Functional Interaction of NF-Y and Sp1 Is Required for Type A
Natriuretic Peptide Receptor Gene Transcription*
Faquan
Liang,
Fred
Schaufele, and
David G.
Gardner
From the Metabolic Research Unit and Department of Medicine,
University of California, San Francisco, California 94143-0540
Received for publication, July 18, 2000, and in revised form, September 13, 2000
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ABSTRACT |
The vasorelaxant and anti-mitogenic activities of
the atrial and brain natriuretic peptides depend upon their binding to
the type A natriuretic peptide receptor (NPR-A) expressed on the
surface of vascular cells. Intervention strategies aimed at controlling NPR-A expression are limited by the paucity of studies in this area.
Here we identify a sequence CCAAT between
141 and
137 of the NPR-A
promoter that, when mutated, reduces promoter activity by 90% in rat
aortic smooth muscle (RASM) cells. Protein/DNA cross-linking and
immunoperturbation of electrophoretically shifted complexes formed
between RASM nuclear extracts and an oligonucleotide surrounding the
CCAAT sequence indicates that the heterotrimeric transcription factor
NF-Y binds specifically to the wild-type, but not mutated, CCAAT
element. Cotransfection of a dominant negative mutant of the NF-YA
subunit results in a concentration-dependent decrease in
the activity of the NPR-A promoter in RASM cells confirming that
endogenous NF-Y is an activator of the promoter. Mutation of the CCAAT
element, in conjunction with mutation of all three Sp1 sites previously
shown to be involved in NPR-A promoter regulation, virtually eliminates
NPR-A promoter activity in RASM cells. Coexpression of all three NF-Y
subunits together with Sp1 in Drosophila cells deficient in
these factors indicates that NF-Y and Sp1 act synergistically to
reconstitute NPR-A promoter activity. A direct physical association between NF-Y and Sp1 can be demonstrated both in vitro by
glutathione S-transferase pull-down assay and in the intact
cell by coimmunoprecipitation and functional studies. Together, these
studies show that NPR-A promoter activity is dominantly regulated
through functional, and possibly physical, interactions of NF-Y and Sp1.
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INTRODUCTION |
The natriuretic peptides are a family of vasoactive hormones that
play an important role in the regulation of blood pressure and
cardiovascular homeostasis (1). Atrial natriuretic peptide and brain
natriuretic peptide are both produced predominantly in the heart and
circulate in plasma. Their natriuretic, diuretic, and vasorelaxant
activities are mediated through the type A natriuretic peptide receptor
(NPR-A)1 (2) (also known as
guanylyl cyclase A) present on the surface of vascular smooth muscle
and other cells. Recent studies by two independent groups (3, 4) showed
that complete absence of NPR-A in mice leads to hypertension, cardiac
hypertrophy, and sudden death, indicating a critical role for NPR-A in
the regulation of cardiovascular homeostasis.
The molecular regulation of NPR-A gene transcription is only poorly
understood. The rat NPR-A gene has been cloned and sequenced. Sequence
analysis identified very few sequence elements for known transcription
factors. However, three putative Sp1 consensus binding sites
(positioned between
341 and
51) and a CCAAT motif (positioned at
137) were present upstream from the promoter of the NPR-A gene (5).
Our earlier studies (6) demonstrated a critical role for the Sp1 family
of transcription factors in regulating NPR-A gene transcription in rat
aortic smooth muscle (RASM) cells, but the role of the CCAAT sequence
remains unknown.
The CCAAT box is present in a number of eukaryotic promoters (7-10)
and has been demonstrated to be important for the transcription of many
of those genes (7-10). In relatively simple, TATA-less promoters,
which, like the NPR-A promoter, contain only one or two additional
cis-acting elements, the CCAAT box is absolutely required for
regulating gene transcription (10-12). In contrast, the CCAAT box is
somewhat less critical for TATA-containing promoters (10-12).
Typically, the CCAAT element is found as a single copy in the forward
or reverse orientation immediately upstream of the transcription start
site. In the TATA-less NPR-A promoter, the CCAAT sequence is positioned
in the reverse orientation between
137 and
141 upstream of the
transcription start site. In the present study, we show that the CCAAT
box is important for NPR-A gene transcription. We have identified the
nuclear proteins that associate with the CCAAT box as the
heterotrimeric NF-Y complex, and we have demonstrated that functional,
and possibly physical, interaction of NF-Y with Sp1 is essential for
optimal transcription of the NPR-A promoter in vascular smooth muscle cells.
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EXPERIMENTAL PROCEDURES |
Materials--
Rabbit polyclonal antibodies directed against
CCAAT/enhancer-binding protein (C/EBP)
, C/EBP
, and C/EBP
, and
goat polyclonal antibody directed against Sp1 were a kind gift from S. McKnight (University of Texas, Southwestern Medical Center, Dallas,
TX). Mouse monoclonal antibody directed against nuclear factor-Y (NF-Y) A was purchased from PharMingen (San Diego, CA). Rabbit polyclonal antibody directed against NF-YB was a gift from M. Roberto (University of Milan, Milan, Italy). Poly(dI-dC), glutathione-SepharoseTM 4B, and
T7 Sequenase were purchased from Amersham Pharmacia Biotech. Schneider
cell medium was obtained from Life Technologies, Inc. All
oligonucleotides were synthesized by Cruachem, Inc. Other reagents were
obtained through standard commercial suppliers.
Plasmid Construction and Site-directed Mutagenesis--
The
construction of
387 rat NPR-A luciferase has been described
previously (6). The NF-YA expression plasmids pNF-YA (wild-type) and
pNF-YA29 (dominant negative form) were provided by R. Mantovani (University of Milan, Milan, Italy). pPacSp1 and Copia
-galactosidase were obtained from R. Tjian (University of
California, Berkeley, CA). pPacNF-YA, pPacNF-YB, and pPacNF-YC were
provided by T. F. Osborne (University of California, Irvine, CA).
pcDNA3/Sp1 was from K.-S. Chang (University of Texas, M. D. Anderson Cancer Center, Houston, TX). Full-length and mutant GST-Sp1 in
a pGex2TKMSC expression vector were provided by E. Wintersberger
(Universität Wien, Vienna, Austria). pCite-CBF-A, pCite-CBF-B,
and pCite-CBF-C were from B. de Crombrugghe (M. D. Anderson Cancer
Center). GST-CBF-C (NF-YC) in the pGEX-4T-3 vector was provided by
S. N. Maity (M. D. Anderson Cancer Center). GST-CBF-A (NF-YB)
and GST-CBF-B (NF-YA) were constructed by PCR amplification of the
corresponding coding sequences from pCite-CBF-A and pCite-CBF-B,
respectively. The full-length CBF-A cDNA was generated with
oligonucleotides containing BamHI (sense) and
XhoI (antisense) restriction sites at their 5' termini and the full-length CBF-B cDNA generated with oligonucleotides
containing EcoRI (sense) and XhoI (antisense) at
their 5' termini. PCR products were cut with the appropriate
restriction enzymes and inserted into the pGEX-4T-3 vector.
Site-directed mutagenesis of the CCAAT site was carried out with
the QuickChange kit from Stratagene (La Jolla, CA). In brief, 10-50 ng
of
387 NPR-A luciferase, 125 ng of two complementary mutagenic
primers (sense, 5'-GTTAAAGAGTCAGGgcgttTTCCCCCGGCTCTC-3'; antisense, 5'-GAGAGCCGGGGGAAaacgcCCTGACTCTTTAAC-3'); mutagenized bases
are indicated by lowercase letters), 0.2 mM dNTPs, and 2.5 units of Pfu DNA polymerase were mixed in the PCR reaction
buffer. PCR was carried out for 18 cycles using 30 s denaturation
at 95 °C, 1 min annealing at 55 °C, and 2 min/kilobase extension
at 68 °C. After PCR, 1 µl of DpnI was added to the
reaction to cut the parental DNA template, and 5 µl of this digest
was used for transformation. Several candidate colonies were screened
by sequencing, and positive colonies were chosen for large scale DNA
preparation. Mutation of three consensus Sp1 sites in the NPR-A gene
promoter has been described previously (6).
Cell Culture--
Embryonic RASM cells (passage 19) were kindly
provided by H. Ives (University of California, San Francisco, CA).
Cells were cultured at 37 °C in a 5% CO2 humidified
incubator in Dulbecco's Modified Eagle's-H21 medium containing
10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 2% (v/v) broth, tryptose phosphate.
Drosophila Schneider cells (SL-2) were obtained from the
Cell Culture Facility at the University of California (San Francisco,
CA). Cells were cultured in Schneider's medium containing 10% fetal
bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at
25 °C.
Transfection, Luciferase, and
-Galactosidase Assays--
RASM
cells were transiently transfected with 10 µg of
387 rat NPR-A
luciferase or the relevant promoter mutant and 1-5 µg of pNF-YA or
pNF-YA29 by electroporation (Gene-Pulser, Bio-Rad) at 250 mV and 960 µF. For Drosophila Schneider cells, 5 µg of
387 NPR-A
luciferase or the relevant promoter mutant and 2 µg of Copia
-galactosidase were cotransfected along with increasing amounts
(1-10 µg) of pPacSp1, and/or plasmids encoding NF-Y subunits (pPacNF-YA, pPacNF-YB, and pPacNF-YC), alone or in combination, by
electroporation at 180 mV and 960 microfarads. After transfection, cells were plated on six-well plastic plates and cultured for 48 h. Cells were harvested and lysed in 100 µl of cell culture lysis
reagent (Promega, WI). Protein concentration of each cell extract was
measured using Coomassie protein reagent (Pierce). Cell lysates were
processed (20 µg of protein/sample) and assayed for luciferase as
described previously. Measurements of
-galactosidase activity were
made using the Galacto-Light PlusTM kit from Tropix, Inc. (Bedford, MA).
Preparation of Nuclear Extracts--
Cells were harvested and
lysed by the addition of 0.5 ml of lysis buffer (containing 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5% Nonidet P-40, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) on ice for 10 min. Lysates
were centrifuged; the pelleted nuclei were resuspended in buffer B
(containing 20 mM HEPES, pH 7.9, 420 mM NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 25%
glycerol, and the above protease inhibitors) and kept on ice for 30 min. Nuclei were centrifuged at 12,000 rpm for 15 min, and the
supernatant extracts were saved. Extracts were stored at
80 °C
prior to use.
UV Cross-linking Analysis--
Purified
32P-end-labeled, double-stranded oligonucleotide spanning
the CCAAT site in the NPR-A promoter or a control oligonucleotide encoding a C/EBP binding site was incubated with 10 µg of RASM nuclear extract in binding reaction buffer (25 mM HEPES, pH
7.5, 50 mM KCl, 1 mM dithiothreitol, 10 µM ZnSO4, 0.2 mg/ml bovine serum albumin,
10% glycerol, and 0.1% Nonidet P-40) containing 0.5 µg of
poly(dI-dC) at room temperature for 30 min. Each reaction mixture was
then pipetted onto parafilm and subjected to irradiation for 10-20 min
at a distance of 5 mm using an ultraviolet lamp with 254-nm emission.
Samples were resolved by electrophoresis on 10% denaturing
polyacrylamide gel and exposed to an x-ray film for autoradiography.
Electrophoretic Mobility Shift Assay (EMSA)--
The
CCAAT oligonucleotides used for EMSAs were as follows: wild-type,
5'-GTTAAAGAGTCAGGATTGGTTCCCCCGGCTCTC-3'; mutant,
5'-GTTAAAGAGTCAGGgcgttTTCCCCCGGCTCTC-3'. Only coding strand
sequence is provided; mutagenized bases are identified by lowercase
letters, and the CCAAT sequence is underlined. The sequence (coding
strand) of the C/EBP binding oligonucleotide is:
5'-TAGCTGAGATCTTGCGTAACCATTGCCCA-3'. Nuclear extracts (10 µg)
were incubated in binding reaction buffer containing 0.5 µg of
poly(dI-dC) at room temperature for 10 min. Purified
32P-end-labeled, double-stranded oligonucleotide was added
for an additional 10 min in a total volume of 20 µl. For competition experiments, a 1-100-fold molar excess of unlabeled double-stranded oligonucleotide was added to the binding reaction. For
immunoperturbation experiments, nuclear extracts were incubated on ice
for 1 h with 2 µg of polyclonal antibody against C/EBP
,
C/EBP
, C/EBP
, NF-YA, NF-YB, DBP, TEF, Sp1, or Sp3 prior to the
addition of labeled probe. Independent studies with the anti-C/EBP
(23) and C/EBP
(data not shown) antibodies demonstrated that each
was capable of disrupting (C/EBP
) or supershifting (C/EBP
) the
relevant DNA-protein complex in the mobility shift assay. All samples
were resolved on 5% nondenaturing polyacrylamide gels. Gels were dried and exposed to film for autoradiography.
GST Pull-down Assay--
35S-Labeled Sp1 protein was
synthesized in vitro using the TnT SP6 quick-coupled
transcription/translation system from Promega (Madison, WI) according
to the manufacturer's instructions. GST fusion protein expression
vectors including pGEX-CBF-A, pGEX-CBF-B, pGEX-CBF-C, and pGEX-Sp1 were
transformed into the BL-21 strain of Escherichia coli
(Stratagene, La Jolla, CA), expanded in suspension culture and induced
(3 h) with 1 mM isopropyl
-D-thiogalactopyranoside. Cells were pelleted, sonicated
in TST buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20, and centrifuged. The
resultant supernatant was then added to 300 µl of
glutathione-Sepharose beads, mixed on a rotating wheel at 4 °C for
1 h, and centrifuged. The pellet containing the bound GST fusion
protein was washed three times with TST buffer, then resuspended in 300 µl of protein binding buffer containing 20 mM HEPES (pH
7.9), 150 mM KCl, 25 mM MgCl2, 10%
glycerol, 1 mM dithiothreitol, 0.1% Triton X-100, and
0.1% Nonidet P-40. Bound protein was quantitated using the Coomassie
protein assay reagent (Pierce).
Ten µg of each GST-CBF subunit bound to glutathione-Sepharose beads
was incubated with 4 µl of in vitro translated
35S-Sp1 in 150 µl of protein binding buffer at 4 °C
for 1 h. The reaction contents were then precipitated by
centrifugation. The precipitate was washed three times with 500 µl of
protein binding buffer, resuspended in 15 µl of SDS sample buffer,
and loaded on a 10% denaturing polyacrylamide gel. The gel was dried
and exposed to x-ray film prior to autoradiography.
Coimmunoprecipitation--
One mg of RASM nuclear extract was
mixed with 2 µg of either anti-Sp1 antibody or anti-Rel A (p65)
antibody in 200 µl of protein binding buffer. As a control, 200 µg
of RASM nuclear extract was mixed with either 10 µg of GST alone or
GST-Sp1 bound to glutathione-Sepharose beads. After incubation at
4 °C for 2 h, the reaction mixtures were pelleted, and the
precipitates were washed three times with protein binding buffer,
resuspended, and boiled with SDS sample buffer. The protein was
resolved on 10% SDS-PAGE gel, transferred to nitrocellulose membrane,
and immunoblotted with anti-NF-YA antibody. The immunoprecipitated
protein signal was detected using the ECLTM Western blot detection
system (Amersham Pharmacia Biotech).
Statistic Analysis--
Data were evaluated by one-way analysis
of variance using Newman-Keul's test for significance.
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RESULTS |
Three Sp1 consensus sites and one CCAAT site have been identified
by DNA sequence analysis in the proximal 5'-flanking sequence of NPR-A
gene (5, 6). All of these regulatory elements are present within a
segment of the NPR-A gene, extending from 387 base pairs upstream to
the transcription start site, which we showed previously to direct the
optimal level of NPR-A promoter activity in RASM cells (6). The
relative location and specific mutations introduced at each of these
sites are presented in Fig. 1A. To determine the relative
contribution of each of these putative regulatory elements to NPR-A
gene transcription, mutations were introduced into each site within the
context of the
387 NPR-A luciferase reporter and transfected into
RASM cells. Our previous studies showed that both Sp1 and Sp3 bind to
each of the three Sp1 consensus sites in the NPR-A promoter and that
mutation of all three Sp1 sites in concert reduced
387 NPR-A
luciferase reporter activity in RASM cells to 10% that of the native
promoter. In the present study, we showed that mutation of the CCAAT
site resulted in a similar ~90% reduction in activity whereas
mutation of the CCAAT and Sp1 sites in combination virtually eliminated
NPR-A promoter activity (Fig. 1B). Thus, a CCAAT-binding
transcription factor is a dominant activator of the NPR-A promoter.

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Fig. 1.
Mutation of Sp1 and CCAAT sites eliminates
rat NPR-A promoter activity in transiently transfected RASM cells.
The CCAAT site is on the minus strand in the NPR-A promoter and reads
as ATTGG in the figure. Panel A, location and
site-directed mutagenesis of the Sp1 and CCAAT sites. WT and
MUT represent the wild-type and mutagenized sites.
Mutagenized bases are indicated by lowercase
letters, and the putative regulatory element is
underlined. Panel B, 10 µg of
wild-type (WT) 387 NPR-A LUC or the mutant
(MUT) indicated were transiently transfected into RASM
cells. After 48 h of culture, cells were lysed for luciferase
assay. The data represent the mean ± S.D. from four experiments
done in triplicate.
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A number of transcriptional activators that bind to the CCAAT motif
have been described. One or more of these may participate in
CCAAT-dependent, NPR-A promoter activity. These factors
include C/EBP (13-16), NF-Y (17-19), mouse y-box protein 1 (MSY-1)
(20), and CCAAT binding transcription factor/nuclear factor-1
(CTF/NF-1) (19, 21). We performed UV cross-linking and electrophoretic mobility shift assays (EMSA) to identify the protein(s) that interact with the CCAAT motif in the proximal NPR-A promoter. UV cross-linking analysis showed at least two protein complexes in RASM nuclear extracts, with molecular mass values of ~117 and ~78 kDa, that associated with a labeled oligonucleotide spanning the CCAAT motif in
the NPR-A promoter, but not with an otherwise identical oligonucleotide containing the mutations that block NPR-A promoter activity (Fig. 2). We also cross-linked the same RASM
extracts with an oligonucleotide that binds to C/EBPs in rat liver
tissue and pituitary progenitor GHFT1-5 cells (22, 23). Three nuclear
proteins of ~42, ~48, and ~52 kDa were identified. Although a low
intensity ~42-kDa band was seen in the RASM extracts, the ~48- and
~52-kDa bands were clearly distinguishable from those interacting
with the NPR-A CCAAT sequence (Fig. 2). This suggests that C/EBP
proteins are present in the RASM nuclear extract but, for the most
part, do not bind to the CCAAT motif in the NPR-A promoter.

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Fig. 2.
UV cross-link analysis of proteins present in
RASM nuclear extracts that bind to the CCAAT site in the rat NPR-A
promoter. Ten micrograms of RASM nuclear extract was incubated
with 32P-labeled, double-stranded CCAAT or CCAAT mutant
oligonucleotide (see "Experimental Procedures" for description) or
a control 32P-labeled, double-stranded oligonucleotide
encoding a known C/EBP binding site. The reaction mixture was
irradiated by UV at 254 nm for 10-20 min, then separated on a 10%
denaturing polyacrylamide gel and exposed to x-ray film.
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EMSA of DNA-binding proteins present in RASM extracts was carried out.
A single slowly migrating band was observed which was effectively
competed by unlabeled wild-type (WT) oligonucleotide but not
by its mutated counterpart (Fig.
3A). EMSA with the
C/EBP-specific oligonucleotide demonstrated a faster-migrating protein
complex supporting our contention (see above) that C/EBP does not
participate in formation of the complex identified on the NPR-A CCAAT
element (Fig. 3B). This conclusion drew further support from
the failure of the C/EBP oligonucleotide to compete with the native
complex on the NPR-A promoter fragment (Fig. 3B). The
117-kDa size of the UV cross-linked protein is similar to the size
predicted for the CCAAT-binding, heterotrimeric NF-Y complex, raising
the possibility that this transcription factor is involved in
regulation of the NPR-A gene. To test this hypothesis, we employed
specific antibodies directed against subunits of the NF-Y protein
complex. Immunoperturbation studies showed that the slowly migrating
band in the EMSA was supershifted or disrupted by antibody directed
against the A or B subunits of NF-Y, but was not recognized by antibody
directed against C/EBP
, C/EBP
, C/EBP
, DBP, or TEF (Fig.
3C). Collectively, these findings support the hypothesis
that the RASM nuclear protein that binds to the CCAAT box of the NPR-A
promoter is NF-Y.

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Fig. 3.
EMSA analysis of the interaction of RASM
nuclear extracts with the CCAAT site in the rat NPR-A promoter.
Ten µg of RASM nuclear extract was incubated with
32P-labeled, double-stranded NPR-A CCAAT oligonucleotide or
32P-labeled, double-stranded C/EBP-binding oligonucleotide
and subjected to EMSA. Panel A, competition of
RASM nuclear protein interaction with 32P-labeled CCAAT
probe by increasing concentrations (1-100-fold excess) of unlabeled
double-stranded NPR-A CCAAT oligonucleotide. Panel
B, competition of RASM nuclear protein interaction with
32P-labeled NPR-A CCAAT probe by increasing concentrations
(1-100-fold excess) of unlabeled, double-stranded C/EBP-binding
oligonucleotide. Panel C, identification of CCAAT
binding complexes by EMSA. Ten µg of RASM nuclear extracts was
preincubated on ice for 1 h with 2 µg of antibody directed
against C/EBP , C/EBP , C/EBP , NF-YA, NF-YB, DBP, TEF, Sp1, Sp3,
or with preimmune serum before the addition of labeled NPR-A CCAAT
probe. The position of the CCAAT oligonucleotide/NF-Y complex is
indicated by the arrow.
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NF-Y (also called CBF or CP1) is a ubiquitous transcription factor that
binds to CCAAT motifs in the proximal promoters of a large number of
mammalian genes (7-12, 17-19). NF-Y/CBF consists of three subunits,
A, B, and C, all of which are required for DNA binding. To confirm that
the CCAAT box is essential for NPR-A gene expression, we examined the
effect of forced overexpression of NF-Y or an NF-Y dominant negative
mutant on NPR-A promoter activity. Overexpression of wild-type NF-YA
failed to activate the NPR-A luciferase reporter (Fig.
4); however, cotransfection of a dominant
negative mutant of NF-YA (pNF-YA29) resulted in a
dose-dependent reduction of
387 NPR-A promoter activity
in RASM cells (Fig. 4). The mutant NPR-A reporter lacking the CCAAT element was unaffected by pNF-YA29. The failure of NF-YA to activate the NPR-A promoter might reflect the fact that the ubiquitously expressed NF-Y protein, or more specifically NF-YA, is not limiting for
NPR-A gene transcription in RASM cells. To confirm that NF-Y is an
activator of the NPR-A promoter, we introduced the NPR-A luciferase
reporter into Schneider cells, a Drosophila cell line that
does not express appreciable levels of a number of mammalian transcription factors, including Sp1 and NF-Y. As shown in Fig. 5A, overexpression of the
three NF-Y subunits (A, B, and C) led to a
concentration-dependent increase in NPR-A-driven reporter activity in Schneider cells.

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Fig. 4.
Effects of overexpression of NF-YA on NPR-A
promoter activity in RASM cells. Cells were cotransfected with 10 µg of 387 NPR-A LUC and 1-5 µg expression vector encoding
wild-type NF-YA or the dominant negative NF-YA29. After 48 h of
culture, cells were harvested, and the cell lysates were assayed for
luciferase. Data represent the mean ± S.D. from three experiments
performed in duplicate. *, p < 0.01 versus
the wild-type (WT) control.
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Fig. 5.
Cooperative interaction of NF-Y and Sp1 is
required for NPR-A gene transcription. Panel
A, overexpression of NF-Y and Sp1 synergistically activates
NPR-A promoter activity. Drosophila Schneider cells were
transiently cotransfected with 5 µg of 387 NPR-A-LUC, 2 µg of
Copia -galactosidase, increasing concentrations (1, 5, or 10 µg) of pPacSp1, and a mixture of equal amounts of pPacNF-YA,
pPacNF-YB, and pPacNF-YC. After 48 h of culture, cells were lysed
and assayed for luciferase and -galactosidase activity. Luciferase
measurements were normalized for -galactosidase activity within a
given sample. The data represent the mean ± S.D. for five
experiments performed in duplicate or triplicate. *, p < 0.01 versus control, Sp1, or NF-Y alone. Panel
B, in vitro interaction of Sp1 and NF-Y. Ten µg
of of each GST-NF-Y subunit bound to glutathione-Sepharose beads was
incubated with 4 µl of in vitro translated
35S-Sp1 in 150 µl of protein binding buffer at 4 °C
for 1 h. Reactions were then precipitated by centrifugation. The
precipitate was washed, resuspended in SDS sample buffer, and resolved
on a 10% denaturing polyacrylamide gel. The gel was dried and exposed
to film for autoradiography. The result shown is representative of two
independent experiments. Panel C, NF-Y associates
with Sp1 in vivo. One mg of RASM nuclear extract was
incubated with anti-Sp1 or anti-Rel A antibody in protein binding
buffer at 4 °C for 2 h. As a control, 200 µg of RASM nuclear
extract was incubated with 10 µg of either GST alone or GST-Sp1 bound
to glutathione-Sepharose beads in protein binding buffer at 4 °C for
2 h. The reactions were pelleted, and the precipitates were
washed, resolved on 10% SDS-PAGE gel, transferred to the
nitrocellulose membrane, and immunoblotted with anti-NF-YA antibody.
The protein signal was detected using a Western blotting kit. The
result presented is representative of three independent experiments.
Panel D, Drosophila Schneider cells
were transiently cotransfected with 5 µg of 387 NPR-A CCAAT mutant
or the triple Sp1 mutant, 5 µg of pPacSp1, and a mixture of equal
amounts of pPacNF-YA, pPacNF-YB, and pPacNF-YC. After 48 h of
culture, cells were lysed and assayed for luciferase activity. The data
represent the mean ± S.D. from four experiments. #,
p < 0.05 versus Sp1 (CCAAT mutant); *,
p < 0.01 versus NF-Y (triple Sp1
mutant).
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Cooperative interactions among transcription factors have been shown to
be important for the regulation of a number of gene promoters. Indeed,
simultaneous mutation of all of the Sp1 sites and the CCAAT motif led
to a reduction in NPR-A promoter activity to background levels (Fig.
1B). To determine whether Sp1 cooperates with NF-Y at the
NPR-A promoter,
387 NPR-A luciferase was cotransfected into Schneider
cells along with Drosophila expression vectors encoding Sp1
and the three NF-Y subunits. Whereas expression of either Sp1 or NF-Y
activated the NPR-A promoter to a moderate degree, simultaneous
expression of both Sp1 and NF-Y together resulted in a dramatic
increase in promoter activity (Fig. 5A). This increase
exceeded that seen with either transcription factor alone, implying
that Sp1 and NF-Y act cooperatively to drive NPR-A gene transcription.
Transcription factors may cooperate at a promoter by separately
contacting different rate-limiting targets, by mutually contacting a
single rate-limiting target or by interacting with each other to form a
novel activity. We performed a GST pull-down assay to probe the
potential interaction of Sp1 and NF-Y in vitro. As shown in
Fig. 5B, [35S]methionine-labeled Sp1 was able
to bind GST-NF-YA and NF-YC, but not GST alone or NF-YB, suggesting
that Sp1 interacts physically with the NF-YA and NF-YC subunits. For
the converse experiment, nuclear extracts from RASM cells were
incubated with GST-Sp1 or GST alone, and the washed beads were
electrophoresed, blotted onto filters, and then probed with anti-NF-YA
antibody. This demonstrated that endogenous NF-YA in RASM cells also
interacted with Sp1 (Fig. 5C). Finally, we conducted
coimmunoprecipitation studies to show that Sp1 and NF-YA interact in
the context of the intact cell. Immunoprecipitation of Sp1 from the
same extracts followed by Western blot analysis for NF-YA revealed a
low intensity but specific band, which migrated in a position the size
of NF-YA (Fig. 5C). As a control, NF-YA was not
coimmunoprecipitated when anti-Rel A antibody was used in place of
anti-Sp1 antibody (Fig. 5C). Thus, NF-Y and Sp1 interact
both in vitro and in the context of the intact cell.
Finally, we asked whether this interaction could be demonstrated to
have functional sequelae in the cell. To address this, we transfected
NPR-A promoter-driven luciferase constructs, with mutations at either
the Sp1 or NF-Y binding sites, into Drosophila Schneider
cells, alone or in combination with Sp1 or NF-Y expression vectors. As
shown in Fig. 5D, the CCAAT mutant promoter displayed a
robust response to cotransfected Sp1 but no response to NF-Y. However,
when Sp1 and NF-Y were cotransfected together, there was a small but
statistically significant increment in promoter activity. We interpret
this increment as reflective of the ability of Sp1 to recruit NF-Y into
the regulatory complex through protein-protein interaction. Similar
findings were observed with the triple Sp1 mutant promoter.
Cotransfection with NF-Y effected a significant increase in promoter
activity while Sp1 was completely ineffective. Once again,
overexpression of NF-Y and Sp1 together led to an increment in promoter
activity that exceeded that seen with either transcription factor alone.
 |
DISCUSSION |
In this study, we demonstrated that a CCAAT box in the proximal 5'
flanking sequence of the NPR-A gene is essential for transcriptional activity. Mutation of the CCAAT motif spanning
142 to
138 relative to the transcriptional start site resulted in 90% reduction in NPR-A
promoter activity. A number of nuclear transcription factors have been
shown to bind to the CCAAT box, including C/EBP, NF-Y, MSY-1, and
CTF/NF-1 (13-21). Only NF-Y requires all five base pairs for binding
(7). UV cross-linking, EMSA competition, and immunoperturbation studies
demonstrated that the trans-acting factor NF-Y, but not C/EBP, bound to
the CCAAT site in the proximal NPR-A promoter. Moreover, forced
expression of a dominant negative mutant of NF-YA resulted in a
dramatic reduction in NPR-A promoter activity. Together, these data
suggest that NF-Y, when bound to the CCAAT element, plays a critical
role in the regulation of NPR-A gene transcription.
NF-Y is a ubiquitous heterotrimeric transcription factor, also referred
as CP1 or CBF, that consists of NF-YA, NF-YB, and NF-YC subunits with
molecular mass values of 42, 36, and 40 kDa, respectively (24-27).
NF-YB and NF-YC contain a conserved histone-fold motif, which forms a
dimer that interacts with NF-YA (26, 27). All three NF-Y subunits are
required for binding to the CCAAT motif (26, 27). Our cross-linking
studies demonstrated that at least two complexes with molecular masses
of 78 and 117 kDa specifically cross-linked to the CCAAT sequence of
the NPR-A promoter. These are very close to the predicted molecular
mass of NF-YB/NF-YC dimers and the heterotrimeric complex of all three
NF-Y subunits, respectively. Functional analysis has shown that NF-Y is
crucial for transcriptional activation and reinitiation on genes like NPR-A, which lack a TATA box (10-12, 28). Interestingly, NF-Y has been
shown to recognize CCAAT boxes in several genes involved in growth
regulation, including murine ribonucleotide reductase R2 (29), murine
E2F-1 (30), cyclin B1 (31), cdc2 (32), cyclin A (32), the protein
phosphatase cdc25C (32), and human thymidine kinase (33). Recent
studies also demonstrated that stable expression of a dominant negative
mutant of NF-Y in mouse fibroblast cells resulted in retardation of
cell growth and inhibition of transcription of various cellular genes
(34), implying that NF-Y may be crucial for cell cycle progression. The
liganded NPR-A has been demonstrated to function in a growth
suppressant mode in vascular smooth muscle, endothelial, and cardiac
fibroblast cells in culture (35-37), implying that transcriptional
control by NF-Y may be more than fortuitous.
Our earlier studies documented the importance of Sp1 to NPR-A gene
transcription (6). We have shown here that NF-Y does not work in
isolation, but rather operates in conjunction with Sp1 to regulate
NPR-A gene transcription. Mutation of the CCAAT and Sp1 sites in
combination completely eliminated NPR-A promoter activity, whereas
overexpression of both Sp1 and NF-Y in Drosophila cells led
to amplification of NPR-A promoter activity beyond that seen with
either transcription factor alone. Furthermore, we demonstrated a
physical interaction between these two transcription factors in
vitro and in vivo that may play a role in their
cooperative functional activity. Gel shift studies in which both Sp1
and NF-Y were examined, either alone or in combination, did not suggest that their functional cooperativity was due to cooperative binding of
these factors to the NPR-A promoter, at least in vitro (data not shown). However, Wright et al. (38) have demonstrated
that the half-life of either NF-Y or Sp1 binding is dramatically
increased when both transcription factors are bound to the proximal
promoter of the major histocompatibility complex class II-associated
invariant chain (Ii) gene. We cannot exclude the possibility that
similar interactions between Sp1 and NF-Y might stabilize their binding on the NPR-A promoter. In addition, Sp1 and NF-Y can bind individually to separate loci on the p300 coactivator (39-41). It is possible that
the functional interaction of NF-Y and Sp1 could involve synergistic
regulation of this important transcriptional coactivator.
Taken together, the studies presented here demonstrate that the bulk of
NPR-A gene expression in RASM cells depends upon functional, and
possibly physical, interactions between two transcription factors, NF-Y
and Sp1. Since regulation of blood pressure depends, in part, upon the
correct expression of NPR-A in relevant target tissues, the intricate
molecular processes controlling NF-Y and Sp1 expression and activity
are likely to be important components in the regulatory machinery
governing cardiovascular homeostasis at the organismal level.
 |
ACKNOWLEDGEMENTS |
We are grateful to S. McKnight, M. Roberto,
R. Mantovani, R. Tjian, T. F. Osborne, K.-S. Chang, E. Wintersberger, B. de Crombrugghe, and S. N. Maity, who provided
important reagents used in this study.
 |
FOOTNOTES |
*
This work was supported by Grant HL45637 from the National
Institutes of Health.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: Metabolic Research
Unit and Dept. of Medicine, 1109 HSW, University of California, San
Francisco, CA 94143-0540. Tel.: 415-476-2729; Fax: 415-476-1660; E-mail: gardner@itsa.ucsf.edu.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M006350200
 |
ABBREVIATIONS |
The abbreviations used are:
NPR-A, type A
natriuretic peptide receptor;
GST, glutathione
S-transferase;
PCR, polymerase chain reaction;
C/EBP, CCAAT/enhancer-binding protein;
RASM, rat aortic smooth muscle.
 |
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