In hind limb muscles, the aldolase A
muscle-specific promoter is specifically expressed in glycolytic
fast-twitch fibers. Here, we show that in addition, it is expressed at
higher levels in trunk and limb muscles than in neck and head muscles
independent of their fiber-type content. We have identified by analysis
of transgenic mice a DNA element that is required for this differential expression and, to a lesser extent, for fiber-type specificity. We show
that members of the nuclear receptor superfamily bind this element in
skeletal muscle nuclear extracts. Interestingly, in gel mobility shift
assays, different complexes were formed with this sequence in tongue
nuclear extracts compared with limb or trunk muscle nuclear extracts.
Therefore, binding of distinct nuclear receptors to a single regulatory
sequence appears to be associated with the
location-dependent expression of the aldolase A
muscle-specific promoter.
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INTRODUCTION |
One of the main characteristics of skeletal muscles is their
diversity. In the adult, this diversity appears with regard to shape,
anatomical position, mechanical function, energy requirement, and nerve
stimulation of each individual muscle. At the cellular level, it is
revealed by the existence of different types of myofibers characterized
by expression of specific isoforms of structural proteins and metabolic
enzymes (1, 2). The usual classification divides muscle fibers in four
major types based on MHC1
isoform expression: type I (slow, oxidative), type IIA (fast, oxidative), type IIX (fast, oxidative and glycolytic), and type IIB
(fast, glycolytic). Several genes show a fiber type-specific pattern of
expression, and for some of them, their regulatory sequences have been
characterized, including troponin I fast (3) or slow (4-6), MLC1f
(myosin light chain 1
fast) and MLC3f (7-9), aldolase A (10, 11), and MHC
(12). Some other genes are active in a subset of muscles, with no
obvious link with a particular fiber type (e.g. ryanodine
receptor-3 in diaphragm (13) or engrailed-2 in jaw muscles (14)). This
kind of muscle diversity has also been revealed by random integration
of transgenes into "special" loci (15) or by deletion analysis of
muscle regulatory sequences (16-18), the most studied example being
the rostro-caudal gradient of expression displayed by MLC1f
transgenes (8, 19). Together, these data show that besides fiber-type
specificity, an additional level of regulation may overlap and modulate
fiber-specific gene expression. Multiple factors are thought to
contribute to muscle diversity like innervation, hormonal influence,
and participation of distinct myoblast lineages (20). But so far, the
molecular basis of this diversity is still poorly documented. In
particular, the transcription factors that activate genes in particular
fibers and/or muscles are not known. Determination of the sequences
involved in the regulation of genes expressed in a restricted subset of muscles and/or in a specific type of muscle fiber is the first step
toward the characterization of these factors.
We have previously shown in a transgenic mouse model that the
muscle-specific promoter of the human aldolase A gene (pM) is specifically expressed in hind limb muscles composed of type IIB fibers
(11). This pattern of expression results from the contribution of at
least two distinct sets of control elements, one of which consists of
an NF-1-binding site (16, 21). Interestingly, this element does not
activate transcription equally in all fast-twitch muscles, revealing
that pM is differentially regulated in different subtypes of
fast-twitch muscles. While our first observations were limited to hind
limb muscles, this result prompted us to investigate the activity of pM
in additional muscles, from head, neck, trunk, hind limb, and fore
limb.
We show here that a pM310CAT construct containing a 355-base pair pM
promoter linked to a chloramphenicol acetyltransferase reporter gene is
active in glycolytic fast-twitch limb and trunk muscles of adult
transgenic mice. Surprisingly, in head and neck muscles, activity is at
least 100-fold lower, even in muscles composed of type IIB fibers. We
identify a sequence, called M1 in previous reports, whose mutation
abolished the differential expression of pM in head/neck and trunk/limb
muscles. This sequence shows similarity to consensus binding sites for
steroid-related nuclear receptors. Indeed, we show that RXR, RAR, and
ARP1 nuclear receptors are part of the complexes formed by nuclear
proteins with M1. Interestingly, the composition and relative amount of these complexes are different in head and body muscles, suggesting that
nuclear receptors are involved in the muscle
location-dependent regulation of a fast-twitch
muscle-specific promoter.
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EXPERIMENTAL PROCEDURES |
Transgenic Mice and Constructs--
The creation of transgenic
mice to obtain constructs pM310CAT, mM1, M-tkCAT,
AT-tkCAT, and
M
B-tkCAT has been described elsewhere (11, 16, 21). The M
E-tkCAT
construct was obtained by introduction of a
HindIII-BglII fragment from the mE construct
(aldolase A pM sequences from base pairs
310 to
55) (22) upstream
from the tk-105 promoter (herpes simplex virus thymidine kinase
truncated promoter) from vector pBLCAT2 (23). The fragment to be
microinjected was isolated on 1% agarose gel after digestion by
AgeI and SacI, followed by electroelution and
purification on an Elutip column (Schleicher & Schull).
Transgenic mice were generated, identified, and propagated as described
previously (21). For chloramphenicol acetyltransferase assays, various
tissues were dissected from adult (at least 7-week-old) F1 transgenic
animals, except for the few founders analyzed, for which transgene
presence in each tissue was verified by Southern blotting.
Chloramphenicol acetyltransferase activity was measured as described
previously (11), with amounts of proteins up to 200 µg and reaction
times up to 4 h. For each line, at least two different mice were
analyzed.
Histochemical Determination of Muscle Fiber
Types--
Gastrocnemius, tongue, masseter superficial, and
digastricus muscles were dissected from adult mice. Muscles were frozen
in isopentane at liquid nitrogen temperature and cross-sectioned in a
microtome-cryostat. The 5-10-µm-thick unfixed serial sections were
reacted with BA-D5 (MHC-I), SC-71 (MHC-IIA), BF-F3 (MHC-IIB), or BF-35
(all MHC types, except IIX) monoclonal antibodies (24) (provided by
Regeneron Pharmaceuticals, Tarrytown, NY) as described (7). Sections
were then treated with anti-mouse digoxigenin antibody (1:300 dilution;
Boehringer Mannheim) for 20 min, rinsed three times with
phosphate-buffered saline and 0.05% Triton X-100, and then incubated
with alkaline phosphatase-conjugated anti-digoxigenin antibody (1:500
dilution; Boehringer Mannheim). Alkaline phosphatase activity was
revealed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sigma). Sections not stained with anti-MHC antibodies were
used as negative controls.
Northern Blots--
Total RNAs were prepared from several
tissues and skeletal muscles of adult B6/CBA mice by the guanidium
thiocyanate single-step procedure (25). Northern blot analysis was done
as described previously (10) using a mouse pM-specific probe (gift from
M. C. Colbert) (26) or an R45 ribosomal probe for
standardization.
Gel Mobility Shift Assays--
Nuclear extracts from rat spleen,
liver, and skeletal muscle were prepared as described previously (16).
Gel mobility shift assays and transcription-coupled translation were
performed as described previously (22). Anti-RXR antibodies (4RX-1D12,
which recognizes RXR
, RXR
, and RXR
) were a gift from Prof. P. Chambon. RAR antisera were from Santa Cruz Inc. COUP-TF/ARP1 antiserum was provided by Dr. M.-J. Tsai. The human ARP1 cDNA (kindly
provided by Dr. S. K. Karathanasis) (27) was subcloned into the
pCR3 vector (Invitrogen) and place under the control of the T7
promoter. In vitro transcription/translation was done as
described previously (22).
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RESULTS |
pM Is Highly Active in Fast-twitch Trunk and Limb Muscles, but Far
Less in Those Localized in Head and Neck--
We investigated pM310CAT
expression in a large number of muscles: from limbs (vastus lateralis,
gastrocnemius, psoas major, soleus, extensor digitorum longus, tibialis
anterior, and triceps brachii), trunk (sternomastoideus, longissimus
dorsi, intercostals muscles, and diaphragm), and neck and head muscles
(digastricus, mylohyoideus, masseter, and tongue) (Fig.
1). For the two independent lines
studied, a high chloramphenicol acetyltransferase activity was detected
in all trunk and limb muscles, with the exception of muscles devoid of
type IIB fibers, soleus (types I and IIA) and diaphragm (types IIA and
IIX) (7, 28), in which activity was at least 100-fold lower. This
result reflects the specific expression of pM in type IIB glycolytic
fast-twitch fibers.2
Surprisingly, in all head and neck (suprahyoid) muscles tested, activity was on the average 100-fold lower (1000-fold for the tongue)
than in trunk and limb muscles. Since this difference may arise from a
lack of type IIB fibers in these muscles, we determined their
fiber-type composition and compared the ratio between pM activity and
percentage of type IIB fibers. As shown in Table
I, this ratio was close to 100 (arbitrary
units) in three muscles of the limb and trunk, whereas it dropped to
2.3 and 1.4 in the masseter and digastricus, respectively, and even to
<0.1 in the tongue. Therefore, in head and neck muscles, the level of
activity of pM is not determined by the proportion of type IIB fibers;
the weak activity of pM reflects an intrinsic difference between these
muscles and those from limb and trunk, and not a different composition
of fiber type (as judged by MHC expression). Below, this difference is
referred to as "muscle location-dependent
expression."

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Fig. 1.
Expression pattern of the pM310CAT transgene
in several skeletal muscles. pM310CAT expression was investigated
in distinct muscles of two different transgenic lines (20 and 98):
tongue (Tg), masseter superficial (MS),
digastricus (Dig), mylohyoideus (Mh),
sternomastoideus (SM), LgD, intercostals (from T2
to T10, numbered from the rostral to caudal positions),
diaphragm (D), triceps brachii (Tri), psoas major
(PsM), vastus lateralis (VL), soleus
(Sol), gastrocnemius (G), extensor digitorum
longus (EDL), and tibialis anterior (TA). Muscles
are grouped according to their localization in the body. *,
soleus and diaphragm are the only two skeletal muscles studied here
that are devoid of type IIB fibers. Values correspond to the mean
activity of at least two transgenic mice.
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We then examined whether the absence of expression in head/neck muscles
for the pM310CAT transgenes reflects the expression of the endogenous
mouse aldolase A muscle-specific promoter. A Northern blot with total
RNAs from different types of muscles was hybridized with a probe
specific for mouse aldolase A exon M (Fig.
2); hybridization with the ribosomal
probe R45 was used as a control for mRNA quantity and quality. The
endogenous M transcripts were highly expressed in fast-twitch limb and
back muscles, weakly in the masseter, and at a very weak level in the
type IIB-rich tongue muscle, as in muscles devoid of type IIB fibers
(soleus and diaphragm). No expression was detected in heart. Therefore, the pattern of expression of pM310CAT coincides with endogenous pM
promoter activity. In addition to a type IIB-specific expression in
trunk and limb muscles, these promoters display a kind of
"location-dependent" regulation since rostral muscles
(neck and head) express pM very weakly despite their high proportion of
type IIB fibers.

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Fig. 2.
Northern blot analysis of mouse M aldolase A
mRNA in various muscles. A Northern blot was hybridized with a
mouse aldolase A muscle-specific probe (pM) or with a probe
complementary to 18 S rRNA (R45) for the standardization of the
quantity of RNA present in each sample (10 µg of total RNA).
H, heart; Tg, tongue; MS, masseter;
D, diaphragm; VL, vastus lateralis; G,
gastrocnemius; Sol, soleus. Exposure times were 16 h
for pM and 3 h for R45.
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The M1 Sequence Is Required for Selective Expression of Aldolase A
Transgenes in Trunk and Limb Skeletal Muscles--
To identify the
sequence(s) involved in the preferential activity of pM in trunk/limb
muscles, we studied the expression pattern of transgenes harboring
distinct segments of the pM regulatory region (Fig.
3A). We compared the activity
of several transgenes in tongue and longissimus dorsi muscles and in
several lines for each transgene (Fig. 3). On average, a 140-fold ratio
of the activity in tongue and LgD muscles was observed for the
construct M-tkCAT, compared with the 1000-fold ratio in pM310CAT
transgenes; pM location-dependent expression is conserved
without the proximal sequences (TATA box and exon M). The ratio of the
activity in tongue and LgD muscles was diminished and probably reflects
the fact that, usually, M-tkCAT transgenes were less active in
trunk/limb muscles than pM310CAT; the M enhancer is probably more
potent when linked to its innate minimal promoter. For the
AT-tkCAT
transgenes, it should be first noticed that there was no detectable
activity in muscles tested in four out of the nine transgenic lines
studied. Nevertheless, in all lines with the "active" transgenes,
we still observed a 220-fold ratio on average between tongue and LgD
muscles. Thus, the sequences sufficient for muscle
location-dependent expression are localized between base
pairs
160 and
35. Further 5
-deletions of the pM regulatory
sequences resulted in total loss of transgene activity (16). All four
transgenic M
E-tkCAT lines (sequences from base pair
235 up to the
M1 site) were active, and there was an average 70-fold differential
expression between tongue and LgD muscles. This result indicates that
the E box and the SP1-binding site are also dispensable for a correct
pattern of expression. Thus, the sequences needed for a graded
expression between head and more caudal muscles were mainly localized
in the region shared by the
AT and M
E fragments: namely, the
MEF3-, MEF2/NF-1-, and M1-binding sites.

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Fig. 3.
Transgene activity in tongue and longissimus
dorsi muscles. Shown is a schematic description of the pM
transgenes used in the study. Binding sites for proteins found by
footprinting experiments (22) on pM proximal regulatory sequences are
represented by boxes. The nucleotide position is numbered
from the pM transcription start site. Mutation in the M1 site is
schematized by a black × on M1, and the tk-105
promoter by a hatched box. The diagram represents the ratio
of transgene activity in tongue (Tg) and longissimus dorsi
muscles on a logarithmic scale. The mean activity is represented by a
bar; each diamond corresponds to the value of a
single expressing line (mean of at least two individual mice from the
same line, except for two mM1 transgenic founder mice). The number of
transgenic lines expressing the transgene and the number of transgenic
lines studied are given for each construct.
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The M1 sequence has been shown to be important for pM expression since
its mutation resulted in a strong diminution of pM activity in myotubes
in primary cell culture and in hind limb muscles of transgenic mice
(21, 22). We tested whether the muscle location-dependent
expression is conserved in constructs with either a mutated M1 site
(mM1) or a deletion of this site (M
B-tkCAT) (Fig. 3). Mutation of
the M1-binding site resulted in the loss of the differential activity
between tongue and trunk muscles (an average of 0.4-fold). With the
thymidine kinase promoter, deletion of M1 from the M
E fragment
resulted in complete absence of expression for three out of the four
transgenic lines obtained (M
B-tkCAT), indicating that in this
context, M1 is critical for promoter activity. In addition, in the only
line with an expressed transgene (M
B-tk-67), the LgD/tongue activity
ratio was only ~4. Comparison of the DNA constructs with an intact M1
sequence to those with a mutation or deletion of this site revealed
that M1 is required for a 20-fold (at least) difference in transgene activity in tongue and LgD muscles (p < 0.001, with
2 test; similar results were obtained with masseter
instead of tongue or vastus lateralis instead of LgD).
From the data presented in Table II, it
appeared that mutation of M1 caused a drop in activity in all
fast-twitch muscles studied down to the level of pM310CAT transgenes in
the tongue. This loss of activity is particularly dramatic in limb and
trunk muscles (from 1200 to 8000-fold), but is also observed (only
70-fold) in masseter, which was the highest expressing head muscle
studied. Taken together, these results suggest that M1 behaves like a
muscle location-dependent cis-activator; M1 is
essential for a high level of expression of pM in fast-twitch trunk and
limb muscles, but it is dispensable in the tongue. Interestingly, this
sequence is conserved in the human, rat, and mouse promoters (see Fig. 5A) (29).
M1-binding Site and Fiber Specificity--
The mutation of M1 also
reduces the fiber specificity of pM transgenes. Expression in diaphragm
(composed of type IIX and IIA fibers) was far less affected (only
50-fold) by M1 mutation than that in type IIB fiber-containing limb and
trunk muscles (Table II). Thus, the mutation or deletion of M1 resulted
in a loss of the differential activity between type IIB fiber-rich trunk and limb muscles and diaphragm (Fig.
4). However, fast-twitch fiber
specificity is conserved since mM1 transgenes were still expressed at a
higher level in gastrocnemius muscles than in slow-twitch soleus
muscles (devoid of type IIB fibers and with rare IIX fibers) (Fig. 4
and Table II). It is unclear whether the ratio of the activity in fast
gastrocnemius and slow soleus muscles was significantly reduced or not
since there is a great variation among the different mM1 lines;
nevertheless, in three lines out of five (four out of six if
M
B-tkCAT-67 was included), this ratio dropped to <10-fold, whereas
for pM310CAT and M-tkCAT transgenes, chloramphenicol acetyltransferase activity was at least 130-fold higher (average of 175-fold) in gastrocnemius than in soleus muscles, suggesting a potential
involvement of M1 in fast-twitch specificity.

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Fig. 4.
Fiber specificity is altered in the absence
of M1. Values are ratios of transgene activity in the soleus to
that in the gastrocnemius (Gastroc./Soleus) or in diaphragm
to the mean of transgene activity in three glycolytic fast-twitch
muscles: LgD, gastrocnemius, and triceps brachii
(IIB/Diaphragm). Points show values for independent
transgenic lines (+M1, pM310CAT ( ) and M-tkCAT ( );
M1, mM1 ( ) and M B-tkCAT ( )), and the mean ratio
is represented by a bar. mM1 differs significantly from pM310CAT for type IIB fiber/diaphragm (p < 0.01 by
Student's t test). The difference for the
gastrocnemius/soleus ratio in transgenes with or without M1 is
significant only if M-tkCAT and M B-tkCAT lines are included
(p < 0.05).
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The M1 Sequence Is Bound by Nuclear Receptors--
Transgenic
studies have shown that M1 is required for the muscle
location-dependent activity of pM310CAT transgenes. A
similar sequence is found in the same position (relative to the
transcription start site) in the mouse pM promoter, which has the same
pattern of expression (Fig. 2). Comparison of M1 sequences in human,
mouse, and rat aldolase A genes revealed a conserved motif (a/gGGt/gCA) directly repeated with a 1-nucleotide-long spacer (DR1) (Fig. 5A); this sequence looks like
a potential binding site for members of the nuclear receptor
superfamily (30). When GMSA were performed with skeletal muscle nuclear
extracts (from back muscles), two main complexes were formed
specifically with M1 (Fig. 5, B and C); the
formation of these two complexes (named A and B) was competed by the
addition of an excess of an M1-binding site, but not by an
oligonucleotide corresponding to the mutation of the M1 site introduced
in transgenic mice. Complex A was more abundant in skeletal muscle
nuclear extracts than in liver or spleen nuclear extracts (Fig.
5B). As an excess of a consensus DR1-binding site for
nuclear receptors prevented the formation of both complexes A and B
(Fig. 5C), we assumed that the main binding activity on M1
is due to nuclear receptor complexes. Other complexes were formed with
M1 (marked by asterisks), but they were poorly competed by
an excess of unlabeled M1 probe and thus corresponded to nonspecific binding to M1; the upper band (marked by a diamond), whose
formation was only moderately competed by an excess of the M1
oligonucleotide, probably corresponds to an SP1-like protein since it
was very efficiently competed by an excess of the SP1 consensus site
(data not shown).

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Fig. 5.
Members of the nuclear receptor superfamily
bind M1. A, the sequence of the M1 site was compared in
human, mouse, and rat aldolase A genes. The three conserved sequences
look like a DR1 site whose consensus sequence is given below.
Arrows indicate the repeated sequence. B, gel
shift assays were carried out on M1 with skeletal muscle
(M), liver (L), and spleen (S) nuclear extracts. The specific complexes A and B are indicated, and
asterisks mark nonspecific binding (see C for
competition experiments). C, the M1 probe was incubated with
muscle nuclear extracts (N.E), and the specificity of
complexes formed was tested by the addition of a 60-fold excess of
unlabeled oligonucleotides. The oligonucleotides used were as follows:
M1, GGCGGGAAAAGGGCAGGGGTCATTAGA; mM1,
GGCGGGAAgAtctCAGGGGTCATTAGA; and DR1,
GGCGGGCCCAGGTCAGAGGTCATTAGA.
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Many nuclear receptors are able to bind to DR1 sequences, including the
retinoic acid receptors RAR and RXR, peroxisome-activated receptors,
and orphan nuclear receptors like COUP-TF/ARP1 and TR2/TR4 (30). As the
M1 site mediated a muscle location-dependent activity, we
investigated the nature of the complexes bound to this sequence with
nuclear extracts from muscles of diverse origins: tongue (head muscle)
and longissimus dorsi, diaphragm, and soleus. For normalization of the
nuclear extracts, binding assays were performed with an upstream
stimulatory factor-binding site; in each case, upstream stimulatory
factor binding activity was detected in similar amounts (Fig.
6A). For the M1 probe, body
muscle nuclear extracts (from trunk or limb, fast or slow twitch)
displayed the same pattern as longissimus dorsi nuclear extracts (Fig.
6B and data not shown for gastrocnemius and vastus
lateralis); complexes A and B were formed at least in equal amount,
with usually more of complex A than complex B (to an extent that
depended on the body muscle considered). With tongue nuclear extracts,
the complexes formed were different. First, complex A was only weakly
detected (Figs. 6B and
7A); in addition, no complex B
was detected, whereas a third complex C appeared, migrating slightly
slower than complex B observed with LgD nuclear extracts (Fig.
7A). Complexes B and C were better separated when GMSA was
performed with a 6% acrylamide gel (Fig. 7A) compared with
a 5% acrylamide gel (Fig. 6B); however, under this
condition, complex A could not be easily distinguished from the
additional SP1-like binding activity. In the following experiments,
GMSA was performed with either 5 or 6% acrylamide gels, depending on
which complex was considered.

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Fig. 6.
Binding activity of different muscle nuclear
extracts. GMSA were performed with nuclear extracts from back
muscle (LgD), diaphragm (D), soleus (Sol), and
tongue (Tg). An upstream stimulatory factor
(USF)-binding site
(GAAGATCGGGGACACATGTGGGGCGAAG) was used to control the
amount and quality of nuclear extracts (A). A 5% acrylamide
gel was used to separate slow migrating complexes A (B).
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Fig. 7.
Presence of RAR, RXR, and ARP1 nuclear
receptors in the complexes formed with M1. M1-bound complexes
formed with nuclear extracts from tongue (Tg), LgD,
diaphragm (D), and soleus (Sol) were separated on
6% (A) and 5% (B) acrylamide gels. M1-labeled oligonucleotide was incubated with nuclear extracts of different muscles in the presence of anti-RAR antibodies (A), anti-RXR
antibodies (B, lanes 1-4), or anti-COUP-TF
antibodies (B, lanes 9-12). The same complexes
were observed with two independent nuclear extracts, and quantification
of the relative amount of supershifted complex B in LgD muscles was
performed in several separate GMSA with a Shimadzu densitomer.
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We then asked if known nuclear receptors participated in complexes A,
B, and C by using specific antibodies. The addition of an anti-RAR
antibody prevented the formation of complex C with tongue nuclear
extracts, but had no effect on complex B formation in body muscles
(Fig. 7A) or on complex A. The addition of an antibody
directed against RXR proteins entirely supershifted this complex C in
the tongue, but only partly (~40% according to quantification performed with a Shimadzu densitometer) supershifted complex B in LgD
muscles (Fig. 7B). In both nuclear extracts, complex A was
not affected by this anti-RXR antibody. When an antibody directed against COUP-TF/ARP1 was added to the nuclear extracts, we observed a
nearly complete supershift of both complexes A and B in body muscles,
whereas this antibody supershifted only poorly (if at all) complex C
formed with M1 by tongue nuclear extracts (Fig. 7B). Nuclear
extracts from soleus, diaphragm, gastrocnemius, and triceps muscles
gave the same results as LgD nuclear extracts (data not shown).
In skeletal muscles, ARP1 transcripts were far more abundant than
COUP-TF1 transcripts (data not shown). Therefore, the
COUP-TF-immunoreactive complexes formed with M1 should correspond to
homo- or heterodimers containing the ARP1 orphan nuclear receptor. We
performed GMSA with in vitro translated ARP1 proteins; the
retarded complex (corresponding to ARP1 homodimers) migrated slightly
faster than complex B (Fig. 8). This
observation suggests that while complex B could perhaps include ARP1
homodimers in addition to RXR-containing complexes, complex A is
probably a heterodimer between ARP1 and another unknown protein.

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Fig. 8.
Binding of ARP1 homodimers to M1. GMSA
with the M1 oligonucleotide was performed with a control reticulocyte
lysate (retic-), with in vitro translated human
ARP1, and with LgD nuclear extracts (N.E.). ARP1 homodimers
bound to M1 are indicated by an arrowhead.
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In summary, these experiments show that nuclear receptors are able to
bind M1 in muscle nuclear extracts. With tongue nuclear extracts,
RAR/RXR heterodimers formed the main binding activity (complex C). In
body muscle nuclear extracts, M1 is mainly bound by complexes
containing ARP1 orphan nuclear receptors; complex B corresponds to
ARP1- and RXR-containing complexes (including ARP1/RXR heterodimers and
perhaps some ARP1 homodimers), whereas complex A probably corresponds
to a heterodimer between ARP1 and another unidentified protein.
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DISCUSSION |
Position-dependent Regulation of Muscle-specific
Genes--
In this paper, we show that the muscle-specific promoter of
the human aldolase A gene is expressed both in a fiber type- and muscle
location-dependent manner. Previous works from other groups have already shown that muscle-specific transgenes could be
differentially expressed in diverse muscles depending on their
position: MLC1f transgenes display a rostro-caudal gradient
of expression together with a preferential activity in fast-twitch
fibers (7, 19), but this pattern was not observed with the endogenous
gene; whereas in our case, the pM310CAT transgene mimics endogenous
promoter expression. In addition, we have identified the sequence (M1) that is responsible for the high level of pM activity in trunk and limb
muscles. Recently, Hauschka and co-workers (17) have shown that a
muscle creatine kinase transgene with all its E boxes mutated was
expressed with a pattern very reminiscent of the pM pattern: higher
expression in fast-twitch muscles than in soleus muscles, except for
the tongue. Thus, activation of the muscle creatine kinase promoter
depends on E boxes in tongue, heart, and, to a lesser extent, soleus
muscles, whereas distinct elements control its activity in fast-twitch
body muscles. It may be worth noting that the aldolase A gene is
regulated in a similar but slightly different way: the gene is
transcribed from two distinct promoters, one active in fast-twitch limb
and trunk muscles (pM; this study) and another (pH) that functions
predominantly in heart, soleus, and tongue
muscles.3 It would be
interesting to investigate whether aldolase A and muscle creatine
kinase share the same kind of regulatory mechanisms in the same type of
muscles.
It appears from studies on chick embryos (31, 32) that head and neck
muscle precursor cells come from unsegmented mesoderm and from the most
rostral somites, whereas more caudal somites give rise to limb and
trunk muscles. Furthermore, recent work by Buckingham and co-workers
(33) demonstrated that myogenesis proceeds differently in somites that
will give rise to body muscles and in head mesoderm that will make head
muscles; mice lacking functional Myf-5 and Pax3
genes do not make body muscles, whereas they still form apparently
normal head muscles. It is therefore tempting to establish a link
between somitic origin and pM activity. Besides, in addition to a
different embryologic origin, head muscles and particularly the tongue
do not have the same physiological role as limb muscles. Since muscle
activity is known to influence muscle gene expression (34, 35), pM body
muscle-selective expression may also result from the contribution of
extrinsic factors that could be regionally expressed or modulated by
the physiological and contractile status of each muscle. Current work is in progress to distinguish between these two nonexclusive
models.
Nuclear Receptors and Muscle Location-dependent
Expression--
We have identified M1 as a DNA-binding site required
for a high level of activity of pM in trunk and limb muscles. With
nuclear extracts from these muscles, the M1 site is recognized by
nuclear receptors. While complexes formed with M1 in GMSA are similar with trunk and limb muscle nuclear extracts, the results are very different with tongue nuclear extracts; in this case, M1 is
predominantly bound by complexes containing RAR/RXR heterodimers
instead of ARP1, RXR, and probably other nuclear receptors. Thus, the
M1 binding activity in muscle nuclear extracts changes in parallel with
the transcriptional activation mediated through this sequence. The GMSA
data suggest that ARP1 may be important for pM regulation as an
activator since an ARP1-immunoreactive activity formed the main
complexes detected in muscles in which M1 is important for pM
expression. This observation may be surprising since ARP1 is known to
behave mainly as an inhibitor of transcription, either as an active
inhibiting factor (36, 37) or more frequently by competition with other
nuclear receptors for a common DNA-binding site (27, 38-40), when
tested in cotransfection studies. However, in other reports, it was
demonstrated that ARP1 could be an activating transcription factor,
depending on promoter context (41, 42), on exogenous factors (like
dopamine or cAMP) (43), or on interaction with cofactors (44). Since
complex A containing ARP1 activity formed with M1 did not migrate in
GMSA like ARP1 homodimers, it suggests that this complex is a
heterodimer between ARP1 and another protein. One can propose that this
ARP1 dimerization partner is a key element for enhancing pM activity;
the observation that complex A was far more abundant in body skeletal
muscle than in tongue muscles or nonmuscle tissues suggests that this
protein may be muscle-enriched. In chick primary myotubes,
overexpression of ARP1 alone has no effect on pM310CAT activity (data
not shown), suggesting that something (this unknown dimerization
partner, activating exogenous signals, or a putative ligand) is missing in this cell system compared with the in vivo situation.
Alternatively, the absence of complex B and the presence of complex C
in tongue compared with body muscles may provide another potential
mechanism of location-dependent regulation for pM.
Identification of proteins involved together with ARP1 in the formation
of complex A or characterization of the RAR/RXR isoforms forming
complexes B and C will be crucial to test functionally the role of
these complexes in pM regulation.
Fiber- or Muscle Subtype-specific Regulatory
Mechanisms?--
Previous works had identified MEF3 and NF-1
DNA-binding sites as key elements for pM activity in hind limb muscles
(16, 21). From the present work, the M1 site appears to be essential to
stimulate transcription from pM in fast-twitch trunk or limb muscles.
We also show that the mutation of M1 alters pM fiber specificity, which
makes M1 one of the few identified cis-acting elements that
may be involved in muscle fiber-specific expression. In previous
in vivo footprinting experiments, dimethyl sulfate accessibility to M1 was shown to be different in soleus and
gastrocnemius muscles (21). However, GMSA did not reveal any difference
for M1-bound proteins in slow- and fast-twitch muscle nuclear extracts. In addition, it should be emphasized that fiber specificity is not
suppressed by the mutation of M1, suggesting that additional fiber-specific elements are present elsewhere in the 310-base pair
enhancer sequence. Our previous observations (16, 21) suggest that NF-1
proteins could be such elements, at least for some hind limb
muscles.
In conclusion, we have shown here that diversity of skeletal muscles is
associated with a great diversity of transcriptional regulatory
mechanisms. In particular, pM is expressed 100-fold more in trunk and
limb fast-twitch muscles than in (fast) head/neck muscles. This muscle
location-dependent activity is mediated through a sequence
(M1) that is bound by nuclear receptors, whose nature and gel mobility
seem to be correlated with M1-mediated transcriptional activation. We
have identified RAR, RXR, and ARP1 proteins as potential members of
these complexes, even if other nuclear receptors could bind M1.
Together with previous results (16, 21), this report shows that the
type IIB fiber-specific expression of pM results not from a single
master element, but from the cooperation of several distinct
fiber-specific cis-acting elements that function predominantly in distinct subsets of muscles. A combination of all
these regulatory mechanisms may enable a fine-tuning of the level of
gene expression, muscle by muscle, as well as a differential adaptation
of distinct muscles to modifications of humoral, neural, and
contractile status.
We are grateful to Prof. P. Chambon and the
IGBMC for the gift of anti-RXR antibodies, to Dr. M.-J. Tsai for the
gift of anti-COUP-TF antibodies, and to Dr. Karathanasis for the human
ARP1 cDNA. We thank M. Raymondjean and B. Viollet for helpful
discussions. We are grateful to M. Salminen for providing some
previously described transgenic lines further analyzed in this study.
We thank J.-P. Concordet and C. Moch for critical reading of the
manuscript. We thank Isabelle Lagoutte, Arlette Dell'Amico, and
Hervé Gendrot for skillful care of the numerous mice used in this
study.