From the Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120
Received for publication, December 21, 2000, and in revised form, January 26, 2001
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ABSTRACT |
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CC(A/T)6GG or serum response
elements represent a common regulatory motif important for regulating
the expression of many smooth muscle-specific genes. They are
multifunctional elements that bind serum response factor (SRF) and are
important for serum induction of genes, expression of muscle-specific
genes, and differentiation of vascular smooth muscle cells. In the
current study, a yeast two-hybrid screen was used to identify proteins
from mouse intestine that interact with SRF. A novel
homeodomain-containing transcription factor, called Barx2b, was
identified that specifically interacts with SRF and promotes the DNA
binding activity of SRF. Northern blotting, RNase protection analysis,
and Western blotting revealed that Barx2b mRNA and protein are
expressed in several smooth muscle-containing tissues, as well as in
skeletal muscle and brain. In vitro binding studies using
bacterial fusion proteins revealed that the DNA-binding domain of SRF
interacts with a region of Barx2b located amino-terminal of the
homeobox domain. The results of these studies support the hypothesis
that interaction of SRF with different homeodomain-containing proteins may play a critical role in determining the
cell-specific functions of SRF.
Analysis of several smooth muscle-specific genes has, thus far,
failed to identify any smooth muscle-restricted transcription factors
that control their expression (reviewed in Ref. 1). Data have suggested
that expression of a single smooth muscle-specific protein may be
regulated by distinct response elements and distinct transcription
factors in different smooth muscle tissues. For example, the telokin
promoter was found to require an estrogen response element for high
levels of expression in uterine smooth muscle, but this element is not
required for expression in intestinal smooth muscle (2). In addition,
several fragments of the SM22 It is likely that SRF bound to CArG elements in smooth muscle-specific
genes may be interacting with other tissue-restricted factors to
mediate tissue-specific gene expression. Given its broad pattern of
expression, it seems likely that the interaction of SRF with other
factors determines whether SRF functions to activate muscle-specific
genes or to confer serum inducibility to a gene. For example the smooth
muscle Yeast Two-hybrid Screen--
To identify proteins that interact
with SRF, we performed a yeast two-hybrid screen of a mouse intestine
cDNA library using the yeast strain Y190 (Matchmaker Two-hybrid
system; CLONTECH). This yeast strain contains
LacZ and His3 reporter genes downstream of a
minimal ADH promoter and three DNA binding sites for GAL4. The human
cDNA for SRF (a generous gift from Dr. R. Prywes, Columbia University, New York, NY) was fused to the GAL4 DNA-binding
domain in plasmid pGBT9 (CLONTECH). A cDNA
library was constructed in pGAD10, from mouse intestinal mRNA,
using a two-hybrid cDNA construction kit according to the
manufacturer's directions (CLONTECH). The resultant cDNAs are fused to the carboxyl terminus of the GAL4 transcription activation domain. In the presence of 45 mM
3-amino-1,2,4-triazole, the SRF-GAL4BD fusion protein did not
result in colony growth or Lambda Library Construction and Screening--
The remaining
cDNA obtained from construction of the mouse intestine pGAD10
cDNA library was ligated to EcoRI-digested and dephosphorylated Northern Blotting--
Total RNA was isolated from adult tissues
using a single-step guanidinium isothiocyanate procedure (16); 15 µg
were separated on a 1.2% formaldehyde agarose gel and transferred to a
nylon membrane under vacuum. Hybridization was carried out at 65 °C overnight with the same Barx2b probe used for lambda library screening. Final wash conditions were 2× SSPE + 1.0% SDS for 10 min at
65 °C.
RNase Protection Assays--
A 536-bp fragment of the Barx2b
cDNA (corresponding to nucleotides 594-1122) was subcloned into
pGEM 7Z (Promega). The plasmid was linearized with SmaI and
a 32P-labeled antisense riboprobe generated using SP6
polymerase and a Maxi Script in vitro Transcription kit
(Ambion, Austin, TX) according to the manufacturer's directions. The
Barx2b riboprobe was gel-purified on a 6% polyacrylamide/8
M urea gel and eluted overnight at 37 °C. Ribonuclease
protection assays were then performed according to the manufacturer's
directions (Standard RPA II kit; Ambion). Briefly, 1 × 106 cpm of gel-purified Barx2b riboprobe was coprecipitated
with 20 µg of RNA and hybridized overnight at 42 °C. Samples were
digested with RNase A/T1 at a 1:150 dilution for 30 min at 37 °C and
then inactivated and precipitated. Samples were solubilized in 8 µl of gel loading buffer, and half the volume was loaded onto a 6% polyacrylamide/8 M urea gel run at 55 W for 2 h.
35S sequencing reactions were run alongside samples
to verify the size of the probe and protected fragments. To verify the
integrity of each RNA sample, a control In Vitro Binding of Recombinant Proteins--
To characterize
the interaction between SRF and Barx2b in vitro, various
fragments of each of the proteins were expressed as fusion proteins in
bacteria. Wild type SRF and three deletion mutants were expressed as
glutathione S-transferase (GST) fusion proteins. Deletion
fragments were generated by polymerase chain reaction and subcloned
into pGEX4T (Amersham Pharmacia Biotech); all constructs were verified
by DNA sequencing. The following fragments of SRF were expressed:
(a) deletion 1, encoding amino acids 1-338; (b)
deletion2, encoding amino acids 1-222; and (c) deletion 3, encoding amino acids 1-132. Wild type Barx2b and six subfragments were expressed as fusion proteins with a His tag and T7 epitope tag at the amino terminus. Deletion fragments were generated by polymerase chain reaction or restriction digestion and
subcloned into pET28 (Novagen, Madison, WI); all constructs were
verified by DNA sequencing. Barx2b fragments expressed encode the
following amino acids: (a) 1-283, wild type; (b)
34-283, deletion 1; (c) 124-283, deletion 2;
(d) 1-133, deletion 3; (e) 26-215, deletion 4;
(f) 1-123, deletion 5; and (g) 143-205, hox domain.
For in vitro binding experiments, GST-SRF fusion proteins
were bound to glutathione-agarose beads and then incubated with bacterial lysates containing Barx2b fusion proteins. GST and GST-SRF protein expression was induced by the addition of 0.4 mM
isopropyl-1-thio- Barx2b Antibody Production--
For generation of a polyclonal
antibody to Barx2, an amino-terminal fragment of Barx2b extending from
amino acid 1 to 133 was expressed in bacteria using the pET expression
system (Novagen). This fragment exhibits very little homology to Barx1
and should thus produce antisera specific for Barx2. The resultant
His-tagged fusion protein was purified to homogeneity using Talon beads
(CLONTECH). The purified protein was used to
generate polyclonal antiserum in rabbits by Covance Research Products
(Richmond, CA). The specificity and titer of the anti-Barx2 antisera
were confirmed by Western blotting.
Western Blot Analysis--
Western blotting of in
vitro binding complexes and Barx2b expression in tissues and cells
was carried out essentially as described previously (17). Tissue
extracts were prepared by homogenizing frozen pulverized tissue in
radioimmune precipitation buffer (1% Nonidet P-40, 1% sodium
deoxycholate, 0.1% SDS, 0.15 M NaCl, 2 mM
EDTA, 100 units/ml aprotinin, and 0.01 M sodium phosphate
pH 7.2).
Gel Mobility Shift Analysis of DNA Binding--
Mobility shift
assays were performed in a final volume of 15 µl. Binding mixtures
contained 0.2 ng (1.5 × 104 cpm) of end-labeled
double-stranded DNA probe, 200 ng of salmon sperm DNA, 4.5 µg of
bovine serum albumin, and various amounts of purified recombinant
protein as indicated in a binding buffer containing 12 mM
HEPES, pH 7.9, 60 mM KCl, 4 mM
MgCl2, 10% glycerol, and 1 mM dithiothreitol.
All binding reactions were incubated for 15 min at room temperature
followed by 1 h at 5 °C, except where indicated. For antibody
supershift experiments, antibody was added after the 15-min room
temperature incubation. For experiments in which the time of incubation
was varied, proteins were allowed to incubate in reaction buffer for 10 min at room temperature before the addition of probe. Incubations were
then performed for the specified times, and the reactions were
immediately loaded onto a 4% polyacrylamide gel (containing 6.75 mM Tris, pH 7.9, 3.3 mM sodium acetate, pH 7.9, 1 mM EDTA, and 2.5% glycerol). Unlabeled competitor
double-stranded oligonucleotides at 200-fold excess were included in
some reactions as indicated in the figure legends. The sequences
of the sense strand of each probe were as follows: (a) CArG
probe, ACCCTATCCCTTTTATGGGAGCTGAAGGGA;
(b) SRE probe, CAGGATGTCCATATTAGGACATCT; and (c)
Barx2b probe, (two binding sites)
CTAGCCATTAGCGAGATGTCCATTAGCGAGCTAG.
Underlined nucleotides are not part of the natural
sequences but were added for labeling purposes. Polyclonal antibodies
to ets and SRF were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA.)
Mammalian Expression and Reporter Gene Assays--
For
expression in mammalian cells, a fragment of the Barx2b cDNA
encoding the coding region was amplified by polymerase chain reaction
and cloned into pcDNA 3.1 His C (Invitrogen, Carlsbad, CA). This
results in the expression of Barx2b as a fusion protein with
amino-terminal 6xHis and X-press epitope tags. For some experiments, wild type Barx2b was expressed from our full-length cDNA without an
additional epitope tag by subcloning the cDNA into the expression vector pCMV5. The resultant plasmids were sequenced to verify the
integrity of the insert.
All promoter reporter genes were constructed by cloning fragments of
promoters into the pGL2B luciferase vector (Promega). The
rabbit telokin promoter-luciferase reporter gene used includes nucleotides
A10 cells were grown in high-glucose Dulbecco's modified Eagle's
medium containing 50 units/ml penicillin, 50 µg/ml streptomycin, and
20% fetal bovine serum. REF52 and COS cells were grown in media
supplemented with 10% fetal bovine serum. Cells to be transfected were
seeded at 1.4 × 105 cells/dish in 35-mm dishes. At
16-18 h after seeding, each dish was washed once with
phosphate-buffered saline, pH 7.4, replaced with 2 ml of complete
media, incubated with a total of 2 µg of plasmid DNA (1 µg of
promoter-luciferase, 0.5 µg of Barx2b expression plasmid, and 0.5 µg of pRL-luciferase as an internal control) and 3 µl of Fugene in
0.1 ml of Dulbecco's modified Eagle's medium (Life Technologies,
Inc.). Twenty-four h later, 10-µl of extracts (400 µl/dish) were
prepared for dual luciferase assays. Assays were performed using a dual
luciferase reporter assay system according to the manufacturer's
directions (Promega). Reporter gene luciferase activities were
normalized to the luciferase activity of the internal control. For
analysis of Barx2b protein expression, COS cell lysates were generated
36 h after transfection using radioimmune precipitation buffer and
analyzed by Western blotting as described above.
Identification of Barx2b as a SRF-binding Protein in Mouse
Intestine--
To identify proteins that interact with SRF, we
performed a yeast two-hybrid screen of a mouse intestine cDNA
library in the yeast strain Y190 (Matchmaker Two-hybrid system;
CLONTECH). In a screen of ~107
cDNAs, 2 of the Barx2 Expressed in Intestine Is Distinct from Barx2 Previously
Cloned from Mouse Day 11.5 Embryos--
The nucleotide sequence of two
cDNA clones isolated from the yeast two-hybrid screen of a mouse
intestinal library spanned nucleotides 364-800 of the previously
described mouse Barx2 cDNA (20). Both of the mouse intestinal
cDNA clones have an insert of 84 nucleotides just 3' of the
homeobox domain (nucleotides 952-1041 in the intestinal clone, Fig.
1A) that is not present in the
previously described Barx2 cDNA. The Barx2 clone isolated was used
as a probe to screen a mouse intestine
Although an open reading frame extends to the 5' end of our clone,
three lines of evidence support the designation of nucleotides 297-299
as the translation start site. First, there is a very high degree of
sequence homology between the mouse and human Barx2 cDNAs 3' of the
predicted translation start site, but 5' of this region, the sequences
diverge (21, 22). Second, the size of the Barx2 mRNA estimated from
Northern blots is 1.8 kb, similar to the size of the cDNA (Fig.
3A). Third, the recombinant protein expression without an
epitope tag has the same apparent molecular mass as the protein
present in tissues (Fig. 3C).
Barx2b Expression Is Tissue-restricted--
The expression pattern
of Barx2b was examined by Northern blotting and by RNase protection
analysis. Together, these data demonstrated that in the adult mouse,
Barx2b mRNA is expressed at high levels in several smooth
muscle-containing tissues including the ileum, stomach, uterus, aorta,
and lung. It is also expressed at significant levels in skeletal muscle
and brain, and no expression is detected in the spleen, kidney, liver
or heart. During mouse embryonic development, there is a large increase
in Barx2b expression between embryonic day 10 and embryonic day
15 (Fig. 3). Results from Western blotting experiments are
generally consistent with the Northern blots and confirm that
Barx2b protein is expressed in a tissue-restricted manner in adult mice
(Fig. 3C). However, it was noted that the levels of
Barx2b protein and mRNA do not correlate perfectly, particularly
in the lung and aorta, suggesting that Barx2b protein levels may also
be regulated by posttranscriptional mechanisms.
Barx2b Binds Directly to SRF in Vitro--
To confirm results
obtained from the yeast two-hybrid analysis, GST pull-down assays were
used to determine whether SRF interacts directly with Barx2b. Fig.
4 shows that GST-SRF binds directly to
Barx2b. This interaction was specific because no Barx2b bound to GST
alone. To map the interaction between SRF and Barx2b, several deletion
constructs were generated, expressed in bacteria, and used in GST
pull-down assays (Figs. 4 and 5).
Deletion of amino-terminal residues from Barx2b results in Barx2b
mutants that still interact with SRF with high affinity (Fig.
4A,
To identify the region of SRF that interacts with Barx2b, three
carboxyl-terminal deletion mutants of SRF were constructed, expressed
as GST-fusion proteins, and analyzed for their ability to bind wild
type Barx2b. Deletion of the carboxyl-terminal transcription activation
domains of SRF had no effect on Barx2b binding. In contrast, deletion
of the MADS box DNA binding and dimerization domain of SRF abolished
Barx2b binding (Fig. 5). Western blotting of bacterial lysates
confirmed that all SRF deletion mutants were expressed at similar
levels (data not shown).
Barx2b Does Not Alter the SRF-dependent Activity of
Reporter Genes--
To determine the effects of Barx2b on
SRF-dependent gene transcription, Barx2b expression
plasmids were co-transfected together with SRF-dependent
reporter genes into mammalian cells, and the effects on reporter gene
activity were determined. Expression of Barx2b was found to have no
significant effect on the activity of SRF-dependent
promoters, including a 400-bp telokin promoter or a (CArG)x2-TK
promoter (5), in A10 smooth muscle cells or REF52 fibroblasts (data not
shown). Similarly, co-expression of Barx2b and SRF also had no
significant effect on telokin reporter gene activity as compared with
co-expression of SRF alone (data not shown).
Barx2b Increases the Affinity of SRF for a CArG Box--
To
directly assess the effects of Barx2b on the ability of SRF to bind to
DNA, gel mobility shift assays were performed. Identical mobility-shifted complexes were observed on the telokin CArG element or
cFos SRE element when purified SRF was incubated alone or together with
purified Barx2b (Fig. 6). The
mobility-shifted complex could be supershifted with antibodies to SRF
but not by antibodies to either Barx2 or ets (Fig. 6). In the absence
of SRF, Barx2b produced a fast-migrating mobility-shifted complex on
the cFos SRE probe but not on the telokin CArG probe. When a consensus
Barx2 binding site was used as a probe, Barx2b but not SRF resulted in
a fast-migrating mobility-shifted complex. A similarly migrating
complex was formed in the presence of both Barx2b and SRF, and this
complex could be supershifted with antibodies to Barx2b but not by
antibodies to either SRF or ets (Fig. 6). Because we were unable to
obtain any evidence for a SRF-Barx2b complex in gel mobility shift
assays, we investigated the ability of Barx2b to alter the kinetics of SRF binding to DNA. Under conditions of limiting SRF protein, Barx2b
increased the affinity of SRF for DNA such that the SRF-DNA complex
formed more quickly in the presence of Barx2b (Fig.
7).
Results show that Barx2b is a novel homeodomain-containing protein
that interacts with SRF and increases the affinity of SRF for DNA. This
function of Barx2b is analogous to that reported previously for Phox or
Mhox (23). The enhancement of DNA binding by Phox1/Mhox was found to be
primarily kinetic in vitro, and additional cellular proteins
such as TFII-I are required in vivo to stabilize the
SRF-homeodomain complexes (24). Similarly, we were unable to
demonstrate the presence of a tertiary Barx2b/SRF/DNA complex in
vitro, suggesting that the enhancement of SRF DNA binding mediated
by Barx2b is also largely kinetic and perhaps requires other endogenous
proteins to stabilize the complex in vivo. Together, these
data suggest that members of several families of homeobox-containing genes can interact with SRF and modulate its ability to bind DNA. The
interaction of different classes of homeodomain proteins with SRF may
thus represent a general mechanism by which the DNA binding activity of
SRF is modulated in different tissues. In further support of this
proposal, Nkx3-1, a member of the NK family of homeodomain-containing proteins, has recently been shown to promote the
interaction of SRF with a CArG element in the smooth muscle Unlike Phox/Mhox, the homeodomain alone of Barx2b was not sufficient to
mediate interaction with SRF (Ref. 23 and Fig. 4). A 10-amino acid
region amino-terminal of the Barx2b homeodomain was found to be
necessary but not sufficient to bind SRF. The ability of two
nonoverlapping fragments of Barx2b (amino acids 1-123 and
124-283) to bind to SRF suggests that there are two distinct
SRF binding sites; however, the 1-123 fragment appears to have a lower
affinity than the 124-283 fragment (Fig. 4). A similar bipartite
binding site has been reported on Nkx2.5 in which two distinct regions
of the Nkx2.5 homeodomain were shown to mediate SRF binding (26). The
10-amino acid region of Barx2b that is critical for SRF binding
(123SESETEQPTPR133) contains several potential
phosphorylation sites for casein kinase II (Ser123,
Ser125, and Thr127) and one potential site
(Thr131) of phosphorylation by proline-directed kinases.
This observation raises the possibility that in vivo the
interaction of Barx2b with SRF may be regulated by signaling cascades.
Barx2b isolated from adult mouse intestine is identical to the
previously described mouse Barx2 except for the presence of an
additional 25 amino acids at the amino terminus and the presence of a
30-amino acid insert close to the carboxyl terminus of the molecule.
The sequence identity between these molecules suggests that they likely
represent alternatively spliced versions of the same gene. During early
mouse embryonic development (embryonic days 9.5-12.5), Barx2 has been
shown to be expressed at high levels in neural and craniofacial
structures and to control the expression of L1 neural adhesion molecule
(20). The probes used for these studies, however, would not distinguish
between Barx2 and Barx2b. In chicken, cBarx2b has also been shown to be
expressed in myogenic cells in the myotome and in the skeletal muscles
of the limb as well as in neural tissues (19). In the current study, we
have shown that in addition to being expressed in adult brain and
skeletal muscle, mouse Barx2b is also expressed at high levels in
several adult smooth muscle tissues including the gut, uterus, and
aorta. We also found that Barx2b is the predominant isoform present
during the later stages of mouse development (embryonic day 15; Fig. 3); we were unable to detect the previously described mouse
Barx2 in adult tissues by RNase protection analysis. These data suggest that Barx2/Barx2b may play roles in regulating gene expression in
several different tissue lineages. The insert present in the carboxyl-terminal region of Barx2b is rich in acidic, glutamine, and
proline residues, suggesting that this region may be involved in
transcription activation and that Barx2b may thus possess functions distinct from those of Barx2.
The mechanisms by which Barx2 and Barx2b regulate gene expression are
likely to be complex, involving both activation and repression
resulting from Barx2/b binding to homeodomain binding sites as well as
indirect regulation of gene expression through its interaction with
SRF. Similar to Nkx2.5, Barx2b not only interacts with SRF, but it also
binds directly to some, but not all, CArG elements. For example, Barx2b
bound directly to the serum response element from the cFos gene, but no
binding was detected to the CArG element from the telokin gene (Fig.
6). The ability of Barx2b to interact with SRF is likely to be shared
by Barx2 because the region of Barx2b shown to interact with SRF is
completely conserved in Barx2. Barx2 and Barx2b may regulate the
activity of SRF by two different mechanisms, enhancement of the ability
of SRF to bind to DNA (Fig. 7) and
regulation of the interaction of SRF with other accessory
factors (27). Recent findings that suggest that Barx2 can also interact
with other transcription factors of the cAMP-response element-binding
protein family further demonstrate the complexities of gene regulation
that can be mediated by Barx2 (28).
The interaction of SRF with tissue-restricted transcription factors is
likely to be important in determining the function of SRF on a given
promoter in specific cell types. The ability of SRF to activate
muscle-specific gene expression in addition to mediating growth factor
responsiveness of genes likely results from complex protein-protein and
protein-DNA interactions that are both promoter- and cell
type-dependent. To confer growth factor responsiveness to
genes, SRF interacts with members of the ets family of transcription
factors, such as p62TCF, and these proteins interact with SRF together
with DNA sequences that flank the SRF binding site (29). In contrast,
in muscle tissues, which express high levels of Phox/Mhox, the
expression of Phox/Mhox prevents the interaction of SRF with SAP-1, a
member of the ets family of ternary complex factors (27). In skeletal
muscle, SRF bound to a CArG element of the cardiac muscle
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
promoter that have been shown to be
sufficient to mediate expression in arterial smooth muscle do not
result in detectable expression in visceral smooth muscle (3, 4).
CC(A/T)6GG (CArG)
1 or serum response elements
(SREs) have been shown to be critical for the activity of most smooth
muscle-specific promoters characterized (4-11); thus, it appears
likely that CArG elements represent a common regulatory motif important
for regulating the expression of smooth muscle-specific genes. CArG or
SRE elements bind serum response factor (SRF), a protein that plays an
important role in both the expression of muscle-specific genes and
serum induction of genes (12). Consistent with its role in regulating
expression of muscle-specific genes, although SRF is widely expressed,
it is present at the highest levels in skeletal, cardiac, and smooth muscle-containing tissues (13). In addition to regulating the activity
of smooth muscle-specific promoters, SRF has also been directly shown
to be required for the differentiation of proepicardial cells into
coronary vascular smooth muscle cells (14).
-actin gene contains several CArG elements that are required
for transcriptional activity; however, the endogenous gene or a
reporter gene containing a 1063-bp fragment of the promoter is not
induced by serum. In contrast, a truncated 191-bp
-actin promoter
fragment is induced by serum (15), suggesting that within this single
gene, the function of SRF bound to CArG elements is dependent on
interactions with factors that bind to other elements within the gene.
Similarly, although SRF has been shown to be required for telokin
promoter activity, endogenous telokin is not induced by
serum.2 In the current study,
we have used a yeast two-hybrid screen to identify proteins from mouse
intestine that interact with SRF. These studies show that a novel
homeodomain-containing transcription factor, Barx2b, specifically
interacts with SRF and promotes the DNA binding activity of SRF.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity. The identity of
positive clones was determined by direct DNA sequencing. To verify that
SRF interacted with the sequenced clones, these clones were
reintroduced into yeast together with SRF-GAL4BD plasmid. True positive
clones were identified by their ability to activate the
LacZ reporter gene.
gt11 arms. The lambda library was packaged, amplified, and screened by standard procedures (16). Nitrocellulose filters were hybridized at 65 °C overnight with a 32P
probe corresponding to the entire Barx2 clone obtained from the yeast
library screen and to nucleotides 364-892 of the previously described
mouse Barx2 cDNA. Filters were washed in 2× SSPE (1× SSPE = 180 mM NaCl, 10 mM
NaH2PO4, 1 mM EDTA, pH 7.4) + 1.0%
SDS at room temperature for 10 min, 2× SSPE + 1.0% SDS at 65 °C
for 10 min, and 0.2× SSPE + 0.1% SDS at 65 °C for 10 min. Lambda
DNA was isolated using Lambdasorb (Promega, Madison, WI) and digested with NotI, and the resulting fragments were subcloned into
pGEM 5Z and sequenced by automated sequencing.
-actin riboprobe (308 bases)
was included in each of the hybridization mixes. A 245-base fragment of
this probe is protected from RNase digestion by endogenous
-actin mRNA.
-D-galactopyranoside for 1 h.
Lysates were prepared by sonicating bacterial pellets in
phosphate-buffered saline containing 10 mg/ml bovine serum albumin,
0.1% Triton X-100, 500 µg/ml phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 1 mM dithiothreitol. Cleared lysates
were incubated with glutathione-agarose beads for 2 h at 4 °C.
Bound proteins were collected by centrifugation and washed twice with 4 ml of lysis buffer before incubation with Barx2b lysates. Barx2b cleared lysates were prepared in the same manner as the GST-SRF lysates. After incubation at 4 °C for 1 h,
glutathione-agarose-bound protein complexes were washed three times in
lysis buffer without bovine serum albumin. Glutathione-agarose-bound
proteins were then analyzed by Western blotting. In some experiments,
SRF was expressed as a His-T7-tagged fusion protein, and Barx2b was
expressed as a GST-fusion protein, and the binding experiments were
carried out as described above.
256 to +147 of the telokin gene, as described previously (18). The minimal TK promoter used comprised nucleotides
113 to +20
of the thymidine kinase gene; CArG-TK comprised two copies of
the telokin CArG element upstream of the minimal TK promoter. Plasmids
were transfected into rat A10 smooth muscle cells, REF52 fibroblasts,
or COS cells using Fugene (Roche).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase-positive colonies were identified as encoding a novel form of the homeodomain-containing protein Barx2
(19-22). These clones were found to be dependent on SRF for activation
of the reporter genes upon retransformation of plasmids into Y190 yeast cells.
gt11 cDNA library to
isolate a full-length cDNA. Five cDNA clones were isolated from
this screen, the longest of which was 1852 bp (Fig. 1A). All
of the cDNA clones isolated contained the same nucleotide insert as
that found in the original clones identified by the two-hybrid screen.
The longest clone extends the published Barx2 cDNA sequence an
additional 240 nucleotides at the 5' end and has a short poly(A) tail
at the 3' end. The 5' region also contains an additional in-frame
translation start site (Fig. 1B). Because of the similarity
between our clone and the previously published chicken Barx2b cDNA
(19) (73% amino acid identity; Fig. 2)
and the expression of both proteins in skeletal muscle (Fig.
3), it is likely that our cDNA
represents the mouse homologue of chicken Barx2b, hence we have
referred to the protein encoded by our cDNA as mouse Barx2b. Human
Barx2 also contains the inserted region present in mouse Barx2b and
is homologous to the mouse protein (87% amino acid identity; Fig. 2)
(21, 22).
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Fig. 1.
A novel Barx2b cDNA was cloned from mouse
intestine. A, the nucleotide sequence of full-length
Barx2b cDNA is shown, and the positions of the putative start and
stop codons are indicated in bold. SalI
restriction sites from the linker are underlined.
B, schematic representation of the relationship between
Barx2b cDNAs isolated from a yeast two-hybrid library screen and
from a gt11 cDNA library and the previously published mouse
Barx2 cDNA. The lambda clone shown at the top
corresponds to the nucleotide sequence shown in A. The
position of the probe used for RNase protection analysis is indicated
at the bottom of the figure.
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Fig. 2.
Barx2b likely represents an alternatively
spliced form of Barx2. Alignment of the amino acid sequences of
mouse Barx2b (M Barx2b), mouse Barx2 (M Barx2;
accession number L77900; Ref. 20), human Barx2 (H
Barx2; accession number AF031924; Ref. 22), and chicken Barx2b
(C Barx2b; accession number AF102555; Ref. 19).
Dashes represent deleted residues, dots represent
identical residues, and alternative residues are indicated in
single-letter amino acid code. The position of the homeodomain is
underlined, and the insert present in mouse Barx2b is
double underlined.
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Fig. 3.
Barx2b is expressed at high levels in smooth
muscle tissues, skeletal muscle, and brain. A, Northern
blot analysis of Barx2 expression. Adult mouse total RNA (15 µg) and
polyadenylated RNA (1 µg) were run on a 1.2% formaldehyde-agarose
gel, transferred to a nylon membrane, and hybridized overnight at
65 °C with the 32P-labeled Barx2b riboprobe indicated in
Fig. 1 (corresponding to nucleotides 662-1122). B, RNase
protection analysis of Barx2b mRNA expression in various adult
mouse tissues, whole mouse embryos, and mouse cell lines, as indicated.
C2C12 MT, differentiated C2C12
skeletal muscle myotubes; C2C12 MB, undifferentiated
C2C12 skeletal muscle myoblasts; GI
SMC, a T-antigen-transformed intestinal smooth muscle cell
line;2 PY4, a mouse endothelial-like cell line.
RNA samples (20 µg (unless otherwise indicated)) and a yeast RNA
control were hybridized for 16-18 h at 42 °C with a gel-purified
32P-labeled antisense Barx2b cRNA probe (corresponding to
nucleotides 662-1122). Samples were digested with RNase A/T1 (1:100),
precipitated, run on 6% acrylamide/8 M urea gel, and
visualized by autoradiography. The positions of the undigested Barx2b
(524 bases) and -actin probes (308 bases) and protected Barx2b
transcripts (460 bases) and
-actin transcripts (245 bases) are
indicated. It would be predicted that mRNA corresponding to the
published mouse Barx2 cDNA would protect fragments of the probe of
290 and 81 bases. C, Western blot analysis of Barx2b
expression in various mouse tissues and in COS cells transfected with
either full-length Barx2b cDNA (rBARX2b) or empty vector
(VECTOR). Tissue extracts were prepared from mouse heart,
aorta, and lung and from transfected COS cells as described in
"Materials and Methods." Fifty µg of extracts was analyzed from
each of the tissues, and 30 µg of extract was analyzed from the
transfected cells. The positions of molecular mass markers are
indicated to the left of the blot.
1 and
2), suggesting that residues 1-124 are not
required for binding to SRF. Similarly, carboxyl-terminal deletions to
amino acid 215 or 133 did not prevent SRF binding to Barx2b (Fig.
4A,
4 and
3); however, deletion of the carboxyl terminus
up to amino acid 123 dramatically decreased SRF binding (Fig. 4A,
5). In addition, the Hox domain alone (amino acids
143-205) was not able to bind SRF. Analysis of Barx2b expression in
the supernatants from GST pull-down assays confirmed that each of the
deletion mutants was expressed at a similar level (data not shown).
These data suggest that residues 123-133 amino-terminal of the Hox
domain are important for the interaction of Barx2b with SRF. To
determine whether the residues identified were sufficient to mediate
SRF binding, amino acids 123-133 were expressed as a GST-fusion
protein, and their ability to bind to SRF was determined. Results from
this analysis demonstrate that GST-WT Barx2b, but not GST-Barx123-133
or GST alone, was able to bind to SRF (Fig. 4B). These data
suggest that residues 123-133 are necessary but not sufficient to
mediate the binding of Barx2b to SRF.
View larger version (42K):
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Fig. 4.
SRF interacts directly with
Barx2b. A and B, full-length human SRF
was expressed as a GST-fusion protein. GST-SRF (+) and GST alone ( )
were immobilized on glutathione-Sepharose beads and incubated with
bacterial lysates containing either wild type or various deletion
mutants of Barx2b, as indicated. After extensive washing of the beads,
bound protein complexes were dissolved in SDS sample buffer and
analyzed by immunoblotting with anti-T7 epitope antisera (top
panels). The presence of GST-SRF in each of the + samples was
confirmed by immunoblotting with anti-SRF antiserum (middle
panels). The presence of GST in each of the
samples was
confirmed by immunoblotting with anti-GST antibodies (data not shown).
C, full-length wild type Barx2b and the
6 fragment of
Barx2b expressed as GST-fusion proteins and GST alone were immobilized
on glutathione-agarose beads and incubated with bacterial lysates
containing full-length human SRF expressed as a T7-tagged protein. SRF
bound to the Barx2b fusion proteins was then visualized by Western
blotting with an anti-SRF antibody (top panel). The presence
of SRF in each of the binding assays was confirmed by Western blotting
of the supernatant fractions from the assays (bottom panel).
The presence of GST-
6 and GST was confirmed by Ponceau stain of the
Western blot (data not shown). The schematic structures of the various
Barx2b deletion mutants, together with a summary of their ability to
bind to SRF, are shown at the bottom of the figure. The
homeobox domain is shaded, and the insert present in Barx2b
but not Barx2 is stippled.
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Fig. 5.
Barx2b interacts with the MADS domain of
SRF. Full-length wild type human SRF and three deletion mutants
were expressed as GST-fusion proteins. GST-fusion proteins and GST
alone were immobilized on glutathione-Sepharose beads and incubated
with bacterial lysates containing wild type Barx2b, as indicated. After
extensive washing of the beads, bound protein complexes
(PELLET) were dissolved in SDS sample buffer and analyzed by
immunoblotting with anti-SRF antisera (top panels). The
presence of Barx2b in each of the binding reactions was confirmed by
immunoblot analysis of the supernatant fractions (SUP). The
structure of each of the SRF deletions is shown schematically at the
bottom of the figure, together with a summary of their
ability to bind to Barx2b (+ indicates detectable binding; indicates
that no binding was detected). The MADS DNA binding and dimerization
domain of SRF is shaded.
View larger version (79K):
[in a new window]
Fig. 6.
SRF and Barx2b do not form
protein complexes in mobility shift assays. Mobility shift assays
were performed using either the telokin CArG (CArG), cFos SRE (SRE), or
consensus Barx2 (BARX2) elements as probes, as indicated. Binding
assays contained 50 ng of either purified SRF (SRF),
purified Barx2b (BARX2), or each of the proteins
(SRF+BARX2). After incubation at room temperature for 15 min, antibodies to either ets, Barx2b, or SRF were added as indicated,
and the incubations were continued for 1 h on ice.
Mobility-shifted complexes were then separated on a 4% acrylamide gel
and visualized by autoradiography as described in "Materials and
Methods." The positions of mobility-shifted complexes corresponding
to SRF and Barx2 are indicated, together with the position of a
supershifted complex and the position of the free probes.
View larger version (47K):
[in a new window]
Fig. 7.
Barx2b enhances the DNA binding activity of
SRF. Gel mobility shift assay using the cFos SRE as a probe.
Probes were incubated with 5 ng of purified SRF, except where indicated
otherwise, with (+) or without ( ) 50 ng of Barx2b for the times
indicated. Mobility-shifted complexes were then separated on a 4%
acrylamide gel and visualized by autoradiography as described in
"Materials and Methods." The position of mobility-shifted complex
corresponding to SRF is indicated by the arrow.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actin
promoter and to potentiate the activity of this smooth muscle-specific
promoter (25). In contrast to the effects of Nkx3-1 on the activity of
the smooth muscle
-actin promoter, Barx2b, in either the absence or
presence of exogenous SRF, did not alter the activity of the telokin
promoter in smooth muscle cells (data not shown). This result implies
that either additional accessory factors are required for Barx2b to
activate the promoter or that the effects of Barx2b may be
promoter-specific and that the telokin promoter may not represent a
physiological target. Additional studies will be required to identify
physiological targets of Barx2b in muscle tissues.
-actin
gene has been shown to cooperate with muscle-specific myogenic factors
bound to an adjacent E box, and this interaction is required for
transcriptional activation (30). Similarly, in cardiac muscle, SRF
bound to CArG elements interacts with the homeodomain-containing
protein Nkx-2.5 to activate transcription of the cardiac
-actin gene (26). In addition, Nkx3-1 but not Nkx2-5 has been shown to activate the
smooth muscle
-actin promoter (25). Together, these data suggest
that the interaction of SRF with tissue-restricted homeodomain proteins, such as Barx2b and members of the Nkx family, may be one
means by which SRF mediates its cell-specific functions.
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ACKNOWLEDGEMENTS |
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We thank Ron Prywes for providing the human SRF cDNA and Patricia Gallagher for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL-58571 (to B. P. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Cellular and
Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5120. Tel.: 317-278-1785; Fax:
317-274-3318; E-mail: pherring@.iupui.edu.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M011585200
2 B. P. Herring, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: CArG, CC(A/T)6GG; SRE, serum response element; SRF, serum response factor; bp, base pair(s); SSPE, saline/sodium phosphate/EDTA; GST, glutathione S-transferase; TK, thymidine kinase.
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