From the Laboratoire de Développement et
Différenciation Cardiaques, Institut de Recherches Cliniques de
Montréal and ¶ Département de Pharmacologie,
Université de Montréal, 110 des Pins Ouest, Montréal
QC, H2W 1R7, Canada and § Department of Medicine, Division
of Experimental Medicine, McGill University, Montréal QC, H3A
1A3, Canada
Received for publication, January 10, 2001
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ABSTRACT |
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YY1, a multifunctional protein essential
for embryonic development, is a known repressor or activator of
transcription. In cardiac and skeletal myocytes, YY1 has been described
essentially as a negative regulator of muscle-specific genes. In this
study, we report that YY1 is a transcriptional activator of the B-type natriuretic peptide (BNP) gene, which encodes one of the heart major
secretory products. YY1 binds an element within the proximal cardiac
BNP promoter, in close proximity to the high affinity binding sites for
the zinc finger GATA proteins. We show that YY1 cooperates with GATA-4
to synergistically activate BNP transcription. Structure-function
analysis revealed that the DNA binding domain of YY1 is sufficient for
cooperative interaction with GATA-4, likely through corecruitment of
the CREB-binding protein coactivator. The results suggest that
YY1 and GATA factors are components of transcriptionally active
complexes present in cardiac and other GATA-containing cells.
The B-type natriuretic peptide
(BNP)1 is a peptide hormone
that is synthesized and secreted from the heart and plays an important role in cardiovascular homeostasis (reviewed in Ref. 1). We have
previously demonstrated that the proximal 114 bp of the rat BNP
promoter are sufficient for maximal BNP expression in cardiomyocytes (1). Deletion analysis established three regulatory regions within this
proximal promoter that are required for maximal transcriptional activity. Further analysis of one of these elements, which contains a
binding site for the GATA family of zinc finger proteins, led to the
isolation and characterization of transcription factor GATA-4 (1). A
cardiac-restricted member of the GATA family of proteins, GATA-4, was
since shown to be a key regulator of several cardiac genes (reviewed in
Ref. 2) and an essential factor for heart development (3-5). A 5'
deletion that removed the GATA motifs resulted in a 3- to 4-fold
decrease in promoter activity; additionally, removal of a 20-bp element
( YY1 is a 65-kDa multifunctional zinc finger DNA binding transcription
factor, belonging to the human GLI-Krüppel family of nuclear proteins (6) and essential for mammalian embryonic development
(7). Identified as an initiator binding protein (6, 8), it can either
activate or inhibit transcription depending on the promoter context
(9-12). A potent transcriptional repressor, YY1 has been associated
with the repression of a variety of cellular and viral genes including
c-fos (13), The mechanisms by which YY1 mediates its pleiotropic responses remain
undefined although it is clear that the promoter context and the
cellular environment, including coactivators and corepressors, influence YY1 action. The cAMP-response element-binding protein (CBP)
belongs to a class of transcriptional cofactors that link upstream
transactivators and the basal transcription machinery (26-28). In
skeletal muscle, the CBP/p300 proteins act as transcriptional adaptors
for MyoD and MEF-2 in the regulation of myogenesis (27, 29-31). In the
heart, CBP/p300 plays an important role in maintaining the terminally
differentiated state of cardiomyocytes by both preventing reinitiation
of DNA synthesis and activating subsets of cardiac-specific genes (32).
Although the exact transcription factors with which CBP/p300 interact
to regulate cardiac gene expression remain unclear, activity of the
GATA proteins is likely to be modulated by these coactivators given
that CBP cooperates with GATA-1 (33) and p300 was recently shown to act
as a coactivator for GATA-5 in the regulation of the cardiac atrial
natriuretic factor gene (34).
In the present work, we demonstrate that YY1 functions as a
transcriptional activator of the cardiac BNP promoter and we provide evidence that YY1 cooperates with GATA-4 to enhance BNP transcription. This synergy requires GATA but not YY1 DNA binding sites and is mediated via CBP that likely serves as a bridge between the two factors
allowing the formation of a transcriptionally competent complex. These
findings suggest that cardiac-specific genes are controlled by complex
transcriptional pathways involving combinatorial interactions between
cell-restricted (GATA-4) and ubiquitous factors (YY1 and CBP); such
multicomponent complexes likely provide additional levels of gene
expression control.
Plasmids--
Rat BNP luciferase constructs were obtained by
cloning appropriate promoter fragments generated using restriction
enzymes or PCR into PXP2, as previously described (1). Point mutations in the proximal BNP promoter region were generated by PCR, and constructs were confirmed by sequencing. The expression vectors for the
intact and mutated YY1 were generous gifts of Dr. Kenneth Walsh and
Dr. Robert Schwartz and have been previously described (10, 13). The
GATA expression vectors used have been described previously
(35, 36). The expression vector for the human CBP was a gift from Dr.
Robert Rehfuss (McGill University, Montréal, CA).
Cell Cultures and Transfections--
HeLa or CV1 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum. Primary cardiocyte cultures were prepared from
1-4-day Harlan Sprague-Dawley rats and kept in synthetic medium
as previously described (37). 24 h after plating, cells were
transfected using the calcium phosphate technique. Rous sarcoma
virus-human growth hormone was included (2 µg per dish) to normalize
for transfection efficiencies. The total amount of DNA was kept
constant (maximum 10 µg per 35-mm dish) by the addition of PCB6+ or
pBluescript DNA. Cells were harvested, and the medium was collected
36 h after transfection. Luciferase activity was measured with an
Amersham Pharmacia Biotech luminometer, and the human growth
hormone was measured in the cell media by radioimmunoassay as
previously described (38). The results reported were obtained from at
least four independent experiments, each carried out in duplicate using
two different DNA preparations.
Nuclear Extracts and Electrophoretic Mobility Shift
Assays--
Nuclear extracts were prepared from cells according to
Schreiber et al. (39). Extracts from HeLa cells
overexpressing YY1 were prepared from 200,000 cells transfected with 10 µg of expression vectors. The probe for YY1 binding,
GGTCTCCATTTTGAAGCG, corresponds to the adeno-associated virus P5 +1
site and has been previously described (6). The probes used from the
BNP promoter correspond to sequences from
Binding reactions were performed at room temperature for 20 min in a
20-µl reaction mixture containing 3-5 µg of nuclear extracts, 10 mM Tris-HCl, pH 7.9, 60 mM KCl, 5 mM MgCl2, 1 µg poly dI-dC oligonucleotides, 1 mM EDTA, 1 mM
dithiothreitol, and 10% glycerol. Unlabeled double-stranded
oligonucleotides were added as competitors at 100-fold molar excess
when required. Binding reactions were analyzed on a 5% (30:1)
polyacrylamide nondenaturing gel in 0.5× Tris borate-EDTA buffer at 10 volts/cm for 3 h at room temperature. As a control, an
oligonucleotide containing an octomer motif was used to detect OCT-1
binding in the nuclear extracts as described previously (4). The
antibody directed against human SP1 was purchased from Santa Cruz
Biotechnology and that for human YY1 was kindly provided by
Dr. Robert Schwartz (Baylor College of Medicine). Supershift
assays were performed to identify antibody-protein interactions. A
reaction mixture containing the binding buffer, nuclear extracts, and
dI-dC was first incubated for 1 h at 4 °C with the
relevant antibody. The labeled probe was then added, and binding was
carried out for an additional 20 min at room temperature. The
DNA-protein complexes were resolved on a 5% nondenaturing gel as
described above.
YY1 Binds to and Transactivates the BNP Promoter--
Deletion
analysis of the proximal rat BNP promoter indicated that sequences
between
The functional consequences of YY1 binding to the BNP promoter were
tested. Transient cotransfection analysis revealed that full-length YY1
is a potent transactivator of the BNP promoter (Fig.
3). Other related zinc fingers such as
Egr-1 and the KRAB domain containing protein ZNF74 (47) had no effect
on BNP promoter activity (data not shown). YY1 transactivation of the
BNP promoter required the proximal YY1 binding site, because 5' or
internal deletions, as well as a point mutant that removes the YY1
site, significantly reduced transactivation (Fig. 3). Other potential YY1 binding sites are present further upstream (
To delineate the regions of YY1 required for transactivation, the
effects of several deletion mutants of YY1 on the BNP promoter were
assayed (Fig. 4A). The results
indicate that an intact zinc finger DNA binding is essential but not
sufficient for maximal transactivation (Fig. 4B, DM4 and DBD
mutants, respectively). Surprisingly, deletions of the putative
N-terminal activation domains of YY1 did not completely abolish
transactivation as these mutants retained the ability to consistently
induce a 2-fold increase in promoter activity. These observations are
consistent with studies indicating that, although the N-terminal region
contains an autonomous activation domain (amino acids 70-143)
(48), it is often dispensable for activation by YY1. For example,
activation of the c-Myc promoter by YY1 is not affected by deletion of
amino acids 89-220 (DM3) (10), and activation of a heterologous
promoter construct containing the YY1 initiator element of the
adeno-associated P5 promoter was maximal when amino acids
154-199 or 69-85 were deleted (49). Together, these data
suggest that a YY1 protein containing essentially the zinc finger
region, as found in cardiomyocytes, can activate target promoters
possibly through recruitment or interaction with coactivators.
YY1 Cooperates with GATA Transcription Factors to Activate the BNP
Promoter--
The BNP promoter is a target for both GATA-4 and YY1,
and the YY1 site at
Next, we tested whether YY1 binding was required for synergy. For this,
we generated the YY1 mutant in the context of the BNP
Next, we tested whether a truncated YY1 protein can functionally
synergize with GATA-4, and we performed a structure-function analysis
to determine the GATA-4 domains required for YY1 interaction. Wild type
and mutant GATA-4 were epitope-tagged, which allowed assessment of the
amount of protein produced using Western blot analysis (Fig.
6A). These were tested on
full-length YY1 and on the shorter YY1 form consisting of the DNA
binding domain; both YY1 proteins were produced at similar levels as
confirmed by gel shift analysis (Fig. 6B). Either one of the
N or C terminus transactivation domains is sufficient for GATA-4
activation of BNP (Fig. 6C). However, deletion of the C
terminus domain totally abrogated synergy with YY1 whereas removal of
the N-terminal domain slightly but consistently enhanced the synergy
with YY1 (Fig. 6C). Consistent with this, the DNA binding
domain of GATA-4 was not sufficient for YY1 synergy. In contrast, the
YY1 DNA binding domain is sufficient for maximal synergy with GATA-4,
and the YY1 N terminus is dispensable in the presence of GATA-4 (Fig.
6D). Thus, in presence of GATA-4, the endogenous cardiac YY1
protein would be a potent transcriptional activator.
CBP Potentiates Synergy between YY1 and GATA-4 on the BNP
Promoter--
To determine whether the synergy between GATA-4 and YY1
reflects physical interaction between the two proteins, we performed a
series of coimmunoprecipitation experiments using in
vitro-translated YY1 and GATA-4 proteins. No detectable
interaction between GATA-4 and YY1 could be observed, although in the
same experiments, we were able to detect other documented interactions
(2) including that between GATA-4 and GATA-6 proteins (data not shown).
These results suggest that either the interaction between the two
proteins was not stable under these conditions or that it was mediated via a third protein.
Both YY1 and members of the GATA family interact with several other
nuclear proteins (35, 50-53) including some coactivators (33). For
example, it was recently demonstrated that the coactivator protein
CBP/p300, which had been shown to associate with YY1 (54), also
interacts with GATA-5 to regulate transcription of the atrial nutriuretic peptide gene (34). We therefore tested whether CBP was a potential mediator of synergy between YY1 and GATA-4. As shown in
Fig. 7, CBP significantly enhanced
GATA/YY1 activation of the BNP promoter (15-fold compared with 6-fold
without CBP). CBP alone or in combination with either GATA-4 or YY1 had
no significant effect on promoter activity supporting the hypothesis
that its role was to mediate the formation of a transcriptionally
competent complex between YY1 and GATA-4.
In this study we demonstrate the role of YY1 as an important
regulator of BNP transcription and provide evidence for the existence of a novel transcriptional pathway involving the combinatorial action
of YY1, GATA-4, and CBP. YY1 is a complex transcription factor that is
conserved across species. In the heart, YY1 has been previously
characterized as a repressor of cardiac gene expression (22). Our
results demonstrate that YY1 is a potent transactivator of the BNP
promoter and suggest that YY1 may be a positive regulator of BNP and
other cardiac genes.
Despite considerable efforts, we were unable to demonstrate a direct
interaction between YY1 and GATA-4, and we suggest that this
interaction is mediated via the CBP/p300 class of proteins. Alone CBP
has no effect on BNP transcription suggesting that it acts as a
mediator that bridges the two factors. The CBP/p300 proteins contain
intrinsic histone acetyltransferase activity (55) and can modify both
the interactions of GATA-4 or YY1 with DNA or the chromatin structure
itself to facilitate the formation of a transcriptionally competent
complex over the BNP promoter. CBP has been previously demonstrated to
regulate the activity of GATA-1 by acetylation (28).
We have mapped distinct regions of YY1 required for GATA-4 synergy or
activation. The N-terminal domain of YY1 is required for maximal
DNA-dependent BNP activation; however, synergy with GATA-4
renders this domain dispensable, and the zinc finger region of YY1 is
sufficient to mediate functional interaction with GATA-4. This finding
may be very relevant to understanding the mechanisms of YY1 in gene
regulation especially, because the major YY1 isoform present in
cardiomyocytes appears to correspond to a truncated form consisting
essentially of the DNA binding domain. The presence of multiple YY1
isoforms is consistent with published reports showing the presence of
several YY1 mRNA isoforms in different cells and tissues including
the heart (6, 8)2 and
YY1 complexes of varying mobility in nuclear extracts from different
cells (56).
Because YY1 binding is required for basal activation but not for GATA-4
synergy, we speculate that YY1 could have a dual role in BNP regulation
depending on the cellular context. In the presence of high levels of
GATA-4 and CBP, recruitment to a GATA-containing complex via CBP may be
the mechanism of action of YY1. In such a complex, YY1 could act as an
architectural factor that functions to enhance protein-protein
interactions between upstream factors and the basal transcriptional
machinery as has been previously demonstrated on the c-fos
promoter (57). This is consistent with the observation that the distal
and not the In addition to adequate protein levels, GATA/YY1/CBP cooperation may
also necessitate post-translational modifications of one or more
component of the complex. In the absence of complex assembly, YY1 may
still activate the BNP promoter through sequence-specific binding to
its site and via its own activation domains. Productive interaction
among GATA factors, YY1, and CBP may be only observed under stimulated
conditions such as stress or growth response or in a cell-specific
manner, e.g. in cardiac but not brain cells, both
of which express the BNP gene.
Finally, it is noteworthy that both GATA-4 and YY1 have been linked to
growth responses. The role of YY1 in cell growth was confirmed by
inactivation of the YY1 gene in mice (7) as discussed earlier.
Furthermore, YY1 has been implicated in mediating
interleukin-1
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 to
60 bp), which contains putative binding sites for YY1 and
CACC box-binding proteins, led to a further 3-fold loss of BNP promoter
activity in cardiomyocytes (1). We therefore undertook to examine the potential role of the YY1 protein in the regulation of BNP transcription.
-casein (14),
-globin (15), serum
amyloid A1 (16), cytomegalovirus (17), and the mouse mammary leukemia
virus (18). In addition to its role as a repressor, YY1 was shown to
act as an activator of some promoters like the c-Myc (9, 10), the
dihydrofolate reductase (11), the IgH intronic enhancer (19), the
ribosomal gene promoters (20), and more recently, the myeloid-specific
gp91 (21). YY1 binding sites have been identified within the regulatory
regions of several cardiac and skeletal muscle genes, including muscle creatine kinase (22),
-skeletal actin, and cardiac actin (10, 23,
24). The finding that deletion of YY1 sites within these promoters
results in a small, albeit reproducible gain in promoter activity,
together with studies showing that YY1 competed with the action of
serum response factor led to the suggestion that YY1 behaves as a
repressor of cardiac genes (22, 25).
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
84 to
60 bp as
follows: wild type,
TCAGAGATCTCCCCACCCCTACTCCATGAGAAGG; YY1m,
TCAGAGATCTCCCCACCCCTACTCTATGAGAAGG; CACCm,
TCAGAGATCTCCTTAGGCCTACTCCATGAGAAGG.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 and
60 bp contribute significantly to BNP promoter
activity. Their deletion decreases promoter activity by 3- to 4-fold in
atrial primary cardiocyte cultures (Fig.
1A). The
80- to
60-bp
region harbors two potential transcription factor binding sites, a
CCACC box, which can interact with two classes of zinc finger proteins,
SP1, and members of the EKLF-Krüppel family (40-42), and
adjacent to it, a CCAT core, which is a putative binding site for the
YY1 zinc finger protein (43, 44). Gel shift analysis showed that both
SP1 and YY1 interact with the BNP sequences (Fig.
2) as evidenced by competition analysis
and antibody recognition. Incubation of a 20-bp oligonucleotide
corresponding to the
80- to
60-bp region with HeLa cell nuclear
extracts resulted in the formation of three major sequence-specific DNA
binding complexes (Fig. 2A). The complex with the slowest
mobility was identified as an SP1-like protein based on competition
with an unlabeled high affinity SP1 site and its interaction with the SP1 antibody (Fig. 2 and data not shown). The two faster migrating complexes were identified as YY1; both complexes are specifically competed with a consensus YY1 site from the adenovirus-associated P5
promoter and blocked by a YY1 antibody (Fig. 2B). In
addition, they show similar mobility as complexes obtained on the well
studied adenovirus YY1 binding site (Fig. 2C). Binding of
SP1 and YY1 to their cognate juxtaposed sites occurs independently,
because mutation of the YY1 site (YY1m) does not affect SP1 binding.
Similarly, mutation of the SP1 site, which abrogates SP1 binding, does
not qualitatively or quantitatively alter the YY1 complexes (Fig. 2A). YY1 is expressed in cardiomyocytes (22, 24, 45),
and it has been shown to negatively regulate several cardiac promoters including
-skeletal actin (10, 24), cardiac actin (23), and muscle
creatine kinase (22). SP1 levels are very low in postnatal
cardiomyocytes but appear to increase following trophic growth
stimulation (46). When gel shift analyses were carried out using
cardiomyocyte nuclear extracts, the predominant complex observed over
the BNP probe corresponded to YY1 (Fig. 2D).
Moreover, the majority of YY1 binding in cardiomyocytes corresponded to the faster migrating YY1 complex observed in HeLa cells. Based on
YY1 antibody recognition and gel migration properties, this complex
likely represents an alternatively spliced YY1 form containing essentially the DNA binding zinc finger region.
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Fig. 1.
A, structural organization of the
proximal BNP promoter. Regulatory elements in the proximal BNP promoter
are highlighted, and their position relative to the start site is
indicated. rBNP, rat BNP. B, mapping regulatory
domains on the BNP proximal promoter. Primary atrial and ventricular
cardiomyocyte cultures obtained from 4-day-old rats were transiently
transfected with BNP-luciferase constructs containing the indicated BNP
promoter. The results are expressed relative to BNP-114 and represent
the mean of three independent experiments carried out in duplicate. The
Rous sarcoma virus-human growth hormone plasmid was used as an internal
control.
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Fig. 2.
Identification of sequence-specific
DNA-binding proteins that interact with the proximal BNP element.
A, three different radiolabeled 20-bp oligonucleotides
corresponding to the sequence between 80 and
60 bp (WT
BNP) and others that contain mutations within the putative YY1
site or the CACC site were incubated with HeLa cell nuclear extracts as
described under "Experimental Procedures." The sequence-specific
binding species obtained were competed with a 100-fold molar excess of
cold competitor oligonucleotide that corresponds to a consensus YY1
site found in the adenovirus associated P+5 (adeno-YY1)
promoter. B, supershift analysis using anti-YY1 or anti-SP1
on the wild type BNP probe revealed that the complex with the slowest
mobility contained an SP1-like species. The two complexes with faster
mobility correspond to YY1 as they are completely abrogated in the
presence of YY1 antibody. C, the oligonucleotide containing
the adenovirus consensus YY1 site was used as probe. Incubation with
HeLa nuclear extracts revealed the presence of two sequence-specific
complexes that were similar in mobility to those obtained on the BNP
wild type probe and correspond to YY1 as confirmed by incubation with
the YY1 antibody. Note that the SP1 antibody had no effect on YY1
binding. D, predominance of the fastest mobility YY1 complex
in cardiac extracts prepared from neonate ventricular cardiomyocyte
cultures. A similar pattern is observed in neonate atrial
cardiomyocytes and in adult heart extracts. Also note the absence of
SP1 binding in cardiac extracts.
209,
220,
374, and
636), which might explain why the
2.2-kilobase pair BNP construct is maximally induced by YY1.
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Fig. 3.
YY1 transactivates the BNP promoter. The
full-length 2200-bp BNP-luciferase (Luc) plasmid, along
with several deletions and constructs containing point mutations, were
transiently cotransfected with 1 µg of cmv-YY1 expression vector. The
reporters were maintained at 6 µg per dish, and the total amount of
DNA was kept constant at 10 µg by the addition of empty vector DNA.
For each construct the results (Fold Activation) correspond
to the change in activity in the presence of YY1 relative to the
control YY1 antisense vector. The data shown represent a mean of four
to six independent experiments each carried out in duplicate.
rBNP, rat BNP.
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Fig. 4.
Structure-function analysis of YY1. To
map the region of YY1 implicated in the transactivation of BNP, 3 µg
of the full-length BNP-2200-bp luciferase construct were cotransfected
with 5 µg of full-length wild type (WT) or various
deletions of YY1, all cloned in the same EMSV-driven expression
vector (10) and depicted in A. Transfections were carried
out in HeLa or CV1 cells. The results shown in B are from
HeLa cells and represent the mean of two independent experiments each
carried out in duplicate.
70 is flanked by functional GATA elements
centered at
90 and
30 (1). We tested whether the two factors
functionally cooperate. Cotransfection of YY1 with limiting amounts of
GATA-4 resulted in synergistic activation of the BNP promoter.
Activation with both YY1 and GATA-4 (10- to 15-fold) was systematically
greater than the additive effect of each factor alone (6- to 7-fold), suggesting that the two proteins cooperate over the BNP promoter (Fig.
5A). Interestingly, YY1 could
also cooperate with other members of the GATA family including the
hemopoietic GATA-1 and -3 and the cardiac GATA-6 proteins suggesting
that GATA-YY1 interaction may be relevant to many cells (Fig.
5A). Removal of the upstream GATA elements (BNP
80)
abolished the YY1/GATA-4 synergy. (Fig. 5A). Activation by
either factor was retained because of the presence of the
60 YY1 site
and the
30 GATA site, but the addition of both YY1 and GATA-4 gave at
best an additive effect. These results indicate that the
80
GATA-containing enhancer is essential for synergy with YY1 and suggest
that binding of GATA-4 (or -6) to the more proximal GATA element (which
is a specialized TATA box) cannot support YY1 cooperation.
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Fig. 5.
YY1 can transcriptionally cooperate with
members of the GATA family. A, cotransfection assays
were performed in HeLa cells using 1 µg of GATA expression vectors in
the presence or absence of 1 µg of cmv-YY1. The reporter BNP
luciferase constructs containing either 114 or
80 bp (
114
BNP and
80 BNP) were maintained at 4 µg per dish.
The data are expressed as -fold activation and are from one
representative experiment performed in duplicate. B, synergy
with GATA-4 does not require DNA binding by YY1. Luciferase reporters,
which contain the wild type BNP sequences (
114 BNP WT) or
a point mutation in the YY1 binding site (
114 BNP mut),
were cotransfected with 1 µg/dish of each YY1 and GATA-4. The results
shown are a mean of two independent experiments each carried out in
duplicate.
114 promoter.
As shown in Fig. 5B, this construct was no longer activated
by YY1 but retained GATA-4 inducibility. Remarkably, the YY1 mutation
did not alter the ability of YY1 to cooperate with GATA-4 and activate
the BNP promoter to nearly maximal levels (Fig. 5B). Thus
the binding of GATA-4 to DNA appears to be sufficient to recruit YY1
into a transcriptionally productive complex.
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Fig. 6.
Domains of GATA-4 required for activation and
synergy. A, 6 µg of the 114-bp BNP-luciferase
construct was cotransfected with wild type or mutant GATA-4 (1 µg/dish) expression vectors in the absence or presence of the cmv-YY1
vector. The results are expressed as -fold activation of the BNP
promoter relative to the control antisense GATA-4 vector and represent
a mean of three independent experiments each carried out in duplicate.
B, the same BNP-luciferase construct was cotransfected with
the various YY1 expression vectors (1 µg/dish) in the presence or
absence of the GATA-4 vector. The results shown in A and
B are the mean of two independent experiments each carried
out in duplicate. Note that removal of the GATA-4 C-terminal activation
domain eliminates synergy and that the DNA binding domain of YY1 is
sufficient for synergy. C, schematic representation of the
GATA-4 constructs used. The two black squares represent the
two zinc fingers. All GATA-4 proteins were epitope-tagged as described
under "Experimental Procedures" and were produced in HeLa or CV1
cells at similar levels as evidenced by Western blotting with the
hemagglutinin antibody. Ctl are control extracts
prepared from cells transfected with the empty vector. D,
gel shift analysis using nuclear extracts prepared from HeLa cells
transfected with empty vector (Control), wild type YY1, or a
YY1 deletion containing only the DNA binding zinc finger domain (shown
in Fig. 4A). Note that both protein forms are expressed at
similar levels.
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Fig. 7.
CBP enhances GATA-4/YY1 synergy.
Limiting amounts (10 ng each) of expression vectors containing
cDNAs for CBP, GATA-4, and the DBD of YY1 were cotransfected in
HeLa or CV1 cells with 1 µg of the 2.2-kilobase pair BNP-luciferase
vector. The results depicted are from a representative experiment in
CV1 cells carried out in duplicate. Similar results were obtained in
two other independent experiments.
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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30-bp GATA element supports synergy.
-induced hypertrophy in cardiomyocytes (58). More
recently, YY1 has also been implicated in endoplasmic reticulum
stress-induced transcription (59); whether in cardiomyocytes YY1 is
involved with GATA-4 in mediating transcriptional regulation in
response to stress or growth stimulation deserves to be investigated.
On the other hand, GATA-4 has been implicated in the activation of
several genes during hypertrophy, including the angiotensin II type 1a receptor and the
-myosin heavy chain genes in response to pressure overload hypertrophy in rats (32, 60, 61). The role of GATA-4 in the
hypertrophic response may reside, in part, in GATA-4 association with
other factors including NFAT-3 (62) or the MADS protein Mef2 and serum response factor (50, 63).
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ACKNOWLEDGEMENTS |
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We thank Frédéric Charron for help with the immunoprecipitation experiments and for carrying out the Western blot shown in this manuscript. We are grateful to Dr. Robert Schwartz for the YY1 expression vectors, Georges Nemer for critical reading of the manuscript, M. Chamberland for technical assistance, L. Laroche for secretarial help, and members of the Nemer laboratory for helpful discussions.
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FOOTNOTES |
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* This work was supported by the Canadian Institutes of Health Research (CIHR; MT-13057). M. N. is a CIHR Senior Scientist.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratoire de
Développement et Différenciation Cardiaques, Institut de
Recherches Cliniques de Montréal, 110 des Pins Ouest,
Montréal QC, H2W 1R7 Canada. Tel.: 514-987-5680; Fax:
514-987-5575; E-mail: nemerm@ircm.qc.ca.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M100208200
2 Unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: BNP, B-type natriuretic peptide; bp, base pair(s); CBP, cAMP-response element-binding protein.
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