Originally published In Press as doi:10.1074/jbc.M006532200 on September 18, 2000
J. Biol. Chem., Vol. 275, Issue 50, 39061-39072, December 15, 2000
The Smooth Muscle 
-Actin Gene Promoter Is a Molecular
Target for the Mouse bagpipe Homologue, mNkx3-1, and
Serum Response Factor*
James A.
Carson
§,
Rebecca A.
Fillmore¶,
Robert J.
Schwartz
, and
Warren E.
Zimmer¶
From the Department of Cellular and Molecular
Biology, Baylor College of Medicine, Houston, Texas 77030 and the
¶ Department of Structural and Cellular Biology, University of
South Alabama, Mobile, Alabama 36688
Received for publication, July 21, 2000, and in revised form, September 5, 2000
 |
ABSTRACT |
An evolutionarily conserved vertebrate homologue
of the Drosophila NK-3 homeodomain gene
bagpipe, Nkx3-1, is expressed in vascular and visceral
mesoderm-derived muscle tissues and may influence smooth muscle cell
differentiation. Nkx3-1 was evaluated for mediating smooth muscle
-actin (SMGA) gene activity, a specific marker of smooth muscle
differentiation. Expression of mNkx3-1 in heterologous CV-1 fibroblasts
was unable to elicit SMGA promoter activity but required the
coexpression of serum response factor (SRF) to activate robust SMGA
transcription. A novel complex element containing a juxtaposed
Nkx-binding site (NKE) and an SRF-binding element (SRE) in the proximal
promoter region was found to be necessary for the Nkx3-1/SRF
coactivation of SMGA transcription. Furthermore, Nkx3-1 and SRF
associate through protein-protein interactions and the homeodomain
region of Nkx3-1 facilitated SRF binding to the complex
NKE·SRE. Mutagenesis of Nkx3-1 revealed an inhibitory domain
within its C-terminal segment. In addition, mNkx3-1/SRF cooperative
activity required an intact Nkx3-1 homeodomain along with the MADS box
of SRF, which contains DNA binding and dimerization structural domains,
and the contiguous C-terminal SRF activation domain. Thus, SMGA is a
novel target for Nkx3-1, and the activity of Nkx3-1 on the SMGA
promoter is dependent upon SRF.
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INTRODUCTION |
Homeobox proteins are a class of developmentally regulated
transcription factors that are important for embryonic patterning and
differentiation (reviewed in Ref. 1). Although first identified in
Drosophila, these regulators have been found in all metazoan species examined to date from fungi to humans. They constitute a family
of transcription factors that are recognized by a conserved segment of
60 amino acids, referred to as the homeodomain, that usually recognizes
a TTAATT-degenerate DNA consensus binding sequence found within the
control elements of target genes (1-3). Drosophila and
vertebrate hox genes are clustered on the genome,
exhibit a high degree of structural conservation across species, and
are expressed in a temporal and spatial sequence that is also conserved (1). Genome and expression analyses have now firmly established that
there are a variety of homeodomain-containing proteins in vertebrates
including a class of unique genes that are dispersed throughout the
genome (1, 4, 5). Among the dispersed class of homeodomain proteins is
the NK family, which was first defined by four genes
(nk-1 through nk-4) identified in
Drosophila. This family exhibits specific homology within
the homeodomain and shares other regions of conserved sequence outside
the homeodomain (4-7). In Drosophila, two nk
genes, tinman and bagpipe, have been found to be
closely linked and are expressed and participate in the specialization
of mesoderm-derived heart and visceral organs (8, 9). Mutations in
tinman block dorsal vessel (Drosophila heart
equivalent) and visceral musculature formation; however, bagpipe mutations lead to only midgut visceral musculature
abnormalities. Thus, bagpipe may be a downstream target of
tinman (8).
nkx3-1 and nkx3-2, two murine homologues
of the bagpipe gene, have recently been identified (10-13).
These genes demonstrate overlapping expression patterns in somites of
early embryos (~8.5-9 embryonic days); however, only the
nkx3-2 gene was expressed in the lateral and splanchnic
mesoderm (10, 12). In later stage embryos and in adults nkx3
genes are differentially expressed. Nkx3-1 is predominantly expressed
in brain, kidney, blood vessels, and the male reproductive system (10,
11, 14), whereas Nkx3-2 is found in the lateral plate mesoderm
surrounding the mid- and hindgut and within cartilagenous condensations
(12, 13). In adult tissues, nkx3 genes retain expression
within the mesoderm-derived structures surrounding vascular (Nkx3-1 in
the blood vessels) and visceral (Nkx3-2 in the mid- and hindgut
mesoderm) organs and may influence smooth muscle cell differentiation.
At present, potential target genes regulated by the Nkx3 family have
yet to be identified.
Smooth muscle cells are integral cellular components of most organs
through their role in controlling vascular tone, gastrointestinal motility, fluids movement, and airway resistance. Differentiated smooth
muscle cells are characterized by their expression of a unique subset
of contractile protein isoforms including smooth muscle 
-actin
(15-18), smooth muscle -actin (17-21), smooth muscle myosin heavy
and light chains (22-24), calponin (25), SM22 (26-28), and telokin
(29-31). Furthermore, these cells possess the capability to express
appropriate levels of these characteristic smooth muscle proteins even
though they are derived from diverse embryonic origins (32). In
contrast to skeletal and cardiac muscle cells, smooth muscle cells
retain their capacity to modulate reversibly their phenotype during
postnatal development (33, 34). This phenotypic modulation includes a
reentry into the cell cycle and an altered expression of the
characteristic proteins of the differentiated cell, effects that have
been implicated in the pathogenesis of certain cardiovascular (35) and
gastrointestinal (36) disease states. Thus, a knowledge of the
molecular mechanisms that control smooth muscle development and
cell-specific gene expression will provide insights into cellular
differentiation and the physiological responses of these cells to
injury and disease states.
We have examined the expression of the smooth muscle -actin
(SMGA)1 gene as a model to
understand the molecular mechanisms that regulate genes during smooth
muscle differentiation. In aves (20, 21, 37) as in mammals
(17-19), SMGA expression is restricted to smooth muscle tissues with
the exception of the post-meiotic spermatocyte (38); thus SMGA provides
an excellent marker for the smooth muscle phenotype. In addition,
tissue-restricted expression of SMGA arises during early embryonic
development of the vasculature and gastrointestinal tract (17, 18, 20,
21, 39), suggesting that the activation of SMGA transcription may
require factors unique to the differentiating smooth muscle cell.
Transcriptional regulation of the SMGA gene appears to require the
interplay of positive- and negative-acting cis elements
within the promoter. Two regions displaying positive acting
transcriptional activity were mapped on the SMGA promoter, referred to
as the specifier and modulator domains (21). A key cis
element for smooth muscle-specific SMGA transcription found in both of
these domains is the CArG/SRE (CC(A/T)6GG) motif. The
positive acting transcriptional activity of the specifier and modulator
domains is derived from the binding of SRF to the SRE sites within
these domains, and we have demonstrated that SRF-containing complexes
play a prominent role in the developmental activation of the SMGA gene
(40). Based upon our studies and the many examples from
SRF-dependent regulation of cardiac (6, 14, 41-43) and
skeletal (44-48) muscle genes, we predicted that SRF requires
association with other factors to regulate the SMGA gene.
Here we demonstrate the cooperative interaction of Nkx3-1 with
SRF-activating SMGA transcription. The conservation of structure and
activity of actin gene transcriptional machinery permits examining these components utilizing a heterologous cotransfection assay. Transcriptional synergy is supported by a complex of regulatory elements that is composed of immediately adjacent NKE-SRE
cis elements within the SMGA proximal promoter. Our studies
demonstrate that the SMGA gene promoter serves as a target for the NK3
family of transcription factors, specifically the Nkx3-1 factor, and supports the hypothesis that appropriate transcriptional regulation of
smooth muscle specific genes requires the combinatorial interactions of
SRF with coaccessory trans-acting factors expressed within the smooth muscle cell.
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MATERIALS AND METHODS |
Recombinant Plasmids--
Approximately 2300 bp of the avian
smooth muscle -actin promoter and 5' deletions have been cloned into
pGL-3 basic plasmid driving the expression of the reporter gene
luciferase (21, 37, 40). The 
1224, 176, and 108 5' deletions of
the smooth muscle -actin promoter were used in transfection
experiments in the current study. Mutations were made to -actin
promoter elements SRE1, SRE2, and NKE1 using multiple primer polymerase chain reaction (PCR) and verified by DNA sequencing. The SMGA SRE1 was
disrupted by changing the wild type element from
5'-CCTATTTAGG-3' to 5'-CCTATCCCGG-3' and the
SRE2 sequence (5'-CCTATATGG-3') mutated to
5'-CCTTATGTTT-3'. The SMGA NKE1 was disrupted
by changing the wild type element from 5'-CACTTAGCCT-3' to
5'-CACCCCCCCT-3'). The NKE1/SRE1 double mutant contained
the mutations to both sites.
PCR was used to isolate the 862-bp Nkx3-1 cDNA from 6-day embryoid
bodies with forward primer 5'-GCTCTAGAATGCTTAGGGTAGCGGAGCC-3' and reverse primer 5'-TTGGATCCAGAGACCCCCAGGGAAGACAG. The isolated cDNA was cloned into pCRII (Invitrogen, TA Cloning Kit) and
verified by sequencing. The Nkx3-1 fragment was excised from pCRII with XbaI and BamHI and cloned into identical sites of
the pCGN vector downstream of the cytomegalovirus promoter and HA
epitope tag. Mutations of the Nkx3-1 sequence were made using PCR-based
techniques (Stratagene, Excite PCR Mutagenesis Kit). The pCGN-Nkx3-1
cDNA clone was the template for these experiments, and all mutants were cloned into the same vector maintaining an HA epitope tag for each
of the mutant proteins. All mutants were confirmed by DNA sequencing
and Western analyses of lysates derived from transfected cells.
The 330-bp cardiac -actin promoter cloned in front of a luciferase
reporter gene has been previously described. The construction of the
consensus NKE reporter construct A20 has also been described previously
(41). Briefly, the 3× (NKE)-tata-luciferase construct was
constructed by inserting a linker containing three copies of an
intermediate strength NKE in front of the -cardiac actin minimal
TATA box driving the expression of luciferase. Human SRF expression
vectors (pCGN-SRF, pCGN-SRF
C, and pCGN-SRFpm1) driven by the
cytomegalovirus promoter have been previously described (41-43) and
were generously supplied by Ron Prywes. The construction and use of the
pCGN-Nkx2.5 expression vector has been previously described
(41-43).
Expression and Purification of Bacterially Expressed His-Nkx3-1
and GST-SRF--
The Nkx3-1 homeodomain cDNA was isolated by PCR
on the full-length cDNA using forward primer
5'-TCACCAAGCAGCCACAGAAG-3' (359-379 bp) and reverse primer
5'-CTGCTTTCGCTTGGTCTTATAGC-3' (529-552 bp). The Nkx3-1 homeodomain
protein consisted of 193 bp, including 13 bp 5' to the homeodomain. To
express Nkx3-1 in bacteria, the cDNAs for the full-length and
homeodomain-only region of Nkx3-1 were isolated by PCR, verified by DNA
sequencing, and cloned into pRSET B (Invitrogen) vector. The
full-length and homeodomain-only Nkx3-1 cDNA PCR products were
cloned into pCRII (Invitrogen, TA Cloning Kit). The cDNA for the
Nkx3-1 homeodomain region was excised from pCRII with
HindIII and XhoI, sites internal to the PCR
primers, and ligated into the HindIII/XhoI sites
of pRSET B, downstream of a 6× histidine tag. These constructs were
used to transform BL21 (DE3) cells (Novagen). Freshly transformed cells
were grown in 500 ml of LB broth containing 100 µg/ml ampicillin at
37 °C to A600 = 0.8. Isopropyl-
-D-thiogalactopyranoside at a final concentration of 1 mM was then added, and growth continued
for another 2 h. Cells were harvested and suspended in 20 ml of
column buffer (20 mM Tris, pH 7.4, 200 mM NaCl,
1 mM EDTA), sonicated, and cellular debris removed by
centrifugation. The fusion protein was purified by binding to a metal
affinity column (CLONTECH), washed extensively, and
then eluted with fractions of column buffer containing 10-200
mM imidazole. The concentration of the protein was
determined by Bradford protein assay, and purity was determined by
Coomassie staining after SDS-polyacrylamide gel electrophoresis. Full-length human SRF was expressed and purified as a glutathione S-transferase (GST) fusion protein as described previously
(41-43). The concentration of the protein was determined by Bradford
protein assay and purity determined by Coomassie staining after
SDS-polyacrylamide gel electrophoresis.
Transfection Assays in CV-1 Fibroblast Cell Cultures--
Monkey
CV-1 fibroblasts were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum. Cells were plated at
an approximate density of 5 × 105 cells per 6-cm
plate. Cells were transfected 24 h post-plating using
LipofectAMINE (Life Technologies, Inc.) as described previously (49).
Briefly, each transfection reaction contained 1 µg of luciferase
reporter plasmid (-actin, A20, or -CA) and various amounts of
transactivator plasmids (pCGN-Nkx3.1 and/or pCGN-SRF). All
transfections were balanced to 2 µg of DNA with empty vector in order
to keep the level of DNA and CMV promoter constant in all transfection
reactions. Cells were transfected 16-18 h after which the transfection
media were removed and replaced with Dulbecco's modified Eagle's
medium supplemented with 2% horse serum and 10 µg/ml insulin for an
additional 48 h. Cells were then harvested by washing with PBS and
then scraped in 400 µl of 1× Reporter Lysis Buffer (Promega).
Cellular debris was removed by centrifugation, and 30 µl of
supernatant was analyzed for luciferase activity by mixing with 100 µl of luciferase substrate (20 mM Tris-HCl, pH 8.0; 4 mM MgSO4, 0.1 mM EDTA, 30 mM dithiothreitol, 0.5 mM ATP, 0.5 mM D-luciferin, 0.25 mM coenzyme
A). Emitted luminescence was measured for 10 s. Protein
concentrations were measured by the Bradford assay (Bio-Rad) and used
to normalize luciferase activity.
In Vivo Immunoprecipitation of the Nkx3-1·SRF
Complex--
Interactions between Nkx3-1 and SRF were examined by
in vivo immunoprecipitation using previously described
methodology (42). Briefly, 3T3 cells plated on 100-mm dishes were
transfected with 2 µg of either pCGN-Nkx3.1 or pCGN-SRF plasmid DNA
using LipofectAMINE (Life Technologies, Inc.). Forty eight hours
post-transfection the cells were washed twice in ice-cold PBS,
harvested in 1 ml of ice-cold PBS, and collected by centrifugation at
4 °C. Cells were resuspended in EBC buffer (50 mM Tris,
pH 8.0; 120 mM NaCl; 0.5% Nonidet P-40; 2 µg ml
leupeptin; 2 µg/ml pepstatin; and 1 mM
phenylmethylsulfonyl fluoride), rocked at 4 °C for 15 min, and
centrifuged at 4 °C. The supernatant was transferred to a new tube
and protein concentration determined (Bio-Rad), and 500 µg of protein
extracts containing SRF and Nkx3-1 were incubated for 2 h at
4 °C with 4 µg of anti-SRF antibody (Santa Cruz Biotechnology) in
a total of 500 µl of NETN buffer (20 mM Tris, pH 8.0; 100 mM NaCl; 1 mM EDTA; 5 mM MgCl; 1 mM dithiothreitol; 0.05% Nonidet P-40; 1 mM
phenylmethylsulfonyl fluoride). Non-transfected 3T3 cell extracts
served as controls. Thirty microliters of protein G PLUS/protein
A-agarose beads (Oncogene Science) equilibrated in NETN buffer were
then added, and the incubation was continued for an additional 2 h. The beads were collected by brief centrifugation, washed three times
with 1 ml of NETN buffer, and suspended in 30 µl of 2× SDS sample
buffer. Samples were then boiled 5 min, briefly centrifuged, and the
supernatant separated by 10% SDS-PAGE. Proteins were visualized by
Western blot analysis using anti-HA antibody and ECL (Amersham
Pharmacia Biotech).
DNA Binding Mobility Shift and DNase I Footprinting
Assays--
Double-stranded oligonucleotides corresponding to the
smooth muscle -actin NKE1/SRE1 elements were constructed consisting of 100 to 74 bp of the promoter
(5'-CCATCACTTAGCCTATTTAGGGTCTT-3'). The oligonucleotide was
end-labeled using the polynucleotide kinase reaction, and band shift
assays were performed as described previously (40-43). Briefly, 20 µl containing 1 µg of either poly(dI-dC) or poly(dG-dC) binding
buffer (10 mM Tris, pH 8.0; 1 mM
dithiothreitol; 1 mM sodium phosphate; 5% glycerol; 50 mM sodium chloride) and nanogram quantities of purified
bacterially expressed proteins were incubated 10 min at room
temperature, and then the probe was added (20,000 cpm/reaction) and
incubated for 15 min at room temperature. Binding complexes were then
run on a 5% polyacrylamide gel in 0.5× TBE buffer. The gel was prerun
for 20 min, followed by sample electrophoreses for 2 h at 180 V. Binding complexes were visualized by autoradiography.
DNase I footprinting was performed on a fragment of the SMGA gene from
108 to +15 using purified SRF and Nkx3-1 proteins. This fragment was
cloned into pGL-3 vector as described previously (21, 40). The DNA was
digested with enzymes present in the vector multiple cloning site that
were unique within the gene promoter/luciferase vector (NheI
for coding strand and HindIII for non-coding strand), and
the DNA construct was then labeled with 32P using Klenow
enzyme. Single end-labeled DNA was then digested with the second enzyme
and the fragment purified by electrophoresis on polyacrylamide gels as
described previously. Each mapping assay contained ~25 fmol of
fragment (25,000 cpm) in 50 µl of gel shift assay buffer described
above. Proteins were added to the assay tubes on ice, and the reaction
was continued on ice for 1 h, after which DNase I was added and
the reaction changed to room temperature for 1 min. Subsequent steps
were carried out essentially as described previously (41-43). In order
to map footprints directly upon the DNA fragment, a ladder of G + A
Maxam-Gilbert sequence was performed on the labeled DNAs, and the
ladder was electrophoresed in adjacent wells of the same gel.
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RESULTS |
Coexpression of SRF and Nkx3-1 Activated the Smooth Muscle
-Actin Promoter--
Smooth muscle cell-specific transcription of
the SMGA gene is controlled through two positive acting DNA domains
within sequences flanking the 5' end of the gene (21). Furthermore,
four of the six CArG elements found inside these domains avidly bind
SRF (37, 40), and SRF alone was capable of modestly activating
transcription from constructs containing the intact domains (~2.3
kilobase pairs) linked to a luciferase reporter gene in heterologous
cotransfection experiments (21). As shown in Fig.
1, deletion of the SMGA flanking DNA to a
point that eliminated the distal positive domain (construct 1224) and
most of the proximal positive domain (construct 176) of the gene did
not appreciably diminish the ability of SRF to activate transcription
from this promoter. The 176 construct contains two CArG/SRE elements
located at 85 (CArG/SRE1) and 120 (CArG/SRE2), both of which were
bound by smooth muscle nuclear complexes (37, 40); however, of the two
CArGs, SRE2 appeared to bind SRF with greater avidity.
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Fig. 1.
A short portion (176 bp) of the SMGA promoter
was sufficient to support synergistic transactivation by Nkx3-1 and
SRF. A, a diagram of deletion mutants of the avian SMGA
promoter used in this study illustrating the positions of an NKE
(closed box) and six SRE (open box) motifs is
shown. The negative numbers refer to the 5' end position of the various
DNA fragments with respect to the SMGA gene. B, CV-1 cells
were transfected with the 1224 SMGA promoter-luciferase reporter gene
(1 µg of DNA) and CMV promoter-directed expression vectors as
described under "Materials and Methods." Expression vectors for SRF
and Nkx3-1 were assayed singularly (0.2 µg of DNA) and in combination
where SRF was maintained at a constant level (0.2 µg), and Nkx3-1 was
provided at increasing content (0.2-0.8 µg). All transactivation
reactions were balanced to 1 µg of DNA content by the addition of
empty CMV promoter vector. These data were generated from a minimum of
four experiments, performed in duplicate. The fold induction represents
the luciferase activity measured in lysates receiving various
transactivation plasmids compared with the activity in lysates derived
from cells that received the 1224 construct and vector control
(vector lane). C, CV-1 cells were transfected as
in B, except the cells received the SMGA 176 reporter
construct. In addition to Nkx3-1, the 176 SMGA construct was assayed
with an Nkx2-5 expression vector (41-43), both singularly (0.2 µg)
and in combination with SRF (0.2 µg of SRF with 0.8 µg of Nkx2-5).
Fold induction was derived from the data as above. D, CV-1
cells were transfected as described with the SMGA 108
promoter-luciferase reporter gene as the target. Fold induction was
calculated from luciferase activities in lysates of cells transfected
with SRF (0.2 µg) or Nkx3-1 (0.2 µg) alone or in combination (SRF
at 0.2 µg; Nkx3-1 at 0.8 µg) compared with the activity measured in
cells receiving empty vector. E, 50 µg of CV-1 cellular
protein extracted from cells transfected with empty vector
(control), pCGN Nkx3-1, and/or pCGN SRF as described above
was sized by 10% SDS-PAGE, and the proteins were transferred to a
nylon membrane. The membrane was probed with an anti-HA epitope
monoclonal antibody, and the HA-tagged fusion proteins visualized by
chemiluminescence. The numbers to the right of
the blot show the migration of protein standards, and the positions of
HA-tagged SRF and Nkx3-1 are indicated by the arrows.
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We then asked if a vertebrate homologue of bagpipe, the
homeodomain factor Nkx3-1, was capable of activating the SMGA promoter. Whereas Nkx3-1 alone was not able to activate appreciably the SMGA
promoter/reporter constructs above vector controls, there was a robust
increase in promoter activity when Nkx3-1 was expressed with SRF (Fig.
1, B and C). The activation of reporter gene
activity obtained in cells expressing both factors was approximately
20-25-fold above vector controls. These values were significantly
enhanced compared with the expression obtained from cells transfected
singularly with SRF or with Nkx3-1 indicating a cooperative and/or
synergistic interaction of these trans-factors upon SMGA
promoter activity. Moreover, this synergistic response was maintained
using constructs containing the first 176 base pairs of 5'-flanking
sequence of the SMGA promoter (Fig. 1C). Deletion of the
distal CArG/SRE, SRE 2, from the SMGA 176 promoter construct (Fig.
1D) eliminated Nkx3-1 and SRF-stimulated transcription,
indicating the positive acting nature of this cis-acting
element in the Nkx3-1/SRF-dependent transcriptional
activation. Western blotting analyses of cellular lysates confirmed
that both SRF and Nkx3-1 were expressed efficiently in the transfected
cells (Fig. 1B).
Previously, we showed that Nkx2-5 cooperates with SRF to transactivate
the cardiac -actin promoter (41-43, 50). We asked if the
tinman homologue, Nkx2-5, could substitute for Nkx3-1 in cotransfection experiments with the SMGA promoter. Unlike Nkx3-1, Nkx2-5 was not able to augment the basal transcriptional response of
the 176-bp SMGA promoter obtained with SRF (Fig.
2B). In addition, we did not
observe Nkx3-1/SRF-dependent transcriptional activation from the NKE target promoter A20 (41; data not shown). However, the A20
promoter, derived from preferred Nkx2-5-binding sequences and a minimal
portion of sequences of the -cardiac actin promoter, was activated
with Nkx2-5 and SRF under the same conditions. Taken together, these
data show that Nkx3-1 exhibits a synergism with SRF to activate
transcription from the SMGA promoter. This activation is specific for
the SMGA promoter and is directed through the initial 176 bases
flanking the SMGA gene.
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Fig. 2.
Evolutionarily conserved SMGA promoter
proximal SRE1 and NKX-binding sites are required for Nkx3-1/SRF
synergistic transcriptional activation. A, the
transcriptional activity of the 176 SMGA promoter (wild type) and
selected point mutants of the promoter by Nkx3-1 and SRF was assayed by
cotransfection analysis in CV-1 cells. The specific base changes that
inactivate SRE 2 (SRE2mut), SRE 1 (SRE1mut), and
NKE 1 (NKE1mut) are shown below the diagram of
the 176 SMGA promoter fragment. Activity of wild type and mutant
promoter constructs was determined in cells overexpressing Nkx3-1 and
SRF singularly or in combination. The fold activation was calculated
from comparison of activities in lysates of cells expressing
transactivator proteins to that of cells treated with empty vector.
Each plot represents a minimum of four experiments assayed in
duplicate. B, an alignment of the proximal promoter
sequences elucidated from the avian (chick) (21), human
(19), and mouse (51) SMGA genes is shown. The positions of SRE
(CArG/SRE 2, CArG/SRE 1) and the NKE (NKE1) motifs are marked
above the sequence.
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Multiple DNA Elements Are Required for
Nkx3-1/SRF-dependent Activation of SMGA
Transcription--
There is a high level of sequence homology
(~70%) that extends over the initial 400 bp flanking the 5' region
of the SMGA gene across species (19, 21, 51). Furthermore, the
structure and spacing of CArG motifs appear to be strictly maintained
across species (21). We focused upon the NKE and CArG sites of the SMGA
176 promoter fragment as potential cis
regulatory elements responsible for Nkx3-1/SRF
transcriptional activation. To assess fully Nkx3-1 and SRF factor
function, we mutated the two CArG/SRE motifs and a potential NKE site.
As illustrated in Fig. 2, a mutation of CArG/SRE2 (120) that
abolished binding with purified SRF (40) also completely inhibited
transcriptional activation of the SMGA-176 promoter by SRF. However,
the mutant CArG/SRE2-containing SMGA promoter did not block SRF and
Nkx3-1-dependent activity. Similarly, mutagenesis of the
85 CArG/SRE1 motif (CCTATTTAGG 
CCTATCCCGG) also abolished SRF-dependent
activation. However, in contrast to mutated SRE2, mutated SRE1 blunted
coactivation by SRF and Nkx3-1 via the mutated 176 promoter (Fig.
2A). The 90-bp element (CATCACTTAG) resembled the
consensus binding sequence for TTF-1/Nkx2-1 and Nkx2-5 factors
(41-43, 52). Mutagenesis of this potential NKE site reduced promoter
SRF and Nkx3-1-dependent activity by approximately 40%
(Fig. 2). Therefore, only CArG/SRE 1 appears to be required for the
Nkx3-1/SRF response. However, the inability of SMGA promoter constructs
containing just the NKE1/SRE1 motif to be activated by Nkx3-1 and SRF
(108, Fig. 1D) demonstrates that although this
motif is necessary it is not totally sufficient to support the
synergistic transcriptional response. Alignment of sequences near the
proximal avian (21), human (19), and mouse (51) SMGA promoters
emphasize the conservation of structure and spacing of the NKE1 and SRE
motifs across diverse species (Fig. 2B). Taken together,
these data are consistent with the hypothesis that multiple DNA-binding
elements are required for SRF and Nkx3-1/SRFdependent
transcriptional activation.
Nkx3-1 Homeodomain and SRF Bound the NKE1/CArG1 Region of the SMGA
Promoter--
We reasoned that in order for SRF and Nkx3-1 to function
together, they might bind in the immediate vicinity of these promoter elements. To test this hypothesis, we performed DNA binding experiments using purified bacterially expressed proteins with a DNA segment containing their potential binding elements. A DNA probe containing sequences 100 to 74 of the SMGA promoter (NKE/CArG probe) was incubated with purified Nkx3-1 homeodomain (Fig.
3A) or SRF (Fig. 3D) proteins, and
the mixtures were separated on polyacrylamide gels to assay for protein
binding. As shown in Fig. 3A, the homeodomain of the Nkx3-1
protein formed two shifted complexes with the NKE/CArG probe fragment.
Both of the Nkx3-1 homeodomain complexes increased in intensity with
added protein; however, the slower migrating complex demonstrated a
more intense signal, indicative of stronger binding. DNA binding
specificity was demonstrated by specific competition using a 50-fold
excess of unlabeled NKE/CArG fragment but not with a DNA
oligonucleotide duplex containing a sequence for GATA factor binding
(53, 54). An excess of three synthetic NKE sites in A20, previously
shown to bind Nkx2-5 with great avidity (41), competed away the more
slowly migrating species (Fig. 3B). This experiment shows
that the Nkx3-1 homeodomain can bind a DNA containing an Nk homeodomain
consensus core-binding site ((C/T)AAG)) (6, 41) as had been previously
suggested (41). Furthermore, the slower migrating complex formed with
the NKE/CArG element represents the interaction of the Nkx3-1
homeodomain at the NKE sequence. Shifts with the NKE/CARG SRE1-mutated
probe eliminated the migration of the more rapidly migrating species but still allowed for the appearance of a single Nkx2-5 and/or Nkx3-1
DNA-binding complex that specifically competed with A20. Another
shifted species, presumably nonspecific, was also observed with Nkx3-1
and the SRE1 mutant probe that could not be competed with multimerized
NKEs.
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Fig. 3.
Nkx3-1 and SRF independently bound to closely
juxtaposed NKE and SRE1 elements. Fusion proteins containing
Nkx3-1 (His Nkx3-1) homeodomain and SRF (GST-SRF) were purified from
bacteria and utilized in gel shift (electrophoretic mobility shift
assay) and DNase I footprinting analyses to localize their binding
sites upon the SMGA promoter NKE/CArG element. A-C
represent experiments utilizing His-Nkx3-1 homeodomain fusion protein.
A, Nkx3-1 homeodomain protein binding was analyzed by
electrophoretic mobility shift assay. Two complexes were formed with
increasing quantities of the Nkx3-1 fusion protein (100, 200, and 400 ng; lanes 2-4, respectively) on the NKE/CArG element
(arrows). These complexes do not form in the presence of a
50-fold excess of unlabeled NKE/CArG element (lane 5);
however, they are not inhibited by excess GATA protein-binding sites
(lane 7). B, lane 1, WT NKCArG probe
only; lane 2, Nkx2-5 homeodomain binding (100 ng);
lane 3, Nkx3-1 homeodomain binding (400 ng); lane
4, Nkx3-1 + self-competitor; lane 5, Nkx3-1 + nonspecific competitor (competes slow complex, not fast); lane
6, NKE/CArGSRE1 mutant probe only; lane 7, Nkx2-5 (100 ng); lane 8, Nkx3-1 (400 ng); lane 9, Nkx3-1 + Nke binding competitor (competes fast complex, not slow); and
lane 10, Nkx3-1 + nonspecific GATA competitor. Note the
NS (denoted with the asterisks) as nonspecific
shifted band that did not compete with any binding site including
unlabeled probe fragment. C shows an autoradiogram of a
DNase I footprinting experiment in which the purified Nkx3-1 fusion
protein was incubated with a DNA probe representing 108 to + 15 of
the avian SMGA gene. The probe was analyzed in parallel reactions as
detailed under "Material and Methods" containing 0, 0.25, 0.5, or 1 µg of protein and displayed on 6% polyacrylamide sequencing gels.
Position of probe protection is indicated by the arrows, and
the positions of DNase I enhancements are shown by the
arrowheads. The negative numbers refer to nucleotide
positions to the gene and were determined by running Maxam and Gilbert
sequence reactions of the labeled fragment run in adjacent wells of the
same gel. D and E show experiments utilizing
purified GST-SRF fusion protein. D is an electrophoretic
mobility shift assay of SRF (100 ng) with the NKE/CArG element as
labeled DNA probe. The position of the SRF shift (lane 2) is
shown to the left of the autoradiogram, which is effectively
competed by excess, unlabeled fragment (lane 3) but not with
excess DNA housing GATA-binding sites (lane 4). E
shows the protection of the SMGA promoter fragment (188 to + 15) by
added SRF protein (0.2-0.8 µg). The protected region of the probe
fragment (nucleotides 93 to 76) is shown between the
arrows to the right of the panel.
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To map precisely Nkx3-1 binding, we performed footprinting experiments
using an end-labeled fragment derived from the SMGA promoter (108 to
+15). The Nkx3-1 homeodomain protected a segment of sequence
corresponding to the NKE1 element (95 to 89) under conditions of a
low protein load (0.25 µg, Fig. 3C). With increasing protein, the Nkx3-1 homeodomain narrowly protected the 5' boundary of
CArG/SRE1 (88 to 79) of the SMGA promoter. Moreover, there is an
appearance of new DNase I cleavage sites 3' to the NKE1 sequence
appearing with Nkx3-1-dependent DNase I protection
(indicated by the arrowheads, Fig. 3B),
suggesting an altered DNA conformation induced by homeodomain binding
to DNA. Taken together, these results show that the homeodomain segment
of the Nkx3-1 factor (amino acids 124-184 of the protein) is capable
of binding to sequences within the proximal promoter of the SMGA gene,
one site over a consensus NKE and a second weaker binding over a
CArG/SRE.
We showed that a DNA fragment containing CArG/SRE sequences 1 and 2 was
capable of binding bacterially synthesized, purified SRF (40). When the
probe containing the NKE1/SRE1 sequence of the SMGA promoter was
incubated with purified SRF a singular, prominent protein-DNA-binding
complex was formed (Fig. 3D). This specific binding complex
was efficiently competed with unlabeled NKE/CArG probe but not with a
fragment bearing GATA-binding sites. SRF bound to the CArG/SRE1 segment
(88 to 79) of the SMGA promoter as indicated by a DNase I
footprinting experiment (Fig. 3D). At higher protein loads,
the purified SRF broadens its DNase I protection of surrounding DNA
sequences. Approximately 10-fold more protein was required to obtain
measurable SRF binding upon SRE1 than the binding observed upon SRE2 in
previous studies (40), suggesting a weak SRE1 binding relative to other
SRF-binding sites within the SMGA promoter. In support of this
observation, minimal DNase I protection was detected over the proximal
SRE1 using DNA fragments containing both SRE1 and SRE2 motifs under
conditions of limiting amounts of SRF (data not shown). These
experiments demonstrated that SRF and Nkx3-1 factors bound to
contiguous sequences within the SMGA proximal promoter and was
buttressed by the cotransfection experiments with wild type and mutant
SMGA promoter fragments (Figs. 1 and 2), which indicated that the
NKE/CArG motif within the proximal promoter is the operative
cis-acting element for Nkx3-1/SRF-mediated SMGA promoter activation.
Nkx3-1 Homeodomain Was Sufficient to Facilitate SRF Binding to the
SMGA Nke/CArG Motif--
In studies of the -cardiac actin gene
where SRF was found to cooperate with Nkx2-5/Csx to activate
transcription, the factors appeared to bind equivalent sequences,
specifically SRE motifs (41-43). Here we have mapped the binding sites
for Nkx3-1 and SRF to adjacent sequences. The Nkx3-1 homeodomain was
found to also exhibit a weak interaction with the SRE1 motif of the
SMGA promoter. Furthermore, we have shown that although SRE1 of the
SMGA promoter conforms to consensus SRE motifs (55), it is not an avid
SRF-binding site. To determine whether the binding of one factor at the
NKE/CArG element influences the binding of the other, we performed DNA binding and DNase I footprinting experiments (Fig.
4). As shown in Fig. 4A, 0.25 µg of Nkx3-1 homeodomain protein produced two complexes with the SMGA
NKE/CArG probe, and 0.1 µg of purified SRF produced a single major
protein-DNA complex. Incubating less SRF along with the probe (0.005 µg, lane 4, Fig. 4A) resulted in reduced
intensity of the SRF-containing complex. In comparison, this amount of
SRF was capable of avid binding to other SRE motifs of the SMGA gene
(40) and of other genes (28, 41, 44, 43, 55).
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Fig. 4.
Nkx3-1 homeodomain facilitates SRF binding to
the NKE/CArG element. A, lanes 1-3
represent controls to show the migration of complexes formed with
Nkx3-1 (lane 2) and SRF (lane 3) on the NKE/CArG
probe fragment. Lane 4 shows the amount of shifted probe
when reduced amounts of SRF (5 ng) are assayed. Lanes 4-7
contain the same amount of SRF (5 ng) to which 100, 200, or 400 ng
(lanes 5-7, respectively) Nkx3-1 homeodomain have been
added. Increased SRF-binding complex in addition to the two formed from
Nkx3-1 is indicated. B represents a similar experiment,
except DNase I footprinting was utilized to localize binding sites of
SRF and Nkx3-1 upon the avian SMGA promoter fragment (108 to +15).
Parallel reactions were performed with no protein added, SRF alone (0.1 µg), or SRF (0.1 µg) and Nkx3-1 (0.25-1 µg) proteins together.
The protected region of the DNA probe is shown between the
arrows (nucleotides 93 to 72), and the positions of
newly formed DNase I cleavage sites induced by Nkx3-1 protein binding
are denoted by the arrowheads.
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The addition of Nkx3-1 homeodomain to the reaction increased the
binding avidity of SRF to the Nke/CArG probe (lanes 5-7, Fig. 4A). We did not observe the formation of ternary higher
order complexes regardless of the amount of Nkx3-1 protein added to the
reaction mix. The same result was obtained in DNase I footprinting analyses (Fig. 4B). Low levels of SRF did not produce a
noticeable footprint upon the 108/+15 SMGA gene fragment (lane
2, Fig. 4B). However, with the addition of the Nkx3-1
homeodomain, the region of the DNA probe corresponding to the Nke/CArG1
element was protected from digestion. Significant binding was observed
even at the lowest amount of Nkx3-1 added into the reaction (0.25 µ g, lane 3, Fig. 4B). Also as the Nkx3-1
homeodomain content was increased, the added Nkx3-1 homeodomain and SRF
completely protected the Nke/CArG portion of the probe (lane
5). Additionally, there was the appearance of new DNase I cleavage
sites with added Nkx3-1 as that observed when the homeodomain was
assayed singularly (Fig. 3C). These experiments demonstrate
that when included in the same reaction Nkx3-1 and SRF bind adjacent
elements within the SMGA promoter, namely the Nke/CArG1 motif, and
there is enhanced SRF binding when Nkx3-1 is added to the reaction.
Evaluation of the reciprocal interaction, adding increasing amounts of
SRF with a constant amount of Nkx3-1 homeodomain, did not enhance
Nkx3-1 binding (data not shown). Thus, SRF binding to the Nke/CArG was
facilitated by the Nkx3-1 homeodomain.
Nkx3-1 Associated with SRF Independent of DNA Binding--
We did
not observe the formation of higher order complexes in gel shift
experiments with Nkx3-1 homeodomain and SRF proteins incubated together
with the NKE/CArG probe. Similar results have been observed for
interactions of SRF with other proteins such as Phox1 (48, 56, 57) and
Nkx2-5 (41-43, 50), indicating that the formation of a ternary complex
if they exist is transient or unstable and could not be resolved under
the binding and gel electrophoresis conditions employed in our
experiments. We wanted to determine if the synergy shared between
Nkx3-1 and SRF in the activation of SMGA transcription was attributable
to protein-protein associations; therefore, we employed
coimmunoprecipitation assays to test if these two factors are capable
of interacting in vivo. Cellular lysates were derived from
NIH 3T3 cells following transfection of the cells with vectors
expressing HA epitope-tagged SRF or Nkx3-1. For these experiments, we
utilized conditions that maximized the expression of the HA-tagged
protein, and control experiments showed that immunoreactive, HA-tagged
protein was easily detected by Western assay (Fig. 1E). The
Nkx3-1 expression vector contained the entire coding sequence of the
protein cloned in-frame with the HA epitope so that we could test for
protein interactions utilizing the complete Nkx3-1 protein. Equivalent
protein content of lysates from cells that were overexpressing SRF or
Nkx3-1 were incubated in the presence of an SRF antibody, and
antibody-SRF complexes were then collected by precipitation with
protein A/G-agarose beads. The SRF antibody was derived from a peptide
antigen representing amino acids 486-505 of the human SRF polypeptide,
and we have demonstrated previously that it reacts specifically with
the native and bacterially expressed protein (40-44, 50). The
collected material was then analyzed by SDS-PAGE, immunoblotted, and
probed with an anti-HA epitope monoclonal specific antibody. As shown in Fig. 5, a band corresponding to
HA-tagged Nkx3-1 was recognized by the HA epitope antibody only when
the SRF antibody was included in the initial incubation reaction. No
Nkx3-1 was present when the SRF antibody was not included in the
initial reaction or when an irrelevant antibody was included in the
reaction (data not shown). Immunoreactive Nkx3-1 was demonstrated by
the presence of an antigen species of the predicted molecular weight in
lysates transfected with the Nkx3-1 expression vector but absent in
untransfected cell lysates and in cells transfected singularly with the
SRF expression vector. These results demonstrate that Nkx3-1 is
precipitated from cellular lysates using an SRF-specific antibody only
when SRF is also present in the lysate, indicating that these two
proteins are capable of forming complexes via protein-protein
interactions.
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Fig. 5.
Nkx3-1 and SRF associate in vivo,
and their SMGA promoter coactivation depends upon DNA binding of
SRF and C-terminal activation domains. A, fibroblasts
were transfected with either pCGN-Nkx3-1 or pCGN SRF expression vectors
as described. 48 h post-transfection cells were harvested, and
cellular lysates were prepared as detailed under "Materials and
Methods." Equivalent quantities of the cell lysates (500 µg) were
mixed, after which the mixtures were incubated with anti-SRF
(lane 2) or preimmune sera (lane 3), and the
immune complexes were collected by incubation with protein A-agarose
beads and centrifugation. Proteins within the pelleted complexes were
fractionated by 10% SDS-PAGE, transferred to membranes, and the
membranes probed with an anti-HA epitope monoclonal antibody to
visualize the HA epitope-tagged Nkx3-1 protein. Lane 1 represents the results obtained from analyzing 50 µg of the lysate
from the Nkx3-1-transfected cells showing the migration of the Nkx3-1
protein (10% Input). B, cotransfection
experiments were performed in CV-1 cells using the 176 SMGA
promoter-luciferase gene reporter as described. In these experiments
the luciferase activity generated with expression vectors encoding
Nkx3-1 and mutant SRF proteins (SRFPM1 and
SRFCT) was compared with the activity
demonstrated from cells transfected with native SRF (SRF WT)
and mNkx3-1 vectors. SRFpm1 is a dominant negative molecule and
contains three mutations that disrupt DNA binding but not dimerization.
The SRF CT mutant lacks the C-terminal activation domain (deleted
amino acids 222-508). All transfections contained equivalent DNA
quantities by the addition of empty CMV promoter vector as described
previously.
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The MADS Box and C-terminal Domains of SRF and the Homeobox Domain
of Nkx3-1 Was Required for Coactivation of the SMGA Promoter--
We
asked if specific domains of these factors are important for functional
coactivation of the SMGA promoter. The MADS box domain of SRF has been
demonstrated to convey gene regulatory activity through associations
with other factors, as well as being the DNA binding domain of the
molecule (42, 43, 55, 58). The SRFpm1 molecule contains
three amino acid substitutions (Arg to Leu at position 143, Lys to Ala
at position 145, and Leu to Gly at position 146) that interrupt DNA
binding but do not alter the ability of the protein to dimerize (59).
Cotransfection of the SRFpm1 mutant blocked SRF-mediated
transcriptional activation of -skeletal and -cardiac actin
promoters (43, 46). Moreover, the SRFpm1 mutant protein has
been demonstrated to inhibit transcription from the SMGA promoter in
differentiated smooth muscle (40) and inhibit the expression of
multiple smooth muscle marker proteins (calponin, SM22, and SMAA) in
developing coronary smooth muscle cells (39). Substitution of
SRFpm1 for native SRF in our cotransfection experiments
completely abolished the Nkx3-1/SRF-mediated transcriptional activation
of the SMGA promoter (Fig. 5B). Therefore, these data indicate that coactivation of the SMGA promoter by SRF and Nkx3-1 required SRF DNA binding activity. We next asked whether the
transactivation domain within the C-terminal region of the molecule was
necessary for the Nkx3-1/SRF-mediated transcriptional activation of
SMGA. Coexpression of an SRF molecule truncated at amino acid 338, lacking the C-terminal activation domain (41, 60), with native Nkx3-1 reduced promoter activity from the SMGA 176-bp construct by ~75% (Fig. 4B), indicating that the coactivation of SMGA
transcription by SRF and Nkx3-1 is directed, at least in part, through
the C-terminal transactivation domain of SRF.
Although there is significant homology between mNkx3-1 and
bagpipe within their homeodomains, which leads to the
classification of mNkx3-1 as an NK3 homologue (10, 11, 14), these
proteins do not exhibit significant homologies at other segments or
domains. This differs from the relationships shown for other
Drosophila nk genes and their mammalian
counterparts, such as tinman and nkx2-5, which
have homologous segments outside the homeodomain thought to be
important for protein-protein interactions and perhaps transactivation
activities (6, 41, 42). To test if there were specific regions of
Nkx3-1 that were important for its transcriptional activation activity,
we generated mutant Nkx3-1 proteins and assayed their activity upon the
SMGA 176 promoter fragment singularly and in the presence of SRF
(Fig. 6). When the 53 amino acids on the
C-terminal side of the homeodomain were deleted from the molecule (Nkx3-1 CT), there was a 15-fold increased activity of the mutant protein on the SMGA promoter compared with the native protein (Fig.
6A). These data indicate that the C terminus of Nkx3-1
contains a transcriptional repressor domain, similar to that located in the C-terminal domain of Nkx2-5 (42, 43). Overexpression of the Nkx3-1
homeodomain resulted in 7-fold greater transcriptional activity upon
the SMGA promoter than observed for the wild type mNkx3-1 molecule.
Lysates of transfected cells analyzed by Western blotting demonstrated
that the mutant proteins were adequately expressed in these experiments
(Fig. 6B). Thus, the DNA binding activity of the Nkx3-1
homeodomain (Fig. 3) is capable of transducing a significant
transcriptional response. The Nkx3-1 mutant containing only the
homeodomain was sufficient to elicit a measurable transcriptional response in experiments where the Nkx3-1 proteins were coexpressed with
native SRF (Fig. 6B). In addition, these cotransfection
assays indicate that the C-terminal region of the mNkx3-1 molecule may play an important inhibitory role that is relieved by the association with SRF. Taken together, our experiments show that regulatory regions
in addition to the SRF and Nkx3-1 DNA binding domains are necessary for
the transcriptional coactivation of SMGA promoter.
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Fig. 6.
The inhibitory C-terminal domain of Nkx3-1
activity is required for SMGA promoter coactivation with SRF. CMV
promoter expression vector pCGN was utilized to encode HA
epitope-tagged fusion Nkx3-1 wild type and mutant proteins. Expressed
mutant proteins are Nkx3-1 NT, a deletion of amino acids 1-124,
Nkx3-1 CT, a deletion of amino acids 184-237 and the homeodomain
alone (deletion of amino acids 1-124 and 184-237). These vectors were
cotransfected with the SMGA 176 promoter-luciferase reporter
construct with (C) or without (A) added SRF
expression vector in CV-1 cells. A, the fold activity was
calculated by comparing the luciferase activity in cells transfected
with the various Nkx3-1 mutant protein vectors with that obtained from
overexpressing the native mNkx3-1 protein (Nkx3.1 WT). The
amount of luciferase activity generated from native mNkx3-1 (WT) was
assigned a value of 1.0, and the activities of the mutant proteins were
compared with the wild type activity. B represents a
Western blot of cellular lysates derived from cells transfected with
empty CMV promoter vector (lane 1), native mNkx3-1
(WT, lane 2), Nkx3-1 HOM (lane 3),
Nkx3-1 CT (lane 4), Nkx3-1 NT (lane 5), and
Nkx3-1 homeodomain (HOM, lane 6) probed with an
anti-HA epitope monoclonal antibody. Antibody-antigen complexes (shown
by the arrows to the left of the autoradiogram)
were revealed by chemiluminescence as described previously.
C, wild type (WT) and mutant mNkx3-1 vectors (0.8 µg) were assayed with added pCGN-SRF (0.2 µg) expression vector as
described previously. In these experiments the fold activity was
calculated from luciferase activities in lysates of cells transfected
with SRF and Nkx3-1 compared with the activity measured with empty pCGN
vector.
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DISCUSSION |
In Drosophila, the NK3 homeodomain protein
bagpipe is expressed in the visceral mesoderm region of the
embryo and has been implicated in the development of visceral and
gonadal mesoderm (8, 9). Although the expression pattern of the
vertebrate bagpipe homologue Nkx3-1 has been described (10,
11, 14), there is little information regarding molecular targets
accorded by this developmentally and androgen-regulated transcription
factor. Previous studies (17-21, 37, 39, 40, 51, 61) demonstrated that
the SMGA isoform is a marker for visceral as well as vascular mesoderm
differentiation. We asked if a vertebrate homologue of bagpipe, murine Nkx3-1, could regulate SMGA gene activity.
As observed in cotransfection experiments, we found that the SMGA promoter was transactivated by SRF with mNkx3-1. The transcriptional activation elicited by Nkx3-1 was significant and specific since this
factor was not capable of stimulating transcription from a vector
bearing response elements derived from the cardiac -actin gene.
Furthermore, another Nkx factor, Nkx2-5, could not substitute for
Nkx3-1 in transactivating the SMGA promoter (Fig. 1). Our results
showed that the initial 176 bp of the SMGA promoter was sufficient for
the synergistic activation of the SMGA promoter by SRF and Nkx3-1. This
short segment of the promoter is highly conserved across vertebrate
evolution (21) and plays a role in cell-specific transcription of the
gene (21, 40, 61). Although both CArG/SRE motifs within the 176-bp
promoter segment influenced SMGA transcription, CArG/SRE1 was
demonstrated to be necessary for the synergistic Nkx3-1/SRF
transactivation, and the proteins were found to bind with adjacent but
overlapping motifs surrounding and including this motif (Fig. 3).
Analysis of this novel and vital binding motif, designated as an
NKE/CArG element, supports earlier studies that predicted a role for
its involvement in directing transcription of the SMGA gene (21, 40).
Therefore, our present studies substantiate SMGA as a Nkx3-1 regulatory
target and define a mechanism by which this homeodomain is capable of
controlling gene transcription. As Nkx3-1 is expressed within the
somatic mesoderm in early embryos (10, 11) and in differing tissues
throughout adulthood (11, 14), our data set a paradigm by which this
gene product may regulate other genes and directly influence
developmental gene programs.
Promoter deletion analyses have previously demonstrated two flanking
regions of the SMGA gene that convey a smooth muscle-specific, positive
transcriptional response (21). These two regions, called the smooth
muscle specifier and the smooth muscle modulator domains (21), are
heavily dependent upon SRF for their positive transcriptional activity
(37, 40). Furthermore, the proximal segment of the smooth muscle
specifier domain (sequences ~200 bp flanking the gene) containing 2 CArG/SRE motifs was observed to bind SRF complexes in smooth muscle
cell nuclear lysates as well as purified SRF (40). We demonstrated here
that both of the CArG/SRE motifs within the initial 200 bp flanking the
SMGA gene are capable of SRF binding, albeit with differing affinities.
CArG/SRE1 of the SMGA promoter weakly bound SRF alone. This binding was
facilitated by the presence of the Nkx3-1 homeodomain via binding to a
DNA sequence within and adjacent to CArG/SRE1 of the SMGA promoter as
shown in Fig. 3. The Nkx3-1-binding site adjacent to CArG/SRE1 on the
SMGA promoter, 5'-CTAAGTG-3' (minus strand) is identical in structure
to the consensus Nkx2 core-binding sequences 5'-TNAAGTG-3' (41, 52,
62). Nkx3-1 has been shown to bind synthetic oligonucleotides bearing
the Nkx2 core-binding sequence (11); therefore, our studies demonstrate
that naturally occurring binding sites for vertebrate NK3 proteins fit
the general NKE consensus. Our results differ from the studies that
evaluated the role of Nkx2-5/SRF-facilitated binding and activation of
the cardiac -actin gene, where both proteins bound the same CArG/SRE
motif (41-43, 50), because there is the juxtaposition of an NKE with a
CArG/SRE element within the SMGA proximal promoter. However, we did
observe a weak, secondary binding of Nkx3-1 to the SRE sequence, and
our data cannot formally eliminate the possibility that this binding is
required for facilitated SRF binding. In addition,
Nkx3-1/SRF-dependent SMGA gene regulation is mediated by
sequences that are highly conserved among species, suggesting a strict
maintenance of this regulatory paradigm throughout evolution for
cell-specific gene transcription.
Although smooth muscle-specific expression of SMGA is strongly
dependent upon SRF activity (40), SRF appears to cooperate with other
factors to regulate SMGA transcription. The interaction of SRF with
accessory factors has been demonstrated in a number of systems. Studies
of c-fos gene regulation has led to the identification of
several accessory factors, including SAP-1, Elk-1, and Phox-1 (48,
63-65). These factors appear to be expressed ubiquitously and together
with SRF potentiate the transcriptional activity of the
c-fos gene, although the exact regulatory mechanisms are somewhat distinct. Additionally, SRF interaction with cell-specific regulatory factors has been characterized. MCM1, the yeast SRF homologue, is influenced by the interactions with an array of accessory
factors that either activate or repress genes in a cell-specific and
temporal pattern (66). In vertebrates, the cardiac-specific homeodomain
Nkx2-5 makes specific physical interactions with SRF producing an
enhanced transcription of cardiac-specific genes (41-43, 53).
Similarly, SRF interaction with skeletal muscle cell-restricted basic
helix-loop-helix proteins of the MyoD family may directly influence the
expression of muscle-specific genes (47) including SRF itself (44, 60).
In many of these systems, SRF is regarded as providing the platform for
the recruitment of accessory factors, thus affecting the regulatory
pattern. It is clear that SRF binds avidly to multiple sites within the
SMGA promoter, any of which may provide the opportunity for
SRF-dependent interactions (21, 37, 40). Nkx3-1 facilitated
the binding of SRF to the SMGA CArG/SRE1 and surrounding sequences to
affect the synergistic activation of SMGA promoter. The fact that SRF binding was required for this activation (Fig. 5) and that SRF activation of an SMGA promoter in the presence of a mutated high affinity SRE (CArG/SRE2) required the coexpression of Nkx3-1 (Fig. 2)
correlates well with Nkx3-1-dependent facilitated SRF
binding to the SMGA promoter. Thus, SRF interactions with accessory
proteins may be more complex than previously anticipated and may depend to a greater extent upon the composition of regulatory elements as a
mechanism to ensure cell-specific gene activation.
Combinatorial synergy of Nkx3-1 and SRF for the regulation of SMGA
transcription might be achieved at several levels including enhanced
protein complex interaction, changes in DNA conformation, and/or
release of a repressor protein activity. The native polypeptides are
able to form protein-protein interactions in the absence of DNA binding
(Fig. 5) which may serve to alter protein complex conformations and
enhance the interaction of other protein domains with general
transcription factors. This appears to be an operational mechanism for
regulation of the -skeletal actin gene via the interaction of SRF,
TEF-1, and Sp1 (67). Alternatively, since a mutation of the NKE
sequence of the Nke/CArG element did not totally obstruct activation of
the 176 SMGA promoter fragment (Fig. 2), it is possible that
protein-protein interactions allow activation without Nkx3-1 binding.
Phox, a human homologue of Mhox, interacts with SRF to enhance the
exchange of SRF with its binding site in the c-fos promoter
and does not require specific homeodomain DNA binding activity (48, 51,
68). However, Mhox and chicken HOXB4 (an avian Dfd paralogue), like
Nkx2-5 (Fig. 1), was incapable of activating the SMGA 176 promoter in
the presence of SRF (data not shown). Thus, SRF is capable of
interacting with homeodomain factors in a way that alters the activity
of the SRF molecule.
We noted an appearance of new DNase I-sensitive cleavage sites with
Nkx3-1 binding upon the SMGA promoter fragment with or without SRF
present (Figs. 3 and 4), indicating that the binding of the Nkx3-1
homeodomain alters the conformation of the DNA 5' to its binding site.
This might provide the means for enhancing SRF binding to the
relatively weak CArG/SRE1 motif, in essence making this motif more
attractive for SRF binding and thus facilitating binding. A variety of
homeodomain (69-71) and MADS box proteins, including SRF (58, 72), are
capable of bending DNA, thereby increasing the specificity or affinity
of DNA binding. DNA bending and the formation of looping structures
appears to be a general mechanism for bringing transcriptional
activator complexes together, providing a mechanism for cooperativity
among complexes separated along the DNA by some distances (73-76).
Therefore, the altered chromatin structure induced by way of Nkx3-1
binding the NKE/CArG element might allow DNA bending that brings
complexes bound along the SMGA promoter together for appropriate
cell-specific transcription. It is also possible that Nkx3-1·SRF
complexes compete for binding with negative acting
trans-factors. Synergistic activation of the -cardiac
actin via Nkx2-5·SRF complexes includes the displacement of the
negative acting factor YY1 from SRE2 of the promoter (50). Analysis of
the SMGA promoter identified CArG/SRE1 as a potential YY1-binding site
(21) which might function to repress SMGA synthesis. When the distal
CArG/SRE was removed from the SMGA 176 promoter fragment, it was not
activated by Nkx3-1·SRF complexes. In these experiments, a high
affinity SRE which is an integral motif of the positive acting SMGA
specifier was removed from the test promoter. Because of the dependence
upon SRF for SMGA transcription (40), the removal of a positive acting
cis element (CArG/SRE2) may allow the dominance of negative
regulatory influences, such as YY1, over the positive acting
Nkx3-1·SRF complex.
It is clear that cell-specific transcription of the SMGA gene requires
complex interactions directed by multiple cis-acting elements. One way of stimulating the SMGA promoter involves the cooperative interactions of SRF and the vertebrate NK3 homologue Nkx3-1
upon a specific segment of the promoter, the NKE/CArG element. Therefore, SMGA gene activation may require increased levels of SRF
with the appearance of Nkx3-1 to foster cooperative
trans-factor complex formation. Consistent with this
hypothesis, we have recently demonstrated that the developmentally
regulated expression of SRF is a key determinant of SMGA gene
regulation during smooth muscle myogenesis (40). In addition, the
appearance of Nkx3-1 within the smooth muscle component of visceral
organ vessels such as the kidney (14) corresponds with the onset of
SGMA expression detected by in situ hybridization analyses
(17). Therefore, the combinatorial interaction of SRF with Nkx3-1 may
directly influence the cell-specific activation of the SMGA gene.
Moreover, Nkx3-1·SRF complexes may be an important part of the
regulatory machinery of other genes during embryogenesis and in adult
tissues. Nkx3-1 is expressed in somites, brain, blood vessels, kidney, and the male reproductive tract (10, 11, 14). Furthermore, the recent
loss of function of nkx3-1 gene knock-out in mouse demonstrated defects in morphogenesis and cellular differentiation of
male reproductive organs, specifically the prostrate (77), indicating
that like other NK-related genes, nkx3-1 plays an essential role in organogenesis. The demonstration of developmentally regulated SMGA expression within the male reproductive tract and germ cells (38)
indicates that SMGA may be regulated within these cells by an
Nkx3-1-dependent mechanism.
| |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants R01HL50422, P01HL49953 (to R. J. S.), 5P60HL38639, and
HL59956 (to W. E. Z.), United States Department of Agriculture Grant
9404341, and American Heart Association Grant AL-G-940003 (to
W. E. Z.).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 an NIA postdoctoral training grant from the National
Institutes of Health.
To whom correspondence should be addressed: Dept. of Cellular
and Molecular Biology, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030. Tel.: 713-798-6649; Fax: 713-798-7799; E-mail:
schwartz@bcm.tmc.edu.
Published, JBC Papers in Press, September 18, 2000, DOI 10.1074/jbc.M006532200
| |
ABBREVIATIONS |
The abbreviations used are:
SMGA, smooth muscle
-actin;
SRF, serum response factor;
SRE, SRF-binding element;
bp, base pair;
PCR, polymerase chain reaction;
GST, glutathione
S-transferase;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel electrophoresis;
HA, hemagglutinin;
WT, wild type;
CMV, cytomegalovirus.
| |
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TOP
ABSTRACT
INTRODUCTION
