From the Department of Molecular Biology and
Oncology, University of Texas Southwestern Medical Center at Dallas,
Dallas, Texas 75235-9148 and the § SUNY Mount Sinai School
of Medicine, New York, New York 10228
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ABSTRACT |
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Serum response factor (SRF) is a MADS box
transcription factor that controls a wide range of genes involved in
cell proliferation and differentiation. The MADS box mediates
homodimerization and binding of SRF to the consensus sequence
CC(A/T)6GG, known as a CArG box, which is found in
the control regions of numerous serum-inducible and muscle-specific
genes. Using a modified yeast one-hybrid screen to identify potential
SRF cofactors, we found that SRF interacts with the high mobility group
factor SSRP1 (structure-specific recognition protein). This
interaction, which occurs in yeast and mammalian cells, is mediated
through the MADS box of SRF and a basic region of SSRP1 encompassing
amino acids 489-542, immediately adjacent to the high mobility group
domain. SSRP1 does not bind the CArG box, but interaction of SSRP1 with
SRF dramatically increases the DNA binding activity of SRF, resulting
in synergistic transcriptional activation of native and artificial
SRF-dependent promoters. These results reveal an important
role for SSRP1 as a coregulator of SRF-dependent
transcription in mammalian cells.
Serum response factor
(SRF)1 belongs to the MADS
(MCM1, Agamous, Deficiens, SRF) box family of transcription factors
(1). The MADS box mediates homodimerization and binding of SRF to the serum response element or CArG box, CC(A/T)6GG, first
identified in the c-fos promoter (1, 2). SRF regulates a
variety of genes whose transcription can be stimulated in the absence
of new protein synthesis (reviewed in Ref. 3) and is essential for the
G1 to S phase transition of the cell cycle (4), presumably because key SRF target genes are required for this process. In addition
to replicative responses, SRF has been implicated in controlling gene
expression in postmitotic cells, particularly in muscle, and CArG boxes
are required for expression of numerous muscle-specific genes in
vitro and in vivo (6-12). Microinjection studies have
demonstrated that neutralizing SRF expression blocks differentiation of
skeletal myoblasts (5). SRF is also highly enriched in skeletal,
cardiac, and smooth muscle cells (12, 13).
Numerous studies have demonstrated that the activity of SRF is
influenced by cofactors that either interact with SRF directly or bind
DNA sequences adjacent to CArG boxes. Among the transcription factors
that influence SRF activity are Ying Yang 1 (YY1) (14), the homeodomain
proteins phox1/MHox (15, 16) and Nkx2.5 (9), NF- HMG transcription factors are the most abundant nonhistone proteins in
the nucleus. These proteins fall into three distinct classes: HMG-I,
HMG 14/17, and HMG-1/2 (22). There are three known members of the HMG-I
family. HMG-I and HMG-Y are derived from alternatively spliced
transcripts from the same gene and show indistinguishable biologic
activities and HMGI-C is derived from a separate gene (22). The HMG-I
factors bind AT-rich DNA in the minor groove through an AT-hook protein
motif (22, 23). These factors influence the DNA binding and
transcriptional activity of other transcription factors, but they lack
intrinsic transcriptional activity. The HMG-1/2 class of transcription
factors is divided into two subfamilies according to the number of HMG
domains, DNA sequence recognition, and evolutionary conservation (22).
One subfamily, which includes yeast ARS-binding factor, ABF-2, RNA polymerase II upstream binding factor, UBF, and the mitochondrial transcription factor, mTF-1, consists of proteins with multiple HMG
domains that bind to structurally modified DNA with little or no
sequence specificity (23). Members of the other subfamily, which
includes the lymphoid enhancer-binding factor, LEF-1, the sex-determining factor, SRY, the related Sox factors (23), and structure-specific recognition protein, SSRP1 (24), possess a single
HMG box and bind DNA with moderate sequence specificity. By themselves,
members of the HMG-1/2 family of factors do not activate transcription,
but are postulated to facilitate DNA binding of other transcription
factors (reviewed in Ref. 23). Specifically, HMG-1/2 factors have been
shown to enhance the transcriptional activity of Oct-1, Oct-2, and
Oct-6 (25), the homeotic protein HOXD9 (26), MLTF (27), p53 (28), and a
variety of steroid hormone receptors (29). Genetic evidence for a role
of HMG-1/2 factors in transcriptional regulation comes from gene
disruption experiments in yeast. Disruption of the yeast genes encoding
the HMG-like factors NHP-6A and -6B results in diminished expression of
a subset of genes in response to stimuli (30). This effect seems to be
transcription factor-specific since only certain genes are affected.
In the present study, using a modified yeast one-hybrid screen to
identify SRF-interacting factors, we demonstrate that the HMG-1/2
protein SSRP1 physically interacts with SRF. Transfection assays reveal
that SSRP1 enhances SRF-dependent transcription in
mammalian cells and that this is mediated by an increase in DNA binding
by SRF in the absence of detectable SSRP1-DNA interaction. These
results suggest that SSRP1 is a positive coregulatory protein involved
in modulating SRF-dependent gene expression in mammalian cells.
Yeast Interactive Screen--
A modified yeast one-hybrid screen
was performed using full-length human SRF as bait to identify potential
interacting factors. The SRF bait plasmid encoded amino acids 1-508
under control of the alcohol dehydrogenase promoter. For this screen,
we created a yeast strain, 4xCArGnear-LacZ, by integration of an
SRF-dependent lacZ reporter into the
URA3 locus. This reporter contains the lacZ gene
under control of 4 tandem copies of the promoter-proximal CArG box,
referred to as CArGnear, from the mouse SM22 promoter (6)
plus 15 flanking nucleotides on each side, in the vector pLacZi. Each
copy of CArGnear contained the sequence (+ strand): 5'-ACTTGGTGTCTTTCCCCAAATATGGAGCCTGTGTGCAGTG-3'. In
addition, this strain contains a plasmid expressing the histidine
marker under control of 4 tandem copies of CArGnear as described above.
This was integrated into the HIS 4 locus allowing for growth
on selective media upon activation of the reporter.
pADH-SRF was co-transformed into the 4xCArGnear-LacZ yeast strain with
an adult rat lung cDNA library (CLONTECH) that
contained the GAL4 transcription activation domain (TAD) fused to
random cDNA. Greater than 4 million primary colonies were screened
for positive interactions and 62 LacZ-positive yeast colonies were initially identified. From each positive colony, the activating plasmid
was rescued, transformed into Escherichia coli DH5 Plasmid Construction--
Deletions of clone 48 were made using
unique restriction sites contained within clone 48 that allowed for
in-frame fusions to the carboxyl terminus of the Gal4 activation domain
in plasmid pGAD10 (CLONTECH). flag-clone 48 was
constructed by fusing the cDNA fragment isolated in the initial
one-hybrid screen in-frame downstream of a flag epitope in vector pECE.
Myc-SSRP1 expression plasmid was provided by M. Baron.2 For plasmids used
for in vitro transcription/translation, the SSRP1
cDNA (amino acids 22-709) plus the NH2-terminal Myc
epitope was excised from vector pMT23-SSRP1 by digestion with
NotI and ligated in-frame into vector pCITE2A creating
pCITE-SSRP1. The cDNA insert of clone 48 was excised with
EcoRI and ligated in-frame into vector pCITE2A, creating
pCITE-clone 48.
Cell Culture, Transfection, and Luciferase Assays--
COS cells
were maintained in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum, 0.01% penicillin, and 0.01% streptomycin. Cells
were seeded at approximately 50% confluence in 6-well plates and grown
for 18 h before transfection. Cells were transfected using Fugene
6 as recommended by the manufacturer (Roche Molecular Biochemicals) and
harvested 24-48 h later. Typically, greater than 40% of the cells
were transfected as detected by LacZ staining (not shown).
Approximately 200 ng of luciferase reporters under control of the
following SRF-dependent promoters were used: the 322-base
pair SRF promoter (31), the 282-base pair SM22
promoter (6), and the 600-base pair Nuclear Extract Preparation, in Vitro Transcription/Translation
and Gel Mobility Shift Assays--
Nuclear extracts were prepared as
described in Ref. 31. In vitro transcription/translation was
performed as recommended by the manufacturer (Promega). SSRP1 products
were generated from pCITE-SSRP1 and pCITE-clone 48. SRF was generated
from plasmid pT7 Coimmunoprecipitation and Western Analysis--
COS cells were
transfected as described above with the following modifications. Cells
were seeded onto 10-cm plates and transfection mixtures (volumes and
amounts of DNA) were increased 10-fold. Two µg of plasmids expressing
Myc-SSRP1 (amino acids 22-709) or flag-clone 48 (amino acids 489-587)
were included in the transfections. Nuclear extracts were prepared as
described above. Approximately 500 µg of nuclear extract was diluted
to 1 ml in nondenaturing immunoprecipitation buffer (25 mM
NaP04, pH 7.5, 100 mM NaCl, 2 mM
MgCl2, 1 mM dithiothreitol, 10% glycerol,
1 × EDTA-free Complete Protease Inhibitor (Roche Molecular
Biochemicals)). Five µg of anti-SRF antisera (Santa Cruz) and 50 µl
of protein A/G beads (Santa Cruz) were added and incubated at 4 °C
on a platform rocker for 30 min. Reactions were spun at 2500 rpm for 3 min and pellets were washed three times with 500 µl of
coimmunoprecipitation buffer. After the final wash, pellets were
resuspended in 50 µl of 2 × SDS sample buffer and boiled for 5 min. Samples were spun briefly and denaturing electrophoresis
performed. Electrophoresis conditions and transfer to membrane were
performed using standard protocols (35). Western blot analyses were
performed using the ECL-PLUS detection kit (Amersham), anti-Myc (Santa
Cruz), anti-flag (Kodak), and anti-SRF (Santa Cruz) antibodies.
Quantitation was performed by densitometry of x-ray films.
Immunocytochemistry--
COS cells were transfected as described
above with the following modifications. Cells were plated onto sterile
glass coverslips before transfection. Thirty-six hours after
transfection, cells were washed with 1 × PBS, fixed in 3.7%
formaldehyde for 5 min at room temperature, washed three times in
1 × PBS, and then pre-blocked in 1 × PBS containing 2%
horse serum, 2% bovine serum albumin (fraction V), and 0.1% Nonidet
P-40 for 30 min at room temperature. Anti-Myc or anti-flag antibody was
added at dilutions of 1:200 and 1:400, respectively, in fresh pre-block
solution and incubated an additional 30 min. Cells were then washed
three times in 1 × PBS. Anti-rabbit or anti-mouse fluorescein
isothiocyanate-conjugated secondary antibody was then added at a
dilution of 1:250 for 30 min in pre-block solution and cells washed
again three times with 1 × PBS before visualization.
Identification of SSRP1 as an SRF-interacting Factor in the Yeast
Modified One-hybrid System--
Our initial goal was to identify
factors from smooth muscle cells that interact with SRF, using a rat
lung cDNA library in a yeast one-hybrid screen. Adult lung was used
as the source of the cDNA library due to its high degree of
vascularization and smooth muscle content. The SRF "bait" consisted
of the entire 508-amino acid human SRF protein. Expression of SRF was
verified by Western analysis of yeast extracts using an anti-SRF
antibody (data not shown). As a reporter, we used a lacZ
gene under control of 4 tandem copies of the proximal CArG box from the
SM22 promoter, referred to as CArGnear (6), plus 30 nucleotides of 5'- and 3'-flanking sequence. CArGnear has been shown to
bind SRF and to be essential for SM22 expression in subsets
of smooth muscle in transgenic mice
(6).3 Although the SRF bait
used in the screen contained the SRF transcription activation domain
(36), it failed to activate transcription of the lacZ
reporter on its own in yeast. The inability of full-length SRF to
activate transcription in yeast has been previously reported (37).
Screening of approximately 4 million primary yeast colonies for lacZ
expression following transformation with the rat lung cDNA library
resulted in identification of two clones, referred to as 48 and 62, that strongly activated lacZ and fulfilled all criteria for
specificity (see "Materials and Methods"). Both clones, which were
isolated from independent yeast transformations, contained cDNA
inserts encoding amino acids 489-587 of the HMG protein SSRP1. This
region encompasses eight amino acids of the acidic region, a basic
region, and approximately the first two-thirds of the HMG box (Fig.
1A).
SSRP1-SRF Interaction Is Mediated by Amino Acids 489-542 of
SSRP1--
The modified yeast one-hybrid screen indicated that a
99-amino acid portion of SSRP1, residues 489-587, physically
associated with SRF. To dissect this region further, we divided clone
48 into two parts, residues 489-542 and 543-587, and tested the
ability of each region, when fused to the GAL4 TAD, to interact with
SRF and activate the integrated CArG box-dependent reporter
genes in yeast. As seen in Fig. 1B, residues 489-542
retained the ability to interact with SRF, while residues 543-587 were
unable to interact with SRF, as measured by growth on selective media
and LacZ activity. Neither clone 48 nor the two deletion derivatives
activated expression of the reporter in yeast in the absence of SRF.
These results demonstrate that activation of CArG-dependent
transcription in yeast by clone 48-GAL-TAD is SRF-dependent
and that this interaction is mediated by amino acids 489-542 of SSRP1.
This region includes the basic region, but excludes the HMG domain.
Recently, residues 50-81 of HMG-I(Y), which include the third AT-hook
DNA-binding domain, were shown to interact with SRF (21). To determine
whether the SRF-binding domain of SSRP1 might share homology with this
region of HMG-I(Y), we compared the two domains using the MacVector
Clustal Alignment program. Indeed, there was substantial homology
between the two regions, particularly within the charged residues (Fig.
1C).
Clone 48 and Full-length SSRP1 Are Localized to the Nucleus
in Mammalian Cells--
To determine whether clone 48 and full-length
SSRP1 colocalize to the nucleus, COS cells were transfected with
expression plasmids encoding clone 48 with a flag-epitope tag or SSRP1
with a Myc-epitope tag and immunocytochemistry was performed.
Full-length SSRP1 was localized to the nucleus of COS cells, as was the
portion of SSRP1 encoded by clone 48 (Fig.
2). The nuclear localization of SSRP1 is
consistent with its ability to interact with SRF and enhance
SRF-dependent transcription (38). These data indicate that
the nuclear localization signal of SSRP1 is contained within amino
acids 489-587. This signal is probably contained within the basic
region amino-terminal to the HMG domain of clone 48 and SSRP1 (Fig.
1A).
SSRP1 Activates SRF-dependent Promoter Activity in
Mammalian Cells--
Our one-hybrid data demonstrated that SSRP1 was
capable of interacting with SRF in yeast. However, because SSRP1 was
fused to the GAL4 transactivation domain, it was unclear whether native SSRP1 might influence SRF-dependent transcription in
mammalian cells. We therefore transfected COS cells with a plasmid
expressing near full-length SSRP1 with or without SRF, and the effect
on SRF-dependent transcription was determined using a
variety of promoters linked to luciferase. Three
SRF-dependent luciferase reporters were used in the
analysis: SRF-luciferase containing the SRF promoter with
two functionally redundant CArG boxes (31), SM22-luciferase
containing 282 nucleotides of promoter sequence including CArGnear and
CArGfar (6), and
To determine if SRF-binding sites are required for
SSRP1-dependent activation of transcription, an
SRF promoter-luciferase reporter containing in-context
mutations of both CArG boxes was used. These mutations have been
demonstrated to disrupt SRF DNA binding and ablate serum-inducible
expression of the SRF promoter in fibroblasts (31). As shown
in Fig. 3A, mutation of the CArG box sequences completely
abolished SRF- and SSRP1-dependent activation. This
demonstrates that SSRP1-dependent activation is mediated by
the CArG boxes and not simply by a general up-regulation of transcription due to SSRP1 recruitment of other regulatory factors to
the promoter.
To further investigate the requirement of SRF in SSRP1-mediated
activation of transcription through the CArG box, we used a dominant
negative SRF mutant (pM1) that lacks DNA binding activity but possesses
the ability to dimerize with endogenous SRF (34). Transfection of
pM1-SRF reduced expression of all three luciferase reporters,
consistent with a primary role of SRF in regulating these promoters.
More importantly, co-transfection of SSRP1 with pM1-SRF severely
hampered the ability of SSRP1 to enhance CArG box-dependent
expression, reflecting interference with endogenous SRF. These data
demonstrate that SSRP1 is able to enhance CArG box-dependent transcription specifically through SRF and
not through other mammalian CArG box-binding factors.
We also examined the role of the MADS box of SRF in
SSRP1-dependent transcriptional activation. The MADS box of
SRF, contained in amino acids 90-222 of SRF and referred to as
SRFcore, was fused to the activation domain of VP16, creating the
chimeric protein SRF-VP16. This chimeric protein also synergized with
SSRP1 to activate SRF-dependent transcription (data not
shown). These data demonstrate that SSRP1 enhances
SRF-dependent transcription from a variety of promoters and
suggest that the MADS box-DNA binding/dimerization domain of SRF is
required to observe the effect.
SSRP1-enhanced Gene Expression Is Not a Generalized MADS
Box-dependent Effect--
Given the ability of SSRP1 to
enhance the transactivation potential of a SRFcore-VP16 chimeric
protein containing the MADS box of SRF, we considered the possibility
that a similar effect might be observed with other MADS box-containing
transcription factors. To test this, we transfected COS cells with a
MEF2-dependent reporter gene in the presence or absence of
SSRP1 and MEF2C. MEF2 contains a MADS box related to that of SRF, but
it binds a different DNA sequence (CTA(T/A)4TAG). As shown
in Fig. 3D, MEF2C activated this reporter, whereas SSRP1 had
no effect either alone or with MEF2C. This demonstrates that SSRP1 is
not simply a MADS box-dependent transcriptional activator.
SSRP1 Increases Binding of SRF to the CArG Box--
To determine
whether SSRP1 increased transcriptional activity of SRF by enhancing
SRF-DNA binding activity, we performed gel mobility shift assays with
in vitro-translated SRF and SSRP1 and a labeled probe
corresponding to the CArGnear sequence from the SM22
promoter. In the presence of SRF, a single DNA-protein complex was
observed (Fig. 4A). SSRP1 did
not show detectable binding to the probe, but it enhanced the binding
of SRF. The SRF-containing complex exhibited the same mobility in the
presence and absence of SSRP1, suggesting that SSRP1 enhanced SRF-DNA
binding activity without forming a stable complex with SRF on DNA. The
same results were observed in the presence of clone 48 protein (data
not shown).
We next examined whether SSRP1 could enhance the DNA binding activity
of SRF in vivo (Fig. 4B). Nuclear extracts from
COS cells gave rise to a DNA-protein complex with the CArGnear probe that comigrated with the complex formed with in vitro
translated SRF (lane 2 and data not shown). This complex was
supershifted in the presence of anti-SRF antibody (compare lanes
2 and 3). By transfecting COS cells with an expression
vector encoding Myc-SSRP1, we were able to assess the affects on DNA
binding activity of SRF. No binding of either SSRP1 or clone 48 protein
to the probe was observed (data not shown), consistent with the
one-hybrid data demonstrating that SSRP1 fused to the GAL4
transactivation domain was incapable of activating CArG
box-dependent transcription in the absence of SRF in yeast.
However, in the presence of Myc-SSRP1 (lanes 4-6), there
was a dramatic enhancement in SRF-DNA binding. Similar enhancement of
SRF DNA binding activity was observed in the presence of clone 48 (lanes 7-9). Consistent with the conclusions from in
vitro translation products, Myc-SSRP1 was not detected as forming
a ternary complex with SRF (lanes 4-6). These data suggest
that the enhancement of SRF-dependent transcription by SSRP1 is due, at least in part, to increased association of SRF with
its binding site via an SSRP1-dependent mechanism.
SRF and SSRP1 Physically Associate in Vivo--
Our results
demonstrated that SSRP1 can enhance the ability of SRF to interact with
the CArG box. However, we were unable to detect ternary complex
formation between SRF and SSRP1 on the CArG box nor SSRP1-DNA
interaction. To investigate whether SSRP1 could interact directly with
SRF in the absence of DNA, nuclear extracts prepared from
SSRP1-transfected COS cells were subjected to coimmunoprecipitation
using an anti-SRF antibody followed by Western blot analysis with
antibody against the epitope contained on SSRP1(Myc) and clone 48(flag)
(Fig. 5A). As shown in Fig.
5B, lane 3, SSRP1 was readily detectable in
transfected COS cells and was coimmunoprecipitated with SRF (lane
2), demonstrating a physical association between the two factors
in vivo. Although clone 48 was expressed at appreciable
levels in COS cells (Fig. 5B, lane 3), clone 48 failed to
coimmunoprecipitate with SRF. In light of the functional data
indicating that clone 48 could interact with SRF in yeast and enhance
SRF-dependent transcription in mammalian cells, the
inability to detect interaction of clone 48 and SRF in
coimmunoprecipitation assays may suggest that the interaction of these
proteins is less stable or more transient than for full-length SSRP1
and is therefore unable to be detected by coimmunoprecipitation.
To control for similar levels of SRF in each sample, immunoprecipitates
were probed with anti-SRF antibody. Interestingly, as seen in Fig.
5C, approximately 3-fold more SRF protein was detected in
SSRP1- and clone 48-transfected cells. Similar increases in SRF protein
were confirmed by Western analysis using extracts from
SSRP1-transfected cells before coimmunoprecipitation (not shown). These
coimmunoprecipitations appear to be quantitative because a second round
of immunoprecipitation failed to recover additional SRF (not shown).
These results suggest that SSRP1 is able to enhance
SRF-dependent transcription from the SRF
promoter as it exists within chromatin. This is consistent with our
transfection studies indicating that SSRP1 enhances expression of the
SRF promoter.
Our results show the HMG factor SSRP1 interacts with SRF in yeast
and mammalian cells and that SSRP1 enhances SRF-dependent transcription from a variety of SRF-dependent promoters,
including the SRF promoter itself. The increased
transcriptional activity of SRF in the presence of SSRP1 is accompanied
by an increase in SRF-DNA binding activity in the absence of stable
SSRP1 interaction with the CArG box or SRF bound to DNA. The enhanced
transcriptional activity of SRF in the presence of SSRP1 is not a
general property of MADS box proteins because the myogenic MADS box
factor MEF2C was not responsive to SSRP1. SSRP1 was also unable to
activate transcription in yeast from a CArG box-dependent
lacZ reporter gene. This suggests that SSRP1 cannot
cooperate with MCM1, the yeast SRF-like MADS box factor. In addition,
Irx1, a yeast SSRP1 protein that confers sensitivity to cisplatin (40),
does not interact with SRF, as evidenced by a lack of autonomous
SRF-dependent activation of the reporter. This suggests that SSRP1-SRF
interaction is highly specific.
SSRP1 is an HMG box protein identified initially by its ability to
recognize and bind cisplatin-modified DNA (39). The functions of
SSRP1-like proteins are only beginning to be elucidated, however, the
evolutionary conservation of these proteins from plants to humans
suggests a role in fundamental cellular processes. In this regard,
recent studies have implicated SSRP1 in cell proliferation. Decreased
expression of SSRP1 by antisense RNA impairs growth of fibroblasts in
culture (41). SSRP1 has also been demonstrated to be up-regulated in
proliferating fetal kidney cells and iron-nitrilotriacetate-induced renal carcinomas (42), but is not readily detectable in normal adult kidney.
Based on its ability to recognize and bind cisplatin-modified DNA,
SSRP1 was postulated to bind specific DNA structures targeting them for
repair or altering their structure and thereby affecting DNA
replication and transcription (39). SSRP1 also binds the positive
regulatory element, PREII, from the human embryonic-like Given the ability of SSRP1 to recognize and manipulate DNA structure
and its lack of a transactivation domain, two general models can be
envisioned whereby SSRP1 enhances SRF-dependent transcription. One possibility is that the CArG box is a combination of
the binding site for SRF and a structure recognized by SSRP1. In this
case, SSRP1 might act as a molecular chaperone, transiently manipulating DNA structure and facilitating DNA binding of SRF. According to this model, SSRP1 would dissociate upon binding of SRF to
DNA and would not be an essential component of the final complex.
However, because SSRP1 does not bind detectably to the CArG box
sequence and can be co-precipitated with SRF in the absence of DNA, we
feel it is unlikely that the potentiation of SRF activity by SSRP1
requires interaction of SSRP1 with DNA. A second possibility is
association of SRF and SSRP1 in the absence of DNA results in a change
in conformation of SRF that increases its DNA binding activity, with a
consequent increase in transcriptional potency. Again, SSRP1 would not
be an essential component of the final complex. Based on our results,
we believe this second model best explains the mechanism for
SSRP1-mediated enhancement of SRF-dependent transcription.
The ability of SSRP1 to increase DNA binding by SRF without forming a
stable complex with SRF on DNA is reminiscent of the activity of the
homeodomain protein phox/MHox (15). However, the possibility exists
that SSRP1 physically associates with the CArG box but is not detected
under the conditions of the assay system used.
Whether the observed stimulatory effect of SSRP1 on
SRF-dependent transcription is entirely due to increased
DNA binding by SRF remains unknown. Like other HMG proteins (22, 23),
SSRP1 may also facilitate the formation of enhancesome complexes by altering DNA conformation, juxtaposing components of the
transcriptional machinery in a more favorable orientation for
transcription. Consistent with this possibility, the yeast SSRP1-like
factor Pob3 has been implicated in forming a protein complex with
Cdc68, manipulating chromatin structure, thereby enhancing
transcription (43). It would be interesting to know if SSRP1 increases
SRF association not only to DNA but to components of the
transcriptional machinery previously shown to associate with SRF
(44).
In SSRP1-transfected cells, we observed an increase in SRF expression.
Given the ability of SSRP1 to increase expression of the SRF
promoter, these results suggest that enhancement of
SRF-dependent gene expression by SSRP1 can occur in the
context of chromatin, as well as from reporter genes. An alternative
explanation for the increase in SRF protein in SSRP1 cells would be an
increase in stability of SRF in the presence of SSRP1. However, we do
not favor this interpretation since SSRP1 enhances SRF promoter
activity likely leading to increased SRF protein expression.
Recently, HMG-I(Y) was shown to interact with SRF and to potentiate
SRF-dependent transcription (21). Although HMG-I(Y) interacts with the CArG box, enhancement or SRF-dependent
transcription does not require interaction of HMG-I(Y) with DNA.
Deletion mapping of HMG-I(Y) showed that amino acids 50-81, which
encompass the third A/T-hook DNA-binding domain, mediate interaction
with SRF. Intriguingly, the region of SSRP1 (amino acids 489-542) that
interacts with SRF shows significant amino acid homology to this region of HMG-I(Y), suggesting that the interaction of these two HMG factors
with SRF may be mediated by a common functional domain. However, it
should be pointed out that although the SRF-interacting region of
HMG-I(Y) maps to the AT-hook domain, SSRP1 does not contain an
AT-hook.
SRF integrates growth factor signals and coordinates programs for
muscle gene expression. These diverse roles in gene regulation are
dependent on interactions with a wide range of
signal-dependent and cell-specific cofactors. While our
initial goal was to identify smooth muscle-specific cofactors for SRF,
SSRP1 is expressed in numerous cell types in addition to smooth
muscle4,5
and is therefore unlikely to confer smooth muscle specificity to SRF.
Rather, given the ubiquitous expression of SSRP1, we believe we have
identified a more general mechanism for potentiation of SRF-dependent transcription. The potential involvement of
SSRP1 in the control of SRF-dependent genes during
proliferation and differentiation is currently being investigated.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B (17), ATF6 (18),
myogenic basic helix loop helix factors (19), and members of the ETS
family of proto-oncogene products (reviewed in Ref. 20). Most recently,
two members of the HMG-I family of nonhistone nuclear proteins,
HMG-I(Y) and HMG-I(C), have also been demonstrated to associate with
SRF (21). Physical association of SRF and HMG-I proteins leads to
enhanced SRF-DNA association and increased SRF-dependent
transcription in yeast and Drosophila cells.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
plasmid DNA was isolated and the cDNA insert sequenced. Clones
containing inserts in the antisense orientation or out-of-frame with
the GAL4 TAD were discarded. The 2 remaining clones were retransformed back into yeast to verify the interaction with SRF. Both clones (48 and
62) encoded amino acids 489-587 of SSRP1.
-myosin heavy chain
promoter (32). A luciferase reporter under control of three tandem
copies of the muscle creatine kinase MEF2 site and the E1b
promoter (33) was also used. Transactivation assays also included 100 ng of pCGN-SRF or pM1 (34), 200 ng of near full-length SSRP1 (amino
acids 22-709) or flag-clone 48 (amino acids 489-587), 100 ng of
pcDNA-MEF2C (33), and 50 ng of pHsp68-LacZ were used in the
transfections where indicated. In transfections not receiving the full
complement of expression plasmids, total plasmid was adjusted to 1 µg
with parental expression plasmid lacking insert. Luciferase assays were
performed as described by the manufacturer (Promega). In each instance,
luciferase data were normalized to
-galactosidase activity to
correct for variations in transfection efficiency.
ATG (2). Gel mobility shift assays were performed
using 32P-labeled complementary oligonucleotides spanning
nucleotides
165 to
125 of the SM22 promoter. The
sequence of the top strand was 5'-
ACTTGGTGTCTTTCCCCAAATATGGAGCCTGTGTGCAGTG-3'. In gel
mobility shift reactions using nuclear extract, approximately 5 µg of
total protein was used. Approximately 5 µl of in vitro
translated SSRP1 or clone 48 were used in the binding reaction with or
without increasing concentrations of in vitro translated
SRF. The gel mobility shift buffers, binding conditions, and
electrophoresis conditions are described elsewhere (31).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Interaction of clone 48 with SRF in
yeast. A, schematic diagram of full-length human SSRP1.
The SSRP1 protein is 709 amino acids and contains several regions of
clustered charged residues. The acidic region spans amino acids
440-496. The HMG box spans amino acids 539-614 and is flanked by two
short basic regions. B, clone 48 and amino- and
carboxyl-terminal deletions fused to the GAL4 transactivation domain
were expressed in yeast with SRF to determine the region of clone 48 that mediates the interaction with SRF. Residues 489-542 interact with
SRF as detected by growth on selective media (minus histidine, minus
leucine, minus tryptophan ( HLT)) and activation of the
lacZ reporter gene, whereas residues 543-587 do not
interact with SRF. No growth on selective media nor activation of the
lacZ reporter was observed in the absence of SRF.
C, comparison of the amino acids of SSRP1 and HMG-I(Y) shown
to interact with SRF. Significant homology is observed between the two
regions of SSRP1 and HMG-I(Y), particularly in the charged
residues.
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Fig. 2.
Nuclear localization of clone 48 and
SSRP1. COS cells were transfected with a plasmid expressing Myc
epitope-tagged near full-length SSRP1, flag epitope-tagged clone 48, or
the parent expression plasmid with no insert. Immunocytochemistry was
performed as described under "Materials and Methods." Both SSRP1
and clone 48 are localized to the nucleus.
-myosin heavy chain-luciferase containing
approximately 600 nucleotides of promoter sequence with a single CArG
box shown to bind SRF (32). As shown in Fig. 3, SSRP1 weakly activated these three
promoters. Similar results were obtained with clone 48 (not shown). SRF
was also able to activate transcription from all three promoters in COS
cells. However, co-transfection of SSRP1 and SRF resulted in
synergistic activation of all three promoters, suggesting a functional
role for the SSRP1-SRF interaction identified in yeast. Since SSRP1 does not contain a transcription activation domain (24), the ability of
SSRP1 to enhance activity of these promoters in the absence of
exogenous SRF is likely to reflect synergy with endogenous SRF.
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Fig. 3.
SSRP1 activation of SRF-dependent
transcription. COS cells were transiently transfected with a
luciferase reporter gene linked to the: A, wild-type or
mutant SRF promoter; B, -myosin heavy chain
promoter; C, SM22 promoter; or D, 3 tandom copies of the MEF2-binding site upstream of the minimal
E1b promoter and expression vectors encoding SRF, dominant
negative SRF (pM1), SSRP1, or MEF2C as indicated. In each instance,
hsp68-lacZ was included in the transfection to normalize for
transfection efficiency. Twenty-four to fourty-eight hours after
transfection, cells were harvested and luciferase activity and
-galactosidase activity were determined. SRF and SSRP1 synergize to
activate expression of all SRF-dependent reporters tested
(A-C). This synergy was not observed using the mutant
SRF promoter construct containing CArG box mutations
(A) nor with dominant negative SRF expression plasmid (pM1).
SSRP1 failed to activate MEF2C-dependent transcription
(D). Experiments were repeated at least three times in
duplicate. Experiments involving MEF2C were done two times in
duplicate.
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Fig. 4.
SSRP1 enhances SRF-CArG box interaction.
A, gel mobility shift assays were performed using in
vitro translated SRF and Myc-SSRP1. The 32P-labeled
oligonucleotide probe used corresponds to nucleotides 165 to
125 of
the SM22 promoter. This region contains the proximal CArG
box (CArGnear) shown to bind SRF (6). Lane 1, unprogrammed
reticulocyte lysate; lane 2, 5 µl of in vitro
translated Myc-SSRP1; lanes 3-5, increasing
concentrations of in vitro translated SRF; lanes
6-8, increasing concentrations of in vitro translated
SRF in the presence of 5 µl of in vitro translated
Myc-SSRP1. In each reaction, the total volume of reticulocyte lysate
was adjusted to 6 µl total with unprogrammed lysate. Myc-SSRP1 does
not bind the CArG box (lane 2) but greatly enhances SRF-CArG
box interaction (3.6-fold ± 0.9) (compare lanes 3-5
with lanes 6-8). Similar results were obtained with clone
48. Similar results were obtained in four independent experiments using
freshly translated protein. B, gel mobility shift assays
were performed using untransfected nuclear extracts,
Myc-SSRP1-transfected nuclear extracts, and flag-clone 48-transfected
nuclear extracts. The 32P-labeled oligonucleotide probe
used corresponds to nucleotides
165 to
125 of the SM22
promoter. Lane 1, probe alone; lane 2,
untransfected nuclear extract; lane 3, untransfected nuclear
extract plus anti-SRF antibody; lane 4,
Myc-SSRP1-transfected nuclear extract; lanes 5 and
6, Myc-SSRP1-transfected nuclear extract plus anti-SRF
antibody and anti-Myc antibody, respectively; lane 7,
flag-clone 48-transfected nuclear extract; lanes 8 and
9, flag-clone 48-transfected nuclear extract plus anti-SRF
antibody and anti-flag antibody, respectively. SSRP1 and clone 48 enhance SRF-DNA interaction (11.3-fold ± 3.6). Only the region of
the gel containing SRF-DNA complexes is shown. Neither SSRP1 nor clone
48 bound the oligonucleotide probe used. Similar results were obtained
in three independent experiments.
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Fig. 5.
SSRP1 coimmunoprecipitates with SRF from
nuclear extracts. A, diagram of the experimental
procedure used in this analysis. COS cells were transiently transfected
with plasmids expressing flag epitope-tagged clone 48 or Myc
epitope-tagged SSRP1. Cells were harvested 24 h later and nuclear
extracts were prepared. SRF was immunoprecipitated from approximately
500 µg of nuclear protein, separated by SDS-polyacrylamide gel
electrophoresis and analyzed by Western blot with the indicated
antibodies. B, approximately 20% of the
nonimmunoprecipitated nuclear extract and the immunoprecipitates were
electrophoresed on a denaturing gel, transferred to membrane, and
subjected to Western analysis using anti-Myc or anti-flag antibody.
Lane 1, nontransfected cells; lane 2, Myc-SSRP1
and flag-clone 48-transfected cells; lane 3, 20% of the
total protein input used in the coimmunoprecipitation. Both clone 48 and SSRP1 are expressed at appreciable levels in COS cells (lane
3). Near full-length SSRP1, but not clone 48, coimmunoprecipitates
with SRF. C, Western analysis was performed on SRF
immunoprecipitates using the anti-SRF antibody. Lane 1,
untransfected cells; lane 2, Myc-SSRP1; lane 3,
flag-clone 48 transfected cells. In SSRP1- and clone 48-transfected
cells, SRF protein levels are increased indicating that SSRP1 can
enhance SRF-dependent expression in the context of
chromatin structure (2.5-fold ± 0.5 for flag-clone 48 and
3-fold ± 1.1 for Myc-SSRP1, respectively). Similar results were
obtained in three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-globin
gene (24), resulting in an increase in transcription. However, SSRP1
does not contain a transcriptional activation domain (24). Thus, it has
been proposed that SSRP1, like other HMG proteins, may play an
architectural role in the control of gene expression by coordinating
the assembly of multiprotein transcriptional complexes.
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ACKNOWLEDGEMENTS |
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We thank the Olson lab for critical input during the course of this work. We also thank M. Baron and P. Hayes for unpublished reagents and Robert Schwartz for SRF mutant pM1.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health, American Heart Association, and Muscular Dystrophy Association (to E. N. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 214-648-1187; Fax: 214-648-1196; E-mail: eolson{at}hamon.swmed.edu.
2 J. A. Spencer, M. H. Baron, and E. N. Olson, unpublished data.
3 L. Li and E. Olson, unpublished observations.
4 J. Spencer and E. Olson, unpublished data.
5 P. Hayes and M. Baron, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: SRF, serum response factor; HMG, high mobility group; TAD, transcription activation domain; PBS, phosphate-buffered saline.
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