From the § Department of Biochemistry, School of
Medicine, the Department of Biomedical Sciences, School
of Veterinary Medicine, and the ¶ Dalton Cardiovascular Research
Center, University of Missouri, Columbia, Missouri 65211
Received for publication, August 24, 2000, and in revised form, September 26, 2000
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ABSTRACT |
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To examine the role of the Regulation at the level of gene transcription represents a
critical control point whereby a cell-specific phenotype can be specified and modulated in response to intrinsic and extrinsic stimuli
throughout development. For example, in the day 17 post-coitus mouse
fetus, To investigate the mechanistic basis controlling both slow fiber
restricted expression and MOV responsiveness of the MCAT regulatory elements (5'-CATTCCT-3') are similar in nucleotide
sequence to the SV40 enhancer GT-IIC (5'-CATTCCA-3'),
SphI (5'-CATGCCT-3', and SphII (5'-CATACCT-3')
elements (12, 13). Earlier studies using protein purification and
cDNA cloning identified transcription enhancer factor 1 (TEF-1) as
the GT-IIC protein binding activity within HeLa cell extracts
(14, 15). Subsequent studies have established the presence of a
multigene TEF-1 family in vertebrates (nominal TEF-1 (NTEF-1), related
TEF-1 (RTEF-1), divergent TEF-1, and embryonic TEF-1). Further
diversity within the TEF-1 family of transcription factors is
accomplished by alternative splicing (12, 13, 16). In addition to
existing as multiple isoforms, distinct TEF-1 isoproteins can modulate
gene transcription as a result of post-transcriptional modification via
different intracellular signaling pathways (17, 18) or by interacting with various cobinding and/or coactivator proteins that may be cell-specific or ubiquitous (12, 13, 19).
MCAT elements are commonly located in the control region of many
striated (skeletal and cardiac) muscle-specific genes (Ref. 13 and
references within). A detailed molecular analysis of the cTnT promoter
has elucidated the mechanistic basis by which two closely positioned
MCAT elements participate in directing embryonic skeletal
muscle-specific gene transcription of the cTnT gene (20, 21). These two
MCAT elements (MCAT1 and MCAT2) bind several protein complexes that
display high, intermediate, and low migrating mobility in
electrophoretic mobility shift assays (EMSA). All binding complexes
contain TEF-1 protein; however, the low mobility complex (LMC) formed
at the MCAT1 element contains an additional protein identified as
poly(ADP-ribose) polymerase (PARP) (20-22). Mutation of the
5'-flanking nucleotides of either the MCAT1 or MCAT2 elements revealed
their integral requirement for achieving both skeletal muscle-specific
expression of cTnT reporter genes, and the formation of a LMC at these
MCAT elements when using embryonic chicken skeletal muscle nuclear
extract (20). Likewise, Gupta et al. (23) have reported that
the positive regulation of The In the current study we have generated multiple independent transgenic
mouse lines harboring either a wild type 293-bp human Transgenes and Site-directed Mutagenesis--
The wild type
The distal MCAT site in the human Transgenic Mice--
Transgenic mice were generated by
microinjection of purified transgene DNA into pronuclei of single cell
fertilized embryos as described previously (26). Transgenic founder
mice were identified by Southern blot analysis. Subsequent
transgene-positive offspring were identified by PCR amplification of
genomic DNA using primers specific for the CAT gene. In the present
study, multiple independent transgenic lines representing each
transgene (wild type
The in vivo presence of the MCAT mutant sequence was
verified by PCR amplification of
Transgene copy number was estimated as described previously (26).
Briefly, 10 µg of transgenic mouse genomic DNA was digested with
EcoRI, followed by fractionation through a 1% agarose gel and transfer onto a nylon membrane. The membrane was hybridized with a
32P-labeled CAT cDNA probe used to identify each
transgene. The intensity of each signal was quantified with the use of
a PhosphorImager (Storm860 with IMAGEQUANT version 5.1 software,
Molecular Dynamics) and was compared with that of a previously reported
single-copy transgenic line (human Animal Care and MOV Surgical Procedure--
The ablation surgery
used in this study was performed as described previously (27). All
procedures were conducted using adult female transgenic mice (22-26 g)
and were approved by the Animal Care Committee for the University of
Missouri-Columbia. MOV was imposed on the hindlimb fast twitch
plantaris muscle by surgical removal of the gastrocnemius and soleus
muscles. All mice demonstrated normal mobility shortly after recovering
from anesthesia. The post-surgical experimental period lasted 8 weeks after which time the overloaded plantaris muscle (MOV-P) was removed. Sham operated mice served as controls for the plantaris (CP) and soleus
(CS) muscles.
CAT Assays--
CAT assays were performed as described
previously (9, 27) and the percent conversion of
[14C]chloramphenicol to the acetylated form was
quantified using a PhosphorImager (Storm860) with IMAGEQUANT version
5.1 software.
Preparation of Nuclear Protein Extract from Adult Skeletal
Muscle--
Nuclear extracts were isolated from adult rat CP, MOV-P,
and CS muscle as described previously (8). Protein concentration was
determined according to Bradford (28).
Electrophoretic Mobility Shift Assay--
All oligonucleotide
probes used in this study are listed in Table
I. EMSAs were carried out as described
previously (8). Binding reactions were performed using either
3.5 µg of CP, MOV-P, or CS nuclear extract and 20,000 cpm of labeled
probe for 20 min at room temperature in a 25-µl total volume. Where
indicated, either in vitro transcribed/translated (TnT)
NTEF-1, RTEF-1, embryonic TEF-1, divergent TEF-1, PARP, or Max protein
were used in place of muscle nuclear extract (see figure legends for
specific quantities). The binding reactions were resolved on a 5%
nondenaturing polyacrylamide gel at 220 volts for 2.5 h at
4 °C. Supershift EMSAs were performed by first preincubating
skeletal muscle nuclear extract with 2 µl of the corresponding
antibody for 30 min at room temperature followed by the addition of the
32P-labeled DNA probe. Following electrophoresis, the gels
were dried, and DNA-protein complexes were visualized by
autoradiography at In Vitro Transcription and Translation--
In vitro
coupled TnT was performed using 1 µg of TEF-1, PARP, or Max
expression plasmids in the T7 TnT rabbit reticulocyte lysate kit
according to the manufacturer's instructions (Promega). The expression
plasmids corresponded to either NTEF-1 (pXJ40-TEF-1A, ORF of human
TEF-1 (15)), RTEF-1 (pXJ41-TEF-3, ORF of human TEF-3 (29)), embryonic
TEF-1 (pXJ41-TEF-4, ORF of mouse TEF-4 (29)), and divergent TEF-1
(pXJ41-TEF-5, ORF of human TEF-5 (30)), PARP (pBS-II SK+ PARP,
full-length human PARP (31)), or Max (pVZ1 p21-Max, full-length human
Max (23)). Parallel TnT reactions were performed in the presence of
[35S]methionine (PerkinElmer Life Sciences). The
integrity and expected molecular weights of the protein products were
verified by resolving the radiolabeled reaction products on a SDS-12%
polyacrylamide gel. Parallel reactions of lysate not programmed with
plasmid DNA were used as negative controls (unprogrammed lysate
(UL)).
Antibodies--
The antibodies used in this study were as
follows: NTEF-1, mouse polyclonal antibody raised against amino acids
86-199 of human TEF-1 (BD Transduction Laboratories); PARP, rabbit
polyclonal antibody raised against full-length human PARP (Roche
Molecular Biochemicals); and Max, rabbit polyclonal antibody raised
against full-length mouse Max (Upstate Biotechnology). All antibodies listed hereafter were purchased from Santa Cruz Biotechnology, Inc. and
include: MyoD, rabbit polyclonal antibody raised against full-length,
(amino acids 1-318) mouse MyoD; Myogenin, rabbit polyclonal antibody
raised against full-length (amino acids 1-225) of rat myogenin; USF-1,
rabbit polyclonal antibody raised against carboxyl terminus amino acids
291-310 of human USF-1; E2A.E12, rabbit polyclonal raised a peptide in
the carboxyl terminus of human E47 and corresponds to amino acids
422-439 of human E12; and HEB, rabbit polyclonal antibody
raised against a peptide in the carboxyl-terminal domain of human HEB
(HTF 4).
Production and Purification of GST Fusion Proteins--
The
GST-TEF-1 expression plasmid has been described elsewhere (23).
Briefly, full-length rat TEF-1 cDNA was subcloned into the
XbaI and XhoI sites of the bacterial expression
vector pGEX-KG. Full-length Max cDNA was amplified from
pVZ1-p21-Max (23) in PCR reactions using the following primers: sense
strand, 5'-CCGCTCCCTGGGCGGATCCAAATGAGCGATACC-3' and antisense
strand, 5'-TGGCCTGCCCCGCTCGAGTTAGCTGGCCTC-3'. The amplified
cDNA was subcloned into the BamHI and XhoI
sites of pGEX-5X-1 (Amersham Pharmacia Biotech). The GST fusion
proteins were expressed in Escherichia coli and purified
according to the manufacturer's (Amersham Pharmacia Biotech)
instructions with modifications. Briefly, bacteria (DH5 In Vitro Protein-Protein
Interactions--
[35S]Methionine labeled TnT proteins
corresponding to NTEF-1, PARP, and Max were incubated with 2 µg of
immobilized GST, GST-TEF-1, or GST-Max with continuous rocking for
3 h at 4 °C in a modified 1× protein interaction buffer
described by Gupta et al. (23); 20 mM HEPES, pH
7.5, 75 mM KCl, 1 mM EDTA, 2 mM
MgCl2, 2 mM dithiothreitol, and 1% Triton
X-100. Unprogrammed lysate that was also transcribed and translated in
the presence of [35S]methionine was used as the negative
control. After incubation the glutathione-Sepharose beads were pelleted
by centrifugation at 500 × g and washed five times
with ice-cold 1× protein interaction buffer. The fusion proteins were
eluted off the beads by heating at 95 °C for 3 min in the presence
of 1× SDS sample buffer and analyzed on a 4-20% gradient SDS-PAGE
gel. The gel was dried and exposed to film for 15 h at room temperature.
Mutation of the Distal MCAT Element Decreased Slow Fiber Expression
of Transgene
To assess whether the distal MCAT element was solely responsible for
directing both muscle-specific and high levels of slow fiber
expression, we measured the CAT specific activity (pmol/µg of
protein/min) in muscle and nonmuscle tissues obtained from transgenic
mice carrying mutant transgene Mutation of the Distal MCAT Element Does Not Eliminate MOV
Responsiveness of Transgene
Collectively, our transgenic analysis demonstrates that the Multiple Nuclear Protein Binding Complexes Form at the The LMC Formed at the
Because adjacent E-box elements have been shown to be important for
nuclear protein binding at the Nucleotides Comprising the NTEF-1, PARP, and Max Comprise the LMC Formed at the TEF-1, but Not PARP or Max, Specifically Binds to the NTEF-1, PARP, and Max Stably Interact--
Our DNA-protein binding
assays revealed that of the three proteins tested, only NTEF-1 binds
the MCAT elements are known to participate in directing
muscle-specific expression. Our transgenic findings herein show that
the control region distal MCAT element ( Transgenic Mouse Analyses of the in Vivo Function of the Human
Because MCAT elements have been shown to function as inducible promoter
elements in response to mechanical stimuli (13)y and the distal MCAT
element resides within the 89-bp region required for MOV responsiveness
of transgene
In addition to our mutant MCAT transgenic findings, our EMSA analysis
provides supportive evidence that the distal MCAT element contributes
to slow fiber expression in the control soleus and MOV-P muscle. In
direct and competition EMSA experiments we observed the formation of a
specific LMC that varied in intensity according to the proportion of
slow type I fibers within the adult stage rat skeletal muscles from
which nuclear extract was isolated (Fig. 3). This EMSA binding pattern
(CP < MOV-P < CS) is relevant to the in vivo
expression pattern of our transgenes because the mouse and rat display
a qualitatively similar pattern of slow type I fibers within these muscles.
Mutant Transgene The
Despite the fact that some of the nuclear proteins that interact at the
Although not an absolute certainty, we suggest that the conditions of
our in vitro binding assays do not adequately emulate the
in vivo biological conditions reflected in our adult stage skeletal muscle nuclear extract, thereby precluding independent binding
by both Max and PARP, as well as the reconstitution of a LMC. For
example, it is possible that something in the rabbit reticulocyte
lysate may have inhibited DNA binding by both PARP and Max.
Alternatively, these proteins may require a post-translational modification that occurs only in the context of intact adult stage skeletal muscle. Nevertheless, our data provide evidence that the
In summary, our results provide convincing evidence that formation of
the -myosin heavy chain
(
MyHC) distal muscle CAT (MCAT) element in muscle fiber
type-specific expression and mechanical overload (MOV) responsiveness,
we conducted transgenic and in vitro experiments. In adult
transgenic mice, mutation of the distal MCAT element led to significant
reductions in chloramphenicol acetyltransferase (CAT) specific activity
measured in control soleus and plantaris muscles when compared with
wild type transgene
293WT but did not abolish MOV-induced CAT
specific activity. Electrophoretic mobility shift assay revealed the
formation of a specific low migrating nuclear protein complex (LMC) at
the
MyHC MCAT element that was highly enriched only when
using either MOV plantaris or control soleus nuclear extract.
Scanning mutagenesis of the
MyHC distal MCAT element revealed that
only the nucleotides comprising the core MCAT element were essential
for LMC formation. The proteins within the LMC when using either MOV
plantaris or control soleus nuclear extracts were antigenically related
to nominal transcription enhancer factor 1 (NTEF-1), poly(ADP-ribose) polymerase (PARP), and Max. Only in vitro translated TEF-1
protein bound to the distal MCAT element, suggesting that this
multiprotein complex is tethered to the DNA via TEF-1. Protein-protein
interaction assays revealed interactions between nominal TEF-1, PARP,
and Max. Our studies show that for transgene
293 the distal MCAT element is not required for MOV responsiveness but suggest that a
multiprotein complex likely comprised of nominal TEF-1, PARP, and Max
forms at this element to contribute to basal slow fiber expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-myosin heavy chain
(
MyHC)1 expression is
detected in the heart, and subsequently its expression becomes
primarily restricted to adult stage type I skeletal muscle fibers
(1-4). Once myofibers have differentiated and acquired a distinct
adult phenotype, the molecular properties of the fiber are not static
but instead are remarkably malleable in response to a broad spectrum of
physiological signals (5). A well documented example of this phenotypic
plasticity is the induction of
MyHC expression in fast type II
fibers in response to increased neuromuscular activity imposed by
mechanical overload (MOV) (5, 6). The regulated control of
MyHC
expression has been shown to involve the summation of inputs from
multiple cis-acting modules located within both the
MyHC
proximal promoter and more distal regulatory regions (3, 6-11).
However, the precise contribution of each discrete element comprising
these regions has not been systematically analyzed in the in
vivo context. In addition, the identity of the cell-specific
and/or ubiquitous transcription factors that direct
MyHC slow fiber
restricted expression or MOV responsiveness in adult stage skeletal
muscle have not been determined as yet.
MyHC gene, we
have conducted an extensive in vivo deletion analysis of the
human
MyHC promoter in the context of the transgenic mouse (4, 6,
8-11). These studies defined a minimal promoter region comprised of
293 bp of the
MyHC promoter (transgene
293WT) that closely mimics
the expression pattern of the endogenous
MyHC gene at all
developmental stages, and in response to MOV (4, 9). Most striking was
the finding that the deletion of an 89-bp region (
293 to
205)
resulted in the loss of transgene
293WT expression and MOV
responsiveness, thus indicating that this 89-bp region contains
regulatory elements important for both basal slow fiber expression and
MOV responsiveness of the
MyHC gene (9). Further analysis of this
89-bp region led to the identification of a putative MOV element
(
A/T-rich element:
269 to
258) that formed an enriched
multiprotein binding complex only when MOV-plantaris nuclear extract
was used (8). In addition to this A/T-rich element, the 89-bp region
also contains a muscle CAT (MCAT) site. Whether this regulatory element
plays a functional role in conferring
MyHC basal slow fiber
expression and/or MOV responsiveness has not been investigated as yet.
MyHC gene expression in cardiomyocytes
requires an E-box/MCAT composite element that involves the cobinding of NTEF-1 and the basic helix-loop-helix leucine zipper transcription factor Max. Additionally, Ojamaa et al. (24) have
demonstrated that the
MyHC E-box/MCAT element can serve as a
contractile/mechanical responsive element in cardiomyocytes and under
conditions of increased contractile activity binds the basic
helix-loop-helix leucine zipper protein, upstream stimulatory factor
(USF).
MyHC gene is regulated by mechanical stimuli and contains within
its proximal promoter two closely spaced MCAT elements; a distal MCAT
(
290/
284) and a proximal MCAT (
210/
203). These two distinct
MCAT elements are highly conserved and located within a region required
for both slow fiber expression and MOV responsiveness of a 293-bp human
MyHC transgene (
293) in adult skeletal muscle (4). Our previous
EMSA analysis revealed that only the distal MCAT element formed a
series of protein complexes displaying low, intermediate, and high
migrating mobility similar to those formed at the cTnT MCAT element,
and in addition, the low mobility complex was enriched only when using
MOV-P nuclear extract (25). On the basis of these data obvious
questions arise. First, does the distal MCAT element (
293/
284) bind
both TEF-1 and PARP? Second, does this element contribute to both basal
slow fiber expression and MOV responsiveness of the minimal 293-bp
human
MyHC transgene?
MyHC
transgene (
293WT) or a 293-bp MCAT element mutant transgene (
293Mm). These mice were used in studies aimed at determining whether the distal MCAT element is required for: 1) basal slow fiber
expression, 2) muscle-specific expression, and 3) MOV
responsiveness. We also conducted a detailed molecular analysis to
determine what nuclear protein(s) comprise the low mobility binding
complex formed at the distal MCAT element. Our findings show that the
MyHC distal MCAT element contributes to basal slow fiber expression
of transgene
293 but is not required for MOV-responsive expression
of transgene
293 in adult transgenic mice. The multiprotein complex
formed at the
MyHC distal MCAT element is comprised of NTEF-1, PARP, and Max. DNA binding studies indicate that PARP and Max are bound to
the distal MCAT element via TEF-1, whereas protein-protein interaction
assays revealed that these three proteins physically interact in
vitro. Collectively, these data support the notion that the
assembly of TEF-1, PARP, and Max at the
MyHC distal MCAT element
(LMC) contributes to basal slow fiber expression of transgene
293 in
adult stage skeletal muscle.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MyHC 293-bp transgene (
293) used in this study has been described
previously (4, 9). Briefly,
MyHC transgenes consist of 293 bp of
human
MyHC 5'-promoter sequence and 120 bp of 5'-untranslated region
(includes exon 1), fused to the 5'-end of the bacterial CAT gene (see
Fig. 1). The transgenes were released free of pSVOCAT vector sequence
by NdeI/BamHI restriction digest, and the digest
products were fractionated by agarose gel electrophoresis. The
transgene insert DNA was electroeluted from the gel, concentrated by
ethanol precipitation, and dialyzed against microinjection buffer (10 mM Tris-HCl, pH 7.4, 0.15 mM EDTA) for 48 h at 4 °C. As a final step in transgene construct purification, the
dialyzed DNA was passed through a 0.2-µm filter.
293 promoter was mutated within
the plasmid p
293CAT using the QuikChangeTM site-directed
mutagenesis kit (Stratagene) according to the manufacturer's instructions. Complementary oligonucleotide primers harboring mutations
within the MCAT site were designed to meet the length and melting
temperature requirements specified by the manufacturer (mutated bases
are underlined):
5'-GCATAGTTAAGCCAGCCAAGCGCGTCTTAGGAGGCCTGGCCTGGG-3'. Base pair substitutions were incorporated at nucleotides determined by
our previous diethylpyrocarbonate interference footprinting to be
crucial DNA-protein contact sites (25). Unintentional transcription
factor recognition sites were not created by these mutations as
assessed by cross-referencing the mutated primers against the
eukaryotic transcription factor data base available on the Wisconsin
Package, version 10.0, Genetics Computer Group (Madison, WI). The
PCR-mediated incorporation of mutant sequence was performed using 5 ng
of double-stranded p
293CAT DNA template utilizing the
manufacturer's recommended conditions. The PCR product was transformed
into Epicurian coli XL1-Blue supercompetant cells (Stratagene), and the resulting plasmid DNA was isolated using anion
exchange columns (Qiagen EndoFreeTM). Successful
incorporation of the mutation was verified via automated sequencing of
both strands (Applied Biosystems model 377). The distal MCAT mutant
transgene (
293Mm) was isolated and purified as described above.
293WT and mutant
293Mm) were analyzed. All
lines were maintained in a heterozygous state by continual outbreeding
to nontransgenic FVB/n mice.
293WT and
293Mm mouse genomic
DNA. PCR conditions were as described above, and the reactions
performed were with both a sense strand primer harboring the mutated
MCAT sequence on the 3' terminus
(5'-GCATAGTTAAGCCAGCCAAGCGCGTCTTAG-3') and an antisense strand primer specific to sequences within the CAT gene (5'-GGATATATCAACGGTGGTAT-3'). Under these conditions, only
genomic DNA harboring the transgene
293Mm sequence was amplified resulting in a 450-bp PCR product (see Fig. 1D).
1285, line 41) (10). The membrane
was stripped and rehybridized with a 32P-labeled
c-myc (single copy gene) cDNA probe (350 bp of exon 2)
to verify that each lane contained equal amounts of DNA.
80 °C.
Oligonucleotide probes and competitors
) containing
the pGEX-KG-TEF-1 and PGEX-5X-1-Max plasmids were grown for 15 h
at 37 °C in LB-Amp medium. Subsequently, the cells were diluted
1:100 in fresh medium, grown for 4 h
(A600 ~0.7), and then induced to
express the fusion protein at 37 °C for 4 h, using 0.4 mM isopropyl-
-D-thiogalactopyranoside. Following induction, the bacteria were pelleted by centrifugation at
5,000 × g for 10 min at 4 °C, and resuspended in
1× phosphate-buffered saline supplemented with protease inhibitors.
The cells were lysed by sonication for two 40-s pulses (while on ice).
The protein was solubilized (30 min, 4 °C) by the addition of Triton
X-100 to a final concentration of 1% in the sonicated lysate and then centrifuged at 20,000 × g for 10 min at 4 °C to
remove insoluble material. Fusion proteins were immobilized by the
addition of glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech)
to the supernatant, and the binding reaction was allowed to proceed at
4 °C for 30 min. The beads were pelleted at 500 × g
for 5 min at 4 °C and washed five times with 1× phosphate-buffered
saline. The proteins were quantified by SDS-PAGE using known
concentrations of bovine serum albumin as a standard and stained with
Coomassie Blue.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
293Mm--
We initiated our investigation into the
in vivo functional role served by the
MyHC distal MCAT
element in adult stage skeletal muscle by generating multiple
independent lines of two classes of transgenic mice (Fig.
1, A-C). The first class of
transgenic mouse carries a wild type transgene comprised of 293 bp of
human
MyHC promoter fused to the CAT reporter gene (termed transgene
293WT), whereas the second class carries the same transgene except that the highly conserved distal MCAT element has been mutated (termed
transgene
293Mm) (Fig. 1, A-C). Nucleotide mutations were introduced into the core distal MCAT element by base pair substitution and reflected sites of strong DNA-protein interaction as
assessed by our previous diethylpyrocarbonate interference footprint
analysis (25).
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Fig. 1.
MyHC distal MCAT sequence and
mutation. A, nucleotide sequence comparison showing
conservation of the distal MCAT element (shading) within the
MyHC proximal promoter of various species. See Vyas et
al. (8) for the accession number and reference of each sequence. A
gap (dot) was inserted in the pig sequence to optimize the
alignment. B, illustration of the 293-bp
MyHC wild type
and MCAT mutant transgenes analyzed in this study. Transgenes consist
of 293 bp of human
MyHC 5'-promoter sequence and 120 bp of
5'-untranslated region linked to the CAT reporter cDNA. The
open box within the 5'-untranslated region represents the
first untranslated exon. C, mutation of the distal MCAT
binding site (shading) with base pair alterations shown in
lowercase and boldface type. D,
in vivo verification of MCAT mutant sequence by PCR
amplification of
293Mm transgenic mouse genomic DNA on a 1% agarose
gel stained with ethidium bromide. Under the described PCR conditions
(see "Experimental Procedures" for details), only genomic DNA
harboring the MCAT mutant transgene sequence (
293Mm lines 4, 6, 7, and 9, lanes 3-6) produced a 450-bp PCR product
(arrow). Because of the 7-bp mismatch (overlapping the
distal MCAT core sequence) at the 3' terminus of the sense strand
primer, no PCR product was amplified in genomic DNA of
293 wild type
transgenic mice (lanes 7-9). +, positive control, amplified
product from purified
293 MCAT mutant transgene DNA construct
(lane 2). MW, DNA size marker (in base pairs) is
shown in lane 1.
293Mm. The CAT specific activity
measured in the CS and CP muscles of mice representing each of the four
independent
293Mm lines was significantly decreased as compared with
CAT specific activities measured in the CS and CP muscles obtained from
mice representing each of the three independent
293WT lines (Fig.
2, A and B, and
Table II). Our analysis of the
293Mm
transgenic lines did not reveal measurable levels of CAT specific
activity in any nonmuscle tissue examined even under conditions where
excess protein and extended incubation times were used (50 µg
protein/overnight incubation) (data not shown). These data indicate
that the distal MCAT element is not required to direct muscle specific
expression of transgene
293 in the chromosomal context. Because this
result was observed for all four independent transgenic lines each
carrying different transgene copy numbers, it is unlikely that
chromosomal position or transgene copy number accounts for these
results.
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Fig. 2.
Effects of MCAT element mutation on
293 CAT specific activity. A, representative
CAT assay demonstrating the expression levels in CS extracts of
293
wild type and MCAT mutant transgenic lines. All samples were incubated
at 30 µg for 17 h to illustrate the low expression levels of the
MCAT mutant lines relative to the wild type lines. Extracts obtained
from nontransgenic (Ntg) mouse soleus muscle served as the
negative control, and purified CAT protein (70 ng) was used as a
positive control. B, bar graph represents comparison of wild
type (black bars) and MCAT mutant (gray bars)
293 transgene expression levels in soleus muscle. Error
bars correspond to the S.E. for six or more independent animals
for each line.
Response of wild-type 293 (
293wt) and MCAT mutant
293
(
293Mm) transgene activity
293wt, line 2, CP/MOV-P, 20 µg/60 min; CS, 10 µg/60 min; line 96, CP/MOV-P/CS 7.5 µg/30 min; Line 99, CP/MOV-P/CS
7.5 µg/30 min.
293Mm, CP/MOV-P for lines 4, 6, 7, and
9, 30 µg/17 h; CS, lines 4 and 7, 30 µg/60 min; line 6, 15 µg/60
min; line 9, 10 µg/17 h. Note that CAT-specific activity for
293
wild-type lines 96 and 99 have been previously reported by McCarthy
et al. (9).
293Mm--
Our previous transgenic
analysis has revealed that the
MyHC distal MCAT element
(
290/
284) is located within an 89-bp region (
293/
205) that is
required for MOV responsiveness of transgene
293WT (4). Therefore,
to determine whether the distal MCAT element functions as an
MyHC
MOV-responsive element, CAT specific activity was measured in sham
operated CP and MOV-P muscles of transgenic mice carrying either
transgene
293WT or
293Mm following an 8-week period of MOV. For
mice representing each of the three independent lines carrying
transgene
293WT, expression assays revealed that the CAT specific
activity measured in MOV-P muscle extract was 2.9-7.5-fold higher than
that measured in sham operated CP muscle extract (Table II).
Interestingly, following the 8-week MOV period, the CAT specific
activity measured in the MOV-P muscle obtained from mice representing
each of the four independent
293Mm transgenic lines was also
1.5-7.1-fold higher than that measured in corresponding CP muscles
(Table II).
MyHC
distal MCAT element is required for basal slow muscle fiber expression
levels of transgene
293 in both soleus and plantaris muscles.
However, this element is not required for MOV responsiveness because
its mutation did not alter the relative fold induction of transgene
293Mm in the MOV-P muscle.
MyHC
Distal MCAT Element--
To determine whether the nuclear protein
binding properties of the distal MCAT element differed between muscles
comprised of different proportions of histochemically classified slow
type I fibers, we performed an EMSA analysis using nuclear extracts isolated from rat CS (78-90% type I fibers), CP (4-8% type I
fibers), and MOV-P (30% type I fibers) muscles. EMSA analysis of
binding reactions containing the 21-bp double-stranded
32P-labeled wild type distal MCAT probe (Table I) and 3.5 µg of either CP, MOV-P, or CS nuclear extract revealed the formation of multiple binding complexes (Fig. 3).
These binding complexes are referred to as LMC, intermediate mobility
complex (IMC), and high mobility complex (HMC). The LMC appeared as a
single band that varied in quantity concordant to the proportion of
slow type I fibers populating the muscles from which the nuclear
extract was isolated, that is, CS > MOV-P > CP. The IMC
consisted of two distinct bands whose intensity appeared to vary
inversely to the amount of LMC that was formed. The high mobility
complex consisted of multiple bands; however, only one band was easily
distinguishable in our experiments regardless of the amount of nuclear
extract used or the length of time the gel was exposed to film.
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Fig. 3.
EMSA analysis of skeletal muscle nuclear
extract binding at the distal MCAT element.
32P-Labeled distal MCAT probe was incubated with 3.5 µg
of either CP, MOV-P, or CS nuclear extract. Binding complexes were
resolved by PAGE as described under "Experimental Procedures." Note
the enriched DNA-protein interactions at the LMC for the MOV-P
(lane 2) and CS (lane 3) nuclear extract but not
for the CP (lane 1) nuclear extract. Free probe represents
excess unreacted radiolabeled oligonucleotide.
MyHC and cTnT Distal MCAT Elements Are
Similar--
Interestingly, Larkin et al. (20) have
recently reported a similar binding pattern (LMC, IMC, HMC) as assessed
by EMSA analysis using the cardiac troponin T (cTnT) MCAT elements
(MCAT1 and MCAT2) and chicken embryonic skeletal muscle nuclear
extract. Therefore, to address the possibility that the same proteins
bind to the
MyHC distal MCAT and cTnT MCAT elements, we performed
competition EMSA using as competitor the cTnT and
MyHC MCAT elements
(Fig. 4). The addition of 100-fold molar
excess cold wild type distal
MyHC MCAT probe to binding reactions
containing either CP (Fig. 4, lane 1 versus
lane 2), MOV-P (lane 6 versus lane 7), or CS (lane 11 versus 12) nuclear extract completely abolished
complex formation, indicating that these complexes are specific.
Interestingly, the addition of 100-fold molar excess cold cTnT MCAT1
probe to binding reactions containing either CP (Fig. 4, lane 1 versus lane 3), MOV-P (lane 6 versus lane 8), or CS
(lane 11 versus lane 13) prevented IMC and HMC formation and
competed away most of the LMC formation. Similarly, competition using
the
MyHC E-box/MCAT composite element completely abolished the
formation of the IMC and HMC and abolished nearly all of the LMC (data
not shown). As an additional competitor, we used the muscle creatine
kinase transcriptional regulatory factor x element that has previously been shown not to bind TEF-1 when using TEF-1-containing MM14 nuclear
extract, despite its relative sequence homology to MCAT consensus
elements (32). The addition of 100-fold molar excess cold
transcriptional regulatory factor x element to binding reactions containing either CP (Fig. 4, lane 1 versus lane 4), MOV-P
(lane 6 versus lane 9), or CS (lane 11 versus lane
14) nuclear extract did not effectively compete for the formation
of the LMC, IMC, or HMC.
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Fig. 4.
Competition EMSA assessment of
sequence-specific DNA-protein binding at the distal MCAT element.
3.5 µg of either CP (lanes 1-5), MOV-P (lanes
6-10), or CS (lanes 11-15) nuclear extract was
incubated in the presence of the 32P-labeled distal MCAT
oligonucleotide. For analysis of sequence specificity of binding at
this MCAT element, the following cold (nonradioactive) competitors were
added to the reaction mixture at a 100-fold molar excess prior to the
addition of the probe: distal MCAT (lanes 2, 7,
and 12), cTnT MCAT (lanes 3, 8, and
13), MCK transcriptional regulatory factor x (lanes
4, 9, and 14), and the MCK high affinity
right E-box (lanes 5, 10, and
15).
MyHC and cTnT MCAT elements, we used
the high affinity MCK E-box as competitor to determine whether the
E-box in the immediate 5'-flanking region of the
MyHC distal MCAT
element is required for complex formation. Addition of 100-fold molar
excess cold MCK E-box element to binding reactions containing either CP
(Fig. 4, lane 1 versus lane 5), MOV-P (lane 6 versus
lane 10), or CS (lane 11 versus lane 15) nuclear
extract slightly abolished formation of the LMC only. Collectively, our direct and competition EMSA data provide evidence that: 1) nuclear protein binding at the
MyHC distal MCAT element is specific, 2)
distinct patterns of nuclear protein binding complexes were formed when
using either CP, MOV-P, or CS nuclear extracts, and 3) the nuclear
proteins forming the LMC preferentially bind the
MyHC distal MCAT
element versus the cTnT and
MyHC MCAT elements. This
difference is likely due to sequence specific differences between these
three MCAT elements (Table I).
MyHC Distal Core MCAT Element Are
Essential for Skeletal Muscle Nuclear DNA-Protein Binding Complex
Formation--
To identify nucleotides within the 21-bp
MyHC distal
MCAT oligonucleotide probe that interact with MOV-P and CS nuclear
protein to form the LMC, IMC, and HMC, we performed competition EMSA
using scanning mutagenesis. In these experiments, we introduced
nucleotide substitutions two base pairs at a time starting within the
immediate 5'-flanking region (E-box) and extending throughout the
core MCAT element and its immediate 3'-flanking region (Fig.
5A). The resulting unlabeled
oligonucleotides were then added in 100-fold molar excess to binding
reactions containing either MOV-P or CS nuclear extract and the
32P-labeled wild type distal MCAT probe (Fig.
5B). As shown previously, the addition of excess wild type
MyHC distal MCAT probe to binding reactions containing either MOV-P
(Fig. 5B, lane 1 versus lane 2) or CS (lane
11 versus lane 12) nuclear extract completely abolished the
formation of all binding complexes. Similarly, all binding complexes
were effectively competed away by the addition of either distal MCAT
mut-1, mut-2 (contain mutations within E-box), or mut-7 (3'-flanking
region) to binding reactions containing either MOV-P (Fig.
5B, lane 1 versus lanes 3, 4, and
9) or CS (lane 11 versus lanes 13, 14,
and 19) nuclear extract. Importantly, mutant MCAT probes
carrying nucleotide substitutions within the core MCAT element (mut-3,
mut-4, mut-5, and mut-8) did not act as effective competitors of
complex formation when added to binding reactions containing MOV-P
(Fig. 5B, lane 1 versus lanes 5-7 and
10) or CS (lane 11 versus lanes 15-17 and
20) nuclear extract. Interestingly, the addition of distal
MCAT mut-6 probe to binding reactions containing MOV-P (Fig.
5B, lane 1 versus lane 8) or CS (lane 11 versus lane 18) nuclear extract completely abolished LMC formation
but not IMC or HMC formation. These data confirm our previous findings using diethylpyrocarbonate interference footprinting (25) by showing
that nucleotides comprising the core
MyHC distal MCAT element are
critical for the formation of all binding complexes when using either
MOV-P or CS nuclear extract (Fig. 5C).
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Fig. 5.
Competition EMSA determination of specific
nucleotides involved in DNA-protein interactions at the distal MCAT
element. A, summary of MCAT mutant oligonucleotides
used in competition EMSA shown in B. wt, wild
type or nonmutated distal MCAT sequence. The core MCAT sequence is
indicated with shading. Mutated bases are
lowercase and in boldface type. +++, competition
was as effective as distal MCAT wild type sequence; ++, partially
effective competition; , no detectable competition. B,
32P-labeled distal MCAT oligonucleotide was incubated in
the presence of either MOV-P (lanes 1-10) or CS
(lanes 11-20). Where indicated, 100-fold molar excess of
either distal MCAT oligonucleotide (lanes 2 and
12) or various distal MCAT mutant oligonucleotides
(harboring base pair mutations shown in A) were added to the
reaction prior to the addition of the probe. C, distal MCAT
oligonucleotide with MCAT core sequence is shaded, and E-box
sequence is in boldface type. Two base pair numbering above
the oligo sequence represents specific nucleotide mutation. The
black box delineates nucleotides whose mutation renders the
distal MCAT an ineffective competitor (especially of the LMC),
suggesting this region as a putative binding site for the LMC.
Diethylpyrocarbonate interference footprint (25) is shown here to
illustrate the DNA-protein binding profile. Open circles
denote partial interference; closed circles depict complete
interference. Sequence numbering begins at the 5'-end of the sense
strand.
MyHC Distal
MCAT Element--
Previous work has shown that TEF-1 and Max interact
at the
MyHC E-box/MCAT composite element, and that TEF-1 and PARP
associate at the cTnT MCAT1 elements (22, 23). In addition, Max has been identified as a component of the cTnT MCAT binding complex formed
when using neonatal cardiomyocyte nuclear extracts (23). Thus, to
assess whether these nuclear proteins were components of the LMC, IMC,
and HMC formed at the
MyHC distal MCAT element, we performed
antibody EMSAs using polyclonal antibodies that recognize each of the
aforementioned proteins. The specific binding complexes formed at the
32P-labeled human
MyHC distal MCAT element when reacted
with CP (Fig. 6, lane 1 versus lane 2), MOV-P (lane 4 versus lane
5), or CS (lane 13 versus lane 14) nuclear extract were
not altered by preincubation with preimmune serum. The addition of
polyclonal NTEF-1 antibody to binding reactions containing CP
(lane 1 versus lane 3), MOV-P (lane 4 versus lane
6), or CS (lane 13 versus lane 15) nuclear extract
supershifted the top band of the IMC doublet and the prominent HMC
band. In addition, the enriched LMC observed when using MOV-P nuclear
extract appeared to be nearly immunodepleted, whereas the highly
enriched LMC formed when using CS nuclear extract was partially
abolished. Interestingly, the addition of either polyclonal PARP or Max
antibody to binding reactions using MOV-P nuclear extract essentially
immunodepleted the LMC, which resulted in an embellishment of the top
band of the IMC, which is comprised entirely of NTEF-1 protein (Fig. 6,
lane 4 versus lanes 7 and 8). Identical results
were obtained when CS nuclear extract was used in binding reactions
with the exception that the highly enriched LMC was again only
partially abolished (Fig. 6, lane 13 versus lanes 16 and
17). When all combinations of the three polyclonal antibodies were added to binding reactions using MOV-P (Fig. 6, lane 4 versus lanes 9-12) or CS
(lane 13 versus lanes 18-21) nuclear extract, the LMC
was immunodepleted. The preincubation of binding reactions containing
either CS or MOV-P nuclear extract with antibodies recognizing other
E-box binding basic helix-loop-helix and basic helix-loop-helix leucine
zipper proteins (MyoD, myogenin, E2A, HEB, and USF) did not alter
MyHC distal MCAT element binding complex formation, or mobility
(data not shown). These data support three notable conclusions: 1) the
LMC formed at the human
MyHC distal MCAT element is comprised of at
least three proteins antigenically related to NTEF-1, PARP and Max, 2)
the prominent HMC band and the top band of the IMC doublet are
comprised of protein antigenically related to NTEF-1 or possibly TEF-1
isoprotein(s) recognized by the NTEF-1 polyclonal antibody used in
these experiments, and 3) the bottom band of the IMC doublet was not
supershifted/immunodepleted by the addition of any antibodies used
herein and thus may be comprised by a TEF-1 isoform not recognized by
the polyclonal NTEF-1 antibody or an as yet unidentified nuclear
protein(s).
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Fig. 6.
Antibody supershift EMSA analysis of muscle
nuclear extract binding complexes at the distal MCAT element.
Supershift EMSAs were performed by preincubation of CP (TEF-1 Ab only,
lane 3), MOV-P, or CS nuclear extract with 1 µl of
anti-Max, anti-PARP, or anti-TEF-1 Ab for 30 min at room temperature
prior to the addition of the labeled distal MCAT probe. Various
combinations of antibodies were used as indicated by the plus
sign above each lane. TEF-1 SS marks the supershifted
TEF-1 components of the IMC and HMC. Autoradiographic exposure time for
the CS complexes (lanes 13-21) was reduced to visualize the
effects of the various antibodies on the LMC without interference from
the supershifted TEF-1 complex. Control reactions were performed with
preimmune serum (PI; lanes 2, 5, and
14). Ab, antibody.
MyHC
Distal MCAT Element in Vitro--
Because our antibody supershift
assays indicate that proteins antigenically related to Max, NTEF-1, and
PARP comprise the LMC formed at the
MyHC distal MCAT element, it was
important for us to determine the ability of these three factors to
independently bind to the 32P-labeled human
MyHC distal
MCAT element. EMSA analysis of binding reactions containing the
32P-labeled human
MyHC distal MCAT element and rabbit
reticulocyte lysate-generated in vitro translated TEF-1
isoproteins (Fig. 7A, inset
shows expected 54-kDa TEF-1 proteins) revealed the formation of
specific binding complexes that were different from the nonspecific complex formed when unprogrammed lysate was used (Fig. 7A,
lane 1 versus lanes 2-5). In contrast, a binding complex
was not formed when either [35S]methionine-labeled
in vitro translated PARP (Fig. 7B,
inset shows expected 120-kDa PARP protein) or Max (Fig.
7C, inset shows expected 22-kDa Max protein) were
reacted with the
MyHC distal MCAT element (Fig. 7B,
lanes 1-4, Max, and data not shown). The inability of PARP
or Max to form a binding complex with the
MyHC distal MCAT element
remained, even when using 10-100 ng of either purified recombinant
baculovirus expressed PARP (data not shown) or Max (Fig. 7C,
lanes 1-3). Further, we were unable to reconstitute the
formation of a
MyHC distal MCAT LMC in our in vitro
binding assays despite the simultaneous use of TEF-1, PARP, and Max, in binding reactions (data not shown). We were, however, able to detect
DNA-protein binding when adding either 5 or 10 ng of purified Max to
binding reaction containing the CM-1 oligonucleotide (contains (33)
previously shown Myc/Max E-box binding site CACGTG (23, 34)) (Fig.
7C, lanes 4-6), indicating that the purified Max protein used in our LMC reconstitution experiments possessed DNA binding capabilities.
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Fig. 7.
EMSA analysis of recombinant TEF-1, PARP, and
Max binding at the distal MCAT element. A,
inset, reveals the correct size of
[35S]methionine-labeled TEF-1. The rabbit reticulocyte
lysate system was programmed with 1 µg of circular TEF-1 expression
plasmids in the presence of [35S]methionine. The TnT
product was resolved by 12% SDS-PAGE and exposed to film. Molecular
mass markers (in kDa) are shown on the left. Main
panel, 32P-labeled distal MCAT probe was incubated
with rabbit reticulocyte lysate corresponding to various TEF-1 isoforms
as indicated (lanes 2-5). , labeled distal MCAT
electrophoresed in the absence of protein extract (lane 6).
NS, nonspecific binding endogenous to the rabbit
reticulocyte lysate system. B, inset, reveals the
correct size of [35S]methionine-labeled PARP. Main
panel, distal MCAT probe was incubated in the presence of UL
(lane 1) or increasing amounts (1, 5, and 10 µl) of PARP
cDNA programmed rabbit reticulocyte lysate. No difference was
observed in sequence-specific DNA-protein binding between the UL and
PARP programmed reactions. NS, nonspecific binding
endogenous to the UL. C, left inset, reveals
correct size of [35S]methionine-labeled Max. Right
inset, Coomassie-stained SDS-PAGE shows correct size of
recombinant baculovirus expressed Max (200 ng). Main panel,
radiolabeled distal MCAT (lanes 1-3) or CM-1 (lanes
4-6) were incubated with purified baculovirus expressed Max in
the amounts indicated.
MyHC distal MCAT element; therefore, the protein-protein
interactions within our LMC remained to be identified. Importantly,
previous work has shown a stable protein-protein interaction between
RTEF-1 and PARP (22) and between NTEF-1 and Max (23), and, therefore,
it was logical to test for protein-protein interactions between NTEF1,
PARP, and Max. To this end, we performed in vitro pairwise
protein-protein interaction assays wherein
[35S]methionine-labeled in vitro translated
Max, NTEF-1, or PARP protein were individually reacted with either
bacterially expressed GST-NTEF-1 or GST-Max fusion proteins that were
bound to glutathione-Sepharose beads (Fig.
8). Following incubation, the pelleted
glutathione-Sepharose beads/fusion protein complex was extensively
washed, and this complex was analyzed for bound
[35S]methionine-labeled in vitro translated
protein by using SDS-PAGE (Fig. 8). Importantly,
[35S]methionine-labeled in vitro translated
PARP protein was found to bind both GST-Max (Fig. 8A,
lane 3) and GST-NTEF-1 (Fig. 8C, lane
3) but not the 26-kDa GST protein (Fig. 8, A and
C, lane 2), revealing that this interaction was
specific between each respective fusion protein and in vitro
translated PARP protein. Similarly,
[35S]methionine-labeled in vitro translated
NTEF-1 and Max were found to bind specifically to GST-Max (Fig.
8B, lane 2 versus lane 3) and GST-NTEF-1 (Fig.
8D, lane 2 versus lane 3), respectively. In all
analyses described above, the [35S]methionine-labeled
in vitro translated protein, bound by the GST fusion
protein, migrated to a position in the SDS-polyacrylamide gel that
aligned with the [35S]methionine-labeled in
vitro translated protein that was not reacted with GST fusion
protein (Fig. 8, A-D, lanes 1 versus lanes 3).
When unprogrammed rabbit reticulocyte lysate was reacted with either
GST, GST-NTEF-1, or GST-Max, a [35S]methionine-labeled
in vitro translated protein product was not observed,
further demonstrating specificity of the interactions between the
fusion protein and the in vitro translated product (Fig. 8,
A-D, lanes 4-6). Collectively, these data
indicate that the assembly of the
MyHC distal MCAT multiprotein LMC
involves DNA binding of TEF-1, and that TEF-1, PARP, and Max are
capable of protein-protein interactions between each other. However, it remains to be determined conclusively whether the LMC involves a
ternary complex comprised of these proteins or alternatively two
subsets of paired protein complexes (see "Discussion").
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Fig. 8.
Protein-protein interactions between TEF-1,
PARP, and Max. Full-length Max and NTEF-1 were prepared as GST
fusion proteins and immobilized on Sepharose beads as described under
"Experimental Procedures." Immobilized GST, GST-Max, or GST-TEF-1
was incubated with [35S]methionine-labeled PARP TnT
(A and C, lanes 2 and 3),
NTEF-1 TnT (B, lanes 2 and 3), or Max
TnT (D, lanes 2 and 3). The labeled
proteins bound to the beads were analyzed by 4-20% SDS-PAGE. Positive
control labeled TnT reactions are shown for each panel in lane
1. Molecular mass markers in kDa are shown to the
left of each panel.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
290/
284) contributes
significantly to basal slow muscle expression of chromosomally
integrated transgene
293 but is not absolutely required for MOV
responsiveness. Furthermore, our in vivo protein-protein
interaction studies elucidate the possibility that in adult skeletal
muscle NTEF-1, PARP, and Max may physically interact to form a ternary
binding complex at the
MyHC distal MCAT element.
MyHC Distal MCAT Element Reveals That It Contributes to Basal Slow
Fiber Expression but Is Not Absolutely Required for MOV
Responsiveness--
In adult mice, basal expression levels of the
endogenous
MyHC gene is high in the slow twitch soleus muscle,
whereas it is not expressed to any appreciable degree in the fast
twitch plantaris muscle, an expression pattern mimicked by wild type
transgene
293WT (4, 9). Our analysis of mutant transgene
293Mm
basal expression revealed that mutation of the distal MCAT element
significantly decreased but did not abolish expression in the slow
twitch soleus muscle (Fig. 2, A and B, and Table
II). In fact, even though the magnitude of mutant transgene
293Mm
expression was decreased, its expression pattern (soleus > plantaris) still mirrored that of wild type transgene
293WT. These
results showing low, yet persistent expression of mutant transgene
293Mm strongly suggest that other cis-acting elements
within the 293-bp human
MyHC basal promoter must be acting in
concert to preserve wild type levels of slow muscle expression.
Candidate sites within the
MyHC proximal control region
(
293/
170) that may act in combination with the distal MCAT element
to direct high levels of slow muscle expression are an A/T-rich
(
269/
258), CCAC/Sp1 (C-rich,
242/
231), and NFAT (
179/
171).
In this regard, our results from EMSA and transgenic mouse analysis
indicate that the
MyHC NFAT and
A/T-rich elements contribute to
slow muscle expression because their independent mutation in the
context of transgene
293 (
293Nm,
293A/Tm) significantly decreased slow muscle
expression.2 Our data
provide evidence that the combined inputs from the distal MCAT,
A/T-rich, and
NFAT elements underlie transgene
293WT slow
muscle gene expression in adult transgenic mice. Indirect support for
this notion comes from recent findings implicating NFAT and MEF2
proteins as downstream mediators of calcium-activated intracellular
signaling pathways that have been proposed to regulate the slow gene
program (Ref. 39 and references within). Additional work is underway to
clarify whether these three elements collaborate to direct slow muscle
specific expression.
293WT, we investigated its role in MOV-inducible
expression. Our analysis revealed that the range of MOV-induced CAT
specific activity measured in the MOV-P muscle of mice harboring mutant
transgene
293Mm was almost identical to those carrying wild type
transgene
293WT, indicating that the distal MCAT element is not
absolutely required for MOV responsiveness of transgene
293 (Table
II). Although MOV induction could still be achieved when the distal
MCAT element was mutated (
293Mm), this element still appears to be
necessary to achieve high levels of transgene
293 induction in
response to MOV (Table II), implicating the requirement for additional
element(s). In support of this notion, we have recently identified by
EMSA analysis a putative MOV element (A/T-rich element (
269/
258))
that demonstrates enriched binding of two unidentified nuclear proteins
(44 and 48 kDa) only under MOV conditions (8). Thus the
A/T-rich and
distal MCAT elements may participate collaboratively to confer MOV
responsiveness to transgene
293, a study currently under investigation.
293Mm Does Not Display a Loss of Muscle
Specific Expression--
Previous studies by Rindt et al.
(7) reported that mutation of the distal MCAT element within a
600-bp mouse
MyHC promoter (
0.6MCAT) led to the loss of
muscle specific expression (7). Regardless of the aforementioned
findings, we did not detect measurable levels of transgene
293Mm in
any nonmuscle tissue examined (data not shown). Several explanations
may account for these different observations. First, the loss of mouse
MyHC transgene
0.6MCAT muscle specific expression was examined in
only one transgenic line; therefore, this result may reflect
chromosomal integration site effects as opposed to the loss of promoter
regulation. Second, the levels of human transgene
293Mm studied
herein are on average lower than those of mouse transgene
0.6MCAT,
which may have resulted in our missing possible aberrant (nonmuscle)
expression. Last, our mutation was restricted to a 5-nucleotide
substitution within the core MCAT element, whereas the mutation within
mouse transgene
0.6MCAT involved substitution of 19 nucleotides that
spanned the core MCAT as well as both 5' and 3' regions (7). Based on
the findings of Larkin et al. (20) concerning the
requirement of the MCAT 5'-flanking nucleotides to achieve cTnT muscle
specific gene expression, it is reasonable to suggest that mutation of the immediate 5'-flanking region may have altered muscle specific expression.
MyHC Distal MCAT LMC Is a Multiprotein Complex and Is Likely
Comprised of TEF-1, PARP, and Max--
Because of the relevance of
previous findings showing physical interactions between NTEF-1 and Max
(23) as well as RTEF-1 and PARP (22), it was important that we
determine whether these same proteins comprise the LMC that forms at
the
MyHC distal MCAT element when using adult stage MOV-P or soleus
nuclear extract. Our EMSA and protein-protein interaction assays
provide multiple lines of evidence that TEF-1, PARP, and Max most
likely comprise a ternary protein complex that binds the
MyHC distal
MCAT element to form the LMC. First, EMSA analysis revealed the
formation of a LMC with a mobility that resembled the LMC formed at the
cTnT element (Fig. 3). Second, in competition EMSAs, oligonucleotides containing either the
MyHC E-box/MCAT site or the cTnT MCAT1 element
abolished the formation of most but importantly not all of the LMC
formed at the
MyHC distal MCAT element. This is most apparent when
using CS nuclear extract (Fig. 4, lane 13). Alternatively, these findings might also suggest that the proteins comprising the LMC
at these three different MCAT elements may be the same but reflect
differences in binding affinity. Third, the use of polyclonal
anti-NTEF-1, PARP and Max, antibodies revealed that the LMC contained
proteins antigenically related to TEF-1, PARP, and Max (Fig. 6).
Fourth, our in vitro protein-protein interaction assays show
that NTEF-1, PARP, and Max all physically interact with each other
(Fig. 7). Alternatively, when considering the third and fourth issues
above, it remains possible that two different populations of LMC exists
in the form of TEF-1/PARP and TEF-1/Max. Although this is possible, it
seems unlikely because there would be a difference of approximately
100-kDa between these two distinct LMCs. For example, a PARP (120-140
kDa)/TEF-1 (54 kDa) protein complex would have a mass of 174-194 kDa,
whereas a TEF-1/Max (22 kDa) protein complex would have a mass of 76 kDa. This 100-120 kDa difference in mass would likely be resolved in
our EMSA assays. Last, in DNA binding assays, our experiments reveal
that of the three proteins tested, only in vitro synthesized
TEF-1 isoproteins bind to the
MyHC distal MCAT element (Fig.
8A), indicating that the putative ternary protein complex is
tethered to this element via TEF-1 DNA-protein binding.
MyHC, cTnT, and
MyHC MCAT elements are the same, our experiments
show that the DNA binding properties of the
MyHC distal MCAT element
differ, possibly explaining why Max and PARP did not bind this element.
This important distinction is supported by differences in footprint
patterns obtained for the
MyHC, cTnT, and
MyHC MCAT elements. Our
footprint analysis (25) in conjunction with our current scanning
mutagenesis results provide evidence that strong DNA-protein
interactions occurred throughout the core MCAT element with weaker
interactions in the flanking regions (Fig. 5). In contrast, similar
analysis of the cTnT MCAT1 element revealed strong DNA-protein
interactions occurring at the 5'-flanking nucleotides, the proposed
site (5'-TGTTG-3') of PARP binding that is conspicuously absent from
the
MyHC distal MCAT oligonucleotide (Refs. 20 and 22 and Table I).
Likewise, analysis of the
MyHC E-box/MCAT hybrid element revealed
strong DNA-protein interactions spanning the 5'-flanking nucleotides
containing the high affinity Max target binding site (CACGTG) that is
also absent from the
MyHC distal MCAT oligonucleotide (Ref. 40 and
Table I). The lack of Max DNA binding to our MCAT element was not
completely unexpected because Max was identified as a neonatal
cardiomyocyte nuclear protein component of a binding complex that
formed at a cTnT core MCAT oligonucleotide devoid of an E-box element
(23). Consequently, this same oligonucleotide was shown to be incapable of binding purified Max protein, showing that Max can participate in
transcriptional regulation without binding DNA.
MyHC distal MCAT element LMC is primarily bound by TEF-1 protein and
that PARP and Max may weakly interact with flanking nucleotide.
MyHC distal MCAT LMC when using either MOV-P or soleus nuclear
extract is comprised of TEF-1, PARP, and Max and that this interaction
correlates with slow muscle expression but is not required for MOV
responsiveness of wild type transgene
293WT. Mechanistically, our
findings suggest that TEF-1 binds to the core MCAT nucleotides where it
serves to activate transgene
293WT transcription. Because Max does
not contain a transactivation domain, its presence in the LMC may serve
to mediate favorable interactions between the transcription initiation
complex, as well as transcription factors bound at adjacent
cis-acting elements via protein-protein interactions. PARP,
on the other hand, has been shown to participate in diverse processes
that include DNA repair, chromatin remodeling, and gene transcription
(41). Thus, it is reasonable to speculate that nuclear PARP is
recruited to the
MyHC distal MCAT element to activate
MyHC gene
expression in slow muscle by altering local chromatin structure, or by
modulating transcription factor activity via poly(ADP)-ribosylation.
Support for the later notion comes from recent evidence showing the
involvement of calcium in the activation of nuclear PARP in neuronal
cells (42) and calcium-activated intracellular signaling pathways in
regulating, in part, the slow skeletal muscle gene program (39). In our
studies, calcium-activated mechanisms should be carefully considered
because slow type I skeletal muscle fibers have been reported to have
2-6-fold higher basal levels of intracellular calcium than fast
type II fibers (see Ref. 39 and references within), and MOV is
associated with an increased proportion of slow type I fibers (6).
Thus, it is plausible that the higher basal levels of intracellular
calcium within the soleus muscle and within the induced slow type I
fibers populating the plantaris following MOV resulted in elevated
levels of activated nuclear PARP and thus enhanced LMC formation (Figs.
3 and 4). The physiological importance of the formation of the
MyHC
distal MCAT LMC is underscored by our transgenic mouse analysis wherein
mutation of the distal MCAT element led to a significant decrease in
slow muscle expression of chromosomally located transgene
293Mm. The
investigation into the involvement of calcium-activated pathways in the
MOV induced increased proportion of slow type I fibers in adult
skeletal muscle is currently underway.
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ACKNOWLEDGEMENTS |
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We thank Dr. Irwin Davidson for TEF cDNAs, Dr. Mahesh Gupta for GST-NTEF-1, Drs. Simbulan-Rosenthal and Smulson for pBS-II SK+ PARP, Dr. R. Eisenman for the Max expression vector, Dr. D. Ayer for purified baculovirus expressed Max protein and the CM-I oligonucleotide, and Dr Hannink for pGEX-5X-1 and pGEX-KG. Also, we thank Drs. Ordahl, Larkin, Butler, and Farrance for oligonucleotides and helpful discussions. Dedicated to the memory of George W. Smith and Marcia Smith.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R01-AR41464 and R01-AR47197 (to R. T.).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: University of
Missouri-Columbia, Dept. of Veterinary Biomedical Sciences and
Department of Biochemistry, 1600 E. Rollins Ave., W112 VET Medicine
Bldg., Columbia, MO 65211. Tel.: 573-884-4547; Fax:
573-884-6890; E-mail: tsikar@missouri.edu.
Published, JBC Papers in Press, September 28, 2000, DOI 10.1074/jbc.M007750200
2 G. L. Tsika and R. W. Tsika, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
MyHC,
-myosin
heavy chain;
MOV, mechanical overload;
MOV-P, overloaded plantaris
muscle;
bp, base pair(s);
CAT, chloramphenicol acetyltransferase;
MCAT, muscle CAT;
TEF-1, transcription enhancer factor 1;
NTEF-1, nominal
TEF-1;
RTEF-1, related TEF-1;
EMSA, electrophoretic mobility shift
assay;
LMC, low mobility complex;
IMC, intermediate mobility complex;
HMC, high mobility complex;
PARP, poly(ADP-ribose) polymerase;
USF, upstream stimulatory factor;
PCR, polymerase chain reaction;
CP, control plantaris;
CS, control soleus;
TnT, troponin T;
cTnT, cardiac
TnT;
ORF, open reading frame;
PAGE, polyacrylamide gel electrophoresis;
UL, unprogrammed lysate;
GST, glutathione
S-transferase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Lyons, G. E., Ontell, M., Cox, R., Sassoon, D., and Buckingham, M. (1990) J. Cell Biol. 111, 1465-1476[Abstract] |
2. | Lyons, G. E., Schiaffino, S., Sassoon, D., Barton, P., and Buckingham, M. (1990) J. Cell Biol. 111, 2427-2436[Abstract] |
3. |
Knotts, S.,
Rindt, H.,
Neumann, J.,
and Robbins, J.
(1994)
J. Biol. Chem.
269,
31275-31282 |
4. |
Wiedenman, J. L.,
Tsika, G. L.,
Gao, L.,
McCarthy, J. J.,
Rivera-Rivera, I. D.,
Vyas, D.,
Sheriff-Carter, K.,
and Tsika, R. W.
(1996)
Am. J. Physiol.
271,
R688-R695 |
5. | Booth, F. W., and Baldwin, K. M. (1996) in Handbook of Physiology (Rowell, L. B , and Shepherd, J. T., eds) , pp. 1075-1123, Oxford University Press, New York |
6. |
Tsika, G. L.,
Wiedenman, J. L.,
Gao, L.,
McCarthy, J. J.,
Sheriff-Carter, K.,
Rivera-Rivera, I. D.,
and Tsika, R. W.
(1996)
Am. J. Physiol.
271,
C690-C699 |
7. | Rindt, H., Gulick, J., and Robbins, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1540-1544[Abstract] |
8. |
Vyas, D. R.,
McCarthy, J. J.,
and Tsika, R. W.
(1999)
J. Biol. Chem.
274,
30832-30842 |
9. |
McCarthy, J. J.,
Vyas, D. R.,
Tsika, G. L.,
and Tsika, R. W.
(1999)
J. Biol. Chem.
274,
14270-14279 |
10. |
Wiedenman, J. L.,
Rivera-Rivera, I.,
Vyas, D.,
Tsika, G.,
Gao, L.,
Sheriff-Carter, K.,
Wang, X.,
Kwan, L. Y.,
and Tsika, R. W.
(1996)
Am. J. Physiol.
270,
C1111-C1121 |
11. |
McCarthy, J. J.,
Fox, A. M.,
Tsika, G. L.,
Gao, L.,
and Tsika, R. W.
(1997)
Am. J. Physiol.
272,
R1552-R1561 |
12. | Jacquemin, P., and Davidson, I. (1997) Trends Cardiovasc. Med. 7, 192-197[CrossRef] |
13. | Larkin, S. B., and Ordahl, C. P. (1999) in Heart Development (Rosenthal, N. , and Harvey, R. P., eds) , pp. 307-329, Academic Press, San Diego, CA |
14. | Davidson, I., Xiao, J. H., Rosales, R., Staub, A., and Chambon, P. (1988) Cell 54, 931-942[Medline] [Order article via Infotrieve] |
15. | Xiao, J. H., Davidson, I., Matthes, H., Garnier, J. M., and Chambon, P. (1991) Cell 65, 551-568[Medline] [Order article via Infotrieve] |
16. | Jiang, S. W., Trujillo, M. A., Sakagashira, M., Wilke, R. A., and Eberhardt, N. L. (2000) Biochemistry 39, 3505-3513[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Ueyama, T.,
Zhu, C.,
Valenzuela, Y. M.,
Suzow, J. G.,
and Stewart, A. F.
(2000)
J. Biol. Chem.
275,
17476-17480 |
18. |
Gupta, M. P.,
Kogut, P.,
and Gupta, M.
(2000)
Nucleic Acids Res.
28,
3168-3177 |
19. | Belandia, B., and Parker, M. G. (2000) J. Biol. Chem. 275, P30801-P30805 |
20. | Larkin, S. B., Farrance, I. K., and Ordahl, C. P. (1996) Mol. Cell. Biol. 16, 3742-3755[Abstract] |
21. |
Farrance, I. K.,
and Ordahl, C. P.
(1996)
J. Biol. Chem.
271,
8266-8274 |
22. |
Butler, A. J.,
and Ordahl, C. P.
(1999)
Mol. Cell. Biol.
19,
296-306 |
23. | Gupta, M. P., Amin, C. S., Gupta, M., Hay, N., and Zak, R. (1997) Mol. Cell. Biol. 17, 3924-3936[Abstract] |
24. |
Ojamaa, K.,
Samarel, A. M.,
and Klein, I.
(1995)
J. Biol. Chem.
270,
31276-31281 |
25. | Vyas, D. R., McCarthy, J. J., Tsika, G. L., and Tsika, R. W. (2000) Basic Appl. Myol. 10, 5-16 |
26. | Tsika, R. W. (1994) Exercise Sport Sci. Rev. 22, 361-388[Medline] [Order article via Infotrieve] |
27. | Tsika, R. W., Hauschka, S. D., and Gao, L. (1995) Am. J. Physiol. 269, C665-C674[Abstract] |
28. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Jacquemin, P.,
Hwang, J. J.,
Martial, J. A.,
Dolle, P.,
and Davidson, I.
(1996)
J. Biol. Chem.
271,
21775-21785 |
30. |
Jacquemin, P.,
Martial, J. A.,
and Davidson, I.
(1997)
J. Biol. Chem.
272,
12928-12937 |
31. |
Simbulan-Rosenthal, C. M.,
Rosenthal, D. S.,
Iyer, S.,
Boulares, A. H.,
and Smulson, M. E.
(1998)
J. Biol. Chem.
273,
13703-13712 |
32. |
Fabre-Suver, C.,
and Hauschka, S. D.
(1996)
J. Biol. Chem.
271,
4646-4652 |
33. | Ayer, D. E., Kretzner, L., and Eisenman, R. N. (1993) Cell 72, 211-222[Medline] [Order article via Infotrieve] |
34. | Blackwood, E. M., and Eisenman, R. N. (1991) Science 251, 1211-1217[Medline] [Order article via Infotrieve] |
35. | Deleted in proof |
36. | Deleted in proof |
37. | Deleted in proof |
38. | Deleted in proof |
39. | Olson, E. N., and Williams, R. S. (2000) Cell 101, 689-692[Medline] [Order article via Infotrieve] |
40. |
Gupta, M. P.,
Gupta, M.,
and Zak, R.
(1994)
J. Biol. Chem.
269,
29677-29687 |
41. | D'Amours, D., Desnoyers, S., D'Silva, I., and Poirier, G. G. (1999) Biochem. J. 342, 249-268[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Homburg, S.,
Visochek, L.,
Moran, N.,
Dantzer, F.,
Priel, E.,
Asculai, E.,
Schwartz, D.,
Rotter, V.,
Dekel, N.,
and Cohen-Armon, M.
(2000)
J. Cell Biol.
150,
293-308 |