From the Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
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ABSTRACT |
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Although the cardiac homeobox gene
Csx/Nkx-2.5 is essential for normal heart development,
little is known about its regulatory mechanisms. In a search for the
downstream target genes of Csx/Nkx-2.5, we found that the atrial
natriuretic peptide (ANP) gene promoter was strongly transactivated by
Csx/Nkx-2.5. Deletion and mutational analyses of the ANP promoter
revealed that the Csx/Nkx-2.5-binding element (NKE2) located at Murine Csx/Nkx-2.5 is a homeobox-containing gene
originally identified as a potential vertebrate homolog of the
Drosophila gene tinman (1, 2). tinman
is expressed in all mesodermal cells during germ-band elongation, but
its expression is subsequently restricted to the dorsal cardiac
mesoderm and, in the later developmental stage, to the dorsal vessel,
an insect equivalent of the vertebrate heart (3). A loss-of-function
mutant of the Drosophila tinman gene exhibits
complete loss of heart formation, indicating that tinman is essential
for Drosophila heart formation (4, 5). The expression of
Csx/Nkx-2.5 is also highly restricted to the heart and the
heart progenitor cells from the very early developmental stage when the
two heart primordia are symmetrically situated in the anterior lateral
mesoderm (1, 2), and targeted disruption of murine
Csx/Nkx-2.5 results in embryonic lethality due to the abnormal looping morphogenesis of the primary heart tube (6). Furthermore, Csx/Nkx-2.5 family genes are also identified in
various vertebrate species from zebrafish to humans (7-10). These
results indicate that Csx/Nkx-2.5 is also essential for
normal heart development and morphogenesis in vertebrates like the
tinman gene in Drosophila and suggest that the
regulatory mechanism of heart development controlled by Csx/Nkx-2.5 is
highly conserved in evolution.
To clarify the molecular framework of vertebrate cardiogenesis, the
identification of upstream regulatory factors that control the
expression of the Csx/Nkx-2.5 gene and the downstream
targets of the Csx/Nkx-2.5 protein is necessary (11-13). In
Drosophila, tinman gene expression is controlled
by several upstream factors including twist, decapentaplegic, and
wingless (3, 14-16), and tinman directly regulates the expression of
D-MEF2 via the cardiac enhancer in the D-MEF2
gene (17). In vertebrates, however, there is only limited information
regarding the upstream factors or the downstream targets of
Csx/Nkx-2.5. Among the possible regulatory factors of cardiac
development, bone morphogenetic proteins have recently been identified
as potential inducing signals of Csx/Nkx-2.5 that are
secreted from the endoderm (18). As for the downstream targets of
Csx/Nkx-2.5, the expression of ventricular myosin light chain 2, CARP
(cardiac ankyrin-repeat
protein), and a basic helix-loop-helix transcription factor
(eHAND) is significantly reduced in Csx/Nkx-2.5 knockout
mice (6, 19, 20), suggesting that Csx/Nkx-2.5 may, either directly or
indirectly, regulate the expression of these genes. In addition, since
CARP promoter activity is down-regulated by the overexpression of the
dominant-negative form of Csx/Nkx-2.5 in cultured cardiac myocytes
(19), CARP may be a direct target of Csx/Nkx-2.5, although the precise
cis-regulatory sequences in the CARP promoter are not known.
In addition to Csx/Nkx-2.5, several transcription factors have also
been identified as potential regulators of cardiac development. Among
these molecules, MEF2C and GATA-4 are thought to be involved in the
early step of cardiac development because both of these factors appear
in the precardiac mesoderm almost simultaneously with Csx/Nkx-2.5 and
earlier than any other known transcription factors implicated in
cardiogenesis (21, 22). MEF2C belongs to the MEF2 (myocyte
enhancer factor-2) subfamily of
MADS box transcription factors and binds to the AT-rich element in the regulatory regions of numerous muscle-specific genes (23). GATA-4 is a
member of the cardiac GATA subfamily, which is composed of GATA-4/5/6
and binds to the WGATAR motif in the promoter regions of cardiac- or
gut-specific genes (24). Targeted disruption of MEF2C
results in embryonic lethal phenotype due to the right ventricular
dysplasia (25), and GATA-4 In this study, to clarify the genetic pathways that control vertebrate
heart development, we tried to find the direct downstream target genes
of Csx/Nkx-2.5. Cotransfection of the expression plasmid of human
Csx/Nkx-2.5 and the reporter genes with promoters of various
cardiac-specific genes linked to the firefly luciferase gene revealed
that the atrial natriuretic peptide
(ANP)1 gene promoter is
strongly transactivated by Csx/Nkx-2.5 via the high affinity
Csx/Nkx-2.5-binding element termed NKE2 (Nkx-2.5 response
element-2) located at Construction of Plasmids--
The expression plasmid of human
Csx/Nkx-2.5 (pEFSA-CSX1) and that of human GATA-4 (pSSRa-hGATA4) were
previously described (10, 28). To construct luciferase reporter genes
for deletion analysis, various length of ANP genomic fragments were
amplified by polymerase chain reaction (PCR) with primers containing a
SacI site in the forward primer and an XhoI site
in the reverse primer. For mutational analyses, two GATA elements
(located at Cell Culture, DNA Transfection, and Reporter Gene
Assay--
COS-7 cells were cultured in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum. Transient transfections were performed 24 h after plating with the standard calcium
phosphate method. For each 35-mm dish, the reporter construct,
expression vectors of CSX1 and/or GATA-4, SV40- In Vitro Transcription and Translation--
PCR-amplified
cDNA fragments corresponding to the coding region of CSX1
derivatives and GATA-4 derivatives were subcloned into pSPUTK vector
(Promega) containing the SP6 RNA polymerase promoter and the sequence
of the Xenopus Electrophoretic Mobility Shift Assay (EMSA)--
Double-stranded
oligonucleotides corresponding to the TTF-1 (thyroid
transcription factor-1)-binding
sequence, NKE, NKE2, and the GATA site with or without mutations were
synthesized with GATC overhanging at the 5' terminus of each
oligonucleotide. The two complementary oligonucleotides were annealed
and labeled with [ Co-immunoprecipitation Experiments--
The expression plasmid
of HA-tagged CSX1 (pEFSA-HA-CSX1) was described previously (10). To
generate the expression plasmid of Myc-tagged GATA-4
(pBK-CMVMyc-GATA4), the corresponding cDNA fragment was amplified
by PCR and subcloned into the XbaI/ClaI site of
modified pBK-CMV vector (Stratagene), which contains the Myc tag
sequence at the NheI/XbaI site. These constructs
were transiently transfected into COS-7 cells by the calcium phosphate method, and 48 h after transfection, nuclear extracts were
prepared as described previously (10). Nuclear extracts were then
incubated with anti-HA monoclonal antibody 12CA5 in NTEN binding buffer (150 mM NaCl, 50 mM Tris (pH 7.5), 0.5 mM EDTA, 0.5% Nonidet P-40, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.25% bovine serum albumin) for 2 h at 4 °C with gentle
rotation, and the immune complex was precipitated with protein
A-Sepharose beads, washed three times with NTEN binding buffer,
resuspended in sample buffer, and subjected to SDS-PAGE. Resolved
proteins were transferred onto Hybond ECL nitrocellulose membrane
(Amersham Pharmacia Biotech) and immunoblotted with anti-Myc polyclonal antibody. Horseradish peroxidase-conjugated anti-rabbit IgG was used as
the secondary antibody, and the immune complex was visualized using the
ECL detection kit (Amersham Pharmacia Biotech).
GST Fusion Protein--
PCR-amplified cDNA fragments
corresponding to full-length CSX1 and CSX1 derivatives were subcloned
in frame into the BamHI/EcoRI site of pGEX-2T
(Amersham Pharmacia Biotech). To express GST fusion proteins in
bacteria, JM109 cells were transformed with pGEX-2T-CSX1 constructs,
and fusion protein expression was induced by 0.1 mM isopropyl- GST Pull-down Assay--
In vitro interaction assays
were performed with GST-CSX1 fusion proteins and in vitro
translated GATA-4 derivatives. GST or GST-CSX1 fusion proteins
immobilized on glutathione-Sepharose 4B beads and in vitro
translated GATA-4 derivatives labeled with [35S]methionine were mixed in NTEN binding buffer,
incubated for 2 h at 4 °C with gentle rotation, and washed
three times with binding buffer. Beads were resuspended in SDS-PAGE
sample buffer, and GATA-4 derivatives bound to GST-CSX1 fusion proteins
were resolved by SDS-PAGE and visualized by fluorography.
Csx/Nkx-2.5 Transactivates the ANP Promoter--
To search for the
direct downstream target genes of the Csx/Nkx-2.5 protein, we
cotransfected luciferase reporter constructs containing various
cardiac-specific gene promoters and the Csx/Nkx-2.5 expression vector
into COS-7 cells and examined whether the transcriptional activation of
native promoters was observed by the overexpression of Csx/Nkx-2.5.
Among the reporter constructs tested, Csx/Nkx-2.5 most strongly
transactivated the ANP-luc reporter containing the 600-bp 5'-flanking
region of the rat ANP gene (Fig. 1),
suggesting that the ANP gene is a downstream target of Csx/Nkx-2.5.
The Csx/Nkx-2.5-binding Site (NKE2) Located at
Because the consensus Csx/Nkx-2.5-binding element (TGAAGTG) was located
at NKE2 Is a High Affinity Binding Site for Csx/Nkx-2.5 and Is Highly
Conserved in Evolution--
To examine whether Csx/Nkx-2.5 binds to
NKE2, EMSA was performed with in vitro translated
Csx/Nkx-2.5 protein and oligonucleotide probes corresponding to the
TTF-1-binding element, wild-type NKE2, mutant NKE2, and wild-type NKE.
Under our experimental conditions, the Csx/Nkx-2.5 protein bound to
NKE2 with almost equal binding affinity compared with the TTF-1-binding
element, although it bound to NKE with much lower binding affinity and
did not bind to mutant NKE2 (Fig. 2, B, right;
and C, right). Comparison of the sequences of the
human, mouse, and rat ANP promoters revealed that NKE2 is highly
conserved in these species (Fig. 2C, left). Together, these results strongly suggest that NKE2, as well as proximal
NKE, plays a critical role in the transcriptional regulation of the ANP
gene by Csx/Nkx-2.5.
Csx/Nkx-2.5 and GATA-4 Synergistically Transactivate the ANP
Promoter--
Because the GATA site was located at Csx/Nkx-2.5-binding Sites, but Not GATA Sites, Are Required for
Csx/Nkx-2.5-GATA-4 Positive Cooperation--
The apparent
cooperativity of Csx/Nkx-2.5 and GATA-4 was further investigated using
deletion and mutant constructs of the ANP promoter. Removal of the
region between
The dependence of Csx/Nkx-2.5-GATA-4 transcriptional synergism on the
Csx/Nkx-2.5-binding site was also investigated with the artificial
reporter construct containing multimerized Csx/Nkx-2.5-binding sites
(4x(TTF-1)-tk-luc). Cotransfection experiments revealed that
4x(TTF-1)-tk-luc is synergistically transactivated by Csx/Nkx-2.5 and
GATA-4 (Fig. 4). All these results
collectively suggest that the Csx/Nkx-2.5-binding site is essential,
but that the GATA-4-binding site is dispensable, for the
transcriptional synergy between Csx/Nkx-2.5 and GATA-4.
Csx/Nkx-2.5 and GATA-4 Exhibit Negative Cooperation on
GATA-4-dependent Promoters--
To further investigate the
Csx/Nkx-2.5-GATA-4 transcriptional cooperation, we examined the effects
of the coexpression of Csx/Nkx-2.5 and GATA-4 on
GATA-4-dependent promoters. For this purpose, we utilized
the artificial promoter that contains multimerized GATA sites
(6x(GATA)-tk-luc) and the IL-5 promoter that has been shown to be
positively regulated by GATA-4 and does not contain the putative
Csx/Nkx-2.5-binding sequence in the proximal promoter region. As
expected, when GATA-4 was cotransfected with the 6x(GATA)-tk-luc reporter gene, modest transcriptional activation was observed, whereas
Csx/Nkx-2.5 did not affect the activity of the reporter gene.
Unexpectedly, however, cotransfection of Csx/Nkx-2.5 with GATA-4
partially inhibited the GATA-4-induced transactivation of the reporter
gene (Fig. 5, left).
Essentially the same results were obtained when the IL-5
promoter-containing reporter gene was examined (Fig. 5,
right). These results suggest that the transcriptional cooperation between Csx/Nkx-2.5 and GATA-4 is promoter
context-dependent; these two factors exhibit positive
cooperation on Csx/Nkx-2.5-dependent promoters and negative
cooperation on GATA-4-dependent promoters.
Csx/Nkx-2.5 and GATA-4 Interact with Each Other Both in Vivo and in
Vitro--
To examine whether Csx/Nkx-2.5 and GATA-4 directly interact
with each other in vivo, co-immunoprecipitation experiments
were performed. COS-7 cells were transiently transfected with HA-tagged Csx/Nkx-2.5 and Myc-tagged GATA-4, and nuclear extracts were
immunoprecipitated with anti-HA antibody, followed by immunoblotting
with anti-Myc antibody. Western blot analysis revealed that Myc-GATA-4
protein was co-immunoprecipitated with HA-Csx/Nkx-2.5 (Fig.
6A), indicating the
interaction of HA-Csx/Nkx-2.5 and Myc-GATA-4 in vivo, either direct or indirect.
To further confirm the direct interaction of these two factors, GST
pull-down assay was performed with GST-Csx/Nkx-2.5 derivatives and
in vitro translated GATA-4 derivatives. Full-length
GST-Csx/Nkx-2.5 immobilized on glutathione-Sepharose beads could retain
in vitro translated GATA-4, whereas GST protein could not
bind to GATA-4, indicating that Csx/Nkx-2.5 directly interacts with
GATA-4 in vitro (Fig. 6B). Examination of the
binding of GATA-4 derivatives to full-length GST-Csx/Nkx-2.5 revealed
that the zinc-finger domain of GATA-4 (GATA-4(ZF)) is sufficient for
interacting with Csx/Nkx-2.5. Since GATA-4(ZF) bound to
GST-Csx/Nkx-2.5(HD), the homeodomain of Csx/Nkx-2.5 is sufficient for
interacting with GATA-4 (Fig. 6C).
Csx/Nkx-2.5 Reduces the DNA-binding Affinity of GATA-4--
To
determine the mechanism of positive and negative cooperation of
Csx/Nkx-2.5 and GATA-4 on Csx/Nkx-2.5- and GATA-4-dependent promoters, respectively, the DNA-binding activities of Csx/Nkx-2.5 and
GATA-4 were examined in the presence or absence of each other. EMSA
revealed that the DNA-binding activity of Csx/Nkx-2.5 was not affected
by GATA-4 (Fig. 7, left),
whereas that of GATA-4 was reduced when the Csx/Nkx-2.5 protein
coexisted (right). These results suggest that the negative
cooperation of Csx/Nkx-2.5 and GATA-4 on GATA-4-dependent
promoters is at least in part due to the reduced DNA-binding affinity
of GATA-4 in the presence of Csx/Nkx-2.5, whereas the positive
cooperation of Csx/Nkx-2.5 and GATA-4 on
Csx/Nkx-2.5-dependent promoters is not due to the
cooperative binding of these factors to DNA.
In this study, we have obtained the following results. (i) The ANP
promoter is strongly transactivated by Csx/Nkx-2.5. (ii) High level
transactivation of the ANP promoter by Csx/Nkx-2.5 is dependent on
NKE2, a Csx/Nkx-2.5-binding element located at The ANP Promoter Is Transactivated by Csx/Nkx-2.5 via Multiple
cis-Elements--
Our initial objective in this study was to identify
the direct downstream targets of Csx/Nkx-2.5 in the genetic pathway of heart formation. Among the various cardiac-specific promoters tested,
ANP was strongly transactivated by Csx/Nkx-2.5, and high level
transactivation of the ANP promoter by Csx/Nkx-2.5 was mediated by
NKE2, the Csx/Nkx-2.5-binding site located at
ANP is one of the few genes whose promoter has been identified to have
Csx/Nkx-2.5-binding elements and to be transactivated by Csx/Nkx-2.5.
In addition, endogenous ANP mRNA is increased in the hearts of
human Csx/Nkx-2.5-overexpressing mice recently generated in our
laboratory,2 strongly
suggesting that ANP is a direct target gene of Csx/Nkx-2.5 in
vivo. In Drosophila, D-MEF2 is
the only known direct target of tinman (17).
D-MEF2 expression in cardiac cells is positively regulated by tinman, and the transactivation of D-MEF2 by
tinman is mediated via the two tinman-binding elements in the cardiac enhancer located in the 5'-flanking region of the D-MEF2
gene. The tinman-binding elements in the D-MEF2 enhancer and
NKE2 completely match the consensus high affinity binding sequence for
Csx/Nkx-2.5, TNAAGTG (30), whereas two Csx/Nkx-2.5-binding sites in NKE
do not (29). Our EMSA analysis also showed that the binding affinity of
NKE2 is much higher than that of NKE. These results suggest that NKE2
is a critical cis-regulatory element in the induction and
activation of ANP gene.
Csx/Nkx-2.5 and GATA-4 Synergistically Transactivate the ANP
Promoter--
The existence of the GATA site near NKE2 prompted us to
examine the possible cooperative action of Csx/Nkx-2.5 and GATA-4. Although GATA-4 was a relatively weak transactivator of the ANP promoter under our experimental conditions, the simultaneous
overexpression of GATA-4 with Csx/Nkx-2.5 markedly potentiated the
Csx/Nkx-2.5-induced transactivation of the ANP promoter, indicating
that there exists a positive cooperation between these two factors.
While this manuscript was in preparation, Durocher et al.
(29) reported that Csx/Nkx-2.5 could cooperate with GATA-4 to activate
transcription of the ANP promoter. Our results are essentially similar
to theirs, but they differ in two respects. (i) They used a reporter
construct containing the 130-bp 5'-flanking region of the rat ANP
promoter and observed the cooperative transcriptional activation by
Csx/Nkx-2.5 and GATA-4, whereas we could not detect such synergism when
NKE2 at Csx/Nkx-2.5 and GATA-4 Synergistically Transactivate
Csx/Nkx-2.5-dependent Promoters--
The finding that the
positive cooperation of Csx/Nkx-2.5 and GATA-4 on the ANP promoter was
independent of GATA-4-DNA interaction prompted us to examine the
effects of coexpression of these two factors on the
Csx/Nkx-2.5-dependent artificial promoter,
4x(TTF-1)-tk-luc. Cotransfection experiments revealed that Csx/Nkx-2.5
and GATA-4 also exhibited positive cooperation on this promoter,
although GATA-4 alone induced no significant increase in the promoter
activity. Essentially the same results were recently reported by
Sepulveda et al. (31) regarding the cardiac Context-dependent Transcriptional Cooperation of
Csx/Nkx-2.5 and GATA-4: Csx/Nkx-2.5 and GATA-4 Exhibit Negative
Cooperation on GATA-4-dependent Promoters--
If the
direct protein-protein interaction of Csx/Nkx-2.5 and GATA-4 mediates
the transcriptional synergy, one might expect that these two factors
also exhibit positive transcriptional cooperation on
GATA-4-dependent promoters. Unexpectedly, however,
Csx/Nkx-2.5 and GATA-4 displayed negative cooperation on both the
artificial and native promoters that are positively regulated by
GATA-4, but not by Csx/Nkx-2.5. Together with the findings on the ANP promoter and the 4x(TTF-1)-tk-luc reporter gene, these results suggest
that the Csx/Nkx-2.5-GATA-4 transcriptional cooperation depends on the
context of the promoter; their cooperation is positive when the
promoter contains Csx/Nkx-2.5-binding sites irrespective of the
presence or absence of GATA sites and negative when the promoter
contains only GATA sites.
Csx/Nkx-2.5 and GATA-4 Directly Interact with Each Other--
GST
pull-down assay and co-immunoprecipitation experiments demonstrated
that Csx/Nkx-2.5 and GATA-4 directly interact with each other both
in vivo and in vitro via the homeodomain of
Csx/Nkx-2.5 and the zinc-finger domain of GATA-4, which is consistent
with recent reports (31, 32). Several transcription factors have been
shown to associate with Csx/Nkx-2.5, and the domains of the Csx/Nkx-2.5
protein required for the association are different depending on the
interacting partner. For example, the serum response factor (SRF)
associates with Csx/Nkx-2.5 via the MADS box of SRF and the homeodomain
of Csx/Nkx-2.5 and synergistically transactivates the cardiac Possible Mechanisms for Csx/Nkx-2.5-GATA-4 Negative Cooperation on
GATA-4-dependent Promoters: Csx/Nkx-2.5 Reduces the
DNA-binding Affinity of GATA-4--
In our EMSA analysis with in
vitro translated GATA-4 and Csx/Nkx-2.5, we detected a reduced
DNA-binding affinity of GATA-4 in the presence of Csx/Nkx-2.5.
Sepulveda et al. (31) recently reported that GATA-4 slightly
increased the DNA-binding affinity of Csx/Nkx-2.5. However, we could
not detect such an increase in the DNA-binding affinity of Csx/Nkx-2.5
in the presence of GATA-4, although Csx/Nkx-2.5 and GATA-4 might affect
each other's DNA-binding affinity in an in vivo situation.
Our results suggest that the negative cooperation of Csx/Nkx-2.5 and
GATA-4 on GATA-4-dependent promoters is at least in part
due to the decrease in the DNA-binding affinity of GATA-4 in the
presence of Csx/Nkx-2.5.
Possible Mechanisms for Csx/Nkx-2.5-GATA-4 Positive Cooperation on
Csx/Nkx-2.5-dependent Promoters--
One general mechanism
for positive transcriptional synergy is the cooperative binding of
activators to DNA. For example, the homeodomain protein Phox directly
binds to SRF and increases the DNA-binding affinity of SRF (34).
Likewise, Drosophila extradenticle associates with
Ultrabithorax and raises the DNA-binding affinity of the
extradenticle-Ultrabithorax protein complex (35-37), and so is the
case with the vertebrate Pbx and Hox products (38). In these cases,
direct contact between two factors increases their binding affinity for
DNA, which can be detected by EMSA analysis. However, in our
experiments, the degree of the cooperative binding of Csx/Nkx-2.5 to
its target site in the presence of GATA-4 seems to be relatively small
and does not seem to fully explain the positive cooperation observed in
this study.
In another model of transcriptional synergy, the interaction between
two factors induces a conformational change in one of the factors and
enables it to efficiently activate transcription. Two recent reports
suggest that GATA-4 relieves the autorepression of Csx/Nkx-2.5 by the
C-terminal inhibitory domain (29, 31). The interaction of GATA-4 with
Csx/Nkx-2.5 enhances the transcriptional activity of Csx/Nkx-2.5 by
displacing the C-terminal inhibitory domain, and therefore,
C-terminally truncated Csx/Nkx-2.5 does not exhibit synergy with
GATA-4. However, in our hands, the Csx/Nkx-2.5-GATA-4 transcriptional
synergy on NKE2 or on multimerized Csx/Nkx-2.5 sites was still observed
when the C-terminally deleted form of Csx/Nkx-2.5 was used instead of
wild-type Csx/Nkx-2.5 (data not shown), suggesting that there exist
multiple modes of cooperative transactivation mediated by Csx/Nkx-2.5
and GATA-4.
An alternative model is that Csx/Nkx-2.5 and GATA-4 create a specific
protein complex that interacts with the basal transcriptional machinery
more efficiently than the individual factors. In this case, the
"efficient" interaction with the basal transcription complex may
result from simultaneous interactions with different binding sites
between the Csx/Nkx-2.5-GATA-4 protein complex and the basal
transcription complex and/or from the stabilization of the basal
transcription complex by the specific assembly of the multiprotein
complex. Further studies are necessary to determine the mechanism of
positive synergistic action mediated by Csx/Nkx-2.5 and GATA-4.
Context-dependent Transcriptional Cooperation Mediated
by Csx/Nkx-2.5 and GATA-4--
Accumulating evidence suggests that
multiple cis-trans interactions between
sequence-specific transcription factors and factor-binding sites in the
enhancer as well as the protein-protein interactions among
transcription factors and cofactors regulate gene transcription during
development or in response to external stimuli. Furthermore, in some
natural enhancers, changes in the relative positions or orientations of
protein-binding sites within the enhancer lead to the inactivation of
the enhancer activity (39), and some transcription factors act as
activators in one promoter and as repressors in other promoters (40).
Our present results suggest that synergistic action between two
transcription factors also depends on the promoter context (Fig.
8) and that such transcriptional regulatory mechanisms fine-tune gene expression in cardiac myocytes and
regulate the process of heart development.
240
was required for high level transactivation by Csx/Nkx-2.5. We also
found that Csx/Nkx-2.5 and GATA-4 displayed synergistic transcriptional
activation of the ANP promoter, and in contrast to previous reports
(Durocher, D., Charron, F., Warren, R., Schwartz, R. J., and
Nemer, M. (1997) EMBO J. 16, 5687-5696; Lee, Y., Shioi,
T., Kasahara, H., Jobe, S. M., Wiese, R. J., Markham, B., and
Izumo, S (1998) Mol. Cell. Biol. 18, 3120-3129), this synergism was dependent on binding of Csx/Nkx-2.5 to NKE2, but not on
GATA-4-DNA interactions. Although GATA-4 also potentiated the
Csx/Nkx-2.5-induced transactivation of the artificial promoter that
contains multimerized Csx/Nkx-2.5-binding sites, Csx/Nkx-2.5 reduced
the GATA-4-induced transactivation of the GATA-4-dependent promoters. These findings indicate that the cooperative transcriptional regulation mediated by Csx/Nkx-2.5 and GATA-4 is promoter
context-dependent and suggest that the complex
cis-trans interactions may fine-tune gene
expression in cardiac myocytes.
INTRODUCTION
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ABSTRACT
INTRODUCTION
REFERENCES
/
mice are also
embryonic lethal because of the failure of the fusion of cardiac
primordia at the midline due to the ventral folding/fusion defect of
the developing embryo (26, 27). All these results indicate that
Csx/Nkx-2.5, MEF2C, and GATA-4 are essential transcription factors
required for normal heart development and suggest that multiple
transcription factors coordinately regulate the process of cardiogenesis.
250 in the 5'-flanking
region of the rat ANP gene. Because the GATA motif is located near
NKE2, we cotransfected the expression plasmids of Csx/Nkx-2.5 and
GATA-4 simultaneously with the 300-bp ANP promoter-containing reporter gene and found that the ANP promoter is synergistically transactivated by these two factors. Deletion and mutational analyses indicated that
this synergism is dependent on NKE2, but not on the two GATA sites
within this proximal promoter. The artificial promoter construct containing multimerized Csx/Nkx-2.5-binding sequences was also synergistically transactivated by Csx/Nkx-2.5 and GATA-4. However, Csx/Nkx-2.5 and GATA-4 negatively cooperated on the promoters that are
dependent on GATA-4. Co-immunoprecipitation experiments and glutathione
S-transferase (GST) pull-down assay demonstrated that
Csx/Nkx-2.5 and GATA-4 interact with each other both in vivo and in vitro, and electrophoretic mobility shift assay
revealed that the DNA-binding affinity of GATA-4 was reduced in the
presence of Csx/Nkx-2.5, whereas that of Csx/Nkx-2.5 was not affected
by GATA-4. Our present results indicate that the Csx/Nkx-2.5-GATA-4 synergism is differentially regulated depending on the context of the
cis-elements with which the two factors interact and suggest that the complex interactions among transcription factors and target
DNAs fine-tune gene expression in cardiac myocytes and regulate
development of the heart.
MATERIALS AND METHODS
120 and
270) and one Csx/Nkx-2.5-binding element (NKE2,
located at
250) within the ANP(300)-luc construct containing the
300-bp 5'-flanking region of the ANP gene were mutated either alone or
in combination. To introduce mutations into NKE2 and/or the GATA
element at
270, forward primers that contained corresponding
mutations were used for PCR. A two-step PCR method was used to
introduce mutations into the GATA element at
120. PCR products were
digested with SacI and XhoI; subcloned into the
SacI/XhoI site of PGV-B (TOYO INKI), a
promoter-less vector containing the luciferase gene; and verified by
sequencing. The 4x(TTF-1)-tk-luc reporter gene (which contains four
tandem copies of Csx/Nkx-2.5-binding elements), the 6x(GATA)-tk-luc
reporter gene (which contains six tandem copies of GATA-4-binding
elements), and a reporter gene containing the proximal 400 bp of the
human IL-5 gene promoter were previously described (10, 28).
gal as an internal
control, and the appropriate amounts of parental expression plasmid
were transfected with the total amount of DNA kept constant at 2.5 µg. Luciferase activities were measured 48 h after transfection with a Berthold Lumat LB9501 luminometer, and differences in
transfection efficiency were corrected relative to the level of
-galactosidase activity. Experiments were repeated at least three
times in triplicate, and representative data are shown.
-globin 5'-untranslated region. Proteins
were transcribed and translated in vitro using the TNT SP6
coupled reticulocyte lysate system (Promega) with or without the
[35S]methionine.
-32P]dCTP using Klenow enzyme.
Labeled probes were incubated with 5 µl of programmed or unprogrammed
reticulocyte lysate and 2 µg of poly(dI-dC) in 20 µl of binding
buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl,
10% glycerol, 0.5 mM dithiothreitol, and 0.05% Nonidet
P-40) for 30 min at room temperature. The protein/DNA mixture was
resolved on 5% polyacrylamide gel in 0.5× Tris borate/EDTA buffer at
4 °C for 2 h at 150 V.
-D-thiogalactopyranoside for 5 h at
25 °C. Cells were harvested, resuspended in phosphate-buffered
saline containing 1% Triton X-100, sonicated, and centrifuged. GST
fusion proteins were purified by binding to glutathione-Sepharose 4B
beads (Amersham Pharmacia Biotech) according to the manufacturer's
directions. The concentrations and purity of the glutathione-bound
GST-CSX1 proteins were estimated by SDS-PAGE and Coomassie Blue staining.
RESULTS
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Fig. 1.
Csx/Nkx-2.5 transactivates the ANP proximal
promoter. COS-7 cells were transiently transfected with the
luciferase reporter construct (0.2 µg/3.5-cm dish) containing the
600-bp 5'-flanking region of the rat ANP gene and the indicated
increasing amounts of the CSX1 expression plasmid. Overexpression of
CSX1 transactivated the ANP promoter in a dose-dependent
manner.
250 Is Required
for High Level Transactivation of the ANP Promoter by
Csx/Nkx-2.5--
To identify the cis-regulatory elements in
the ANP promoter that mediate the transcriptional activation by
Csx/Nkx-2.5, luciferase reporter constructs containing various lengths
of the ANP promoter region were cotransfected with the Csx/Nkx-2.5
expression vector. Deletion of the ANP 5'-flanking region from
600 to
390 did not significantly change the -fold activation of the ANP
promoter by Csx/Nkx-2.5. However, deletion between
390 and
160
markedly reduced the -fold induction of the promoter activity, although further deletion up to
125 had little effect (Fig.
2A), indicating that the
Csx/Nkx-2.5-responsive element is situated between
390 and
160 of
the ANP 5'-flanking region.
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Fig. 2.
The Csx/Nkx-2.5-binding element (NKE2)
located at 250 is a developmentally conserved high
affinity binding sequence for Csx/Nkx-2.5 and mediates high level
transactivation of the ANP promoter by Csx/Nkx-2.5. A,
deletion analysis of the ANP promoter. Luciferase reporter constructs
(0.2 µg/3.5-cm dish) containing various lengths of the ANP promoter
were cotransfected into COS-7 cells with an equal amount of CSX1
expression plasmid. Deletion of the promoter region between
390 and
160 significantly decreased the -fold activation of the ANP promoter
activity by CSX1, and this region contains the consensus
Csx/Nkx-2.5-binding element (NKE2), as shown. B, deletion
and mutational analyses of NKE2. The luciferase reporter constructs
(0.2 µg/3.5-cm dish) shown (left) were transfected with
the CSX1 expression plasmid. The deletion or mutation of NKE2 decreased
the -fold induction of the ANP promoter activity by CSX1. EMSA with
in vitro translated CSX1 protein demonstrated that NKE2 is a
high affinity binding element of the Csx/Nkx-2.5 protein, and mutations
introduced into NKE2 dramatically decreased the binding affinity of
NKE2. C, binding affinity of NKE and NKE2 for the
Csx/Nkx-2.5 protein. The sequence comparison of NKE2 with different
species indicated that this element is identical for the rat, mouse,
and human ANP promoters (right). EMSA with in
vitro translated CSX1 protein revealed that NKE2 has a much higher
binding affinity for the Csx/Nkx-2.5 protein than NKE. wt,
wild-type; mt, mutant.
250 in the ANP promoter, we speculated that this Csx/Nkx-2.5-binding element might mediate the transcriptional activation of the ANP promoter by the Csx/Nkx-2.5 protein. To examine
this possibility, the deletion and mutant constructs shown in Fig.
2B were cotransfected with the expression vector of
Csx/Nkx-2.5. Deletion or mutation of the Csx/Nkx-2.5-binding element
located at
250 in the ANP promoter markedly reduced the -fold
induction of transactivation by Csx/Nkx-2.5, whereas deletion up to
270 of the ANP promoter, which did not affect the Csx/Nkx-2.5-binding element, had little effect on the transactivation by Csx/Nkx-2.5 (Fig.
2B). Because previous reports have shown that Csx/Nkx-2.5 binds to another Nkx-2.5-responsive element called NKE located at
80
of the ANP promoter, we designated the Csx/Nkx-2.5-binding element
located at
250 as NKE2. Our present data suggest that NKE2 is
required for high level transactivation of the ANP promoter by
Csx/Nkx-2.5.
280 just distal
from NKE2, we examined whether Csx/Nkx-2.5 and GATA-4 cooperatively regulate the transcription of the ANP gene. When the ANP(300)-luc construct containing the 300-bp 5'-flanking region of the ANP promoter
was cotransfected with the Csx/Nkx-2.5 expression plasmid alone, modest
transactivation of the ANP promoter was observed. When ANP(300)-luc was
cotransfected with the GATA-4 expression plasmid alone, no significant
increase in the promoter activity was observed, suggesting that GATA-4
is a weak transactivator of the ANP promoter under our assay
conditions. However, overexpression of both GATA-4 and Csx/Nkx-2.5
induced much stronger activity of the ANP promoter than that induced by
the expression of Csx/Nkx-2.5 alone (Fig.
3A, left),
suggesting that Csx/Nkx-2.5 and GATA-4 synergistically transactivate
the ANP promoter.
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Fig. 3.
Csx/Nkx-2.5 and GATA-4 synergistically
transactivate the ANP promoter, and NKE2 is required for the
Csx/Nkx-2.5-GATA-4 synergism. A, analysis of the
effect of deletions in the ANP promoter on Csx/Nkx-2.5-GATA-4
synergism. The luciferase reporter constructs (0.2 µg/3.5-cm dish)
containing the 300-, 270-, and 240-bp 5'-flanking regions of the ANP
promoter were cotransfected with the expression plasmids of CSX1
and/or GATA-4 (0.2 µg each/3.5-cm dish). Deletion of NKE2 diminished
the synergy between Csx/Nkx-2.5 and GATA-4 (right), whereas
deletion of the GATA site alone had no effect on Csx/Nkx-2.5-GATA-4
synergism (middle). B, analysis of the effect of
mutations of NKE2 and the two GATA sites in the ANP proximal promoter
on Csx/Nkx-2.5-GATA-4 synergism. Mutations were introduced into the two
GATA sites and NKE2 either alone or in combination, and the resultant
eight reporter constructs (wild-type (wt) and mutants 1-7
(m1-m7); 0.2 µg/3.5-cm dish) were cotransfected with the
expression plasmids of CSX1 and/or GATA-4 (0.2 µg each/3.5-cm dish).
The results obtained could be divided into two cases, positive for
synergism as observed with the reporter construct containing the 300-bp
ANP promoter (A, left) or negative for synergism
as observed with the reporter construct containing the 240-bp ANP
promoter (A, right), and are therefore shown on
the right as + and , respectively.
300 and
270 of the ANP promoter, which resulted in
the deletion of the GATA site at
280, had little effect on the
Csx/Nkx-2.5-GATA-4 synergism as well as Csx/Nkx-2.5-induced activation
(Fig. 3A, middle). However, deletion of the
promoter region between
270 and
240, in which NKE2 is located,
abolished not only Csx/Nkx-2.5-induced activation of the ANP promoter,
but also the cooperativity between Csx/Nkx-2.5 and GATA-4 (Fig.
3A, right), suggesting that NKE2, but not the GATA site at
280, is required for the Csx/Nkx-2.5-GATA-4
transcriptional synergism. To test whether NKE2 (but not GATA elements)
is important for the Csx/Nkx-2.5-GATA-4 synergism, mutations were
introduced into NKE2 and/or two GATA sites within the ANP(300)-luc
construct either alone or in combination, and the effects of
coexpression of Csx/Nkx-2.5 and GATA-4 on these reporter genes were
examined. Cotransfection of the expression plasmids of Csx/Nkx-2.5 and
GATA-4 with wild-type or mutant ANP(300)-luc constructs revealed
that NKE2, but not the two GATA sites, was required for the synergistic transactivation of the ANP promoter by the two factors (Fig.
3B). For example, even when the two GATA sites were mutated,
Csx/Nkx-2.5-GATA-4 synergism was still observed if NKE2 remained intact
(Fig. 3B, ANP(300-m5-)-luc), whereas
Csx/Nkx-2.5-GATA-4 synergism was abolished by the mutation of NKE2
irrespective of the presence or absence of the intact GATA sites
(ANP(300-m2-)-luc, ANP(300-m3-)-luc, ANP(300-m6-)-luc, and ANP(300-m7-)-luc).
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Fig. 4.
Csx/Nkx-2.5 and GATA-4 synergistically
transactivate the Csx/Nkx-2.5-dependent promoters.
COS-7 cells were cotransfected with the luciferase reporter construct
(0.6 µg/3.5-cm dish) containing multimerized Csx/Nkx-2.5-binding
sites (4x(TTF-1)-tk-luc) and the expression plasmids of CSX1 and/or
GATA-4 (0.2 µg each/3.5-cm dish). Csx/Nkx-2.5 and GATA-4
synergistically transactivated the reporter construct, whereas GATA-4
alone had little effect.
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Fig. 5.
Csx/Nkx-2.5 and GATA-4 exhibit negative
cooperation on GATA-4-dependent promoters. COS-7 cells
were cotransfected with the luciferase reporter construct (0.6 µg/3.5-cm dish) containing multimerized GATA sites (left)
or the 400-bp proximal promoter region of the human IL-5 gene
(right) and the expression plasmids of CSX1 and/or GATA-4
(0.2 µg each/3.5-cm dish). In both cases, coexpression of CSX1 with
GATA-4 reduced the -fold activation of the reporter gene induced by
GATA-4 alone, indicating that there exists negative cooperation between
Csx/Nkx-2.5 and GATA-4 on GATA-4-dependent promoters.
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Fig. 6.
Csx/Nkx-2.5 and GATA-4 interact with each
other. A, co-immunoprecipitation. COS-7 cells were
transiently transfected with the expression plasmids of HA-tagged CSX1
and Myc-tagged GATA-4. Nuclear extracts were prepared,
immunoprecipitated (IP) with anti-HA antibody, subjected to
SDS-PAGE, and immunoblotted with anti-Myc antibody. The
arrow indicates the co-immunoprecipitated GATA-4 protein.
B, GST pull-down assay. In vitro translated
GATA-4 protein labeled with 35S was incubated with GST
alone or GST-CSX1 immobilized on glutathione-Sepharose beads, and
bound proteins were analyzed by SDS-PAGE and fluorography. The
arrow indicates the GATA-4 protein. C, schematic
illustration of GST-CSX1 derivatives and GATA-4 derivatives used in the
GST pull-down assay. GST-CSX(HD) interacted with GATA-4(ZF), indicating
that CSX1 and GATA-4 interact with each other via the homeodomain of
CSX1 and the zinc-finger domain of GATA-4.
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Fig. 7.
Csx/Nkx-2.5 reduces the DNA-binding affinity
of GATA-4. EMSA was performed with in vitro translated
CSX1 protein and GATA-4 protein. When NKE2 was used as a probe, the
binding affinity of the CSX1 protein was not affected by the presence
of the GATA-4 protein in the reaction mixture (left). On the
other hand, when the GATA element was used as a probe, the binding
affinity of the GATA-4 protein for the GATA element was reduced by the
presence of the CSX1 protein (right). NS
represents the nonspecific binding of the GATA probe to the protein
contained in the reticulocyte lysate.
DISCUSSION
250. (iii) NKE2 is a
high affinity binding site for Csx/Nkx-2.5 and is highly conserved in
evolution. (iv) Csx/Nkx-2.5 and GATA-4 synergistically transactivate
the ANP promoter. (v) Cooperative transcriptional activation of the ANP
promoter by Csx/Nkx-2.5 and GATA-4 is mediated by NKE2, but is
independent of GATA-4-DNA interaction. (vi) Cooperative transactivation
by Csx/Nkx-2.5 and GATA-4 is also observed on the artificial
promoter containing multimerized Csx/Nkx-2.5-binding sites. (vii)
Csx/Nkx-2.5 and GATA-4 exhibit negative cooperation on the
GATA-4-dependent promoters such as the human IL-5 gene or
the artificial promoter containing multimerized GATA sites. (viii)
Csx/Nkx-2.5 and GATA-4 directly interact with each other both in
vivo and in vitro, and the homeodomain of Csx/Nkx-2.5
and the zinc-finger domain of GATA-4 are required for the interaction.
(viii) Csx/Nkx-2.5 reduces the DNA-binding affinity of GATA-4.
250 in the rat ANP
promoter. Recently, other investigators have shown that Csx/Nkx-2.5 transactivates the ANP promoter via NKE, the proximal
Csx/Nkx-2.5-binding element located at
80 (29). Consistent with their
results, modest transcriptional activation of the ANP promoter by
Csx/Nkx-2.5 was observed when NKE2 was deleted or mutated. Together,
these results suggest that the activity of the ANP promoter is
regulated by Csx/Nkx-2.5 via multiple cis-elements.
250 was deleted or mutated; and (ii) they showed that DNA
binding of the two factors is required for synergistic transcriptional activation, whereas we could detect the cooperative action even when
GATA-4 sites were deleted or mutated. Although the reasons for such
discrepancies are unclear at present, they may be due to the
differences in the cell types or the reporter and effector constructs
used in the transfection experiments.
-actin
promoter, which has no functional GATA elements and is not
significantly activated by overexpression of GATA-4. These results
suggest that the Csx/Nkx-2.5-GATA-4 synergism depends on
Csx/Nkx-2.5-DNA interaction and Csx/Nkx-2.5-GATA-4 association, but not
on GATA-4-DNA interaction.
-actin
gene promoter, and this cooperative action of SRF and Csx/Nkx-2.5
requires SRF-DNA interaction, but is independent of Csx/Nkx-2.5-DNA
interaction (33). Our previous study (10) suggested that the N-terminal
domain of human Csx/Nkx-2.5 interacts with other nuclear factor(s) and
mediates the transactivation activity without directly binding to DNA.
Furthermore, we have recently isolated a multiple zinc
finger-containing putative transcription factor that interacts with the
N-terminal domain and the homeodomain of Csx/Nkx-2.5 using a yeast
two-hybrid interaction
screen.3 Taken together,
these results suggest that the transcriptional regulation by
Csx/Nkx-2.5 may be controlled by multiple protein-protein interactions
mediated by distinct interaction domains of Csx/Nkx-2.5.
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Fig. 8.
Several modes of transcriptional
cooperativity between Csx/Nkx-2.5 and GATA-4. A, when
the promoter contains both NKE and GATA elements, Csx/Nkx-2.5 and
GATA-4 exhibit positive transcriptional cooperation. In this case, the
interaction between GATA-4 and DNA may be either direct or indirect.
B, when the promoter contains only NKE elements, Csx/Nkx-2.5
and GATA-4 also exhibit positive transcriptional cooperation. In this
case, the interaction between GATA-4 and DNA is indirect and possibly
mediated by the protein-protein interaction between Csx/Nkx-2.5 and
GATA-4. C, when the promoter contains only GATA elements,
Csx/Nkx-2.5 and GATA-4 exhibit negative transcriptional cooperation.
Coexistence of Csx/Nkx-2.5 and GATA-4 reduces the GATA-4-induced
promoter activity, possibly due to the reduced DNA-binding affinity of
GATA-4 in the presence of Csx/Nkx-2.5.
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ACKNOWLEDGEMENTS |
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We thank Chika Masuo and Kaoru Kuwabara for technical assistance and Hisamaru Hirai and Tetsuya Yamagata for plasmids.
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FOOTNOTES |
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* This work was supported in part by grants from the Japanese Ministry of Education, Science, and Culture; the Japan Heart Foundation; Yamanouchi Pharmaceuticals; and Tanabe Medical Frontier Conference.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.
Supported by the Sankyo Foundation of Life Science.
§ To whom correspondence should be addressed. Tel.: 81-3-3815-5411; Fax: 81-3-3818-6673; E-mail: komuro-tky{at}umin.ac.jp.
2 E. Takimoto, I. Komuro, I. Shiojima, and T. Mizuno, unpublished observations.
3 Y. Hiroi, S. Kudoh, I. Shiojima, and I. Komuro, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: ANP, atrial natriuretic peptide; bp, base pair(s); IL-5, interleukin-5; GST, glutathione S-transferase; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; SRF, serum response factor.
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REFERENCES |
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