From the Cardiovascular Division and the
¶ Division of Endocrinology, Beth Israel Deaconess Medical
Center, Department of Medicine, Harvard Medical School, Boston,
Massachusetts 02215, the ** Section of Cancer Biology, Radiation
Oncology Center, Washington University School of Medicine, St. Louis,
Missouri 63108, and the
Department of Molecular
Cardiovascular Biology, Yamaguchi University School of Medicine,
1-1-1 Minami-kogushi, Ube 755-8505, Japan
Received for publication, June 8, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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Csx/Nkx2.5 is an evolutionarily conserved
homeodomain (HD)-containing transcription factor that is essential for
early cardiac development. We found that the HD of Csx/Nkx2.5 binds as
a monomer as well as a dimer to its DNA binding sites in the promoter
of the atrial natriuretic factor (ANF) gene, an in vivo
target gene of Csx/Nkx2.5. Csx/Nkx2.5 physically interacts with each
other in vitro as well as in cells, and the HD is critical
for homodimerization. Lys193 and Arg194,
located at the COOH-terminal end of HD, are essential for dimerization. Lys193 is also required for a specific interaction with the
zinc finger transcription factor GATA4. Csx/Nkx2.5 can heterodimerize
with other NK2 homeodomain proteins, Nkx2.3 and Nkx2.6/Tix, with
different affinities. A single missense mutation, Ile183 to
Pro in the HD of Csx/Nkx2.5, preserved homodimerization function, but
totally abolished DNA binding. Ile183 The homeodomain
(HD)1-containing
transcription factors, characterized by their 60-amino acid DNA binding
domain, play critical roles in developmental patterning and
differentiation. The HD forms three Homo- or heterodimerization of transcription factors has been proposed
to regulate transcriptional activity of many transcription factors.
Combinatorial use of a limited number of transcription factors allows
the regulation of a larger number of biological processes, increasing
both the diversity as well as the specificity of control. However, a
very limited number of studies have addressed the homo- and
heterodimerization of HD-containing transcription factors among more
than 400 members of HD proteins from yeast to human. Homodimerization
ability has been demonstrated for Oct1 (10), Paired (11), Cdx2 (12),
Even-skipped (13), Mix.1 (14), and Pit1 (15). Heterodimerization was
shown for HNF1 The biological significance of dimerization of paired-like class HD
proteins has been demonstrated in Xenopus Mix.1, which regulates dorsal-ventral patterning (14). To date, the significance of
homodimerization of HD-containing transcription factors has not been
well established in mammals. Interestingly, 10 heterozygous mutations
of human CSX/NKX2.5 were recently identified in patients with
congenital heart disease. These patients show progressive atrioventricular conduction defects, left ventricular
dysfunction, atrial septal defect, ventricular septal defect, and
tetralogy of Fallot (22, 23). Four mutations are single missense
mutations in the HD that result in markedly reduced DNA binding (24), raising the possibility that if Csx/Nkx2.5 forms homodimers, the mutants with DNA binding defects may dominantly inhibit CSX/NKX2.5 function in human cardiac development and maturation. In
Xenopus, injections of mRNA encoding non-DNA binding
mutants of Xenopus XNkx2.3 and XNkx2.5 suppressed normal
heart formation and resulted in a small heart or no heart formation in
the most severe cases (25). This in vivo evidence suggests
that a non-DNA binding mutant of Csx/Nkx2.5 may act in a dominant
inhibitory manner on wild type Csx/Nkx2.5 through protein-protein
interaction. Therefore, it is critical to examine whether Csx/Nkx2.5
proteins homo- or heterodimerize to regulate their transcriptional activity.
The NK2 class of HD proteins, first described in Drosophila
(26), is highly conserved from nematode to human and is
characterized by a unique Tyr residue at position 54 in the third helix
of the HD. The most frequently observed HD binding motif is
T/AAAT, but the NK2 class HD binds to the unique
T/CAAG motif. The guanine nucleotide at the fourth
position is distinct from all other HD·DNA complexes that
usually have thymidine at this position, and 54Tyr is
responsible for the unique DNA recognition (27-30).
Several NK2 class HD proteins are expressed in the heart. In
Xenopus heart, XNkx2.3 and XNkx2.5 are coexpressed in
precardiac mesoderm as well as in the underlying anterior endoderm from
the gastrulation stage (stage 13) through adult stage (31, 32), whereas
XNkx2.9 expression is restricted to the cardiogenic region of the
embryo prior to differentiation, but transcript levels decrease rapidly
in the heart (33). In other species, cNkx2.3, cNkx2.5, and cNkx2.8 are
coexpressed in chick heart (34-36); and nkx2.3, nkx2.5, and nkx2.7 are
coexpressed in zebrafish heart with distinct, but overlapping,
spatio-temporal patterns (28, 37). In contrast, Csx/Nkx2.5 is the
predominant NK2 class HD protein expressed in mouse cardiac myocytes
from 7.5 days postcoital to adulthood (38-40), and another NK2
HD gene, Nkx2.6/Tix expression is restricted to the sinus venosus,
dorsal pericardium, and outflow tract from 8 to 10 days
postcoital (41).
The ANF promoter has been proposed to be a direct target of Csx/Nkx2.5
(42-46). Csx/Nkx2.5 binds at Plasmid Construct--
FLAG epitope (GAC TAC AAA GAC GAT GAC GAC
AAG) or HA epitope (TAC CCA TAC GAT GTT CCA GAT TAC GCT) was tagged at
the amino terminus of Csx/Nkx2.5 (40). pcDNA3-Csx/Nkx2.5 were
digested with SacII-NotI,
PflI-NotI, KpnI (partial
digestion)-NotI, or AccI-NotI,
Klenow-treated, and then religated to produce
pcDNA3-Csx/Nkx2.5-(1-230), -(1-199), -(1-159), and
-(1-149). PCR amplifications were performed using a forward
primer 5'-CCAAGTGCTCCTGCTTC-3' with the following reverse primers:
5'-GGAATTCCTACTGTCGCTTGCACTTGTAGC-3' for 1-196, 5'-GGAATTCCTATCGCTTGCACTTGTAGCGA-3' for 1-195,
5'-GGAATTCCTACTTGCACTTGTAGCGACGGT-3' for 1-194,
5'-GGAATTCCTAGCACTTGTAGCGACGGTTCT-3' for 1-193,
5'-CCAAGCTTCTACTTGTAGCGACGGTTCTGG-3' for 1-191,
5'-CCAAGCTTCTACTGGAACCAGATCTTGACC-3' for 1-186, and 5'-CCAAGCTTCTAGACCTGCGTGGACGTGAGC-3' for 1-181. PCR
products digested with BstEII-EcoRI, or
Klenow-treated BstEII-digested, were replaced with that of
pcDNA3-Csx/Nkx2.5. Mutations at Lys193,
Arg194, and Lys193-Arg194 were
introduced using the following primers Lys193 (F,
5'-GCTACAAGTGCattCGACAGCGGC-3'; R, 5'-TGCCGCTGTCGaatGCACTTGTAGC-3'); Arg194 (F, 5'-TACAAGTGCAAGatcCAGCGGCAGGAC-3'; R,
5'-GTCCTGCCGCTGgatCTTGCACTTGTA-3'); Lys193-Arg194 (F',
5'-GCTACAAGTGCattgatCAGCGGCAGGAC-3'; R,
5'-GTCCTGCCGCTGatcaatGCACTTGTAGC-3'). Initial PCRs were performed
with the combination of each forward (F) primer and C2 reverse (R)
primer, as well as each reverse primer and P4 forward primer, then
equal amounts of these two PCR products were applied to a second PCR
using C2 and P4 primers as described previously (47).
BstEII-PflMI-digested PCR fragments were replaced
with that of pcDNA3-Csx/Nkx2.5.
Maltose-binding protein (MBP)-HD plasmid was kindly provided by R. Schwartz (48). Using pMAL-malE primer (5'-GGTCGTCAGACTGTCGATGAAGCC-3') and Csx/Nkx2.5 internal primer (5'-ATCTTGACCTGCGTGGACGTGAGC-3'), fragments of MBP-HD plasmid were amplified, digested with
SacI-KpnI, then replaced with that of
MBP-Csx/Nkx2.5 plasmid (47). To construct MBP-(1-250),
SphI-EcoRI (filled) MBP-Csx/Nkx2.5 was replaced
with SphI-digested PCR fragment using forward
5'-CCAAGTGCTCCTGCTTC-3' and reverse
5'-CTCTAGACTAGGGTAGGCGTTGTAGCC-3' primers.
MBP-Csx/Nkx2.5 and pcDNA3-Csx/Nkx2.5 were mutated with two primers
(5'-CAGGTCAAGccaTGGTTCCAG-3', 5'-CTGGAACCAtggCTTGACCTG-3') to construct
MBP-Csx/Nkx2.5(Ile183 Electrophoretic Mobility Shift Assay (EMSA)--
MBP fusion
proteins were prepared as described previously (47). Briefly, cultured
Escherichia coli BL21(DE3) (Novagen) induced with 0.3 mM IPTG were lysed by sonication in lysis buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1% Triton, 2 µg/ml aprotinin, 0.7 µg/ml pepstatin, 0.1 mM PMSF, 1 mM DTT, 10%
glycerol), and lysates were incubated with amylose resin (New England
Biolabs). Fusion proteins were eluted from the beads with the lysis
buffer containing 10 mM maltose.
Two pM end-labeled oligonucleotides,
5'-TCACACCTTTGAAGTGGGGGCCTCTTGAGGCAAATC-3'
(
Nuclear extracts of neonatal cardiac myocytes were prepared as follows:
cells on 10-cm plates were washed with HBS buffer (25 mM
HEPES, pH 7.6, 130 mM NaCl) and soaked in 3 ml of low salt buffer (25 mM HEPES, pH 7.6, 1 mM DTT, 0.1%
Triton X-100, 0.5 mM PMSF, 1 mM sodium
orthovanadate, 2 mM sodium pyrophosphate) for 10 min on
ice. Cells were scraped with the low salt buffer and lysed by Dounce
homogenizer. Nuclei pelleted by centrifugation at 200 × g for 5 min were resuspended in 100 µl of extraction buffer (20 mM HEPES, pH 7.6, 450 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 25% glycerol, 0.1 mM PMSF, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate) and incubated for 30 min at
4 °C. After centrifugation at 15,000 × g for 10 min, the supernatant was used as a nuclear extract for EMSA using the
same methods described above with 66 mM NaCl in the reaction.
Protein-DNA complexes analyzed by electrophoresis in 5% native
polyacrylamide gel were transferred to polyvinylidene difluoride membrane with Tris-glycine-methanol buffer (25 mM Tris, 192 mM glycine, 20% methanol). The membrane was blotted with
anti-Csx/Nkx2.5 Ab (40), anti-FLAG Ab (Sigma), or anti-HA Ab (Roche
Molecular Biochemicals). EMSA in which proteins were transferred
to membranes contains 0.3 µg/µl final concentration of nuclear
extract from noninfected cardiac myocytes or 0.044 µg/µl final
concentration of nuclear extract from adenoviral infected cardiac
myocytes and 0.16 pmol/µl final concentration of nonradiolabeled
double-stranded oligonucleotides.
Protein-DNA binding affinity (Kd) was estimated by
the protein concentration at which 50% of the DNA probe has become bound (49). Molecular mass of MBP fusion protein was estimated by addition of MBP protein (molecular mass = 42 kDa) and HD (aa 122-212, molecular mass = 10.9 kDa) or full length (molecular mass = 32.8 kDa) or Recombinant Adenoviruses--
FLAG- or HA-tagged wild type or
FLAG-tagged Ile183 Protein-Protein Interaction--
Bacterially produced
MBP-Csx/Nkx2.5, MBP-HD, MBP, glutathione S-transferase
(GST)-GATA4 (provided by D. Wilson), and GST protein were made as
described previously (47). In vitro transcribed and
translated proteins were generated by using TNT-coupled reticulocyte lysate systems (Promega). 1 µl of reticulocyte lysate containing 35S-labeled wild type or mutant Csx/Nkx2.5, Nkx2.3, or
Nkx2.6/Tix protein was mixed with fusion proteins in a 400 µl of
binding buffer (20 mM HEPES, pH 7.5, 100 mM
NaCl, 5 mM MgCl2, 0.1% Triton X-100, aprotinin
(2 µg/ml), pepstatin (0.7 µg/ml), 0.1 mM PMSF, 1 mM DTT, and 1% bovine serum albumin) at 4 °C for 2 h. Beads were washed with binding buffer (without bovine serum albumin) and subjected to SDS-PAGE.
To perform coimmunoprecipitation assay, 293 cells in 100-mm plates were
transfected with 9 µg of pcDNA3-FLAG-Csx/Nkx2.5 and/or 9 µg of
pcDNA3-HA-Csx/Nkx2.5 using the calcium phosphate method. Total
plasmid amount was adjusted with pcDNA3 empty vector to 18 µg.
Cells were lysed in the lysis buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.5%
Nonidet P-40, aprotinin (2 µg/ml), pepstatin (0.7 µg/ml), 0.1 mM PMSF, 1 mM DTT) and precleared with normal
mouse IgG-bound protein G. Approximately 1 mg of protein in 1 ml of
lysis buffer was incubated with 3 µg of anti-FLAG mAb affinity gel
(Sigma), washed five times with lysis buffer, and resolved on SDS-PAGE
and subjected to Western blotting using peroxidase-conjugated anti-HA
Ab (Roche Molecular Biochemicals).
Reporter Gene Assays--
10T1/2 fibroblast cells cultured in
six-well plates were cotransfected with 1.0 µg of
ANF(
Rat neonatal cardiac myocytes cultured in six-well plates were
cotransfected with 1.8 µg of ANF( Csx/Nkx2.5 Forms a Homodimer on a Palindromic DNA Sequence in the
ANF Promoter--
The ANF promoter contains three specific binding
sites for Csx/Nkx2.5 that are located upstream of the transcription
start site at positions
To confirm that the appearance of the second shifted band is a result
of the simultaneous occupation of two specific binding sites of the ANF
Homodimerization of Csx/Nkx2.5 in Vitro and in Cells--
We next
examined whether Csx/Nkx2.5 proteins physically and specifically
interact with each other in the absence of DNA. MBP-fused Csx/Nkx2.5
(Fig. 2A, lane 1),
HD-MBP (lane 2), and MBP alone (lane 3) were
mixed with in vitro translated 35S-labeled
Csx/Nkx2.5 protein. After extensive washing, the protein complexes were
resolved on SDS-PAGE and visualized by autoradiography. Approximately
25% of input 35S-labeled Csx/Nkx2.5 protein associated
with MBP-Csx/Nkx2.5 (Fig. 2A,
lane 1) as well as MBP-HD (lane 2), but not with
MBP alone (lane 3). To examine whether Csx/Nkx2.5
homodimerizes in cells, we cotransfected FLAG epitope-tagged Csx/Nkx2.5
expression plasmid with HA epitope-tagged Csx/Nkx2.5 expression plasmid
into the human embryonic kidney carcinoma cell line 293 and confirmed
that both Csx/Nkx2.5 proteins coimmunoprecipitated with anti-FLAG Ab (Fig. 2B, lane 1). Thus, Csx/Nkx2.5 can
homodimerize in solution as well as in cells, and binding to DNA is not
required for this interaction.
Homodimerization of Endogenous Csx/Nkx2.5 on the ANF
To ascertain whether Csx/Nkx2.5 protein forms dimers with DNA in
cardiac myocytes, adenovirus-encoding FLAG-tagged Csx/Nkx2.5 and/or
HA-tagged Csx/Nkx2.5 were coinfected into cardiac myocytes, and the
nuclear extracts were mixed with the DNA probe for EMSA analysis (Fig.
3C). The protein-DNA complex was transferred to a membrane
followed by Western blot analysis (Fig. 3D). We detected the
signal at the monomeric (M) and dimeric (D)
protein-DNA complex, similar to Fig. 3B. Additional slow
migrating bands observed in these experiments (* in Fig. 3D)
corresponded to Csx/Nkx2.5 protein unbound DNA (lanes 4-6
in Fig. 3D). Both FLAG-tagged and HA-tagged Csx/Nkx2.5 were
detected at the dimeric protein-DNA complex when both proteins were
coexpressed in cardiac myocytes (Fig. 3D, lane 2), suggesting that two Csx/Nkx2.5 molecules homodimerize on DNA.
Lys193-Arg194 within the HD Is Required for
Dimerization--
To confirm the specificity and to identify the
regions that are required for dimerization, we mapped the dimerization
domain of Csx/Nkx2.5 using in vitro binding assays.
Initially, four [35S]methionine-labeled COOH-terminal
deletion mutants were mixed with MBP-HD (Fig.
4A). Two COOH-terminal
deletion mutants of Csx/Nkx2.5, 1-230 and 1-199, associated with
MBP-HD, whereas the further deletions to 1-159 or 1-149 abolished the
association (Fig. 4B, top panel). These results
indicate that amino acids between 159 and 199 are necessary for
dimerization. Next, 5 amino acid serial deletion mutants from the
carboxyl terminus of HD, 1-196, 1-191, 1-186, and 1-181, were
examined. The 1-196 protein interacted with the HD, but the 1-191,
1-186, and 1-181 proteins did not (Fig. 4B, middle
panel). Further single amino acid deletions revealed that 1-193
dramatically reduced the interaction, and 1-192 completely abolished
the interaction (Fig. 4B, bottom panel).
Therefore, two basic amino acids, Lys193 and
Arg194 are necessary for the interaction with the HD.
We further mutated Lys193 and Arg194 into
neutral or acidic amino acids (Lys193 to Ile,
Arg193 to Ile, and Lys193-Arg194 to
Ile193-Asp194) and examined them for
dimerization with the HD as well as with full-length Csx/Nkx2.5 (Fig.
4C). The Lys193 Involvement of the Region(s) Outside of HD for
Dimerization--
To identify the domain(s) outside of the HD of
Csx/Nkx2.5 that are involved in homodimerization on DNA, we examined
DNA binding affinity of HD and full-length protein on the palindromic
ANF
To further examine the regions responsible for dimeric DNA binding, we
constructed two deletion mutants, a carboxyl terminus deletion mutant
() and an amino-terminal deletion mutant () and examined
their DNA binding on the ANF Lys193 Is Necessary for Association with GATA4--
We
and others have reported that Csx/Nkx2.5 interacts with the
transcription factor GATA4 (42-45). It was demonstrated that the
second zinc finger of GATA4 is involved in the specific interaction with the HD of Csx/Nkx2.5, and amino acids between 182 and 199 are
responsible for the direct interaction with GATA4 (43). Our data
presented in Fig. 4 revealed that this domain is also responsible for
homodimerization. Therefore, we examined whether GATA4 associates with
Lys193-Arg194 mutants (Fig.
6A). As shown in Fig.
6A, lane 1, the wild type Csx/Nkx2.5
(1-318) associated with GATA4-GST protein, whereas Lys193 Heterodimerization of Csx/Nkx2.5 with Other NK2 Class HD
Proteins--
Since Xenopus XNkx2.3 and XNkx2.5 are
coexpressed in the heart, and Csx/Nkx2.5 and Nkx2.6/Tix are coexpressed
in restricted areas in the mouse heart (31, 32, 41), we examined the
potential interaction of Csx/Nkx2.5 with Nkx2.3 and Nkx2.6/Tix by using in vitro binding assay. As shown in Fig. 6B,
full-length Csx/Nkx2.5 associated with Csx/Nkx2.5 as well as
Nkx2.6/Tix, and weakly with Nkx2.3. Therefore, Csx/Nkx2.5 demonstrates
the potential to interact with other NK2 class proteins with varying
binding affinity depending on the partner.
Generation of an Inhibitory Mutant--
To examine the effects of
protein dimerization on transcriptional activity, we attempted to
create a mutant protein that does not dimerize, but does bind, DNA. As
shown in Fig. 4, we constructed Csx/Nkx2.5 mutants that do not dimerize
to the HD. We next examined the DNA binding of these mutants using ANF
As an alternative to examine the effect of protein dimerization, we
generated a converse mutant in which protein dimerization is preserved,
but DNA binding is abolished. By mutating Ile183 in the
third helix of HD into Pro, DNA binding of
Csx/Nkx2.5(Ile183
We tested the function of the Ile183 Ile183
Transfection efficiency of primary cardiac myocytes is known to be very
low when plasmid vectors are used. Therefore, we infected cardiac
myocytes with adenoviral vectors, which exhibit a high efficiency of
gene transfer. More than 90% of cardiac myocytes expressed either
Ile183 The NK2 class homeobox-containing transcription factor Csx/Nkx2.5
is one of the earliest cardiogenic markers from insects to vertebrates
(28, 31, 34, 38, 39, 57, 58). Recently, human CSX/NKX2.5 mutations were
identified in patients with congenital heart disease (22, 23). These
patients show progressive conduction delays and in some cases left
ventricular dysfunction after birth, suggesting that Csx/Nkx2.5 also
functions in the later stages of heart development and maturation.
While evidence is accumulating that Csx/Nkx2.5 mutations inhibit normal
cardiac development and maturation in both humans and
Xenopus (22, 23, 25), the molecular mechanisms for these
phenotypes remain to be explained. In this study, we report the new
finding of protein dimerization of Csx/Nkx2.5, which may yield insights
into the dominant effects of CSX/NKX2.5 mutations found in humans.
Csx/Nkx2.5 binds to the palindromic Csx/Nkx2.5 consensus binding sites
in the ANF promoter as a monomer as well as a dimer, and dimer
formation increases the protein-DNA binding affinity. Csx/Nkx2.5
physically interacts with each other in vitro as well as in
a cell. Two basic amino acids, Lys193-Arg194,
located in the third helix of HD are necessary for dimerization, and
Lys193 is also indispensable for the association with
GATA4, which is a cofactor for Csx/Nkx2.5 function. To examine the
functional significance of dimerization, we generated a converse mutant
(Ile183 Protein Dimerization of Csx/Nkx2.5 through the HD--
By using
various deletion and point mutants, we found that two positively
charged amino acids, Lys193-Arg194 at the
COOH-terminal end of HD (Lys57-Arg58 in HD),
are critical for protein-protein interaction. As expected, these two
amino acids were highly conserved within NK2 class HD (29). It was
shown that three amino acids
(Arg58-Val59-Lys60) located at the
carboxyl end of the HD of Pit1 are involved in the homodimerization on
DNA by forming a protein-protein interface with the POU-specific domain
(15). In contrast to Csx/Nkx2.5, Pit1 requires DNA to homodimerize
(59).
The Lys193-Arg194 mutation, located at the
carboxyl end of the HD in Csx/Nkx2.5, markedly reduced DNA binding,
which is consistent with the NMR structure of another NK2 class HD
protein Drosophila NK-2. The third Regions Outside the HD Facilitate Dimerization on
DNA--
Cooperative dimerization of HD proteins has been
characterized in paired and paired-like HD proteins (11). Paired HD
proteins cooperatively bind DNA with a palindromic TAAT sequence
separated by 3 bp. The presence of Arg28 or
Arg43 prevents cooperative dimerization, and paired class
HD proteins do not have Arg residues at the 28 and 43 positions (11).
In contrast, 50% of HD proteins have conserved Arg28 or
Arg43 residues among ~350 HD proteins (53). NK class HD
proteins, as well as engrailed, bcd, POU, and msh class proteins do not have Arg28 or Arg43, suggesting the
possibilities for cooperative dimerization in these classes of HD proteins.
In Csx/Nkx2.5, regions outside of the HD, particularly the region
carboxyl terminus to the HD (aa 251-318), appear to facilitate cooperative dimerization on DNA. Compared with the DNA binding of HD of
Csx/Nkx2.5 or the COOH-terminal deletion mutant, we found that the
full-length protein facilitated dimerization, and the monomer-dimer
transition occurred at ~13-fold lower protein concentrations than
that of the HD or the carboxyl terminus deletion mutant. Our
observation that Csx/Nkx2.5 homodimerizes through the HD as well as
outside of the HD supports the hypothesis that protein-protein interactions play important roles in cooperative dimerization. Alternatively, a full-length Csx/Nkx2.5 molecule may "bend" DNA to
facilitate the binding of a second Csx/Nkx2.5 molecule to DNA, or
full-length Csx/Nkx2.5 proteins may be more stable than HD proteins in
a dimerized form on DNA. It is also possible that these effects may
function cooperatively to regulate the transcriptional activation of
target genes.
Palindromic NK2-specific binding sites are also identified in the
enhancer of the Drosophila ind gene, which is the target of
another NK2 class HD protein Vnd (61). Also, Tinman, the Drosophila homologue of Csx/Nkx2.5, binds the sequence
located at Heterodimerization with Other NK2 Class Proteins--
Several NK2
class HD proteins are coexpressed both temporally and spatially,
suggesting that NK2 class HD proteins may heterodimerize. As shown in
Fig. 6, Csx/Nkx2.5 and Nkx2.6/Tix associated with each other, but the
association of Csx/Nkx2.5 and Nkx2.3 was significantly weaker. Although
further quantitative analyses are necessary, these results suggest that
NK2 class HD proteins potentially interact with each other, and the
affinity of the interaction is different depending on heterodimer
pairs. In this study, we did not examine the possible
heterodimerization of Csx/Nkx2.5 with other classes of HD proteins. Of
note, mouse Nkx2.3 is not expressed in the heart, and Nkx2.6/Tix
expression is restricted to the sinus venosa and outflow tract of the
mouse heart. Other NK2 class HD proteins coexpressed temporally and
spatially similar to Csx/Nkx2.5 in the heart have not been described in
mouse and human (29, 41, 63).
The Effect of Non-DNA Binding Mutant on Wild Type
Csx/Nkx2.5--
Based on the studies of phenotypes caused by the
non-DNA binding mutants of Csx/Nkx2.5 in patients and
Xenopus (22-25), and on the evidence for the
formation of homodimers of Csx/Nkx2.5 in our study, non-DNA binding
mutants might act in a dominant inhibitory manner. We generated a
single missense mutation in the third helix of the HD,
Csx/Nkx2.5(Ile183
ANF(
Protein homodimerization of Csx/Nkx2.5 yields the potential for it to
precisely regulate a number of genes by utilizing monomeric and dimeric
binding. It is possible that the genetically dominant effect of the
human CSX/NKX2.5 missense mutations (22-24) may in part be due to an
inhibitory effect of the mutant protein over the wild type protein on
target genes that require dimeric binding.
Pro mutant
acts in an inhibitory manner on wild type Csx/Nkx2.5 transcriptional
activity through the ANF promoter in 10T1/2 cells. However,
Ile183
Pro mutant does not inhibit wild type Csx/Nkx2.5
function on the ANF promoter in cultured neonatal cardiac myocytes,
possibly due to failure of dimerization in the presence of the target
DNA. These results suggest that complex protein-protein interactions of
Csx/Nkx2.5 play a role in its transcriptional regulatory function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices and contacts the major
groove of DNA through the third helix (1). Contrary to the highly
specific biological functions of individual homeobox genes, in
vitro DNA binding studies have demonstrated that most HD proteins
bind to similar short consensus sequences containing the TAAT motif (1,
2). This apparent discrepancy may result from target gene's
specificity for each HD protein in vivo being achieved by
multiple mechanisms, such as interaction with other factors (3), small
differences in DNA binding affinities to target sites (4),
translational regulation of homeobox gene expression (5, 6),
subcellular localization (7), or protein phosphorylation (8, 9).
-HNF1
(16), Oct1-Oct2 (15), Mix.1-Siamois (14),
MCM1-MAT
2 (17, 18), and Extradenticle-Ultrabithorax (19). The
monomer of HD proteins is sufficient to interact with DNA, and the
DNA-bound monomer recruits other partners to the complex (11, 20). Most likely, the monomer HD proteins regulate through the monomeric DNA
binding site, whereas homo- or heterodimerized HD proteins regulate
through the dimeric sites. These differential interactions would
provide more precise gene regulation at each developmental stage
(21).
87 and
242 bp sites, and
transactivates the ANF gene synergistically with the zinc finger
transcription factor GATA4 (42-45). In this study, we demonstrate the
homodimerization of Csx/Nkx2.5 at ANF
242 site and determine the
critical amino acid residues for homodimerization as well as
heterodimerization with GATA4. In addition, we generated a single
missense mutation, Ile183 to Pro in the HD of Csx/Nkx2.5,
which preserved homodimerization function, but totally abolished DNA
binding. Ile183
Pro mutant acted in an inhibitory
manner on wild type Csx/Nkx2.5 transcriptional activity through the ANF
promoter depending on the cellular context.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Pro) and
pcDNA3-Csx/Nkx2.5(Ile183
Pro). All the
PCR-amplified fragments were sequenced for sequence confirmation.
264 to
227), was annealed with
5'-GATTTGCCTCAAGAGGCCCCCACTTCAAAGGTGTGA-3'; 5'-TCACACCTTTGAAGTGGGGGCCT-3' was annealed with
5'-AGGCCCCCACTTCAAAGGTGTGA-3'; 5'-GCCGCCGCAAGTGACAGAATGGGGA-3' (
93 to
65) was annealed
with 5'-TCCCCATTCTGTCACTTGCGGCGGGCCA-3' and used for
EMSA. 3-Fold serial dilutions of 66 ng of bacterially expressed fusion
proteins were incubated with 50,000 cpm of probe, 50 µg of bovine
serum albumin, 0.5 µg of poly(dG-dC) in 10 mM HEPES, pH
8.0, 50 mM KCl, 1 mM EGTA, 10% glycerol, 2.5 mM DTT, 7 mM MgCl2 in 15-µl
reaction volume for 20 min at room temperature, separated in 5%
native polyacrylamide gel with 0.5 × Tris-glycine buffer at 15 mA
for ~20 min.
C (aa 1-250, molecular mass = 27.5 kDa)
or
N (aa 122-318, molecular mass = 21.6 kDa).
Pro mutant was inserted into the
shuttle vector pADloxp vector (50), creating pADloxp-Csx/Nkx2.5(FLAG-wild) or pADloxp-Csx/Nkx2.5(HA-wild) or pADloxp-Csx/Nkx2.5(FLAG-IP). One µg of plasmids was cotransfected with 1 µg of
5 viral DNA into Cre8 cells to produce adenoviruses according to the methods reported previously (50). For control,
5
viral DNA expressing no transgene was infected to 293 cells. The viral
particle number was determined by plaque assays, and 5-15 multiplicity
of infection was used for infection to neonatal rat cardiac myocytes
prepared as described previously (51). The expression of wild type or
Ile183
Pro mutant protein was determined by Western
blotting and immunostaining using anti-FLAG mAb (Sigma) or anti-HA mAb
(Roche Molecular Biochemicals).
638)-Luc reporter construct (provided by K. R. Chien), 0.4 µg of Rous sarcoma virus
-galactosidase (provided by
B. Markham), 0.4 µg of pcDNA3-Csx/Nkx2.5 with or without 0.4 µg
or 0.8 µg of pcDNA3-Csx/Nkx2.5(Ile183
Pro) using
the calcium phosphate method. 0.4 µg of pAT2-GATA4 expression vector
(provided by B. Markham) was cotransfected together with the
plasmids listed above. Total plasmid amount was adjusted to 3.0 µg
with pcDNA3 vector plasmid. After glycerol shock using 1× HEPES
buffer containing 15% glycerol, cells were cultured for another
48 h, lysed with 300 µl of reporter lysis buffer (Promega), and
assayed for luciferase activity (Promega) and
-galactosidase activity.
638)-Luc reporter, 0.6 µg of murine sarcoma virus (MSV)
-galactosidase, 0.75 µg of
pcDNA3-Csx/Nkx2.5(Ile183
Pro) with or without 0.75 µg
of pcDNA3-Csx/Nkx2.5(Wild) using 10 µl of LipofectAMINE 2000 reagents
(Life Technologies, Inc.). Total plasmid amount was adjusted to 4.65 µg with pcDNA3 vector plasmid. Plasmids were also cotransfected into
cardiac myocytes using the calcium phosphate method in a total amount
of 3.1 µg: 1.2 µg of ANF(
638)-Luc reporter, 0.4 µg of
MSV-
-galactosidase, 0.5 µg of pcDNA3-Csx/Nkx2.5(Ile183
Pro) with or without 0.5 µg of pcDNA3-Csx/Nkx2.5(Wild). The adenovirus encoding Ile183
Pro mutant, wild type, or
control empty virus were infected on the next day of primary culture
(day 2). On day 3, 2.4 µg of ANF(
638)-Luc reporter and 0.6 µg of MSV-
-galactosidase were cotransfected using the calcium
phosphate method for 2 h, and luciferase activity (Promega) and
-galactosidase activity were measured after 48 h.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
408,
242, and
87 bp. At position
242
bp, the promoter contains two binding sites spaced by 5 nucleotides (Fig. 1A, panel a).
Because Csx/Nkx2.5 contacts DNA through its HD, we analyzed and
compared the HD binding affinity for two sites in the ANF promoter at
positions
242 bp (ANF
242) and
87 bp (ANF
87). Applying EMSA,
we determined that the HD protein binds to the ANF
242 site
with a Kd = 1 × 10
9
M (Fig. 1A, panel b), which is
slightly lower than the previously reported Kd for
the related Drosophila HD protein NK2 (52), but in the
affinity range for the HD protein (Kd in the range
of 10
9 to 10
10
M) (53). However, for the protein-DNA interaction with the ANF
87 site, we estimated a Kd = 8.2 × 10
8 M (Fig. 1A,
panel c). Therefore, the HD bound to the ANF
242 site with
more than 80 times higher affinity than to the ANF
87 site.
Interestingly, we also observed that Csx/Nkx2.5 forms an additional
specifically shifted band with a migration most likely corresponding to
the occupation of the two specific DNA binding sites of the ANF
242
at higher protein concentrations (Fig. 1A, panel
b, lanes 3-6). We asked whether the protein
concentration at which the second band appears is physiologically
relevant. Csx/Nkx2.5 forms a dimer at a protein concentration of
3.1 × 10
9 M (lane
3), which is comparable with the concentrations reported for other
transcription factors (54-56).
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Fig. 1.
HD of Csx/Nkx2.5 homodimerizes on palindromic
DNA sequences in the ANF promoter. A, schematics of two
Csx/Nkx2.5 consensus binding sites in the ANF promoter (panel
a). A palindromic DNA binding 242 bp site (panel b)
and monomeric
87 bp sites (panel c) were used for gel
shift analysis. The first lane shows free probe (F) without
protein, followed by 3-fold serial increases in HD protein
concentrations shown in lanes 1-6 (panels b and
c). The HD forms monomer (M) as well as dimer
(D) at
242 bp site with a binding affinity
Kd = 1.0 × 10
9
M (panel a) and forms only monomers on
87 bp
site with a Kd = 8.2 × 10
8 M (panel b).
B, a mutated monomeric binding site was used for EMSA. HD
protein binds as a monomer (M) on the mutated
242 bp site
with a Kd = 1-3.0 × 10
9 M. C, HD protein
was mixed with full-length Csx/Nkx2.5 to identify heterodimers. The HD
protein (3.0 × 10
9 M)
(lane 1) was mixed with 3-fold serially increased
full-length Csx/Nkx2.5 protein (0.71, 2.1, or 6.4 × 10
9 M) (lanes 2-4).
The HD and full-length Csx/Nkx2.5 form stable complexes
(HD+Full) and migrated to an intermediate position between
HD dimer (HD D) and full-length dimer (Full D).
M, monomer; D, dimer; F, free
probe.
242, we performed EMSA using an oligonucleotide in which one of the
DNA binding sites was deleted from the ANF
242 site. We found that HD
bound to the mutated site (converted to a single DNA binding site) as a
monomer (M in Fig. 1B) without showing the slow
migrating bands. The binding affinity to the monomeric DNA binding site
was slightly reduced (Kd = 1-3.1 × 10
9 M). Furthermore, we found
that when the HD was mixed with the full-length Csx/Nkx2.5, the HD and
the full-length protein produced newly shifted bands migrating to an
intermediate position between the HD and full-length homodimers (Fig.
1C, HD+Full). These data indicate that the
Csx/Nkx2.5 protein binds with a higher affinity to the palindromic ANF
242 site than to the mutated monomeric ANF
242 site or the
monomeric ANF
87 site. Most likely Csx/Nkx2.5 forms a dimer on ANF
242 site, and dimerization stabilizes the protein-DNA interaction
(see below).
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Fig. 2.
Homodimerization of Csx/Nkx2.5 without
DNA. A, 35S-labeled Csx/Nkx2.5 protein was
mixed with full-length (lane 1), HD (lane 2)
fused to MBP, or MBP alone (lane 3). Bound protein was
resolved on SDS-PAGE and autoradiographed. 50 and 25% input of
35S-labeled Csx/Nkx2.5 protein were loaded on lanes
4 and 5, respectively. MBP-full-length Csx/Nkx2.5
protein (lane 1), MBP-HD (lane 2), and MBP alone
(lane 3) used for the assay are shown on the bottom
panels. B, human embryonic kidney carcinoma 293 cells
were transfected with two expression plasmids encoding FLAG-tagged
Csx/Nkx2.5 and HA-tagged Csx/Nkx2.5. Cell lysates were
immunoprecipitated with anti-FLAG Ab, and immunoprecipitants were
blotted with anti-HA Ab to detect coimmunoprecipitated HA-Csx/Nkx2.5.
Two transfectants expressing either FLAG-Csx/Nkx2.5 (lane 2)
or HA-Csx/Nkx2.5 (lane 3) were also analyzed as
controls.
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Fig. 3.
Csx/Nkx2.5 in the nuclear extract
homodimerizes on DNA. A, endogenous Csx/Nkx2.5 protein
from rat neonatal cardiac myocytes forms monomers (M) as
well as dimers (D) at 242 bp site (lane 1).
These bands correspond to the bands shifted by the nuclear extract from
adenoviral infected rat cardiac myocytes expressing FLAG-tagged
Csx/Nkx2.5 (lanes 2-4). The amounts of nuclear extracts
used are shown. B, Western blot of the protein-DNA complex
with anti-Csx/Nkx2.5 mAb. Endogenous Csx/Nkx2.5 (lane 1) was
detected as monomer and dimer forms on DNA. Higher expression of
Csx/Nkx2.5 was detected in adenoviral infected cardiac myocytes
(lane 2). The amounts of nuclear extracts used are shown.
C, EMSA of nuclear extracts from adenoviral infected cardiac
myocytes. Cardiac myocytes were infected with adenoviral constructs
encoding either FLAG-tagged Csx/Nkx2.5 (lane 1) or HA-tagged
Csx/Nkx2.5 (lane 3) and mixture of two constructs
(lane 2). D, protein-DNA complexes were
transferred to a membrane followed by Western blot analysis. In the
presence of DNA (lanes 1-3), FLAG-tagged Csx/Nkx2.5 or
HA-tagged Csx/Nkx2.5 protein migrated faster than that without DNA (*
in lanes 4-6) and was detected in the monomeric
(M) or dimeric (D) protein-DNA complex. Both
FLAG-tagged and HA-tagged Csx/Nkx2.5 were detected in the dimeric
protein-DNA complex (lane 2) when both proteins were
coexpressed in cardiac myocytes.
242
Site--
To examine whether endogenous Csx/Nkx2.5 homodimerizes on
the ANF
242 site, nuclear extracts prepared from neonatal rat cardiac myocytes were used for EMSA. As shown in Fig. 3A, lane
1, endogenous Csx/Nkx2.5 forms monomers (M) as well as
dimers (D). These two bands corresponded to the bands
shifted by the nuclear extract from adenovirus infected rat cardiac
myocytes that expressed FLAG-tagged Csx/Nkx2.5 (Fig. 3A,
lanes 2-4). Because of the high expression levels of
Csx/Nkx2.5 in the adenoviral vector-infected cardiac myocytes, 20-fold
dilution of the nuclear extracts was required to shift the DNA probe to
a similar level compared with that of the endogenous Csx/Nkx2.5 (Fig.
3A, lane 1 versus lane 3). When the
protein-DNA complex was transferred to the membrane and blotted with
anti-Csx/Nkx2.5 Ab, we detected the signal at the monomeric (M) and dimeric (D) protein-DNA complex both in
the uninfected and the virus infected nuclear extracts (Fig.
3B).
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Fig. 4.
Lys193 and Arg194 are
required for homodimerization with HD. A, schematics of
deletion mutants used for the in vitro binding assays and
summarized results. B, various 35S-labeled
Csx/Nkx2.5 deletion mutant proteins were mixed with MBP-HD protein, and
bound proteins were resolved on SDS-PAGE and autoradiographed. Bound
35S-labeled Csx/Nkx2.5 mutants to HD or 50% input of
35S-labeled protein are shown. Top panel shows
that the deletion between aa 199 and aa 159 abolished the association.
Middle panel shows the 5 amino acid deletion series from the
carboxyl terminus of HD. Amino acids between aa 196 and aa 191 are
necessary for the association. Bottom panel shows single
amino acid deletions between aa 196 and aa 191. 1-195 and 1-194
associated with HD; however, 1-193 markedly reduced the association,
and 1-192 further decreased the association. C, two basic
amino acids Lys193 and Arg194 were mutated into
Ile or Asp and examined for protein-protein interactions with MBP-HD
and MBP-full-length Csx/Nkx2.5. Bound 35S-labeled
Csx/Nkx2.5 mutants to HD or full-length Csx/Nkx2.5 or 50% input of
35S-labeled protein are shown on the upper
panels, and MBP-HD and MBP-full-length Csx/Nkx2.5 protein used for
the assay are shown on the bottom panels.
Ile mutant markedly reduced
the interaction with the HD, and an ~50% reduction was observed in
Arg194
Ile mutant. The interaction with the HD was
undetectable when both amino acids were mutated (Fig. 4C,
HD). However, we still detected a weak interaction between
Lys193-Arg194 mutant and full-length Csx/Nkx2.5
(Fig. 4C, Full). These findings confirm that two
amino acids Lys193 and Arg194, are required for
the dimerization of the HD, and additional protein domain(s) outside of
HD are also likely to be involved in dimerization.
242 site or a mutated monomeric ANF
242 site shown in Fig. 1. The HD protein bound DNA predominantly as a monomer at a low protein concentration (Fig. 5A,
panel a, lanes 1-3) and dimerized more at a
higher protein concentration (Fig. 5A, panel a,
lanes 5 and 6). The monomer to dimer transition
was observed between lanes 4 and 5 at a protein
concentration of 0.91-2.7 × 10
8
M in the HD protein (arrow in Fig.
5A, panel a). However, with the full-length
Csx/Nkx2.5 (Fig. 5A, panel b), the monomer-dimer transition occurred more abruptly between lane 2 and
lane 3 (protein concentration 0.71-2.1 × 10
9 M). We tested and confirmed
that HD and full-length protein bound to the mutated monomeric site
with similar affinity (Kd = 1.0-3.0 × 10
9 M for HD; 0.71-2.1 × 10
9 M for full-length Csx/Nkx2.5)
(Fig. 5A, panels c and d).
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Fig. 5.
Regions outside the HD facilitate
homodimerization on DNA. A, HD and full-length
Csx/Nkx2.5 were analyzed for DNA binding using palindromic ANF 242
site (panels a and b) and mutated monomeric site
(panels c and d). Panel a, HD
protein bound ANF
242 site predominantly as a monomer in low protein
concentrations and gradually transitioned into a dimer. An equivalent
monomer-dimer ratio was observed between lanes 4 and
5 in the HD (approximate protein concentration,
0.91-2.7 × 10
8 M).
Panel b, with full-length protein, the monomer to dimer
transition occurred rather abruptly between lanes 2 and
3 (protein concentration, 0.71-2.1 × 10
9 M). HD (panel c)
and full-length Csx/Nkx2.5 (panel d) bound a mutated
monomeric binding sites with similar affinity (Kd = 1.0-3.0 × 10
9 M for HD;
Kd = 0.71-2.1 × 10
9
M for full-length Csx/Nkx2.5). B, in the
COOH-terminal deletion mutant (panel a), the transition from
monomer to dimer occurred between lanes 4 and 5,
similar to HD (protein concentration, 0.66-2.1 × 10
8 M). In the amino-terminal
deletion (panel b), the transition occurred between
lanes 3 and 4 (protein concentration,
2.4-7.4 × 10
9 M). 3-Fold
serial increases in protein concentration (0.018-4.4 µg/ml) were
used. M, monomer; D, dimer; F, free
probe.
242 site (Fig. 5B). In the
COOH-terminal deletion mutant (Fig. 5B, panel a),
the monomer-dimer transition was observed between lanes 4 and 5 (0.66-2.0 × 10
8 M),
which was similar to that of HD protein. The amino-terminal deletion
showed the monomer-dimer transition between lanes 3 and 4 (2.4-7.3 × 10
9 M) (Fig.
4B, panel b); therefore it required 3-fold lower
protein concentration than that of the HD or the carboxyl terminus
deletion, but still required 3-fold higher protein concentration than
that of the full-length protein. Taken together, although the HD and full-length Csx/Nkx2.5 binds the monomeric DNA binding site with a
similar affinity, full-length Csx/Nkx2.5 preferentially forms dimers at
~13-fold lower protein concentration than the HD alone. Thus, regions
outside of the HD, particularly the COOH-terminal region of Csx/Nkx2.5,
seem to facilitate protein-protein interactions involved in the
dimerization on DNA.
Ile (lane 2) and Lys193
Ile/Arg194
Asp (lane 4) mutants
abolished the interaction. Interestingly, the Arg194
Ile mutant (lane 3) associated with GATA4 with an apparent higher affinity than wild type Csx/Nkx2.5, in contrast to its lower
homodimerization ability (Fig. 4C). These data demonstrate that Lys193 in the HD of Csx/Nkx2.5, which is critical for
homodimerization, is also essential for the interaction with GATA4.
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Fig. 6.
Protein interactions of Csx/Nkx2.5 between
GATA4, Nkx2.3, and Nkx2.6/Tix: Lys193 is required for
interaction with GATA4. A, 35S-labeled wild
type, Lys193, Arg194, and
Lys193-Arg194 mutants were mixed with GST-GATA4
(lanes 1-4) or GST protein (lanes 5-8) to
examine whether Csx/Nkx2.5 Lys193-Arg194
mutations affect the protein interaction with GATA4. Bound proteins
were resolved on SDS-PAGE. Wild type (lane 1),
Arg194 mutant (lane 3) interacted with GATA4,
whereas Lys193 (lane 2) and
Lys193-Arg194 mutant (lane 4) did
not interact. B, 35S-labeled Csx/Nkx2.5
(lanes 1 and 4), Nkx2.3 (lanes 2 and
5), and Nkx2.6 (lanes 3 and 6) were
mixed with MBP-full-length Csx/Nkx2.5 (lanes 1-3) and MBP
fusion proteins (lanes 4-6) to detect heterodimers. 50%
input of proteins is also shown (lanes 7-9).
242 site and found that the Lys193
Ile mutant
completely abolished DNA binding (Fig. 6A).
Lys193-Arg194 mutant bound DNA, but the binding
affinity was significantly lower than that of wild type Csx/Nkx2.5
(Fig. 7A).
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Fig. 7.
A non-DNA binding mutant with preserved
homodimerization acts in an inhibitory manner in
vitro. A, two mutants with protein
dimerization defects were examined for DNA binding. EMSA shows
Lys193 Ile completely abolished DNA binding
(middle panel), and Lys193
Ile/Arg194
Asp significantly decreased DNA binding
(right panel) compared with wild type (left).
B, an Ile183
Pro mutant was created by
mutating Ile183 into Pro in the third helix of HD (see Fig.
3A). EMSA shows Ile183
Pro completely
abolished DNA binding (right panel). C, protein
dimerization of wild type (W) and Ile183
Pro
mutant (IP). 35S-Labeled wild type
(W) (lanes 1, 3, 5,
7) or Ile183
Pro mutant (IP)
protein (lanes 2, 4, 6, 8)
were mixed with MBP-wild type Csx/Nkx2.5 (lanes 1 and
2), MBP (lanes 3 and 4), GATA4-GST
(lanes 5 and 6), or GST fusion protein
(lanes 7 and 8). Complexes were resolved by SDS-PAGE and
autoradiographed. The Ile183
Pro mutant associated with
MBP-Csx/Nkx2.5 protein (lane 2) similar to wild type
Csx/Nkx2.5 (lane 1). The association between
Ile183
Pro mutant and GATA4 (lane 6) was
markedly reduced compared with wild type (lane 5).
D, inhibition of Csx/Nkx2.5-dependent
transactivation in the presence of increasing amount of
Ile183
Pro expression plasmid. 10T1/2 cells were
transiently transfected with 0.4 µg of pcDNA3-wild type
Csx/Nkx2.5 expression plasmid, ANF-Luc, and RSV-
-GAL plasmid and the
indicated amount of pcDNA3-Csx/Nkx2.5(Ile183
Pro)
expression plasmid. Approximately 44% reduction in
transactivation was seen upon transfection with 0.4 µg, and ~53%
reduction with 0.8 µg of pcDNA3-Csx/Nkx2.5(Ile183
Pro) (left panel). The right panel shows the
inhibitory effect of Ile183
Pro in the presence of
GATA4 expression vector. Cotransfection with GATA4 plasmid increased
ANF-Luc activity as reported previously (43). The Ile183
Pro mutant reduced ANF-Luc by ~20% at a 1:1 ratio (0.4 µg) and
by 44% at a 2:1 ratio (0.8 µg). Values are means ± S.E.
Pro) mutant was completely abolished
(Fig. 7B), but this mutant associated with MBP-Csx/Nkx2.5
protein with a similar affinity as that of wild type protein (Fig.
7C, lane 1 versus lane 2). In
contrast, this Ile183
Pro mutant markedly reduced
the interaction with GATA4 (Fig. 7C, lane
6).
Pro mutant by
transient transfection assays in 10T1/2 fibroblasts using
ANF(
638)-luciferase reporter construct (ANF-Luc), which
includes the
87 and
242 bp sites shown in Fig. 1A. The
Ile183
Pro mutant did not bind DNA (Fig.
7B), and the mutant itself did not activate or repress the
ANF-Luc (data not shown). When we cotransfected the expression plasmid
encoding Ile183
Pro mutant protein with wild type
Csx/Nkx2.5 at 1:1 ratio (0.4 µg), the luciferase activity of wild
type Csx/Nkx2.5 decreased by ~44%. A slight further reduction
(~53%) of ANF-Luc activity was observed when the Ile183
Pro expression plasmid was increased to 2:1 (0.8 µg) (Fig. 7D). In the presence of GATA4 expression plasmid, we
observed a further increase of ANF-Luc activity from the ANF promoter
as reported previously (43). We found that the Ile183
Pro mutant reduced luciferase activity by ~20% at 1:1 ratio of
plasmid amount and by ~44% at 2:1 ratio. These data demonstrate that
the non-DNA binding mutant, Ile183
Pro, acts in an
inhibitory manner on wild type Csx/Nkx2.5 in transient transfection
assays in 10T1/2 cells.
Pro Mutant Does Not
Inhibit the Csx/Nkx2.5-dependent ANF Promoter Activation in
Neonatal Cardiac Myocytes--
We further examined the inhibitory
effect of the Ile183
Pro mutant on endogenous
Csx/Nkx2.5 as well as wild type Csx/Nkx2.5 in cultured neonatal cardiac
myocytes. In rat neonatal cardiac myocytes, the base-line ANF-Luc
activity was high. When we used the LipofectAMINE transfection method,
the base-line ANF-Luc activity was approximately the same as that
detected in 10T1/2 cells transfected with the wild type Csx/Nkx2.5
expression plasmid. ANF-Luc activation was suppressed by the
cotransfection of Ile183
Pro expression plasmid by
29%. (Fig. 8A,
183I-P). When we cotransfected the wild type
Csx/Nkx2.5 expression plasmid, ANF-Luc activity was increased by 50%
(Fig. 8A, Wild). However, cotransfection of
Ile183
Pro expression plasmid did not inhibit the wild
type Csx/Nkx2.5 function (Fig. 8A,
183I-P+Wild). Similar results were obtained using
the calcium phosphate methods (data not shown).
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Fig. 8.
Ile183 Pro mutant does not inhibit coexpressed wild type Csx/Nkx2.5
function in cardiac myocytes. A, rat neonatal cardiac
myocytes cultured in six-well plates were cotransfected with 1.8 µg
of ANF(
638)-Luc, 0.6 µg of MSV-
-GAL, 0.75 µg of
pcDNA3-Csx/Nkx2.5(Ile183
Pro) and/or 0.75 µg of
pcDNA3-Csx/Nkx2.5(Wild) using 10 µl of LipofectAMINE 2000 reagents. pcDNA3-Csx/Nkx2.5(Ile183
Pro) reduced
ANF-Luc activation by 29%; in contrast pcDNA3-Csx/Nkx2.5 increased
the activation by 50% (Wild). Cotransfection of
pcDNA3-Csx/Nkx2.5(Ile183
Pro) with
pcDNA3-Csx/Nkx2.5(Wild) did not reduce ANF-Luc activation
(183I-P+Wild). Total plasmid amount was adjusted to
4.65 µg with pcDNA3 vector plasmid. Values are means ± S.E.
B, adenovirus encoding either Csx/Nkx2.5(Ile183
Pro) or wild type Csx/Nkx2.5 or empty vector were transfected and
stained with anti-FLAG Ab. In most of the cardiac myocytes infected
with either Csx/Nkx2.5(Ile183-Pro) mutant or wild type
Csx/Nkx2.5 adenoviral vector, FLAG staining was colocalized with
nuclear staining (arrows in b, c,
e, and f). A few cells without FLAG staining were
observed (* in b, c, e, and
f). The virus titer was between 5 and 15 multiplicity of
infection in each construct Bars = 20 µm. C,
Western blot of cardiac myocyte cell lysates expressing
Csx/Nkx2.5(Ile183
Pro, lane 2), wild type
Csx/Nkx2.5 (Wild, lane 3) and both
(183I-P+Wild, lane 4). D, the
next day of adenoviral infection, 2.4 µg of ANF(
638)-Luc
reporter and 0.6 µg of MSV-
-GAL were cotransfected using the
calcium phosphate method for 2 h, and luciferase and
-galactosidase activity were measured after 48 h. When the
Ile183
Pro mutant protein was expressed, ANF-Luc
activity was suppressed by 31% (183I-P). In
contrast, wild type Csx/Nkx2.5 activated the ANF-Luc reporter by 250%
(Wild), which was not suppressed by Ile183
Pro mutant (183I-P+Wild). Values are means ± S.E. E, DNA binding affinity of wild type Csx/Nkx2.5
expressed by adenoviral vector was not modified by coexpressed
Ile183
Pro mutant protein. EMSA of nuclear extracts
from adenoviral infected cardiac myocytes expressing wild type
Csx/Nkx2.5 alone (panel a) or coexpressing wild type and
Ile183
Pro mutant proteins (panel b). 3-Fold
serial increases in protein concentration (0.015-0.13 µg/µl) were
used. M, monomer; D, dimer; F, free
probe.
Pro mutant or wild type Csx/Nkx2.5 (Fig.
8B), and each construct expressed a similar protein amount
determined by Western blotting (Fig. 8C). Twenty-four hours
after adenovirus infection, ANF-Luc reporter gene was transfected into
cardiac myocytes, and the transcriptional activation was measured by
luciferase activity. When Ile183
Pro mutant protein was
expressed by the adenoviral vector, ANF-Luc activity was suppressed by
31% (Fig. 8D, 183I-P). In contrast, wild
type Csx/Nkx2.5 activated the ANF-Luc reporter by 3.5-fold (Fig.
8D, Wild), which was not suppressed by
coexpression of Ile183
Pro mutant (Fig. 8D,
183I-P+Wild). When we examined DNA binding of the
nuclear extract from cardiac myocytes expressing wild type alone (Fig.
8E, panel a) or wild type with Ile183
Pro mutant (Fig. 8E, panel b), there was no
significant difference in DNA binding of wild type Csx/Nkx2.5 either as
monomers or dimers. These data indicate that the expression of
Ile183
Pro mutant weakly suppresses ANF-Luc activity in
cardiac myocytes; however, Ile183
Pro mutant does not
suppresses ANF-Luc activity or reduce DNA binding of wild type
Csx/Nkx2.5 when coexpressed with wild type Csx/Nkx2.5 (see
"Discussion").
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Pro) that does not bind DNA, but has preserved
homodimerization ability. In transient transfection assays in 10T1/2
fibroblast cells, this mutant acts in an inhibitory manner on wild type
Csx/Nkx2.5. Also, this mutant suppresses the ANF-Luc activity in
cardiac myocytes, but does not inhibit the ANF-Luc activity that is
further induced by wild type Csx/Nkx2.5.
-helix (helix III) of
NK-2 extends up to amino acid 62 in the presence of DNA (52, 60). We
demonstrated that Lys193 is required for the Csx/Nkx2.5 and
GATA4 interaction as well as for homodimerization of Csx/Nkx2.5.
5.4 kilobases of the Dmef2 gene,
which contains two NK2 binding sites separated by 165 bp (62).
Mutations that disrupt either one of two Tinman binding sites caused
loss of activation of the Dmef2 gene, leading to the
hypothesis that the physical interaction of Tinman molecules occurs by
looping of the 165-bp intervening segment (62). Although it has yet to
be shown that Tinman protein homodimerizes, our data are consistent
with this hypothesis.
Pro), which abolishes DNA binding,
but preserves dimerization ability. The interaction between GATA4 and
Csx/Nkx2.5(Ile183
Pro) was significantly weaker than
that of wild type Csx/Nkx2.5; therefore, it is likely that
Csx/Nkx2.5(Ile183
Pro) will not sequester GATA4 from
wild type Csx/Nkx2.5 when it is overexpressed. The transcriptional
activation of wild type Csx/Nkx2.5 on ANF(
638) promoter was
indeed suppressed by the coexpressed Csx/Nkx2.5(Ile183
Pro) in a dose-dependent manner in 10T1/2 cells.
638) promoter activity was slightly suppressed by
Csx/Nkx2.5(Ile183
Pro) in neonatal cardiac myocytes
where endogenous Csx/Nkx2.5 is expressed. However, when both wild type
and Csx/Nkx2.5(Ile183
Pro) mutants were overexpressed
in cardiac myocytes, transcriptional activation by wild type was not
suppressed by the Csx/Nkx2.5(Ile183
Pro) mutant. Thus,
unlike in 10T1/2 cells, Csx/Nkx2.5(Ile183
Pro) does not
seem to act as a typical dominant inhibitory mutant on the
ANF(
638) promoter in cultured cardiac myocytes. The EMSA
using cell lysates prepared from adenovirus-infected cardiac myocytes
revealed that coexpression of Csx/Nkx2.5(Ile183
Pro)
mutant does not inhibit the specific binding of wild type Csx/Nkx2.5 to
the ANF
242 site (Fig. 8E). Since the
Csx/Nkx2.5(Ile183
Pro) mutant expressed in cardiac
myocytes did not bind to the ANF
242 site (data not shown), it is
possible that Csx/Nkx2.5(Ile183
Pro) mutant loses the
ability to form dimers with wild type Csx/Nkx2.5 on DNA and, therefore,
does not inhibit the function of wild type Csx/Nkx2.5 on the ANF
promoter in cardiac myocytes. It is also possible that the inhibitory
effect of Csx/Nkx2.5(Ile183
Pro) observed in 10T1/2
cells may occur through a mechanism independent of wild type
Csx/Nkx2.5. Mutant Csx/Nkx2.5 protein may squelch a transcription
factor that is critical for the ANF(
638) promoter activity in
10T1/2 cells.
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ACKNOWLEDGEMENTS |
---|
We thank E. O. Weinberg for critical reading of the manuscript; S. Hardy, T. Shioi, and A. Horwitz for valuable suggestions; H. Liao for Tix/Nkx2.6 plasmid; M. Schinke for Nkx2.3 plasmid; and B. Lee and J. Hampe for technical help.
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FOOTNOTES |
---|
* This work was supported by the Charles H. Foundation and American Heart Association Massachusetts Affiliate Fellowship and Beginning Grant-in-aid (to H. K.), an American Heart Association National Grant (to A. U.), and by National Institutes of Health (NIH) Grant R01-HL51253 and a Specialized Center for Research in Atherosclerosis in Congenital Heart Disease grant from NIH Grant P50-HL61036 (to S. I.).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 may be addressed: Beth Israel Deaconess Medical Center, 330 Brookline Ave. SL215, Boston, MA 02215. Tel.: 617-667-4862; Fax: 617-975-5268; E-mail: hkasahar@caregroup.harvard.edu.
To whom correspondence may be addressed: Beth Israel Deaconess
Medical Center, 330 Brookline Ave. SL201, Boston, MA 02215. Tel.:
617-667-4858; Fax: 617-975-5268; E-mail: sizumo@caregroup.harvard.edu.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M004995200
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ABBREVIATIONS |
---|
The abbreviations used are: HD, homeodomain; bp, base pair(s); ANF, atrial natriuretic factor; HA, hemagglutinin; PCR, polymerase chain reaction; MBP, maltose-binding protein; EMSA, electrophoretic mobility shift assay; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; Ab, antibody; mAb, monoclonal antibody; aa, amino acid(s); GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MSV, murine sarcoma virus.
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