|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 50, 48889-48898, December 13, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, July 10, 2002, and in revised form, October 4, 2002
Expression of many skeletal muscle-specific genes
depends on TEF-1 (transcription enhancer factor-1) and MEF2
transcription factors. In Drosophila, the TEF-1 homolog
Scalloped interacts with the cofactor Vestigial to drive
differentiation of the wing and indirect flight muscles. Here, we
identify three mammalian vestigial-like genes,
Vgl-1, Vgl-2, and Vgl-3, that share
homology in a TEF-1 interaction domain. Vgl-1 and
Vgl-3 transcripts are enriched in the placenta, whereas
Vgl-2 is expressed in the differentiating somites and
branchial arches during embryogenesis and is skeletal muscle-specific
in the adult. During muscle differentiation, Vgl-2 mRNA levels
increase and Vgl-2 protein translocates from the cytoplasm to the
nucleus. In situ hybridization revealed co-expression of Vgl-2 with myogenin in the differentiating muscle of embryonic myotomes
but not in newly formed somites prior to muscle differentiation. Like
Vgl-1, Vgl-2 interacts with TEF-1. In addition, we show that Vgl-2
interacts with MEF2 in a mammalian two-hybrid assay and that Vgl-2
selectively binds to MEF2 in vitro. Co-expression of Vgl-2
with MEF2 markedly co-activates an MEF2-dependent promoter through its MEF2 element. Overexpression of Vgl-2 in MyoD-transfected 10T1/2 cells markedly increased myosin heavy chain expression, a marker of terminal muscle differentiation. These results identify Vgl-2
as an important new component of the myogenic program.
Commitment of pluripotent cells to the skeletal muscle lineage
involves multiple inductive signals and the hierarchical activation of
several transcription factors (1). Differentiation of skeletal muscle
cells is coupled to withdrawal from the cell cycle and is accompanied
by transcriptional activation of muscle-specific genes. The MyoD family
of basic-helix-loop-helix transcription factors, including MyoD, Myf5,
myogenin, and MRF4, plays a central role in activating the muscle
differentiation program (2). Activation of muscle gene expression by
the MyoD family of factors is dependent on their association with
members of the MEF2 family of MADS box transcription factors (3).
Together, MyoD and MEF2 account for a large part of muscle-specific
gene activation. In addition, many muscle genes contain muscle-specific
cytidine-adenosine-thymidine cis-elements
(5'-CATDSH-3') that are also required for muscle-specific expression
(4, 5). The proteins that bind to these elements belong to the
transcription enhancer factor-1
(TEF-1)1 family of
transcription factors (6).
TEF-1 factors regulate tissue-specific gene expression in muscle and
placenta (7, 8) and are considered to be general transcription factors
in other tissues (for review see Ref. 9). TEF-1 factors are not
tissue-restricted in their expression, suggesting that additional
cofactors are required to confer tissue-specific function. Proteins
that interact with TEF-1 include the basic-helix-loop-helix leucine
zipper protein Max (10), the nuclear protein poly(ADP-ribose)polymerase (9), the steroid receptor co-activator proteins (11), the Src/Yes-associated protein YAP65 (12), and a serum response factor
(13). Some of these cofactors are known to participate in muscle
differentiation. For example, the steroid receptor co-activator GRIP-1
is necessary for MEF2C-dependent gene expression and muscle differentiation (14). In addition, poly(ADP-ribose)polymerase interaction with TEF-1 contributes to the muscle-specific activity of a
muscle-specific cytidine-adenosine-thymidine element (9). However, no TEF-1 cofactor has been identified that has a
tissue-restricted pattern of expression.
In Drosophila, the TEF-1 homolog Scalloped is required for
the development of chemosensory organs on the wing blade (15). The
finding that TEF-1 could functionally substitute for Scalloped during
Drosophila development (16) suggested that evolutionarily conserved cofactors might be present in both Drosophila and
vertebrates. The nuclear protein Vestigial is a necessary cofactor of
Scalloped in Drosophila (17-19). Moreover,
Drosophila Vestigial was shown to physically interact with
both Scalloped and human TEF-1 (19), suggesting the existence of
vertebrate homologs of Vestigial. The Drosophila vestigial
gene plays a central role in the development and patterning of the
wing: loss of Vestigial results in a failure of wing development, and
ectopic expression of Vestigial in tissues of the eyes, legs, and
antennae leads to the development of ectopic wings (20). Binding of the
Vestigial cofactor switches the DNA-target selectivity of the Scalloped
protein (21) and activates wing-specific genes. vestigial
expression is regulated by inputs from three signaling pathways:
Wingless, Decapentaplegic, and Notch/Suppressor of Hairless and by its
own expression in a positive feedback loop (22). These signals are also
known to play an important role in skeletal muscle differentiation in
vertebrates. Recently, vestigial was shown to specify the
differentiation of the indirect flight muscles, providing direct
evidence that Vestigial plays a role in muscle differentiation (23).
However, the mechanism of Vestigial function remains poorly understood.
A human protein related to Vestigial was identified from an expressed
sequence tag (EST) isolated from a fetal heart cDNA library and
named tondu (TDU) (24). Northern blot analysis of fetal tissues also
revealed tondu mRNA in fetal kidney and lung, however, expression
was not examined in adult tissues. tondu was shown to physically and
functionally interact with TEF-1. We also identified this cDNA in a
search of the GenBankTM data base for genes related to
Drosophila vestigial, but gave it the name Vestigial-like 1 (Vgl-1), based on its homology to the previously described
Drosophila vestigial. In addition, we found two human
genomic sequences that encode different mRNAs, which we call Vgl-2
and Vgl-3. Here, we show that Vgl-2 is a muscle-specific cofactor of
TEF-1 in adult tissues, that Vgl-2 is up-regulated and translocates
from the cytoplasm to the nucleus upon muscle differentiation, that
Vgl-2 interacts with MEF2 and co-activates an
MEF2-dependent muscle-specific promoter. Moreover,
co-expression of Vgl-2 with MyoD significantly increases the expression
of myosin heavy chain, a marker of terminally differentiated muscle.
Taken together, our results identify Vgl-2 as an important new
transcriptional cofactor of the muscle-specific program.
Cloning of Vestigial-like cDNAs--
The sequence of
Drosophila vestigial was used in a BLAST search of
GenBankTM to identify vestigial-like genes. Three genomic
clones, one on Xq26.1-27.2, one on 6q21, and a third on chromosome 3, contained a sequence motif related to the TEF-1 interaction domain of
Vestigial. ESTs related to these genomic clones were identified in the
human EST data base. EST 347406, initially isolated from a human fetal heart cDNA library, corresponded to the Vgl-1 cDNA
and was obtained from Genome Systems, Inc. (St. Louis, MO). The
sequence of this EST has been reported by others (24). For Vgl-2, an
antisense oligonucleotide corresponding to the sequence homologous to
the TEF-1 interaction domain (GenBankTM accession #798880,
nucleotides 88263-88320),
5'-GGCCCTGCTGAAATGTTCATCCACCACGGAGCTGATGTCCCCCTGGAAATAAGTGAAG-3', was used to probe a human multiple-tissue Northern blot.
Vgl-2 expression was restricted to adult human skeletal
muscle. To isolate the Vgl-2 cDNA, human skeletal muscle
cDNA was obtained (a gift from Prof. Eric Hoffman) and used for
reverse transcription-PCR. The following oligonucleotides were used,
5'-CCTGAGCTCCGGGGAAGGAGAG-3' and 5'-CAGCTTTGCCCACGCACAGACC-3',
corresponding to sequences in the 5'-untranslated region predicted
from the genomic sequence (GenBankTM accession
#798880, nucleotides 90979-90958 and 83166-83187) and to sequence in
the 3'-untranslated region (GenBankTM accession
#798880, nucleotides 83166-83187) in an EST (1089328) that maps to the
same genomic locus and that produced the same profile when used to
screen the Northern blot. An antisense oligonucleotide specific for the
Vgl-3 gene on human chromosome 3 (GenBankTM
accession #AC012221, nucleotides 113762-113718,
5'-GCTCTTGAGAAGTGTTCATCCACTACTGACCCAATGTCTCCCTGG-3') corresponds to the
TEF-1 interaction domain. The mouse Vgl-2 cDNA was
isolated from an adult diaphragm cDNA library (Stratagene, La
Jolla, CA). All cDNAs were sequenced on both strands.
Northern and Dot Blot Analysis--
A human
multiple-tissue Northern blot and a dot blot containing RNA from
50 human tissues (Clontech Laboratories, Palo Alto, CA) were sequentially probed with 32P-end-labeled antisense
oligonucleotides corresponding to the sequence encoding the TEF-1
interaction domain in Vgl-2 and Vgl-3. All oligonucleotides were
synthesized and high pressure liquid chromatography-purified by Operon
Technologies (Alameda, CA). Theses blots were also probed with a
32P-labeled random primed full-length Vgl-1
cDNA. RNA was also prepared from mouse
C2C12 cells and transfected 10T1/2 cells
cultured in 10% fetal bovine serum or induced to differentiate by
changing the medium to 2% horse serum. This RNA was used for Northern
blot analysis. The mouse Vgl-2 cDNA was used to screen
the C2C12 and 10T1/2 Northern blots.
Blots were visualized on a PhosphorImager (Amersham Biosciences)
or by autoradiography on x-ray film.
In Situ Hybridization--
Parasagittal sections of mouse
embryos were purchased from Calbiochem-Novabiochem (San Diego, CA).
Swiss Webster mouse embryos for whole mount in situ
hybridization were dissected from timed pregnancies with embryonic day
(e) 0.5 being noon on the day of the vaginal plug. Sense and antisense
digoxigenin-labeled riboprobes for mouse Vgl-2 and
myogenin (gift of Drs. Bill Klein and Anita Myer) were
synthesized according to a supplier protocol (Roche Molecular
Biochemicals, Indianapolis, IN). Whole mount in situ hybridization was performed according to D. Wilkinson (25). Hybridization and post-hybridization washes were carried out at 60 °C for sections and at 63-65 °C for whole mounts.
Construction of Expression Vectors--
The CMV enhancer-driven
human TEF-1 plasmid pXJ40-TEF-1A containing human TEF-1 with a
synthetic initiator codon and the parental plasmid pXJ40 have been
described previously (26). The pXJ40-RTEF-1 expression plasmid has also
been described previously, where we designated the leucine codon 7 codons 5' to the isoleucine codon in RTEF-1, which is replaced by
methionine in pXJ40-TEF-1A, as the initiator of human RTEF-1 (27). The
pACT-MyoD vector from the Promega CheckMateTM mammalian
Two-Hybrid System was digested with BamHI, and the 1.7-kb
MyoD fragment was ligated into the pXJ40 expression vector. The mouse
The MatchmakerTM mammalian two-hybrid system
(Clontech, Palo Alto, CA) was used initially to
test for interaction between TEF-1 and Vgl factors, using the pG5CAT
reporter plasmid. We subsequently used the pG5luc luciferase reporter
plasmid from the CheckMateTM mammalian two-hybrid kit
(Promega, Madison, WI). We first tested for TEF-1/Vgl interactions by
making fusion constructs between the pM vector (containing the coding
sequence for the yeast GAL4 DNA binding domain and a multiple cloning
site) and Vgl factors tested with the pVP16 vector (encoding the
activation domain of the herpes simplex virus VP16 protein) fused to
the activation domain of TEF-1 or RTEF-1.
The pM-Vgl-1 vector was constructed by linearizing the pM vector with
BamHI, filling-in with Klenow, digesting with
PstI, and ligating in-frame to the filled-in
EcoRI/PstI fragment of Vgl-1. The pM-Vgl-2 vector
was constructed by linearizing the pM vector with EcoRI,
filling-in with Klenow, digesting with HindIII and ligating
in-frame to the filled-in BspHI/HindIII fragment of Vgl-2. The pVP16-TEF-(80-426) vector was made by linearizing the pVP16 vector with EcoRI, filling in with Klenow,
digesting with XbaI, and ligating in-frame to the filled-in
BspEI/XbaI fragment of a PCR-generated mutant of
TEF-1 carrying a BspEI site, reported previously (31). The
pVP16-TEF-(168-426) vector was made by linearizing pVP16 with
MluI, filling-in with Klenow, digesting with
XbaI, and ligating in-frame to the filled-in
BamHI/XbaI TEF-1 fragment from pXJ40-TEF-1A.
pVP16-RTEF-(89-434) was made by linearizing the pVP16 vector with
EcoRI, filling-in with Klenow, digesting with
XbaI, and ligating in-frame with the filled-in
BspEI/XbaI fragment of RTEF-1 from
pXJ40-RTEF-1.
Because the pM and pVP16 vectors have the same in-frame multiple
cloning sites, the converse constructs were made by swapping the
inserts from the pM vectors to the pVP16 vectors and vice versa. Thus,
the EcoRI/XbaI fragments of pM-Vgl-1 and pM-Vgl-2 were ligated into pVP16 to make pVP16-Vgl-1 and pVP16-Vgl-2. Similarly, the EcoRI/XbaI fragments of pVP16-TEF-(80-426)
and pVP16-TEF-(168-426) were ligated into pM to make pM-TEF-(80-426)
and pM-TEF-(168-426). The pMRTEF-(89-434) vector was made by
releasing an EcoRI fragment from pVP16-RTEF-(89-434) and
ligating to the EcoRI-linearized pM plasmid. In addition,
pM-RTEF-(214-434) was made by linearizing pM with MluI,
filling-in with Klenow, digesting with XbaI, and ligating to
the filled-in NcoI/XbaI fragment of pXJ40-RTEF-1. The pM-RTEF-(234-434) vector was made by linearizing pM with
XmaI, filling-in with Klenow, digesting with
HindIII, and ligating to the filled-in
XhoI/HindIII fragment of a PCR-generated mutant of RTEF-1 carrying an XhoI site, reported previously (31).
The pM-TEF-(225-426) vector was made by linearizing pM with
MluI, filling-in with Klenow, digesting with
XbaI, and ligating to the filled-in
XhoI/XbaI fragment of pXJ40-TEF-1A.
The pM-MEF2 expression vectors were obtained as follows:
pM-MEF2-(1-178) was generated by PCR amplification of a fragment containing an XmaI site (underlined) 4 codons upstream of
the MEF2C initiator codon, using the oligonucleotide
5'-GAGAGAAGAAACCCGGGGACTATGGGGAG-3' and an oligonucleotide
containing the PstI site (underlined) in the MEF2C cDNA,
5'-CATACTATTCCTCTGCAGAGACG-3'. The full-length pM-MEF2 was
generated by ligating a PstI/PstI fragment of
MEF2C cDNA encoding amino acids 178-466 into the
PstI-linearized pM-MEF2-(1-178) vector. The
pM-MEF2-(178-436) vector was made by subcloning a PstI/EcoRI fragment into the pM vector. The
pM-MEF2-(1-302) and pM-MEF2-(302-466) vectors were made by releasing
an EcoRV/XbaI or an
XmaI/EcoRV fragment from the full-length vector, respectively.
The myc-tagged mouse Vgl-2 expression vector was made by subcloning
in-frame a PvuII/XhoI fragment containing the
full-length mouse Vgl-2 sequence from pXJ40-Vgl-2 into a pBScmyc vector
linearized at SmaI/XhoI. The cmyc-Vgl-2 cDNA
was then released with ClaI/XhoI and recloned
into pBluescript linearized with ClaI/XhoI to
obtain a useful BamHI site at the 5'end. The
BamHI/XhoI cmyc-Vgl-2 fragment was released from
pBluescript and subcloned into pXJ40. Deletion of the C-terminal domain
of Vgl-2 was obtained following release of an
SfiI/XhoI fragment from pXJ40-cmycVgl-2, by
filling-in with Klenow fragment and religation to generate Tissue Culture and Transfection--
Neonatal rat cardiac
myocytes were isolated as described previously (32) and cultured at low
density (106 viable cells per 60-mm dish) in Hanks'
minimal essential medium supplemented with 5% calf serum (HyClone,
Logan, UT) and bromodeoxyuridine (100 µM) to prevent
non-myocyte proliferation. Twenty-four hours after plating, the medium
was replaced with serum-free minimal essential medium supplemented with
human insulin (10 µg/ml, Sigma), human transferrin (10 µg/ml,
Sigma), and bovine serum albumin (1 mg/ml, Sigma). The following day,
cardiac myocytes were transfected using the calcium phosphate
precipitation method for 2 h with serum-containing medium. A total
of 20 µg of DNA was added per plate, with 5 µg of CAT and Luciferase Assays--
Chloramphenicol acetyl
transferase (CAT) activity was assessed as described previously (27).
Activities (mean ± S.E.) of the SKA and MLC2v reporters were
expressed relative to the activity in the presence of the empty CMV
expression vector, set at 1-fold. By independent samples t
test, mean -fold activities of the SKA promoter's response to TEF-1
and RTEF-1 and to Vgl-1 and Vgl-2 expression were considered different
from control at the p < 0.05 significance level. In
the mammalian two-hybrid assay, activity of the pG5CAT reporter was
compared when co-transfected with empty expression vectors pM and pVP16
as a negative control (no interaction) and with the pM-P53 and
pVP16-TAg vectors as a positive control. Interaction was set at 100%
by the transactivation of pG5CAT by the positive control. When pG5luc
was used as a reporter, luciferase activity was assessed on a
luminometer following the manufacturer's instructions.
In Vitro Protein-Protein Interaction--
In vitro
binding studies were performed using purified MBP-Vgl-2 immobilized on
an amylose-Sepharose resin (New England BioLabs) and in
vitro transcribed/translated MEF-2 protein. Typically, 5 µl of
35S-labeled MEF2 protein was incubated in the presence of 5 µg of immobilized Vgl-2 fusion protein in 500 µl of binding buffer
(150 mM NaCl, 50 mM Tris-Cl, pH 7.5, 0.3%
Igepal CA-630, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, and 0.25% bovine serum albumin) for
2 h at 4 °C with agitation and then centrifuged for 2 min at
15,000 r.p.m. at room temperature. The resin was washed three times in
500 µl of binding buffer without bovine serum albumin. The protein
complexes were released from the resin after boiling in Laemmli buffer
and resolved by SDS-PAGE. Labeled proteins were visualized and
quantified by autoradiography on a PhosphorImager.
Isolation of Vestigial-like-1, -2, and
-3--
Drosophila Vestigial was recently shown to
physically interact with human TEF-1, thereby suggesting the presence
of vestigial homologs in mammals (16). To identify these genes, we used
the sequence of Drosophila vestigial in a BLAST search of
GenBankTM. Three human genomic clones, one on Xq26.1-27.2,
one on 6q21, and a third on chromosome 3, contained a sequence motif
related to the TEF-1 interaction domain of Vestigial. These genes were named vestigial-like 1 (Vgl-1), Vgl-2,
and Vgl-3. ESTs related to these genomic clones were
identified in the human EST data base. EST 347406, initially isolated
from a human fetal heart cDNA library, corresponded to the Vgl-1
cDNA. The sequence of this EST has been reported by others (24). A
human Vgl-2 cDNA was amplified from human skeletal muscle cDNA
by reverse transcription-PCR. The mouse Vgl-2 cDNA was isolated
from an adult diaphragm cDNA library. For Vgl-3, only partial
sequence was identified on human chromosome 3 that corresponds to the
TEF-1 interaction domain. The sequence of human (AY056583) and mouse
(AF542181) Vgl-2 cDNAs have been deposited in
GenBankTM, and the corresponding amino acid sequences are
shown in Fig. 1A. The human
cDNA likely represents a splicing isoform that deletes most of the
C terminus of Vgl-2, because the 3'-untranslated repeat of human and
mouse Vgl-2 are highly similar, and sequences corresponding to the
mouse C-terminal domain are present on human chromosome 6q21 (data not
shown). The existence of alternatively spliced isoforms of human Vgl-2
would be consistent with the presence of multiple transcripts detected
by Northern blot analysis (Fig. 1B). The highly conserved
TEF-1 interaction domains of Vgl-1, Vgl-3, and Drosophila
Vestigial are aligned with the same sequence of Vgl-2. Less than 10%
sequence identity was observed outside the TEF-1 interaction domain
between different members of the vestigial family.
Vgl-2 Expression in Adult Tissue Is Muscle-specific--
Northern
blot analysis of RNA from adult human tissues revealed that
Vgl-1 and Vgl-3 transcripts are
placenta-enriched, with trace levels of Vgl-1 expression
detected in the kidney and Vgl-3 in the pancreas. In
contrast, Vgl-2 expression is skeletal muscle-specific in
adult tissues (Fig. 1B). These results were confirmed with a
dot blot containing RNAs derived from 50 human tissues and with an
adult mouse multi-tissue Northern blot (data not shown). A previous
report examined the expression of Vgl-1 in human fetal tissues, where it was detected in the kidney and lungs but not the
brain or liver (24). Thus, if Vgl factors functionally interact with
TEF-1 factors in these tissues, as suggested by their highly conserved
TEF-1 interaction domains (Fig. 1A), they likely contribute to the tissue-specific functions of the TEF-1 factors. Given that TEF-1
factors are implicated in cardiac muscle gene regulation, it is
interesting to note the absence of Vgl-1, Vgl-2,
or Vgl-3 transcripts in the adult heart or in neonatal rat
cardiac myocytes (data not shown). Because Vgl-2 expression
in adult tissues was limited to skeletal muscle, we next examined its
expression during muscle differentiation using the mouse cell line
C2C12.
Vgl-2 mRNA Is Up-regulated upon Muscle
Differentiation--
The mouse cell line
C2C12 remains as proliferative and
undifferentiated myoblasts when cultured in high serum but
differentiates into multinucleated myotubes when serum is partially
withdrawn. Northern blot analysis of RNA isolated from sub-confluent
C2C12 cells maintained in high serum revealed
that Vgl-2 is present in these cultures (Fig.
1C). However, this likely reflects the presence of a small
number of differentiated muscle cells, as the differentiated muscle
marker myosin heavy chain was also expressed at low levels (data not
shown). Upon serum withdrawal, Vgl-2 mRNA levels were
progressively up-regulated in differentiating myotubes. Thus, muscle
differentiation is associated with an increase in Vgl-2 expression.
Vgl-2 mRNA Is Detected in Differentiating Fetal
Muscle--
Because Vgl-2 expression is muscle-specific in adult
tissues, we determined the timing of Vgl-2 expression in relation to a
known marker of the myogenic differentiation program using in situ hybridization of whole mount embryos and in sections (Fig. 2). During embryonic development, the
epaxial skeletal muscles of the back are derived from the myotome
compartment of the somites, whereas all hypaxial limb muscles are
derived from cells that migrate from the lateral dermomyotome of the
somites, orderly arranged blocks of mesodermal tissue located on both
sides of the neural tube (33). Expression of the myogenic regulatory factors proceeds sequentially, with MyoD and myf5 activated early during the commitment of pluripotent mesoderm of the somite and myogenin and MRF4 activated later during muscle differentiation in the
myotome and limb muscles (2). Whole mount in situ
hybridization was carried out on mouse embryos from embryonic day (e)
7.5 to e11.5 (Fig. 2). Vgl-2 expression is not detected
until e8.5 in the 5- to 6-somite stage embryo, where transcripts are
detected in the first branchial arch (Fig. 2A,
arrowhead). By the 7- to 8-somite stage, Vgl-2 expression
was detected in the first two branchial arches (Fig. 2, B
and C) but was notably absent in somites (Fig. 2,
A-C). Because somite differentiation occurs in an anterior to posterior fashion, with more anterior somites formed earlier than
the more posterior ones, a gradient of differentiation can be observed
along the anterior-posterior axis. At later e9.5 stages, embryos with
26-28 somites showed clear expression of both Vgl-2 and the
late marker of muscle differentiation myogenin in the myotome of differentiated somites (Fig. 2D).
Vgl-2 and myogenin expression is absent in the
most posterior somites where the myotome has not yet differentiated. At
this stage, Vgl-2 expression can also be detected in the
first three branchial arches, which appears to be limited to the
surface epithelium and not to the core of the arches. Even at later
stages, e10.5 (Fig. 2E, 34 somite-stage) and e11.5 (Fig.
2F), Vgl-2 and myogenin transcripts
were detected in the myotome in more anterior somites, but were absent
in the most recently formed posterior somites. In e11.5 embryos,
Vgl-2 and myogenin transcripts were also detected
in differentiating muscle of the forelimb buds (Fig. 2G).
Expression of Vgl-2 in the branchial arches and their
derivatives decreases with differentiation (compare Fig. 2,
D-F).
In situ hybridization of embryo sections revealed
Vgl-2 mRNA in the myotome (M) of a tail
somite at e11 (Fig. 2H). At e16, Vgl-2 mRNA
was present in the hypoglossal muscle of the tongue (T) and
branchial arch-derived maxillary and mandibular cartilages (Fig.
2I, arrows). The functional significance of
Vgl-2 expression in branchial arches is not known. Given
that Vgl-2 is specifically expressed in fetal and adult skeletal
muscles, and because its expression is up-regulated during muscle
differentiation, these results are consistent with a role for Vgl-2 in
the myogenic differentiation program.
Vgl-2 Protein Translocates from the Cytoplasm to the Nucleus upon
Muscle Differentiation--
In Drosophila, Vestigial is a
nuclear protein (34). We constructed a myc-tagged mouse Vgl-2
expression vector to confirm that Vgl-2 is also localized in the
nucleus in C2C12 cells. Surprisingly, in
myoblasts, mycVgl-2 was excluded from the nucleus and was abundantly localized in the cytoplasm (Fig. 3,
A and C). However, 1 day after serum was
withdrawn, mycVgl-2 was localized in the aligned nuclei of myotubes
(Fig. 3, B and D). As shown in Fig.
1A, mouse Vgl-2 contains putative nuclear localization (NLS)
and nuclear export signals (NES) in the C-terminal domain. Deletion of
the C-terminal domain in mouse Vgl-2 was associated with loss of
appropriate nuclear localization upon muscle differentiation (Fig. 3,
E and F). Although this result does not yet
resolve the question of whether Vgl-2 is actively exported from the
nucleus in myoblasts through the nuclear export signal or whether
nuclear import requires the nuclear localization signal in
myotubes, this result is consistent with a role for the C
terminus in mediating nuclear shuttling of Vgl-2. In CV-1 cells, the
myc-tagged Vgl-2 protein was detected in both the cytoplasm and nucleus
(data not shown). Clearly, translocation of Vgl-2 to the nucleus upon
muscle differentiation suggests that it participates in regulating
muscle gene expression. Although Drosophila Vestigial and
human Vgl-1 (TDU) were already shown to interact with TEF-1 physically
in vitro and functionally in vivo in yeast and in
Drosophila S2 cells (24), functional interaction in
mammalian cells or between Vgl-2 and TEF-1 factors had not yet been
demonstrated.
Vgl and TEF-1 Factors Functionally Interact in Mammalian
Cells--
To test for functional interaction between TEF-1 and Vgl
factors, a mammalian two-hybrid assay was used in neonatal rat cardiac myocytes and in CV-1 cells (Fig. 4). When
the activation domain of TEF-1 or RTEF-1 was fused with the GAL4 DNA
binding domain, pMTEF-(168-424) or pMRTEF-(214-434), and tested with
either Vgl-1 or Vgl-2 fused to the activation domain of VP16
(pVP16-Vgl-1 and pVP16-Vgl-2), a strong interaction was detected,
particularly in CV-1 cells (Fig. 4, A and B). The
GAL4-TEF-1 or GAL4-RTEF-1 fusion constructs, pMTEF-(168-424) or
pMRTEF-(214-434), were significantly more active in cardiac myocytes
than in CV-1 cells. The stronger activity in cardiac myocytes suggests
the presence of a myocyte-specific cofactor of TEF-1 that remains to be
identified, because none of the Vgl factors were detected in these
cells. These results demonstrate a functional interaction between TEF-1
and Vestigial-like factors.
Mapping the Vestigial Interaction Domain in TEF-1 Factors--
To
identify sequences required for the heterodimerization between TEF-1
and Vgl factors, a series of N- and C-terminal truncations of the
activation domains of TEF-1 and RTEF-1 were fused to the GAL4 DNA
binding domain (Fig. 4C). As reported previously for a
deletion to amino acid 205 in TEF-1 (24), a similar deletion to amino
acid 214 in RTEF-1 fused to GAL4 could still interact with Vgl-1 and
Vgl-2. However, a further truncation to amino acid 225 in TEF-1 or the
corresponding amino acid 234 in RTEF-1 was no longer able to interact
with either Vgl-1 or Vgl-2. Thus, the sequences C-terminal to residues
225 in TEF-1 and 234 in RTEF-1 are not sufficient to productively
interact with Vgl factors. A C-terminal deletion to amino acid 329 in
TEF-1 was previously shown to retain the ability to interact with
Vestigial (24). We attempted to further delineate the C-terminal end of
the Vestigial interaction domain in TEF-1 by testing truncated
fragments from 168-225, 168-280, and 168-294 in cardiac myocytes
(data not shown). None of these additional constructs interacted with
either Vgl factor. Thus, the Vestigial interaction domain
(VID) in TEF-1 must lie between 205 and 329 (Fig.
4C).
The N-terminal truncated constructs TEF-1-(225-424) and
RTEF-1-(234-434) did not transactivate the GAL4-dependent
pG5luc luciferase reporter in cardiac myocytes. Thus, this C-terminal
domain lacks an intrinsic transactivation function. In addition, these
results suggest that a cofactor that interacts with the sequence
between amino acid 205 and the C terminus of TEF-1 exists in neonatal rat cardiac myocytes that is required for tissue-specific
transactivation. It is also important to note that the TEF-1 and RTEF-1
constructs pMTEF168 and pMRTEF214 were 10 times less active in CV1
cells than in cardiac myocytes (Fig. 4), suggesting that CV-1 cells lack the necessary cofactor present in cardiac myocytes.
Vgl-1 Squelches a TEF-1-dependent Promoter in CV-1 and
C2C12 Cells--
Because Vgl-1 was initially
isolated from a human fetal heart cDNA library, we wanted to test
whether overexpression of Vgl-1 would affect the activity of a known
TEF-1-dependent promoter equally in different cell types.
The Vgl-2 Activates a TEF-1-dependent Promoter upon Muscle
Differentiation--
We next tested the function of Vgl-2 in the
myogenic cell line C2C12 before and after the
induction of myogenesis by serum withdrawal (Fig. 5C). Prior
to differentiation, Vgl-2 had no effect on the
TEF-1 overexpression profoundly squelched the SKA promoter in
C2C12 cells either before or after
differentiation. This squelching effect is believed to involve
titration of a limiting co-activator related to the Src/Yes-associated
protein YAP65 (12). Surprisingly, RTEF-1 squelched the SKA promoter in
C2C12 cells prior to differentiation but had
little effect after differentiation. Vgl-1 further squelched the SKA
promoter in the presence of RTEF-1, whereas Vgl-2 had little effect.
RTEF-1 mRNA expression is activated by myogenesis in
C2C12 cells (35, 36) and is highly expressed in
skeletal muscle (37). These results suggest that the activity of Vgl-2, like that of Vgl-1, is dependent on the cell background and on the
differentiation status of the cell.
Because Vgl-2 is a muscle-specific transcriptional cofactor, we next
asked whether other transcription factors of the myogenic differentiation program could functionally partner with Vgl-2. The
myogenic differentiation factor MyoD did not interact with Vgl-2 in a
two-hybrid assay (data not shown). However, other members of the MyoD
family that are expressed later during muscle differentiation have not
been tested. Vestigial-like factors also did not functionally interact
with the serum response factor (data not shown). We recently showed
that TEF-1 factors interact with MEF2 factors through their respective
DNA binding domains (30). Thus, given the partnering of TEF-1 and MEF2
factors, we asked whether Vgl-2 might also interact with MEF2. To test
whether Vgl-2/MEF2 functionally interact, the response of a
MEF2-dependent promoter to Vgl-2 and MEF2 overexpression was examined in CV-1 cells.
MEF2 and Vgl-2 Co-activate a MEF2-dependent
Promoter--
We used the MEF2-dependent chicken myosin
light chain 2v (MLC2v) promoter (29) in transient transfection assays
to determine what effect Vgl-2 has on MEF2 function (Fig.
6). In CV-1 cells, MEF2 activated the
MLC2v promoter about 3-fold. Vgl-2 also activated this promoter about
5-fold. In combination, MEF2 and Vgl-2 co-activated the MLC2v promoter
12-fold (Fig. 6A). These results suggest that Vgl-2 is a
MEF2 co-activator and activates the MLC2v promoter through its
interaction with MEF2. Indeed, mutation of the MEF2 site in the MLC2v
promoter partially blocked its activation by Vgl-2 and completely
blocked the co-activation by MEF2. It is important to note that neither
Vgl-1 nor Vgl-2 demonstrated an intrinsic transactivation function when
fusion constructs made between the yeast GAL4 DNA binding domain and
full-length cDNAs for either Vgl-1 or Vgl-2 were co-transfected
with the GAL4-dependent pG5-luc reporter in CV-1 cells
(data not shown). Thus, rather than acting as a co-activator, Vgl-2
might act to relieve interference by a MEF2 co-repressor.
We next tested the effect of Vgl-2 expression in
C2C12 cells before and after muscle
differentiation (Fig. 6B). Vgl-2 significantly activated the
MLC2v promoter in myotubes but not in myoblasts, likely reflecting the
cytoplasmic localization of Vgl-2 in myoblasts and the nuclear
localization in myotubes (Fig. 3). Interestingly, MEF2 overexpression
activated the MLC2v promoter less strongly in myotubes than in
myoblasts. This result may reflect competition with endogenous MEF2
expression in C2C12 cells.
Vgl-2 Interacts with MEF2--
Functional interaction between
Vgl-2 and MEF2 was further demonstrated using the
mammalian two-hybrid assay (Fig. 7). By
testing a series of truncated constructs, the Vestigial interaction
domain of MEF2 was identified in the C terminus (Fig. 7, B
and C), where a bipartite nuclear localization sequence is
found (38). Physical interaction between Vgl-2 and MEF2 was confirmed
using a maltose binding protein/Vgl-2 fusion protein pull-down assay,
where Vgl-2 selectively bound to MEF2 in vitro (Fig.
7D). Thus, because Vgl-2 physically interacts with MEF2 and
this interaction is independent of the MADS domain in MEF2 that we
showed mediated TEF-1 interaction with MEF2 (30), Vgl-2 does not
require TEF-1 to interact with MEF2.
Vgl-2 Augments Myosin Heavy Chain Expression Induced by
MyoD--
When the myogenic differentiation factor MyoD is expressed
in the pluripotent C3H 10T1/2 cells, they undergo myogenic
differentiation. Because Vgl-2 acted as a co-activator of TEF-1- and
MEF2-dependent promoters in differentiated muscle cells, we
sought to determine whether Vgl-2 overexpression in 10T1/2 cells
could induce muscle differentiation on its own and what effect Vgl-2
overexpression would have on muscle differentiation induced by MyoD.
Although Vgl-2 was not sufficient to induce muscle differentiation in
10T1/2 cells, we observed a marked increase in the expression of
myosin heavy chain by immunofluorescence (Fig.
8A) and by Western blot analysis (Fig. 8B) in 10T1/2 cells induced to
differentiate into muscle cells by MyoD. Western blot analysis of
myosin light chain 2 expression revealed a similar up-regulation,
whereas sarcomeric actin showed only a 2-fold increase in expression
(data not shown). These results suggest that Vgl-2 contributes to
muscle differentiation. Because Vgl-2 expression is
muscle-specific in adult tissues, and because Vgl-2 is
co-expressed with myogenin in somitic myotomes and limb
muscle mass, we next asked whether Vgl-2 is a downstream target of
MyoD. RNA was extracted from 10T1/2 cells transfected with MyoD
and induced to differentiate into muscle by serum withdrawal. Whereas
Vgl-2 mRNA was detected in C2C12
cells and seen to increase upon serum withdrawal, no Vgl-2 was detected
in MyoD-transfected 10T1/2 cells at any time (Fig.
8C). This result may reflect the low sensitivity of this
assay. A very similar result has been reported for MEF2C up-regulation
in the pluripotent mouse P19 stem cells: in contrast to myogenin, MyoD
does not activate detectable levels of MEF2C mRNA in these cells
(39). Thus, the lack of Vgl-2 induction by MyoD does not mean that
Vgl-2 is dispensable for muscle differentiation. The ability of Vgl-2
to augment myosin heavy chain expression in MyoD-transfected
10T1/2 cells suggests that MyoD and Vgl-2 are independent
components of the muscle differentiation program.
Taken together, the ability of Vgl-2 to functionally interact with
TEF-1 and MEF2, to activate TEF-1- and MEF2-dependent
promoters in differentiated muscle cells, to serve as a MEF2
co-activator, to become localized to the nucleus upon muscle
differentiation, and to augment the expression of myosin heavy chain in
MyoD-transfected cells committed to myogenic differentiation, these
results strongly implicate Vgl-2 in muscle-specific gene expression.
We have identified a novel family of transcriptional
cofactors that share DNA homology with the TEF-1 interaction domain of the Drosophila protein Vestigial. In addition, these
Vestigial-like factors are expressed in a tissue-restricted pattern in
the human adult. Vgl-1 and Vgl-3 are both highly
expressed in the placenta and likely confer the placenta-specific
functional properties that have been ascribed to the TEF-1 factors (8,
40). Vgl-1 mRNA was also detected at very low levels in
the adult kidney but not in the lung. Previously, Vgl-1
(TDU) was reported to be expressed at about equal levels in the human
fetal lung and kidney (24), and the Vgl-1 EST was initially
cloned from a human fetal heart cDNA library. Thus, Vgl-1 may be
more broadly expressed in fetal tissues and serve a specialized
function in the placenta.
A Role for Vgl-2 in Skeletal Muscle
Differentiation--
Vgl-2 expression was detected only in
adult skeletal muscle by Northern blot analysis and in situ
analysis detected Vgl-2 mRNA in differentiating muscle cells of the
myotomes and limb bud. In addition, Vgl-2 mRNA was detected in
C2C12 myoblasts and was further activated upon
muscle differentiation. In C2C12 cells, a
transiently transfected myc-tagged Vgl-2 protein translocated to the
nucleus upon muscle differentiation. The C-terminal domain of Vgl-2
contains putative nuclear localization and nuclear export signals, and
deletion of these sequences disrupts nuclear localization. Vgl-2
activated TEF-1- and MEF2-dependent promoters only after C2C12 cells were induced to differentiate into
muscle. Moreover, Vgl-2 co-expression with MyoD in the pluripotent
10T1/2 cells markedly increased the expression of myosin heavy
chain, a marker of muscle differentiation. Taken together, these
results strongly implicate Vgl-2 in the transcriptional regulation of
muscle-specific genes.
Our results present the first evidence for a physical and functional
interaction between Vestigial-like factors and MEF2. In CV-1 cells,
Vgl-2 activated the MEF2-dependent MLC2v promoter and
potentiated its activation by MEF2 through the MEF2
cis-element. CV-1 cells express detectable levels of MEF2
protein by gel shift assay, but do not express detectable levels of
Vgl-2 mRNA by Northern analysis (data not shown). Given that Vgl-2
has no intrinsic transactivation function on its own, the Vgl-2/MEF2
co-activation of the MLC2v promoter suggests that Vgl-2 might recruit a
co-activation function to MEF2 or might relieve inhibition by a MEF2
co-repressor. MEF2 factors are known to interact with many other
proteins that bind at or near the N-terminal MADS and MEF2 domains (see
Ref. 41 for review). To our knowledge, this is the first time a MEF2
cofactor has been identified that interacts with the C-terminal nuclear localizing domain.
Because MEF2 expression is activated during muscle
differentiation and MEF2 physically and functionally interacts with
MyoD to co-activate muscle gene expression (42), through its
interaction with MEF2, Vgl-2 may facilitate the assembly of a
multiprotein complex at muscle-specific promoters. Several lines of
evidence are consistent with this model. First, we showed here that
Vgl-2 interacts with TEF-1 and MEF2 transcription factors. Second, we observed that Vgl-2 interaction with TEF-1 and MEF2 factors is orientation-specific. When a chimeric protein containing the yeast GAL4
DNA binding domain was made with Vgl-1 or Vgl-2, co-transfected with a
chimeric construct containing the activation domain of TEF-1, RTEF-1,
or MEF2 fused to the activation domain of VP16, together with a
GAL4-dependent reporter construct, no interaction was
detected between Vestigial-like and TEF-1 or MEF2 factors (data not
shown). Thus, the spatial orientation of TEF-1, MEF2, and
Vestigial-like factors appears to be important in determining whether
they can functionally interact in mammalian cells. This is in contrast
to what was reported for Vestigial and Scalloped in the yeast
two-hybrid assay (24, 43). These results suggest that TEF-1 and MEF2
factors must bind to DNA to productively interact with the
Vestigial-like cofactors. Third, we showed previously that TEF-1
factors interact with MEF2 through its MADS domain (30). Fourth, TEF-1
factors also interact with all three members of the p160 family of
steroid receptor co-activators, including GRIP-1 (11). GRIP-1 is a
known co-activator of MEF2C and is required for muscle differentiation
(14). The domain of GRIP-1 that interacts with both TEF-1 and MEF2C
resides in the basic-helix-loop-helix/Per-Arnt-Sim domain located in
the N terminus of all the steroid receptor co-activators (11, 14).
Other domains of the p160 proteins include a hormone receptor
interaction domain and activation domains that recruit the histone
acetyltransferases CBP/p300 (44, 45). Thus, Vgl-2 might function to
optimize co-activator recruitment to TEF-1 and MEF2 during muscle differentiation.
Alternatively, Vgl-2 might function to relieve inhibition
by a co-repressor. MEF2 factors interact with the class II histone deacetylases (HDAC4, HDAC5, and HDAC7) that are implicated in muscle
differentiation (46). Histone acetylation/deacetylation is a
fundamental mechanism for the control of gene expression. Histone
acetyltransferases stimulate transcription through acetylation of
histones, resulting in relaxation of nucleosomes; HDACs antagonize this
activity and repress transcription (47). HDAC4 and HDAC5 interact with
the MADS domain of MEF2 and repress MEF2-dependent genes
(48, 49). HDAC5 shuttles to the cytoplasm when myoblasts are induced to
differentiate (46), whereas HDAC4 behaves like Vgl-2, shuttling from
the cytoplasm to the nucleus upon muscle differentiation (49). Nuclear
import of HDAC4 requires the trans-acting nuclear localization domain
of MEF2 (50), although it remains unclear why MEF2 should import its
own co-repressor. Because Vgl-2 interacts with the same domain in MEF2,
Vgl-2 might act to displace HDAC4 from MEF2. It will be interesting to
test whether HDAC4 interacts with Vgl-2. This mechanism would be
consistent with the observation that Vgl-2 appears to act as a
co-activator of MEF2 at the MLC2v promoter despite Vgl-2 having no
intrinsic transactivation function.
Is There a Cardiac-specific Cofactor of
TEF-1?--
Here, we showed that Vgl-2 is a skeletal muscle-specific
cofactor of TEF-1. However, none of the Vestigial-like factors
identified so far are expressed in the adult heart, and exhaustive
screening of an adult human heart cDNA library did not identify Vgl
homologous clones (data not shown). Alternatively, among the known
cofactors of TEF-1, YAP65 is a general transcriptional co-activator
that interacts with the C-terminal domain of TEF-1 (12) that we suggest contains a cardiac-specific transactivation function (see Fig. 6). A
cardiac-specific isoform of YAP65, or the related protein TAZ (51),
might account for TEF-1 activity in cardiac myocytes. Recently, a
cardiac-specific cofactor for the serum response factor (SRF) has been
identified and called myocardin (52). Myocardin belongs to the SAP
domain family of nuclear proteins and activates cardiac muscle
promoters by associating with SRF. Expression of a dominant negative
mutant of myocardin in Xenopus embryos interferes with
myocardial cell differentiation. Myocardin provides a mechanism whereby
SRF can convey myogenic activity to cardiac muscle genes. Given that
TEF-1 and SRF physically and functionally interact (13) and that Vgl
factors did not interact with SRF, it is tempting to speculate that
myocardin might also confer cardiac-specific function to the TEF-1
factors. The identification of Vgl-2 as a skeletal muscle-specific
cofactor of TEF-1 and MEF2 highlights the importance of tissue-specific
cofactors in conferring tissue-specific function to broadly expressed
transcription factors. These conclusions, although supported by several
criteria, need to be viewed as tentative, pending a loss-of-function
mutation and co-precipitation of the two endogenous proteins.
We are grateful to Prof. Pierre Chambon for
the pXJ40 and PXJ40-TEF-1A plasmids, to Prof. Paul Simpson for the
*
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) AY056583 and AF542181.
¶
Supported by Grant-in-Aid 50282N from the American Heart
Association and by Grant HL57211 from the National Institutes of Health. To whom correspondence should be addressed: Cardiovascular Institute, School of Medicine, University of Pittsburgh, BST 1704.3, 200 Lothrop St., Pittsburgh, PA 15213. Tel.: 412-383-9761; Fax: 412-383-8997; E-mail: stewartaf@msx.upmc.edu.
Published, JBC Papers in Press, October 9, 2002, DOI 10.1074/jbc.M206858200
The abbreviations used are:
TEF-1, transcription
enhancer factor-1;
EST, expressed sequence tag;
Vgl-1, Vestigial-like
1;
TDU, human Vgl-1;
CMV, cytomegalovirus;
MLC2v, chicken myosin light
chain 2v;
e, embryonic day;
SKA, skeletal muscle
Mammalian Vestigial-like 2, a Cofactor of TEF-1 and
MEF2 Transcription Factors That Promotes Skeletal Muscle
Differentiation*
,¶
Cardiovascular Institute, School of
Medicine, and § Department of Biological Sciences,
University of Pittsburgh, Pennsylvania 15213
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
113 bp SKA/CAT reporter construct was a gift of Professor Paul
Simpson (28). The mouse 113-bp SKA promoter fragment was subcloned
into the pGL2 basic luciferase vector. The 397-bp fragment of the
chicken skeletal 
-actin promoter was a gift of Professor Mahesh
Gupta (13). A 250-bp fragment of the chicken myosin light chain 2v
(MLC2v) promoter was generated by PCR from published sequences (29) and
subcloned into the pGL2 basic luciferase vector as reported previously
(30). The MEF2 site in the MLC2v promoter was mutated to a
non-functional site using the QuikChangeTM site-directed
mutagenesis kit (Stratagene, La Jolla, CA) and the following
oligonucleotide primers:
5'-CATGGGGTTACCTTTAGCCTGGAATGGGGTG-3' and
5'-CACCCCATTCCAGGCTAAAGGTAACCCCATG-3' that contain two
mutated sites (underlined) that prevent MEF2 binding, as shown
previously (30).
C-Vgl-2.
All constructs were sequenced at the University of Pittsburgh core facility.
113
SKA-CAT reporter plasmid and 100 ng of CMV-driven expression plasmid,
and the mixture was adjusted to 20 µg with pBluescript plasmid. In
the case of the mammalian two-hybrid assay, the pG5luc reporter was
co-transfected with the pGAL4 and pVP16 plasmids in 20 µg of total
DNA adjusted with pBluescript. After transfection, the plates were
rinsed several times in fresh medium, maintained in serum-free medium
for 2 days, and then harvested for CAT or luciferase assays. CV-1
fibroblasts, C3H 10T1/2 cells, or the myogenic
C2C12 cells were maintained in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum.
C2C12 cells were induced to differentiate by
changing the medium to 2% horse serum. Cells were cultured in six-well
plates and transfected with 0.7 µg of the luciferase reporter
plasmids and 0.1 µg of a pXJ40-derived expression plasmid using the
LipofectAMINE reagent (Invitrogen, Carlsbad, CA) in serum-free medium.
To test for the effects of Vgl and TEF or Vgl and MEF2 factors at
muscle-specific promoters, 0.7 µg of the luciferase reporter plasmids
and 0.05 µg each of the pXJ40-Vgl and pXJ40-TEF or pXJ40-Vgl and
pXJ40-MEF2 expression plasmids were used. For the two-hybrid assay, 0.7 µg of the pG5lux reporter plasmid was transfected with 0.1 µg of the pM expression vectors and 0.1 µg of the pVP16 vectors. Three hours after transfection, medium was replaced with serum-containing medium for 24 h.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (53K):
[in a new window]
Fig. 1.
Northern blot analysis of Vgl-1,
Vgl-2, and Vgl-3 expression in adult human tissues. A,
aligned sequences of mouse and human Vgl-2. Dots indicate
sequence identity. The TEF-1 interaction domains of Vgl-1, Vgl-2, and
Vgl-3 are compared with the same domain in Drosophila
Vestigial. Putative nuclear localization and nuclear export signals are
underlined. The SfiI site in the mouse Vgl-2
cDNA was used to generate CVgl-2, a Vgl-2 expression construct
that lacks the C-terminal domain (see Fig. 3). B, a human
multitissue Northern blot probed with Vgl-1 cDNA revealed a major
transcript at 1.35 kb in placenta. A faint band was also
detected in kidney RNA. Vgl-2 expression was strictly skeletal
muscle-specific. Signals in the placenta lane are cross-hybridization
of the Vgl-2 oligonucleotide probe to Vgl-1 and Vgl-3 transcripts.
Using an EST cDNA probe for Vgl-2, signal was only detected in
skeletal muscle (data not shown). The Vgl-3 oligonucleotide probe
revealed a transcript at 8.0 kb predominantly in placenta RNA. A trace
Vgl-3 signal was also seen in pancreas. C, Vgl-2 mRNA is
up-regulated following muscle differentiation. A representative
Northern blot analysis of RNA from mouse C2C12
cells harvested at the time of muscle differentiation when medium was
switched from 10% fetal bovine serum to 2% horse serum (0) or at 12, 24, and 48 h following muscle differentiation. The blot was
sequentially probed with mouse Vgl-2 and glyceraldehyde-3-phosphate
dehydrogenase (G3PDH). The -fold induction of Vgl-2 mRNA
was determined from three separate experiments and normalized to G3PDH
levels.
View larger version (86K):
[in a new window]
Fig. 2.
Vgl-2 is co-expressed with myogenin in
differentiated muscle. Whole mount in situ
hybridization (A-G) with antisense riboprobes for
Vgl-2 or myogenin was carried out using staged
embryos at e8.5 (A-C), e9.5 (D), e10.5
(E), and e11.5 (F and G). A. Vgl-2 expression is first detected in the first branchial
arch (arrowhead) at the 5- to 6-somite stage. B,
by the 7- to 8-somite stage, expression can be detected in the first
two branchial arches. C, dorsal view of the embryo in
B, clearly showing robust expression in the first branchial
arch. In panels A-C, no expression is detected in the
cranial somites. D, e9.5 embryos at the 26- to 28-somite
stage show clear expression of both Vgl-2 (left)
and myogenin (right) in the myotome of the
differentiated somite. Vgl-2 and myogenin
expression is absent in the most posterior somites and not detected
until somite 7+ (arrow). At this stage Vgl-2
expression can also be detected in the epithelial layer of the first
three branchial arches. E, e10.5 embryos (34-somite stage)
with Vgl-2 and myogenin transcripts detected in
the myotome in more anterior somites but absent in the most recently
formed posterior somites. In D and E, the most
posterior somite showing Vgl-2 or myogenin
expression is indicated by an arrow. F, e11.5
whole embryos or dissected forelimb buds (G) with expression
of Vgl-2 and myogenin. In the myotome, expression
of Vgl-2 appears as broad stripes as compared with the thin
bands of myogenin expression (G). Transcripts for
both Vgl-2 and myogenin can be detected in the
forelimb bud at this stage. Expression of Vgl-2 in the
branchial arches and their derivatives decreases with differentiation.
H, in situ hybridization of embryo sections
revealed Vgl-2 mRNA in the myotome (M) of a
tail somite at e11, but not in the neural tube (NT) or
notochord (Nc). I. At e16, Vgl-2 mRNA was
present in the hypoglossal muscle of the tongue (T) and
branchial arch-derived maxillary and mandibular cartilages
(arrows).
View larger version (91K):
[in a new window]
Fig. 3.
Vgl-2 protein translocates to the nucleus
following muscle differentiation. A myc-tagged Vgl-2 protein was
expressed by transient transfection of C2C12
cells cultured in high serum. Nuclei are stained in blue with the
fluorescent dye 4',6-diamidino-2-phenylindole, whereas transfected
cells expressing the myc-tagged Vgl-2 protein are labeled
green at low magnification (A and B,
scale bar = 100 µm) and high magnification
(C-F, scale bar = 20 µm). A
and C, Vgl-2 is a cytoplasmic protein in myoblasts.
B and D, Vgl-2 is a nuclear protein in myotubes.
Three aligned nuclei of a myotube (arrows) are shown
following fusion of two transfected (green nuclei) and one
untransfected (blue nucleus) myoblast. E and
F, Vgl-2 with a C-terminal deletion ( CVgl-2) does not
localize appropriately to the nucleus upon muscle differentiation
(F). The arrow indicates cytoplasmic retention of
CVgl-2 in a differentiated muscle cell.
View larger version (27K):
[in a new window]
Fig. 4.
Functional interaction between TEF-1 and Vgl
factors in mammalian cells. The GAL4-dependent
reporter pG5luc was co-transfected with expression vectors carrying
chimeric cDNAs encoding the DNA binding domain of yeast GAL4 fused
to sequences encoding different domains of TEF-1 and RTEF-1 together
with expression constructs carrying chimeric cDNAs encoding the
activation domain of VP16 fused to the cDNAs encoding either Vgl-1
or Vgl-2. The activity of the pG5luc reporter was normalized to the
level of transactivation obtained when co-transfected with the pMP53-
and pVP16TAg-positive control plasmids (mean ± S.D.,
n 
3 experiments). Note the different scales for the
normalized interaction (%) in cardiac myocytes (A) and CV-1
cells (B). Significance: *, p < 0.05 compared with TEF-1 or RTEF-1 fragment. C, mapping of the
Vestigial interaction domain. The results of the two-hybrid analysis
are shown schematically. Data for constructs labeled with an
asterisk are shown in A and B; for all
other constructs, data not shown. Fragments of TEF-1 and RTEF-1 that
interact with Vgl-1 or Vgl-2 are indicated by a plus sign
(+), whereas fragments that do not are indicated by a minus
sign ().
113-bp mouse skeletal muscle -actin (SKA) promoter has been
well characterized in terms of its interaction with TEF-1 transcription
factors (27, 31). Vgl-1 and Vgl-2 both had a modest effect on the
activity of the SKA promoter when co-transfected into neonatal rat
cardiac myocytes (Fig. 5A). In
addition, the squelching effect of TEF-1 overexpression was partially
relieved by either Vgl-1 or Vgl-2, and the transactivation observed
with RTEF-1 overexpression was further activated in the presence of
Vgl-1 or Vgl-2. These findings suggested that TEF-1 and Vgl factors
could productively interact in cardiac myocytes to alter the expression
of a muscle-specific TEF-1-dependent promoter. In contrast,
Vgl-1 significantly squelched the SKA promoter in CV-1 and
C2C12 cells, whereas Vgl-2 did not (Fig. 5,
B and C). Provided Vgl-1 and Vgl-2 are expressed
at similar levels, this result suggests a functional difference between
different members of the Vgl family.
View larger version (20K):
[in a new window]
Fig. 5.
Effects of Vgl factors on a
TEF-1-dependent promoter are
cell-dependent. The TEF-1-dependent mouse
113 SKA promoter was co-transfected with CMV enhancer-driven
expression vectors containing no cDNA insert (a dash,
), or the Vgl-1, Vgl-2, TEF-1, and RTEF-1 cDNAs. The activity of
the SKA promoter is expressed as a -fold of the activity when
co-transfected with the empty pXJ40 (CMV) expression vector
(mean ± S.E.). A, in cardiac myocytes, Vgl-1 and Vgl-2
tended to activate the SKA promoter. Asterisks (*) indicate
mean -fold activities that differ from control (p < 0.05, n 7 experiments). B, in CV-1 cells,
Vgl-2 had little effect, possibly due to promoter competition of the
co-transfected empty pXJ40 expression vector as a control for the TEF-1
expression vectors. Vgl-1 significantly squelched the SKA promoter on
its own (*) or in the presence of TEF-1 and RTEF-1 (#,
p < 0.05, n = 4 experiments).
C, in the myogenic C2C12 cells prior
to differentiation, Vgl-1 significantly squelched the SKA promoter on
its own and in the presence of RTEF-1 overexpression. Vgl-2 had
little effect in undifferentiated C2C12 cells
(n = 3 experiments). After differentiation, Vgl-1
squelched whereas Vgl-2 activated the SKA promoter (n = 4 experiments). Significance: *, p < 0.05; **,
p < 0.01; ***, p < 0.001.
113 mouse SKA
promoter. After differentiation, Vgl-2 weakly but significantly
activated the SKA promoter, suggesting that it participates in the
muscle-specific activation of this TEF-1-dependent promoter.
View larger version (22K):
[in a new window]
Fig. 6.
MEF2 and Vgl-2 co-activate a
MEF2-dependent promoter. The activity of the
MEF2-dependent chicken myosin light chain 2v (MLC2v)
promoter was set at 1-fold in the presence of 100 ng each of the empty
pXJ40 and pCMV expression vectors in transient transfection assays in
CV-1 cells (A) or C2C12 cells
(B) assayed before and after myogenic differentiation.
A, in CV-1 cells, MEF2 activated the wild type
(WT) MLC2v promoter 3-fold, Vgl-2 also activated this
promoter about 5-fold, and co-expression of MEF2 and Vgl-2 had a
synergistic effect, co-activating the MLC2v promoter 12-fold. Mutation
of the MEF2 site blocked MEF2-dependent activation, reduced
Vgl-2 activation, and abrogated MEF2/Vgl-2 co-activation of the MLC2v
promoter. B, in C2C12 myoblasts,
Vgl-2 weakly activated the MLC2v promoter (not significant), whereas
MEF2 activated the promoter nearly 10-fold. The effects of MEF2 and
Vgl-2 co-expression were additive. In C2C12
myotubes, Vgl-2 significantly activated the MLC2v promoter and MEF2
activated less strongly than in myoblasts. Significance: *,
p < 0.05, mean ± S.D., n 3 experiments.
View larger version (20K):
[in a new window]
Fig. 7.
Functional interaction between MEF2C and Vgl
factors in CV-1 cells. Using the mammalian two-hybrid assay, the
GAL4-dependent reporter pG5luc was co-transfected with
expression vectors carrying chimeric cDNAs encoding the DNA binding
domain of yeast GAL4 fused to sequences encoding different domains of
MEF2C together with expression constructs carrying chimeric cDNAs
encoding the activation domain of VP16 fused to the cDNAs encoding
either Vgl-1 or Vgl-2. The activity of the pG5luc reporter was
normalized to the level of transactivation obtained when co-transfected
with the pMP53- and pVP16TAg-positive control plasmids (mean ± S.E., n 5 experiments). Significance: *,
p < 0.05 compared with MEF2C fragment. A,
full-length MEF2C interacts with Vgl-1 and Vgl-2. B, the C
terminus of MEF2C interacts with Vgl-1 and Vgl-2. Fragments containing
the MADS domain of MEF2-(1-178) and -(1-302) did not interact with
either Vgl-1 or Vgl-2. A fragment including the activation domain of
MEF2-(178-436) but lacking the 30 C-terminal residues also did not
interact. Only when the C-terminal fragment was included was a robust
interaction detected. C, summary of the two-hybrid analysis
mapping the Vestigial interaction domain (VID) of MEF2.
D, Vgl-2 physically interacts with MEF2C in
vitro. Radiolabeled MEF2C protein synthesized in vitro
preferentially bound to a MalE-Vgl-2 fusion protein when compared with
MalE. The MEF2C lane represents 20% of the protein loaded
onto the column.
View larger version (57K):
[in a new window]
Fig. 8.
Vgl-2 augments MyoD-induced myosin heavy
chain expression in 10T1/2 cells, but its own expression is
MyoD-independent. A, C3H 10T1/2 cells were
transiently transfected with a MyoD expression vector, induced to
differentiate by lowering serum in the culture medium, and stained for
myosin heavy chain with the MF20 monoclonal antibody. B,
10T1/2 cells co-transfected with MyoD and Vgl-2 expression
vectors reveal a more robust myosin heavy chain induction upon
differentiation. C, Western blot quantitation of myosin
heavy chain expression in transfected 10T1/2 cells reveals a 6- to 7-fold greater induction with Vgl-2/MyoD co-expression than with
MyoD alone (n = 3, p < 0.05).
D, Northern blot analysis shows that MyoD does not induce
Vgl-2 mRNA expression in transfected 10T1/2 cells, whereas
Vgl-2 mRNA is detected in C2C12 myoblasts
and up-regulated with differentiation. Endogenous MyoD mRNA in
C2C12 cells was not visible at the exposure
shown. Days following serum withdrawal (0, 1, and 2) and shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
113 mouse SKA-CAT reporter, to Prof. Mahesh Gupta for the 295
chicken SKA-luciferase reporter, to Prof. Chaker Adra for providing the
RNA dot blots, and to Prof. Eric Hoffman for the human skeletal muscle
cDNA. We thank Dr. Hsiao-Huei Chen for the
C2C12 cells and Dr. Jorge Sepulveda for the
CV-1 cells. Jun Li and Jin Fu are acknowledged for their excellent
technical assistance.
FOOTNOTES
ABBREVIATIONS
-actin;
HDAC, histone deacetylase;
SRF, serum response factor;
CAT, chloramphenicol
acetyl transferase.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1.
Cossu, G.,
and Borello, U.
(1999)
EMBO J.
18,
6867-6872[CrossRef][Medline]
[Order article via Infotrieve]
2.
Molkentin, J. D.,
and Olson, E. N.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
9366-9373 3.
Molkentin, J. D.,
Black, B. L.,
Martin, J. F.,
and Olson, E. N.
(1995)
Cell
83,
1125-1136[CrossRef][Medline]
[Order article via Infotrieve]
4.
Mar, J. H.,
and Ordahl, C. P.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
6404-6408 5.
Mar, J. H.,
and Ordahl, C. P.
(1990)
Mol. Cell. Biol.
10,
4271-4283 6.
Farrance, I. K.,
Mar, J. H.,
and Ordahl, C. P.
(1992)
J. Biol. Chem.
267,
17234-17240 7.
Larkin, S. B.,
Farrance, I. K. G.,
and Ordahl, C. P.
(1996)
Mol. Cell. Biol.
16,
3742-3755[Abstract]
8.
Jiang, S.-W.,
and Eberhardt, N. L.
(1994)
J. Biol. Chem.
269,
10384-10392 9.
Butler, A. J.,
and Ordahl, C. P.
(1999)
Mol. Cell. Biol.
19,
296-306 10.
Gupta, M. P.,
Amin, C. S.,
Gupta, M.,
Hay, N.,
and Zak, R.
(1997)
Mol. Cell. Biol.
17,
3924-3936[Abstract]
11.
Belandia, B.,
and Parker, M. G.
(2000)
J. Biol. Chem.
275,
30801-30805 12.
Vassilev, A.,
Kaneko, K. J.,
Shu, H.,
Zhao, Y.,
and DePamphilis, M. L.
(2001)
Genes Dev.
15,
1229-1241 13.
Gupta, M.,
Kogut, P.,
Davis, F. J.,
Belaguli, N. S.,
Schwartz, R. J.,
and Gupta, M. P.
(2001)
J. Biol. Chem.
276,
10413-10422 14.
Chen, S. L.,
Dowhan, D. H.,
Hosking, B. M.,
and Muscat, G. E.
(2000)
Genes Dev.
14,
1209-1228 15.
Campbell, S.,
Inamdar, M.,
Rodrigues, V.,
Raghavan, V.,
Palazzolo, M.,
and Chovnick, A.
(1992)
Genes Dev.
6,
367-379 16.
Deshpande, N.,
Chopra, A.,
Rangarajan, A.,
Shashidhara, L. S.,
Rodrigues, V.,
and Krishna, S.
(1997)
J. Biol. Chem.
272,
10664-10668 17.
Halder, G.,
Polaczyk, P.,
Kraus, M. E.,
Hudson, A.,
Kim, J.,
Laughon, A.,
and Carroll, S.
(1998)
Genes Dev.
12,
3900-3909 18.
Paumard-Rigal, S.,
Zider, A.,
Vaudin, P.,
and Silber, J.
(1998)
Dev. Genes Evol.
208,
440-446[CrossRef][Medline]
[Order article via Infotrieve]
19.
Simmonds, A. J.,
Liu, X.,
Soanes, K. H.,
Krause, H. M.,
Irvine, K. D.,
and Bell, J. B.
(1998)
Genes Dev.
12,
3815-3820 20.
Kim, J.,
Sebring, A.,
Esch, J. J.,
Kraus, M. E.,
Vorwerk, K.,
Magee, J.,
and Carroll, S. B.
(1996)
Nature
382,
133-138[CrossRef][Medline]
[Order article via Infotrieve]
21.
Halder, G.,
and Carroll, S. B.
(2001)
Development
128,
3295-3305 22.
Klein, T.,
and Arias, A. M.
(1999)
Development
126,
913-925[Abstract]
23.
Sudarsan, V.,
Anant, S.,
Guptan, P.,
VijayRaghavan, K.,
and Skaer, H.
(2001)
Dev. Cell
1,
829-839[CrossRef][Medline]
[Order article via Infotrieve]
24.
Vaudin, P.,
Delanoue, R.,
Davidson, I.,
Silber, J.,
and Zider, A.
(1999)
Development
126,
4807-4816[Abstract]
25.
Wilkinson, D. G.
(1992)
in
In Situ Hybridization: A Practical Approach
(Wilkinson, D. G., ed)
, pp. 75-83, IRL Press, Oxford
26.
Xiao, J. H.,
Davidson, I.,
Matthes, H.,
Garnier, J. M.,
and Chambon, P.
(1991)
Cell
65,
551-568[CrossRef][Medline]
[Order article via Infotrieve]
27.
Stewart, A. F. R.,
Suzow, J.,
Kubota, T.,
Ueyama, T.,
and Chen, H. H.
(1998)
Circ. Res.
83,
43-49 28.
Karns, L. R.,
Kariya, K.,
and Simpson, P. C.
(1995)
J. Biol. Chem.
270,
410-417 29.
Zhou, M. D.,
Goswami, S. K.,
Martin, M. E.,
and Siddiqui, M. A.
(1993)
Mol. Cell. Biol.
13,
1222-1231 30.
Maeda, T.,
Gupta, M. P.,
and Stewart, A. F. R.
(2002)
Biochem. Biophys. Res. Commun.
294,
791-797[CrossRef][Medline]
[Order article via Infotrieve]
31.
Ueyama, T.,
Zhu, C.,
Valenzuela, Y. M.,
Suzow, J. G.,
and Stewart, A. F. R.
(2000)
J. Biol. Chem.
275,
17476-17480 32.
Simpson, P.
(1983)
J. Clin. Invest.
72,
732-738[Medline]
[Order article via Infotrieve]
33.
Ordahl, C. P.
(1992)
Curr. Topics Dev. Biol.
26,
145-168[Medline]
[Order article via Infotrieve]
34.
Zider, A.,
Paumard-Rigal, S.,
Frouin, I.,
and Silber, J.
(1998)
J. Neurogenet.
12,
87-99[Medline]
[Order article via Infotrieve]
35.
Yockey, C. E.,
Smith, G.,
Izumo, S.,
and Shimizu, N.
(1996)
J. Biol. Chem.
271,
3727-3736 36.
Hsu, D. K. W.,
Guo, Y.,
Alberts, G. F.,
Copeland, N. G.,
Gilbert, D. J.,
Jenkins, N. A.,
Peifley, K. A.,
and Winkles, J. A.
(1996)
J. Biol. Chem.
271,
13786-13795 37.
Stewart, A. F. R.,
Richard, C. W., 3rd,
Suzow, J.,
Stephan, D.,
Weremowicz, S.,
Morton, C. C.,
and Adra, C. N.
(1996)
Genomics
37,
68-76[CrossRef][Medline]
[Order article via Infotrieve]
38.
Yu, Y. T.
(1996)
J. Biol. Chem.
271,
24675-24683 39.
Rogerson, P. J.,
Jamali, M.,
and Skerjanc, I. S.
(2002)
FEBS Lett.
524,
134-138[CrossRef][Medline]
[Order article via Infotrieve]
40.
Jacquemin, P.,
Oury, C.,
Belayew, A.,
and Martial, J. A.
(1994)
DNA Cell Biol.
13,
1037-1045[Medline]
[Order article via Infotrieve]
41.
McKinsey, T. A.,
Zhang, C. L.,
and Olson, E. N.
(2002)
Trends Biochem. Sci.
27,
40-47[CrossRef][Medline]
[Order article via Infotrieve]
42.
Black, B. L.,
Molkentin, J. D.,
and Olson, E. N.
(1998)
Mol. Cell. Biol.
18,
69-77 43.
Nagel, A. C.,
Wech, I.,
and Preiss, A.
(2001)
Mech. Dev.
109,
241-251[CrossRef][Medline]
[Order article via Infotrieve]
44.
Ogryzko, V. V.,
Schiltz, R. L.,
Russanova, V.,
Howard, B. H.,
and Nakatani, Y.
(1996)
Cell
87,
953-959[CrossRef][Medline]
[Order article via Infotrieve]
45.
Bannister, A. J.,
and Kouzarides, T.
(1996)
Nature
384,
641-643[CrossRef][Medline]
[Order article via Infotrieve]
46.
McKinsey, T. A.,
Zhang, C. L., Lu, J.,
and Olson, E. N.
(2000)
Nature
408,
106-111[CrossRef][Medline]
[Order article via Infotrieve]
47.
Kuo, M. H.,
and Allis, C. D.
(1998)
Bioessays
20,
615-626[CrossRef][Medline]
[Order article via Infotrieve]
48.
Lu, J.,
McKinsey, T. A.,
Zhang, C. L.,
and Olson, E. N.
(2000)
Mol. Cell
6,
233-244[CrossRef][Medline]
[Order article via Infotrieve]
49.
Miska, E. A.,
Langley, E.,
Wolf, D.,
Karlsson, C.,
Pines, J.,
and Kouzarides, T.
(2001)
Nucleic Acids Res.
29,
3439-3447 50.
Borghi, S.,
Molinari, S.,
Razzini, G.,
Parise, F.,
Battini, R.,
and Ferrari, S.
(2001)
J. Cell Sci.
114,
4477-4483[Medline]
[Order article via Infotrieve]
51.
Kanai, F.,
Marignani, P. A.,
Sarbassova, D.,
Yagi, R.,
Hall, R. A.,
Donowitz, M.,
Hisaminato, A.,
Fujiwara, T.,
Ito, Y.,
Cantley, L. C.,
and Yaffe, M. B.
(2000)
EMBO J.
19,
6778-6791[CrossRef][Medline]
[Order article via Infotrieve]
52.
Wang, D.,
Chang, P. S.,
Wang, Z.,
Sutherland, L.,
Richardson, J. A.,
Small, E.,
Krieg, P. A.,
and Olson, E. N.
(2001)
Cell
105,
851-862[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  A. C. T. Teng, K. Adamo, F. Tesson, and A. F. R. Stewart Functional characterization of a promoter polymorphism that drives ACSL5 gene expression in skeletal muscle and associates with diet-induced weight loss FASEB J, June 1, 2009; 23(6): 1705 - 1709. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Deng, S. C. Hughes, J. B. Bell, and A. J. Simmonds Alternative Requirements for Vestigial, Scalloped, and Dmef2 during Muscle Differentiation in Drosophila melanogaster Mol. Biol. Cell, January 1, 2009; 20(1): 256 - 269. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ota and H. Sasaki Mammalian Tead proteins regulate cell proliferation and contact inhibition as transcriptional mediators of Hippo signaling Development, December 15, 2008; 135(24): 4059 - 4069. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sawada, H. Kiyonari, K. Ukita, N. Nishioka, Y. Imuta, and H. Sasaki Redundant Roles of Tead1 and Tead2 in Notochord Development and the Regulation of Cell Proliferation and Survival Mol. Cell. Biol., May 15, 2008; 28(10): 3177 - 3189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yoshida MCAT Elements and the TEF-1 Family of Transcription Factors in Muscle Development and Disease Arterioscler. Thromb. Vasc. Biol., January 1, 2008; 28(1): 8 - 17. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Gan, T. Yoshida, J. Li, and G. K. Owens Smooth Muscle Cells and Myofibroblasts Use Distinct Transcriptional Mechanisms for Smooth Muscle {alpha}-Actin Expression Circ. Res., October 26, 2007; 101(9): 883 - 892. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Hucl, J. R. Brody, E. Gallmeier, C. A. Iacobuzio-Donahue, I. K. Farrance, and S. E. Kern High Cancer-Specific Expression of Mesothelin (MSLN) Is Attributable to an Upstream Enhancer Containing a Transcription Enhancer Factor Dependent MCAT Motif Cancer Res., October 1, 2007; 67(19): 9055 - 9065. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. B. Kim, G. J. Porreca, L. Song, S. C. Greenway, J. M. Gorham, G. M. Church, C. E. Seidman, and J. G. Seidman Polony Multiplex Analysis of Gene Expression (PMAGE) in Mouse Hypertrophic Cardiomyopathy Science, June 8, 2007; 316(5830): 1481 - 1484. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Garg, A. Srivastava, M. M. Davis, S. L. O'Keefe, L. Chow, and J. B. Bell Antagonizing Scalloped With a Novel Vestigial Construct Reveals an Important Role for Scalloped in Drosophila melanogaster Leg, Eye and Optic Lobe Development Genetics, February 1, 2007; 175(2): 659 - 669. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Anbanandam, D. C. Albarado, C. T. Nguyen, G. Halder, X. Gao, and S. Veeraraghavan Insights into transcription enhancer factor 1 (TEF-1) activity from the solution structure of the TEA domain PNAS, November 14, 2006; 103(46): 17225 - 17230. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Zhao, G. Caretti, S. Mitchell, W. L. McKeehan, A. L. Boskey, L. M. Pachman, V. Sartorelli, and E. P. Hoffman Fgfr4 Is Required for Effective Muscle Regeneration in Vivo: DELINEATION OF A MyoD-Tead2-Fgfr4 TRANSCRIPTIONAL PATHWAY J. Biol. Chem., January 6, 2006; 281(1): 429 - 438. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Azakie, L. LaMont, J. R. Fineman, and Y. He Divergent transcriptional enhancer factor-1 regulates the cardiac troponin T promoter Am J Physiol Cell Physiol, December 1, 2005; 289(6): C1522 - C1534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sawada, Y. Nishizaki, H. Sato, Y. Yada, R. Nakayama, S. Yamamoto, N. Nishioka, H. Kondoh, and H. Sasaki Tead proteins activate the Foxa2 enhancer in the node in cooperation with a second factor Development, November 1, 2005; 132(21): 4719 - 4729. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Zhu, B. Ramachandran, and T. Gulick Alternative Pre-mRNA Splicing Governs Expression of a Conserved Acidic Transactivation Domain in Myocyte Enhancer Factor 2 Factors of Striated Muscle and Brain J. Biol. Chem., August 5, 2005; 280(31): 28749 - 28760. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Allen, J. N. Weber, L. K. Sycuro, and L. A. Leinwand Myocyte Enhancer Factor-2 and Serum Response Factor Binding Elements Regulate Fast Myosin Heavy Chain Transcription in Vivo J. Biol. Chem., April 29, 2005; 280(17): 17126 - 17134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-H. Chen, S. J. Mullett, and A. F. R. Stewart Vgl-4, a Novel Member of the Vestigial-like Family of Transcription Cofactors, Regulates {alpha}1-Adrenergic Activation of Gene Expression in Cardiac Myocytes J. Biol. Chem., July 16, 2004; 279(29): 30800 - 30806. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Srivastava, A. J. Simmonds, A. Garg, L. Fossheim, S. D. Campbell, and J. B. Bell Molecular and Functional Analysis of scalloped Recessive Lethal Alleles in Drosophila melanogaster Genetics, April 1, 2004; 166(4): 1833 - 1843. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Gunther, M. Mielcarek, M. Kruger, and T. Braun VITO-1 is an essential cofactor of TEF1-dependent muscle-specific gene regulation Nucleic Acids Res., February 3, 2004; 32(2): 791 - 802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Leask, A. Holmes, C. M. Black, and D. J. Abraham Connective Tissue Growth Factor Gene Regulation. REQUIREMENTS FOR ITS INDUCTION BY TRANSFORMING GROWTH FACTOR-beta 2 IN FIBROBLASTS J. Biol. Chem., April 4, 2003; 278(15): 13008 - 13015. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |