From the ¶ Department of Biochemistry and Cell Biology, SUNY
at Stony Brook, Stony Brook, New York 11794-5215, the
Cardiovascular Research Center, Massachusetts General
Hospital, Charlestown, Massachusetts 02129-2060, the
§ Samsung Biomedical Research Institute, Seoul, Korea
135-230, and the
Institute of Neuroscience, Yang-Ming Medical
University, Taipei, Republic of China
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ABSTRACT |
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Motor activity blocks the extrasynaptic
expression of many genes in skeletal muscle, including those encoding
ion channels, receptors, and adhesion molecules. Denervation reinduces
transcription throughout the multinucleated myofiber, restoring the
developmental pattern of expression, especially of the genes coding for
the acetylcholine receptor. A screen for trans-acting
factors binding to the enhancer region of the -subunit gene of the
acetylcholine receptor identified CTF4, a ubiquitously expressed and
alternatively spliced chicken homologue of the human E protein
transcription factor HTF4/HEB. Expression of the CTF4 locus
closely parallels that of myogenin and acetylcholine receptor during
development and maturation of skeletal muscle, but transcription is not
similarly regulated by neuronal cues. Alternative splicing within the
region encoding the transactivation domain generates two CTF4 isoforms with different tissue distributions, but similar binding affinities for
the acetylcholine receptor
-subunit enhancer and similar transcriptional potential when complexed to myogenin. Direct injection of a myogenin, but not a MyoD, antisense expression vector into denervated skeletal muscle caused a significant decrease in the transcriptional activation of a depolarization-sensitive reporter gene.
Similarly, injection of a CTF4, but less so of an E12, antisense expression vector impaired the denervation response, further
implicating the involvement of a myogenin/CTF4 heterodimer in the
expression of AChR genes in vivo.
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INTRODUCTION |
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Vertebrate skeletal muscle development proceeds by fusion of undifferentiated mononucleated myoblasts to form multinucleated myofibers, with a concomitant activation of muscle-specific genes. Although much is known about the cis-elements and the trans-acting factors that confer muscle-specific gene expression in an in vitro cell culture system, the mechanisms responsible for the modulation of the more complex patterns of gene expression present within the myofibers found in muscle have remained elusive. Many of the activated muscle genes, especially those coding for the contractile apparatus and metabolic pathways, are expressed in all nuclei of every myofiber, whereas others are activated in only a subset of fibers or even a fraction of the nuclei within a single cell, or are restricted to a particular developmental period. Upon innervation and maturation of the myofiber, expression of proteins required for the formation of the neuromuscular junction and the myotendinous junction becomes restricted; thus, subsynaptic nuclei are stimulated by a trophic factor released from the nerve terminal to express acetylcholine receptor (AChR)1 genes, counteracting the ongoing electromechanical activity which eliminates the AChR from extrajunctional regions (1, 2).
Like many skeletal muscle-specific genes, the genes coding for the subunits of the AChR contain E boxes in their regulatory regions that myogenic factors recognize and activate in heterologous expression systems. E boxes are cis-regulatory elements that contain the sequence CANNTG and are found in the enhancers of many developmentally regulated genes from a diverse array of tissues (3). E boxes have been implicated in the dramatic stimulation of receptor gene transcription that is seen during myogenesis in vivo and in vitro and may also play a role in denervation supersensitivity, i.e. receptor gene activation resulting from denervation of the adult muscle fiber (4-7). Similarly, they have been found in the promoters of human (8), mouse (9), and chicken (10) myogenin which are likewise responsive to electrical membrane activity. Denervation does not trigger up-regulation of other genes such as the muscle creatine kinase and myosin light chain 1f/3f genes, both of which clearly contain E boxes as crucial control elements in their regulatory regions (11, 12). The context of an E box, i.e. its flanking regions and the trans-acting factors bound to them, may govern whether a particular E box-containing gene will respond to denervation.
The helix-loop-helix (HLH) motif in the eponymous super-family of transcription factors is a conserved structure that mediates homo- and hetero-dimerization (13); members of this protein family play an important role in differentiation. In basic-helix-loop-helix (bHLH) proteins the HLH motif is immediately preceded by a region rich in basic amino acids that is responsible for site-specific DNA binding to E boxes. The E proteins form one important family of bHLH transcription factors that bind such cis-regulatory elements. Three vertebrate loci have been reported: E2A/PAN/SEF2 (13-15), E2-2/ITF2 (16) and HTF4/CTF4/HEB (17-19). E proteins are widely expressed throughout the organism and are involved in the differentiation of many tissues, including the central nervous system, skin, heart, and skeletal muscle; their dimerization with members of another bHLH family, the myogenic determination factors (MDFs), results in the transactivation of a wide array of muscle-specific genes. Each of the primary transcripts arising from the three E protein loci is alternatively spliced to give rise to multiple isoforms with distinct differences in DNA binding and dimerization domains, nuclear localization signals, and consensus sites for phosphorylation. This also holds for CTF4. As in the case of its rat homologue REB (20)/SCBP (21), alternative splicing of the primary transcript produces two gene products, one with 24 amino acids inserted in the middle of the LH domain, a well conserved transcription-activation motif unique to E proteins (22). The transcripts encoding the two proteins exhibit characteristic spatial and temporal distribution patterns as well as subtle differences in their gene activation capabilities.
CTF4 was originally identified by its ability to recognize a potent
enhancer in the AChR -subunit promoter, prompting us to investigate
its potential role in AChR gene regulation, both during muscle cell
differentiation and the subsequent innervation-induced restriction of
receptor expression to the neuromuscular junction. Myogenin, one of the
four MDFs, is itself regulated by innervation and the
innervation-dependent electrical activity of the sarcolemma (reviewed in Ref. 23), and therefore a potential participant in the
regulation of AChR subunit genes. Whether myogenin exclusively or
preferentially interacts with a specific E protein partner when
activating AChR gene expression is not known.
The differences in depolarization sensitivity of E boxes in muscle-specific promoters may be a result of specific target preferences among bHLH proteins; individual factors and factor combinations have been shown to select specific sequences (3, 24, 25). To investigate the possible participation of CTF4 in the up-regulation of AChR genes in denervated muscle, we combined an antisense strategy with the technique of intramuscular plasmid injection. Here we present in vivo antisense suppression results, and show that myogenin and to a lesser extent CTF4 contribute to the denervation-triggered activation of AChR expression in skeletal muscle.
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EXPERIMENTAL PROCEDURES |
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Animal Experiments--
White Leghorn cockerels (Hall's
Brothers Hatchery, North Brookfield, MA), were anesthetized 3-4 days
after hatching with an intraperitoneal injection of ketamine (50-100
mg/kg), and section of the sciatic nerve was performed unilaterally as
described previously (26). At the desired time after denervation,
animals were sacrificed; the shank muscles of the operated and control
legs were isolated and rinsed with phosphate-buffered saline, then
processed for the preparation of muscle extracts; nuclear extracts were
prepared by the method of Dignam et al. (27). The effects of
electrical stimulation were measured 6-7 days after denervation; the
denervated leg musculature was stimulated in 100-Hz trains, 2-s
duration, applied once every min. Plasmid injection was carried out as
described by Wolff et al. (28). The AChR -subunit
regulatory region drives expression of reporter enzymes following AChR
induction by nerve section; thus, the denervation-induced expression of
p
2kbLuc resembles that of the endogenous AChR subunit genes.
Reporter plasmids were injected 24 h prior to denervation. Three
days after denervation, when luciferase is maximally expressed, animals
were sacrificed, and muscle tissue frozen quickly in liquid nitrogen and pulverized with pestle and mortar. Powdered specimens were resuspended in lysis buffer (100 mM potassium phosphate, pH
7.8, 1 mM DL-dithiothreitol, 1% Triton X-100)
and incubated on ice for 1 h with frequent vortexing. After
centrifugation at 14,000 rpm for 15 min, the supernatant was assayed
for enzyme activity. All animal experimentation followed protocols
approved by the Stony Brook Institutional Animal Care and Use
Committee. For chicken embryos, fertilized White Leghorn eggs were
obtained from local hatcheries and incubated at 38 °C in a
humidified incubator.
Measurement of mRNA-- Total RNA was extracted by the guanidinium isothiocyanate procedure of Chirgwin et al. (29). RNA was quantified spectrophotometrically and checked for integrity by electrophoresis. Messenger RNA levels were measured by ribonuclease protection assays essentially as described previously (30).
Enzyme Assays-- Chloramphenicol acetyltransferase (CAT) and luciferase assays followed established procedures (31).
Plasmids--
Expression vectors were constructed by cloning
cDNAs for chicken MyoD (32), myogenin (33), and the two isoforms of
CTF4 into the plasmid pEMSVscribe (34). A cDNA encoding E12 was
inserted into the vector pBKCMV (Stratagene, La Jolla, CA). To assess
tissue and stage specificity of the expression of CTF4, a
riboprobe synthesized from a 198-base template corresponding to the
3'-untranslated region was used for mRNA analysis. In order to
determine the relative abundance of the two CTF4 transcripts in the
same assay, another riboprobe template, covering the proximal -
splice junction, was generated by polymerase chain reaction. The
upstream primer was located 5' to the splice donor site; the downstream
primer was in the alternatively spliced region (Fig. 1). The resulting fragment is 203 bp in length, of which 171 are common to both forms.
pSK+/
50, the plasmid used for generating the electrophoretic mobility assay probe, was constructed by cloning 50 bp of annealed synthetic oligonucleotides, containing the 36-bp
-subunit enhancer with its 2 E boxes
(GGCGGCCCTCAGCTGTCATGCCTGGAACAGGTGGTG) (35) and
flanking BamHI linkers, into the SmaI site of
Bluescript SK+ (Stratagene). Labeled probes, either 49 or 97 bp long,
were generated by excision with BamHI or a combination of
HindIII and XbaI, respectively, followed by
Klenow fill-in. An 83-bp fragment of the chick AChR
upstream
sequence (
207 to
125) which contains a single E box was cloned into
the HindIII and XhoI sites of pBR322. p
2kbCAT and p
2kbLuc contain ~2 kb of AChR
-subunit upstream region
inserted into the pCAT-Basic and pGL2-Basic vectors (Promega, Madison, WI), respectively. Plasmids for in vitro expression:
pChMyoG/SK
contains 1.1 kb of full-length chicken myogenin cDNA,
and pCTF4#10/SK
, 4.2 kb of CTF4 cDNA (clone no. 10), both
inserted into the EcoRI site of pBluescript SK
(Stratagene). pCMV-E12 encoding the chick E12 protein was obtained from
Bruce Paterson.
Immunohistochemistry-- The crural musculature from an animal that had been denervated 3 days previously was removed from both ipsi- and contralateral limbs and fixed in 4% paraformaldehyde in phosphate-buffered saline. Immunohistochemistry was performed on paraffin-embedded sections, which were incubated with the CTF4-specific antibody 251, a biotinylated anti-rabbit antibody, alkaline phosphatase-conjugated avidin, and finally the enzyme substrate Fast Red (Sigma).
Cell Culture and Transfections--
NIH/3T3 cells were
maintained in growth medium (Dulbecco's modified Eagle's medium
containing 10% supplemented calf serum) before transfection. Cells
(1.5-3.0×105) were plated 2 days prior to transfection on
100-mm dishes and refed with growth medium 2 h prior to
transfection. Transient transfections (10 µg of reporter plasmids,
p2kbCAT or p
2kbLuc, 10 µg of effector plasmids, and 2 µg of
internal control plasmids, pRSVCAT or pRSVLuc) were performed by the
calcium phosphate precipitation method as described previously (35)
except that medium was changed 12 h after introduction of DNA
without glycerol shock. Twenty-four h after transfection, the medium
was changed to serum-deficient medium (Dulbecco's modified Eagle's
medium containing 2% heat-inactivated horse serum). Three days after
transfection, the cells were rinsed twice with 5 ml of
phosphate-buffered saline and harvested in 1 ml of 40 mM
Tris-HCl, pH 7.8, 1 mM EDTA, and 150 mM NaCl.
The cell suspension was centrifuged for 7 s at 14,000 rpm, and the pellet resuspended in 100 µl of lysis buffer. After a 1-h incubation on ice, the cell lysis mixture was spun again, and the supernatant assayed or stored at
70 °C.
In Vitro Transcription and Translation-- In vitro translation was performed using the TNTTM reticulocyte lysate system (Promega) in 50 µl of buffer mixture containing 1 µg of DNA template, 25 µg of rabbit reticulocyte lysate, 1 mM amino acid mixture minus methionine, 4 µl of [35S]methionine at 10 mCi/ml, 5 units of RNA polymerase, and 40 units of RNase inhibitor (Boehringer Mannheim) at 30° for 1 h. The 35S-labeled proteins were resolved on 10% SDS-polyacrylamide gel.
Bacterial Expression of Fusion Proteins-- Chicken myogenin and CTF4 cDNA fragments were cloned into the pGEX vector (Pharmacia Biotech Inc.) encoding glutathione S-transferase. The induced fusion proteins were affinity-purified on glutathione-agarose beads and used in binding studies and for the generation of polyclonal antibodies.
Electrophoretic Mobility Shift Assay--
Appropriate amounts of
bacterial fusion proteins, in vitro translated factors, or
tissue extracts were incubated with typically 10,000 cpm of the
32P-labeled -subunit enhancer probe in a total volume of
20 µl. The binding reactions contained 20 mM Hepes (pH
7.9), 100 mM potassium chloride, 0.2 mM
ethylene diamine tetraacetic acid, 20% glycerol, 0.1 mM
phenylmethylsulfonyl fluoride, 0.1 mM dithiothreitol, 3 µg of poly(dA-dT) or poly(dI-dC), and 0.5 µg of yeast tRNA, and were incubated at 37 °C for 15 min. The protein-DNA complexes were
resolved by electrophoresis through 3.5-5% (w/v) nondenaturing polyacrylamide gels at 100 V for 1.5 h at room temperature. The gel and electrophoresis buffer contained 106 mM Tris, 89 mM boric acid, and 0.1% Nonidet P-40. For supershifts, 0.3 µl of anti-CTF4 or preimmune serum was added to these mixtures.
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RESULTS |
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CTF4 Contains Alternative Transactivation Domains--
The core
enhancer sequence of the chicken AChR -subunit gene contains two E
boxes that are the primary elements responsible for mediating
transcriptional control both during development and upon denervation
(2). A double-stranded oligonucleotide containing four copies of the
enhancer was used as a probe in an affinity screening protocol to
isolate factors involved in neuron-regulated muscle gene transcription.
Of the four DNAs isolated from an E10 whole chicken embryo
gt11
cDNA library, three encoded the E protein CTF4 (18), a homologue of
the human gene HTF4 (17)/HEB (19) (Fig.
1). All three CTF4 cDNAs contained a
72-bp region encoding a 24-residue peptide which is absent from the human protein. A search for additional chicken E protein isoforms was
performed with degenerate oligonucleotide primers targeting regions
conserved in all known vertebrate E proteins, in a polymerase chain
reaction with muscle cDNA as template. Gel electrophoresis of the
polymerase chain reaction product indicated two fragments which were
subsequently cloned and sequenced. The longer fragment corresponds to
the cDNA isolated by the affinity screening protocol above, while
the shorter fragment is derived from an alternatively spliced mRNA
encoding an otherwise identical polypeptide except for lacking the 24 amino acids found in the original CTF4 clones, but absent in HTF4/HEB
(Fig. 1). As a similar splicing pattern has been seen in rat (see
above), we have adopted the nomenclature proposed for REB (20) and
designated cDNAs encoding the shorter and longer proteins CTF4
and CTF4
, respectively. The 24-residue insertion interrupts the
loop-helix (LH) motif transactivation domain contained by all other E
proteins (22).
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Expression Is Especially Abundant in Brain and Skeletal Muscle of the Early Fetus-- The transcriptional regulation of the CTF4 gene was examined by ribonuclease protection assay, utilizing a probe that partly spans the alternatively spliced exon located within the transactivation domain (Fig. 1). During the development of the chicken embryo, CTF4 is continuously transcribed at high levels, but steady-state transcript levels drop considerably after embryonic day 12 (Fig. 2). The two splice variants are present at significant levels in the early developing embryo from the first time point examined, E5.5, with little overall change in the ratio of the two mature transcripts throughout development. The expression of the two CTF4 transcripts was then examined in several different tissues of E10 embryos. Both transcripts were found in all tissues assayed, but their concentration ratios vary by over an order of magnitude. Absolute levels of the CTF4 transcripts vary widely also, with high expressing tissues such as brain and muscle containing about ten times more than liver. The processed transcript lacking the alternatively spliced exon predominates in the majority of tissues, including liver, skeletal muscle, bone marrow, gizzard, and in particular lung. Only in the brain is the long form more abundant; expression levels in the developing brain are higher than in any other embryonic tissue examined and remain moderately high, though fluctuating, throughout development. CTF4 transcripts are most abundant in the telencephalon and cerebellum of the neonate animal, but rare in the brain stem and optic lobe. The CNS also contains a minor band of about 180 nucleotides; this size is consistent with a CTF4 isoform containing the the three amino-terminal residues encoded by the alternatively spliced exon. Although this region contains a splice-donor consensus sequence, we were unable to demonstrate the existence of such a cDNA utilizing reverse transcription-polymerase chain reaction (data not shown).
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Developmental Regulation of Expression in Skeletal Muscle--
The
AChR is expressed along the entire length of the muscle fiber surface
during early stages of development. As the muscle matures,
transcription of the genes encoding the AChR receptor becomes
restricted to the newly formed neuromuscular junctions as a result of
repression of gene activity in extrajunctional muscle nuclei (reviewed
in Ref. 36). In the crural muscles, restricted expression occurs during
early fetal development (E13-15), resulting in significantly fewer
receptor transcripts. Temporal expression patterns of the myogenic
factors and CTF4 were measured in developing muscle. The
down-regulation of the myogenic factors MyoD, myogenin, and myf5 (but
not of herculin/MRF4) shortly after the shift to the fetal stage (E13)
is accompanied by a drop in mRNAs encoding AChR and
subunits,2 and a decline in
CTF4 transcripts (Fig. 3A).
Both the short and long CTF4 transcripts were present throughout the
embryonic and early fetal period of muscle development. To determine if
changes in transcript levels are accompanied by changes in protein
concentrations, we performed supershift analysis. As is shown in Fig.
3C, CTF4-like immunoreactivity is abundant in the early
embryo leg, but disappears toward the second week in ovo,
accompanying a general decline in E box binding activity as measured
with a probe derived from the AChR
-subunit enhancer (Fig.
3B). These changes parallel, and possibly precede, the long
known and pronounced reductions in AChR protein levels in the embryonic
hind limb musculature (36).
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Response in Skeletal Muscle to Neuronal Signals--
As
CTF4 is developmentally expressed in the hind limb muscle in
a pattern similar to the AChR -subunit and the myogenic factors MyoD, myogenin, and myf5, we investigated whether these genes are all
under similar mechanistic control. The myogenin gene in particular
responds to the establishment and disruption of neuromuscular transmission (33, 38, 39). The response of the CTF4 gene to
denervation was therefore determined in the crural muscles of neonate
chickens (Fig. 4, top panel).
Whereas AChR
-subunit message levels increase 80-fold by day 3 postdenervation (37) and myogenin mRNA rises by more than 200-fold
(33), CTF4 transcript levels remain fairly constant (~2-fold
increase), and CTF4 protein, as measured by supershift analysis, also
increases only modestly over a 3-day period (data not shown),
indicating that the motor neuron affects CTF4 gene
expression relatively little. Similarly, whereas electrical stimulation
of chronically denervated skeletal muscle causes an immediate decline
in transcript levels for AChR
subunit and myogenin (33), the
CTF4 gene does not respond to the depolarization signal
under these conditions (Fig. 4, bottom panel). This could
result from an mRNA half-life that significantly exceeds the
duration of the experiment or may indicate independence from neuronal
signals.
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E Box Binding of CTF4 in Homo- and Heterodimeric Form--
We next
tested the ability of the two CTF4 isoforms to bind to the 50 probe
which encompasses the
-subunit enhancer and its two E boxes (35).
Both CTF4
and CTF4
are capable of binding the probe; for the
longer isoform this result was expected, as it had originally been
identified by a protocol selecting for single gene products. Two other
E proteins, the E2A products E12 and E47, and the MDF myogenin were
also examined for their binding activity. While E47 alone displayed
binding activity, E12 failed to efficiently bind to
50 as a
homodimer, presumably because of the inhibitory domain at the
N-terminal which is absent in other E proteins (41). Similarly,
myogenin displays low affinity and binds the probe only at high
concentrations. As seen in Fig. 5,
combination of an E protein with myogenin (in amounts that by
themselves do not generate band shifts) potentiated formation of
complexes that migrate to a position intermediate between that seen
with either bHLH protein alone, indicating the formation of
heterodimers; this was confirmed by antibody disruption experiments (not shown). No significant difference between CTF4
and CTF4
heterodimers in the amounts and patterns of shifted bands were observed, suggesting that the additional 24 amino acids at the LH
domain do not affect DNA binding. These experiments show that the CTF4
proteins, like E12, efficiently form heterodimers with myogenin.
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Transactivation of the AChR -Subunit Promoter--
To determine
the activation potential of CTF4, expression plasmids were transfected
into NIH/3T3 fibroblasts along with the reporter plasmid p
2kbCAT
which contains the CAT gene under the control of the AChR
-subunit
promoter. The NIH/3T3 cell line was chosen as the transfection
recipient as it contains low levels of endogenous E proteins. Results
of these experiments are shown in Fig. 6.
Only CTF4
was capable of efficiently transactivating p
2kbCAT
without additional cofactors. Enzymatic activity due to transactivation
of the reporter construct by CTF4
was detectable only with extended
incubation of the assay reaction. Since gel shift analysis of the two
isoforms revealed little difference in their DNA binding abilities, it
is likely that the additional 24 residues of CTF4
modulate the
transactivating function. The further addition of myogenin to
fibroblasts increased reporter gene activity. Synergistic effects were
particularly apparent when limiting amounts of effector plasmids were
used. Under such conditions, whereas either myogenin or CTF4 by
themselves failed to activate the reporter gene, their combination
resulted in activation. Transactivation is more pronounced with the
short isoform, suggesting that an intact transactivation domain is
required for optimal activity.
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Expression of CTF4 Is Required for Maximal
Myogenin-dependent Gene Activity in Denervated
Muscle--
To determine whether CTF4 participates in AChR gene
activation in either innervated or denervated muscle we adopted a
strategy of CTF4 elimination in muscle fibers through intramuscular
injection of plasmid DNA expressing antisense RNA. Since only a small
fraction of the muscle fibers of the injected muscle take up the
plasmid (41), the effect of an inhibitory transgene on endogenous AChR expression would remain undetectable. The p2kbLuc reporter, whose activity in denervated muscle mimics and parallels the activity of the
endogenous AChR
-subunit gene, was co-injected as a reporter to
permit monitoring the effect of antisense RNA expression on the
targeted transcription factor. To test the validity of the antisense
approach, the effect of suppressing myogenin which is likely to be the
major activator of the AChR subunit genes (33) was examined first.
Administration of the anti-myogenin expression vector pCMVantiMG
reduced denervation-induced activity to as low as 16% of control,
depending upon the amount of antisense plasmid used (Fig.
7A). In contrast, exogenous
myogenin induced the reporter by about 10-fold in innervated muscle
(Fig. 7B). The specificity of the inhibitory effect of the
antisense construct was established by the rescue of p
2kbLuc
expression with exogenous myogenin (Fig. 7C) and by the
relative inefficiency of MyoD suppression (Fig. 7D). The
possible involvement of E proteins in the denervation response was
examined in the same fashion. Specifically, the effects of
antisense-CTF4 and antisense-E12 were analyzed; as shown in Fig. 7,
E and F, the targeting of either E protein
affects the denervation response to some extent. Antisense-CTF4
suppresses reporter gene activity over 5-fold, suggesting a significant
involvement of this factor in the transcriptional activation of the
AChR
-subunit gene upon denervation; treatment with the antisense
construct targeted at E12 produces a less dramatic effect.
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DISCUSSION |
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In this study, we characterize the expression pattern of the gene
encoding the chicken E protein CTF4 and describe its ability to
transactivate, in concert with myogenin, the gene encoding the
-subunit of the AChR. As the chicken homologue of the human, rat,
and mouse proteins HEB, REB, and ME1, CTF4 is a member of the A class
of bHLH proteins and ubiquitously expressed. The primary transcript of
CTF4 is alternatively spliced, with the longer mRNA containing an
additional 72-bp segment encoding 24 amino acids whose presence
disrupts a transactivation domain characteristic of the gene products
of all three E protein loci. This region, including the
flanking residues of the transcriptional activation domain, is
perfectly conserved at the amino acid level when compared with its
homologues in the rat REB (20)/SCBP (21) and a Torpedo californica protein,3
attesting to the evolutionary antiquity and presumptive significance of
this domain. The CNS also contains a minor band of about 180 nucleotides which may arise from alternate splicing at Cga/GTtG (splice
junction consensus in capitals) within the intron shown in Fig. 1.
Three different transcripts of the homologous gene have also been
observed in the rat (21).
Basic-HLH proteins must dimerize before becoming functional
transcription factors capable of binding an E box-containing DNA sequence. As a rule such protein complexes are heterodimers; in fact
some bHLH-containing proteins, such as c-myc and in particular the
MDFs, do not efficiently form homodimers. This does not hold for CTF4;
homodimers of both isoforms of CTF4 were equally capable of binding to
the E box-containing -subunit enhancer. In addition, homodimers of
the shorter, but less so of the longer, isoform of CTF4 were able to
transactivate a reporter gene under control of the
-subunit promoter
in NIH/3T3 fibroblasts, a cell line containing very low levels of
endogenous E proteins. As homodimers of each CTF4 isoform are equally
able to bind to the E boxes of the
-subunit enhancer, differences in
reporter enzyme levels are likely due to the relative effectiveness of
the respective transactivation domains. When myogenin was co-expressed
in the same experiment to allow for the formation of CTF4 heterodimers, only minimal differences in reporter activity were observed for the two
E protein isoforms. The transactivation domain of myogenin apparently
obviates the need for the E protein to provide such a domain itself. As
has been pointed out previously, the alternative domain is predicted to
form an ankyrin motif (20) which is likely to mediate protein-protein
interactions. Ankyrin motifs exclude members of the Rel family of
transcription factors from the nucleus during particular phases of the
cell cycle by sequestering them in an inactive cytoplasmic complex with
I
B (42). Although E proteins are abundantly expressed in both
proliferating and recently differentiated cells, they possess potent
growth-suppressive activity that must be carefully regulated to prevent
premature exit from the cell cycle at the G1 restriction
point (43). It is possible that the ankyrin motif might allow for
precise regulation of nuclear E protein concentrations during the cell
cycle, an aspect that would not be reflected in our assays for gene
expression in differentiated myofibers.
CTF4 exhibits tissue-specific regulation during development.
The tissue distribution of the two isoforms in the 10-day embryo is not
unlike the one described for REB and REB
in the adult rat (20)
colon, a predominance of the long form in the brain; a prevalence of
the short form in liver, lung, and skeletal muscle; and comparable
amounts in the heart. The transcript is expressed at high levels at the
early fetal stage (embryonic day 8/9) in both brain and muscle, two
tissues that are late to mature. The brain continues to express high
levels in the neonate chicken; however, in hind limb skeletal muscle,
CTF4 mRNA undergoes a sharp decline at the transition from early to
late fetus (embryonic day 14/15). It is at this period that
neuromuscular junctions mature in chicken embryo leg muscle (44), and
transcription of MyoD and myogenin is reduced to low levels in all
muscle nuclei. Since the genes that encode the extrajunctional form of
the AChR and the myogenic factors MyoD and myogenin lose activity at
about the time of the down-regulation of CTF4, one might expect similar mechanisms to be at play. However, this does not seem to hold for the
adult myofiber where denervation dramatically up-regulates, and
membrane activity strongly suppresses, AChR and myogenin genes (33, 40)
without similarly affecting CTF4 expression.
AChR expression in skeletal muscle depends on the developmental stage
of the tissue. While receptor genes are silent in myoblasts, they
become activated during differentiation and myofiber formation; later,
innervation of the myofiber and the ensuing electromechanical activity
suppress the genes. This responsiveness to neuronal cues can be
demonstrated by experimental manipulation: Section of the motor nerve
causes cessation of activity and de-repression of AChR genes, while
imposition of electrical activity again silences them. Consequently,
AChR genes are much more active (~5-10-fold) in denervated than in
innervated muscle (45). Since in the chicken the genes for MyoD, myf5,
and herculin are little affected by innervation, it is likely that AChR
gene expression is mainly dependent on myogenin. Our antisense
experiments suggest that this is also true for denervation-triggered
stimulation of AChR expression. It has previously been shown by
Brunetti and Goldfine (46) that expression of the AChR subunit in
BC3H-1 cells can be suppressed with antisense oligonucleotides directed
against myogenin. The observation of these authors is in agreement with our findings, although it does not rule out a role, in muscle tissue,
for other MDFs, especially MyoD (which is absent from BC3H-1
cells).
The parallel expression of myogenic factors and receptor genes during
embryonic development and in adult muscle suggests a functional
relationship, as the transcription factors are expressed when they
might be active in stimulating receptor gene expression. The regulatory
role of CTF4 during denervation-triggered receptor expression is not as
clear, since the denervation response of the CTF4 gene is
less pronounced. Nevertheless, transfection of an antisense expression
vector leads to significant reduction in the activation of AChR
promoters which otherwise is the hallmark of the denervation response.
Perhaps the presence of CTF4 is necessary, but by itself not sufficient
to stimulate AChR expression; being expressed constitutively, it may
eventually become the limiting factor in denervation-induced AChR
up-regulation. A possible explanation of the relatively low
effectiveness of antisense-CTF4 (compared with the suppression of
myogenin) is that, in the absence of CTF4, other E proteins which are
still abundant in the nuclei have an opportunity to dimerize with, and
activate, myogenin. A similar effect was observed with antisense-E12,
even though the suppression of the reporter gene is a little less
pronounced than the effect of antisense-CTF4. Perhaps the two E
proteins share the task of dimerizing with myogenin; the importance of
combined gene dosage effects for the function of E proteins has been
pointed out before (47). A role for CTF4 in receptor expression is
further suggested by its ability to associate with the -subunit
enhancer, and to enable myogenin and MyoD to do likewise.
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ACKNOWLEDGEMENTS |
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We thank Marlies Schmidt for excellent technical assistance, and Bruce Paterson for the cDNAs encoding chicken MyoD and E12.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant NS20233 and a grant from the Muscular Dystrophy Association.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: Dept. of Biochemistry and Cellular Biology, SUNY at Stony Brook, Stony Brook, NY 11794-5215; Tel.: 516-632-8561; Fax: 516-632-8575; E-mail: jschmidt{at}life.bio.sunysb.edu.
1 The abbreviations used are: AChR, acetylcholine receptor; bHLH, basic helix-loop-helix; CAT, chloramphenicol acetyltransferase; HLH, helix-loop-helix; LH, loop-helix; MDF, myogenic determination factor; bp, base pair(s); kb, kilobase pair(s).
2 H.-J. Tsay and Y.-S. Lee, unpublished results.
3 C. Neville, unpublished results.
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REFERENCES |
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