From the Department of Neuroscience (D13), Biomedical
Research Center, Osaka University Graduate School of Medicine,
Yamadaoka 2-2, Suita City, Osaka 565-0871, Japan and the
§ Second Department of Internal Medicine, Ehime
University School of Medicine, Ehime 791-0295, Japan
Received for publication, February 6, 2001, and in revised form, February 26, 2001
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ABSTRACT |
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Tropomyosin (TM) is a regulatory protein of
actomyosin system. Muscle type-specific expression of TM isoforms is
generated from different genes and by alternative splicing. The vertebrate muscular system consists of skeletal, cardiac, and
smooth muscles, all of which are derived from the mesoderm. The
molecular basis for muscle contraction is the actomyosin system, which
converts the chemical energy of ATP into mechanochemical force (1).
However, the actomyosin system is differentially regulated depending on
the presence of different contractile proteins (2-4), whose expression
patterns are muscle-type dependent. Therefore, these contractile
proteins are considered to be muscle-specific molecular markers.
Despite intensive study in this area, only one general concept
concerning the transcription of muscle-specific marker gene has been
agreed upon. Serum response factor
(SRF)1 and its DNA binding
sequence, CArG box (CC(A/T)6GG), are thought to comprise
one of the core machineries for the muscle-specific transcription of
the skeletal Tropomyosin (TM), which is a regulatory protein of the actomyosin
system, has several isoforms that are generated from different genes
and by alternative splicing in a muscle-specific context (reviewed in
Ref. 20). Three distinct chicken TM genes, Here, we investigated the SMC-specific transcription of the Construction of Reporter Genes and Expression
Vectors--
Promoter region of the Antibodies--
An anti-Barx1b antibody was purified from
anti-GST-Barx1b-C (amino acids 195-247) rabbit serum by the sequential
application of GST and GST-Barx1b-C affinity columns. Anti-human SRF
polyclonal, anti-GST polyclonal, and anti-Myc monoclonal antibodies
were purchased from Santa Cruz Biotechnology. An anti-FLAG M2 antibody
was obtained from Sigma.
Cell Culture and Transfection--
Differentiated chicken
gizzard SMCs were cultured as described previously (26). Mouse
C3H10T1/2 fibroblasts grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum were transfected for 48 h
with 1 µg of Purification of GST-SRF and Barx1b--
GST-SRF or GST-Barx1b
derivatives were transformed into Escherichia coli BL21.
Protein expression was induced by 1 mM
isopropyl- Pull-down Assay--
A series of GST-SRF proteins were purified
and coupled to glutathione-Sepharose 4B gels. Barx1b derivatives were
translated in vitro using TNT-quick coupled transcription
and translation system kit with [35S]methionine
(Promega). The binding reaction was carried out with 2 µg of GST-SRF
and 10 µl of in vitro translated Barx1b in 500 µl of
binding buffer (20 mM Tris-HCl, pH 7.5, 80 mM
KCl, 10% glycerol, 0.05% Nonidet P-40, 1 mM EDTA, and 5 mM MgCl2) at 4 °C for 2 h. GST-SRF
bound Sepharose was washed three times, treated with 2% SDS sample
buffer, and analyzed by SDS-PAGE. Labeled proteins were visualized by autoradiography.
Gel Mobility Shift Assay--
Oligonucleotide probes were
subcloned into pBluescript SK(+) (Stratagene) and excised by
BamHI and HindIII digestion. The probes were
labeled with [32P]dCTP and purified with QIA quick
nucleotide removal kit (Qiagen). Nuclear extracts from chicken gizzards
at embryonic day 15 (E15) were prepared by the method of de Jonq
et al. (27). Chicken gizzard nuclear extracts and the
labeled In Situ Hybridization--
Whole chicken embryos or gizzards at
E15 were embedded with Tissue-Tek (SAKURA) and cut into 12-15-µm
thick by cryostat (Bright, Huntingdon, United Kingdom). The
sections were fixed and hybridized with cRNA probes as described
previously (8). The fluorescent signal was detected using the TSA Plus
DNP AP system (PerkinElmer Life Sciences Inc.) and HNPP/Fast Red TR
(Roche Molecular Biochemicals) according to the manufacturer's
recommendation. A Immunoprecipitation--
10T1/2 cells were seeded at a density
of 9 × 105 cells per 10-cm plate, and transfected
with 2 µg of pcDNA3.1-wt-SRF-FLAG and 2 µg of
pCS2+MT-wt-Barx1b. Transfected cells were washed three times with
phosphate-buffered saline and lysed with 600 µl of IP buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2,
100 mM KCl, 0.5 mM EDTA, 1 mM
dithiothreitol, 0.5% Nonidet P-40, 50 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin, and 2 µg/ml aprotinin). Cell lysates were incubated with 200 µg/ml DNase I and 10 µg/ml RNase A for 30 min at 26 °C, and centrifuged at 100,000 × g for 15 min. The supernatant (250 µl) was rocked with 2.5 µg of anti-Barx1b
polyclonal or 2 µg of anti-SRF polyclonal antibody for 12 h at
4 °C, then protein A-Sepharose gels (Amersham Pharmacia Biotech)
were added. After 6 h incubation, the gels were washed three times
and treated with 2% SDS sample buffer. Samples were separated by
SDS-PAGE and subjected to Western blotting with mouse anti-FLAG M2 or
anti-Myc monoclonal antibody. Target proteins were detected with
peroxidase linked anti-mouse IgG (Amersham Pharmacia Biotech) and
Supersignal West reagent (Pierce).
SRF-CArG-dependent Transcription of the Chicken Barx1b, A Novel Homologue of Barx Homeoprotein
Family--
To elucidate the mechanism of SMC-specific transcription,
we screened the homeobox genes from a gizzard SMC cDNA library and cloned the gax, hox cluster (hoxA5 and
hoxB3-B5), and a novel clone of Barx family
genes. This clone was 1,170 base pairs in length and encoded a single
open reading frame of 247 amino acids (Fig.
2A). Barx1 cDNAs have been
recently isolated from mouse (28), human (29), and chicken (30). The
novel clone shows 82-84% total identity with known Barx1 homologues.
The homeodomain of this clone is completely identical with that of
other Barx1 family members, including unique Thr and Tyr residues in
the third helix. It also shares four leucine repeats and a
Barx-specific domain at the COOH terminus (Fig. 2A). On the
other hand, the novel clone has a long NH2-terminal
sequence and shows a substitution of 13 amino acids in the
NH2 terminus (amino acids 102-130) and 10 amino acids in
the COOH terminus (amino acids 228-247) compared with other Barx1
family members. Based on these findings, we called this novel homologue
chicken barx1b. In addition, we noticed that there is a
sequence similarity between the Barx1b and Nkx-2.5 families (see
"Discussion"). They share the consensus sequence of the FIL domain
(31) at their NH2 terminus, and contain basic residue-rich
sequence around the NH2-terminal boundary of the homeodomain (Fig. 2, B and C).
Expression of Barx1b in Chicken Embryos--
The expression
patterns of the cloned homeobox genes, including
barx1b in E15 chicken embryos, were compared with
those of Coordinated Activation of the Interaction between SRF and Barx1b--
The interacting domains of
SRF and Barx1b were mapped by a pull-down assay.
35S-Labeled Barx1b derivatives were incubated with GST-SRF
immobilized on glutathione-Sepharose gels (Fig.
5A). Wild-type Barx1b (wt) and
its COOH terminus deletion (
We then examined whether the minimum interaction domain of SRF and
Barx1b are actually involved in Ternary Complex Formation between SRF, the CArG Box, and
Barx1b--
To study the interaction of SRF and Barx1b with the Here, we demonstrated the SMC-specific transcription of the Although the transcription of muscle-specific genes has been
extensively studied, no general mechanism explaining muscle-specific transcription has been revealed except for the SRF and CArG box interaction. SRF is dominantly expressed in all three muscle types, which are derived from mesoderm (Fig. 3B and Ref. 16) and is involved in muscle-specific gene transcription. However, SRF also plays
a vital role in the expression of immediate-early genes (32). These
diverse roles of SRF might be regulated by different co-activators for
different genes. In fact, ternary complex factor recruits SRF and
potentiates the transactivation of the c-fos gene (32).
Therefore, it is likely that other co-activators of SRF confer
muscle-specific transcription. Currently, candidates for such
co-activators include members of the homeoprotein family. Grueneberg
et al. (18) cloned PHOX1 from a human
glioblastoma cDNA library, and found it to enhance the affinity of
SRF for DNA. Mhox, a murine homologue of PHOX1,
is widely distributed in mesoderm-derived cells, including those of
skeletal and smooth muscles and the heart (33). Hautmann et
al. (34) reported that the angiotensin-II-stimulated smooth
muscle Several studies have reported the isolation of homeobox genes from
smooth muscle tissues. Two independent groups cloned the Hox
cluster genes from rat and human aorta cDNA libraries (41, 42), but
the possible involvement of such Hox cluster genes in the
transactivation of SMC marker genes remains unknown. Walsh et
al. (43) isolated Gax from cultured rat aortic vascular
smooth muscle cells. Gax is a homeobox gene expressed
in all three muscle lineages, and its expression is rapidly
down-regulated during the G0 to G1 transition
in vascular smooth muscle cells (43). In this study, we screened a
chicken gizzard cDNA library using degenerative probes targeted
against a most conserved third helix region of the homeodomain and
cloned six homeobox genes; barx1b, gax, and
hox cluster genes (hox A5, B3, B4, and
B5). Of these, Barx1b mRNA exclusively localized to the
SMC layer of the upper digestive organs, their attached arteries, and
to craniofacial structures (Fig. 3, C and F). The
distribution of Barx1b completely overlapped that of The Bar family was first identified in the Drosophila Bar
locus (BarH1 and BarH2) (44). A vertebrate homologue of the
Drosophila Bar family, Barx, is composed of Barx1 and Barx2
(28, 45). The homeodomain of Barx1 is completely conserved among
species (Fig. 2A) and is 87% identical to those of the Barx2 family
(45). The expression pattern of Barx1b is similar to that of other
Barx1 family members, and includes the craniofacial structures,
stomach, and limb bud (28, 30). Barx1b and chicken Barx1,
which was cloned from a chicken head cDNA library, show completely
different sequences at their NH2 and COOH termini (Fig.
2A). Barx1b has a long NH2 terminus compared
with other Barx1 family members, therefore, it is a novel homologue of
the Barx1 family. Saito et al. (31) defined a characteristic
domain consisting of Phe, Iso, and Leu at the NH2 terminus
of the Bar family, and named this sequence a "FIL" domain. Chicken
Barx1b also contains a FIL domain at its NH2 terminus (Fig.
2B). It has been reported that NK2 family members share a
"TN" domain at their NH2 terminus (reviewed in Ref.
46). Ranganayakulu et al. (47) reported that a cardiogenic domain of tinaman is mapped to NH2-terminal
amino acids 1-52, which include the TN domain. Interestingly, we
noticed that the FIL consensus sequence is also present in the TN
domain of the NK2 family proteins. Furthermore, the Barx1b homeodomain
shows about 70% similarity with that of the NK2 family. In addition to
these sequence similarities, both Barx1b and Nkx-2.5/Csx show unique
tissue-restricted expression patterns, respectively. Therefore, there
might be some cross-relationships between the Barx and NK2 families.
We investigated the direct interaction between Barx1b and SRF using
pull-down (Fig. 5, A and B), immunoprecipitation
(Fig. 5C), and gel mobility shift (Fig. 6) analyses. The
results demonstrated that Barx1b and SRF bind to each other, and their
binding involves 11 residues of the NH2-terminal arm plus
the homeodomain of Barx1b and the MADS domain of SRF, and is
independent of DNA (Fig. 5, A and B). A region
around the NH2-terminal boundary of the Barx1b homeodomain
contains an Arg/Lys-rich sequence and is conserved among all Barx1
family members (Fig. 2A). Interestingly, NK2 family also
contains these basic residues around the NH2 boundary of the homeodomain. Chen et al. (7) reported that Nkx-2.5/Csx requires the NH2-terminal arm plus the homeodomain for SRF
binding. Thus, the basic residues around the NH2 terminus
of the homeodomain of Barx1b (and possibly all Barx1 family members)
and of Nkx-2.5/Csx might be critical for SRF binding. Our gel mobility
shift assay revealed the involvement of Barx1b in a ternary complex
with SRF and CArG box (Fig. 6). Although Barx1b alone did not directly bind to Recently, GATA family transcription factors have been shown to take
part in muscle gene regulation, especially in the heart. At present,
there are six known GATA proteins (GATA-1 to -6), which are divided
into two subgroups. One includes GATA-1, -2, and -3, which are
important for the differentiation of hematopoietic cells (49), and the
other consist of GATA-4, -5, and -6, which are expressed in variety of
tissues, including the heart, gut, lung, and urogenital tracts (50).
Among the latter group, GATA-4 is restrictively expressed in the heart
and potentiates cardiac muscle genes with Nkx-2.5/Csx (48) or SRF (51).
GATA-6 is also expressed in the heart, but cannot substitute for GATA-4 in interacting with Nkx-2.5/Csx (52). Among the GATA family members,
only GATA-6 is known to be expressed in vascular SMCs (53), but no one
has reported its involvement in the transcription of SMC genes. In a
preliminary experiment, we found that co-expression of GATA-6 with SRF
and Barx1b potentiated the In this study, we showed the SMC-specific transcription of the -TM
isoforms in chicken skeletal and smooth muscles are encoded by a single gene and transcribed from the same promoter. We previously reported a
smooth muscle cell (SMC) phenotype-dependent change in
-TM expression (Kashiwada, K., Nishida, W., Hayashi, K., Ozawa, K., Yamanaka, Y., Saga, H., Yamashita, T., Tohyama, M., Shimada, S., Sato,
K., and Sobue, K. (1997) J. Biol. Chem. 272, 15396-15404), and identified
-TM as an SMC-differentiation marker.
Here, we characterized the transcriptional machinery of the
-TM gene
in SMCs. Promoter and gel mobility shift analyses revealed an
obligatory role for serum response factor and its interaction
with the CArG box sequence in the SMC-specific transcription of the
-TM gene in differentiated SMCs. We further isolated a novel
homologue of the Barx homeoprotein family, Barx1b, from chicken
gizzard. Barx1b was exclusively localized to SMCs of the upper
digestive organs and their attached arteries and to craniofacial
structures. Serum response factor and Barx1b bound each other directly,
coordinately transactivated the
-TM gene in differentiated SMCs and
heterologous cells, and formed a ternary complex with a CArG probe.
Taken together, these results suggest that SRF and Barx1b are
coordinately involved in the SMC-specific transcription of the
-TM
gene in the upper digestive organs and their attached arteries.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actin (5),
caldesmon (6), cardiac
-actin (7),
1-integrin (8), SM22
(9),
telokin (10), smooth muscle myosin heavy chain
(11), smooth muscle
-actin (12),
calponin (13), and desmin (14) genes. SRF was
originally identified as a transcription factor of the c-fos gene (15). It is expressed at high levels in all three muscles (16),
and at a far lower level in non-muscle cells (15). A recent study using
SRF-deficient mice revealed the misformation of mesoderm leading to
fetal death at around embryonic day 12.5; no skeletal, cardiac, and
smooth muscle
-actin transcripts were detected in these mice (17).
These findings suggest the possible involvement of co-activators in the
SRF and CArG interaction that confer the muscle specificity on the
transactivation of muscle-specific marker genes. One set of candidates
for such co-activators includes members of the homeoprotein family.
Grueneberg et al. (18) first reported a paired
class homeoprotein, Phox1, which enhances the DNA binding ability of
SRF (18). In addition, two NK family homeoproteins have been reported
as co-activators of SRF. Nkx-2.5/Csx and SRF activate the
cardiac
-actin transcription (7) and Carson
et al. (19) reported that Nkx-3.1 potentiates the
transcription of the smooth muscle
-actin gene
with SRF.
-TM,
-TM, and
cardiac-TM, have been identified (20). In chicken smooth muscle cells
(SMCs), TM forms an
/
heterodimer. We reported previously that
-TM exhibits a change in its isoform from a smooth muscle-type
(
-TMsm) to a fibroblast one (
-TM-F1 and
-TM-F2), and that the
expression of smooth muscle-type
-TM is down-regulated at the
mRNA and protein levels during the de-differentiation of vascular
and visceral SMCs. Consequently,
- and
-TMs are also regarded as
SMC-differentiation molecular markers (21). The chicken
-TM gene
contains muscle and non-muscle promoters, and both smooth (
-TMsm)
and skeletal (
-TMsk) muscle isoforms are transcribed from the same
muscle promoter (22). Furthermore,
-TMsm and
-TMsk are generated
by alternative splicing and contain exons 6a/9d and exons 6b/9a,
respectively (20). Toutant et al. (23) reported the E, C,
and CArG boxes as putative cis-elements of the
-TM muscle
promoter in skeletal myoblasts. However, there are no other reports
concerning the tissue-specific transcription of the
-TM gene.
-TM gene
and demonstrated that CArG box, but not other cis-elements, is critically involved in the SMC-specific transcription. We further cloned a novel homologue of Barx1 homeoprotein family,
Barx1b, as a partner of SRF. Barx1b is predominantly
expressed in the SMC layer of the upper digestive organs, their
attached arteries, and in craniofacial structures. In visceral SMCs and
heterologous cells, SRF and Barx1b coordinately transactivated the
-TM gene. Gel mobility shift analysis showed a ternary complex
between the SRF/CArG probe and Barx1b, and immunoprecipitation analysis
revealed a DNA-independent interaction between SRF with Barx1b in
vivo.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-TM gene was cloned from a
chicken genomic library, then sequenced and subcloned into pGL3 basic
luciferase plasmid (Promega) (BTM1272). A series of promoter constructs
(BTM785, 230, 192, 117, and 81) containing deletions was then generated from BTM1272. Mutations were introduced into the E
(GGTGCCTCGAGCCGG), C (CCGAGGATCCCGTCC), and CArG
(TGTCCCTAAGCTTTG) boxes of BTM230. The chicken
barx1b, gax, and hox cluster genes were cloned from a chicken gizzard cDNA library using degenerative oligoprobes encoding the consensus sequence of the third helix of the
homeodomain (QVKIWFQNRRMK) (24). Human SRF and SRF mutant (SRFpm),
which lacks CArG binding ability, were introduced FLAG tag at their
COOH termini and were expressed as described previously (25). GST-SRF
derivatives were constructed in a pGEX 6P-1 vector (Amersham Pharmacia
Biotech). A series of Barx1b constructs, wild type (wt; amino acids
1-247), NH2 terminus deletion (
N; amino acids
135-247), COOH terminus deletion (
C; amino acids 1-194), 31 residues of NH2-terminal arm plus the homeodomain (31+HD;
amino acids 104-194), 11 residues of NH2-terminal arm plus
the homeodomain (11+HD; amino acids 124-194), the homeodomain alone
(HD; amino acids 135-194), and with the homeodomain deleted (
HD;
amino acids 1-134 and 195-247), were amplified by polymerase chain
reaction, and subcloned into pGEX6P-1, pCS2+MT, or pcDNA3.1
FLAG vector. The coding region of chicken mhox was amplified
by reverse transcriptase-polymerase chain reaction from chicken gizzard
total RNA. Full-length of gax and mhox were
subcloned into pcDNA3.1 FLAG vector.
-galactosidase control plasmid and 1.5 µg of
luciferase reporter plasmid with or without SRF/Barx1b expression
vectors using TransIT LT1 lipofection reagent (Pan Vera Corp.). The
cell lysates were measured for luciferase and
-galactosidase
activities. The transfection efficiencies were normalized to the
-galactosidase activity.
-D-thiogalactopyranoside. The cells were
resuspended with lysis buffer (50 mM Tris-HCl, pH 8.3, 500 mM NaCl, 1 mM EDTA, 2 mM
dithiothreitol, and 50 µg/ml phenylmethylsulfonyl fluoride) and lysed
with a French pressure cell press (SIM-AMINCO). The recombinant
proteins were purified with glutathione-Sepharose 4B gels. The purity
and quantity of the recombinant proteins were determined by
SDS-PAGE after Coomassie blue staining.
-TM CArG probe were incubated in 10 µl of binding buffer
(20 mM Tris-HCl, pH 8.0, 75 mM KCl, 0.5 mM EDTA, 0.05% Nonidet P-40, 50 µg/ml bovine serum albumin, 5 mM dithiothreitol, 5% glycerol, and 25 µg/ml
herring sperm DNA). Purified fusion protein(s) and the labeled
-TM
CArG probe were incubated in 10 µl of binding buffer (20 mM HEPES-KOH, pH 7.9, 75 mM KCl, 0.05% Nonidet
P-40, 50 µg/ml bovine serum albumin, 5 mM dithiothreitol,
5% glycerol, and 1 µg/ml herring sperm DNA) at room temperature for
20 min, then the mixtures were loaded onto a 4% polyacrylamide gel.
Electrophoresis was carried out under constant current of 10 mA in
0.5 × TBE buffer. The gel was dried and visualized by autoradiography.
-TM cRNA probe encoding exons 1a and 2, which are
common to both the smooth and skeletal muscle isoforms, was used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-TM Gene in
Chicken Gizzard SMCs--
To elucidate the essential
cis-elements of the
-TM gene, we cloned the chicken
-TM promoter region, and analyzed its promoter activity in
differentiated gizzard SMCs (Fig.
1A). A series of deleted
reporter genes (BTM1272-BTM117) showed only a slight reduction in their
promoter activities, even when the E and C boxes were deleted (BTM117).
In contrast, loss of the CArG box (BTM81) resulted in a marked decrease
in the promoter activity. Mutation of the CArG box (CArG box MUT), but
not the E and C boxes (E box MUT and C box MUT), in BTM230 also
markedly decreased the promoter activity. Gel mobility shit analysis
was then performed using nuclear extracts prepared from differentiated
gizzard SMCs and 32P-labeled
-TM CArG probe (Fig.
1B). The labeled CArG probe and nuclear protein complex
(Fig. 1B, lane 1), which was displaced by an excess amount
of unlabeled probe (Fig. 1B, lane 2), was supershifted with
anti-SRF antibody (Fig. 1B, lane 4). These results suggested
that CArG box is the critical cis-element of the
-TM gene
in visceral SMCs, and that SRF serves as a trans-factor of the CArG box.
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Fig. 1.
SRF- and CArG box-dependent
transcription of the -TM gene in chicken
gizzard SMCs. A, schematic diagram showing the
-TM
promoter activity using a series of
-TM reporter constructs.
Mutations were introduced in the E, C, and CArG boxes of BTM230 (E, C,
and CArG box MUT, respectively). A luciferase reporter plasmid (1.5 µg) and pSV-
-galactosidase control vector (1.0 µg) were
co-transfected into differentiated gizzard SMCs. Cell lysates were
recovered at 48 h after transfection. Raw luciferase activities
were normalized to the
-galactosidase activities and represented as
fold activation relative to BTM81. The results are taken from one
representative experiment (out of at least three) performed in
triplicate. The error bars correspond to the standard
deviation. B, identification of SRF as a
-TM CArG box
binding transcription factor. 32P-Labeled CArG probe was
incubated with NE prepared from differentiated gizzard SMCs (lane
1). DNA-protein complex was competed with a 50-fold molar excess
of unlabeled CArG probe (lane 2). Supershift assays were
performed with rabbit control IgG (lane 3) or anti-SRF
polyclonal antibody (lane 4). Arrow, DNA-protein
complex; asterisk, the shifted complex with anti-SRF
antibody.
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Fig. 2.
Sequence alignment of Barx1b and Barx family
members. A, primary structures of chicken Barx1b,
chicken Barx1, human Barx1, and mouse Barx1. Numbers
represent positions of residues. The FIL domain is
underlined, the leucine repeat region is indicated by
dots, the homeodomain is boxed, and the
Barx-specific domain is hatched. B, sequence similarities of
the FIL domain in the Bar family and the TN domain of mouse
Nkx-2.5/Csx. cBarx1b, chicken Barx1b; cBarx2b, chicken Barx2b;
D.BarH1, Drosophila BarH1; D.BarH2,
Drosophila BarH2; mNkx-2.5, mouse Nkx-2.5/Csx. C,
basic regions around the NH2-terminal boundary of the
homeodomain in Barx1b and Nkx-2.5/Csx are indicated in bold.
The NH2 termini of the homeodomains are
boxed.
-TM and SRF by in situ hybridization. The Barx1b
mRNA was exclusively expressed in upper digestive tissues such as
the esophagus, crop, and gizzard, and in craniofacial structures (Fig.
3C). gax was expressed in all muscle lineages, and hox genes were
ubiquitously expressed (data not shown). The
-TM mRNA was seen
in all smooth and skeletal muscle tissues, but was faint in cardiac
muscle (Fig. 3A). The SRF mRNA was ubiquitous but
prominent in all three muscle types (Fig. 3B). The
-TM,
SRF, and Barx1b mRNAs in gizzard were then precisely localized by
fluorescent in situ hybridization. The
-TM and Barx1b
mRNAs were only seen in the SMC layer of the gizzard (Fig. 3,
D and F), but the SRF mRNA was detected from the glandular to the SMC layers (Fig. 3E). Signals of all
three transcripts were also seen in the adjacent arteries (Fig. 3,
D-F). As a control, we performed the same analysis using
sense probes, and found no signals in the E15 whole embryo (data not
shown). Northern blotting revealed that the expressions of Barx1b and SRF mRNAs in gizzards preceded that of
-TM mRNA and were
most prominent at E10-13. In addition, the expression patterns of
Barx1b, SRF, and
-TM were sustained after hatching. The
-TM
mRNA was up-regulated during development of the gizzard (data not
shown).
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Fig. 3.
Expression of -TM,
SRF, and Barx1b mRNAs in chicken whole embryo and
gizzard. In situ hybridization was performed
with 35S-labeled cRNA probes of chicken
-TM
(A), SRF (B), and Barx1b (C). cRNA
probes were labeled with 35S-UTP and hybridized with whole
body sections of E15 chicken embryo. Sk, skeletal muscle;
th, thigh; he, heart; es, esophagus;
cr, crop; gz, gizzard; and cf,
craniofacial structures. Scale bar indicates 1 cm.
Fluorescent in situ hybridization was performed with cRNA
probes of chicken
-TM (D), SRF (E), and Barx1b
(F). cRNA probes were labeled with digoxigenin-UTP and
hybridized with E15 chicken gizzard. ar, arteries;
ml, muscular layer; gl, glandular layer.
Scale bar indicates 1 mm.
-TM Promoter with SRF and
Barx1b--
We next investigated the possible involvement of
Barx1b in the SRF-dependent activation of the
-TM
promoter. The
-TM reporter gene (BTM117), which contains a CArG box,
was co-transfected with SRF and/or Barx1b expression vectors in
differentiated gizzard SMCs (Fig. 4).
When SRF or Barx1b was transfected alone, the activation rates of the
reporter gene were doubled. However, co-transfection with Barx1b and
SRF showed an approximately 5-fold increase. The co-transfection of
other homeoproteins, Gax or MHox, with SRF, which was done as a
control, failed to show the coordinated activation of the reporter
gene. When a mutation was introduced into the CArG box of the reporter
gene (CArG MUT), no significant activation was observed even in the
presence of SRF and Barx1b (Fig. 4, right four columns).
Taken together, these results indicate that SRF and Barx1b coordinately
transactivate the
-TM gene through the CArG box in differentiated
SMCs.
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Fig. 4.
Coordinated transactivation of the
-TM gene with Barx1b and SRF. BTM117 (1.5 µg) (left eight columns) or CArG MUT (1.5 µg)
(right four columns) reporter gene and pSV-
-galactosidase
control vector (1.0 µg) were co-transfected with 25 ng of Barx1b,
Gax, or MHox and/or 25 ng of SRF expression vector(s) in differentiated
gizzard SMCs. The total amount of DNA was kept constant with empty
expression vector. Raw luciferase activities were normalized to the
-galactosidase activity and represented as the fold activation
relative to BTM117 alone.
C) bound to SRF, but its NH2 terminus deletion (
N) did not. Interestingly, deletion mutants composed 31 or 11 residues of the NH2-terminal arm plus the
homeodomain (31+HD or 11+HD) retained their binding activity with SRF,
but the homeodomain (HD) alone or the homeodomain deletion (
HD)
lacked the activity. These results suggest the critical involvement of 11 residues of the NH2-terminal arm plus the homeodomain of
Barx1b in its interaction with SRF. An essential domain of SRF required for Barx1b binding was also determined (Fig. 5B). SRF
derivatives fused with GST were incubated with the
35S-labeled minimum binding domain of Barx1b (11+HD).
Wild-type (wt) and the MADS domain (MADS) of SRF interacted with
Barx1b, but the NH2 (N) and COOH (C) termini of SRF did
not. Thus, the domains of Barx1b and SRF essential for their physical
interaction were mapped to 11 residues of the NH2-terminal
arm plus the homeodomain of Barx1b and the MADS domain of SRF. To
confirm the direct interaction between SRF and Barx1b in
vivo, FLAG-tagged SRF and Myc-tagged Barx1b were co-expressed in
10T1/2 cells and immunoprecipitated (Fig. 5C). The proteins
precipitated with anti-SRF or anti-Barx1b antibodies were analyzed by
Western blotting using anti-FLAG (Fig. 5C, lanes 1-4) or
anti-Myc (Fig. 5C, lanes 5-8) antibodies. FLAG-SRF and
Myc-Barx1b were successfully co-immunoprecipitated with anti-Barx1b and
anti-SRF antibodies, respectively (Fig. 5C, lanes 4 and
7).
View larger version (28K):
[in a new window]
Fig. 5.
Interacting domains of Barx1b and SRF.
A, the binding domains of Barx1b and SRF were determined by
pull-down assay. 35S-Labeled Barx1b derivatives were
incubated with GST-SRF/Sepharose gels, and the bound proteins were
visualized by autoradiography. The left panel shows the
structure of wild-type Barx1b (wt), and its mutants (NH2
terminus deletion, N; COOH terminus deletion,
C; 31 residues of
the NH2-terminal arm plus the homeodomain, 31+HD; 11 residues of the NH2-terminal arm plus the homeodomain,
11+HD; homeodomain alone, HD; and homeodomain deletion,
HD). For the
middle panel, the gel shows 10% input of each translated
Barx1b products. The right panel shows the proteins
bound to GST-SRF/Sepharose. B, the binding domain of SRF for
35S-labeled Barx1b-11+HD was determined. The left
panel shows the structure of SRF. Wt, wild-type SRF;
N, NH2 terminus domain; C, COOH
terminus domain; and MADS, MADS domain. The right
panel shows 35S-labeled Barx1b-11+HD bound to each
GST-SRF mutant/Sepharose. C, mouse C3H10T1/2 fibroblasts
were co-transfected with Myc-tagged Barx1b (pCS2-MT-Barx1b) and
FLAG-tagged SRF (pcDNA3.1 SRF-FLAG). The tagged proteins were
immunoprecipitated with anti-SRF polyclonal (lanes 3 and
7) or anti-Barx1b (lanes 4 and 8)
polyclonal antibodies, then immunoblotted with anti-FLAG (left
panel) or anti-Myc (right panel) monoclonal antibodies.
Lanes 1 and 5, mock transfected; lanes
2 and 6, 5% input. D, Barx1b deletion
mutants (25 ng) and SRF or SRFpm (12.5 ng) were co-transfected with
BTM117 reporter plasmid (1.5 µg) and pSV-
-galactosidase control
vector (1.0 µg) in mouse C3H10T1/2 fibroblasts. Raw luciferase
activities were normalized to the
-galactosidase activity and
represented as fold activation relative to BTM117 alone. SRF wild type,
wt; and mutant, pm.
-TM transcription in 10T1/2 fibroblasts (Fig. 5D). Expression of wt-Barx1b or SRF alone
showed a 2- or 5-fold increase in the promoter activity, respectively. Co-expression of SRF and wt-Barx1b, however, showed a striking increase
in activity about 17-fold. Consistent with the results of pull-down
assays (Fig. 5, A and B), SRF and Barx1b-
C or
Barx1b-11+HD, which directly binds to SRF, coordinately activated the
-TM promoter by 16- or 13-fold, respectively. Barx1b-
N,
Barx1b-HD, or Barx1b-
HD, which lack SRF binding activity, failed to
potentiate the promoter activity, even when SRF was present. This
coordinated activation was not seen with an SRF mutant (SRFpm) that
lacks DNA binding ability.
-TM
cis-element, gel mobility shift analysis was performed using
32P-labeled putative cis-elements. In this
experiment, we used Barx1b-
C, which fully retained the SRF binding
ability and the
-TM transcription activity with SRF. To reveal the
binding site(s) in the
-TM promoter region for Barx1b, we examined
the interactions of putative homeoprotein binding AT-rich sequences
(
1272 to
1242,
1270 to
1040,
431 to
401, and
437 to
417) with Barx1b (24). None of these 32P-labeled AT-rich
probes, however, bound to GST-Barx1b-
C. The same result was obtained
using GST-Barx1b-wt (data not shown). This finding was consistent with
promoter analyses, which showed that no deletions of AT-rich sequences
(BTM1013, BTM785, and BTM230) affected the
-TM promoter activity
(Fig. 1A). Furthermore, mutation of the CArG box efficiently
abolished the transactivation of the
-TM gene with SRF and/or Barx1b
(Fig. 4). These results suggest that Barx1b acts as a DNA-independent
co-activator of SRF, and that it requires the SRF and CArG interaction
as a base foothold. Finally, we examined the interaction between SRF,
the 32P-labeled CArG probe, and Barx1b (Fig.
6). SRF bound to the labeled CArG probe
in a dose-dependent manner (Fig. 6A, lanes 6 and
7, arrowhead) and the SRF-DNA complex was supershifted with
anti-SRF antibody (Fig. 6A, lane 9, two
asterisks). In contrast, GST-Barx1b-
C alone did not bind to the
labeled CArG probe (Fig. 6A, lanes 2-5). The same result
was obtained using GST-Barx1b-wt (data not shown). When both SRF and
GST-Barx1b-
C were incubated with the labeled CArG probe, a band
shift was observed (Fig. 6B, lanes 3 and 4, arrow). Because this slowly migrating band was supershifted with anti-GST or anti-SRF antibodies (Fig. 6B, lanes 5 and
6, two asterisks), the band was identified as a ternary
complex between SRF, CArG probe, and Barx1b. In addition, as
GST-Barx1b-
C amounts were increased, free probes were gradually
reduced (Fig. 6B, lanes 3 and 4, asterisk). No
ternary complex was observed, when GST was incubated with SRF and CArG
probe (data not shown).
View larger version (43K):
[in a new window]
Fig. 6.
Ternary complex formation between Barx1b,
SRF, and -TM CArG probe. A,
32P-labeled CArG probe (lane 1) was incubated
with indicated amounts (ng) of GST-Barx1b-
C (lane 2, 2.5 ng; and lanes 3-5, 10 ng). Labeled DNA and 10 ng of
GST-Barx1b-
C were incubated with a 50-fold molar excess of CArG
unlabeled probe (lane 4) or 200 ng of anti-GST polyclonal
antibody (lane 5). Purified SRF (lane 6, 2.5 ng;
and lanes 7-9, 5.0 ng) was also incubated with labeled
probe. DNA·SRF complex (arrowhead) was competed with a
50-fold molar excess of CArG unlabeled probe (lane 8) or
supershifted with 200 ng of anti-SRF polyclonal antibody (lane
9). B, 32P-labeled
-TM CArG probe was
incubated with GST-Barx1b-
C alone (lane 1), SRF alone
(lane 2), or the indicated amounts (ng) of SRF and
GST-Barx1b-
C (lanes 3-6). The ternary complex
(lanes 3 and 4 and inset; arrow) was
supershifted with 200 ng of anti-GST polyclonal antibody (lane
5) or anti-SRF polyclonal antibody (lane 6). The
inset shows an enlarged view of SRF-DNA and
SRF-DNA·GST-Barx1b-
C complexes in lanes 2-4.
Arrowhead, DNA·SRF complex; arrow,
DNA-SRF/GST-Barx1b-
C complex; asterisk, free probes;
two asterisks, shifted complex with anti-GST or anti-SRF
antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-TM
gene. Promoter analyses using a series of deleted and mutated
-TM
reporter genes and gel mobility shift assays revealed an obligatory
role of the SRF and CArG interaction for
-TM expression in SMCs
(Fig. 1). We also isolated a novel homologue of the Barx family,
Barx1b, and found it to act as a co-activator of SRF (Figs. 2 and 4).
Barx1b was exclusively localized to the SMC layer of the upper
digestive organs and its attached arteries and in craniofacial structures (Fig. 3). We further demonstrated that SRF and Barx1b, which
directly bound each other, coordinately transactivated the
-TM gene
(Figs. 4 and 5), and formed a ternary complex with a CArG probe (Fig.
6).
-actin transcription is mediated by SRF and
MHox. However, whether there is a physical interaction between SRF and
MHox and the details of their involvement in coordinated
transactivation remains unclear. Nkx-2.5/Csx, a member of
the NK2 family, was originally cloned from a mouse heart cDNA
library as a mammalian homologue of Drosophila tinman, which is a critical transcription factor for cardiogenesis in the fly (35-37). Chen et al. (7) reported a physical interaction
between Nkx-2.5/Csx and SRF and their coordinated transactivation of
the cardiac
-actin gene. Recent genetic
analysis showed that NKX-2.5/CSX is also involved in
development of the septum and the atrioventricular conduction in humans
(38). However, no expression of Nkx-2.5/Csx is observed in a smooth
muscle lineage. In Drosophila, bagpipe controls
the midgut formation under the control of tinman (35). bagpipe belongs to the NK3 family, and two mammalian
homologues, Nkx-3.1 and Nkx-3.2, have been
recently reported (39, 40). Carson et al. (19) reported the
coordinated activation of the smooth muscle
-actin gene by SRF and Nkx-3.1. However, Nkx-3.1 is not
expressed in the muscular layer of smooth muscle tissues (39). Thus,
other homeoproteins are likely to be involved in the SMC-specific transcription.
-TMsm mRNA
in the SMC layer of gizzards and their attached arteries (Fig. 3,
D and F). At present, barx1b is the
only known homeobox gene whose expression is restricted to SMCs.
-TM CArG probe, it increased the DNA binding ability of SRF,
resulting in formation of a ternary complex. The same DNA independence
is observed with Nkx-2.5/Csx. Nkx-2.5/Csx does not require its own DNA
binding ability for the transactivation of the cardiac
-actin
promoter when it is co-expressed with SRF (7). Thus, Barx1b and
Nkx-2.5/Csx seem to function similarly, but they are also different in
that the COOH terminus of Nkx-2.5/Csx serves as an inhibitory domain
for the cardiac gene transcription (48), but Barx1b-wt or Barx1b-
C
alone did not significantly change the transactivation of the
-TM
gene (Fig. 5D). Lastly, we identified the essential domain
of Barx1b (11+HD) for SRF binding that was sufficient for the
transactivation of the
-TM gene (Fig. 5D). Barx1b
markedly increased the
-TM promoter activity only in the presence of
SRF (Figs. 4 and 5D). In addition, the coordinated transactivation was abolished by the overexpression of an SRF mutant
that lacks DNA binding ability (Fig. 5D). We also examined the effect of other homeoproteins, Gax and MHox, on the
-TM
promoter. None of them, however, showed a similar cumulative
transactivation with SRF (Fig. 4). Thus, the coordinated
transactivation of the
-TM promoter with SRF is a characteristic
function of Barx1b, and other mesodermally restricted homeoproteins
lack this activity.
-TM promoter activity even more than SRF
and Barx1b alone (data not shown). However, GATA-6 is expressed not in
the SMC layer, but in the glandular layer of the gizzard (data not
shown). These findings suggest that there may be another novel GATA
family protein in visceral SMCs.
-TM
gene by a ubiquitous transcription factor, SRF, and an SMC-restricted
homeoprotein, Barx1b. Barx1b is highly expressed in the SMC layer of
the upper digestive organs and their adjacent arteries, but in neither
lower digestive organs nor the aorta (Fig. 3C). It is well
known that there are at least two distinct SMC populations in the aorta
(54). SMCs in the aortic arch and the upper thoracic aorta are derived
from neural crest cells, and thus are of ectodermal origin. In
contrast, SMCs in the abdominal aorta are derived from lateral plate
mesoderm. The fact that Barx1b is expressed in the SMC layer of the
upper digestive organs and their adjacent arteries, but is absent in
the aorta, indicates that homeoproteins might play a role in
tissue-specific gene regulation in a homeotic fashion. It is possible
that other unknown homeoproteins are expressed in SMCs of the aorta,
lower digestive organs, and visceral organs, and act as co-activators
of SRF. So far, the muscle-specific transcription had been considered
in a lineage-dependent context, but there would also be
genetic hierarchies in other tissues. In fact, MyoD family had been
thought to be a master transcription factor determining the skeletal
muscle lineage. Tajbakhsh et al. (55) reported the
agenesis of body wall muscles in Pax-3/Myf-5-deficient mice. The
skeletal muscles in the head were, however, not affected in these mice.
Their findings indicate that MyoD expression is definitely controlled
by the homeoprotein Pax-3 in the trunk, but that there is another
Pax-3-independent pathway in the head. Our findings also suggest the
involvement of homeoproteins in tissue-dependent gene
transcription within the SMC lineage.
![]() |
Addendum |
---|
While this article was under review, a report by Herring et al. (56) appeared. The authors reported that Barx2b physically interacts with SRF and enhances DNA binding affinity of SRF. In addition, Barx2b localized in smooth muscle, skeletal muscle, and brain, but did not transactivate the telokin promoter with SRF.
![]() |
FOOTNOTES |
---|
* This work was supported by grants-in-aid for Research on Brain Science from the Ministry of Health and Welfare of Japan (to K. S.) and in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (to K. S.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB044371 (chicken barx1b) and AB054298 (chicken
-tropomyosin promoter).
¶ To whom correspondence should be addressed: Dept. of Neuroscience (D13), Biomedical Research Center, Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita city, Osaka 565-0871, Japan. Tel.: 81-6-6879-3680; Fax: 81-6-6879-3689; E-mail: sobue@nbiochem. med.osaka-u.ac.jp.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1075/jbc.MM101127200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
SRF, serum response
factor;
TM, tropomyosin;
SMC, smooth muscle cell;
CArG box, CC(A/T)6GG;
HD, homeodomain;
NE, nuclear extracts;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel
electrophoresis;
-TMsm, smooth muscle
-tropomyosin;
-TMsk, skeletal
-tropomyosin.
![]() |
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