GATA-4 and Serum Response Factor Regulate Transcription of the
Muscle-specific Carnitine Palmitoyltransferase I
in Rat
Heart*
Meredith L.
Moore
,
Guo-Li
Wang
,
Narasimhaswamy S.
Belaguli§,
Robert J.
Schwartz§, and
Jeanie B.
McMillin
¶
From the
Department of Pathology and Laboratory
Medicine, Medical School, University of Texas-Houston Health
Science Center and the § Department of Cell Biology, Baylor
College of Medicine, Houston, Texas 77030
Received for publication, October 13, 2000, and in revised form, October 17, 2000
 |
ABSTRACT |
Transcriptional regulation of nuclear
encoded mitochondrial proteins is dependent on nuclear
transcription factors that act on genes encoding key components of
mitochondrial transcription, replication, and heme biosynthetic
machinery. Cellular factors that target expression of proteins to the
heart have been well characterized with respect to
excitation-contraction coupling. No information currently exists that
examines whether parallel transcriptional mechanisms regulate nuclear
encoded expression of heart-specific mitochondrial isoforms. The muscle
CPT-I
isoform in heart is a TATA-less gene that uses Sp-1 proteins
to support basal expression. The rat cardiac fatty acid response
element (
301/
289), previously characterized in the human gene, is
responsive to oleic acid following serum deprivation. Deletion and
mutational analysis of the 5'-flanking sequence of the carnitine
palmitoyltransferase I
(CPT-I
) gene defines regulatory regions in
the
391/+80 promoter luciferase construct. When deleted or
mutated constructs were individually transfected into cardiac
myocytes, CPT-I/luciferase reporter gene expression was significantly
depressed at sites involving a putative MEF2 sequence downstream from
the fatty acid response element and a cluster of heart-specific
regulatory regions flanked by two Sp1 elements. Each site demonstrated
binding to cardiac nuclear proteins and competition specificity (or
supershifts) with oligonucleotides and antibodies. Individual
expression vectors for Nkx2.5, serum response factor (SRF), and GATA4
enhanced CPT-I reporter gene expression 4-36-fold in CV-1 cells.
Although cotransfection of Nkx and SRF produced additive luciferase
expression, the combination of SRF and GATA-4 cotransfection resulted
in synergistic activation of CPT-I
. The results demonstrate that SRF
and the tissue-restricted isoform, GATA-4, drive robust gene
transcription of a mitochondrial protein highly expressed in heart.
 |
INTRODUCTION |
Expression of nuclear and mitochondrial encoded expression of
respiratory chain subunits occurs despite physical separation of
transcriptional events within separate genomes. Stimulation and
coordination of mitochondrial gene expression from these two sites is
accomplished by the nuclear respiratory factors, NRF-1 and NRF-2 (1,
2). Using electrical stimulation to produce hypertrophic growth of
neonatal cardiac myocytes, the transcriptional activation of cytochrome
c is preceded by induction of NRF-1 mRNA (3). This
observation is consistent with NRF-1 induction as a prerequisite for
synthesis of respiratory chain components. These basic insights into
cellular factors that link nuclear events to mitochondrial gene
activation are critical for adaptation to environmental stresses and
the necessity for enhanced energy production.
In contrast to subcellular coordination of mitochondrial biogenesis and
respiratory chain synthesis, less is known concerning tissue-specific
transcriptional regulation of nuclear encoded genes involved in energy
metabolism. These genes are particularly important in cardiac muscle
where contractile activity must be supported by a high rate of aerobic
ATP production. There are examples of muscle- or heart-specific
mitochondrial proteins that contain sites for ubiquitous transcription
factors as well as striated muscle-specific motifs in the proximal
promoter, e.g. muscle-specific cytochrome oxidase genes (4).
Increases in contractile activity induced by electrical stimulation of
neonatal rat cardiac myocytes in culture results in increased mRNA
levels of the striated muscle-specific, energy-metabolizing enzymes, carnitine palmitoyltransferase I
(CPT-I
)1 (3), and
adenylate-succinate synthase I, a component of the purine nucleotide
cycle (5).
CPT-I
(the muscle isoform) has been cloned and is the predominant
isoform in rat heart and the sole enzyme expressed in skeletal muscle
(6). CPT-I
is expressed in most tissues, including the heart. The
latter isoform is present at very low levels in the adult cardiac
myocyte and is absent in skeletal muscle (6). CPT-I is an example of an
enzyme for which the isoforms are very different kinetically. The
muscle isoform exhibits an affinity for carnitine that is at least an
order of magnitude higher in Km and a
KI for malonyl-CoA that is an order of magnitude
lower than the KI measured for CPT-I
(6). High
expression of the muscle isoform of CPT-I is characteristic of adult
heart, so that this particular isoform appears adapted for efficient
derivation of energy from long chain fatty acids in an active
contracting myocyte.
Very little is known about the heart-specific elements that drive
transcription of CPT-I
in heart. Recent work has demonstrated the
presence of a fatty acid response element (FARE) in the human CPT-I
promoter (7). After exposing neonatal rat cardiac myocytes to
serum-free conditions, addition of exogenous oleate increases expression of the human CPT-I
reporter gene 8-20-fold and CPT-I
mRNA levels rise 4-5-fold (7). Peroxisome proliferator-activated receptor
and the retinoid X receptor act to activate
CPT-I
through the FARE site. Peroxisome proliferator-activated
receptor
may also play a pivotal role in the expression of enzymes
of
-oxidation (8). The physiological impact of fatty acid induction of CPT-I
in heart is less certain since cardiac CPT-I is resistant to fasting, a condition that enhances serum fatty acid concentrations (9, 10).
We hypothesized that factors that increase expression of tissue
specific proteins involved in contractility and energy-utilizing reactions in heart would also be important in regulating the expression of an enzyme involved in transformation of its major energy substrate, long chain fatty acids. Our studies demonstrate for the first time that
CPT-I
is regulated by the muscle-specific factor, GATA-4, and by
combinatorial interactions between GATA-4 and the nuclear factor, serum
response factor (SRF). Interaction between MADS box and C4 zinc finger
proteins represents a novel coregulator mechanism of the cardiac actin
promoter (11). SRF is especially abundant in embryonic and adult
cardiac, skeletal and smooth muscle cells (12-15). The recent
homologous recombinant knockout of the murine SRF gene locus
demonstrated that SRF is absolutely required for the appearance of
mesoderm and muscle lineages during mouse embryogenesis (12). The
identification of CPT-I
as a GATA-4-dependent gene that
is coactivated by SRF is the first suggestion that this paradigm may
represent an important mechanism by which expression of cardiac
specific genes is synchronized between subcellular compartments.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transfections--
Primary cultures of neonatal
rat cardiac myocytes were prepared as described previously (16). Cells
were plated at a density of 6 × 105 cells/well in
six-well plates (Primeria, Fisher), and maintained in Dulbecco's
modified Eagle's medium (DMEM, CellGro, Fisher) with 1%
penicillin/streptomycin (Life Technologies, Inc.) and 10% calf serum
(HyClone Laboratories, Inc.). The cells were incubated at
37o in the presence of 95% O2 and 5%
CO2 for 36 h before transfection. Myocytes were
transfected using calcium phosphate precipitation in the presence of
serum as described previously (18). The calcium phosphate precipitate
contained 1.0 µg of CPT-I
firefly luciferase vector and 0.25 µg
of a CMV-driven Renilla luciferase expression vector
(Promega) as a control for transfection efficiency. Six hours following
transfection, the myocytes were washed twice with phosphate-buffered
saline and maintained in DMEM with serum for an additional 48 h.
For treatment with oleic acid, the myocytes were transfected with the
CPT-I
(
318/+80) reporter gene and maintained in DMEM + serum for
12 h, washed with phosphate-buffered saline and incubated in
serum-free DMEM ± 0.5 mM oleate (2:1 molar ratio
oleate:bovine serum albumin) for an additional 24-30 h. CV-1 monkey
kidney fibroblasts (ATCC no. CCL-70) were maintained as above but were
trypsinized 24 h before transfection and plated to reach 85%
confluence by the start of the experiment. CV-1 cells were transfected
using LipofectAMINE Plus reagent system (Life Technologies, Inc.) in
serum-free medium. After 3 h, the transfection medium was replaced
with fresh serum-containing medium for 48 h. Cotransfections
included 1.0 µg of the wild-type, truncated, or mutant CPT-I
firefly luciferase reporter constructs and various combinations of the
following CMV expression vectors: 0.4 µg of Nkx2.5, 0.4 µg of
GATA-4, 0.1 µg of SRF, 0.1 µg of SRF-
C, and 0.1 µg of SRF-pm.
Total DNA for each transfection was corrected to a final concentration
of 2.0 µg by addition of empty CMV vector. Total protein in each well
was measured using the BCA protein assay reagent kit (Pierce). CPT-I
firefly luciferase was corrected for protein and normalized to that of
the CMV/Renilla expression for each separate experiment.
Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclear
extracts from primary rat neonatal cardiac myocytes were prepared as
described previously (17). Double-stranded DNA probes containing
sequences from the rat CPT-I
promoter were synthesized by Operon
Technologies, Inc. (Alameda, CA) as shown in Table I. EMSA reaction
mixtures included 2-15 µg of nuclear extract, 25 mM
Hepes, 100 mM KCl, 0.1% Nonidet P-40 (v/v), 1 mM dithiothreitol, 5% glycerol, and 50 ng of poly(dI·dC)
as a nonspecific competitor in a 20-µl reaction volume. After
incubation for 10 min at room temperature, 0.3 ng of radiolabeled probe
was added and the reaction incubated for 20 min. When included,
specific antibodies (Santa Cruz) or 100-fold molar excess of cold probe was added during the first incubation. Protein-DNA complexes were separated on a 4% nondenaturing polyacrylamide gel at 25 °C.
Plasmids and Constructs--
Construction of the rat CPT-I
promoter fragment
391/+80 has been reported previously (18). The
expression vector pCMV-Nkx 2.5 was a gift from Dr. Janet Mar.
Construction of the expression vectors for pCGN-SRF, pCGN-SRFpm,
pCGN-SRF
C, and pCDNA3-GATA-4 has been described (15). A series
of CPT-I
promoter constructs with the 5' end between
315 and
31
and the 3' end from
1 to +80 were created by PCR amplification from
the plasmid template,
361/+80. Specific primers were designed with an
artificial restriction site and the desired region of the rat CPT-I
promoter. For deletion of the MEF2 region contained in the
306/+80
construct, 10 bp (
281 to
270) were deleted from the artificial
primer. The PCR products were restricted and cloned into the multiple
cloning site of the promoterless firefly luciferase vector, pGL3 basic (Promega, Madison, WI). The inserted CPT-I
fragment in each
construct was confirmed by DNA sequencing. The Renilla
expression vector, pRL, was purchased from Promega. Mutations were
introduced to the
391/+80 CPT-I construct with the QuickChange
site-directed mutagenesis kit (Stratagene, La Jolla, CA). Briefly, high
performance liquid chromatography-purified primers containing the
desired mutation were used in concert with the high fidelity polymerase Pfu-turbo for PCR amplification of the
391/+80 plasmid.
Wild-type template was digested with DpnI, and the remaining
mutated product was transformed into Escherichia
coli and sequenced for correct insertion of the mutant primer.
Quantification of CPT-I
Transcripts--
Specific
quantitative assay of rat CPT-I
was performed using real time PCR
(7700 Prism, PerkinElmer Life Sciences) based on hydrolysis of a
specific fluorescent probe at each amplification cycle by the
endonuclease activity of Tac polymerase as described previously (19). The sequence for the muscle-specific CPT-I was
obtained from GenBankTM (accession no. D43623, nucleotide numbers
869-889 (forward primer) and 932-952 (reverse primer)). The level of
transcripts for the constitutive housekeeping gene product,
cyclophilin, was quantitatively measured for each sample to control for
sample-to-sample differences in RNA concentrations. The PCR data are
reported as the number of transcripts per number of cyclophilin
mRNAs (19).
Statistics--
Each experiment was performed in triplicate, and
the reported values represent the mean of three to five separate
cultures ± standard error. The significance of the differences
was determined using Student's t test for nonpaired and
paired variates (SigmaPlot statistics software).
 |
RESULTS |
A region within 391 bases 5'of the first exon is sufficient to
drive the heart-specific expression of the CPT-I
luciferase reporter
gene (18). By normalizing the
391/+80 reporter construct to 1.00, the
ratio of the larger genomic fragment,
1188/+80, to
391/+80 is
0.93 ± 0.075. Therefore, we conclude that the promoter elements
that regulate CPT-I
expression to the heart are within 390 bp of the
transcription start site. A FARE was located in the rat gene at
303
to
296. When 0.5 mM oleic acid (2:1 molar ratio with
bovine serum albumin) was added to serum-free medium and neonatal
cardiac myocytes cultured for 24-30 h, CPT-I
/luciferase expression
was increased from 0.63 ± 0.02 relative luciferase units (in the
absence of oleate) to 1.88 ± 0.02 in the presence of oleate.
Partial deletion of this region (
bases
301 to
293) resulted in
a diminished fatty acid effect, i.e. 0.80 ± 0.01 relative luciferase units (in the presence of oleate) from 0.65 ± 0.06 (in the absence of oleate). Electrophoretic mobility shift assays of this region (Table I) demonstrated
binding of myocyte nuclear protein to this site, and this binding was
competed by 100- fold excess of cold oligonucleotides and antibodies to
the binding factor COUP-TF (20), but not by mutant oligonucleotides
(data not shown).
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Table I
Oligonucleotides for EMSA and reporter gene analysis
Double-stranded oligonucleotides were synthesized for use in DNA EMSAs.
The consensus sequences for factor binding are underlined and the
mutational changes shown in capitals. The quick-change primers
represent the sequence-specific mutations, shown in capitals, of the
indicated sites used for transfection studies of the luciferase
reporter gene.
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To map the regions in the CPT-I
gene that influence expression of
the luciferase reporter construct,
391/+80 was progressively deleted
from the 5' end and analyzed for luciferase expression following
transfection into neonatal cardiac myocytes (Fig.
1). Compared with the full-length
391/+80, deletion of 85 bp to
306/+80 produced minimal changes in
luciferase expression. Deletion of the MEF2 site within the
306/+80
reporter gene resulted in an overall decrease in luciferase expression
of 34%. With further deletion of the 5'-flanking sequence to
270/+80, luciferase expression progressively decreased by 60%.
Subsequent deletion of a consensus E box (
252/
247) led to a
significant (85%) enhancement of luciferase expression. In the
presence of neonatal cardiac myocyte nuclear protein, EMSA analysis of
the
252 E box demonstrated a shift (Table I) that was competed by
100× wild-type oligonucleotide, but not by 100× mutant
oligonucleotide (Fig. 2). The shift was supershifted by antibodies to either USF1 or USF2 and supershifted to a
higher molecular weight complex in the presence of both antibodies (Fig. 2A). The data suggest that the
252 E box interacts
with USF1 and USF2 and may function as a suppressor of CPT-I
.
Deletion of 165 bp of flanking sequence diminishes full-length CPT-I
expression by 50% (
126/+80). This truncation removed one Sp1 site at
136/
131 and a GATA site at
129/
126; deletion of 15 more bp
containing a CA box (
117/
112) resulted in a dramatic decrease in
reporter gene expression by 90%. Further deletion resulted in partial
restoration of activity and an extended deletion produced a minimal
promoter containing an Sp-1 site at
74/
68 (Fig. 1).

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Fig. 1.
Deletion analysis of the
CPT-1 391/+80 promoter fragment. Serial
deletions of the 391/+80 promoter fragment ligated to the luciferase
reporter were transfected into neonatal cardiac myocytes. Transfection
reactions included 1.0 µg of reporter and 250 ng of CMV-driven
Renilla luciferase expression vector as a control for
transfection efficiency. Following transfection, cells were maintained
in DMEM containing 10% fetal calf serum for 48 h. Firefly
luciferase was normalized to Renilla and is expressed as a
ratio multiplied by 100. Bars represent the means ± S.E. of two
to three experiments performed in triplicate.
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Fig. 2.
Identification of specific transcription
factors binding to the CPT-1 promoter.
Protein binding to sequences in the CPT promoter was characterized by
gel-mobility shift assay. Oligomers containing binding sites from the
CPT-1 promoter were end-labeled with [ -32P]ATP and
incubated with neonatal rat heart-myocyte, nuclear-protein extracts
(see "Experimental Procedures"). Unlabeled competitor oligomers or
antibodies were included as indicated above each lane. A,
labeled oligomers corresponding to Ebox 252 incubated with rat
myocyte nuclear extract (RMNE) and 2 µl of either USF1 or USF2
antibodies or 1 µl of each for a total of 2 µl. B,
sequences representing MEF2 280 incubated with 4 µg of RMNE and
MEF2A antibody (2 µl) or with TNT proteins (1 µl of final TNT
reaction). C, labeled probes for the Sp1 74 binding site
incubated with 10 µg of RMNE and 2 µl of either Sp1 or Sp3
antibody. D, Labeled oligomers representing GATA-129 and
GATA-96 sites incubated with 10 and 15 µg of RMNE, respectively;
GATA4 antibodies added as indicated. E, labeled probes for
the SRE-112 binding site incubated with 5 µg of RMNE; SRF antibody (2 µl) added as shown.
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To examine potential regulatory elements in the context of the intact
promoter, mutations were targeted to known sequences contained in the
deletion constructs that appear to serve as regulators of CPT-I
expression. The mutated constructs were transfected into neonatal
cardiac myocytes for measurement of luciferase expression. In agreement
with the deletion data, mutation of the MEF2 site (
280/
271)
produced a 46% decrease in reporter gene activity (Fig.
3). Gel mobility-shift assays were
conducted to examine nuclear protein binding to double-stranded
oligonucleotides corresponding to
280/
271 in the CPT-I
gene. Two
bands were shifted and were identical in mobility to TNT-produced
proteins corresponding to MEF2C and MEF2A (Fig. 2B). Both
bands were competed by 100× wild-type but not 100× mutant oligomers
(Fig. 2B), and both were supershifted by antibodies to MEF2.
These data suggest that cardiac specificity of CPT-I
expression is
partly conferred by the presence of a MEF2 site at the distal end of
the promoter construct.

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Fig. 3.
Effects of individual site mutations on
promoter activity. A model of the protein binding sites indicates
the location of the mutated regions. Each region is represented by an
X on a labeled line to the left of the results
from each separate transfection. Mutated promoter constructs were
transfected into rat neonatal cardiac myocytes as described under
"Experimental Procedures." Luciferase activity is compared with the
wild-type 391/+80 promoter after normalization to protein content and
Renilla expression. Bars represent three to five
experiments in triplicate reported as means ± S.E. Except for the
NKE mutation, all transfections of mutated constructs were
significantly different from wild type (10 3 > p > 10 6).
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Although mutation of the Sp1 (
137/
131) did reduce CPT-I
expression by 60%, mutations in the CA box (CACCC) at
117/
112 and
the Sp1 site (
74/
68) produced strong repression of luciferase expression, i.e. 75% and 76%, respectively (Fig. 3). These
data suggest that factors binding to these sites are most likely to be
responsible for basal transcription of the CPT-1
gene.
Electrophoretic mobility shift assay of the oligomer containing the
74/
68 region demonstrated two bands, both of which were competed by
wild-type oligonucleotide but not by mutant oligomers (Fig.
2C and Table I). Similarly, antibodies to Sp1 and Sp3
abolished formation of the protein/DNA complex (Fig. 2C),
suggesting that Sp1 and Sp3 proteins bind to the proximal promoter at
74/
68 and may also play a role in binding to the CA box at
117/
112 (21).
A region flanked by the two Sp1 sites at
137/
131 and
74/
68
contains a cluster of potential cardiac regulatory elements including
two GATA binding sites, an SRE, and one potential Nkx site (
94/
88)
that partially overlaps with a second GATA binding motif at
96/
93.
Mutation of the SRE (
112/
104) results in a 56% decrease in
CPT-I
expression in cardiac myocytes (Fig. 3). Serum withdrawal for
44 h dramatically decreases the mRNA concentration of CPT-I
in cardiac myocytes (Fig. 4). This
decrease in mRNA content is reversed by replacement of serum-free
DMEM with media containing 10% bovine calf serum (Fig. 4), supporting
a role for serum-containing factors in the up-regulation of CPT-I
transcription. Mutation of either GATA site results in a 43-38%
decrement in gene expression (
129/
126 and
96/
93,
respectively), whereas mutation of both sites (double GATA mutant)
causes a slightly greater fall in luciferase activity (55%). The
binding of SRF to the SRE (
112/
104) and GATA-4 to the two potential
GATA sites at
96/
93 and
129/
126 was confirmed by EMSA (Fig. 2,
D and E). Mutation of the Nkx site (
94/
88)
has no effect on reporter gene expression in cardiac myocytes (Fig.
3).

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Fig. 4.
Quantitative RT-PCR of
CPT-1 transcripts in response to serum
deprivation and restoration. Total RNA from rat neonatal cardiac
myocytes was isolated and analyzed by quantitative RT-PCR. , freshly
isolated myocytes were maintained in DMEM with 10% serum for 12 h. The medium was then removed and replaced with serum-free DMEM for an
additional 8, 32, or 44 h. , freshly isolated myocytes were
placed in serum-free medium immediately after plating for 12, 20, or
44 h. Fresh DMEM with 10% serum was added, and the cells were
maintained for an additional 44, 36, or 12 h for a total of 56 h/treatment. Results represent CPT-I transcripts normalized to
cyclophilin mRNA and are presented as mean ± S.E. of
triplicate determinations.
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Although homeodomain factors achieve specificity via protein binding to
DNA, Nkx, SRF, and GATA-4 have been demonstrated to exert regulatory
control over cardiac gene expression via protein-protein interactions
(11, 22, 23). To study biological activity of these proteins
independent of DNA binding, Nkx, GATA-4, and/or SRF expression vectors
were cotransfected into CV-1 cells with the full-length CPT-I
reporter gene construct. Transfection of Nkx 2.5 or SRF alone produces
modest expression of CPT-I
/luciferase in CV-1 cells (~4-fold
activation, Fig. 5). Cotransfection of Nkx and SRF with the promoter gene construct produces additive effects
on luciferase expression (Fig. 5A), suggesting that both factors contribute independently to CPT-I
gene activation. GATA-4 transfection alone stimulated CPT-I
36-fold, making GATA-4 the most
potent tissue-specific regulatory element thus far described for this
gene. Cotransfection of GATA-4 and Nkx 2.5 with the CPT-I
reporter
construct diminished the response of the CPT-I
gene to this
combination of factors (Fig. 5B). An additive effect would be predicted if both factors were acting independently. It is possible
that Nkx/GATA4 protein-protein interactions (22-24) decreased the
amount of GATA4 (and Nkx) available for DNA binding to GATA elements on
the CPT-I
gene (25). In contrast to the GATA-4/Nkx cotransfections,
coexpression of GATA-4 and SRF produced a synergistic response of the
CPT-I
gene (Fig. 5C). This combination of factors resulted in a synergism that was significantly different from the
predicted value if the actions of these two proteins were independent.
These data provide the first example of a nuclear encoded, cardiac
mitochondrial gene where SRF and GATA-4 act as mutual coregulators
(11).

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Fig. 5.
Combinatorial interactions of Nkx2.5, SRF,
and GATA4. Subconfluent CV-1 cells were transfected as described
with 1 µg of the 391/+80 CPT-1 promoter luciferase reporter
construct. Total DNA concentrations were adjusted to 2 µg with empty
pcDNA vector. A, transfection reactions included 0.4 µg of Nkx2.5 or 0.1 µg of SRF CMV-driven expression vectors alone
or in combination. B, the CMV-Nkx2.5 (0.4 µg) and
CMV-GATA4 (0.4 µg) expression constructs were used alone or in
combination to influence the 391/+80 reporter. C,
CMV-driven SRF and GATA4 expression were combined to regulate
luciferase expression. Cells were maintained in DMEM with 10% serum
for 48 h after transfection and then harvested. Luciferase
activity was measured and normalized to total protein concentration and
Renilla luciferase. Results are reported as mean ± S.E. of three to five experiments performed in triplicate.
Cotransfection of all expression vectors with CPT-I reporter gene
produced significant changes compared with wild type alone. GATA-4 + SRF compared with GATA-4 alone (*, p < 0.002).
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To confirm the role of the predicted regulatory domains and protein/DNA
interactions in reporter gene expression, point mutations in SRE, the
two GATA-4 sites (double mutation) and Nkx2.5 in the
391/+80 CPT-I
construct were cotransfected with SRF or GATA-4 alone or in
combination. Mutation of the SRE (and the Nkx), but not of the GATA
sites, significantly reduced CPT-I
induction by SRF (Fig.
6A). The induction of CPT-I
reporter expression by GATA-4 alone was significantly reduced by
~50% by all three point mutations (Fig. 6B). The point
mutation in the Nkx site (
94/
88) adjacent and overlapping the GATA
site likely represents interruption of GATA-4 binding at
96/
93. The
remaining reporter gene activity in the presence of the GATA double
mutation may represent physical association of the transfected GATA-4
with endogenous factors or basal transcriptional complexes in the CV-1 fibroblasts (11, 25). The combinatorial effects of SRF and GATA4 on the
CPT-I
reporter gene were abolished by mutation of either the SRE or
the two GATA sites (Fig. 6C). These results reinforce the
SRE dependence of these coregulators for synergism (11). Moreover, the
results again demonstrate that dramatic induction in CPT-I
gene
expression is also regulated in large part by GATA-4 interactions
alone, some of which appear independent of binding to traditional GATA
sites. The drop in synergism due to the point mutation in the Nkx site
(Fig. 6C) appears to reflect a requirement for appropriate
flanking sequences to facilitate GATA-4 interaction with the DNA at
96/
93.

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Fig. 6.
Induction and synergistic activation of the
CPT-1 promoter by SRF and GATA4 is reduced with DNA binding site
mutations. Subconfluent CV-1 cells were transfected with 1 µg of
wild-type or mutant reporter constructs with expression vectors for SRF
and GATA4 alone or in combination. Mutated promoters are schematically
represented to the left. Total DNA concentrations were
adjusted to 2 µg with empty pcDNA3 vector. Cells were harvested
48 h after transfection, and luciferase activity was recorded and
normalized to protein content and Renilla expression.
Bars represent mean ± S.E. of three to five
experiments in triplicate. Except for the SRF induction of the
CPT-I -GATA double mutant, all results are significantly decreased
compared with wild-type reporter activity (0.01 > p > 10 5).
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Transfection of a DNA binding mutant SRFpm abolishes the synergistic
response between GATA-4 and SRF (Fig. 7).
This finding is consistent with requirement for SRF binding to the SRE
to produce cardiac
actin promoter coactivation (11). Induction of
the GATA-dominated CPT-I
reporter gene expression remains elevated in the presence of transfected GATA-4 and SRFpm. A small reduction in
the normal GATA-4 up-regulation may reflect altered affinities of the
gene for the SRFpm/GATA-4 complex, including direct binding of the
complex to the GATA site(s). A deletion of the C-terminal activation
domain of SRF (SRF
C) inhibits the combinatorial action of SRF and
GATA-4 and dramatically reduces the induction of gene expression due to
GATA-4 (Fig. 7). The promoter activation that remains (13-fold) is on
the order of induction suggestive of independent GATA binding to the
DNA/protein complexes (Fig. 6B, 15-fold induction). The data
confirm coactivation of CPT-I
by GATA-4 and SRF that augments the
enhancement of gene expression seen in the presence of GATA-4
alone.

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Fig. 7.
Disruption of SRF protein structure prevents
SRF/GATA4 synergy. Subconfluent CV-1 cells were transfected as
described under "Experimental Procedures." Where indicated,
expression vectors for wild-type SRF were replaced with 100 ng of
either SRF-PM or SRF- C. GATA4 concentrations remained constant (0.4 µg). Cells were harvested after 48 h in serum containing DMEM
and luciferase activity measured. Reporter activity was normalized to
protein content and Renilla luciferase. Results are
presented as mean ± S.E. for three to five experiments performed
in triplicate. Both the SRF-PM (*, p < 0.005) and the
SRF- C (*, p < 10 4) are
significantly decreased compared with the GATA-4 and SRF coinduction of
CPT-I .
|
|
 |
DISCUSSION |
Basal expression of the CPT-I
gene is analogous to the CPT-I
gene in that it contains no TATA box and uses Sp proteins 1 and 3 to
drive expression (26). The factors that identify and separate the
tissue location of these two isoforms are of particular interest to
cardiac muscle where the muscle isoform is dominant, but the liver
isoform (CPT-I
) is also expressed (6). We have identified a region
within
391 base pairs of the transcription start site of the muscle
isoform that is sufficient to drive CPT-I
transcription and that
contains MEF2 and E box elements as well as a cluster of other
heart-specific sites. This cluster of CPT-I
regulatory elements is
located between
137 and
68 bp of the proximal promoter and includes
GATA, Nkx2.5, and SRF DNA-binding sites. Identification of these
important cis-trans regulatory domains suggests a
multiplicity of pathways that may be expressed and are potentially
interactive depending on the CPT-I
gene context and the
developmental environment of the myocyte.
The GATA family of transcription factors, represented by heart-specific
GATA-4, are potent transactivators of several cardiac promoters. These
include atrial natriuretic factor (27), cardiac sarcolemmal
Na+,Ca+ exchanger (28), B-type natriuretic
peptide (29), and cardiac troponin I (30). GATA-4 (and GATA-6)
colocalize in postnatal cardiomyocytes and are believed to act in the
differential control of various cellular processes (29). The present
results are the first to identify a heart-specific protein, carnitine
palmitoyltransferase I
, as a downstream mitochondrial target of
GATA-4. Two GATA binding sites are present in this gene although
transcriptional activation by GATA does not always require DNA binding
(25). Together with Nkx 2.5, GATA-4 has been demonstrated to be a
powerful transcriptional coactivator of the ANF promoter (31) and the
cardiac
actin promoter (23). In the context of the CPT-I
gene,
however, coexistence of GATA-4 and Nkx 2.5 decreases reporter gene
expression in CV-1 cells. Compared with a significant, but small,
transactivation by Nkx 2.5, GATA-4 expression in CPT-I
-transfected
CV-1 cells produces a greater than 30-fold increase in CPT-I
gene
reporter activity, indicating GATA domination of the CPT-I
promoter.
Nkx 2.5 may act to sequester GATA factors away from GATA-binding sites in GATA-dependent promoters (25). A DNA-independent
interaction between GATA and Nkx 2.5 could therefore remove GATA-4 from
its interaction sites in CPT-I
, resulting in the decreased reporter gene expression observed.
In cardiac myocytes, the double GATA site mutation reduces reporter
gene expression by greater than 50%, supporting the importance of DNA
binding in the cumulative GATA effects. Cotransfection of the reporter
gene with the double GATA mutant into CV-1 cells also reduces reporter
gene expression by GATA-4 by 43%, but significant gene induction is
still retained. These data again suggest that GATA-4 may also influence
CPT-I
gene expression by protein-protein interactions, interacting
with endogenous levels of factors that affect basal transcription (25).
Among these factors, SRF has recently been shown to be a mutual
coregulator with GATA-4 in numerous myogenic SRE-dependent
promoters (11). In the absence of GATA-DNA binding, SRF binds to the
CArG box sequence and physically associates with GATA-4 through the
MADS box of SRF and the second zinc finger domain of GATA-4 (11). In
the CPT-I
promoter, the presence of serum has dramatic effects on
CPT-I
mRNA content in the neonatal myocyte cultures. SRF binds
to one SRE (
112/
104), and mutation of this site also dramatically
decreases CPT-I
reporter gene expression. Although cotransfection of
SRF with CPT-I
has small inductive effects comparable to those seen
with Nkx 2.5, the presence of GATA-4 factors synergistically activates
CPT-I
to levels that are significantly greater than expression of
GATA-4 and SRF independently. The data suggest that even in a
background of high GATA-4-induced gene expression, SRF binding to SRE
and association with GATA-4 is a quantitatively important mechanism to
up-regulate further expression of the muscle isoform of CPT-I. It is
possible that the postnatal expression of CPT-I
is dependent on the
presence of serum factors and GATA-4 interacting physically and
functionally to increase the myocardial content of this isoform to
adult levels (6). Supporting a possible physiological importance of
this mechanism, we have demonstrated that electrical stimulation of
neonatal cardiac myocytes in culture induces CPT-I
mRNA
accumulation (3) subsequent to up-regulation of GATA-4 gene expression
(5).
A requirement for MEF2 in CPT-I
gene expression is consistent in the
known role for the MEF2 proteins in the differentiation of muscle cell
lineages. MEF2 has also been reported to mediate hypertrophic signaling
as well as synergistic transcriptional responses (32). These pathways
gain additional significance in the heart where cardiac hypertrophy and
failure is linked to down-regulation of the enzymes of the
-oxidation pathway (33), including CPT-I
(34). The CPT-I
promoter contains a MEF2/DNA binding site, and mutational analysis
reveals a 46% depression in CPT-I
gene expression when the MEF2
site is mutated. Nuclear protein extracts of the neonatal cardiac
myocytes demonstrate binding of both MEF2A and MEF2C in electrophoretic
mobility shift assays. The ability of the MEF2 proteins to activate
transcription in vivo depends on the dimer composition of
the binding complex and the cellular context.
Twenty base pairs downstream of the MEF2 binding element, we have
identified an E box that acts as a suppressor of CPT-I
in the
promoter deletion analysis. The consensus E box binds basic helix-loop-helix regulatory proteins and are contained in the regulatory regions of most developmentally controlled, muscle-specific genes. In the CPT-I
gene, we have identified the E box-binding proteins as the upstream stimulatory factors, USF1 and USF2. Although a
role for rat USF1 has been suggested in contractile-mediated activation
of
myosin heavy chain gene (35), USF can either positively or
negatively regulate promoter activity via independent cis regulatory
elements (36). E boxes are also frequently associated with adjacent
MEF2 sites with a spacing that promotes protein-protein interaction
between E box and MEF2 basic helix-loop-helix factors bound to the DNA.
The interaction of this E box site with MEF2 and adjacent sites in the
promoter is currently under investigation.
Finally, the presence of a FARE site in the rat CPT-I
promoter has
been confirmed by these studies. The activity of this site in our hands
produces a small induction of CPT-I
gene expression (2-3-fold) at
high physiological concentrations of oleate (2:1 molar ratio) in the
cell medium. This fold induction is less than previously reported at a
7:1 oleate to albumin molar ratio (8-20-fold) (7). Although a role for
fatty acids in the induction of CPT-I
gene transcription is an
attractive regulatory mechanism physiologically, other studies have not
been able to demonstrate a role for elevated serum fatty acids in
altering the cardiac content of CPT-I
mRNA and protein (9, 10).
These studies suggest that, like other heart-specific proteins,
CPT-I
contains the same pattern of muscle-specific control regions.
Its expression in the heart is likely GATA-4-dominated and is subject
to protein-protein interactions that can regulate its expression in a
manner that is context-dependent. Interestingly, these
studies also verify that gene expression of a major controlling enzyme
in mitochondrial oxidative metabolism is coordinate with expression of
proteins important in contractile function and energy consumption.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants RO1 HL38863 (to J. B. M.) and PO1-HL49953 (to R. J. S.), and by United States Department of Agriculture Grant ARS6250-6100 (to R. J. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Pathology
and Laboratory Medicine, Medical School, University of Texas-Houston Health Science Center, 6431 Fannin, Houston, TX 77030. Tel.:
713-500-5335; Fax: 713-500-0730; E-mail:
jeanie.b.mcmillin@uth.tmc.edu.
Published, JBC Papers in Press, October 18, 2000, DOI 10.1074/jbc.M009352200
 |
ABBREVIATIONS |
The abbreviations used are:
CPT-I, carnitine
palmitoyltransferase I;
CMV, cytomegalovirus;
EMSA, electrophoretic
mobility shift assay;
PCR, polymerase chain reaction;
bp, base pair(s);
FARE, fatty acid response element;
SRF, serum response factor;
SRE, serum response element;
DMEM, Dulbecco's modified Eagle's medium;
USF, upstream stimulatory factor;
RMNE, rat myocyte nuclear extract;
TNT, transcription/translation.
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