From the Department of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania 19104 and the
¶ Department of Medicine, University of Chicago,
Chicago, Illinois 60637
Received for publication, January 23, 2001, and in revised form, February 7, 2001
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ABSTRACT |
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Serum response factor (SRF) plays an important
role in regulating smooth muscle cell (SMC) development and
differentiation. To understand the molecular mechanisms underlying the
activity of SRF in SMCs, the two CArG box-containing elements in the
arterial SMC-specific SM22 The unique contractile properties of smooth muscle cells
(SMCs)1 distinguish this
muscle cell lineage from cardiac and skeletal myocytes (for review, see
Refs. 1-3). A distinguishing feature of the SMC lineage is its
capacity to proliferate and modulate its phenotype during postnatal
development (4, 5). This presumably evolved to facilitate adaptive and
reparative processes such as those that occur in response to arterial
injury. However, the capacity of SMCs to modulate their phenotype has
also been implicated in the pathogenesis of atherosclerosis and
bronchial asthma (6, 7). Ultimately, SMC phenotype is dependent upon the expression of genes encoding SMC-specific contractile proteins, cell surface receptors, and intracellular enzymes. In cardiac and
skeletal myocytes, gene expression is regulated primarily at the level
of transcription and skeletal and cardiac muscle-specific and
-restricted transcription factors have been identified (8-10). In
contrast, relatively little is currently understood about the transcriptional programs that regulate SMC development and
differentiation and SMC-specific transcription factors have not as yet
been identified.
Accumulating evidence suggests that the MADS box transcription factor
SRF plays an important role in regulating SMC specification and
differentiation (for review, see Ref. 3). SRF is a 508-amino acid
protein that binds to the serum response element (SRE), or CArG box,
corresponding to the consensus nucleotide sequence
CC(AT)6GG (11). Five alternatively spliced isoforms of SRF
have been identified; at least one of which acts in a dominant negative
fashion and represses SRF-dependent transcription (12, 13).
SRF binds DNA and physically associates with other transcription
factors through its conserved MADS box domain (14, 15). Functionally important SRF-binding sites were identified originally in
cis-acting regulatory elements controlling expression of
growth responsive (11, 16) and striated muscle-specific genes (17-20).
Subsequently, functionally important CArG boxes have been identified in
multiple SMC-restricted transcriptional regulatory elements including
the SM22 Because of it SMC lineage-restricted pattern of expression, our group,
and others, have utilized the murine SM22 In the studies described in this report, we show that SRF binding is
required for activity of the SM22 Plasmids--
All transgenic vectors were prepared in the
modified BluescriptII KS (Stratagene) plasmid containing the bacterial
LacZ gene subcloned in the HindIII site of the
AscI-flanked polylinker described previously (21). The
p-441SM22.LacZ transgenic vector in which LacZ is placed
under the transcriptional control of the 441-bp mouse SM22 Transgenic Mice--
Transgenic mice harboring the
-441SM22.LacZ, -441SM22µSME-1.LacZ, -441SM22µSME-4.LacZ,
-441SM22 µCArG.LacZ, -441SM22 Electrophoretic Mobility Shift Assays (EMSAs)--
Primary rat
aortic SMCs, A7r5 SMCs, NIH3T3 fibroblasts, HepG2 cells, HeLa cells,
C2C12 myoblasts, and myotubes and EL4 T cells were grown in culture,
harvested, and nuclear extracts prepared as described previously (21,
29). EMSAs were performed in 0.25 × TBE (1 × TBE is 100 mM Tris, 100 mM boric acid, and 2 mM EDTA) as described previously (21). The following
complementary oligonucleotides were synthesized with BamHI
and BglII overhanging ends: SME-1
(5'-CAAGGAAGGGTTTCAGGGTCCTGCCCATAAAAGGTTTTTCCCGGCCGC-3'); SME-4 (5'-CTCCAACTTGGTGTCTTTCCCCAAATATGGAGCCTGTGTGGAGTG);
c-fos (5'-CTTACACAGGATGTCCATATTAGGACATCTGCG).
For cold competition experiments, 10 to 200 ng of unlabeled
competitor oligonucleotide were included in the binding reaction mixture. For antibody supershift experiments, 1 µl of rabbit
preimmune affinity purified IgG (Santa Cruz), UV Cross-linking Analyses--
EMSA reactions performed with
32P-labeled deoxybromouridine-substituted SME-4
oligonucleotide probes (bp An Intact CArG Box Is Required for Activity of the SM22
To determine whether SME-1 and SME-4 mediate distinct or redundant
functions, site-directed mutations in SME-1 and SME-4 (Fig. 1,
upper panel), respectively, were generated within the
context of the intact 441-bp SM22 SME-1 and SME-4 Are Necessary and Sufficient to Restrict Transgene
Expression to Arterial SMCs--
To determine whether SME-1 is
necessary and sufficient to restrict transgene expression to arterial
SMCs, SM22.SME-1.LacZ embryos were generated containing the
LacZ reporter gene under the transcriptional control of the
minimal SM22 CArG Box Specificity Restricts Gene Expression to Arterial
SMCs--
Functionally important CArG boxes have been identified in
transcriptional regulatory elements controlling expression of
growth-related genes, most notably c-fos (16). To determine
whether the c-fos SRE (Fig. 2, upper panel) could
be substituted for the two CArG boxes in the SM22 Nucleotides Flanking the SME-4 CArG Box Are Required for
Transcriptional Activity in Arterial SMCs--
To determine whether
the SME-4 CArG box alone is necessary and sufficient to restrict
transgene expression to arterial SMCs, SM22.SME4.CArG.LacZ transgenic
mice were generated containing a transgene encoding LacZ
under the transcriptional control of four copies of the SME-4 CArG box
linked to the minimal SM22 Characterization of Nuclear Protein Complexes That Bind to SME-4
and c-fos SRE--
To determine whether SME-4 and c-fos SRE
differentially bind nuclear protein complexes expressed in SMCs, a
series of EMSAs was performed. SME-4 binds four specific nuclear
protein complexes, designated A-D, each of which is competed by
unlabeled SME-4 competitor oligonucleotide (Fig.
4A, lanes 3-5). Of note,
several nonspecific binding activities were also observed that were not
cross-competed with high concentrations of unlabeled SME-4
oligonucleotide. In addition, complexes A-D were efficiently competed
by increasing concentrations of unlabeled c-fos SRE
oligonucleotide (Fig. 4A, lanes 6-8). Each of these nuclear
protein complexes was also observed in EMSAs performed with nuclear
extracts prepared from primary rat aortic SMCs, NIH3T3 cells, C2C12
myoblasts, and myotubes, EL4 T cells, HepG2 cells, HeLa cells, and
C3H10T1/2 fibroblasts (data not shown). Antibody supershift experiments
revealed that complex A contains SRF or an antigenically-related
protein (Fig. 4A, lane 10, arrow), while complexes C and D
contain YY1 (Fig. 4A, lane 11, arrows). To determine whether
Ets family members and/or ternary complex factors (TCFs) expressed in
SMCs bind to the radiolabeled SME-4 oligonucleotide (and/or SRF), the
binding reactions were preincubated with polyclonal antibodies that
recognize SAP-1A, Elk-1, and Ets-1, respectively. However, none of the
nuclear protein complexes, including SRF-containing complex A, were
supershifted or abolished (Fig. 4A, lanes 12-14).
To characterize SMC nuclear protein complexes that bind to the
c-fos SRE, an EMSA was performed with a radiolabeled
oligonucleotide corresponding to the c-fos SRE and nuclear
extracts prepared from A7r5 SMCs. The c-fos SRE also bound
specifically to four nuclear protein complexes, designated A-D, each of
which is competed with increasing concentrations of unlabeled
competitor c-fos SRE oligonucleotide (Fig. 4B, lanes
2-5). Once again, several nonspecific binding activities were
identified that were not cross-competed by unlabeled c-fos
SRE oligonucleotide. In addition, complexes A-C were competed with
unlabeled SME-4 (Fig. 4B, lanes 6-8, and data not shown). EMSAs performed with the radiolabeled c-fos SRE probe and
increasing concentrations of unlabeled competitor c-fos SRE
and SME-4 oligonucleotide (5, 10, 25, 50, and 100 ng) revealed that the
c-fos oligonucleotide more efficiently competes for SRF
binding activity (data not shown). Antibody supershift experiments
demonstrate that the low mobility complex A contains SRF (Fig.
4B, lane 10, arrow), while complex B contains YY1 (Fig.
4B, lane 11, arrow). Despite the fact that the
c-fos SRE is flanked by a consensus TCF-binding site (CAGGA) (see Fig. 2, upper panel), none of the nuclear protein
complexes were supershifted or abolished when the binding reactions
were preincubated with SAP-1A, Elk-1, and Ets-1 antisera (Fig.
4B, lanes 11-14). Taken together, these data confirm that
SME-4 and the c-fos SRE cross-compete for binding of SRF and
YY1, although their relative affinity SRF binding to the
c-fos SRE and SME-4 vary. In addition, SME-4 and the
c-fos SRE bind at least two unidentified nuclear protein
complexes that are also expressed in other cell lineages.
Biochemical Characterization of Nuclear Protein Complexes That Bind
Directly to SME-4--
As discussed above, EMSAs failed to reveal an
SMC-specific nuclear protein complex that binds to SME-4. However, an
inherent limitation of EMSAs is that the band shifts observed may
contain a single nuclear protein or a multiprotein complex. Therefore, to biochemically characterize each nuclear protein that binds directly
to SME-4, with particular emphasis on identification of SMC-specific
nuclear proteins, UV cross-linking analyses were performed with a
deoxybromouridine-substituted SME-4 oligonucleotide and nuclear
extracts prepared from primary rat aortic SMCs, A7r5 SMCs, and multiple
other cell lines. As shown in Fig. 5,
SME-4 bound six nuclear proteins ranging in size from 40 to 130 kDa that are expressed in arterial SMCs. As expected, a 68-kDa band (arrow), corresponding to the size of SRF was observed in
all lanes. In addition, a 44.5-kDa band (arrow)
corresponding to the expected size YY1 was identified. Moreover, 130-, 80-, 56-, and 40-kDa proteins were also identified. p80 and p56
(open arrowheads) were enriched in SMC-nuclear extracts
(compare lanes 1 and 2 versus lanes 3-7). In addition, several faint bands of very low
molecular weight were detected in A7r5 smooth muscle cells and in
several of the non-SMC lines. These data demonstrate that in addition to SRF and YY1 at least four additional nuclear proteins, two of which
are enriched in SMCs, bind to SME-4.
Accumulating evidence suggests that SRF plays a critical role in
regulating SMC specification, differentiation, and phenotype (21-28).
However, the molecular basis underlying activity of SRF in SMCs remains
to be elucidated. Mice harboring targeted mutations in SRF die at the
onset of gastrulation (E7.5) and do not form detectable mesoderm
precluding assessment of the function of SRF in smooth muscle cells
(34). SRF is a member of the MADS box family of transcription factors
which have evolved to control cell fate decisions and regulate cellular
differentiation (for review, see Ref. 35). The pattern of SRF
expression in the developing embryo remains controversial. Although
originally characterized as a ubiquitously expressed gene product (36),
detailed analyses of avian and murine embryos revealed that SRF is
preferentially expressed in the developing neural tube, heart, skeletal
muscle, and smooth muscle (37-39). Consistent with this finding, the
SRF promoter activity is activated in a muscle-restricted fashion (39).
However, the pattern of SRF expression alone cannot explain how
SMC-restricted genes are activated and repressed in the developing embryo and in response to injury. SMC-restricted genes are expressed in
a temporally coordinated pattern in the developing embryo that is
distinct from the pattern of SRF expression (for review, see Ref. 2).
Epicardial cells derived from the proepicardial organ expressing SRF do
not differentiate into SMCs and express genes encoding SMC markers
until they undergo mesenchymal transformation (28). Given the
plasticity that distinguishes the SMC lineage, it is not surprising
that molecular mechanism(s) have evolved to precisely modulate activity
of SRF in SMCs.
Our group and others reported that CArG box-containing transcriptional
regulatory elements restrict gene expression to tissue-restricted subsets of smooth muscle cells (21-25, 40). In fact, every
SMC-specific transcriptional regulatory element characterized to date
contains one, and usually several, CArG boxes (21-27). Most
SMC-specific genes, including SM22 Mutational and deletion analyses of the SM22 How then does a transcription factor, such as SRF, restrict gene
expression to SMCs? In the developing embryo, SMC-restricted genes are
expressed in distinct temporal and spatial patterns (for review, see
Ref. 2). In the mouse embryo, SM22 Alternatively, SMC lineage-restricted transcription factors may not
exist and SRF-dependent gene expression could be regulated via intracellular signaling pathways that activate or repress SRF
and/or by the expression of alternative SRF isoforms. Consistent with
this model, SRF-dependent genes encoding vascular
SMC-specific contractile proteins are down-regulated in response to
arterial wall injury (21, 22, 24, 25), concomitant with activation of
intracellular signaling pathways which repress activity of SRF
(44-47). Alternatively, alternative splicing and generation of an
alternative SRF isoform that antagonizes activity of the native
(full-length) SRF protein (12, 13) may repress
SRF-dependent transcription of SMC-restricted genes.
Consistent with this hypothesis, the alternatively spliced SRF promoter, SME-1 and SME-4, were
functionally and biochemically characterized. Mutations that abolish
binding of SRF to the SM22
promoter totally abolish promoter
activity in transgenic mice. Moreover, a multimerized copy of either
SME-1 or SME-4 subcloned 5' of the minimal SM22
promoter (base
pairs
90 to +41) is necessary and sufficient to restrict
transgene expression to arterial SMCs in transgenic mice. In contrast,
a multimerized copy of the c-fos SRE is totally inactive in
arterial SMCs and substitution of the c-fos SRE for the
CArG motifs within the SM22
promoter inactivates the 441-base pair
SM22
promoter in transgenic mice. Deletion analysis revealed that
the SME-4 CArG box alone is insufficient to activate transcription in
SMCs and additional 5'-flanking nucleotides are required. Nuclear
protein binding assays revealed that SME-4 binds SRF, YY1, and four
additional SMC nuclear proteins. Taken together, these data demonstrate
that binding of SRF to specific CArG boxes is necessary, but not
sufficient, to restrict transgene expression to SMCs in
vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
promoter (21-23), the smooth muscle myosin heavy chain
(SM-MyHC) promoter and intragenic enhancer (24), the SM
-actin
promoter and intragenic enhancer (25), the telokin promoter (26), and the
-enteric actin promoter (27). In addition, SRF has been shown to
play an important role in specification of SMCs from undifferentiated
mesenchyme (28).
promoter as a model system
to examine the transcriptional programs that regulate SMC development
and differentiation (21-23, 29-31). SM22
is a single copy gene
encoding a 22-kDa protein with two potential EF-hand calcium-binding
domains, an actin-binding domain, and one calponin-repeat homology
domain (29, 32). Preliminary characterization of the SM22
promoter
revealed six nuclear protein-binding sites, designated smooth muscle
elements (SME) 1-6, respectively (21). Two of these
cis-acting elements, SME-1 and SME-4, contain consensus CArG
boxes and bind specifically to SRF (21). In transgenic mice, the
SM22
promoter restricts expression of a transgene encoding LacZ to arterial smooth muscle cells, the myotomal component
of the somites, and the bulbocordis region of the embryonic heart (21-23). However, in contrast to the endogenous SM22
protein, SM22
promoter-driven transgene expression is not observed in visceral or venous SMCs (21-23).
promoter in arterial SMCs, but
that SME-1 and SME-4 function in a partially redundant fashion in
vivo. Most importantly, we demonstrate that a multimerized copy of
SME-1 (bp
279 to
256) or SME-4 (bp
171 to
135) is necessary and
sufficient to confer arterial SMC-restricted gene expression in
transgenic mice, while a multimerized copy the c-fos SRE is
not. Consistent with these data, substitution of the c-fos SRE (16) for the two CArG boxes in the SM22
promoter totally inactivates the SM22
promoter in transgenic mice. Deletion analyses revealed that the SME-4 CArG box and additional 5'-flanking nucleotides are required for transcriptional activity in arterial SMCs. Nuclear protein binding assays demonstrated that SME-4 binds six nuclear proteins, two of which are enriched in SMC nuclear extracts. Taken together, these data demonstrate that binding of SRF to specific CArG
boxes is necessary, but not sufficient, to restrict gene expression to
tissue-restricted subsets of SMCs.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
promoter
was described previously (21). p-441SM22 µSME-1.LacZ is identical to
p-441SM22.LacZ except three nucleotides (underlined) in SME-1
(5'-TCCTGCCCATAGATCTTTTTTCCC-3') were mutated to abolish
SRF and Sp1 binding activity (21). The
441SM22µSME-4.LacZ transgenic plasmid is identical to p-441SM22.LacZ except two
nucleotides in SME-4
(5'-GCTCCAACTTGGTGTCTTTCCCCGGATATGGAGCCT-3') were mutated to abolish SRF binding activity (21). p-441SM22µCArG.LacZ is identical to p-441SM22.LacZ except it contains the mutations in both
SME-1 and SME-4 described above. p-441SM22
c-fos.LacZ is identical to
p-441SM22.LacZ except the nucleotide sequence of the c-fos
SRE (5'-CCATATTAGG-3') has been substituted for the CArG boxes within
SME-1 and SME-4, respectively. The pSM22.LacZ plasmid encodes
LacZ under the transcriptional control of the 90-bp mouse
SM22
promoter (21). pSM22.SME4.LacZ contains four copies of SME-4
(bp
171 to
136) subcloned 5' of the SM22
promoter into
SmaI-digested pSM22.LacZ. pSM22.SME1.LacZ contains three copies of SME-1 (bp
279 to
256) subcloned into
SmaI-digested pSM22.lacZ. pSM22.c-fos.LacZ contains three
copies of the c-fos SRE and its 5'- and 3'-flanking
sequences
(5'-CCCCCCCTTACACAGGATGTCCATATTAGGACATCTGCGTCACAGGTTTCCACGGCC-3') (11, 16) subcloned into SmaI-digested pSM22.LacZ.
pSM22.SME4.CArG.LacZ contains four-copies of the SME-4 CArG box
(bp
150 to
141) subcloned into SmaI-digested pSM22.LacZ.
pSM22.SME4.5'CArG.LacZ and pSM22.SME4.3'CArG.LacZ contain 4 copies of
the SME-4 CArG box including its 5'-flanking sequence (bp
171 to
141) or 3'-flanking sequence (bp
151 to
136), respectively,
subcloned into SmaI-digested pSM22.LacZ.
c-fos.LacZ, SM22.LacZ,
SM22.SME4.LacZ, SM22.SME1.LacZ, SM22.c-fos.LacZ, SM22.SME4.CArG.LacZ, SM22.SME4.5'CArG.LacZ, and SM22.SME4.3'CArG.LacZ were generated according to standard techniques as described previously (21). To
identify transgenic embryos, high molecular weight DNA was prepared
from the yolk sacs of embryonic day (E)11.5 embryos and Southern blot
analyses were performed as described previously (21). The number of
copies per cell was quantitated by comparing the hybridization signal
intensity to standards corresponding to 1, 10, and 100 copies/cell
using a Molecular Dynamics PhosphorImager. E11.5 embryos were fixed,
stained for
-galactosidase activity, and cleared as described
previously (21). Photography was performed using a Nikon SMZ-U
dissecting microscope, Nikon 6006 camera, and Kodak EPT160 film.
-SRF rabbit polyclonal
IgG (Santa Cruz sc-335X),
-YY1 rabbit polyclonal IgG (Santa Cruz, sc-281X),
-SAP1A rabbit polyclonal IgG (Santa Cruz sc-1426X),
-Ets-1 rabbit polyclonal IgG (Santa Cruz sc-350X), or
-Elk-1 rabbit polyclonal IgG (Santa Cruz sc-355X) was incubated with the
indicated nuclear extract at 4 °C for 20 min prior to the binding
reaction as described previously (21).
190 to
110) were scaled up four times as
described (33). The binding reactions were exposed to UV light in an UV
Stratalinker 2400 (Stratagene Inc.) and treated with 4 µg of DNase I
(Worthington) and 3 units of micrococcal nuclease (Worthington).
DNA-bound proteins were fractionated by SDS-polyacrylamide gel
electrophoresis and autoradiographically visualized.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Promoter
in Arterial SMCs in Vivo--
The arterial SMC-specific 441-bp SM22
promoter contains six nuclear protein-binding sites, two of which
(SME-1 and SME-4) contain embedded CArG boxes that bind SRF (21). To
determine whether binding of SRF to the 441-bp SM22
promoter is
required for transcriptional activity in SMCs, the transcriptional
activity of the 441-bp SM22
promoter containing site-directed
mutations in SME-1 and SME-4 that abolish SRF binding was assayed in
F0 transgenic mice (Fig. 1,
upper panel). Consistent with our previous report (21), in
E11.5-441SM22.LacZ embryos, the 441-bp SM22
promoter restricted
activity of the LacZ transgene to arterial SMCs, the
bulbocordis region (BC) of the heart (future right ventricle), the
cardiac outflow tract (OFT), and the myotomal component of the somites
(S) (Fig. 1, A-C). LacZ-positive cells
(blue-stained cells) were observed in presumptive SMCs underlying the
endothelium of major arteries (Fig. 1B). In addition,
-galactosidase activity was reproducibly observed in regions of the
cardiac outflow tract including the aortopulmonary septum (Fig.
1C). In contrast, in 11 independent
E11.5-441SM22µCArG.LacZ transgenic embryos, containing mutations in
both SME-1 and SME-4 that abolish SRF binding to the SM22
promoter,
-galactosidase activity was not observed (Fig. 1D). These
data demonstrate that binding of SRF to the mouse SM22
promoter is
required for transcriptional activity in all three embryonic muscle
cell lineages.
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Fig. 1.
In vivo mutational analyses of
CArG boxes in the arterial SMC-restricted SM22
promoter. Upper panel, nucleotide
sequences of the CArG box-containing transcriptional regulatory
elements SME-1, µSME-1, SME-4, and µSME-4. The SME-1 and SME-4
nuclear protein-binding sites in the mouse SM22
promoter were
identified by DNase I footprint analyses (21). The site-directed
mutations generated in SME-1 (µSME-1) and SME-4 (µSME-4) that
abolish SRF binding are shown below each respective element.
A, E11.5 transgenic embryos were harvested and stained for
-galactosidase (LacZ) activity as described previously
(21). In -441SM22.LacZ embryos, LacZ activity was observed
in the dorsal aorta (Ao) and smaller branch arteries, the
myotomal component of the somites (S), the cardiac OFT, and
the BC region of the embryonic heart. B, cross-section
through the iliac artery of an E11.5-441SM22.LacZ embryo.
-Galactosidase activity is restricted exclusively to the layer of
cells (SMC) underlying the endothelium. Bar, 1 µm. C, cross-section through the chest cavity of an
E11.5-441SM22.LacZ embryo at the level of the cardiac outflow tract.
-Galactosidase activity is restricted to cells within the
aortopulmonary septum (APS). Bar, 1 µm.
D, in E11.5-441SM22.LacZµCArG embryos, LacZ
activity was not observed. E, in E11.5-441SM22 µSME-1
embryos LacZ activity was observed in the dorsal aorta
(Ao) and smaller branch arteries, myotomal component of the
somites (S), cardiac OFT, and BC region of the heart.
F, in four E11.5-441SM22 µSME-4 embryos, LacZ
activity was observed in the dorsal aorta (Ao), cardiac OFT,
and BC region of the heart but not in the somites.
promoter and the transcriptional
activity of these mutant promoters were assessed in F0
transgenic mice. In four independent -441SM22 µSME-1.LacZ embryos,
containing a mutation that abolishes binding of SRF and Sp1 to SME-1,
blue staining was observed in arterial SMCs, the myotomal component of
the somites (S), the cardiac OFT, and the BC region of the embryonic heart (Fig. 1E). This pattern closely resembled
that observed in the control -441SM22.LacZ embryos although the
intensity of staining was somewhat diminished (compare Fig. 1,
A and E). In contrast, in four independent
-441SM22 µSME4.LacZ transgenic embryos, containing a promoter
mutation that selectively abolishes binding of SRF to SME-4,
-galactosidase activity was observed in arterial SMCs and the
cardiac outflow tract, but not within the myotomal component of the
somites (Fig. 1F). Of note, blue staining in the dorsal
aorta was consistently less intense than that observed in -441SM22.LacZ
and -441SM22.µSME1.LacZ transgenic embryos (compare Fig. 1,
A and F). Taken together, these data demonstrate
that mutations that abolish binding of SRF to SME-1 and SME-4,
respectively, attenuate, but do not abolish, activity of the mouse
SM22
promoter in arterial SMCs. In addition, mutation of SME-4
abolished transgene expression in the somites, while mutation of SME-1
did not.
promoter (bp
90 to +41) and three copies of SME-1 (bp
279 to
256). In three independent SM22.SME-1.LacZ embryos, dark
blue staining indicative of
-galactosidase activity was observed
within arterial SMCs, the myotomal component of the somites (S), the BC
region of the primitive heart and cardiac OFT. This pattern
recapitulated the pattern of
-galactosidase activity observed in
control -441SM22.LacZ transgenic embryos, but the blue staining was
consistently more intense (compare Figs.
2A and 1A). To
determine whether SME-4 is necessary and sufficient to restrict
transgene expression to arterial SMCs, SM22.SME-4.LacZ transgenic
embryos were generated harboring the LacZ transgene under
the transcriptional control of the minimal SM22
promoter linked to
four copies of SME-4. In four independent SM22SME-4.LacZ transgenic
embryos, pale blue staining was restricted to arterial SMCs (Fig.
2B). LacZ activity was not observed within the
somites or bulbocordis region of the embryonic heart. In contrast, in
three independent SM22SME-4.LacZ embryos,
-galactosidase activity
was also observed within the myotomal component of the somites
suggesting that the site of transgene integration into the host genome
may alter the muscle-restricted pattern of transgene expression (Fig.
3A). Of note, inclusion of
additional nucleotides flanking SME-1 (bp
292 to 250) and SME-4 (bp
190 to
110), respectively, did not alter the pattern of transgene
expression observed (data not shown). Taken together, these data
demonstrate that a multimerized copy of either SME-1 or SME-4 is
necessary and sufficient to restrict transgene expression to arterial
SMCs in F0 transgenic embryos. However, differences in the
level of expressed transgene as well as differences in the
muscle-restricted pattern of transgene expression were observed between
SME-1 and SME-4 promoter-driven constructs.
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Fig. 2.
SME-1 or SME-4 is necessary and sufficient to
restrict transgene expression to arterial SMCs. Upper
panel, the nucleotide sequences of SME-1, SME-4, and the
c-fos SRE. The respective CArG box sequences are indicated.
A, SM22.SME1.LacZ embryos demonstrated intense
LacZ activity restricted to the dorsal aorta (Ao)
and smaller branch arteries, the myotomal component of the somites, the
cardiac outflow tract and the bulbocordis region of the embryonic
heart. B, in four of seven SM22.SME4.LacZ embryos
LacZ activity was restricted to arterial SMCs and the
cardiac outflow tract. In three of seven SM22.SME4.LacZ embryos
staining was also observed in the somites (see Fig. 3B).
C, LacZ activity was not observed in any
SM22.c-fos.LacZ embryo.
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Fig. 3.
The SME-4 CArG box alone is not sufficient to
restrict gene expression to arterial SMCs in transgenic mice.
Upper panel, the nucleotide sequence of SME-4 and SME-4
deletion mutants SME-4.CArG, SME-4.5'CArG, and SME-4.3'CArG are shown.
Four copies of each oligonucleotide were subcloned immediately 5' of
the 90-bp SM22 promoter and the activities of each respective
promoter were analyzed in F0 transgenic mice. A,
three of seven SM22.SME4.LacZ embryos demonstrated LacZ
activity in the dorsal aorta (Ao), the myotomal component of
the somites (S), and the cardiac OFT. In four of seven
SM22.SME4.LacZ embryos LacZ activity was restricted to
arterial SMCs and the cardiac outflow tract (see Fig. 2B).
B, SM22.SME4.CArG.LacZ embryos did not express the
LacZ transgene. C, SM22.SME4.5'CArG embryos
demonstrated LacZ activity restricted to arterial SMCs, the
myotomal component of the somites and the cardiac outflow tract.
promoter,
-441SM22
c-fos.LacZ transgenic mice were generated containing a
transgene encoding LacZ under the transcriptional control of
the 441-bp SM22
promoter in which the c-fos SRE has been
substituted for the CArG boxes within SME-1 and SME-4, respectively.
Surprisingly, in five independent -441SM22
c-fos.LacZ embryos,
-galactosidase activity was not observed (data not shown). In
theory, nucleotides flanking the c-fos SRE may also be
required for activity of this element in arterial SMCs. To examine this possibility, SM22.c-fos.LacZ transgenic embryos were generated containing the LacZ reporter gene under the transcriptional
control of the minimal SM22
promoter linked to three copies of the
c-fos SRE and its flanking sequences including the
TCF/Ets-binding site (Fig. 2, upper panel). Once again,
-galactosidase activity was not observed in five of six independent
SM22.c-fos.LacZ embryos (Fig. 2C). Of note, in one
SM22.c-fos.LacZ embryo faint blue staining was observed at the caudal
region at the base of the neck and in the limb buds (data not shown).
These data demonstrate that binding of SRF to CArG box sequences alone
is not sufficient to activate transcription in arterial SMCs and that
nucleotides within and flanking specific CArG box motifs distinguish
their respective activities in SMCs.
promoter (Fig. 3, upper
panel). Of note, this 10-bp sequence (CCAAATATGG) binds SRF in
EMSAs (data not shown). However, in six independent SM22.SME4.CArG.LacZ
embryos,
-galactosidase activity was not observed (Fig.
3B). Therefore, to determine whether 5' or 3' sequences flanking the SME-4 CArG box are required for transcriptional activity in arterial SMCs, transgenic mice were generated in which the LacZ gene was placed under the transcriptional control of
the minimal SM22
promoter linked to four copies of SME-4 deletion mutants (Fig. 3, upper panel). In two independent E11.5
SM22.SME4.5'CArG.LacZ embryos, containing four copies of the SME-4 CArG
box and its 5'-flanking sequences (bp
171 to
142),
-galactosidase activity was observed in a pattern recapitulating
that observed in the SM22.SME4.LacZ embryos (compare Fig. 3,
A and C). In contrast, LacZ activity
was not observed in four independent SM22.SME4.3'CArG.LacZ embryos,
containing 4 copies of the SME-4 CArG box and its 3'-flanking sequence
(bp
151 to
136) (data not shown). These data demonstrate that the
SME-4 CArG box sequence alone is not sufficient to restrict gene
expression to arterial SMCs in transgenic embryos and that additional
5'-flanking sequence in SME-4 (bp
171 to
152) is required to
activate transcription in arterial SMCs.
View larger version (49K):
[in a new window]
Fig. 4.
EMSAs of the SME-4 and c-fos
SRE nuclear protein-binding sites. A,
identification of nuclear protein complexes that bind to SME-4.
Radiolabeled oligonucleotides corresponding to the SME-4-binding site
were subjected to EMSAs with 10 µg of nuclear extracts prepared from
A7r5 SMCs. Some binding reaction mixtures included 10-100 ng of the
indicated unlabeled competitor oligonucleotides or 1 µl of the
indicated antiserum. Antibody supershift assays were performed by
preincubating the binding reactions with either preimmune serum
(PI), or polyclonal antiserum that recognize SRF, YY1,
SAP-1A, Elk-1, or Ets-1. Four specific nuclear protein complexes were
detected and designated A to D to the left of the
autoradiogram. Nuclear protein complexes were ablated and supershifted
with -SRF and
-YY1 antiserum. B, identification of
nuclear protein complexes that bind to the c-fos SRE. EMSAs
were performed as described above. Four specific nuclear protein
complexes, designated A to D, are identified to the left of
the autoradiogram. Nuclear protein complexes that were ablated and
supershifted with
-SRF antiserum and
-YY1 antiserum are
indicated.
View larger version (77K):
[in a new window]
Fig. 5.
UV cross-linking analyses of SMC proteins
that bind SME-4. EMSAs performed with nuclear extracts prepared
from primary rat aortic SMCs (VSMC), A7r5 SMCs, NIH 3T3
cells, HepG2 cells, C2C12 myoblasts and myotubes (C2B and C2T), and EL4
T cells were incubated with radiolabeled deoxybromouridine-substituted
SME-4 oligonucleotides (bp 190 to
110), exposed to UV light, and
treated with DNase I and micrococcal nuclease. DNA-bound proteins were
fractionated by SDS-polyacrylamide gel electrophoresis and visualized
autoradiographically. 68- and 44.5-kDa proteins corresponding in size
to SRF and YY1 were identified. Two unidentified proteins, p80 and p56
(open arrows) were enriched in VSMC and A7r5 cell
extracts.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, are also transiently expressed
in the embryonic heart and skeletal muscle (2). In skeletal and cardiac myocytes, SRF functions in concert with muscle-restricted transcription factors, including myogenic basic helix loop helix factors,
Nkx-2.5 and GATA-4, to activate cell lineage-restricted transcription (37-39). Despite the fact that the 441-bp SM22
promoter activates transgene expression in the somites and embryonic heart, it does not
bind directly to any previously described muscle-restricted transcription factors.2
Moreover, a multimerized copy of SME-1 or SME-4 is necessary and
sufficient to restrict gene expression to arterial SMCs as well as the
myotomal component of the somites, the bulbocordis region of the heart,
and the cardiac outflow tract. These data demonstrate that the minimal
transcriptional regulatory program that restricts gene expression to
arterial SMCs includes those factors that bind directly or indirectly
to the CArG box-containing elements in the SM22
promoter, SME-1 and
SME-4. Moreover, these data suggest that this transcriptional
regulatory program shares common features with the regulatory
program(s) that restricts gene expression to embryonic skeletal muscle
and the embryonic heart (at least the bulbocordis and outflow tract).
promoter demonstrate
conclusively that nucleotides within, and flanking, CArG boxes
distinguish the activity and specificity of SRF in arterial SMCs. These
data suggest that a common nucleotide sequence within and/or flanking
CArG boxes distinguish their activity in arterial SMCs. However, a
computer-assisted homology search failed to reveal conserved
nucleotides within or flanking SMC-specific CArG
boxes.3 In this regard it is
noteworthy that SME-1 and SME-4, as well as all other previously
identified SMC-specific CArG box-containing transcriptional regulatory
elements (21-26), are not flanked by TCF-binding sites. Consistent
with this observation, antibodies that recognize Ets factors including
Ets-1, Elk-1, and SAP-1A, failed to supershift SMC-derived nuclear
protein complexes that bind to SME-1, SME-4, or the c-fos
SRE. Association of ternary complex factors with SRF increases the DNA
binding affinity of SRF (41). Thus, it is possible that differences in
DNA binding affinity of SRF mediated through its association with TCFs
distinguishes the activities of SMC-specific CArG boxes
(i.e. low affinity SRF-binding sites) from CArG boxes that
regulate expression of growth-responsive and immediate early genes
(i.e. high affinity SRF-binding sites).
is expressed as early E8.0 in the
cells surrounding the dorsal aorta (42), while the CArG
box-dependent smooth muscle myosin heavy chain (SM-MyHC) gene is not expressed until much later in
embryonic development (24, 43). One possibility is that activity of SRF
is modulated by its capacity to differentially heterodimerize with
other transcriptional activators and/or repressors. However, despite
intense investigation, to date no SMC-specific transcription factor(s)
have been identified. Consistent with these data, EMSAs failed to
reveal an SMC-specific nuclear protein complex that binds specifically
to SME-1 or SME-4. However, UV cross-linking analyses revealed four
nuclear proteins that bind to SME-4 in addition to SRF and YY1. While
none of these proteins was expressed exclusively in SMCs, at least two
proteins appeared to be enriched in SMC nuclear extracts. Thus it
remains possible that SRF may function in a combinatorial fashion with
other ubiquitously expressed and potentially novel SMC
lineage-restricted transcription factors to regulate expression of
genes encoding SMC-restricted proteins. Alternatively, one or more
ubiquitously expressed factors may be post-translationally modified in
a cell lineage-specific fashion and in turn activate or repress
SRF-dependent transcription in SMCs.
5
isoform represses activity of the SM22
promoter (13). Moreover,
stretch of bronchial SMCs alters the pattern of SRF isoform splicing
resulting in induction of the SRF
5 isoform that acts in a dominant
negative fashion to repress transcription (48). Finally, it should be
noted that these molecular mechanisms (intracellular signaling and
alternative splicing) are not mutually exclusive and, in theory,
explain how a single transcription factor could serve as a nuclear
sensor integrating multiple signals from the cell surface and
translating these signals into a gradient of SMC phenotypes.
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ACKNOWLEDGEMENTS |
---|
We thank Eric Olson for sharing data prior to publication and helpful discussions. We thank Lisa Gottschalk for expert preparation of figures for this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant R0156915 (to M. S. P.).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.
§ Contributed equally to the results of this manuscript.
Established Investigator of the American Heart
Association. To whom correspondence should be addressed: University of
Pennsylvania School of Medicine, 9123 Founders Pavilion, Philadelphia,
PA 19104-4283. Tel.: 215-662-3140; Fax: 215-349-8017; E-mail:
parmacek@mail.med.upenn.edu.
Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M100631200
2 S. Kim and M. Parmacek, unpublished observation.
3 M. Parmacek, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SMC, smooth muscle cell; SRE, serum response element; SRF, serum response factor; bp, base pair(s); EMSA, electrophoretic mobility shift assay; BC, bulbocordis; OFT, outflow tract; TCF, ternary complex factors.
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