From the Department of Neurobiology, The Scripps
Research Institute, La Jolla, California 92037 and ¶ The
Neurosciences Institute, San Diego, California 92121
Received for publication, July 29, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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The homeobox protein Barx2 is expressed in both
smooth and skeletal muscle and is up-regulated during differentiation
of skeletal myotubes. Here we use antisense-oligonucleotide inhibition
of Barx2 expression in limb bud cell culture to show that Barx2 is required for myotube formation. Moreover, overexpression of Barx2 accelerates the fusion of MyoD-positive limb bud cells and C2C12 myoblasts. However, overexpression of Barx2 does not induce ectopic MyoD expression in either limb bud cultures or in multipotent C3H10T1/2
mesenchymal cells, and does not induce fusion of C3H10T1/2 cells. These
results suggest that Barx2 acts downstream of MyoD. To test this
hypothesis, we isolated the Barx2 gene promoter and identified DNA regulatory elements that might control Barx2
expression during myogenesis. The proximal promoter of the
Barx2 gene contained binding sites for several factors
involved in myoblast differentiation including MyoD, myogenin, serum
response factor, and myocyte enhancer factor 2. Co-transfection
experiments showed that binding sites for both MyoD and serum response
factor are necessary for activation of the promoter by MyoD and
myogenin. Taken together, these studies indicate that Barx2 is a key
regulator of myogenic differentiation that acts downstream of muscle
regulatory factors.
Most skeletal muscle develops from mesenchymal premuscle
condensations that are determined early in development (1). Several types of transcription factors control the specification and
differentiation of skeletal myoblasts within these condensations.
Critical among these are four basic helix-loop-helix myogenic
regulatory factors (MRFs)1:
MyoD, Myf5, myogenin, and MRF4, each of which binds to E-box sequences
in the promoters of skeletal muscle genes (see Ref. 2 for review). Each
of the four MRFs can induce skeletal muscle differentiation when
expressed in nonmuscle cells (3-7). However, during normal skeletal
muscle development, the MRFs function in a hierarchical fashion: MyoD
and Myf5 are involved in myoblast specification, whereas myogenin and
MRF4 control the terminal differentiation of myoblasts into myotubes
(8, 9). MyoD also promotes myoblast differentiation by inducing the
expression of myogenin and MEF (myocyte enhancer factor) 2 family
proteins. In addition, several homeobox genes, including Msx1, Pax3,
and Pax7 influence the specification of myoblasts by regulating the expression of particular MRFs (10-12). However, few homeobox genes have been described that act downstream of MRFs or that influence myotube fusion directly without influencing the expression of MRFs.
One component of the morphogenetic program specified by homeobox genes
is the control of cell adhesion, a process that is essential to
condensation, migration, and fusion of cells in several developmental
contexts (13, 14). Barx1 and Barx2 are two closely related homeobox
proteins that were found in separate efforts to identify factors that
control the expression of cell adhesion molecules (CAMs) (15, 16). In
an earlier study, we discovered Barx2 and showed that it is expressed
in precise patterns during mouse embryonic development in the nervous
system, neural crest-derived craniofacial structures, lung buds, and
limb mesenchyme (16). More recently, other researchers have shown that
chicken and murine Barx2 variants are expressed in both skeletal and
smooth muscle (17, 18). For example, the chicken Barx2 homologue
(Barx2b) is expressed in the early myotome and persists after
differentiation of muscle groups in the limb, neck, and cloaca (17). In
addition, Herring et al. (18) showed that murine Barx2 is
expressed in adult skeletal muscle and smooth muscle-containing
tissues. Barx2 is also expressed in C2C12 myoblasts, and its expression
increases dramatically when myoblasts fuse to form myotubes (18).
Collectively, these observations suggest roles for Barx2 in both
skeletal and smooth muscle development.
In this study we examined the role of Barx2 in muscle development.
Inhibition of Barx2 expression in limb bud cell cultures inhibits
myotube formation, whereas overexpression of Barx2 accelerates the
appearance of myotubes. However, overexpression of Barx2 does not
induce ectopic MyoD expression in limb bud cultures, nor is it
sufficient to specify a myoblast fate in multipotent C3H10T1/2 mesenchymal cells that do not express MyoD. These results suggest that
Barx2 acts downstream of MyoD to promote myogenic differentiation but
cannot substitute for MyoD function. In support of this conclusion, we
found that both MyoD and myogenin can bind to and activate the
Barx2 promoter in C2C12 myoblasts. In C3H10T1/2 cells
overexpression of Barx2 induces a smooth muscle-like appearance with
increased smooth muscle- Morpholino Antisense Oligonucleotide Inhibition of Barx2
Expression in Limb Bud Cultures--
Morpholino oligonucleotides
(ODNs) that are antisense to a region of the Barx2 mRNA near the
initiation codon were designed and synthesized by Gene Tools LLC.
Control sense ODNs were also prepared. Dissociated mesenchymal cells
were prepared from E10.5 mouse limb buds and 2 × 106
cells were plated in 10-µl aliquots at a density of 2 × 107 cells/ml in CMRL-1066 (Invitrogen) containing 2% fetal
bovine serum (FBS) in four-well culture dishes. After 1 h
incubation to allow the micromass cultures to adhere, sense and
antisense ODNs were introduced using LipofectAMINE 2000 (Invitrogen). The cells were cultured for an additional 24 h before fixing with 4% paraformaldehyde and staining with either
anti-MyoD or anti-Barx2 antibodies as described below.
Retroviral Transduction of Barx2 in Limb Bud
Cultures--
Retroviral vectors were constructed that contain either
full-length mouse Barx2 cDNA or a cDNA fragment that encodes
the homeodomain, Barx basic region, and COOH-terminal activation domain
(HD-BBR-C) (19). A control retroviral vector was constructed that
contains the green fluorescent protein gene. The retroviral vector was based on the murine embryonic stem cell virus with modifications as
described previously (20). The vector contains a truncated version of
the phosphoglycerate kinase promoter flanked by the chicken lysozyme
insulator sequence, driving expression of the green fluorescent protein
or Barx2 cDNAs. Retroviral particles were packaged in COS1 cells as
described previously (21). Supernatant containing retroviral particles
was collected after 48 h, filtered, and used to infect primary
limb bud cells. Aliquots of 2 × 106 dissociated E10.5
mouse limb bud cells were incubated with 2 ml of filtered retroviral
supernatant for 2 h at 4 °C. The cells were plated in micromass
conditions as described above, incubated for 1 h, and then
supplemented with 200 µl of CMRL-1066 containing 10% FBS and 200 µl of retroviral supernatant. After 24 h the medium was changed
to CMRL-1066 containing 2% FBS and the cells were cultured for a
further 24 h. Cells were then fixed with 4% paraformaldehyde and
either photographed at ×10 magnification using phase-contrast optics,
or stained with anti-MyoD antibodies as described below.
Immunohistochemistry--
Primary limb bud cultures and
C3H10T1/2 cell cultures were fixed with 4% paraformaldehyde and then
permeabilized for 1 min with ice-cold acetone. Cultures were then
stained with polyclonal antibodies to either Barx2 or MyoD (Santa
Cruz), or with monoclonal antibodies to smooth muscle (SM)- Stable Transfection of Barx2 in C2C12 Cells and Differentiation
of C2C12 Cells into Myoblasts--
1 × 107 C2C12
cells were electroporated with either 10 µg of linearized
Barx2/pcDNA3 expression vector or pcDNA3 control plasmid. The
Barx2 expression plasmid contains an in-frame NH2-terminal Myc tag and was described previously (16). Cells were selected for 14 days with 1 mg/ml G418 to establish a population of cells that carried
the plasmid at varying integration sites. Cells were tested for
expression of Barx2 after 3-4 passages by Western blotting and reverse
transcriptase-PCR. Experiments were performed with low passage stocks,
as Barx2 levels declined significantly after six passages. To examine
differentiation of the transfected C2C12 cells, cultures were incubated
in Dulbecco's modified Eagle's medium with 2% horse serum and
examined for myotubes at 24, 48, and 96 h. At 48 h, the
cultures were fixed for 10 min with 4% paraformaldehyde and
photographed at ×5 magnification using phase-contrast optics.
Quantitative Western Blotting--
To examine protein expression
in pcBarx2- and pcDNA3-transfected C2C12 cultures, proliferating
cells were harvested at 80% confluence and lysed in a hypotonic buffer
containing 1% Nonidet P-40 (22). Protein concentration was determined
by Bradford assay (Bio-Rad) and 20-µg aliquots were separated by
SDS-PAGE, transferred to polyvinylidene difluoride membrane
(Invitrogen), and blotted using either polyclonal antibodies to Barx2
(Santa Cruz) or monoclonal antibodies to SM- Isolation of the Murine Barx2 Gene--
The murine
Barx2 gene was isolated from a 129/SvJ murine genomic BACmid
library in the pveloBAC vector after screening with a PCR probe derived
from the 5' end of the Barx2 cDNA sequence (Genome Systems Inc).
Several overlapping restriction fragments containing the
Barx2 gene were mapped using Southern blot analysis and sequenced.
Construction and Mutagenesis of Barx2 Promoter/Luciferase
Reporter Plasmids and MRF Expression Plasmids--
Barx2
promoter/luciferase reporter constructs were generated in the
promoterless pGL3basic vector (Promega). The longest promoter construct
contained a 1812-base pair KpnI-SacI fragment
from the 5' end of the murine Barx2 gene. Additional
reporter plasmids containing truncated versions of the Barx2
promoter, or internal deletions of promoter regions, were generated by
cleavage at restriction sites or by PCR amplification. Mutation of the
E-box element (E2) and the CArG box/SRE within the 0.44-kb
Barx2 promoter construct was performed using the QuikChange
protocol (Stratagene). All constructs were confirmed by
sequencing. Synthetic promoter constructs were prepared by cloning
oligonucleotides corresponding to a 58-bp segment of the
Barx2 promoter (referred to here as the myogenic regulatory
region (MRR)) into a modified version of the pGL3basic vector
containing a minimal TATA box promoter and initiator sequence (21). In
addition, two mutant versions of the MRR were generated in which either
the E2 element or the CArG box/SRE were mutated. The sequences of these
oligonucleotides with the E-box and CArG box motifs shown in bold, are
as follows: MRR
5'-gctccgcacctggccctgcaggaagtgcgcgctgattgacagctgcggtgtcccaaaaaggct-3';
To construct the MyoD and myogenin expression plasmids, we first
isolated full-length cDNAs from E14 embryonic mouse limb RNA by
reverse transcriptase-PCR. The cDNAs were cloned into a modified
version of the pcDNA3 vector (Invitrogen) that contained an
NH2-terminal Myc epitope tag. Proteins of the appropriate
molecular weights were produced by in vitro translation
using the Promega TNT coupled transcription/translation kit.
Cellular Transfection of C3H10T1/2 and C2C12
Cells--
C3H10T1/2 were cultured in Dulbecco's modified Eagle's
medium containing 10% FBS (Invitrogen) and antibiotics (penicillin and
streptomycin). C2C12 cells were cultured in Dulbecco's modified Eagle's medium with 20% FBS and antibiotics. Both cell lines were obtained from American Type Culture Collection. C3H10T1/2 cells were
placed in 6-well tissue culture plates at an initial density of 1 × 105 cells/well and transfected with 0.5 µg of each
luciferase reporter construct. C2C12 cells were placed in 24-well
tissue culture plates at an initial density of 1 × 104 cells/well and co-transfected with 200 ng of each
luciferase reporter construct and 200 ng of each pcDNA3 expression
plasmid. All transfections used FuGENE reagent (Roche Molecular
Biochemicals) and included the LacZ reporter CMV Gel Mobility Shift Experiments--
Oligonucleotide probes were
synthesized that corresponded to each of the two E-box elements, and
the CArG box/serum response element (SRE) from the murine
Barx2 gene. In addition, mutant versions of these sequences
were designed in which the core motifs were disrupted. Double-stranded
probes were end-labeled with polynucleotide kinase and
[ Inhibition of Barx2 Expression Prevents Myotube Formation in
Primary Limb Bud Cell Cultures--
To determine whether Barx2 is
involved in the differentiation of skeletal myoblasts, we inhibited
Barx2 expression in primary myoblasts using morpholino antisense ODNs
and then examined their ability to form myotubes. Morpholino ODNs are
RNase H-independent, resistant to nucleases, and have a good targeting
predictability (24, 25). For this study, limb bud mesenchymal cells
from E10.5 mouse embryos were cultured in micromass conditions; these micromass cultures have been shown to contain cells that are committed to either a myoblast or chondrocyte fate (26). The limb buds were
dissected, dissociated, and cultured in low serum under micromass conditions as described under "Experimental Procedures." The
cultures were treated immediately after plating with either Barx2
antisense ODNs or control sense ODNs and examined 24 h later for
formation of myotubes and for expression of Barx2 and MyoD.
As shown in Fig. 1, cultures treated with
ODNs antisense to Barx2 showed negligible expression of Barx2 (Fig. 1,
top right panel) and formed few if any myotubes within the
24-h culture period. Many cells showed nuclear expression of MyoD,
however, these cells were not fused and did not show the characteristic elongated nuclei of myotubes (Fig. 1, bottom right panel).
In contrast, cultures that were treated with the sense ODNs contained many cells that expressed Barx2 and MyoD and that were fused into multinucleated myotubes (Fig. 1, left panels). These results
indicate that Barx2 expression is required for myoblasts to
differentiate into myotubes in culture. Immunohistochemical staining
showed that Barx2 antisense-treated cultures do not express Barx2, but retain appreciable, albeit diffuse, expression of MyoD. The observation that MyoD staining in antisense-treated cultures is diffuse relative to
the control cultures may be due in part to the condensed and elongated
morphology of nuclei in myotubes.
Barx2 Overexpression Accelerates Myotube Formation in Primary
Limb Bud Cells--
To determine whether overexpression of Barx2 can
promote myotube formation, a retroviral system was used to overexpress
Barx2 proteins in limb bud cultures. Micromass cultures were infected with retroviral constructs that express either full-length Barx2 protein or a fragment of Barx2 containing the homeodomain, Barx basic
region (BBR), and the carboxyl-terminal domain (Barx2/HDBBRC). The
Barx2/HDBBRC fragment contains the DNA binding and transactivation domains of Barx2 and was previously shown to activate the promoter of
the Barx2 target gene N-CAM, which is a cell adhesion molecule known to
influence myotube fusion (19, 27). Control cultures were infected with
a retroviral construct expressing green fluorescent protein. The cells
were cultured in micromass conditions (28) for 24 h in media
containing 10% serum to allow integration of the virus into dividing
cells and the expression of Barx2 proteins. The cultures were then
incubated for a further 24 h in media containing 2% serum to
induce myotube formation.
As shown in Fig. 2, micromass cultures
that were infected with retroviral constructs expressing either
full-length Barx2 (not shown) or the Barx2/HDBBRC fragment (Fig. 2,
top right panel) formed many myotubes within 24 h of
culture. In contrast, cells infected with the control retrovirus
produced very few myotubes within 24 h (Fig. 2, top left
panel). Immunohistochemical staining with antibodies to MyoD
revealed that only MyoD-positive cells fused to form myotubes. Thus
Barx2 cannot promote myotube fusion in the absence of MyoD (Fig. 2,
middle panels). Moreover, when the number of MyoD-positive
cells was counted in three separate fields, their numbers did not
change after infection with the Barx2 construct (data not shown). Thus
Barx2 does not induce ectopic expression of MyoD. Together these
results indicate that Barx2 acts downstream of, but does not substitute
for, MyoD activity. The myotubes that formed after infection of limb
bud cells with the Barx2/HDBBRC-expressing retrovirus also showed
increased expression of SM- Barx2 Promotes Myotube Formation in C2C12 Myoblasts but Not in
C3H10T1/2 Fibroblasts--
The results of both Barx2
inhibition and overexpression experiments in primary limb bud cultures
suggest that Barx2 acts downstream of MyoD to promote the
differentiation of cells that are already committed to the myoblast
fate. To formally test whether Barx2 acts upstream or downstream of
MyoD, we compared the effects of overexpressing Barx2 in MyoD-positive
C2C12 myoblasts and in MyoD-negative C3H10T1/2 mesenchymal cells.
A population of C2C12 myoblasts that overexpress Barx2 was generated by
stable transfection of a pcBarx2 expression plasmid, and a control
population was established by stable transfection of the empty
pcDNA3 vector. Overexpression of Barx2 protein in pcBarx2-transfected cells was confirmed by Western blotting with anti-Barx2 antibodies (see Fig.
3D). The effect of Barx2
overexpression on myotube formation was examined by plating
pcBarx2-transfected and control cells at the same initial density and
culturing in media containing 2% horse serum. Under these conditions,
cultures of pcDNA3-transfected C2C12 cells differentiated
relatively slowly, forming myotubes after 4 days (Fig. 3A,
left panel). In contrast, C2C12 cells that were transfected
with the pcBarx2 plasmid showed significantly accelerated
differentiation, forming myotubes within 2 days of serum withdrawal
(Fig. 3A, right panel). These results are similar
to those obtained in primary limb bud cultures and provide further
evidence that expression of Barx2 promotes the differentiation of
MyoD-expressing myoblasts.
To test whether Barx2 is also involved in myoblast determination, we
next examined the effect of overexpressing Barx2 in the mesenchymal
progenitor cell line C3H10T1/2 that does not express MyoD. Ectopic
expression of MyoD was previously shown to convert C3H10T1/2 cells into
skeletal myoblasts that fuse into myotubes when cultured in low serum
conditions (31). If Barx2 were able to induce the expression of MRFs
such as MyoD it may be expected to convert C3H10T1/2 cells into
myoblasts. C3H10T1/2 cells were transiently transfected with either the
pcBarx2 plasmid, a pcDNA expression plasmid containing the MyoD
cDNA (pcMyoD), or the empty vector. To identify transfected cells,
a plasmid encoding enhanced yellow fluorescent protein (EYFP) was
co-transfected with the expression vectors. The cells were then
incubated in media containing 2% horse serum and the formation of
myotubes by the EYFP-labeled cells was examined over the next 4 days.
Consistent with previous reports (31), ectopic MyoD expression induced
myotube formation in nearly all of the transfected cells after 4 days
of serum deprivation (Fig. 3B, right panel). In
contrast, Barx2-transfected C3H10T1/2 cells did not form myotubes (Fig.
3B, middle panel). These results indicate that,
unlike MyoD, Barx2 is not sufficient to specify a skeletal myoblast
fate when expressed in multipotent mesenchymal cells.
Interestingly, when pcBarx2-transfected and control
pcDNA3-transfected C3H10T1/2 cells were grown in high serum conditions, pcBarx2-transfected cells showed morphological changes that are consistent with the differentiation of smooth muscle cells (SMCs) or
myofibroblasts, including cell spreading and formation of stress fibers
(Fig. 3C) (32, 33). C3H10T1/2 cells and other fibroblastic cell lines have been previously reported to differentiate into both
SMCs and myofibroblasts in response to particular signals (34-36).
Using immunohistochemistry, we found that Barx2-transfected C3H10T1/2
cells contained increased amounts of SM-
In these studies, Barx2 overexpression increased the expression of
SM- Identification of the Murine Barx2 Gene--
The results of the
studies described above suggest that Barx2 acts downstream of MyoD
during myoblast differentiation. To determine whether MyoD or other
MRFs may directly regulate the expression of Barx2, we isolated the
murine Barx2 gene and characterized its promoter. The
Barx2 gene was isolated by screening a BACmid murine genomic
library using a probe from the 5' end of the Barx2 cDNA sequence.
Two clones of ~80 kb containing the Barx2 gene were
analyzed by restriction endonuclease digestion and Southern blotting.
Several overlapping restriction fragments containing the
Barx2 gene were mapped and sequenced, revealing four exons spanning greater than 30 kb (Fig. 4). The
size of the first intron was not determined precisely; however,
Southern blots indicated that it spans at least 20 kb. In addition, a
3-kb segment upstream of the first exon was isolated and sequenced.
Comparison of the sequence of the murine Barx2 gene with the
recently published human Barx2 gene (37) revealed
considerable similarity over a 1-kb region upstream of the translation
initiation codon. In particular, the sequence that corresponds to the
transcription start site (TSS) in the human gene (37) is conserved,
suggesting that transcription begins at the same site in the murine
gene (Fig. 5A). DNA sequence
analysis of the region upstream of the predicted TSS revealed potential
binding sites for a number of transcription factors, including
sequences that match the Sp1 consensus motif (G(A/G)GGC(A/G)GGG(A/T)).
A sequence containing a perfect match to the Sp1 consensus sequence
(GGGGCGGGGT) is located ~340 nucleotides upstream of the TSS; an
additional cluster of motifs that partially match the Sp1 consensus is
located ~80 nucleotides upstream of the TSS. Because no TATA box
elements are contained within the 3-kb region upstream of the first
exon, this GC-rich region is likely to represent the core promoter. Several other conserved sequence elements were identified within a
400-bp segment upstream of the TSS. These include two E-box motifs,
recognition sites for MEF and Ets family proteins, and a noncanonical
CArG box/SRE (Fig. 5A).
The Murine Barx2 Promoter Contains Enhancer and Repressor Sequences
That Function in Mesenchymal Cells--
To identify positive and
negative regulatory regions of the Barx2 promoter, six
deletion constructs were generated, either by PCR amplification or by
cleaving the promoter at natural restriction sites (Fig.
5B). These promoter fragments were cloned into the promoterless pGL3basic luciferase reporter vector and their activities were examined after transfection into C3H10T1/2 cells.
The shortest Barx2 promoter construct tested (construct
0.44), containing the region between nucleotides MyoD and Myogenin Bind to an E-box Element within the Barx2
Promoter; Binding to this Element Is Increased during Differentiation
of C2C12 Cells--
The proximal region of the Barx2 promoter contains
recognition motifs for several transcription factors that are
associated with muscle development. Among these elements are two
sequences corresponding to the E-box consensus (CANNTG) that may be
recognized by myogenic basic helix-loop-helix factors such as MyoD or
myogenin (see Fig. 5A). To determine whether these motifs
can bind to MyoD or myogenin, we prepared two probes, designated E1 and
E2, corresponding to each of the motifs, and tested their binding to
in vitro translated MyoD and myogenin proteins in gel
mobility shift experiments. MyoD and myogenin formed complexes with the
E2 probe that were supershifted by antibodies to a Myc epitope tag
located at the NH2 terminus of the proteins (Fig.
6A). In contrast, neither MyoD nor myogenin bound to the E1 probe (data not shown).
The E1 and E2 probes were also tested for binding to nuclear extracts
of C2C12 cells in gel mobility shift assays. Nuclear extracts were
prepared from both proliferating C2C12 myoblasts and C2C12 cells
cultured for 4 days in media containing 2% horse serum to induce
myotube formation. The E2 probe bound to both myoblast and myotube
C2C12 nuclear extracts; mutation of the CANNTG motif within the E2
probe abolished this binding (Fig. 6B). In contrast, the E1
probe showed negligible binding to C2C12 nuclear extracts (data not shown).
As shown in Fig. 6B, the complexes formed between the E2
probe and the myotube nuclear extract appeared more intense than those
formed with the myoblast nuclear extract. To quantify the difference in
binding, we measured the intensities of the probe-protein complexes
that formed with the E2 probe using equivalent amounts of nuclear
protein prepared from myoblast or myotube cultures in four separate gel
mobility shift experiments (see "Experimental Procedures"). On
average, the intensity of the E2 probe-protein complex formed with
myotube nuclear extract was 1.9-fold greater than that formed with
myoblast nuclear extract (Fig. 6B). These data indicate that
the E2 element binds to proteins that are enriched during myogenic
differentiation of C2C12 cells, and that these proteins are likely to
include MyoD and myogenin.
Binding of Serum Response Factor (SRF) to the Barx2Promoter--
The proximal Barx2 promoter contains a noncanonical CArG
box/SRE immediately adjacent to the MyoD/myogenin binding site. This element is a recognition motif for SRF, which has been shown in previous studies to interact with MyoD and myogenin at the promoter of
particular muscle-specific genes (38, 39). To determine whether SRF
binds to the Barx2 promoter, we prepared a probe
corresponding to the Barx2 CArG box/SRE and tested its
ability to bind to nuclear extracts from C2C12 cells. The CArG box/SRE
probe formed complexes with nuclear extracts from both C2C12 myoblasts
and myotubes; this binding was eliminated by mutation of the CArG box
consensus motif (Fig. 6C). Because the sequence of the
Barx2 CArG box/SRE (CCCAAAAAGG) diverges from the canonical
recognition motif for SRF (CC(A/T)6GG), we examined whether
antibodies to SRF could supershift or block the formation of complexes
between the Barx2 CArG box probe and C2C12 nuclear extracts.
The predominant probe-protein complex was supershifted by antibodies to
SRF, suggesting that the complex contains SRF (see lane 5 in
Fig. 6C).
In C2C12 cells, SRF regulates both mitogenic genes such as
c-fos, as well as muscle-specific genes (40, 41). However, SRF has been reported to bind with higher affinity to the CArG box/SRE
from the c-fos gene promoter than to the noncanonical CArG
boxes/SREs present in many muscle-specific genes (40). To examine the
relative strength of binding of SRF to the Barx2 CArG
box/SRE, we compared binding of C2C12 nuclear extracts to the
Barx2 CArG box/SRE probe as well as to a control probe
corresponding to the c-fos CArG box/SRE (40). The
Barx2 CArG box/SRE probe formed much less intense complexes
than the c-fos CArG box/SRE probe when incubated with
equivalent amounts of nuclear protein from C2C12 cells. Collectively
these data suggest that both E-box binding proteins and SRF may
regulate expression of Barx2 in skeletal muscle cells.
MyoD and Myogenin Activate the Proximal Barx2 Promoter in C2C12
Cells--
The results of the gel mobility shift analyses described
above indicated that the proximal Barx2 promoter region is
bound by factors involved in muscle-specific gene regulation. To
examine the role of these elements in the regulation of the
Barx2 gene, C2C12 myoblasts were transfected with various
Barx2 promoter constructs, together with expression plasmids
that encode MyoD and myogenin (Fig. 7).
Constructs 1.0 and 0.44 showed lower levels of basal activity in C2C12
cells than in C3H10T1/2 cells (see Fig. 5). However, co-transfection of
a MyoD expression plasmid activated these constructs approximately 13- and 11-fold, respectively (Fig. 7). Expression of myogenin activated
these two constructs 6- and 5-fold, respectively (Fig. 7). Deletion of
a 220-bp SmaI restriction fragment from construct 0.44 (0.44 The E2 and CArG Box/SRE Motifs Are Required for
Activation of the Barx2 Promoter by MyoD--
To examine the role of
the MyoD/myogenin binding site (E2) in activation of the
Barx2 promoter by MyoD and myogenin, this element was
mutated within the 0.44-kb Barx2 promoter construct. Because
SRF has been shown to act cooperatively with MyoD (38), the SRF binding
site (CArG box/SRE) was also mutated within the 0.44-kb construct.
C2C12 cells were co-transfected with pGL3basic, the 0.44-kb
Barx2 promoter construct, the 0.44
To determine whether these two elements are sufficient for activation
by MyoD, we generated a synthetic construct containing a segment of the
Barx2 promoter that spans the E2 and CArG box/SRE motifs.
This 58-bp segment (hereafter referred to as the myogenic regulatory
region, or MRR) was synthesized and inserted upstream of a minimal TATA
box promoter driving the luciferase reporter gene (see Fig.
8B). When transfected into C2C12 cells, this synthetic promoter construct (MRR) showed ~7-fold greater activity than that of
the minimal promoter alone (construct pLuc). Co-transfection with a
MyoD expression plasmid activated the MRR construct ~6-fold, relative
to transfection of the empty pcDNA3 plasmid (Fig. 8B). In contrast, co-transfection of a myogenin expression plasmid did not
significantly activate the MRR construct (data not shown), suggesting
that activation of the Barx2 promoter by myogenin (see Fig.
7) involves additional sequences outside of the MRR.
To examine whether the E2 or CArG box/SRE elements are required for
activation of the MRR by MyoD, we prepared constructs in which either
the E2 or CArG box/SRE motif was mutated, and tested their ability to
be activated by MyoD in cellular transfection experiments. Mutation of
either the E2 or CArG box/SRE motif reduced the activity of the MRR
promoter construct to the level of the minimal promoter alone. Similar
to the results obtained when the E2 and CArG box/SRE elements were
mutated in the native promoter construct, mutation of either element
with the MRR was sufficient to prevent activation by MyoD. These
results indicate that activation of the Barx2 promoter by
MyoD requires an accessory factor that binds to the CArG box/SRE
element, most likely SRF.
The homeobox protein Barx2 is expressed in several phases of
mesenchymal tissue morphogenesis, including the formation of pre-chondrogenic and pre-muscle mesenchymal condensations and the
differentiation of skeletal and smooth muscle (16-18). Barx2 is also
expressed in adult skeletal and smooth muscle, and its expression in
C2C12 myoblasts is increased during differentiation into myotubes (18).
In this study we showed that expression of Barx2 is required for the
differentiation of MyoD-positive limb bud cells into myotubes, and that
overexpression of Barx2 accelerates myotube formation by both primary
myoblasts and C2C12 cells. However, overexpression of Barx2 did not
increase the number of MyoD-positive cells in limb bud cultures. This
result, together with the observation that overexpression of Barx2 does
not convert MyoD-negative C3H10T1/2 cells into skeletal myoblasts,
strongly suggests that Barx2 does not activate MyoD but rather acts
downstream of MyoD. Importantly, the latter observation also indicates
that Barx2 cannot substitute for MyoD activity.
Taken together, our results suggest that the role of Barx2 in myoblast
differentiation may be distinct from that of other homeobox genes
previously shown to influence muscle development, such as Pax3, Pax7,
Lbx1, and Msx1. Each of these factors has been shown to act upstream of
MRFs in myoblast determination. For example, Pax3 is expressed in
myogenic progenitor cells and proliferating myoblasts and is required
for the activation of MyoD and thus myogenesis (10). Lbx1 also acts
upstream of MRFs to specify myoblasts and both induces, and is induced
by, Pax3 (11). Msx1 antagonizes the activity of Pax3 by direct
interaction leading to repression of myogenesis (12, 42) and ectopic
expression of Msx1 in C2C12 myotubes reduces the expression of MRFs
leading to de-differentiation of myotubes (43). In contrast to the
activities of these factors, our study indicates that Barx2 promotes
myotube formation without inducing the expression of MyoD; thus
Barx2 is the first homeobox gene shown to have a significant
role in myoblast differentiation downstream of MyoD expression.
The mechanism by which Barx2 promotes myotube formation is yet to be
determined. However, because the portion of the Barx2 protein that
contains the DNA binding and activation domains was sufficient to
promote myotube formation, it is likely that Barx2 activates particular
genes that are required for the differentiation of skeletal myoblasts.
For example, in this study we found that Barx2 up-regulated
SM- Our analysis of the Barx2 gene promoter provides a molecular
mechanism for the control of Barx2 expression during myogenic differentiation. We identified a proximal region of the
Barx2 promoter containing an E-box (E2) that binds to both
MyoD and myogenin, and additional binding sites for SRF and MEF2
proteins. Binding of nuclear proteins to the E2 element was found to
increase during differentiation of C2C12 myoblasts, suggesting that the binding of MRFs to this promoter element mediates the previously reported induction of Barx2 expression during myotube formation (18).
Both the E2 element and an adjacent CArG box/SRE, which binds to the
SRF are required for activation of the Barx2 promoter by
MyoD. This result is similar to previous observations that both E-box
and CArG/SRE motifs are involved in activation of the SM- SRF also plays a central role in the differentiation of smooth muscle
(41, 49, 50) and has been shown to interact with various homeodomain
proteins including variants of both Barx1 and Barx2 (18, 51-54). In
particular, Barx2 can stimulate the binding of SRF to its cognate CArG
box/SRE motif (18), and interaction of chicken Barx1b with SRF
regulates smooth muscle cell-specific expression of the Overall, our studies suggest that Barx2 regulates multiple muscle
differentiation pathways. In committed myoblasts Barx2 expression can
be induced by MRFs such as MyoD and myogenin and increased expression
of Barx2 then promotes myotube formation. In uncommitted mesenchymal
cells that do not express MRFs, Barx2 may promote differentiation into
other contractile cell types such as SMCs. The mechanisms by which
Barx2 influences the differentiation of both skeletal and smooth muscle
could be elucidated by chromatin immunoprecipitation or microarray
analysis to identify targets of Barx2 that are specifically regulated
in each of these contexts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin expression and formation of stress
fibers. Overall our data suggest two roles for Barx2 in muscle
development: in MyoD-positive skeletal myoblasts Barx2 controls myotube
fusion, whereas in MyoD-negative mesenchymal cells Barx2 may promote
differentiation of other contractile phenotypes such as smooth muscle
cells or myofibroblasts.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin
(Sigma) at 1:200 dilutions. Secondary antibodies were conjugated with
either fluorescein isothiocyanate or rhodamine (Molecular Probes).
-actin,
-actin, or
desmin (Sigma). Blots were developed using chemiluminescent substrate (Novex) and autoradiographed. The intensity of the protein bands was
determined by densitometry using NIH Image software and a ratio of
either SM-
-actin or desmin protein to
-actin protein was
determined for each sample. The average increase in SM-
-actin expression was determined from three replicate experiments.
E2
5'-gctccgcacctggccctgcaggaagtgcgcgctgattgagtcctgcggtgtcccaaaaaggct-3';
CArG box
5'-gctccgcacctggccctgcaggaagtgcgcgctgattgacagctgcggtgttttcaaaaattct-3'.
gal
(Clontech) at one-tenth of the total DNA amount to
provide an internal reference for transfection efficiency. Cellular
transfection and assay conditions were otherwise as described
previously (21). All experiments were performed in duplicate and the
data shown were derived from at least three independent experiments.
For co-transfection of C3H10T1/2 cells with the pEYFP1 plasmid
(Clontech) and pcDNA3, pcBarx2, or pcMyoD plasmids, 1 × 105 cells were seeded in 60-mm plates
and transfected with 2 µg of each plasmid using FuGENE (Roche
Molecular Biochemicals).
-32P]ATP (3000 Ci/mmol) (DuPont). Probes were
purified by elution from an 8% polyacrylamide gel and their specific
activity was determined. 25,000 cpm (~10 fmol) of each probe was used
in gel mobility shift experiments with C2C12 cell nuclear extracts, or in vitro translated proteins, as described previously (19). C2C12 cell extracts were prepared from proliferating C2C12 myoblasts cultured in high serum conditions (20% FBS) or from myotubes that were
prepared by culture in low serum conditions (2% horse serum) for 4 days. Although C2C12 cells have been reported to differentiate in as
little as 24 h after serum withdrawal, the slow rate of differentiation of our C2C12 cells is consistent with the known variation in the differentiation capacity of different stocks of C2C12
cells (23).2 Nuclear extracts
were prepared as described previously (22). For semiquantitative gel
shift experiments, equal amounts of C2C12 nuclear protein from C2C12
myoblasts or myotubes were used in each binding reaction. Relative
binding of the protein to the E-box probes was determined by measuring
the intensity of the probe-protein complexes formed in gel
mobility-shift experiments using a PhosphorImager (Amerhsam
Biosciences). The relative binding data shown in Fig. 6 were
derived from four independent experiments using different C2C12
extract preparations.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Barx2 is required for the formation of
myotubes in primary limb bud mesenchymal cultures. Limb bud
cells from E10.5 embryos were cultured in micromass conditions and
treated for 6 h with morpholino ODNs that were antisense to Barx2,
or with control sense ODNs. Cells were then cultured for 24 h in
low-serum conditions, fixed, and stained with polyclonal antibodies to
either Barx2 or MyoD. Cultures treated with sense ODNs formed myotubes
and expressed both Barx2 and MyoD at high levels (left
panels). In contrast cultures treated with Barx2 antisense ODNs
did not form myotubes and did not express Barx2, however, MyoD
expression is still apparent (right panels).
-actin, an early marker of skeletal
muscle differentiation (29, 30) (Fig. 2, bottom panels).
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Fig. 2.
Barx2 promotes the formation of myotubes in
primary limb bud mesenchymal cultures. Retroviral constructs that
express either green fluorescent protein (control), or a
fragment of Barx2 containing the homeodomain, Barx basic region, and
the carboxyl-terminal activation domain
(Barx2/HDBBRC), were used to infect dissociated
limb bud cells from E10.5 mouse embryos. After infection, limb bud
cells were cultured in micromass conditions for 24 h in high-serum
media and then incubated for a further 24 h in low-serum media to
induce myotube formation. Cultures that were infected with the
Barx2/HDBBRC-expressing retrovirus formed many myotubes after 24 h
of culture in low-serum, whereas those infected with the control
retrovirus did not (top panels). Immunostaining of cultures
with polyclonal antibodies to MyoD showed that only MyoD-positive cells
fused to form myotubes and that the number of MyoD-positive nuclei is
similar in both control and Barx2/HDBBRC-infected cultures
(middle panels). Immunostaining with monoclonal antibodies
to SM- -actin (green) showed that cells infected with the
Barx2/HDBBR-expressing retroviral construct express higher levels of
SM-
-actin. Nuclei are counterstained with
4,6-diamidino-2-phenylindole (blue).
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Fig. 3.
Barx2 promotes myogenic differentiation of
C2C12 myoblasts but not of C3H10T1/2 fibroblasts.
A, two populations of stably transfected C2C12 cells
were generated by transfection with either the pcBarx2 or pcDNA3
plasmid and selection for 14 days with 1 mg/ml G418. Transfected cells
were incubated in reduced serum media for 2 days, and then examined for
myotube formation using phase-contrast optics. The pcBarx2 transfected
cell population formed myotubes within 2 days of serum withdrawal,
whereas pcDNA3 transfected cells did not. B,
C3H10T1/2 cells were transiently co-transfected with either pcBarx2 or
pcDNA3, and a reporter plasmid that encodes EYFP. The morphology of
live transfected (fluorescent) cells was examined after 4 days of
culture in reduced serum media. Under these conditions, MyoD, but not
Barx2, was sufficient to induce differentiation of C3H10T1/2 cells into
myotubes. C, C3H10T1/2 cells were transiently
transfected with either a Barx2 expression plasmid (pcBarx2) or empty
pcDNA3 plasmid, and stained with monoclonal antibodies to
SM- -actin (red) after 4 days of culture in high serum
media. Nuclei were counterstained with 4,6-diamidino-2-phenylindole
(blue). Cells transfected with pcBarx2 are flattened,
contain stress fibers, and stain more intensely for SM-
-actin.
D, equal aliquots of protein from C2C12 cells stably
transfected with either pcDNA (
) or pcBarx2 (+) were analyzed by
Western blotting using Barx2 antibodies for increased expression of
Barx2 (arrowhead), and for expression of the muscle markers
SM-
-actin and desmin.
-actin was used as a standard. The
expression of SM-
-actin in pcBarx2 (+) transfected cells was an
average of 1.7-fold higher than in pcDNA3 transfected cells (
) in
three replicate experiments (see arrow at
left).
-actin, which is also
consistent with these cellular phenotypes (Fig. 3C). These results suggest that in the absence of MyoD expression, Barx2 influences the differentiation of mesenchymal progenitors into contractile cell types other than skeletal myoblasts.
-actin in all three cellular systems tested: primary myoblasts,
C2C12 myoblasts, and C3H10T1/2 cells. To quantify this effect, we
examined the expression of two cytoskeletal proteins that are
regulated during myogenesis, SM-
-actin and desmin, in Barx2- and
pcDNA3-transfected C2C12 cells. Quantitative Western blotting was
used to measure the amount of SM-
-actin, desmin, and
-actin
protein in extracts of cells grown in high serum conditions (Fig.
3D). After normalization to
-actin, the amount of
SM-
-actin was, on average, 1.7-fold higher in pcBarx2-transfected
cells than in pcDNA3-transfected cells in three separate
experiments. In contrast the expression of desmin did not change
significantly (Fig. 3D). This indicates that Barx2 can
activate particular genes that are associated with the differentiation
of multiple contractile cell types. Collectively our studies in primary
limb bud cultures and cell lines suggest two functions for Barx2 in
muscle differentiation. In committed myoblasts that express MyoD, Barx2
promotes, and is required for, formation of myotubes, whereas in
multipotent mesenchymal cells that lack MyoD, Barx2 may induce a smooth
muscle or myofibroblast phenotype.
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Fig. 4.
The intron/exon structure and splice junction
sequences of the mouse Barx2 gene. The
Barx2 gene spans greater than 30 kb and contains four exons
encoding a protein of 279 amino acids. Coding regions of exons are
depicted as solid black boxes; the 5'- and 3'-untranslated
regions of exons 1 and 4 as open boxes. The predicted
transcription start site (+1) is indicated by the
arrow.
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Fig. 5.
Analysis of the Barx2
promoter and 5'-untranslated region. A,
sequence comparisons show extensive homology between the mouse and
human Barx2 genes within the 1-kb region immediately
upstream of the first exon. Nucleotides that are identical in the two
genes are highlighted in boldface type. The region between
0.44 and
0.08 kb contains putative binding sites for Sp1 and a
cluster of regulatory elements that are involved in muscle-specific
transcription, including binding motifs for basic helix-loop-helix
proteins (E-box), MEF2, Ets proteins, and serum response factor (CArG
box/SRE) (indicated by boxes). The arrow
indicates the predicted transcription start site. This sequence
corresponds to GenBankTM accession number AY188085,
nucleotides 1953-3014. B, six Barx2
promoter/luciferase reporter plasmids were constructed in the
luciferase reporter plasmid pGL3basic by deletion of a 1.7-kb promoter
fragment that contains the transcription start site. C,
C3H10T1/2 cells were transfected with each promoter construct, together
with the lacZ reporter plasmid CMV
gal to provide an internal
reference for transfection efficiency. Luciferase and
-galactosidase
activities were measured 48 h post-transfection. The data are the
average of four experiments, performed in duplicate. D,
schematic representation of the Barx2 promoter depicting the
core promoter, enhancer, and repressor activities that were identified
in transfection experiments. Regulatory elements within the core
promoter region are indicated by polygons: Sp1
(circle), E-box (rectangle), CArG box/SRE
(triangle), and MEF2 (square).
438 and +80 relative to the predicted transcription start site, showed 7-fold greater activity than the pGL3basic plasmid suggesting that this region contains a core promoter (Fig. 5C). A fragment spanning
nucleotides
1012 to +80, relative to the TSS (construct 1.0), showed
~2-fold greater activity than construct 0.44, indicating that the
region between nucleotides
438 and
1012 contains an enhancer.
Addition of the segment between nucleotides
1012 and
1214
(construct 1.2) led to a 2-fold decrease in promoter activity, relative
to construct 1.0, indicating that this 200-bp region contains a
repressor (Fig. 5C). Two additional constructs, 1.5 and 1.7, spanning nucleotides
1446 to +80 and
1732 to +80, respectively,
showed levels of activity similar to that of construct 1.2. However,
deletion of the segment between nucleotides
1012 and
1462 from
construct 1.7 (construct 1.7
KspI) increased activity ~8-fold.
These results suggest that the region between
1012 and
1214
nucleotides contains a repressor that masks an enhancer activity
located between
1462 and
1732 nucleotides upstream of the TSS (Fig.
5D).
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Fig. 6.
The Barx2 promoter contains
binding sites for transcription factors involved in myogenic
differentiation. A, a probe corresponding to the
second E-box element (E2) of the proximal Barx2 promoter was
tested for binding to in vitro translated, Myc-tagged MyoD
or myogenin (MyoG) proteins in gel mobility shift assays. The E2 probe
formed complexes with both MyoD and myogenin translation products that
were not formed with a control (c) translation mixture
(lanes 1-3, arrow at left). Antibodies to the
Myc tag supershifted these complexes (lanes 4-6,
arrows at right). B, the E2 probe was also
tested for binding to nuclear extracts prepared from C2C12 myoblast
(MB) or myotube (MT) cultures. The probe formed
more intense complexes with myotube extracts than with myoblast
extracts (lanes 1 and 2) whereas a mutated
version of the probe in which the E-box probe was eliminated (mE2) did
not bind to either extract (lanes 3 and 4). The
average increase in binding was determined by measuring the relative
intensities of the complexes formed between the E2 probe and the
myoblast or myotube extracts in four independent experiments.
C, a probe corresponding to the CArG/SRE within the
Barx2 promoter (CArG), and a mutant version in which the
core motif was eliminated (mCArG), were tested for binding to C2C12
extracts. As a control, a probe containing the consensus CArG/SRE from
the c-fos promoter was also tested for binding. The
Barx2 CArG probe formed a complex with the C2C12 myoblast
extract (MB) that was not formed with the mCArG probe (lanes 1 and 2, arrow at left).
This complex was supershifted by antibodies to SRF (lanes
3-5, see arrowhead at right). The Barx2
CArG probe formed a much less intense complex with the C2C12 extract
than the c-fos CArG box/SRE, suggesting that the
Barx2 CArG box/SRE has a lower affinity for SRF (compare
lanes 3 and 6).
Sma) prevented its activation by both MyoD and myogenin. The
220-bp SmaI fragment alone (construct Sma) also functioned
as a promoter in C2C12 myoblasts and was activated by MyoD and myogenin
7- and 4-fold, respectively (Fig. 7). These results indicate that the
SmaI restriction fragment within the Barx2
proximal promoter, which contains Sp1, E-box, and CArG box/SRE motifs,
is necessary and sufficient for promoter activation by MyoD, and to a
lesser extent, by myogenin.
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Fig. 7.
MyoD and myogenin activate the mouse
Barx2 promoter in C2C12 myoblasts.
A, Barx2 promoter constructs used to examine
activation by MyoD and myogenin. The 0.44 Sma plasmid was generated
by deleting a 220-bp internal SmaI fragment that contains
Sp1, E-box, and CArG box elements. The Sma plasmid contains the
220-bp SmaI fragment upstream of the luciferase gene in
pGL3basic. B, C2C12 cells were co-transfected with each of
the Barx2 promoter reporter plasmids and either empty
pcDNA plasmid or a pcDNA3 expression plasmid encoding MyoD or
myogenin. The CMV
gal plasmid provided an internal reference for
transfection efficiency. Luciferase and
-galactosidase activities
were measured 48 h post-transfection. The data are the average of
six experiments, performed in duplicate.
E2 construct, or the
0.44
CArG construct, together with pcDNA3, MyoD, or myogenin expression plasmids. As shown in Fig.
8A, mutation of either the E2
or CArG box/SRE element prevented activation of the 0.44-kb Barx2 promoter construct by MyoD and myogenin.
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Fig. 8.
The E2 and CArG/SRE elements are required for
activation of the Barx2 promoter by MyoD and
myogenin. A, constructs used to examine the role
of the E2 and CArG elements in the Barx2 promoter. Each of these
elements was eliminated from the 0.44 Barx2 promoter
luciferase construct by site-directed mutagenesis. B,
C2C12 cells were transfected with each of the Barx2 promoter
constructs shown in A, together with the expression
plasmids, pcDNA3, pcMyoD, or
pcMyogenin. The CMV gal plasmid provided an internal
reference for transfection efficiency. C, a synthetic
promoter construct was prepared containing the E2 and CArG elements
upstream of a TATA box promoter, together with two mutated versions of
this construct in which each motif was eliminated. D,
C2C12 cells were co-transfected with each of the promoter constructs
shown in C, together with pcDNA3 or pcMyoD and the
CMV
gal plasmid. For all experiments, luciferase and
-galactosidase activities were measured 48 h post-transfection
and the data shown are the average of four experiments, performed in
duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin expression in both primary limb bud cultures as well as in
the C2C12 myoblast cell line. Moreover, the observation that Barx2
increased SM-
-actin expression in proliferating myoblasts suggests
that Barx2 induces cellular changes that prime skeletal myoblasts for
differentiation. Other downstream targets of Barx2 that are likely to
influence myoblast fusion include CAMs. Barx2 was previously shown to
regulate the N-CAM promoter and to induce cadherin-6 expression (19,
44). Previous studies have shown that both N-CAM and particular
cadherins, such as N-cadherin, can promote myoblast fusion (27, 45). In
addition, two new Ig family CAMs, CDO and BOC, were shown recently to
play an essential role in myoblast fusion via their heterophilic
interaction (46, 47). In future studies, it will be important to
determine whether these and other CAMs are direct targets of Barx2 in myoblasts.
-actin and
dystrophin genes by MyoD (38, 48), and suggests that MyoD and SRF
cooperatively activate Barx2 expression in C2C12 cells.
-tropomyosin
gene (54). Our studies also suggest a role for Barx2 in SMC
differentiation. Overexpression of Barx2 in C3H10T1/2 cells increased
SM-
-actin expression and promoted cell spreading and formation of
stress fibers. These morphological changes are consistent with
differentiation of SMCs or myofibroblasts, cells that have contractile
properties like smooth muscle, but retain characteristics of
fibroblasts (33, 55, 56). Because the SM-
-actin gene also contains a
CArG box that binds to SRF (41), it is possible that induction of SM-
-actin after overexpression of Barx2 is mediated by a complex of
Barx2 and SRF. Thus both the regulation of Barx2 by SRF, and the
interaction of Barx2 with SRF, may be part of the muscle
differentiation program.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Tom Moller for excellent technical assistance. We appreciate critical readings of the manuscript by Drs. Kathryn Crossin, Bruce Cunningham, Vince Mauro, Joe Gally, and Gerald Edelman.
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FOOTNOTES |
---|
* This work was supported by a grant from the G. Harold and Leila Y. Mathers Foundation, National Science Foundation Grant IBN-9816896 (to F. S. J.), NIH Grant NS39837, and a grant from the Charles and Mildred Schnurmacher Foundation (to D. B. E.).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/EBI Data Bank with accession number(s) AY188085.
§ To whom correspondence should be addressed: Dept. of Neurobiology, The Scripps Research Institute. Tel.: 858-784-2621; Fax: 858-784-2646; E-mail: rmeech@scripps.edu.
Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M207617200
2 The Blau Lab Homepage www.stanford.edu/group/blau/.
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ABBREVIATIONS |
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The abbreviations used are: MRF, myogenic regulatory factor; MEF2, myocyte enhancer factor 2; CAMs, cell adhesion molecules; SMC, smooth muscle cell; TSS, transcription start site; SRF, serum response factor; MRR, myogenic regulatory region; ODN, oligonucleotides; FBS, fetal bovine serum; SM, smooth muscle; SRE, serum response element; EYFP, enhanced yellow fluorescent protein.
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REFERENCES |
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