1 Department of Biology, Wake Forest University, Winston-Salem, North Carolina 27109; and 2 Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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E-box/basic
helix-loop-helix (bHLH)-dependent regulation of promoters for skeletal
muscle-specific genes is well established, but similar regulation of
smooth muscle-selective promoters has not been reported. Using
transient transfection assays of smooth muscle -actin (SM
A)
promoter-chloramphenicol acetyltransferase (CAT) reporter constructs in
rat vascular smooth muscle cells (SMCs) and L6 skeletal myotubes, we
identified two activator elements, smE1 and smE2, with sequences
corresponding to E-box (5'-CAnnTG-3') motifs. In L6
myotubes, 4-bp mutations of smE1 or smE2 E-box motif alone completely
abolished promoter activity. In contrast, mutation of smE1 and smE2 was
required to reduce promoter activity in SMCs. Supershift analyses
identified a myogenin-containing complex as the predominant smE1 and
smE2 binding activity in skeletal muscle, and myogenin overexpression
transactivated the promoter. Supershift analyses with SMC extracts
demonstrated that the bHLH protein upstream stimulatory factor (USF)
bound smE1, and USF overexpression transactivated the promoter in an
smE1-dependent manner. In summary, our results provide novel evidence
implicating E-box elements in directing expression of the SM
A
promoter through distinct bHLH factor complexes in skeletal vs. smooth muscle.
-actin promoter; basic helix-loop-helix protein; E-box; upstream
stimulatory factor; myogenin
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INTRODUCTION |
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SMOOTH MUSCLE -actin (SM
A) is the earliest known
marker of differentiated smooth muscle cells (SMCs) expressed during
development of the arterial wall (21). Conversely, reduced expression
of SM
A is a hallmark of the vascular response to injury and of the relatively dedifferentiated SMCs present in atherosclerotic lesions (34, 35). Activity of the SM
A promoter appears to be closely (but
not inseparably) linked to expression of the larger repertoire of
proteins that are characteristic of the differentiated phenotype of SMCs.
The precise mechanisms for transcriptional regulation of the SMA
promoter in vascular SMCs remain unknown. A number of
cis elements have been identified in
the SM
A promoter that contribute to tissue-specific expression
patterns. These include two separate CArG motifs (7) and a transforming
growth factor-
(TGF-
) controller element (17). However, much
remains to be elucidated about how these and other unidentified
elements within the promoter regulate specificity of expression in SMCs
as well as alterations in their response to injury or atherogenic stimuli.
Expression of the SMA gene is not restricted to SMCs alone. Although
expressed primarily in differentiated SMCs of adult animals, SM
A is
also transiently expressed in fibroblasts, cardiomyocytes, and skeletal
muscle cells during development, after tissue wounding, or in culture
(4, 10, 25, 37, 41, 42). Expression of SM
A by all three muscle types
suggests that the SM
A promoter may be activated by a similar set of
transcription factors and cis
elements, such as binding of basic helix-loop-helix (bHLH) transcription factors to E-box elements. The E-box motif
(5'-CAnnTG-3') binds dimers of bHLH transcription factors
(30). Tissue-specific bHLHs are exemplified by the MyoD family
(including Myf-5, Mrf-4, and myogenin) and are the primary
transcription factors coordinating expression of skeletal
muscle-restricted genes (13). Similarly, cardiac development is
partially regulated by the bHLH factors Th-1/dHAND and Th-2/eHAND (9,
18, 45). Similar to striated muscle, several widely expressed bHLH
factors, including E12/E47 (22, 36), Id (24, 28), and members of the
c-Myc-related, bHLH-leucine zipper (bHLH-ZIP) family (3, 12, 44) are
expressed in SMCs. General mesodermal and neuroectodermal
lineage-determinant bHLH factors such as M-twist, Meso-1, and
HES-1-5 (6, 23, 39, 48) also appear transiently in the aortic
primordia. In contrast to striated muscle, however, regulation by bHLH
factors of a gene associated with the SMC-differentiated phenotype has not been demonstrated (35). Deletion analyses of the rat SM
A promoter by our laboratory showed that the first 125 bp (
1 to
125) 5' to the transcriptional start site drove high-level
expression of a linked chloramphenicol acetyltransferase (CAT) reporter
gene in transient transfection studies with cultured aortic SMCs (28, 41) but not with L6 skeletal myoblasts or myotubes. Additional promoter
sequences from
125 to
271 bp resulted in substantial reporter expression in L6 myotubes but decreased activity by one-half in SMCs. Thus positive and negative regulatory elements are present in
the rat SM
A promoter between
125 and
271 bp. Several
candidate regulatory elements reside within this region, including
three potential elements at
214 bp (smE1),
252 bp (smE2),
and
260 bp (smE3) that correspond to the canonical E-box motif.
In the SM
A promoters of other species, smE1 and smE2 are highly
conserved, whereas the putative E-box at smE3 is present in the rat
promoter only (7, 41).
Given the general importance of E-box elements in differentiation of
striated muscle and their presence in a transcriptionally active region
of the SMA promoter, the goals of this study were 1) to determine whether the three
5'-CAnnTG-3' motifs within the SM
A promoter function as
true E-boxes within the context of rat L6 skeletal myotubes and/or rat
aortic SMCs and, if so, 2) to identify transcription factors that bind the active sites. Our results
demonstrated that, in skeletal myotubes, smE1 and smE2 elements were
required for SM
A promoter activity and bound myogenin-containing transcription factor complexes present in L6 myotube nuclear extracts. Furthermore, myogenin overexpression could transactivate the SM
A promoter. In SMCs, smE1 and smE2 could mutually compensate for each
other in transient transfection assays but bound different transcription factor complexes. smE1 acted as an E-box that was recognized by two bHLH factors, upstream stimulatory factors USF-1 and
USF-2, and overexpression of USFs could transactivate the SM
A
promoter in an smE1-dependent manner. In contrast, mutagenesis data
indicated that smE2 did not appear to function as an E-box in SMCs,
whereas data from gel-shift analyses indicated that smE2 was not bound
by any of several bHLH factors, including USFs. Our data are the first
to demonstrate regulation of an SMC differentiation marker gene via an
E-box-dependent mechanism.
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EXPERIMENTAL PROCEDURES |
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Cell culture. Rat aortic SMCs and endothelial cells were cultured as described previously (41). All other cell lines were obtained from American Type Culture Collection (Manassas, VA). L6 myoblasts were routinely maintained in Ham's F-12 + 10% FCS and fused into myotubes as needed using DMEM-1% FCS for 48 h. COS-7 cells were grown in DMEM + 10% FCS. Rat PC-12 pheochromocytoma cells were maintained in RPMI 1640 + 10% horse serum and 5% FCS. 10T1/2 cells were routinely grown in basal medium Eagle-10% FCS.
Gel-shift analysis of E-box binding proteins.
Nuclear and whole cell extracts were prepared as described previously
(11, 29). Double-stranded oligonucleotides containing 6 bp of native or
mutant smE1, smE2, or smE3 plus 5 bp of native SMA promoter sequence
5' and 3' to each E-box were end labeled with
32P and purified. DNA-protein
binding reactions were separately optimized for L6 skeletal myotube and
SMC nuclear extracts. SMC binding reactions contained 5 µg of nuclear
extract protein and 2 × 105
cpm of DNA probe in a 20-µl final reaction volume of 15 mmol/l HEPES,
pH 7.9, 80 mmol/l NaCl, 25 mmol/l KCl, 1 mmol/l EDTA, 1.25 mmol/l
CaCl2, 1.2 mmol/l dithiothreitol,
15% glycerol, 200 µg/ml BSA, 0.05 mmol/l
4-(2-aminoethyl)benzenesulfonyl fluoride, 0.2 µg/ml each aprotinin
and leupeptin, and 0.2 µg of polydeoxyinosinic-deoxycytidylic acid.
For L6 myotubes, binding was optimal in 10 mmol/l Tris, pH 7.5, 100 mmol/l KCl, 1 mmol/l EDTA, 1 mmol/l dithiothreitol, and 5%
glycerol, with 2 µg of polydeoxyinosinic-deoxycytidylic acid. In cold competitor assays, 10- to 1,000-fold molar excess unlabeled oligonucleotide was added to the binding reaction 30 min
before the radiolabeled probe. Samples were then resolved on 5% 29:1
acrylamide-bis-0.5× Tris base-EDTA-boric acid gels. For
supershift assays, 1-2 µg of antibodies against candidate bHLH
proteins were incubated with standard gel-shift reactions for 30 min
before loading. Antibodies were as follows: Yae monoclonal antibody
against E2A gene products, A20 polyclonal antibody against HEB, C33
monoclonal and C17 polyclonal antibodies against c-Myc and Max,
respectively, M-225 polyclonal antibody against myogenin, and C20 or
N18 polyclonal antibodies against USF-1 and USF-2 (all from Santa Cruz
Biotechnologies, Santa Cruz, CA), G108-391 monoclonal antibody against
human E2-2 (Pharmingen, San Diego, CA), and an additional antibody
against murine E2-2-related gene products (1).
Site-directed mutagenesis of 271-bp SM
A
promoter-CAT reporter and functional analysis by transient
transfection.
To determine whether the putative E-boxes at
214,
252,
and
260 bp of the rat SM
A promoter are required for
transcriptional activity, 1- to 4-bp mutations were made
within each of the three sites by use of the Altered Sites protocol
(Promega, Madison, WI) and PCR (19). The
271- to +43-bp region
of native or E-box mutant promoters was then subcloned into the
Hind
III/Sal I sites of pCAT.Basic reporter
(Promega; Fig. 1). SMCs and L6 myotubes were then transfected with CsCl-purified DNA constructs, and CAT reporter assays were performed as described previously (41). Briefly,
cells were plated at 2 × 104
cells/cm2 in six-well plates; SMCs
were plated 24 h before transfection in DMEM-10% FCS, and
undifferentiated myoblasts were plated 72 h before transfection in
Ham's F-12-10% FCS. At time 0,
cells were washed three times with serum-free DMEM, then transfected with 4 µg of DNA construct mixed with 30 µg of
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (Boehringer Mannheim, Indianapolis, IN) in 50 mM HEPES
buffer, pH 7.9. In all assays the promoterless pCAT-Basic plasmid and
the simian virus 40 (SV40)-driven constitutively active pCAT.Control
plasmid were included as baseline and positive controls for the
transfection and CAT assays. Five hours after addition of DNA, cells
were refed serum-containing medium to a final concentration of 10% FCS
for SMCs or 1% for L6 myoblasts. For L6 myoblasts, this reduction in
serum concentration was sufficient to induce differentiation into
myotubes. After 18 h, SMCs or L6 myotubes were refed fresh DMEM plus
10% or 1% FCS, respectively. Cells were harvested, postnuclear
lysates were prepared 72 h after transfection, and total protein in
lysates was determined by the Bradford method (Bio-Rad, Hercules, CA).
CAT expression per milligram of protein in the postnuclear cell lysate
was determined as described previously (41), and reporter activity was
normalized to a percentage of pCAT.Basic activity. Three replicates per
DNA construct were assayed in each experiment, with each experiment
replicated at least twice. In all cases, CAT activity for test reporter
constructs was less than the expression observed for cells transfected
with the SV40-driven CAT-positive control.
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Transactivation of SMA promoter by USF or myogenin
overexpression.
To determine whether overexpression of USFs or myogenin increased
SM
A promoter activity, SMCs or 10T1/2 fibroblasts were cotransfected
with native or E-box-mutated CAT constructs plus USF-1, USF-2, or
myogenin expression plasmids. The 1.7-kb
EcoR I fragment encoding human USF-1
from plasmid d12 and the 2-kb fragment encoding murine USF-2 from
pM2-2 (26, 27) were subcloned into
pcDNA3.1+ (InVitrogen, San Diego,
CA) under control of the cytomegalovirus (CMV) promoter or pSG-5
(Stratagene, La Jolla, CA) under the SV40 promoter. Myogenin in pEMSV
is an SV40-driven constitutive expression vector. For SMC
cotransfection assays, 1 µg of either empty vector (baseline
control), USF-1, or USF-2 expression constructs was mixed with 4 µg
of pCAT.271/native, smE1 mutant, or smE2 mutant promoter constructs,
and cells were transfected using
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate. For transactivation assays in 10T1/2 cells, 1.9 × 105 cells in six-well plates were
transfected with 3 µg of reporter constructs plus 1 µg of myogenin
or empty expression vectors with use of calcium phosphate.
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RESULTS |
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Mutations of putative E-boxes in the SMA promoter
differentially inhibited transcriptional activity in L6 myotubes vs.
SMCs.
A schematic of the pCAT.271/native construct containing 3 putative
SM
A promoter E-box elements, designated smE1, smE2, and smE3, is
shown in Fig. 1A; native vs. mutated
sequences are listed in Table 1. Four-base
pair mutations were generated in each of the three E-boxes, which, on
the basis of previous studies (5, 13, 20, 23, 30, 46), would maximally
disrupt E-box interactions with bHLH factors. Effects of these
mutations on promoter activity were assayed by transient transfection
into rat aortic SMCs and L6 skeletal myotubes. As shown in Fig.
1B, disruption of smE1 or smE2 alone
nearly abolished CAT reporter activity in L6 myotubes, whereas mutation
of smE3 had no consistent effect. In contrast to skeletal myotubes,
disruption of smE1 or smE2 alone had no significant effect on CAT
activity in SMCs (Fig. 1C). Mutation of smE3 alone did not produce consistent effects on CAT activity; the
4-bp smE3 mutation modestly increased promoter activity (as shown) or,
equally often, had no effect on activity (data not shown).
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Identification of distinct smE1 and smE2 binding protein complexes
in skeletal vs. smooth muscle cells by gel-shift analysis.
To identify SMC and L6 myotube nuclear proteins that bound to smE1 and
smE2, gel-shift assays were performed with double-stranded oligonucleotides containing native or mutated smE1 or smE2 sequences alone. L6 myotube nuclear extracts formed DNA-protein complexes with
smE1- or smE2-containing oligonucleotides that exhibited identical
electrophoretic mobilities, and smE1 and smE2 binding proteins were
competed by an excess of unlabeled probe (Fig.
3A). Unlike smE1 and smE2, an smE3 binding complex could not be detected in
nuclear extracts of skeletal myotubes. Addition of 1-2 µg of a
neutralizing antibody against myogenin (one of the skeletal muscle-specific bHLH factors) inhibited formation of the major smE1 and
smE2 binding complexes (Fig. 3B, smE1
data only shown), implicating myogenin as the smE1 and smE2 binding
factor in L6 myotube nuclear extracts. When gel shifts were performed
using the conditions described for L6 myotubes, but using SMC nuclear extracts, there was no evidence of binding by MyoD or other specific bHLH factors (data not shown).
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Cotransfection of USF-1, USF-2, or myogenin expression constructs
with pCAT.271/native increased promoter activity in SMCs and non-SMC
lines.
Because results of gel-shift analyses with L6 myotube extracts
demonstrated binding of myogenin to smE1 and smE2 (Fig. 3), we used
10T1/2 fibroblasts [which are permissive for transactivation of
E-box-dependent skeletal muscle promoters (31, 33, 38)] to
determine whether myogenin overexpression would transactivate the
p271.CAT/native reporter. 10T1/2 fibroblasts were cotransfected with
p271.CAT reporter plus an SV40-driven constitutive myogenin expression
construct, then induced to differentiate and analyzed for CAT reporter
activity, as described above. Our results demonstrated that myogenin
induced an ~30-fold increase in CAT reporter activity in 10T1/2
fibroblasts (Fig.
6A).
Moreover, mutation of smE1 or smE2 alone each reduced myogenin
transactivation by one-half or more, indicating that transactivation by
myogenin was E-box dependent.
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DISCUSSION |
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Previous deletion analyses of the rat SMA promoter have demonstrated
the presence of multiple positive and negative
cis-acting regulatory elements that
establish tissue-restricted expression of this gene in cultured cells
(7, 28, 41). The present studies provide novel evidence indicating that
two E-box motifs within the 5'-region of the SM
A promoter
contribute to activity in skeletal myotubes and SMCs, although via
distinct mechanisms and bHLH transcription factors. In L6 myotubes,
smE1 and smE2 functioned as canonical E-boxes that were required for
promoter activity. Moreover, smE1 and smE2 bound and were
transactivated by the skeletal muscle-specific bHLH factor myogenin. In
contrast, smE1 or smE2 alone was dispensable for promoter activity in
cultured SMCs, although simultaneous mutation of smE1 and smE2 resulted in reduced promoter activity. In addition, in SMCs, smE1 but not smE2
appeared to function as a canonical E-box that bound the ubiquitously
expressed bHLH-ZIP factor USF, which could transactivate the promoter.
The results of this study demonstrate that, similar to the previously
described cardiac and skeletal -actin genes (32, 38), SM
A
promoter activity is in part controlled by E-box elements. As we
reported previously, the
125 to +43 region of the
SM
A promoter (which does not contain smE1 or smE2) is not active in skeletal myotubes, suggesting that essential promoter elements for
skeletal muscle expression lay upstream of
125.
This report identified paired E-box elements within the upstream region
that bind myogenin-containing transcription factor complexes and
regulate SM
A expression in skeletal muscle cells. This regulatory
pattern matches several previous reports which showed that expression of skeletal and cardiac
-actins in skeletal muscle cells is
dependent on paired E-box elements binding skeletal muscle
lineage-specific bHLHs (16, 38, 43). Thus transient skeletal muscle
expression of SM
A, as occurs during embryonic development, appears
to share at least some of the same regulatory signals that modulate
skeletal
-actin. Indeed, it is interesting to speculate that
sequence conservation of smE2 and, to a lesser extent, smE1 across
multiple mammalian SM
A promoters may be due to their requirement for
paired E-boxes for SM
A promoter activity in skeletal muscle rather
than an essential regulatory function in SMCs. We believe that this observation is potentially quite significant, since unlike most other
targets of skeletal muscle-specific bHLHs, the SM
A gene is only
transiently expressed within skeletal myotubes during early
differentiation but is subsequently suppressed. As such, we believe
that this gene may be particularly powerful for elucidating regulatory
modules that differentially control the temporal and spatial expression
of smooth muscle
-actin in skeletal vs. smooth muscle.
Earlier studies have established a model in which expression of
tissue-specific genes during skeletal muscle differentiation is
controlled primarily by MyoD, myogenin, and other bHLH factors that are
specific for the muscle lineage and bind to E-box elements (1, 13, 32,
33); similar contributions of the bHLH factors dHAND and eHAND have
been shown to be critical during cardiac development (18, 45). However,
more recent studies have shown that, even in skeletal muscle,
cell-specific gene regulation is not simply conferred by a single
cell-specific transcription factor such as MyoD. Rather, it is
dependent on a complex combination of interactions between multiple
regulatory regions or modules and their transacting factors, including
many factors that are not cell specific (14). In contrast to striated
muscle, however, our evidence suggests that bHLH factors may not be the
primary regulatory factor driving SMC-selective expression from the
SMA promoter. First, unlike myogenin, USF is ubiquitously expressed and alone cannot explain the high degree of cell selectivity exhibited by the SM
A gene. Second, the effects of the smE1 and smE2 double mutations on promoter activity in cultured SMCs were relatively modest
compared with the effects in skeletal myotubes. However, our results
provide clear evidence that the smE1-USF interaction has functional
activity. Together, these data indicate that combinatorial interactions
between multiple, non-tissue-specific factors may also contribute to
smooth muscle-specific gene expression in a manner similar to many
other cell types including cardiac and skeletal muscle (14).
An alternative interpretation of our data would be that USF contributes
in other ways to the overall activity of this promoter rather than
controlling tissue-specific gene expression per se. For example, USFs
may modulate SMA expression during different phenotypic states, as
has been suggested for the MLC-2v gene (31) or in response to growth
factors, e.g., TGF-
(43). Consistent with this hypothesis,
expression of c-Myc (a second bHLH-ZIP class factor) has been shown to
be associated with downregulation of SM
A expression in SMCs during
proliferation (3). As members of the c-Myc-related family, USFs can
compete with c-Myc for similar DNA binding sites (2, 27, 46) and so may
indirectly stimulate SM
A expression by inhibiting c-Myc-induced gene
repression. It is also possible that activation of the SM
A promoter
by USFs is essential in vivo but is dispensable in vitro, where there is compensatory promoter activation associated with phenotypic modulation that, in turn, masks a requirement for USF. Clearly, elucidation of the potential role of the smE1 (and smE2) element in
modulation of SM
A expression in SMCs will be dependent on mutational
studies in transgenic mice. Such studies have just recently been made
possible by our identification of sufficient regions of the SM
A
promoter necessary to drive expression of a LacZ reporter gene in vivo
in a manner that recapitulates expression of the endogenous SM
A gene (27a).
Our initial observations (Fig. 2B)
indicated that the presence of smE1 or smE2 alone was sufficient to
maintain SMA promoter activity in SMCs. Thus we had originally
hypothesized that smE1 and smE2 represented redundant canonical E-box
elements in SMCs and that binding of a similar bHLH factor complex to
smE1 or smE2 was sufficient for maintaining promoter activity in SMCs.
However, supershift and immunoblot analyses did not detect USFs, other c-Myc-related proteins, or ubiquitously expressed E2A, E2-2, and HEB-related type 1 bHLH factors within the smE2 binding complexes. Although not every possible tissue-restricted bHLH factor was exhaustively tested, the tissue-specific bHLH factors that have been
identified previously are absent from mature arteries. Other candidates
such as TAL1 and members of the HES family also have very stringent
binding site specificities (5, 23, 49) and can be eliminated on the
basis of lack of any effect of mutations in the 5'-CA or
3'-TG of smE2 in transfection assays (Fig.
2B). Moreover, given our current
knowledge of the E-box/bHLH regulatory system, even a novel, smooth
muscle-specific bHLH factor would be predicted to utilize one of the
ubiquitously expressed type I factors as its dimerization partner. Thus
it is more likely that the smE2 shift complex reflects binding of
non-bHLH proteins to a cis element
that overlaps the E-box region. One analogous example would be the
E-box binding repressor protein ZEB (15), a zinc-finger protein with a
binding site that overlaps an E-box motif within the IgH promoter. ZEB
is displaced from the IgH promoter by E2A binding and appears to be a
critical step in B cell-specific control of the immunoglobulin heavy
chain promoter. Obviously, further studies are required to resolve the
nature and identity of the smE2 binding factor(s) and the mechanism
whereby it can compensate for abolition of the SM
A smE1 motif within
an SMC context. Given the divergence of SMCs from striated muscle cells in the utilization of the bHLH/E-box regulatory pathway, these future
studies may help define the complex interactions between USFs and other
transcription factors that establish the unique smooth muscle phenotype.
In summary, the studies described here provide the first evidence for
novel, E-box-dependent regulation of the SMA gene in skeletal muscle
cells as well as SMCs. Importantly, however, the mechanisms of
regulation are very different in these two cell types. Further studies
are required to elucidate the role of the SM
A E-box elements in
regulation in vivo, during normal development, and in
pathophysiological states characterized by alterations in SM
A
expression in SMC (34, 35, 40) and non-smooth muscle cell types (4, 10,
25, 37, 41, 42).
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ACKNOWLEDGEMENTS |
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We thank Dr. Stephen Hauschka (University of Washington, Seattle, WA) for the kind gift of antibodies against E2-2, Dr. Michèle Sawadogo (M. D. Anderson Cancer Center, Houston, TX) for clones of the human USF-1 and murine USF-2, Dr. Stephen Konieczny (Purdue University, West Lafayette, IN) for the myogenin expression plasmids, and Dr. Paul DiCorleto (Cleveland Clinic, Cleveland, OH) for the rat endothelial cells.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants R01 HL-38854 and P01 HL-19242 awarded to G. K. Owens. A. D. Johnson was supported by NHLBI Training Grant T32-HL-07355; American Heart Association, Virginia Affiliate, fellowship VA-96-F-26; and NHLBI National Research Service Award F32-HL-09648-01.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. K. Owens, Dept. of Molecular Physiology and Biological Physics, University of Virginia, Box 449, Rm. 2-29 Jordan Hall, Charlottesville, VA 22908 (E-mail: gko{at}virginia.edu).
Received 25 January 1999; accepted in final form 29 March 1999.
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