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INTRODUCTION |
Serum response factor
(SRF)1 belongs to a super
family of transcription factors that contain a highly conserved
DNA-binding and dimerization domain termed as MADS box, because of its
homology among yeast (MCM1, Agamous), plant
(Deficiens), and vertebrate (SRF) proteins (1,
2). MADS proteins play a wide range of functions from development in
plants to muscle cell differentiation and growth response in mammalian
cells (reviewed in Ref 2). SRF is a 67-kDa phosphoprotein that
was first identified as a major regulatory protein controlling the
serum-activated expression of the c-fos gene. SRF
regulates gene transcription through binding to a consensus DNA
sequence, the serum response element (SRE), CC(A/T)6GG also
known as CArG box (2-4). Functional SREs are found in the promoter
region of many immediate early genes involved in the mitogenic response
of proliferative cells (2-4). Paradoxically, SREs are also found in
the promoters of several muscle genes expressed in post-replicative
myocytes, where they are involved in controlling the gene tissue
specificity as well as their response to hypertrophic growth stimuli
(5-8). SREs between muscle and nonmuscle gene promoters can be
interchanged without loss of their function (9), thus suggesting that
in addition of SRF binding to SRE in both types of promoters, its
activity is very much governed by other cell type- and
promoter-specific factors interacting with SRF.
Several transcription factors have been identified that either interact
directly with SRF or bind DNA sequences adjacent to SRE to influence
the SRF activity. These include Ying Yang 1 (YY-1) (10, 11),
homeodomain protein Phox-1/MHox (4, 12) and Nkx 2.5 (6), NF-
B (13),
ATF6 (14), ternary complex factors of Ets family (2), myogenic basic
helix-loop-helix proteins (15), and high mobility group factor, SSRP1
(16). In skeletal myoblasts SRF interacts with myogenin-E12 (or
MyoD-E-12) heterodimer, and this interaction has been implicated by
which SRF activity is modulated from proliferative myoblast to control
muscle gene expression in differentiated myocytes (15). However, there
are many other muscle genes that have a functional SRE but do not require myogenic basic helix-loop-helix family of factors for their
tissue-specific regulation. For example, genes expressed in
cardiac myocytes such as cardiac
-actin (7), skeletal
-actin (17), MLC-2 (18),
-MHC (19), atrial natriuretic factor (20),
muscle creatine kinase (21), as well as cardiac troponin T (22,
23) all possess SREs that are critical for their activation in cardiac
muscle cell context but are independent of MyoD regulatory mechanisms.
Therefore, it is likely that the SRE of these promoters could be
interacting with other muscle-specific regulatory elements for gene
trans-activation. Recently, a considerable amount of evidence has
indicated that SREs of skeletal
-actin and
-MHC gene promoters
cooperate positively with another muscle-specific element M-CAT that is
recognized by transcription enhancer factor-1 (TEF-1) (17, 19, 24). A
combinatorial interaction between both these elements is found to be
necessary for
1-adrenergic, transforming growth
factor-
, and stretch-induced activation of skeletal
-actin gene
expression in cardiac myocytes and slow twitch skeletal muscle fibers
(17, 24, 25). These findings raised a possibility of a mutual
cooperation between SRF and TEF-1 proteins.
TEF-1 is a member of a new family of transcription factors that are
characterized by a structurally conserved DNA-binding domain, TEA/ATTS
(26, 27). Proteins containing TEA domain have been shown to control
function in a variety of animals and plant phyla (for review see Ref.
26). In humans, at least four different TEF-1 genes
have been identified that encode hTEF-1, hTEF-3, hTEF-4, and hTEF-5
isoforms (26). Homologues of these isoforms have also been isolated
from mice (28) and chicks (29). Members of the TEF-1 family have been
found to be important for muscle-specific expression of several cardiac
and smooth muscle genes and are targets of hypertrophic stimuli (23,
24, 29-31). A TEF-1 homologue, scalloped, in Drosophila,
has been shown to play an important role in the lineage progression of
sensory neuronal development (32). In mammals, functional TEF-1 is
found as early as 2-8 cell stage in the mouse zygote development, and
inactivation of TEF-1 gene has been shown to result
in embryonic lethality due to myocardial defects (33, 34).
In this paper we tested the hypothesis whether SRF can physically
interact with TEF-1. We demonstrate that SRF forms a stable complex
with TEF-1 both in vitro and in cultured cells. The
formation of this complex requires MADS and TEA DNA-binding domains of
SRF and TEF-1, respectively. This interaction discloses a novel
mechanism by which SRF activity could be modulated to control the
expression of muscle genes in differentiated cells where
MyoD-dependent regulatory mechanisms are not in play.
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MATERIALS AND METHODS |
Overexpression and Purification of GST Fusion Proteins--
The
GST fusion proteins were expressed in bacteria and purified as
described previously (31, 35). In brief, bacteria harboring plasmids
GST-SRF and GST-TEF-1 were grown overnight in LB ampicillin medium. The
next morning, cells were diluted 1:10 with fresh medium, grown to an OD
of 0.6-0.75, and induced with 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside to direct
expression of fusion proteins. After 3-7 h of expression of the GST
fusion proteins, cells were harvested, and the fusion proteins were
isolated as follows. Cells were pelleted at 4,000 × g
at 4 °C, resuspended in phosphate-buffered saline (PBS) containing protease inhibitors, and sonicated for a total elapsed time of 120 s. The bacterial lysate was solubilized by the addition of Triton X-100
to a final concentration of 1% and centrifuged at 13,000 × g at 4 °C to remove insoluble material.
Glutathione-agarose beads were added to the soluble supernatant
fraction, and the binding of GST fusion proteins was allowed to occur
at 4 °C for 30 min. The beads were pelleted in an Eppendorf
centrifuge at 4,000 × g for 2 min, and the GST fusion
proteins bound to the glutathione-agarose beads were washed thoroughly
with PBS containing 0.1% Triton X-100. The integrity of the GST fusion
proteins bound to the beads was analyzed by resolving of proteins on
SDS-PAGE and Coomassie Blue staining, along with known amounts of
bovine serum albumin on the same gel to calculate the yield of
full-length fusion proteins.
Preparation of Nuclear Extract and Electro-mobility Gel Shift
Assay--
Nuclear extract was prepared from neonatal rat hearts by
the method of Dignam et al. (36), with slight modifications
as described previously (35). For the electro-mobility gel shift assay
(EMSA), double-stranded oligonucleotides were 5'-end-labeled with T4
polynucleotide kinase (Life Technologies, Inc.) and
[
-32P]ATP. The analytical binding reaction was carried
out in a total volume of 25 µl containing ~10,000 cpm (0.1-0.5 ng)
of the labeled DNA probe, 2-5 µg of the nuclear extracts, and 1 µg
of poly(dI-dC) (Sigma) as a nonspecific competitor. The binding buffer
consisted of 10 mM Tris-HCl (pH 7.4), 100 mM
NaCl, 0.1 mM EGTA, 0.5 mM dithiothreitol, 0.3 mM MgCl2, 8% glycerol, and 0.5 mM
phenylmethylsulfonyl fluoride. After incubation at room temperature for
20 min, the reaction mixtures were loaded on 5% native polyacrylamide
gels (44:1, acrylamide/bisacrylamide), and electrophoresis was carried
out at 150 V in a 0.5× TBE buffer, in a cold room. For competition and
antibody experiments, unlabeled competitor DNAs or the antibody were
preincubated with nuclear extracts at room temperature for 15-20 min
in the reaction buffers prior to addition of the labeled DNA probe. The
Jurkat T cell nuclear extract, anti-SRF, and anti-FLAG
antibodies used in this study were obtained commercially from
Stratagene, and a monoclonal anti-TEF-1 antibody was purchased from
Transduction Laboratories, Lexington, KY. Sense strand sequences of
double-stranded oligonucleotides used in EMSA are as follows:
-MHC
C(Ar)G, 5'GTCCCAGCAGATGACTCCAAATTTAGGCAGCAGGCA3'; cTNT M-CAT,
5'AGTGTTGCATTCCTCTCTGG3'.
Affinity Precipitation of SRF with the GST-TEF-1
Protein--
Five micrograms of GST or GST-TEF-1 bound to
glutathione-agarose beads was incubated with 40 µg of neonatal rat
heart or Jurkat T cell nuclear extract in 1× DNA-binding buffer for
3 h at 4 °C with continuous rocking. Glutathione-agarose beads
without GST fusion protein were also incubated with nuclear extract to
serve as a negative control. After 3 h of incubation, beads were
pelleted at 14,000 × g for 2 min, and the supernatant
was collected and used directly for the mobility gel shift assay. To
detect the interaction of SRF with GST-TEF-1 protein, the pelleted
beads were washed five times with 1 ml of 1× DNA-binding buffer,
suspended in 2× Laemmli buffer, and subjected to subsequent Western
blot analysis.
In Vitro Characterization of SRF Binding to TEF-1--
The
TNT-coupled rabbit reticulocyte lysate system (Promega, Madison, WI)
was used to translate pBS-Max, pBS-SRF, pBS-MyoD, and pBS-myogenin
plasmids. After translation, the specific incorporation of
[35S]methionine into proteins was determined by
trichloroacetic acid precipitation, and the integrity of translated
proteins was checked by SDS-PAGE and autoradiography. For the in
vitro binding assay, 35S-labeled proteins were
incubated with 2-3 µg of GST or GST fusion proteins on
glutathione-agarose beads in 1× protein interaction buffer (PIB) (20 mM HEPES (pH 7.5), 75 mM KCl, 1 mM
EDTA, 2 mM MgCl2, 2 mM
dithiothreitol, and 0.5% Nonidet P-40) for 2 h at 4 °C with
continuous rocking. The beads were pelleted and washed five times with
1× PIB. The bound proteins were eluted with Laemmli sample buffer and
analyzed on SDS-PAGE.
Coimmunoprecipitation of SRF with TEF-1--
Jurkat T cells at a
density of 1 × 106 cells/tube were transfected with
the expression plasmid pCMV.FLAG-TEF-1 (35) by using the
electroporation procedure (BTX, San Diego, CA). Following 36 h of
transfection, cells were collected, washed with ice-cold PBS, and lysed
in a high salt (100 mM NaCl) lysis buffer. The cells were
allowed to lyse on ice for 15 min and then were further disrupted by
forcing them through a 22-gauge needle several times. The lysed cell
extract was spun to remove cell debris, and the supernatant (whole cell
extract) was transferred to a fresh tube. It was checked for the
expression of ectopically expressed FLAG.TEF-1 protein by Western blot
analysis. For immunoprecipitation of proteins the whole cell extract
was incubated with 2 µl of anti-FLAG or anti-SRF antibodies (Santa
Cruz Biotechnology) conjugated with agarose beads (1.8 mg of
antibody/ml) in a total volume of 1 ml for an hour with continuous
rocking at 4 °C. The beads were pelleted, washed 5 times in the
lysis buffer, suspended in 2× Laemmli buffer, and subsequently
subjected to Western blot analysis using either anti-FLAG or anti-SRF
antibodies. To determine SRF-TEF-1 interaction in cardiac myocytes, the
neonatal rat heart nuclear extract (300-500 µg of protein) was
incubated with 0.5 µg of control mouse or rabbit IgG (species depends
upon the source of the primary antibody) together with 20 µl of
protein A/G-agarose (Santa Cruz Biotechnology) at 4 °C for 30 min.
The pre-cleared nuclear extract (supernatant) obtained by
centrifugation of reaction mixtures was incubated with 20 µg of
either anti-TEF-1 antibody or anti-SRF antibody at 4 °C for 60 min.
Protein A/G-agarose (20 µl) was then added to each sample and
incubated for 4-6 h at 4 °C on a rotating platform. The absence of
primary antibody in a parallel reaction mix served as negative control.
Agarose beads were pelleted by centrifugation, washed four times in
phosphate-buffered saline (PBS), and reconstituted in 50 µl of PBS.
Proteins were removed from the beads by boiling in a 2× Laemmli sample
buffer and analyzed by Western blot analyses using either anti-TEF-1 or
anti-SRF-antibodies.
Far Western Analysis--
Far Western analysis was performed
essentially according to the protocol as described before (37). A
bacterial expression plasmid, pGEX-KG-cAMP-TEF-1, was generated to
synthesize GST-cAMP-TEF-1 fusion protein. A double-stranded
oligonucleotide that encodes two cAMP-dependent protein
kinase A sites was introduced into BamHI-EcoRI
sites of pGEX-KG to create the pGEX-KG-cAMP plasmid. Subsequently, an
NcoI-SacI fragment containing full-length TEF-1 cDNA was subcloned into the NcoI-SacI sites
of the plasmid pGEX-KG-cAMP to direct expression of GST-cAMP-TEF-1. The
GST-cAMP-TEF-1 fusion protein was overexpressed in DH5
cells, and
then it was isolated and labeled with catalytic subunit of the bovine
cAMP-dependent protein kinase A as described before (37).
The labeled GST-cAMP-TEF-1 fusion protein was eluted from beads, and
2 × 510 cpm of protein probe per ml was added to the
nitrocellulose (NC) blot that contained 50 ng of each protein (GST-SRF,
SRF, GST-Max, Max, and GST) in 1× hybridization buffer (20 mM HEPES (pH 7.7), 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 1 mM dithiothreitol, and 0.05% Nonidet P-40). The NC blot
was incubated with the labeled GST-cAMP-TEF-1 protein overnight in a
cold room. The unbound labeled protein was removed by four washes with
1× hybridization buffer at room temperature. The NC blot was wrapped
in Saran wrap and subjected to autoradiography at
70 °C for 4-12 h.
Cell Culture and Transfection--
Primary myocytes were
cultured from 18-day-old fetal rat hearts (see Ref. 41). After
differential plating to eliminate nonmuscle cells, myocytes were plated
at a density of 2 × 106 cells/100-mm culture dish
(Falcon brand, Becton Dickinson Labware) pre-coated with 0.1% gelatin
in Ham's F-12 medium (Life Technologies, Inc.) with 5% calf serum.
Cultures generally consisted of more than 90% myocytes, as measured by
immunocytofluorescence with anti-myosin antibody. More than 90% of the
cells began to contract spontaneously within 24 h after plating.
Jurkat and COS1 cells were grown in growth medium containing
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum in an atmosphere of 5%
CO2. All culture media contained penicillin (5 mg/ml),
streptomycin (5 mg/ml), and neomycin (100 mg/ml).
Primary cultures of cardiac myocytes were transfected after 48 h
in culture with 5 µg of DNA/plate by use of a lipotaxi reagent (Stratagene). COS1 cells were transfected by using calcium phosphate procedure. Typically cells were plated in 100-mm plates, and after reaching 50% confluency they were transfected with 5 µg of total DNA. All transfections contained 1 µg of the pCMV-
gal reference plasmid. The next morning (~18 h after transfection) medium was changed, and after an additional 48 h cells were harvested, and cell lysate was prepared and assayed for luciferase,
-galactosidase activities, and protein content. The luciferase activity for each construct was corrected for protein content of each extract and normalized to the activity of
-galactosidase activity in the same
cell extract.
Construction of Plasmids--
The bacterial expression plasmid
pGEX-KG was utilized to direct overexpression of full-length TEF-1 or
TEF-1 mutants. Briefly, the full-length TEF-1, truncated or
point-mutated versions of TEF-1 were amplified from pBS-TEF-1 (28) by
polymerase chain reaction, digested with
XbaI/XhoI, and subcloned into the
XbaI/XhoI sites of the pGEX-KG vector. The
GST-TEF-1 expression plasmids were subsequently transformed into HB101
cells for inducible production of the protein. The skeletal
-actin
reporter,
394/+24 Sk-
-actin-luc was constructed by subcloning
nucleotides
394 to +24 base pairs RsaI-HindIII
fragment of chicken Sk-
-actin into pXP1 vector. Linker-scanning
BglII mutants of the Sk-
-actin promoter have been
described before (39). The p5xSREluc reporter plasmid is directed by an
artificial promoter containing five copies of the SRF-binding sites (no
Ets-binding sites) upstream of a minimal TATA box (provided by Dr. J. Solway, University of Chicago). The 5× M-CAT-luc reporter plasmid
containing five copies of cTNT M-CAT-1 element was generated by
amplifying five M-CAT-1 sites from an artificial 5× M-CAT
promoter/reporter plasmid (supplied by Dr. C. P. Ordahl University
of California, San Francisco, see Ref. 40) and subsequently cloning
into PGL-3 vector containing luciferase reporter gene (Promega,
Madison, WI).
Oligonucleotide primers used for generating TEF-1 mutants are as
follows: forward primer containing XbaI site,
5'TCTAGAGATTGAGCCCAGCAGCTGGAGCGGC3'; reverse primers with or without
XhoI site; for TEF-(1-210), 5'CTCGAGTCACCAGGCAGGAACTGAGGGGGC3'; for TEF-(1-123), 5'CTCGAGTCACAGGGCCTTGTCCTTGGCAGT3'; for TEF-(1-113), 5'CTCGAGTCAGCTTGTTACCTTCAGCTTGGA3'; for TEF-(1-103),
5'CTCGAGTCAACGAGATTTCCTTCTGGCAAAACCTGAATGTGACTAGACACCTGCTT3'; for
TEF-(1-88), 5'CTCGAGTCACCTTGTCTTTCCCGTCCTGAGTTTGATGTATCTGGCTAT3'; for
TEF (Helix-3mt),
5'CTCGAGTCAACGAGATTTCCTTCTGGCAAGAACCTGAATGGGACTAGACACCGGCTT3'; for TEF
(Helix-2mt), 5'CCTTGTCTTTCCCGTCCTGAGTCCGATGTATCTGCCTATCAA3'; for
TEF-(114-430) a forward primer, 5'TCTAGAGATGGATCAGACTGCCAAGGAC3', and
a reverse primer, 5'GGCCGGCTCGAGTCAGTCCTTCACAAGCCTGTAGATATGGTG3', were
used; for TEF-(27-113), the forward primer
5'TCTAGAGGTCTGGAGTCCTGATATTGAGCAG3' and the reverse primer same as used
for TEF-(1-113) construct; and for TEF (Helix-1mt) the forward primer
5'TCTAGAGGTCTGGAGTCCTGATATTGGGCAGAG TTTCGGGGAG3' and the reverse primer
same as used for TEF-(1-103) construct.
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RESULTS |
Physical Interaction of TEF-1 with SRF--
Our initial goal was
to determine whether TEF-1 can physically associate with SRF. To test
this possibility we first attempted to deplete the nuclear extract of
SRF by affinity precipitation of the protein with GST-TEF-1 beads. The
neonatal rat heart nuclear extract or Jurkat T cell nuclear extract,
containing high levels of SRF, was incubated with GST-TEF-1 or GST
proteins that were immobilized on the glutathione-agarose beads. After
removal of GST beads from nuclear extract, the supernatant was analyzed
by an EMSA using C(Ar)G oligo as a labeled probe. As shown in Fig. 1A, the specific
SRF-DNA complex that could be supershifted by SRF antibody was
found to be abolished completely in nuclear extract samples treated
with GST-TEF-1 beads but not with GST beads alone. These results
suggested that a protein physically interacting with TEF-1 segment of
the GST fusion protein must be involved in generating the
C(Ar)G-protein complex. To find out whether SRF was indeed removed from
the nuclear samples, we analyzed the presence of SRF on
glutathione-agarose beads by Western blot analysis using an anti-SRF
antibody. As shown in Fig. 1B, SRF was present on GST-TEF-1
beads but not on GST beads alone, thus indicating a direct interaction
between SRF and TEF-1 proteins.

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Fig. 1.
Affinity precipitation of SRF with
GST-TEF-1. A, 40 µg of nuclear extract
(N.E.) was incubated in a binding buffer with 5 µg of GST
or GST-TEF-1-linked glutathione-agarose beads for 3 h at 4 °C.
Beads were pelleted, and EMSA was performed with an equal volume of
supernatant from each tube. In lanes 3 and 7,
nuclear extract was preincubated with 3 µl of anti-SRF antibody.
SS represents supershifted complex. In lane 2, a
100-fold excess unlabeled oligonucleotide (same as probe) was used as a
competitor. B, pelleted beads from Jurket T cell nuclear
extract were subjected to Western blot analysis using an anti-SRF
antibody.
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These results were confirmed in vivo by
coimmunoprecipitation of SRF with TEF-1 from both Jurkat T cells as
well as cardiac myocytes. TEF-1 expression has been shown to be
undetectable in lymphocytes (27). Therefore, to see TEF-1/SRF
interaction in Jurkat T cells, we overexpressed TEF-1 in these cells by
transfecting them with an expression plasmid (pCMV.FLAG.TEF-1)
encoding the full-length TEF-1 tagged with a FLAG peptide. For the
control, cells were transfected with a parental plasmid (pCMV.FLAG)
lacking the TEF-1 cDNA. The 2nd day after transfection, cells were
harvested, and cell lysate was examined for expression of the
FLAG.TEF-1 protein by Western blot analysis using an anti-FLAG
antibody. Cell lysate that showed a high level expression of FLAG.TEF-1 was then incubated with anti-FLAG affinity gel beads. These beads were
separated by centrifugation, washed repeatedly, and analyzed by Western
blot analysis using an anti-SRF antibody to examine whether SRF was
coimmunoprecipitated with the FLAG.TEF-1 protein. As shown in Fig.
2, an ~67-kDa SRF protein was pulled
down from cells transfected with the pCMV.FLAG.TEF-1 but not from
control cells that received the plasmid pCMV.FLAG. To confirm these
results we performed an inverse experiment, in which the cell lysate
was subjected to immunoprecipitation with an anti-SRF antibody-linked agarose beads, and beads were analyzed by Western blot analysis using
an anti-FLAG antibody. As expected a band of ~56 kDa of the
FLAG.TEF-1 protein was found precipitated with SRF antibody (Fig. 2).
To determine interaction of TEF-1 with SRF in cardiac myocytes, cardiac
nuclear extract was first pre-cleared with IgG, and the resulting
supernatant was subjected to immunoprecipitation with either anti-TEF-1
or anti-SRF antibodies conjugated with agarose beads. Beads were
pelleted, washed repeatedly, and analyzed by Western blot analysis. As
shown in Fig. 3, whereas an expected band
of ~54 kDa of TEF-1 was pulled down by an anti-SRF antibody, two
bands of ~67 and ~53 kDa of SRF were precipitated by an anti-TEF-1 antibody, whereas neither protein was precipitated with IgG or agarose
beads alone. This lower band of SRF is likely to be an alternatively
spliced variant of SRF that has been shown highly expressed in the
heart (57). These results strongly demonstrate an in vivo
TEF-1/SRF association.

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Fig. 2.
Characterization of in vivo
TEF-1-SRF interaction as determined by coimmunoprecipitation of
proteins from Jurkat T cells. Cells were transfected either with
pCMV.FLAG-TEF-1 encoding a FLAG-TEF-1 fusion protein or parental
vector, pCMV.FLAG. The whole cell lysate was subjected to
immunoprecipitation (IP) with either anti-FLAG or anti-SRF
antibodies conjugated agarose beads. Beads were pelleted, washed, and
subsequently analyzed by Western blot analysis using anti-FLAG or
anti-SRF antibodies. For SRF blot 5 µg of nuclear extract
(N.E.) of Jurkat T cells was used as a positive
control.
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Fig. 3.
Coimmunoprecipitation of TEF-1 and SRF from
cardiac myocytes. Neonatal rat hearts nuclear extracts (500 µg
protein) were incubated with either 0.5 µg of control mouse IgG for
TEF-1 immunoprecipitation or rabbit IgG for SRF immunoprecipitation,
together with 20 µl of protein A/G-agarose beads at 4 °C for 30 min. The pre-cleared nuclear extract (supernatant) obtained by
centrifugation was incubated with 20 µg of either anti-TEF-1 or
anti-SRF antibodies at 4 °C for 60 min for immunoprecipitations of
proteins. Absence of primary antibody in parallel reaction mixtures
served as negative controls (indicated by control IgG). Resuspended
protein A/G-agarose (20 µl) was then added to each sample and
incubated at 4 °C for 6 h, on a rotating device. Beads were
pelleted by centrifugation, washed four times in PBS, and reconstituted
in 50 µl of PBS. Proteins were removed from the beads by boiling in
Laemmli sample buffer and subsequently analyzed by Western analyses
using anti TEF-1 or anti-SRF antibodies. Samples incubated with protein
A/G agarose alone were utilized as an additional negative control
(beads).
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Myogenic bHLH proteins, myogenin and MyoD, have been shown to
heterodimerize with E-12 proteins to bind to SRF (15). Therefore, we
were interested to test whether TEF-1 also requires a cofactor to
interact with SRF. For this purpose we carried out another protein-protein interaction assay (far Western analysis) in which proteins were resolved by SDS-PAGE, transferred to NC membrane, and
then hybridized with 32P-labeled GST-TEF-1 fusion protein.
In this assay protein-protein interaction takes place only when they
are binding directly to each other without a cofactor. Previously,
using the same strategy we have shown that N-terminal region of TEF-1
binds to Max protein (35). Therefore, in this experiment we utilized
interaction of TEF-1 with Max and no interaction with GST as positive
and negative controls, respectively. As shown in Fig.
4, by this analysis also we found that
TEF-1 strongly binds to SRF even when the protein is denatured and
immobilized on the NC membrane. Together, these experiments demonstrate
a direct association between TEF-1 and SRF both in vitro and
in vivo assay conditions.

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Fig. 4.
TEF-1-SRF interaction as determined by a far
Western analysis. Fifty-nanogram amounts of different proteins, as
indicated above each lane, were resolved by SDS-PAGE,
transferred to nitrocellulose membrane, and hybridized with
32P-labeled GST-TEF-1 fusion protein. Membrane was washed
and exposed to x-ray film at 70 °C for 6 h for
autoradiography.
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Mapping Regions of SRF and TEF-1 Necessary for Their
Interaction--
To map the region of SRF required for binding to
TEF-1, we examined the ability of different in vitro
translated SRF segments to interact with bacterially expressed
GST-TEF-1 fusion protein. In vitro translation reaction was
programmed with plasmids encoding either full-length or different
deletion mutants of SRF. After translation, glutathione-agarose beads
bound with GST or GST-TEF-1 fusion protein were incubated with
translated proteins. Beads were removed, washed extensively, and
analyzed by SDS-PAGE. As shown in Fig.
5C, the
[35S]methionine-labeled full-length SRF (wild type) was
retained on beads that contained GST-TEF-1 but not on beads with GST
alone, thus further confirming a physical association between SRF and TEF-1 in in vitro conditions. Removal of C-terminal 337-508
amino acids of SRF retained the ability of the protein to bind to
GST-TEF-1. However, when the N-terminal 1-244-amino acid region that
contains MADS box of SRF was deleted, no TEF-1 binding was observed. To test whether MADS box sequences are involved in TEF-1 binding, another
SRF mutant was analyzed that contains 144-244-amino acid region
consisting of MADS box. Results showed that these sequences are
sufficient to bind to TEF-1. To further narrow down the TEF-1-binding region of SRF, we analyzed an additional SRF mutant containing amino
acids 1-204. This SRF mutant was incapable of binding to TEF-1, thus
indicating that within the MADS box amino acids from position 204-244
are obligatory for SRF/TEF-1 association. Results of this analysis are
summarized in the Fig. 5A.

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Fig. 5.
Mapping the region of SRF necessary for TEF-1
binding. A, schematic representation of different SRF
constructs analyzed and summary of their interaction ability with
GST-TEF-1. The positions of MADS box containing DNA-binding and
dimerization domains of SRF are depicted by different
shadings. B, in the rabbit reticulocyte lysate
(R.R.) pBS-SRF constructs were transcribed/translated with
[35S]methionine, and 20% input of labeled proteins was
resolved by SDS-PAGE. C, the remaining portion of labeled
SRF was divided into two equal halves and incubated with either
GST-TEF-1 or GST linked glutathione-agarose beads. Beads were pelleted
and washed, and proteins bound to beads were analyzed on SDS-PAGE in
the lanes indicated. Wt, wild-type full-length SRF.
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To define the region of TEF-1 that was necessary for SRF binding, we
analyzed several TEF-1 deletion mutants for their ability to interact
with in vitro translated SRF (Fig.
6). Each TEF-1 mutant was synthesized as
a GST recombinant protein, and only those preparations that showed more
than 50% of the expected size of GST fusion protein molecules were
used for a further study. As shown in Fig. 6C, GST-TEF-1
mutants in which C-terminal region was deleted up to 113 amino acids
retained the ability to bind to 35S-labeled SRF. Likewise,
when the 113-amino acid N-terminal region of TEF-1 was omitted
(GST-TEF-(114-430)), no binding of SRF was observed. Another
deletion mutation was generated in which N-terminal first 27 amino
acids were deleted. This GST-TEF-(27-113) mutant was also able to
retain successfully in vitro translated SRF. Thus, these
data indicated that the 27-113-amino acid segment of TEF-1 consisting
of the TEA/ATTS DNA-binding domain of TEF-1 is required for TEF-1/SRF
association. In this assay condition we utilized interaction of TEF-1
with Max and an absence of interaction with bHLH myogenic factors,
MyoD, or myogenin as a positive and negative controls, respectively
(31).

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Fig. 6.
Identification of TEF-1 interacting domain
with SRF. A, schematic diagram of different TEF-1
mutants and a summary of their observed binding activity with SRF. The
hatched box indicates the position of TEA/ATTS domain of
TEF-1. B, in the rabbit reticulocyte lysate pBS-SRF, -MyoD,
-myogenin, and -Max plasmids were transcribed and translated with
[35S]methionine. C and D, the
35S-labeled SRF (C), MyoD, or Max (D)
were incubated with GST or GST-TEF-1 fusion peptides on beads as
indicated above each lane, and proteins bound to beads were
analyzed on SDS-polyacrylamide gels.
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The TEA/ATTS DNA-binding domain of TEF-1 is located between 28 and 97 amino acids. It is predicted to consist of either three
-helices or
one
-helix and two
-sheet structures (42). To determine the role
of these regions and their potential
-helicity in SRF binding, we
generated additional TEF-1 mutants, in which amino acids were either
deleted or those with high probability of
-helicity were replaced by
either proline or glycine, having very low probability of
-helix
structure (43). Each mutant was synthesized as GST fusion protein in a
comparable amount and used to test for their ability to bind to SRF. As
shown in Fig. 7, the N-terminal 103-amino
acid region of TEF-1 was sufficient to bind to in vitro
translated 35S-labeled SRF, and changing two amino acids,
Glu and Gln, to Gly in the first putative
-helix structure had no
effect on their SRF-binding ability. In contrast, replacement of two
amino acids, Ala and Lys, to Gly in the putative 2nd
-helix and Gln
and His to Pro in the 3rd
-helix completely abolished their ability
to bind to SRF. A TEF-1 mutant with deletion of amino acids
corresponding to 3rd putative
-helix structure was also unable to
bind to SRF, even when an excess amount of protein was used (Fig.
7C). These results revealed that amino acids of the 2nd
and 3rd
-helix/
-sheet, but not 1st
-helix, of the TEA/ATTS
domain of TEF-1 mediate interaction with SRF. We also analyzed
DNA-binding ability of these TEF-1 mutants using a M-CAT
oligonucleotide as a labeled probe. As shown in Fig. 7B,
TEF-1 mutant having only 1-103-amino acid region has the ability to
bind to DNA, albeit severalfold lower than the binding ability of the
full-length TEF-1. Substitution mutation of two amino acids in the
putative 1st and 3rd configuration of the
-helices completely
abolished their DNA-binding ability (Fig. 7B). However,
replacement of two amino acids in the putative 2nd
-helix/
-sheet
structure resulted in weak but detectable DNA-binding activity,
consistent with a previous report (42). From these results we conclude
that whereas amino acids comprising the 1st
-helix and 3rd
-helix/
-sheet of TEA/AATTS domain are absolutely required for DNA
binding, the 2nd and 3rd
-helices/
-sheet portions are sufficient
to bind to SRF.

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Fig. 7.
Fine mapping of the region with in TEA/ATTS
domain of TEF-1 that binds to SRF. A, diagrammatic
representation of TEA/ATTS domain of TEF-1 containing three -helices
or one -helix and two -sheet structures. Amino acids that have
been mutated are shown with single letter symbols with their
positions in the protein. B, EMSA showing the DNA-binding
ability of different TEF-1 mutants as indicated above each
lane. C, interaction of different GST-TEF-1 mutants with
in vitro translated 35S-labeled SRF is
shown.
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Positive Cooperation between TEF/SRF Interaction--
To determine
the physiological significance of TEF-1/SRF interaction, we carried out
a transient transfection analysis with primary cultures of cardiac
myocytes or COS1 cells by using reporter plasmids containing either
394/+24-base pair promoter region of the skeletal
-actin gene
consisting of both SRE and M-CAT sites or artificial promoters
containing multiple copies of SREs or M-CAT sites. Cells were
transfected with various combinations of the reporter plasmids and
expression vectors encoding either full-length SRF or TEF-1 (Fig.
8). When an SRF expression vector was
cotransfected with the reporter plasmid, a modest increase (1.5-fold)
in activity was observed. Although the overexpression of TEF-1 tended
to suppress the activity of the reporter plasmid, with combination of
both TEF-1 and SRF expression vectors the luciferase activity was found
to be elevated by 5-6-fold. This combinatorial effect of TEF-1 and SRF
was concentration-sensitive, and maximal effect could be observed when
equal amounts of both expression plasmids were used, and raising the
concentration of one over the other had a negative effect. To see
whether this effect was mediated through binding of SRF and TEF-1 to
their cognate DNA-binding sites, we utilized a reporter plasmid in
which SRE1 sequences of Sk-
-actin gene were mutated. As shown in
Fig. 8B, mutation of SRE-1 that can no longer bind to SRF
(39) significantly reduced the synergistic effect of SRF and TEF-1.
Mutation of an adjacent YY-1-binding site that has been shown before to
be exerting a negative effect on SRE1 (17) further enhanced
trans-activation activity of SRF and TEF-1 (data not shown). When a
reporter plasmid was used in which the M-CAT site of the skeletal
-actin gene was mutated, the positive cooperative effect of
TEF-1/SRF was found to be completely abolished. These results indicated
involvement of both SRE and M-CAT sites of the Sk-
-actin gene in
this synergistic effect of TEF-1 and SRF. To confirm these results
further, we analyzed two other reporter plasmids containing either
multiple SREs or M-CAT sites. As shown in Fig. 8, neither SREs nor
M-CAT sites alone could support the synergistic effect of the SRF and TEF-1. To see that a lack of synergistic effect of SRF and TEF-1 was
not contributed by their inability to bind to each other, we also
coimmunoprecipitated both proteins from these cells. The results
indicated that SRF and TEF-1 efficiently associated with each other in
these transfections (Fig. 8C). Next we asked whether DNA
binding by SRF was necessary for a cooperative effect with TEF-1. To
address this point we examined the activity of a SRF mutant, SRF-pm1,
which is defective in DNA binding because of mutations in DNA
recognition sequences (44), but it still retains the ability to bind to
GST-TEF-1 (data not shown). As shown in Fig. 8, SRF-pm-1 plasmid alone
showed a negative effect on Sk-
-actin gene transcription, and when
combined with TEF-1 this mutant was found incapable of supporting the
synergistic effect of SRF and TEF-1 on gene regulation, even when a
large range of concentrations of SRF-pm-1 and TEF-1 plasmids was
tested. Collectively, these results indicated that both SRE and M-CAT
sites together are required for the positive cooperative effect between
TEF-1 and SRF for gene regulation.

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Fig. 8.
Positive cooperation between SRF and TEF-1
for gene activation. A, schematic representation of
different reporter plasmids used in this study. B, COS1
cells were cotransfected with 3 µg of reporter plasmid and 0.1 µg
of each expression plasmid (pCGN-SRF, pCGN-SRF-pm-1, and pCMV-TEF-1)
either alone or in combination as given below the bar
diagram. In each instance a -galactosidase expression plasmid was
included to normalize for transfection efficiency. For each reporter
plasmid luciferase activity (mean ± S.E.) is derived from 6 to 10 different transfection experiments. C, coimmunoprecipitation
of TEF-1 and SRF from COS1 cells transfected with 5xSRE-luc reporter
plasmid and expression vectors encoding TEF-1 and SRF. Nuclear extracts
(100 µg of protein) pre-cleared with rabbit IgG were incubated with
10 µg of anti-SRF antibody and protein A/G-agarose beads (20 µl)
for immunoprecipitations of proteins, as described in the legend of
Fig. 3. Beads were pelleted, washed in PBS, and boiled in 2× Laemmli
sample buffer and subsequently analyzed by Western blot analyses using
an monoclonal anti-TEF-1 antibody. Samples cleared with normal rabbit
IgG (no SRF antibody) or incubated only with protein A/G-agarose were
utilized as negative controls.
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The requirement of DNA-binding sites of TEF-1 and SRF for their
synergistic effect indicated that these two factors may co-occupy M-CAT
and/or SRE sites. To test this possibility an EMSA was performed in
which labeled M-CAT oligonucleotide was incubated with either GST-TEF-1
or GST-TEF-1 combined with in vitro translated cold SRF. As
shown in Fig. 9A, although SRF
alone was incapable of binding to M-CAT oligonucleotide, in the
presence of GST-TEF-1 it did produce a concentration-dependent
slow migrating complex and that was accompanied with disappearance of
fast migrating complexes generated by smaller fragments of GST-TEF-1
proteins (Fig. 9A, arrow), thus suggesting that SRF has the
ability to associate with TEF-1-M-CAT complex. Since in this experiment
we did not see reduction of the complex generated by the full-length GST-TEF-1, we performed another experiment to test whether full-length TEF-1 and SRF have ability to co-occupy a single M-CAT site. In a
scaled up binding reaction in vitro translated cold TEF-1
was incubated with the labeled M-CAT oligonucleotide. After completion of reaction the TEF-1-M-CAT complex was separated out from the unincorporated radioactivity by a gel shift analysis. The complex was
visualized by autoradiography, eluted from the gel by electrophoresis, and counted for the M-CAT radioactivity. In a protein binding buffer
the equal counts of M-CAT complex were then incubated with resin beads
linked either with GST-SRF, GST-MyoD, GST-myogenin, or GST for 2 h
in a cold room. The beads were separated by centrifugation, washed four
times with 1× binding buffer, and analyzed for the trapped M-CAT
oligonucleotide radioactivity with beads. As shown in Fig.
9B, the GST-SRF beads retained tremendously greater amount of the radioactivity than any other beads tested, thus indicating that
TEF-1-M-CAT complex is being retained on beads through binding to the
SRF segment of the GST-SRF resin. We also analyzed the ability of
SRFpm-1 mutant to retain the TEF-1-MCAT complex, and results were
positive (data not shown). These results provided further support that
SRF was capable of binding to M-CAT-bound TEF-1 protein, and such an
interaction may be partly important for the synergistic activation of
gene transcription. An identical experiment to test whether SRF-SRE
complex could be retained by GST-TEF-1 beads remained unsuccessful in
these assay conditions.

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Fig. 9.
Co-occupation of SRF-TEF-1 complex on a
single M-CAT site. A, EMSA was performed using M-CAT
oligonucleotide as a labeled probe and different combinations of
proteins as indicated above each lane. In the 3rd
lane 50× unlabeled M-CAT oligonucleotide was included as
competitor. Arrow indicates position of fast migrating TEF-1
complex that disappears upon inclusion of SRF in the binding reaction.
B, in a protein binding buffer the radiolabeled M-CAT
oligonucleotide was incubated with in vitro translated
TEF-1, and the unincorporated probe was separated from the DNA-protein
complex by a gel shift analysis. The radioactive M-CAT-TEF-1 complex
was eluted from the gel and incubated with beads linked either to GST,
GST-SRF, GST-MyoD, or GST-myogenin. Beads were separated, washed
extensively, and counted for the trapped M-CAT radioactivity.
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DISCUSSION |
A considerable amount of evidence has indicated an essential role
of the SRF protein in muscle cell differentiation. For instance, (i) an
SRF-related protein (SL-1) was found expressed in the presumptive heart
region of the Xenopus early-bud embryo prior to detection of
any marker of cardiac muscle differentiation (45). (ii) SRF transcripts
are found enriched in the developing myocardium during mouse
embryogenesis (7). (iii) SRF binding activity is increased almost
40-fold during avian myoblast differentiation that correlated with the
appearance of the sarcomeric
-actin transcripts (8). (iv) Ectopic
expression of SRF together with Nkx2.5 was found sufficient to induce
expression of the endogenous cardiac
-actin gene in the 10T1/2
pluripotent fibroblast (6). (v) Neutralizing the SRF activity by
micro-injection of SRF antibodies or SRF antisense oligonucleotides
prevents differentiation of skeletal myoblast (46). These studies
strongly suggest an obligate role of SRF in myogenic differentiation
and muscle gene expression. During skeletal muscle cell development SRF
has been reported to bind to DNA-binding domain of myogenic basic
helix-loop-helix factors related to MyoD (15). Since the cardiac
myogenic program develops independently of MyoD-related protein it was
postulated that SRF must be interacting with other cardiac myogenic
factors. In this study we tested this hypothesis and demonstrated that
identical to SRF/MyoD interaction in skeletal muscle cells, SRF
associates with TEF-1 in cardiac myocytes utilizing again DNA-binding
domains of both proteins, and this collaboration activates
transcription of a cardiac muscle gene. This type of collaboration can
also provide an explanation why TEF-1 null mice show the defect only in
the heart, whereas TEF-1 is expressed both in cardiac and skeletal muscle cells (34).
Several lines of evidence obtained from our protein-protein interaction
assays have shown that binding of SRF to TEF-1 occurs independently of
their DNA binding activity. For instance SRF was pulled down from
solution by GST-TEF-1 when DNA binding of either factor was not
possible, and both proteins were coimmunoprecipitated by antibodies
against either protein, and SRF/TEF-1 mutants defective in DNA binding
interacted as efficiently as their wild-type counterparts. Furthermore,
data obtained by the far Western analysis, in which radiolabeled
GST-TEF-1 was found to interact with SRF and GST-SRF even when these
proteins were denatured and immobilized on the NC membrane, indicated
that this is a direct and stable interaction between two proteins, and
secondary structures of SRF preserved in this assay are sufficient for
SRF-TEF-1 association. Results of this assay also demonstrate that
SRF/TEF-1 interaction occurs without need of any cofactor, which is in
contrast to requirement of E-12-MyoD heterodimer formation prior to
binding to SRF (15). By analyzing different deletion mutants of SRF we
have found that C-terminal half (amino acids 204-244) of the MADS box
is sufficient to bind to TEF-1. Previously, the core region of SRF
comprising MADS box has been found to be necessary for binding to
several other transcription factors including bHLH myogenic factors,
ternary complex factors, Nkx2.5, and Phox-1 (Mhox) as well as for the response to certain signal transduction pathways, thus suggesting multiple roles of this region in SRF function. The homeodomain proteins
Nkx2.5 (and perhaps Phox-1) has been shown to interact with N-terminal
subdomain of the MADS box (6). However, the region of SRF necessary for
binding to tertiary complex factors of Ets family (Elk-1) has been
mapped to the C-terminal half of MADS (amino acids 175 to 217), in
which dimerization of SRF was found to be a prerequisite for the
formation of the Elk-SRF complex (47). Previously, Shore and Sharrocks
(48) have suggested that Elk-1 interacts with SRF either with a
composite surface involving portions from both halves of the SRF dimer
or with a motif that is exposed only upon dimerization. Our data
presented in this study would indicate that dimerization of SRF was not a prerequisite for its binding to TEF-1, as we could observe TEF-1/SRF association even when SRF was denatured and immobilized on the NC
membrane. These findings could also explain why SRF binding to
TEF-1-M-CAT complex could be seen, while under identical conditions we
were unable to detect binding of TEF-1 to SRF-SRE complex. We believe
that a single SRF molecule directly binds to TEF-1 to co-occupy the
M-CAT site. However, when SRF is bound to SRE as a dimer it is
accessible to make tertiary complex with Ets members, as reported
before, but not to TEF-1. It is also possible that binding of TEF-1 to
SRF reduces its affinity to the SRE site, and thus both factors could
not be seen occupying SRE simultaneously under the conditions applied
here. Yet, in vivo they may co-occupy the SRE site with a
lower affinity and that may be important for their coordinated
trans-activation function. Recently, a reduced affinity of SRE to SRF
has been shown to facilitate atrial natriuretic factor gene
transcription in cardiac myocytes (20).
TEF-1 is a prototype member of a large family of transcription factors
containing a highly conserved TEA/ATTS DNA-binding domain. Amino acid
sequences of the TEA/ATTS domain have been predicted to form three
-helices, or one
-helix and two
-sheet configurations (26,
42). Previously, by mutagenesis, amino acids of helix1 and helix3 have
been documented to be essential for protein-DNA contact; however, the
role of the 2nd helix amino acids remained unknown (42). In the present
study, by using deletion and amino acid substitutions, we have shown
that amino acids of 2nd and 3rd
-helix/
-sheet configuration of
TEA/ATTS domain are necessary for SRF binding. Recently, certain
cellular proteins that bind to TEF-1 have been identified, and these
include bHLH-LZ protein, Max (35); TATA-binding protein (49);
poly(A)DP-ribose polymerase (50); and Drosophila vestigial
(51). However, until now no cellular protein binding directly to the
TEA/ATTS domain has been found (except this study). A viral protein,
SV40T-antigen (TAg), has been shown to bind directly to the
TEA/ATTS domain of TEF-1 and that leads to inhibition of TEF-1 DNA
binding activity, yet it activates transcription of the target viral
gene (52). However, this does not appear to be the case with TEF-1/SRF
interaction as we did not observe inhibition of TEF-1 DNA binding
activity upon SRF binding. These findings may indicate that binding of SRF to the TEA/ATTS domain of TEF-1 follows a different mechanism of
gene activation than that exerted by TEF-1/TAg interaction for
SV40 late promoter (52).
How does the TEF-1/SRF association activate gene transcription? Results
obtained from our transfection analysis have shown that both SRE and
M-CAT sites were required for a cooperative trans-activation effect of
these two factors; either site alone was not sufficient to yield
synergistic effect. This is in agreement with previous reports where a
coordination between SRE (or MEF-2 site) and M-CAT sites was shown to
be important for the tissue-specific and signal transduction-induced
expression of the troponin T (22) and skeletal
-actin genes (17, 24,
25). Several possibilities could be envisioned for the synergistic
effect of TEF-1/SRF interaction. First, binding of SRF to TEF-1 might
reduce affinity of the factor to its cognate DNA-binding site, and a
weaker and transient binding to DNA may allow more rapid
transcriptional initiation to take place. There are examples for both
SRF and TEF-1 where a lower affinity to DNA-binding site has been
implicated for the activation of target gene transcription (20, 31, 38,
53). Second, overexpression of both TEF-1 and SRF has been shown to
result in repression of gene transcription due to squelching
phenomenon, and this negative phenotype could be relieved by
overexpression of the subunits of TATA box-binding proteins (49, 54).
Thus, the negative phenotype of TEF-1 and SRF is likely to be a result of their binding to TATA box associated proteins, which might lead to
inhibition of transcription due to disruption of preinitiation complex
formation. In this context SRF/TEF-1 association might serve as a
coactivator for each other, and thus by antagonizing each other's
inhibitory effect on the basal transcription complex formation they may
lead to gene activation. Third, binding of TEF-1 to SRF might exclude
binding of negative acting factors such as Ets members, ERP/Net/Sep2
and/or YY-1, factors to SRF and thus allowing the DNA site to be
occupied by more positive acting SRF complex to activate gene
transcription (11, 55). Recently, a dominant negative isoform of SRF
(
5 SRF) that possesses MADS box sequences but lacks most of the
C-terminal portion of the protein has been shown to be expressed in
skeletal and cardiac myocytes (57). This isoform has the ability to
heterodimerize with full-length SRF and suppress its gene activation
potential. It is likely that TEF-1 through binding to the MADS box of
5SRF prevents it from binding to SRF and that this results in gene activation. Fourth, SRF has been shown to bind to other cardiac myogenic factors, Nkx2.5 and GATA-4, via interaction with N-terminal domain of the MADS box (7, 56). Thus, a possibility exists that binding
of TEF-1 to the C-terminal half of the MADS box may influence the
binding of NKx2.5 and/or GATA-4 to other half of the MADS box, and such
a Nkx2.5/GATA4-SRF-TEF1 ternary complex activates gene transcription.
This type of ternary complex could be analogous to the Phox1-SRF-Elk1
complex that has been implicated in controlling the c-fos
gene expression in proliferating cells (47). Future studies directed
toward determining exact amino acids of TEF-1 and SRF involved in their
synergistic effect should be able to make a distinction among these possibilities.
In summary, in this study we have demonstrated that SRF and TEF-1
collaborate to activate muscle gene expression and that this
cooperativity is mediated by direct protein-protein interaction between
MADS and TEA/AATS DNA-binding domains of these two heterologous family
of transcription factors. Since MADS and TEA/ATTS domains are highly
conserved such an interaction indicates that it would be a common
mechanism for these two groups of members to control diversified
biological functions, and specificity of this interaction may lie with
tissue-specific TEF-1 isoforms, other factors binding to these proteins
and/or signaling mechanisms that influence their mutual interaction.