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INTRODUCTION |
The murine linear heart tube begins to contract spontaneously by
8 d.p.c.1 (1) and pumps
continually throughout the lifetime of the animal; therefore, the
metabolic pathways that provide energy must be properly activated and
regulated. Several genes that encode metabolic enzymes are activated at
low levels during early cardiogenesis, then are up-regulated around
birth to reach adult levels during postnatal development to deal with
the increasing cardiac energy demands (2, 3). Numerous metabolic
pathways also utilize cardiac-specific isozymes that are present at
high levels in the heart (4-6). Although the biochemistry of cardiac
metabolism is well defined, the genetic signaling pathways that control
expression of the cardiac metabolic genes are not well understood. Few
cardiac cis-regulatory elements that function in
vivo have been identified and preliminary studies are just
beginning to describe transcription factor interactions as genetic
regulatory mechanisms. The subset of cardiac regulatory regions that
has received the most attention has been those that control contractile
gene transcription (7-9). Two of the best characterized cardiac
structural gene enhancer and promoter regions regulate the
- and
-myosin heavy chain genes, which have been used to direct expression
of several other genes to the murine heart (10-12). Less well
characterized cardiac structural gene enhancers include those that
control expression of the myosin light chain 1/3 (13) and 2v genes
(14). Another class of cardiac elements, those regulating genes that
encode enzymes of energy metabolism, is the least understood group of cardiac regulatory regions. Since the murine linear heart tube begins
to beat early in development (1), these energy metabolizing pathways
must be well established during cardiac formation and morphogenesis.
Synthesis and control of the metabolic enzymes that provide the massive
supply of energy to the constantly beating heart are areas of study
that can provide further information about the molecular pathways
controlling heart development and function. Analysis of only two
cardiac metabolic gene flanking regions by the transgenic assay,
specifically those regulating the transcription of the creatine kinase
(15-17) and adenylosuccinate synthetase 1 genes (this report), has
delineated some heart-specific control elements. Consequently,
characterization of several energy metabolic regulatory regions is
necessary and a comparison of the metabolic versus the
structural cardiac enhancers will help to define the mechanisms of
tissue-specific activation and enhancement.
We chose to study the adenylosuccinate synthetase 1 (Adss1)
gene to characterize further heart-specific cis-acting
control elements and to analyze trans-regulatory mechanisms.
ADSS1, a striated-muscle specific energy metabolic enzyme, is a member of the purine nucleotide cycle (PNC), which regulates adenine nucleotide pool levels during periods of increased workload. During intense muscle contraction, the forward myokinase and AMP deaminase reactions generate additional ATP. When the myocyte recovers, ADSS1 and
ADSL restore adenine nucleotide levels (18). The PNC may support energy
metabolism differently in the heart. We propose that this cycle is
activated under hypoxic conditions when oxidative phosphorylation
pathways cannot function efficiently. The PNC generates additional ATP,
while cycle intermediates positively regulate anaerobic glycolysis for
substrate level phosphorylation. When oxygen levels return to normal,
the return pathway of the PNC restores adenine nucleotide levels.
Previously, we showed that 1.9 kb of Adss1 5'-flanking DNA
can direct high levels of reporter gene expression to the adult
transgenic mouse heart (19). In the present paper, we report that this
Adss1 1.9-kb fragment regulates reporter expression in
transgenic hearts similar to the endogenous Adss1 gene. This
cardiac enhancer directs copy number-dependent, integration
site-independent expression to the hearts of transgenic mice at levels
comparable with the endogenous gene. Also, a 700-bp fragment within the
1.9-kb 5'-flanking fragment activates a heterologous promoter
specifically in transgenic hearts. This region contains a cluster of
potential cardiac transcription factors that specifically bind proteins
from a cardiac nuclear extract. Using mutational analyses, we show that
these transcription factor binding sites play important regulatory
roles in cardiac reporter gene expression. Finally, we report that
several cardiac transcription factors trans-activate
1.9HSCAT expression in a noncardiac background specifically through
these sites. Combinations of transcription factors lead to enhanced
reporter expression. These results are the first to identify a
potential target gene for the cardiac bHLH factors, Hand1 and Hand2.
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MATERIALS AND METHODS |
Generation of Riboprobes and in Situ Hybridization
Analysis--
In situ hybridization to sections was
performed as described (20). Antisense and sense riboprobes were
generated from the Adss1 cDNA and from the CAT cDNA.
All of the probes were acid-hydrolyzed to an average size of 750 bp.
Sections were viewed with an Olympus BX60 microscope, and photographs
were captured using a SPOT digital camera (Diagnostic Instruments
Inc.).
Tissue Extracts and CAT Assays--
Protein concentration was
determined by the Bradford method (21). CAT assays were performed as
described (22). CAT specific activity was determined under linear conditions.
Isolation of RNA and Northern Blot Analysis--
Total cellular
RNA from tissues was isolated by the Trizol procedure as described
(Life Technologies, Inc.). Northern blot analysis was performed as
described previously (19).
Nuclear Extract Preparation and Electrophoretic Mobility Shift
Assay--
Neonatal rat cardiac nuclear extract was prepared from
primary cardiomyocyte cultures as described (23). Double-stranded oligonucleotides corresponding to various regions of the
Adss1 5'-flanking region were synthesized as follows: Nkx
2.5 (nt
1523 to
1504), MEF2 (nt
1713 to
1694), E box A (nt
1914 to
1894), E box B (nt
1893 to
1874), E box C (nt
1863 to
1834), and E box D (nt
1773 to
1754), GATA site A (nt
1423 to
1402), GATA site B (nt
1407 to
1384), GATA site C (nt
1483 to
1354), GATA site D (nt
1353 to
1324), and GATA site E (nt
1063
to
1043). The Nkx 2.5 site was mutated to taaTAtg, the MEF2 site was
mutated to cGaCCtCtCg, GATA sites A-C were mutated to CAGTGC, GATA
sites D and E were mutated to GCGTTG, and E boxes A-D were mutated to
GGTAAC (mutated sites are capitalized). These oligonucleotides were
used for electrophoretic mobility shift assay analysis (24).
Generation of Constructs--
The 1.9HSCAT construct was
described previously (19). The 0.6XhSCAT construct was generated by
digesting the HSCAT/pSG5 vector with XhoI and
SalI, blunt-ending with T4 polymerase, and was cloned into
the EcoRV site of the Bluescript KS+ vector.
The 700-bp Adss1 HindIII-XbaI fragment was
blunt-ended with Klenow and cloned in the SmaI site in front
of the SPAC (short promoter Ada CAT) (22) construct in both the forward
and the reverse orientations. The SPAC construct is present in the
Bluescript KS+ vector backbone and consists of an 800-bp minimal
Ada promoter, the CAT cDNA, and a SV40 intron and
polyadenylation signal.
The Nkx 2.5, MEF2, and GATA site-directed mutants were generated by a
PCR megaprimer procedure. The primers that were used are as follows
(mutated nucleotides are capitalized, and transcription factor sites
are underlined): universal site sequencing primer, mutant MEF
oligonucleotide reverse,
5'-tcaaagcctgagacGaGaGGtCgccaagggcccacataga-3'; mutant Nkx 2.5 forward,
5'-caagggcaagcattaaTAtgacctgaccaa-3'; mutant GATA A-D
forward spanned nucleotides
1423 to
1324 in which sites A-C were
changed to CAGTGC, and site D was changed to
GCGTTG and primer B, 5'-tctctacctcactcagtgcca-3'. The first PCR reac- tion generated the fragment that contained the mutant site. The PCR fragment was gel-purified by the QiaexII kit (Qiagen, Inc.). That fragment was then used as the megaprimer for the second PCR
reaction. PCR cycling conditions were as follows: hot start at 94 °C
for 10 min, then 94 °C for 1 min; 55 °C for 1 min, and 72 °C
for 2 min for 30 cycles; a final extension at 72 °C for 10 min; and
hold the reactions at 4 °C. The large fragment was gel-purified by
the QiaexII kit (Qiagen, Inc.) and was then digested with
XbaI and cloned into the XbaI-digested, calf
intestinal phosphatase-treated HSCAT backbone. All constructs were
isolated by cesium gradient purification (25). The orientation of the
cloned fragment was determined by restriction enzyme digestion, and the
presence of the mutant site was confirmed by sequencing (U. S.
Biochemical Corp.).
The 5' deletion HSCAT construct was generated by a partial
ApaI digestion of the 1.9HSCAT construct. The deletion
fragment was gel-purified by the QiaexII kit (Qiagen, Inc.),
blunt-ended with T4 polymerase, and religated.
The expression constructs for CV1 cotransfection analysis; pCGN (26),
pCGNkx 2.5 (26), pCGGATA 4 (27), CMV MEF2C (28), and CMV E12 (28) were
described previously. Expression constructs CMV Hand1 and CMV Hand2
were generated by cloning the respective cDNAs into the
EcoRV site of the pCDNA3 vector (Invitrogen).
Transient Transfections into Rat Primary
Cardiomyocytes--
Transient transfection into neonatal rat primary
cardiomyocytes were performed as described (29). CAT assays and
-gal
assays were performed as described (22, 30). Experimental data are presented as the mean of three independent transfection assays done in
duplicate and normalized to
-gal activity.
Production and Analysis of Transgenic Mice--
Transgene
constructs were isolated by agarose gel electrophoresis and purified by
either a QiaexII kit or a Qiaquick kit (Qiagen, Inc.). Transgenic mice
were prepared by injecting the constructs in fertilized FVB/N
oocytes (31). Founder (F0) transgenic animals were identified as
described previously (19).
Cell Culture and Transfections--
Cotransfections into CV1
fibroblasts were performed as described previously (27). Cells were
transfected with a total of 3 µg of plasmid DNA, which included 500 ng of 1.9HSCAT reporter plasmid, 1 µg of CMV
-gal, and various
concentrations of expression vector plasmids (250 ng of CMV Nkx 2.5, 250 ng of CMV GATA 4, 150 ng of CMV MEF2C, 150 ng of CMV E12, 250 ng of
Hand1, and 250 ng of CMV Hand2). All transfections were balanced with a
CMV empty vector. The cells were washed with phosphate-buffered saline
and scraped from dishes in 400 µl of a CAT assay buffer. The cells were lysed by sonication and were centrifuged at 14,000 rpm for 15 min
at 4 °C. The supernatant was transferred to a clean tube, and
protein concentration was determined by the Bradford method (21). CAT
assays and
-gal assays were performed (22, 30). Values were the
average of at least three experiments, each with duplicate plates and
were normalized to
-gal activity.
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RESULTS |
1.9 kb of Adss1 5' Flank Controls Proper Spatiotemporal Expression
of a Reporter in Transgenic Hearts--
Previously, we reported that
1.9 kb of Adss1 5'-flanking DNA possessed the ability to
direct high levels of reporter gene expression to the adult murine
transgenic heart (19). To determine whether this 1.9-kb
Adss1 fragment possesses the capability to control correct
spatiotemporal expression of a reporter, 1.9HSCAT transgenic embryos
were analyzed at different developmental times. Both the endogenous
Adss1 gene and the 1.9HSCAT transgene were expressed in both
the atrium and ventricle of the heart at 9.0 d.p.c. (Fig.
1, A and B). At
11.5 d.p.c., expression of both the endogenous gene and the
transgene were detected in both chambers of the heart and in the
developing facial and jaw muscles (Fig. 1C). Interestingly,
the level of CAT mRNA was lower than the level of Adss1
mRNA in the atrium (Fig. 1D). Faint expression of both the Adss1 gene and the 1.9HSCAT transgene was also detected
in the fetal liver (Fig. 1, C and D). No signal
above background was detected with sense riboprobes (data not shown).
To determine whether the 1.9-kb 5'-flanking region has the capability
to maintain reporter gene expression during late embryogenesis, a
founder 1.9HSCAT transgenic litter was dissected at 15 d.p.c., and
cardiac and liver tissues were analyzed for CAT enzyme activity. In two transgenic embryos (11 and 12), CAT enzyme was strongly expressed in
the heart, but negligible levels were detected in the liver (Fig.
1E). This experiment shows that transgene expression in the
liver ceased, while transgene expression in the heart remained high. No
CAT activity was detected in these tissues in nontransgenic embryos (9 and 10). Thus, the 1.9-kb Adss1 5' fragment possesses the
capabilities to activate gene expression in cardiac progenitors and to
sustain expression throughout embryonic cardiac development.

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Fig. 1.
Prenatal expression pattern of the 1.9HSCAT
transgene compared with the endogenous Adss1
gene. A, cross-section of a 9.0 d.p.c.
1.9HSCAT embryo showing Adss1 expression in the atrium
(a) and the ventricle (v) of the beating heart.
B, serial section of a 9.0 d.p.c. 1.9HSCAT embryo
showing CAT expression in the atrium (a) and the ventricle
(v) of the heart. Negative structures in A and
B are the hindbrain (h), the foregut
(f), and the midgut (m). C, sagittal
section of a 11.5 d.p.c. 1.9HSCAT embryo showing Adss1
expression in the atrium (a), ventricle (v),
developing facial muscles (fm), and liver (l).
D, serial section of a 11.5 d.p.c. 1.9HSCAT embryo
showing CAT expression in the atrium (a), ventricle
(v), facial muscles (fm), and liver
(l). Negative tissues include the branchial arch
(ba) in C and D. In situ
hybridizations were performed as described under "Materials and
Methods." Bar in A: 100 µm for A
and B. Bar in C, 500 µm for
C and D. E, a CAT assay showing four
representative embryos out of the 12 embryos dissected in the 15 d.p.c. 1.9HSCAT litter (see "Materials and Methods"). Tissues
examined for CAT expression were liver (liv) and heart
(Ht). Embryos 9 and 10 were nontransgenic; embryos 11 and 12 were transgenic.
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Several cardiac and muscle energy metabolic genes experience a
transcriptional up-regulation during perinatal development to support
the increased energy demands of the growing, mobile organism (41, 42).
To determine whether Adss1 cardiac expression also increases
during neonatal development, total RNA was isolated from 1.9HSCAT
transgenic hearts at different developmental timepoints (Fig.
2A), and Northern analysis was
performed using both Adss1 and CAT probes. Adss1
transcripts were expressed at low levels on embryonic day 17, and
expression continued to steadily increase through day 21 of postnatal
development. Expression of the 1.9HSCAT construct was not detected at
embryonic day 17 by Northern analysis, but transcripts were visualized
by day 1, and 1.9HSCAT expression continued to be up-regulated through
day 21. However, the expression of the 1.9HSCAT construct appeared to
be up-regulated more slowly than the expression of the endogenous
Adss1 gene. The expression of both genes is depicted
graphically in Fig. 2B. In conclusion, both the endogenous
Adss1 gene and the 1.9HSCAT construct undergo a phase of
transcriptional enhancement during perinatal cardiac development that
is similar to several other muscle metabolic genes.

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Fig. 2.
Postnatal regulation of the Adss1
gene and the 1.9HSCAT transgene. A, total RNA (10 µg) from 1.9HSCAT transgenic hearts was fractionated on a 1% agarose
gel and transferred to a nylon membrane. The 1.9HSCAT transgenic line
contained eight copies of the transgene. The Northern blot was probed
with the 1.9-kb Adss1 cDNA and with a 200-bp
HindIII-EcoRI fragment from the CAT cDNA (see
"Materials and Methods"). The 28 and 18 S ribosomal RNA markers
were visualized by ethidium bromide staining prior to and following
transfer to ensure equal loading and efficient transfer of RNA.
B, a graphical representation of the developmental increase
of mRNA levels of the Adss1 gene and the 1.9HSCAT
transgene. C. Total CAT activity of nine 1.9HSCAT transgenic hearts was
plotted against the copy number. The graph revealed a linear
relationship. D, total RNA (10 µg) from various tissues of
an adult 1.9HSCAT mouse was fractionated on a denaturing 1% agarose
gel and transferred to a nylon membrane. The Northern blot was probed
with the 1.9-kb Adss1 cDNA and with a 200-bp
HindIII-EcoRI fragment from the CAT cDNA (see
"Materials and Methods"). The various tissues are skeletal muscle
(Sk M), heart (Ht), kidney (Ky), liver
(Liv), small intestine (Sm I), stomach
(Sto), and spleen (Spl). The 28 and 18 S
ribosomal RNA markers were visualized by ethidium bromide staining
prior to and following blot transfer to ensure equal loading and
efficient transfer of RNA.
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Important characteristics that define an enhancer element are the
ability to confer integration site independence and copy number
dependence (32). To determine whether the 1.9-kb Adss1 5'
flank can confer dominant expression capability, the total CAT activity
of the 1.9HSCAT construct was compared with the copy number (Fig.
2C). Copy numbers ranged from 1 to 16. The graph resulted in
a linear relationship with 9/9 transgenic mouse hearts. Therefore, the
1.9 kb of Adss1 5' flank appears to confer both integration
site independence and copy number dependence on the CAT reporter gene.
Since expression of the 1.9HSCAT construct can be reasonably predicted
based on copy number, we determined whether this 1.9-kb flanking region
contains all the information necessary to direct the proper levels of
reporter expression to the adult transgenic heart. Total RNA was
isolated from various tissues of an adult 1.9HSCAT mouse containing 16 copies of the transgene (Fig. 2D). Northern analysis showed
the expected pattern of Adss1 mRNA expression in
skeletal muscle and heart, with the highest levels being expressed in
skeletal muscle tissue (4). No expression was detected in nonmuscle
tissues. A CAT probe revealed 1.9HSCAT expression specifically in adult
transgenic heart tissue, but not in skeletal muscle. Therefore, the
1.9HSCAT construct directs expression specifically to the adult
transgenic heart, and the levels of CAT mRNA appear to be
comparable with the endogenous levels of Adss1 mRNA in
cardiac tissue (see "Discussion"). In conclusion, the 1.9-kb
Adss1 5' flank properly activates cardiac reporter
expression, controls correct developmental cardiac up-regulation, and
directs levels of reporter expression to the adult heart that are
similar to endogenous Adss1 levels.
The 700-bp HindIII-XbaI Fragment of the Adss1 5'-Flanking Region
Can Activate a Heterologous Promoter in Either the Forward or Reverse
Orientation Specifically in Transgenic Hearts--
We demonstrated
that the 700 base pairs of Adss1 5' flank between 1.9 and
1.2 kb upstream of the transcriptional start site are essential for
expression of the reporter construct in cardiac tissue (19). To
determine whether this 700-bp HindIII-XbaI
region can act as a cardiac enhancer, this fragment was cloned in front of the SPAC construct (see "Materials and Methods") in both the forward and reverse orientations (Fig. 3,
A and B). This construct contained an 800-bp
adenosine deaminase promoter (Ada), which is expressed at
negligible levels in murine cardiac and skeletal muscle tissues (22).
This minimal Ada promoter alone is not activated in tissues
of transgenic mice, but when controlled by a placental, thymic, or
forestomach enhancer, the promoter is activated specifically in those
respective tissues (22, 33, 47). Both
HX.SPAC + and
constructs directed highest levels of expression
of the reporter construct to cardiac tissue (Fig. 3, A and
B). However, the levels of CAT enzyme activity were
500-1000-fold lower than the 1.9HSCAT levels in the transgenic heart
(data not shown). Therefore, the 700-bp Adss1 fragment
possesses the ability to activate a heterologous Ada
promoter in both the forward and reverse orientations in the transgenic
murine heart.

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Fig. 3.
The 700-bp
HindIII-XbaI Adss1
fragment in either the forward or reverse orientation directs
expression of a heterologous promoter to the heart of transgenic
mice. The 700-bp HindIII-XbaI fragment from
the Adss1 5' flank was cloned in front of an 800-bp minimal
Ada promoter construct (SPAC) in both the forward
(A) and reverse (B) orientations. Adult
transgenic mice were analyzed for CAT expression in various tissues
(see "Materials and Methods"). The tissues were skeletal muscle
(Sk M), heart (Ht), tongue (To),
esophagus (Eso), kidney (Ky), liver
(Liv), small intestine (Sm I), stomach
(Sto), spleen (Spl), thymus (Thy), and
bladder (Bl). Three HX.SPAC+ mice and two HX.SPAC- mice were
analyzed for CAT expression.
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Proteins from a Neonatal Rat Cardiac Nuclear Extract Interact with
Potential Cardiac Transcription Factor Binding Sites within the
700-Base Pair Adss1 Enhancer--
To begin to determine whether
cardiac proteins interact with potential cardiac binding sites present
in the 700-bp HindIII-XbaI fragment from the
Adss1 5'-flanking region, electrophoretic mobility shift
assays were performed (Fig. 4). Each of
the possible transcription factor binding sites was end-labeled and
used as a probe to determine whether proteins present in a neonatal rat
primary cardiac nuclear extract bind to the sites. First, a shifted
complex was evident with an Adss1 Nkx 2.5 probe (Fig.
4A, lane 1). This complex was competed away by
25 × and 50 × (lanes 2 and 3) of cold
wild type Nkx oligonucleotide, but only partially by 100 × of
mutant Nkx oligonucleotide (lane 4). Next, the
Adss1 MEF2 probe bound proteins from the rat cardiac nuclear
extract (Fig. 4B, lane 5). 50 and 100 × cold wild type MEF2 oligonucleotides competed away binding (lanes
6 and 7), but 100 × cold mutant MEF2
oligonucleotides did not compete (lane 8). Third, each of
the four E boxes from the Adss1 5' flank bound proteins
present in the rat cardiac nuclear extract (Fig. 4C,
lanes 9, 12, 15, and 18).
Binding was competed away by 100 × each of the cold wild type E
box oligonucleotides (lanes 10, 13,
16, and 19), but not by 100 × cold mutant E
box oligonucleotides (lanes 11, 14,
17, and 20). Multiple protein complexes formed on
the E box C binding site (lanes 15-17). Finally, proteins
present in the cardiac nuclear extract interacted with each of the
Adss1 GATA transcription factor binding sites (Fig. 4D, lanes 21, 24, 27,
30, and 33). Binding was competed away by 100 × cold wild type GATA oligonucleotides (lanes 22,
25, 28, 31, and 34), but
not by 100 × cold mutant GATA oligonucleotides (lanes
23, 26, 29, 32, and
35). Again, several different protein complexes were
apparent on GATA sites B (lanes 24-26) and E (lanes 33-35). In summary, proteins present in a neonatal rat cardiac nuclear extract can specifically bind to each of the potential Adss1 transcription factor binding sites, but do not bind to
the mutant transcription factor sites.

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Fig. 4.
Electrophoretic mobility shift assay analysis
of the potential transcription factor binding sites from the 700-bp
cardiac enhancer of the Adss1 gene. A,
10 µg of neonatal rat cardiac nuclear extract was incubated with the
labeled Adss1 Nkx 2.5 site (lane 1). The extract
was preincubated with either 25 × (lane 2) or 50 × (lane 3) unlabeled wild type Adss1 Nkx2.5
oligonucleotides or with 100 × (lane 4) unlabeled
mutant Adss1 Nkx 2.5 oligonucleotides. B, 10 µg
of the rat cardiac extract was incubated with the labeled
Adss1 MEF2 site (lane 5). The extract was
preincubated with either 50 × (lane 6) or 100 × (lane 7) unlabeled wild type Adss1 MEF2
oligonucleotides or 100 × (lane 8) unlabeled mutant
Adss1 MEF2 oligonucleotides. C, 10 µg of the
rat cardiac extract was incubated with each of the Adss1 E
boxes (lanes 9, 12, 15,
18). The extract was preincubated with either 100 × unlabeled wild type Adss1 E box oligonucleotides
(lanes 10, 13, 16, 19) or
with 100 × unlabeled mutant Adss1 E box
oligonucleotides (lanes 11, 14, 17,
20). The letters A, B, C,
and D under the lanes refer to each of the E boxes present
in the 700-bp Adss1 fragment. D, 10 µg of the
rat extract was incubated with each of the Adss1 GATA
oligonucleotides (lanes 21, 24, 27,
30, 33). The extract was preincubated with either
100 × unlabeled wild type Adss1 GATA oligonucleotides
(lanes 22, 25, 28, 31, 34)
or with 100 × unlabeled mutant Adss1 GATA
oligonucleotides (lanes 23, 26, 29,
32, 35). The letters A, B,
C, D, and E under the lanes refer to
each of the GATA sites within the 700-bp Adss1
fragment.
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Mutational Analysis of the 1.9-kb Flanking Region Reveals the
Importance of Several Potential Transcription Factor Binding Sites for
Cardiac Gene Expression--
Mutations were generated in the 1.9HSCAT
construct to identify binding sites that regulate Adss1
expression in cardiac muscle (Fig.
5A). The Nkx, MEF2, and GATA
site mutations used for electrophoretic mobility shift assay analysis
were introduced into the 1.9HSCAT construct, while the distal four E
boxes were deleted from the 1.9HSCAT construct. Adss1 is
endogenously expressed in rat primary neonatal cardiomyocytes (data not
shown), so the 1.9-kb 5' flank is recognized in these cells. Each of
the 1.9HSCAT mutant constructs, the wild type 1.9HSCAT construct, a
minimal 0.6XhSCAT construct, and the minimal SPAC construct were
transiently transfected into rat neonatal primary cardiomyocytes (Fig.
5B). The 0.6XhSCAT construct was a negative control, because
it did not contain the Adss1 700-bp cardiac enhancer. The
SPAC construct served as a control to ensure that a noncardiac promoter
would not be ectopically activated in the cardiomyocytes. The primary
cardiomyocytes offer an environment that mimics early cardiac
development, since a number of embryonic genes are expressed under
culture conditions (34). Activation levels of each of the 1.9HSCAT
mutants were decreased compared with the wild type 1.9HSCAT construct.
Both the Nkx mutant and the GATA mutant constructs were activated to
only about 40-60% of the wild type level. The MEF2 mutant was
activated to only around 25% of the 1.9HSCAT level. Essentially no
expression was detected from the 5' E box deletion construct.

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Fig. 5.
Mutational analysis of the Adss1
1.9-kb 5' flank. A, schematic representation of
the mutations introduced into the 1.9HSCAT construct. B, the
wild type 1.9HSCAT construct and the various mutations were transiently
transfected into neonatal rat primary cardiomyocytes and analyzed for
levels of CAT reporter activation. The 0.6XhSCAT construct and SPAC
construct were transfected as base-line and negative controls,
respectively. The data are expressed as an average of three
experiments, each performed with duplicate wells. Transfection
efficiency was normalized to -gal. Transfections were performed and
analyzed as described under "Materials and Methods." C,
wild type and mutant 1.9HSCAT transgenic mice were generated and analyzed as described under
"Materials and Methods." Four-week-old founder transgenics were
dissected and analyzed for levels of CAT expression in the heart. CAT
activities are the average of expression levels in eight 1.9HSCAT mice,
three Nkx mutants, two MEF2 mutants, three E box deletion mutants, and
three GATA mutants.
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Next, we analyzed each of the 1.9HSCAT mutant constructs in transgenic
mice, because the construct is integrated into the chromatin
superstructure and must undergo the same developmental influences that
the endogenous Adss1 gene undergoes to be activated and
expressed. Cardiac expression of each of the 1.9HSCAT mutants was
decreased compared with the wild type 1.9HSCAT construct (Fig. 5C). Expression of the Nkx 2.5 mutant was lowered by
approximately 50%, while expression of the MEF2 mutant was lowered by
about 90%. No expression of either the GATA mutant (see
"Discussion") or the 5' E box deletion mutant was seen. In
conclusion, both the cardiomyocyte transfections and the transgenic
mouse assay defined important roles for each of the transcription
factor binding sites in the cardiac expression of the 1.9HSCAT
reporter, especially for the GATA sites and the E boxes.
Combinatorial Interactions between Several Cardiac Transcription
Factors Activate CAT Reporter Gene Expression in CV1
Fibroblasts--
We have characterized several
cis-regulatory sites within the 700-bp Adss1
fragment that contribute significantly to cardiac expression of this
gene, using both biochemical and functional analyses. The next step was
to assess whether candidate cardiac transcription factors can
contribute to Adss1 cardiac expression. Using noncardiac CV1
fibroblasts as a "test tube", several candidate cardiac
transcription factors were cotransfected with the 1.9HSCAT reporter to
test their trans-activation potential. Nkx 2.5 (26), MEF2C
(28), and GATA 4 (27) were chosen as candidate factors, because they
consistently bind their consensus sequences that are present in the
Adss1 700-bp fragment, and they are expressed throughout
prenatal cardiac development (35). Information about binding
specificities and activation capabilities of Hand1 and Hand2 bHLH
factors is sparse; therefore, we wanted to determine whether
Adss1 could be a potential target gene. Fig.
6A shows that individual
transcription factors modestly trans-activate the reporter
3-7-fold over background. When each transcription factor is
cotransfected with the specific mutant 1.9HSCAT construct, the
expression of the reporter construct is decreased. Therefore, each
factor appears to act through a specific binding site within the
Adss1 flank, and each protein appears to contribute a
distinct level of transcriptional regulation to cardiac gene
expression.

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Fig. 6.
Combinatorial interactions between cardiac
transcription factors trans-activate the 1.9HSCAT
reporter in CV1 fibroblasts. A, wild type and mutant
1.9HSCAT reporter genes (500 ng) were cotransfected with various
cardiac transcription factor expression constructs in CV1 fibroblasts.
The expression plasmids were CMV Nkx 2.5 (250 ng), CMV GATA 4 (250 ng),
CMV MEF2C (150 ng), CMV E12 (150 ng), CMV Hand1 (250 ng), and CMV Hand2
(250 ng). Each transfection mixture contained 3 µg of DNA and was
balanced using a CMV empty construct. B, combinations of
transcription factors were cotransfected into CV1 fibroblasts with the
1.9HSCAT reporter construct. Each transfection mixture contained 6 µg
of DNA and was balanced using a CMV empty construct. CAT values were
normalized to -gal activities. Fold activation is expressed as
relative increase over that of the reporter gene transfected only with
the CMV empty. Each bar is an average of at least three
separate experiments, each performed with duplicate plates.
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It is a fact that several transcription factors are expressed
simultaneously at any given point in cardiac development. We determined
what happens to the level of gene expression when several cardiac
transcription factors are introduced together. The combinations of Nkx
2.5 and GATA 4 or MEF2C, E12, and Hand1 or Hand2 provide a higher level
of 1.9HSCAT expression, about 10-20-fold. However, the combinations
utilizing all of the transcription factors, Nkx 2.5, GATA 4, MEF2C,
E12, and Hand1 or Hand2, result in a substantial 35-65-fold
enhancement of reporter expression, respectively. These results
represent the first identification of a potential target gene for the
Hand bHLH factors and demonstrate that each transcription factor
confers a defined level of transcriptional control. Combinatorial interactions between four families of cardiac transcription factors appear to strongly trans-activate the 1.9HSCAT construct.
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DISCUSSION |
This report and our previous paper (19) show that the 1.9 kb of
Adss1 5' flank provides proper developmental activation of
reporter genes in cardiac muscle precursors. Atrial CAT expression appeared low in the 11.5 d.p.c. transgenic embryo, which could mean that the 1.9-kb 5' flank directs higher reporter levels to the
embryonic ventricles than the atria. This Adss1 1.9-kb flank also confers proper up-regulation of reporter expression during perinatal cardiac development. Interestingly, the endogenous
Adss1 gene reached a higher level of expression earlier
in postnatal development than the 1.9HSCAT transgene did. This
discrepancy could be attributed to a lack of elements that fully
control perinatal cardiac up-regulation. Overall, we show that the
Adss1 1.9-kb fragment confers proper developmental
activation and neonatal enhancement in cardiac lineages.
Elements within the 1.9-kb Adss1 5'-flanking fragment not
only control proper developmental expression, but also direct correct adult cardiac expression levels in a dominant fashion over the surrounding chromatin environment. The characteristics of copy number
dependence and integration site independence have been associated with
classical enhancers, such as the globin locus control region (32).
However, elements that open chromatin are being separated from the
elements that direct high levels of expression (36). Regulatory
elements do not always possess both abilities, like the globin locus
control region and the Adss1 cardiac enhancer. The murine
Ada placental enhancer directs reporter gene expression specifically to the trophoblast lineages of the placenta, but expression levels vary significantly depending on the integration site
of the transgene (33). Interestingly, the 1.9HSCAT graph did not pass
through zero on the x axis; it appeared to pass closer to 2. Two or more copies of a transgene may be necessary to "insulate" each other from the surrounding chromatin (37, 38). Overall, elements
within the 1.9-kb Adss1 5' flank confer copy number
dependence as well as integration site independence on the CAT reporter
construct in the transgenic murine heart. When comparing endogenous and transgenic expression in an adult, a contrasting pattern results. The
endogenous Adss1 gene is most highly expressed in skeletal muscle, with slightly lower levels being expressed in the heart. However, the 1.9HSCAT construct expresses specifically in the adult
transgenic heart, but not in skeletal muscle. Therefore, regulatory
elements for adult skeletal muscle expression must be located outside
of the 1.9-kb region. By correcting for a variety of factors, we
propose that the 1.9-kb flank directs expression levels comparable with
the endogenous levels in the adult transgenic heart. Unfortunately, we
cannot make any conclusive statements about mRNA stability.
We demonstrated previously that the 700-bp fragment between 1.9 and 1.2 kb upstream of the Adss1 transcriptional start site was
essential for cardiac reporter expression (19). This fragment directs
the highest levels of expression of a heterologous promoter to the
transgenic heart from both the forward and reverse orientations. Comparatively, though, the level of CAT activity in the HX.SPAC transgenic hearts was 500-1000-fold lower than the levels of CAT activity in the 1.9HSCAT transgenic hearts (data not shown). These results demonstrate that the 700-bp Adss1
HindIII-XbaI fragment confers cardiac specificity on a
noncardiac promoter, but information within the remaining 1.2 kb of
Adss1 5'-flanking region is necessary to provide the high
level of expression. This ability, in conjunction with the
characteristics of proper developmental control, copy number
dependence, and integration site independence, define the Adss1 5'-flanking region as a cardiac enhancer with
characteristics of an locus control region.
Few cardiac regulatory elements have been identified that possess these
features. Two of the most extensively characterized cardiac enhancers
control the
- and
-myosin heavy chain genes' expression.
Specific elements control cardiac expression, such as TRE, MEF2, MCAT,
and Be3 (7-9). Flanking regions of the myosin light chain 2v (13) and
1/3 (14) genes have also been analyzed in transgenic mice. Only one
other cardiac metabolic gene, muscle creatine kinase, has been analyzed
in vivo. Transgenic analysis described a 1.2-kb 5'-flanking
region of MCK that directs both cardiac and skeletal muscle
expression, while preliminary mutational analysis revealed the
importance of only three E boxes for cardiac expression (17).
Definition of the cis-acting elements that regulate cardiac
gene expression can provide a starting point to construct genetic
signaling pathways. Within the 700-bp Adss1 cardiac
enhancer, multiple potential cardiac transcription factor binding sites
were defined by homology, specifically E boxes (39), an Nkx 2.5 site
(40), a MEF2 site (41), and GATA sites (42). Each of the possible
Adss1 transcription factor binding sites interacted
specifically with proteins from a neonatal rat primary cardiac nuclear
extract. Interestingly, multiple protein complexes were noticed on the
E box C oligonucleotide, which may consist of ubiquitous E proteins
and/or MEF family members, along with the Hand transcription factors
(39). Several complexes also formed on the GATA B and E
oligonucleotides. GATA factors bind DNA as homo- and heterodimers and
have been shown recently to interact with Nkx 2.5 (27) and SRF (43)
transcription factors to regulate cardiac gene expression. These
biochemical experiments revealed protein-DNA interactions within the
700-bp cardiac enhancer that may control Adss1 expression.
Unfortunately, protein-DNA complexes identified by biochemical assays
may not be the same complexes that actually regulate a target gene.
Therefore, the effects of specific mutations on 1.9HSCAT reporter
expression levels were analyzed in both neonatal primary cardiomyocyte
cell culture and in transgenic mice. Cardiomyocytes in culture can express numerous embryonic markers and may offer an early developmental environment to study gene activation (34). The transgenic mouse assay
provides an adult environment to analyze enhancement. The results of
the two experiments generally agree; mutation of transcription factor
binding sites decreases 1.9HSCAT reporter expression in the heart.
Interestingly, the GATA mutant construct retained activity in cells,
but did not express in transgenic mice. This result could mean that the
GATA mutant can be activated, but cannot be up-regulated to adult
levels. In summary, each of the cardiac transcription factor binding
sites present in the Adss1 700-bp enhancer specifically
interacts with proteins present in a cardiac nuclear extract, and these
cis-acting sites confer varying degrees of control on the
expression of the 1.9HSCAT construct in the murine transgenic heart.
Since cardiac-specific cis-acting sites that control the
Adss1 gene were delineated, we began to identify
trans-acting factors that regulate expression. Based on the
biochemical and functional data, we chose candidate transcription
factors to analyze trans-activation potential on the
1.9HSCAT construct. Nkx 2.5, MEF2C, GATA 4, and the Hand1 and Hand2
proteins are all expressed in the precardiac mesoderm and in the
developing heart (35), therefore they may play a role in genetic
activation. The Adss1 Nkx 2.5, MEF2, and GATA sites are
consensus sequences, and the respective factors can consistently bind
them to regulate numerous cardiac target genes (26, 41, 42). Little is
known about the DNA binding sites or the regulatory ability of the Hand
factors, but it has been suggested that they may bind nonconsensus E
boxes (44). To date, no target genes have been identified for the Hand
factors. The results of the cotransfection experiments demonstrate that individual cardiac transcription factors moderately
trans-activate, and mutation of specific binding sites
abolishes trans-regulatory ability. Therefore, each factor
appears to regulate Adss1 expression through a specific
site, and each protein confers a distinct level of transcriptional
control. Combinations of factors strongly activate the 1.9HSCAT
reporter in CV1 fibroblasts. Other interactions have been identified
(Nkx 2.5 and GATA 4 (27, 45); Nkx 2.5 and SRF (43); MEF2 and TR (46);
and MEF2, E12, and MyoD (28)) that regulate short or synthetic
promoters. The activation of the 1.9HSCAT construct may not be as
strong as these reports, but to date, this is the longest endogenous
DNA flanking region that responds to trans-activation by
multiple cardiac transcription factors. The Adss1 gene is
one of the first reported potential targets of the cardiac bHLH factors
Hand1 and Hand2. It appears to respond most strongly to Hand2, either
alone or in combination with other factors. The combinations of the
factors may provide insight into mechanisms of transcriptional
activation in the heart. As the heart must respond to changing energy
demands, numerous signaling pathways are activated. The cardiomyocyte
can modulate gene expression by regulating the levels and modifications
of various transcription factors. The specific type of protein complex that is formed may confer different levels of gene expression. This is
the largest protein-protein interaction that has been shown to
trans-activate, utilizing four different families of cardiac
transcription factors. How this protein-DNA complex interacts with
accessory proteins and with proteins at the proximal promoter remains
as the elusive question in gene regulation.
In conclusion, the 5'-flanking region of the Adss1 gene
contains the information to regulate gene expression in cardiac
lineages. Multiple cis-regulatory sites play important roles
in cardiac expression of the Adss1 gene, while combinations
of several cardiac transcription factors interact to strongly activate
Adss1 in the heart. Future aims include definition of the
factors and/or complexes that control Adss1 gene activation
versus perinatal enhancement in the heart. Several cardiac
transcription factors are expressed in the precardiac mesoderm and in
early cardiac development, such as Hand1 and Hand2, GATA 4, GATA 5, and
GATA 6, MEF2A to MEF2D, and Nkx 2.5 (35), which may be responsible for
Adss1 activation. Some of these factors are shut off by late
embryogenesis and are replaced by other factors that continue to be
expressed throughout postnatal development (35). Different combinations
of transcription factors at certain developmental times and places may
specifically control gene activation versus enhancement.
Definition of the mechanisms by which cardiac metabolic genes are
activated and enhanced during murine development will aid in
understanding how the heart responds to changing energy demands.