From the Ottawa Hospital Research Institute, Ottawa, Ontario K1H 8L6, Canada
Received for publication, March 8, 2001
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ABSTRACT |
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In vivo studies in
the mouse have revealed that the muscle promoter of the mouse
dystrophin gene can target the right ventricle of the heart only,
suggesting the need for other regulatory elements to target the
skeletal muscle as well as other compartments of the heart. In this
study we report the identification of the mouse dystrophin gene
enhancer that is located ~8.5 kilobases downstream from the mouse
dystrophin gene muscle promoter. The enhancer was tested in myogenic
G8, H9-C2, and nonmyogenic 3T3 cell lines and is mostly active in G8
myotubes. Sequence analysis of the mouse dystrophin gene enhancer
revealed the presence of four E-boxes numbered E1-E4, a putative mef-2
binding site, and a serum response element. Site-directed mutagenesis
studies have shown that E-boxes 1, 2, and 3 as well as the serum
response element are required for enhancer activity. Gel shift analysis
revealed two binding activities at binding sites E1 and E3 which were
specific to myotubes, and supershift assays confirmed that myoD binds
at both these sites. Our study also shows that werum response factor
binds the serum response element but in myoblasts and fibroblasts only, suggesting that serum response factor may repress enhancer function.
The 2.5-megabase gene encoding the cytoskeletal protein,
dystrophin (1, 2) is the largest gene yet identified and is complex
with a minimum of seven promoters. Three promoters, muscle (3), brain
(4), and Purkinje (5), express full-length dystrophin isoforms that
localize in the skeletal/cardiac muscle, the cerebral cortex, and in
cerebellar Purkinje cells, respectively. Four other promoters located
in introns 29, 55, 59, and 68 encode shorter isoforms Dp 260 (6), Dp
140 (7), Dp 116 (8), and Dp 71 (9, 10), which are expressed in retina,
the central nervous system, the peripheral nervous system, and
non-muscle tissues, respectively. The exact role of dystrophin has yet
to be determined, but many studies suggest that in muscle it maintains the cytoarchitecture of the cell by bridging intracellular F-actin filaments to the extracellular matrix via contacts with the
dystroglycan complex (11, 12). What is known is that mutation in the
gene resulting in loss of dystrophin is the major cause of Duchenne muscular dystrophy, whereas mutations that alter or reduce the amount
of the protein generally cause the milder Becker muscular dystrophy.
Although many mutations in the dystrophin gene affect coding sequences,
a few of the known mutations affect non-coding sequences, and often
these impact on transcription of the gene. This type of mutation is not
frequently observed in Duchenne muscular dystrophy or Becker muscular
dystrophy but is more common in a group of X-linked dilated
cardiomyopathy patients (13), in which promoter deletions (14) or
splice site mutations (15) abolish expression of the muscle isoform of
dystrophin in skeletal and cardiac muscle. These individuals continue
to express full-length dystrophin in their skeletal muscle from the
brain and/or Purkinje promoters (15, 16), normally silent in this
tissue. This suggests that different regulatory elements are involved
in regulation of dystrophin gene expression in skeletal and cardiac
muscle. This conclusion is supported in mouse by in vivo
studies showing that the muscle core promoter of the mouse dystrophin
gene is capable of expressing a reporter gene only in the right
ventricle of the heart (17), suggesting that other other
cis-acting elements are required for expression in other
compartments of the heart and in skeletal muscle.
The intron-1 enhancer described previously in intron-1 of the human
gene is a good candidate for this regulatory function. Because detailed
regulatory studies of the dystrophin gene cannot be done in human, we
have turned to the mouse gene and have mapped an intron-1 enhancer
element located 8.5 kilobases
(kb)1 downstream from the
mouse dystrophin muscle promoter which shows 65% homology with its
human counterpart located 6.5 kb downstream (18) from the human
dystrophin muscle promoter. We have characterized the mouse enhancer in
G8 and H9-C2 myogenic and 3T3 non-myogenic cell lines and found that
enhancer activity is specific to differentiating myoblasts. Deletion
and site-directed mutagenesis studies have confined the enhancer
activity to a minimum of four putative binding sites. These include
three E-boxes, E1-E3, and a serum response element (SRE). E1 and E3
match the consensus 5'-AACAc/g c/g TGC a/t while E2 and a sequence
contained in the SRE resemble the consensus 5'-GGa/cCANGTGGc/gNa/g.
Factor binding studies by mobility shift analysis suggest that
ubiquitous factors bind E2 and the SRE whereas myotube-specific factors
complexed with myoD bind E1 and E3.
Isolation of the Mouse Dystrophin Muscle Promoter and the Mouse
Dystrophin Enhancer (MDE)--
A bacterial artificial chromosome (BAC)
(20) insert that contains the mouse dystrophin muscle promoter was
identified by screening a BAC genomic library (Genomic Systems Inc.)
with a probe that extends from position
To identify restriction fragments that include the mouse counterpart to
the human enhancer, the BAC clone was incubated with restriction
endonucleases EcoRI, BamHI, and
HindIII. Restriction fragments were separated by agarose gel
electrophoresis and transferred to a GeneScreen Plus membrane. The
latter was hybridized to a SpeI-SacI 195-base
pair (bp) fragment that includes the human dystrophin intron-1 enhancer
in the presence of 6 × sodium citrate, 5 × Denhardt's,
0.5% SDS, 10% dextran sulfate, and 100 µg/ml herring sperm at
50 °C for 20 h. Subsequently the membranes were washed in
6 × sodium citrate, 0.1% SDS at 50 °C for 20 min following a
wash at room temperature. Positive fragments include a
HindIII fragment of 7.0 kb and a BamHI fragment
of 11 kb which were subcloned into pBluescript SK+ to generate pmdeH
and pmdeB, respectively (Fig. 1A). Both fragments were
mapped by partial cleavage using EcoRI, BamHI,
and HindIII restriction endonucleases, and a 3.0-kb EcoRI that hybridized to the human dystrophin enhancer was
subcloned from pmdeB into the EcoRI site of pBluescript SK+
to generate pmdeE (Fig. 1B).
Luciferase Constructs--
Recombinant plasmids that were used
to characterize the dystrophin muscle enhancer activity are derived
from the pGL3 vector series containing the firefly luciferase gene
(Promega). The C3 construct (Fig. 2) was
made by inserting a 3.0-kb SacI-XhoI fragment that was isolated from pmdeE between the SacI and
XhoI sites of the pGL3-P vector (Promega) upstream of the
SV40 early promoter and the luciferase gene. To generate the constructs
C1 and C2, a 3.0-kb BamHI fragment that was isolated from
pmdeE and contains the MDE was inserted in either orientation at the
BamHI site of the pGL3-P vector.
In constructs of the A series, a fragment of 500 bp was tested for
enhancer activity. In A1 and A2, the 500-bp fragment was amplified by
polymerase chain reaction (PCR) using oligonucleotide primers
5'-ATCGTAACGCGTGTCTGACTTCTCAGTTCAGACTTTCACCTTGG
and 5'-ATCGTAACGCGTATAACACTTGATGCGTGCTGAAATG, which
contain the MluI restriction site (underlined). In A3 and A4, the 500-bp fragment was amplified using oligonucleotide primers 5'-ATCGTAGGATCCGTCTGACTTCTCAGTTCAGACTTTCACCTTGG and
5'-ATCGTAGGATCCATAACACTTGATGCGTGCTGAAATG, which
contain a BamHI restriction site. PCR products were gel purified using QIAQUICK gel extraction cartridges (Qiagen Inc.) and
subsequently cleaved with either MluI or BamHI.
MluI fragments were inserted at the MluI site of
pGL3-P uspstream of the of the SV40 promoter to generate A1 and A2.
BamHI fragments were inserted at the BamHI site
of pGL3-P downstream from the luciferase gene to generate A3 and A4.
The constructs B1 and B2 were made by deleting 220 bp from the MDE
contained in constructs A1 and A2. Thus oligonucleotide primers that
define the deletion end points
5'-ATCGTAGAATTCCTTTCCAAGGTGAAAGTCTGAACTGAG and
5'-ATCGTAGAATTCAAGTGAACGAAGACAAAATGTGACC and contain an
EcoRI restriction site (underlined) were used to amplify a
DNA fragment of 5.2 kb which is missing 220 bp of the 500 enhancer
sequencer. Subsequently the fragment was cleaved with EcoRI,
self-ligated, and transformed into Max Efficiency Escherichia
coli DH5 Site-directed Mutagenesis of Enhancer Protein Binding
Sites--
Binding determinants within the putative protein binding
sites of the MDE were replaced with restriction sites by PCR. The oligonucleotide primers that were used to introduce base changes in the
MDE binding sites E1-E4, mef-2, and SRE are listed in Table I. As a result, the bindings sites E1-E4
were replaced by a BamHI restriction site, whereas the mef-2
binding site and the SRE were replaced by MluI and
PstI restriction sites, respectively. The template used was
the B1 construct in which the BamHI site was destroyed by
filling in the cohesive ends with Klenow DNA polymerase (New England
Biolabs Inc.). The PCR products that encode mutations in E-boxes E1-E4
were cleaved with BamHI, and products encoding mutations of
the putative mef-2 and SRE binding sites were cleaved with
MluI and PstI. Cleaved DNA products were gel
purified using QIAQUICK cartridges, self-ligated using T4 DNA ligase
(New England Biolabs Inc.), and transformed into E. coli
DH5 Transfections and Biochemical Assays--
For liposome-mediated
transfections, 5 µg of recombinant plasmid and 2 µg of pGK
Luciferase activity was measured in a 5-µl aliquot of cellular
extract mixed with 100 µl of luciferin reagent (20 mM
Tricine, 1 mM (MgCO2)4
Mg(OH)2.5H2O, 2.7 mM
MgSO4.7H2O, 0.1 mM EDTA, 33.3 mM DTT, 270 µM coenzyme A, 470 µM luciferin, 530 µM ATP) using a ECG
Berthold Lumat 9700 luminometer. The protein concentration of the
cellular extracts was determined with the Bio-Rad protein assay kit.
Nuclear Extracts and Gel Mobility Shift Assays--
Nuclear
extracts from all cell lines were prepared as follows. Cells from a
10-cm dish were washed twice with 3 ml of phosphate-buffered saline and
harvested by scraping in 500 µl of cold phosphate-buffered saline.
Cells were centrifuged at 3,000 rpm at 4 °C for 3 min, resuspended
in 400 µl of buffer A (10 mM Hepes, pH 7.8, 1.5 mM MgC12, 10 mM KCI, and l
mM DTT). Cytoplasmic membranes were disrupted by passing
cells through a 28-gauge needle on a 1-ml insulin syringe ~10
times. Nuclei were centrifuged at 14,000 rpm for 15 s at 4 °C.
The supernatant was discarded, and the nuclei pellet was resuspended in
3 volumes of buffer B (20 mM Hepes, pH 7.8, 25% glycerol,
420 mM KCI, 1.5 mM MgC12, 0.2 mM EDTA, 1 mM DTT) containing protease inhibitors (50 ng/ml phenylmethylsulfonyl fluoride, 5 µg/ml
aprotinin, 5 µg/ml leupeptin, and 5 µg/ml pepstatin). The nuclei
were incubated on ice for a minimum of 20 min and then passed four to
eight times through a 28-gauge needle on a 1-ml insulin syringe. The
debris was centrifuged at 14,000 rpm for 15 s. The protein
concentration of the supernatant was determined using the Bio-Rad
protein assay kit, and the supernatant was stored into aliquots at
To generate a binding substrate, 10 pmol of a single stranded
oligonucleotide was end labeled at the 5'-end using T4 polynucleotide kinase (New England Biolabs Inc.) and Isolating the Mouse Enhancer
Because of the high degree of conservation between mouse and human
(19), we hypothesized that a mouse counterpart to the human dystrophin
intron-1 enhancer would lie at a similar distance downstream from the
mouse promoter. To identify a large mouse clone that might contain the
mouse promoter plus exon-1 and intron-1, we screened a mouse BAC
genomic library (20) using as probe a DNA fragment containing sequences
that extend from Characterization of the Mouse Dystrophin Intron-1
Enhancer
To determine whether the mouse counterpart to the human enhancer
could enhance transcription, a 3.0-kb EcoRI fragment (Fig. 1B) that was isolated from pmdeE and contains the putative
mouse enhancer was inserted in reverse orientation upstream of the SV40 early promoter and in both orientations downstream from the firefly luciferase reporter gene of the pGL3-P vector series (Promega) to
generate recombinant constructs C1, C2, and C3 (Fig. 2). The latter
were transfected into mouse skeletal muscle-derived G8 myoblasts (21)
as well as into rat heart-derived H9-C2 myoblasts (22) and mouse NIH
3T3 (23) embryonic fibroblasts using DODAC:DOPE liposomes. Myoblasts
were harvested after 24 h or induced to differentiate into mature
myotubes over 4 days. Fibroblasts were harvested 24 h after
transfection. Our results show that in construct C2 and C3
transcription increases by ~11 fold in differentiated G8 myotubes but
not in myoblasts (Fig. 2). Interestingly in the C1 construct, transcription from the SV40 promoter increases more than 25- fold. In
lines H9-C2 and NIH 3T3 cells, the enhancer has no effect on transcription of the luciferase gene (Fig. 2).
Smaller fragments of 500 and 280 bp (Fig. 1B) which contain
the putative MDE were used to generate the constructs of the A and B
series, respectively. These were all tested for enhancer activity. Our
results show that both the 500- and 280-bp fragment increase
transcription by a factor of 5-10-fold in differentiated G8 myotubes
and 2-3-fold in differentiated H9-C2 myotubes (Fig. 2). No enhancer
activity was observed in G8 or H9-C2 myoblasts or 3T3 fibroblasts (Fig.
2). Our results suggest that sequence elements contained in the 3-kb
fragment are required for full enhancer activity and restrict enhancer
activity to G8 myotubes. Our results also show quite clearly that
sequence elements contained in a shorter fragment of 280 bp contain the
minimal elements for enhancer activity.
Identification of Sequences Responsible for Enhancer
Activity
Sequence analysis of the MDE revealed a putative mef-2 binding
site (24), four E-boxes (25-30) numbered E1-E4, a SRE (31, 32) (Fig.
3A). E-boxes 1 and 3 match the
consensus 5'-AACAc/gc/gTGCa/t, whereas E-box 2 matches the consensus
5'-GGa/cCANGTGGc/gNa/g (33). E4 does not appear to match either of
these two consensus sequences. Comparison of mouse and human enhancer
sequences reveals a 65% homology. The analysis also reveals
that the mef-1/mef-2 box (21) of the human enhancer is replaced by a
single mef-2 site in the mouse enhancer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
500 to
900 relative to the transcriptional start site of the mouse dystrophin muscle promoter. To
map the promoter within the BAC clone, the BAC insert was digested with
EcoRI, BamHI, or HindIII restriction
endonuclease, and fragments were separated by agarose gel
electrophoresis, transferred to a GeneScreen Plus membrane (NEN Life
Science Products). This was hybridized to the same probe in the
presence of 6 × sodium citrate, 5 × Denhardt's,
0.5% SDS, 10% dextran sulfate, and 100 µg/ml herring sperm
at 65 °C for 20 h. The membranes were washed in 1 × sodium citrate, 0.1% SDS at 65 °C for 20 min following a wash at
room temperature. A positive EcoRI fragment of 7.0 kb was
subcloned into pBluescript SK+ (pmdp) and mapped by partial cleavage
using EcoRI, BamHI, and HindIII
restriction endonucleases (Fig.
1A).
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Fig. 1.
Mapping the region that encompasses the mouse
dystrophin promoter and enhancer. Panel A, three
fragments were subcloned into pBluescript SK+ to yield subclones pmdp,
pmdeH, and pmdeB. Inserts were mapped by partial cleavage with
EcoRI (E), HindIII (H),
and BamHI (B) restriction endonucleases and
aligned to generate a contig of 20 kb. The first exon (EX1)
of the muscle isoform of the mouse dystrophin gene is located ~8.5 kb
upstream of the MDE (MDE). Panel B, a 3-kb
fragment (plain line) that contains the enhancer was
subcloned from pmdeB into the EcoRI site of the pBluescript
SK+ (bold line) (see "Experimental Procedures") to
generate the pmdeE recombinant plasmid. The positions of the
SacI (S) and XhoI (X)
restriction sites and the T7 promoter (T7) are as indicated.
0.5- and 0.28-kb fragments that include the MDE were amplified by PCR
and tested for enhancer activity as described in Fig. 2.
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Fig. 2.
Activation of the SV40 early promoter by the
MDE in myogenic and non-myogenic cell lines. Fragments of 3, 0.5, and 0.28 kb which contain the MDE were positioned either upstream of
the early SV40 promoter (SV40) or downstream the SV40
polyadenylation signal (PA) of the firefly luciferase
reporter gene. The luciferase activity was measured in G8 myotubes
(G8 mt), G8 myoblasts (G8 mb), H9 C2 myotubes
(H9 mt), H9-C2 myoblasts (H9 mb), and 3T3
fibroblasts. Transcription activation was determined by comparing the
activity of each construct with the activity of a construct that
expresses the luciferase gene from the early SV40 promoter. The values
obtained are expressed as the means ± S.E., and those higher than
2.5 are boxed. Experiments were performed in triplicate
between two and six times.
(Life Technologies Inc.). Deletions of the enhancer
sequences were confirmed by sequence analysis with an automatic Applied
Biosystems 373 sequencer.
. All mutations that were introduced in the MDE were confirmed by
sequence analysis.
Oligonucleotide primers used to mutagenize putative binding sites of
mouse dystrophin enhancer
-galactosidase were incubated with .0014 M DODAC:DOPE
liposomes (Inex Pharmaceuticals Inc.) in 0.9% NaCl at room temperature
for 10 min in a final volume of 50 µl. 750 µl of
-minimal
Eagle's medium and 25% fetal bovine serum was added, and the mix was
added to ~2 × 105 cells on the surface of a well
(six-well dish). Cells were incubated with the transfection mix for
3 h at 37 °C, 5% CO2. The mix was removed, and
-minimal Eagle's medium containing 10% fetal bovine serum, 10%
horse serum was added, and the cells were incubated at 37 °C, 5%
CO2 for 20 h. Luciferase activity was measured in both
myoblasts and myotubes. Myoblasts were harvested after 24 h with a
cell scraper in luciferase lysis buffer (25 mM Tris
phosphate, pH 7.8, 2 mM DTT, 2 mM CDTA,
10% glycerol, 1% Triton X-100). Alternatively, cells were induced to
differentiate into mature myotubes in
-minimal Eagle's medium
containing 1% horse serum over 4 days and were harvested as above.
Fibroblasts were harvested 1 day after transfection and harvested as
above. Cellular extracts were prepared as described by the manufacturer (Roche).
-Galactosidase activity was determined by diluting 10-30 µl of
the extract in luciferase lysis buffer in a final volume of 150 µl.
The diluted extract was then mixed with 150 µl of 2 ×
-galactosidase buffer (200 mM sodium phosphate, pH 7.3, 2 mM MgCl2, 100 mM
-mercaptoethanol, 1.33 mg/ml o-nitrophenyl
-D-galactopyranoside). The luciferase activity of each
extract was normalized for protein concentration and
-galactosidase. The luciferase activity obtained from constructs that contain the SV40
promoter/dystrophin enhancer was expressed relative to the activity
obtained from constructs that express the luciferase gene from the SV40 promoter.
80 °C.
-ATP (Amersham Pharmacia Biotech) in kinase buffer (70 mM Tris-HCI, pH 7.6, 10 mM MgCl2, 5 mM DTT) in a final
volume of 10 µl. 10 pmol of the complement strand was prepared in 10 µl of 1 × annealing buffer (10 mM Tris-HCl, pH 7.8, 50 mM NaCl, l mM EDTA) and added to the labeled
oligonucleotide. Both strands were heat denatured by boiling for 5 min
and allowed to cool slowly at room temperature. Competitor duplexes
were generated by heat boiling 500 pmol of complementary strands for 5 min and allowing the strands to cool slowly to room temperature at a
final concentration of 1 pmol/µl in 1 × annealing buffer.
Binding reactions were carried out on ice for 30 min by incubating 0.05 pmol of labeled oligonucleotide with 1 pmol or 20-fold molar excess of competitor DNA and 3 µg of extract in binding buffer (10 mM Hepes, pH 7.8, 50 mM potassium chloride, 5 mM MgC12, 1 mM EDTA, 5% glycerol). Supershift reactions were carried out by preincubating myoD, E12, or
serum response factor (SRF) antibodies (Santa Cruz Biotechnologies Inc.) with the extract for 60 min at 4 °C. The remainder of the binding reaction mix was subsequently added, and binding reactions were
carried out as described earlier. Reaction products were loaded on a
6% polyacrylamide 0.5 × TBE gel and run at 4 °C at 150 V.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
500 to
900 of the mouse dystrophin muscle
promoter. A positive clone of ~120 kb was cleaved with
EcoRI, HindIII, and BamHI and
hybridized to the mouse muscle promoter or the human enhancer (see
"Experimental Procedures"). A 7-kb EcoRI fragment that
hybridizes with the promoter sequence was subcloned into Bluescript SK+
(Fig. 1A). Two fragments hybridized with the human
dystrophin enhancer and therefore contain the putative MDE. A 7-kb
HindIII fragment and an 11-kb BamHI fragment that
hybridize with the human enhancer were subcloned into pBluescript SK+
to yield pmdeH and pmdeB, respectively (Fig. 1A). All three clones were restriction mapped by partial digestion with
EcoRI, BamHI, and HindIII, and the
alignment of restriction sites from the three fragments allowed the
fine mapping of a contig of ~20 kb (Fig. 1A). According to
this restriction map, the putative MDE resides in the region of overlap
of clones pmdeH and pmdeB. Digestion of pmdeB with EcoRI
resulted in the subclone pmdeE containing this region (Fig.
1B).
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Fig. 3.
The nucleotide sequence of the MDE.
Panel A, the MDE (MOUSE) was aligned
with the human dystrophin enhancer (HUMAN).
Asterisks (*) show homology between the two sequences.
Putative binding sites (boxed) that interact with mef-2 and
E-boxes 1-4 (E1-4) were disrupted by the insertion of
MluI and BamHI restriction sites, respectively.
The boxed sequence that contains the SRE was deleted from
the enhancer. Panel B, all enhancer mutations were
introduced in a 280-bp fragment using the B1 construct as template and
tested for enhancer activity in G8 myotubes as described in Fig.
2.
To determine which of the putative binding sites is required for enhancer activity we replaced the binding sites E1-E4 by a BamHI restriction site and replaced the A-T-rich sequences of the mef-2 site with a MluI restriction site by performing site-directed mutagenesis on the 280-bp enhancer (34). A 27-bp region that contains the SRE was deleted and replaced with a PstI site. The resulting constructs were transfected into G8 myoblasts, and enhancer activity was monitored in differentiated myotubes. Our results show that disruption of E1, E2, E3, and SRE abolishes enhancer activity, whereas disruption of the mef-2 or E4 binding site had no effect on transcription (Fig. 3B). Thus, the MDE requires a minimum of four binding sites to activate transcription in G8 myotubes.
Characterization of the Binding Activities That Define MDE Function
The binding activities at the four putative binding sites were investigated further by gel shift assays (35) using three labeled oligonucleotides that contain E-box 1, E-boxes 2 and 3, or the SRE (Table II). The oligonucleotides that contain each of these sequences were incubated in the presence of nuclear extracts (36) that were prepared from non-myogenic NIH 3T3 fibroblasts, undifferentiated myoblasts, or differentiated myotubes of the G8 myogenic line. The specificity of binding was determined by performing binding reactions in the presence of a series of competitors that were added in 20-fold molar excess relative to the substrate.
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Binding at E1 of the MDE--
Binding reactions were carried out
by incubating a labeled oligonucleotide that contains the E1 site in
the presence of G8 myotube, G8 myoblast, or fibroblast extracts. In the
presence of myotube extracts three protein-DNA complexes A1, A2, and A3 are apparent (Fig. 4A),
whereas in the presence of myoblast and fibroblast extracts only A1 and
A3 can be detected (Fig. 4B). A protein-DNA complex that
migrates slightly faster than A3 is detected in all of the extracts in
the presence every competitor. This suggests that this particular
complex is not specific for any of the competitor duplexes used. The
lower amounts of A1 in myotubes may come from myoblasts that have not
differentiated. Thus, factors in the A1 and A3 complexes are likely to
be ubiquitous, whereas certain factors in the A2 protein-DNA complex
are likely to be specific to myogenic cells. Because the E1 binding
site features an E-box of the mef-1 type, the factors that bind at this
site are likely to be part of a mef-1 complex (37, 38). To confirm
further that the A2 protein-DNA complex included factors that were
specific to an E-box of the mef-1 type, we performed binding reactions
in the presence of oligonucleotide competitors that feature E2, E3,
mef-2, and the SRE. We notice that A2 is competed out by excess amounts
of E3 and E1 competitors. Because both E1 and E3 feature an E-box of
the mef-1 type, our results suggest that a mef-1 complex binds to
E1.
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Because mef-1 complexes are known to include myogenic differentiation factors of the myoD family, we repeated the binding reactions by using E2 competitor in the presence G8 myotube extracts that were preincubated with antibodies to myoD (39) or to E-box-binding factor, E12, which forms heterodimers with myoD (40, 41). The results show that antibodies to myoD block the formation of the protein-DNA complex A2 (Fig. 4C), whereas antibodies to E12 have little or no effect. Because myoD binds to E-boxes by forming heterodimers with E-box-binding factors, our results suggests that myoD heterodimerizes with an E-box-binding factor other than E12.
Binding to the Paired E-boxes, E2 and E3, in the MDE-- Binding reactions aimed at characterizing protein-DNA complexes that occur at E2 and E3 were carried out by incubating a labeled oligonucleotide that contains the two putative binding sites in the presence of G8 myotube, G8 myoblast, and fibroblast extracts. In these experiments four protein-DNA complexes were detected which we have labeled B1, 2, 3, and 4. B1 and B4 appear with all three extracts and are therefore ubiquitous. B3 is specific to myoblasts and 3T3 and may therefore represent a complex that dissociates upon differentiation into myotubes. B2 is specific to myotubes and therefore represents a complex that forms upon differentiation into myotubes.
To determine which of the two E-boxes generated these complexes we carried out binding reactions in the presence of competitors that feature either the E2 or E3 boxes (Table III). Binding reactions were also carried out in the presence of competitors that contain the mef-2 binding site and the SRE of the MDE. The mef-2 binding site differs significantly from the E-box consensus and therefore should not compete for the binding of factors that recognize E2 or E3. The SRE, however, features a sequence that resembles the consensus 5'-GGa/cCANGTGGc/gNa/g shared by the E2 binding site and is therefore expected to compete for the binding of factors that recognize E2.
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Protein-DNA complexes B3 and B4 are competed out by oligonucleotide
competitors that contain either E2 or E3 (Fig.
5, B and C),
whereas B1 complexes are competed out by oligonucleotide competitors that contain E2 and are partially competed by an oligonucleotide containing the SRE (Fig. 5, A-C). Thus, our results suggest
that factors in the B1 protein-DNA complex are likely to bind E2 and that factors in the B3 and B4 complexes can bind both E2 and E3. The B2
protein-DNA complex is competed out by the competitors that feature E3,
suggesting that factors that compose the B2 complex are likely to bind
to E3 (Fig. 5A).
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Because B2 complexes can be detected as B1 complexes are competed out
by oligonuleotide competitors that include the SRE or E2 binding sites
(Fig. 5A), we examined whether the dissociation of B1
complexes is required for the formation of B2 complexes. To this end,
labeled oligonucleotides that contain both E2 and E3 were incubated
with myotube extracts and increasing amounts of oligonucleotide
competitor that contains the E2 site only. The results obtained clearly
show that as the amounts of B1 complexes decrease, the amounts of B2
complexes increase (Fig. 6). Therefore, as factors bind E2, E3 is unavailable for binding by other factors. As
factors that bind E2 are competed out, however, E3 can interact with
factors that yield the B2 protein-DNA complex, indicating that the
binding of protein factors to E2 may prevent the binding of protein
factors to the E3 binding site.
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According to sequence comparisons between the muscle creatine kinase (41) and myosin light chain 1/3 (42) muscle-specific enhancers, the E3 binding site of the MDE matches a consensus 5'-AACAc/gc/gTGCa/t that is recognized by myogenic differentiation factors of the myoD family. Myogenic differentiation factors, however, do not recognize the consensus 5'-Gga/cCANGTGGc/gNa/g shared by the E2 binding site of the MDE. Because B1 and B2 protein-DNA complexes are proposed to occur at binding sites E2 and E3, respectively, the presence of myoD in these complexes was investigated further by supershift analysis. Thus, we carried out two distinct sets of binding reactions in which a labeled oligonucleotide that contains the E2 and E3 binding sites was incubated with G8 myotube extracts in the presence of competitors NS and E2 that yield protein-DNA complexes B1 and B2, respectively. The extracts used were preincubated with antibodies to myoD or to E-box-binding factor, E12, which forms heterodimers with myoD. Our results indicate that B2 but not B1 complexes are blocked by the presence of antibodies to myoD but unaffected by presence of antibodies to E12, suggesting that myoD recognizes the E3 binding site (Fig. 5D). Thus, our results suggest that myoD interacts with the E3 binding site by forming heterodimers with E-box-binding factors other than E12.
Binding to the SRE of the MDE--
Binding reactions that were
carried out in the presence of myotube extracts yield a protein-DNA
complex C1 (Fig. 7A). Binding reactions that were carried out in the presence of myoblasts and fibroblasts extracts yield complexes C2, C3, C4 (Fig. 7, B
and C) and C5, and an additional complex that migrates below
C1 is detected in the presence of fibroblasts extracts only. The C1 complex is competed out by the E2 binding site as well as by the SRE.
This may be explained by the fact that both the SRE and the E2 binding
site resemble the consensus 5'-GGa/cCANGTGGc/gNa/g. Thus, factors that
bind the E2 site may also bind the SRE. To determine whether SRF was
present in complexes C1-C5, we performed binding reactions by
incubating a labeled oligonucleotide that contains the SRE with
extracts prepared from myotubes and myoblasts in the presence of SRF
antibodies. The results show that SRF antibodies block C5 complexes but
do not affect C1-C4 complexes, suggesting that C5 complexes result
from SRF interacting with the SRE (Fig. 7D). Protein factors
involved in the C1 complex, however, interact with the E2 binding site
but not with the antibodies to SRF, indicating that protein factors in
the C1 complex may differ from those in the C5 complex. Thus, SRE may
interact with E-box-binding factors as well as with SRF. The
protein-DNA complexes C2, C3, C4 were not competed out by any of the
competitors.
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DISCUSSION |
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Recent studies have shown that the mouse dystrophin muscle promoter targets expression in the right ventricle of the heart only, suggesting that other sequences are needed to target the skeletal muscle and/or other compartments of the heart. Our earlier studies in human have identified an enhancer element downstream from the muscle promoter which was specific to cell lines derived from rat heart. We now report the identification of a MDE that maps in the first intron ~8.5 kb downstream from the muscle promoter of the mouse dystrophin gene. The MDE shows 65% homology with its human counterpart, but the sequence elements that confer enhancer function in both species differ. In human the enhancer is defined by overlapping mef-1/mef-2 binding sites, whereas in the mouse the enhancer is defined by a minimum of four binding sites. These include three E-boxes, two (E1 and E3) of which are of the mef-1 type, and a SRE. A sequence comparison among the mouse dystrophin, the muscle creatine kinase, and myosin light chain-1/3 muscle-specific enhancers reveals that all three feature an E-box that resembles the consensus 5'-AACAc/gc/gTGCa/t paired to an E-box that resembles the consensus 5'-GGa/cCANGTGGc/gNa/g. The conservation of paired E-boxes in the three enhancers suggests that other enhancers that target skeletal and/or cardiac muscle may contain these two sites as well (44).
Transfection experiments show that the MDE increases transcription from the early SV40 promoter mostly in myotubes derived from skeletal muscle. Because the muscle promoter alone targets the expression of lacZ in the right ventricle only, our results suggest that the MDE may be required for targeting expression in skeletal muscle. This hypothesis is strengthened further by observations from our laboratory which show that the MDE can target lacZ expression in both cardiac and skeletal muscle in vivo.2
The protein factors most likely to interact with the sites that confer enhancer activity were characterized further by gel shift analysis. Using myotube extracts, we have identified protein-DNA complexes that occur at the E1 and E3 sites, both of which resemble the mef-1 consensus 5'-AACAc/gc/gTGCa/t. Because myogenic differentiation factors are known to interact with this consensus, we were able to confirm that myoD/myogenin differentiation factors can bind at both these sites. The protein-DNA complex that was detected at the E3 binding site, however, occurs only when factors that bind to the adjacent site, E2, are competed away, suggesting that factors that bind E2 may regulate the binding of factors to E3. Factors that bind E2, however, are not yet known because they bind a consensus 5'-GGa/cCANGTGGc/gNa/g that is not recognized by the known myogenic differentiation factors. Although our study points out the importance of mef-1 type E-boxes for enhancer function, in vivo studies must be carried out to verify this hypothesis.
The SRE may also play a role in regulating enhancer function. In G8 myotubes, the SRE is recognized mostly by E-box-binding factors. In G8 myoblasts, however, the SRE is also recognized by the SRF. The latter is a transcription factor of the MCM1, agamous, deficiens, serum response factor (MADS) box family that regulates gene expression of several muscle-specific genes (45). Because the activity of the MDE is specific to myotubes, E-box-binding factors may act as positive regulators, whereas SRF may act as a negative regulator by preventing the binding of E-box-binding factors to the SRE. Because the latter includes an E-box consensus 5'-GG a/c CANGTGGc/gN a/g which is not recognized by any of the known myogenic differentiation factors, it remains to be determined which E-box-binding factors actually bind the SRE. To this end, previous studies on the muscle creatine kinase enhancer have demonstrated that the methylation protection patterns that occur at this particular sequence are similar but not identical to myoD, confirming that factors do bind at this E-box (33). We are currently investigating the Duchenne muscular dystrophy protection patterns that occur in vivo in order to identify the factors that are most likely to interact with the SRE in both myotubes and myoblasts. (46)
A third factor that may be involved in enhancer function is mef-2. Mef-2 belongs to the family of transcription factors with a MADS box and plays a key role in the regulation of many muscle-specific genes (24, 47). Interestingly, mutations of the mef-2 site in the MDE had no effect on enhancer activity. More surprising is the fact that mef-2 appears to bind the human dystrophin enhancer (48). Although mef-2 acts in combination with myogenic basic helix-loop-helix factors to activate transcription, studies have shown that mef-2 does not require direct binding to DNA (49). Thus, mutations that abolish mef-2 binding to DNA do not necessarily prevent mef-2 from activating transcription. Because mef-2 is frequently associated with functional E-boxes bound by myogenin, we suspect mef-2 may act in combination with transcription factors bound to the E1 or E3 binding sites to activate transcription in skeletal muscle. Because myogenic differentiation factors are absent from the heart, mef-2 may also interact with factors such as SRF (50) or GATA-4 (51) to activate transcription in the cardiac muscle.
Our study also suggests that the genetic environment that surrounds the MDE affect its specificity. Our results show that all of the enhancer fragments that have been tested increase transcription from the SV40 promoter in G8 myotubes. In H9-C2 myotubes, however, the 500-bp enhancer has a moderate effect, whereas the 3-kb or 280-bp enhancers have little or none. This suggests that the 3-kb fragment may contain sequence elements that repress the enhancer function in the H9-C2 line or that the 280-bp fragment may lack sequence elements that enhance transcription from the SV40 promoter. Such elements may include DNA bending motifs or binding sites for proteins that may influence protein-protein interactions via conformational changes in the DNA which affect enhancer and/or promoter activity (52-54). The role of DNA conformation in transcription regulation of muscle-specific genes was demonstrated in studies that showed that the binding of the bHLH factors myoD, Twist, and E2A to their respective sites is mediated by the topology of these sites (55). Another study showed that the proper conformation of a TATA box is required by activator proteins to activate transcription of the myosin heavy chain gene (56). Finally, a third study has reported that transcription activation of the human dystrophin gene depends upon DNA bending of its promoter to activate gene expression (57, 58).
The understanding of how transcription factors control the MDE is
likely to shed more light on the mechanisms that regulate transcription
of the dystrophin gene in skeletal and cardiac muscle. Such findings
may help elucidate complex phenotypes such as those observed in
X-linked dilated cardiomyopathy patients who fail to produce dystrophin
in the heart but up-regulate the expression of the dystrophin gene in
skeletal muscle from Purkinje and brain promoters that are normally
silent in skeletal and/or cardiac muscle. The characterization of the
MDE is also likely to assist the engineering of therapeutic vectors
aimed expressing genes in skeletal and cardiac muscle.
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ACKNOWLEDGEMENTS |
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We are very grateful to Dr. H. J. Klamut for providing the plasmid SA195 and to Inex Pharmaceuticals Inc. for providing liposomes throughout this study. We thank Drs. David Picketts and Karen Copeland for helpful advice on gel mobility shifts.
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FOOTNOTES |
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* This work was supported by the Canadian Genetic Diseases Network and the Heart and Stroke Foundation of Canada.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: Ottawa Hospital
Research Institute, 501 Smyth, Ottawa, Ontario K1H 8L6, Canada. Tel.: 613-737-8802; Fax: 613-737-8803; E-mail: rworton@ohri.ca.
Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M102100200
2 Philip Marshall, Nathalle Chartrand, Yves de Repentigny, Rashmi Kothary, Louise Pelletier, and Ronald G. Worton, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: kb, kilobase(s); SRE, serum response element; MDE, mouse dystrophin enhancer; BAC, bacterial artificial chromosome; bp, base pair(s); PCR, polymerase chain reaction; DTT, dithiothreitol; CDTA, 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; SRF, serum response factor; contig, group of overlapping clones.
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