is selective for MEF-2 over SRF (Fig. 5, lanes
19 and 20).
View larger version (97K):
[in a new window]
Fig. 7.
p300 is present at other cardiac promoter
MEF-2 sites, but not at the hSA SRF site. Homologous AT-rich sites
in the cardiac -myosin heavy chain (
-MHC,
Ref. 48) and muscle creatine kinase (M-CK) promoters
were synthesized as shown in Table II and used as probes in EMSAs. The
hSA p300 responsive element (hSA ATr) and a
proximal serum response element centered at
93, CArG I
(hSA-SRE), were used for comparison. Complexes 1, 2, and 3 can be identified on all three MEF-2 sites but not on the SRE.
Black arrowheads, MEF-2 antibody-supershifted
bands. White arrowheads, p300
antibody-supershifted bands. Lanes are labeled as in Fig. 5
except as follows: Ab, antibody used for supershift;
M, MEF-2 antibody; P, p300 antibody.
View larger version (33K):
[in a new window]
Fig. 8.
Cardiac MEF-2 and p300 interact in a
DNA-dependent manner. A, independent
immunoprecipitation of MEF-2 and p300. A MEF-2 antibody recognizing
MEF-2A, -C, and -D was used to immunoprecipitate cardiac nuclear
extracts. Immunoprecipitates were subjected to Western analysis using
the same MEF-2 antibody (lanes 1-4) and a
polyclonal p300 antibody (lanes 5-9).
Lane 10 shows a p300 Western analysis of cardiac
nuclear proteins immunoprecipitated with a p300 antibody for
comparison. MEF-2 is nearly quantitatively precipitated from extracts
by this method and appears as a doublet at around 70 kDa
(black arrowhead). p300 is not detectable in the
MEF-2 immunoprecipitates, but is clearly seen in the p300
immunoprecipitates (black arrow,
right). T, total nuclear extract; U,
unbound proteins; W1 and W2, proteins in wash
fractions 1 and 2, respectively. Positions of size markers are shown to
left of autoradiogram. E, proteins specifically
eluted from beads. B, MEF-2 and p300 associate in the
presence of the ATr site. A "pull-down" assay was performed as
described under "Experimental Procedures," using a biotinylated hSA
ATr oligonucleotide linked to streptavidin-coated beads for affinity
purification of cardiac nuclear proteins. Nuclear extracts were
incubated with the DNA-linked beads for defined intervals and samples
of the total input protein (T), unbound protein
(U), wash fractions (W3), and specific eluates
(E) were subjected to Western analysis with MEF-2 and p300
antibodies as in panel A. Nuclear extracts from
C2C12 myoblasts were used as positive controls (T (C2)).
Upper panel is a MEF-2 Western blot. Cardiac
MEF-2 is eluted as a narrow doublet from the beads (arrows).
Note the presence of additional MEF-2 species in the C2C12 extract.
Lower panel is a p300 Western blot. A band
corresponding to p300 is visible in the total cardiac nuclear extract
(T) and in the specific eluate (E). Note a second
band of smaller size also recognized by the p300 antibody and
specifically eluted from the affinity beads. The identity of this
protein is not known, but it may represent a degradation product of
p300.
View larger version (20K):
[in a new window]
Fig. 9.
MEF-2 but not GATA-4 is synergistic with
p300. A, functional interaction between MEF-2D and
p300. Transcriptional activities of the intact hSA promoter
(p2130luc1) and a mutant lacking the p300-responsive site
(mATr) were compared in cardiac myocytes and HeLa cells as
indicated, in the presence and absence of p300, and with or without one
of the four MEF-2 isoforms (A-D). 35-mm dishes of cardiac
myocytes were transfected with the indicated constructs at a ratio of
reporter:p300:MEF = 2:1:1. Total transfected DNA was kept constant
by addition of appropriate amounts of blank CMV expression vector. This
graph summarizes three independent experiments in which substantially
similar data were obtained. B, GATA-4 and p300 are not
synergistic for skeletal actin promoter transcription. Cardiac myocytes
were transfected with the intact hSA promoter (p2130luc1,
black bars) or the p300 responsive site mutant
(mATr, light bars), alone or in the presence of
p300, GATA-4, or both. Transfections were performed as in
panel A and represent three independent
determinations. *, p < 0.05; **, p < 0.01, both for comparison with control.
1256.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-myosin heavy chain (50), or
angiotensin type I receptor promoters (51) under equivalent conditions
(data not shown). Our results do not exclude the possibility that
GATA-4 and p300 interact independently of DNA or under defined
conditions in vitro. Further work will be required to
establish the relationship between GATA-4- and
MEF-2-dependent transcriptional activation in the heart.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. R. Eckner, D. Livingston, and E. Olson for the generous gift of cDNA clones used in this paper. Dr. G. Q. Zeng assisted in construction of several of the promoter mutants. We especially appreciate the excellent technical support of Daryl Discher and Mary Gardiner, and the helpful comments of Dr. Gary Grotendorst.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL49891 (to N. H. B.) and HL44578 (to K. A. W.), by an Established Investigator grant from the American Heart Association (to N.H.B.), by a grant from the Miami Heart Research Institute (to N. H. B.), by a grant from the British Heart Foundation (to H. P.), and by a Wellcome Trust Foundation collaborative travel grant (to H. P., K. A. W., and N. H. B.).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) AF288779.
To whom correspondence should be addressed: Dept. of
Molecular and Cellular Pharmacology, University of Miami, P.O. Box
106189 (R-189), Miami, FL 33101. Tel.: 305-243-6775; Fax: 305-243-6082; E-mail: nhb@chroma.med.miami.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M004625200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CBP, cAMP-responsive
element binding protein-binding protein;
MEF, myocyte enhancer
factor;
bp, base pair(s);
MEM, minimal essential medium;
MEM-TIB, minimal essential medium supplemented with insulin, transferrin,
vitamin B12, penicillin, and streptomycin;
MEM-FBS, minimal
essential medium with Eagle's salts, penicillin, streptomycin, and 5%
fetal bovine serum;
-MHC,
-myosin heavy chain;
hSA, human
skeletal actin;
PCR, polymerase chain reaction;
EMSA, electrophoretic mobility shift assay;
SRE, serum response element;
MADS, MCM-1, agamous, deficiens, serum response factor.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Struhl, K.
(1998)
Genes Dev.
12,
599-606 |
2. | Lee, D. Y., Hayes, J. J., Pruss, D., and Wolffe, A. P. (1993) Cell 72, 73-84[Medline] [Order article via Infotrieve] |
3. |
Yao, T. P.,
Ku, G.,
Zhou, N.,
Scully, R.,
and Livingston, D. M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10626-10631 |
4. |
Xu, J.,
Liao, L.,
Ning, G.,
Yoshida-Komiya, H.,
Deng, C.,
and O'Malley, B. W.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
6379-6384 |
5. | Leo, C., and Chen, J. D. (2000) Gene (Amst.) 245, 1-11[CrossRef][Medline] [Order article via Infotrieve] |
6. | Lowell, B. B., and Spiegelman, B. M. (2000) Nature 404, 652-660[Medline] [Order article via Infotrieve] |
7. |
Vega, R. B.,
Huss, J. M.,
and Kelly, D. P.
(2000)
Mol. Cell. Biol.
20,
1868-1876 |
8. |
Tanaka, Y.,
Naruse, I.,
Maekawa, T.,
Masuya, H.,
Shiroishi, T.,
and Ishii, S.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10215-10220 |
9. | Kawasaki, H., Eckner, R., Yao, T. P., Taira, K., Chiu, R., Livingston, D. M., and Yokoyama, K. K. (1998) Nature 393, 284-289[CrossRef][Medline] [Order article via Infotrieve] |
10. | Yao, T. P., Oh, S. P., Fuchs, M., Zhou, N. D., Ch'ng, L. E., Newsome, D., Bronson, R. T., Li, E., Livingston, D. M., and Eckner, R. (1998) Cell 93, 361-372[Medline] [Order article via Infotrieve] |
11. | Giles, R. H., Dauwerse, H. G., van Ommen, G. J., and Breuning, M. H. (1998) Am. J. Hum. Genet. 63, 1240-1242[CrossRef][Medline] [Order article via Infotrieve] |
12. | Arany, Z., Sellers, W. R., Livingston, D. M., and Eckner, R. (1997) Cell 77, 799-800 |
13. | Giordano, A., and Avantaggiati, M. L. (1999) J. Cell. Physiol. 181, 218-230[CrossRef][Medline] [Order article via Infotrieve] |
14. | Snowden, A. W., and Perkins, N. D. (1998) Biochem. Pharmacol. 55, 1947-1954[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Bishopric, N. H.,
Zeng, G.-Q.,
Sato, B.,
and Webster, K. A.
(1997)
J. Biol. Chem.
272,
20584-20594 |
16. |
Kirshenbaum, L. A.,
and Schneider, M. D.
(1995)
J. Biol. Chem.
270,
7791-7794 |
17. |
Hasegawa, K.,
Meyers, M. B.,
and Kitsis, R. N.
(1997)
J. Biol. Chem.
272,
20049-20054 |
18. | Sartorelli, V., Huang, J., Hamamori, Y., and Kedes, L. (1997) Mol. Cell. Biol. 17, 1010-1026[Abstract] |
19. | Bishopric, N. H., and Kedes, L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2132-2136[Abstract] |
20. |
Bishopric, N. H.,
Jayasena, V.,
and Webster, K. A.
(1992)
J. Biol. Chem.
267,
25535-25540 |
21. | Izumo, S., Nadal-Ginard, B., and Mahdavi, V. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 339-343[Abstract] |
22. | Parker, T. G., Chow, K. L., Schwartz, R. J., and Schneider, M. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7066-7070[Abstract] |
23. | Boheler, K. R., Carrier, L., de la Bastie, D., Allen, P. D., Komajda, M., Mercadier, J.-J., and Schwartz, K. (1991) J. Clin. Invest. 88, 323-330[Medline] [Order article via Infotrieve] |
24. | Adachi, S., Ito, H., Tamamori, M., Tanaka, M., Marumo, F., and Hiroe, M. (1998) Life Sci. 63, 1779-1791[CrossRef][Medline] [Order article via Infotrieve] |
25. | Tanaka, M., Hiroe, M., Ito, H., Nishikawa, T., Adachi, S., Aonuma, K., and Marumo, F. (1995) J. Am. Coll. Cardiol. 26, 85-92[CrossRef][Medline] [Order article via Infotrieve] |
26. |
MacLellan, W. R.,
Lee, T. C.,
Schwartz, R. J.,
and Schneider, M. D.
(1994)
J. Biol. Chem.
269,
16754-16760 |
27. |
Karns, L. R.,
Kariya, K.,
and Simpson, P. C.
(1995)
J. Biol. Chem.
270,
410-417 |
28. |
Paradis, P.,
MacLellan, W. R.,
Belaguli, N. S.,
Schwartz, R. J.,
and Schneider, M. D.
(1996)
J. Biol. Chem.
271,
10827-10833 |
29. | Sugden, P. H., and Clerk, A. (1998) J. Mol. Med. 76, 725-746[CrossRef][Medline] [Order article via Infotrieve] |
30. | Taylor, A., Erba, H. P., Muscat, G. E. O., and Kedes, L. (1988) Genomics 3, 323-336[Medline] [Order article via Infotrieve] |
31. |
Miska, E. A.,
Karlsson, C.,
Langley, E.,
Nielsen, S. J.,
Pines, J.,
and Kouzarides, T.
(1999)
EMBO J.
18,
5099-5107 |
32. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Press, Cold Spring Harbor, NY |
33. | Muscat, G. E., Perry, S., Prentice, H., and Kedes, L. (1992) Gene Exp. 2, 111-126 |
34. | Fisher, C. L., and Pei, G. K. (1997) BioTechniques 23, 570-590[Medline] [Order article via Infotrieve] |
35. | Prentice, H., Kloner, R. A., Prigozy, T., Christensen, T., Newman, L., Li, Y., and Kedes, L. (1994) J. Mol. Cell Cardiol. 26, 1393-1401[CrossRef][Medline] [Order article via Infotrieve] |
36. | Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051[Medline] [Order article via Infotrieve] |
37. | Dignam, J. D., Lebovitz, R. M., and Roeder, R. (1983) Nucleic Acids Res. 11, 1475-1489[Abstract] |
38. | Semenza, G. L., and Wang, G. L. (1992) Mol. Cell. Biol. 12, 5447-5454[Abstract] |
39. |
Ebert, B. L.,
and Bunn, H. F.
(1998)
Mol. Cell. Biol.
18,
4089-4096 |
40. | Molkentin, J. D., Black, B. L., Martin, J. F., and Olson, E. N. (1995) Cell 83, 1125-1136[Medline] [Order article via Infotrieve] |
41. | Abraham, S. E., Lobo, S., Yaciuk, P., Wang, H. G., and Moran, E. (1993) Oncogene 8, 1639-1647[Medline] [Order article via Infotrieve] |
42. | Black, B. L., and Olson, E. N. (1998) Annu. Rev. Cell Dev. Biol. 14, 167-196[CrossRef][Medline] [Order article via Infotrieve] |
43. | Grepin, C., Dagnino, L., Robitaille, L., Haberstroh, L., Antakly, T., and Nemer, M. (1994) Mol. Cell. Biol. 14, 3115-3129[Abstract] |
44. |
Molkentin, J. D.,
and Markham, B. E.
(1993)
J. Biol. Chem.
268,
19512-19520 |
45. | Molkentin, J. D., Jobe, S. M., and Markham, B. E. (1996) J. Mol. Cell Cardiol. 28, 1211-1225[CrossRef][Medline] [Order article via Infotrieve] |
46. | Molkentin, J. D., and Markham, B. E. (1994) Mol. Cell. Biol. 14, 5056-5065[Abstract] |
47. |
Ramirez, S.,
Ait-Si-Ali, S.,
Robin, P.,
Trouche, D.,
Harel-Bellan, A.,
and Ait Si Ali, S. A. S. A.
(1997)
J. Biol. Chem.
272,
31016-31021 |
48. |
Blobel, G. A.,
Nakajima, T.,
Eckner, R.,
Montminy, M.,
and Orkin, S. H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2061-2066 |
49. |
Kakita, T.,
Hasegawa, K.,
Morimoto, T.,
Kaburagi, S.,
Wada, H.,
and Sasayama, S.
(1999)
J. Biol. Chem.
274,
34096-34102 |
50. |
Hasegawa, K.,
Lee, S. J.,
Jobe, S. M.,
Markham, B. E.,
and Kitsis, R. N.
(1997)
Circulation
96,
3943-3953 |
51. |
Herzig, T. C.,
Jobe, S. M.,
Aoki, H.,
Molkentin, J. D.,
Cowley, A. W.,
Izumo, S.,
and Markham, B. E.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
7543-7548 |
52. | Eckner, R., Yao, T. P., Oldread, E., and Livingston, D. M. (1996) Genes Dev. 10, 2478-2490[Abstract] |
53. |
Puri, P. L.,
Avantaggiati, M. L.,
Balsano, C.,
Sang, N.,
Graessmann, A.,
Giordano, A.,
and Levrero, M.
(1997)
EMBO J.
16,
369-383 |
54. |
Lin, Q.,
Schwarz, J.,
Bucana, C.,
and Olson, E. N.
(1997)
Science
276,
1404-1407 |
55. |
Naya, F. J.,
Wu, C.,
Richardson, J. A.,
Overbeek, P.,
and Olson, E. N.
(1999)
Development
126,
2045-2052 |
56. | Molkentin, J. D., Kalvakolanu, D. V., and Markham, B. E. (1994) Mol. Cell. Biol. 14, 4947-4957[Abstract] |
57. |
Molkentin, J. D.,
Li, L.,
and Olson, E. N.
(1996)
J. Biol. Chem.
271,
17199-17204 |
58. |
Yang, S. H.,
Galanis, A.,
and Sharrocks, A. D.
(1999)
Mol. Cell. Biol.
19,
4028-4038 |
59. |
Molkentin, J. D.,
and Olson, E. N.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
9366-9373 |
60. | Naya, F. J., Stellrecht, C. M., and Tsai, M. J. (1995) Genes Dev. 9, 1009-1019[Abstract] |
61. | Lee, J. E., Hollenberg, S. M., Snider, L., Turner, D. L., Lipnick, N., and Weintraub, H. (1995) Science 268, 836-844[Medline] [Order article via Infotrieve] |