From the Istituto di Biologia dello Sviluppo del
Consiglio Nazionale delle Ricerche, Via Ugo La Malfa 153, 90146 Palermo, Italy, the ¶ Dipartimento di Biologia
Cellulare e dello Sviluppo, Università di Palermo, 90128 Palermo,
Italy, and the
Dipartimento di Istologia ed Embriologia Medica,
Università di Roma La Sapienza, 00161 Roma, Italy
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ABSTRACT |
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We have previously identified a muscle-specific
enhancer within the first intron of the human enolase gene. Present
in this enhancer are an A/T-rich box that binds MEF-2 protein(s) and a G-rich box (AGTGGGGGAGGGGGCTGCG) that interacts with ubiquitously expressed factors. Both elements are required for tissue-specific expression of the gene in skeletal muscle cells. Here, we report the
identification and characterization of a Kruppel-like zinc finger
protein, termed
enolase repressor factor 1, that binds in a
sequence-specific manner to the G-rich box and functions as a repressor
of the
enolase gene transcription in transient transfection assays.
Using fusion polypeptides of
enolase repressor factor 1 and the
yeast GAL4 DNA-binding domain, we have identified an amino-terminal
region responsible for the transcriptional repression activity, whereas
a carboxyl-terminal region was shown to contain a potential
transcriptional activation domain. The expression of this protein
decreases in developing skeletal muscles, correlating with lack of
binding activity in nuclear extract from adult skeletal tissue, in
which novel binding activities have been detected. These results
suggest that in addition to the identified factor, which functionally
acts as a negative regulator and is enriched in embryonic muscle, the
G-rich box binds other factors, presumably exerting a positive control
on transcription. The interplay between factors that repress or
activate transcription may constitute a developmentally regulated
mechanism that modulates
enolase gene expression in skeletal
muscle.
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INTRODUCTION |
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The muscle-specific or isoform of the glycolytic enzyme
enolase (EC 4.2.1.11) is encoded by a member of the enolase gene family, the expression of which is regulated in a tissue-specific and
development-specific manner (1-4). The gene is primarily expressed in
the cardiac and skeletal muscles, where, during embryonic development,
the
isoform progressively replaces the nearly ubiquitous
isoform (5). Indeed, the message for the
enolase is first detectable in the cardiac tube and in the myotome (11); expression remains extremely low in skeletal primary fibers, whereas a striking increase occurs in the second generation of fibers at the fetal stage
of development (6, 11), and a further increase is observed after birth.
In the adult,
enolase is expressed in both cardiac and skeletal
muscles, with higher levels of expression detected in fast-twitch
fibers than in slow-twitch fibers (11). In vitro studies on
myogenic cell lines and primary myoblasts have shown that the level of
enolase expression increases with terminal differentiation but, at
variance with the majority of the muscle-specific genes, expression
already occurs in proliferating myoblasts (6-8). Interestingly,
enolase, like desmin (9), belongs to a relatively small group of
muscle-specific genes expressed in proliferating myoblasts, as well as
in differentiated myotubes, and it has been suggested that it might be
a marker of adult satellite cells in humans (8, 10).
From these data, it can be presumed that regulation of the enolase
gene expression may take place at multiple levels and involve complex
molecular mechanisms, making the gene a suitable model to investigate
various aspects of muscle-gene transcriptional control.
In the last few years, a great number of transcription factors and DNA regulatory elements have been identified as contributors to the activation of the muscle differentiation program. In skeletal muscle, the MyoD family of basic helix-loop-helix proteins (MyoD, myogenin, Myf-5, and MRF4), the ectopic expression of which can activate skeletal muscle gene expression in a wide range of non-muscle cell types, plays a pivotal role during development (12). The MEF-2 family of MADS-box transcription factors, which bind an A/T-rich element found in the promoters and enhancer of the majority of skeletal and cardiac muscle genes, is involved both in a direct and indirect mechanism of transcriptional activation (13, 14).
Recently, the scenario has become more complex, because several reports have outlined the importance of ubiquitously expressed factors in association with tissue-restricted factors to maintain tissue-specific expression. Functional cooperation between elements that bind ubiquitous factors and tissue-restricted factors has been demonstrated for the regulatory regions of both cardiac and skeletal muscle genes, and in almost all cases, these sequences are located in relatively close proximity (less than 60 bp), suggesting that protein-protein interactions might be involved in the cooperation (reviewed in Ref. 15). Furthermore, it has recently been reported that an apparently ubiquitous binding activity consists itself of a complex composed of a ubiquitous factor and a tissue-restricted cofactor, thus expanding the number of potential regulatory combinations required for the control of tissue-specific transcription during myogenesis (16).
In our previous studies, we identified several distinct regulatory
regions in the human enolase gene and characterized a muscle-specific enhancer present in the first intron of the gene (15).
This element interacts through an A/T-rich box with members of the
MEF-2 family of transcription factors and through a G-rich box,
AGTGGGGGAGGGGGCTGCG, with a ubiquitous factor(s). Mutation of either
the G-rich box, termed
enolase element 1 (BEE-1),1 or the A/T-rich box
resulted in a significant reduced activity of the enhancer in
transient-transfection assays, indicating that MEF-2 and BEE-1 binding
factors are each necessary for tissue-specific expression of the
enolase gene in skeletal muscle cells. Furthermore, sequences
homologous to the BEE-1 site in association to a MEF-2 binding site are
present in the transcriptional regulatory regions of several skeletal
and cardiac muscle-specific genes (15), suggesting the existence of a
widespread pathway of muscle-gene transcriptional control.
This article reports the isolation and characterization of a protein
that binds in a sequence-specific manner to the BEE-1 element. The
deduced amino acid sequence revealed the presence of four C2H2 zinc
finger domains, which identify the protein as belonging to the family
of transcription factors resembling the Kruppel segmentation gene
product of Drosophila (17), and high similarity with a human
zinc finger protein, which has been shown to bind the promoter of the
gene for the V8.1 chain of the T-cell receptor (18). Analysis of
both transcripts and proteins expression in myogenic cells and tissues,
coexpression of the protein with several
enolase reporter
constructs, and the identification of a transferable repression domain
indicated that the zinc finger factor acts as a repressor of
enolase gene transcription.
Furthermore, the data reported indicate that in addition to the
identified zinc finger protein that we have designated enolase repressor factor 1 (BERF-1) and the expression of which is enriched in
embryonic muscle, other factors bind to the BEE-1 element in adult
muscle. These results support the hypothesis of regulatory pathway
involving positive and negative regulators that bind to the same site
or overlapping binding sites within the muscle-specific enhancer of the
enolase gene.
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EXPERIMENTAL PROCEDURES |
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Southwestern (DNA-protein) Screening of the Skeletal Muscle
cDNA Expression Library--
A Zap II expression cDNA
library was prepared with mRNA from limbs of 12-day mouse embryos
using a cDNA synthesis kit (Stratagene). The amplified expression
library was screened by the method of Vinson et al. (19)
using as a probe double-stranded BEE-1 oligonucleotides (sense,
5
-AGCTGTTCTGAGTGGGGGAGGGGGCTGCGCCTGC-3
) that had been end-labeled and ligated into concatamers. One positive clone out of
2.0 × 106 plaques survived three rounds of plaque
purification. The pBluescript phagemid was excised from the
Zap
expression vector by helper phage coinfection (R408 strain) according
to the instructions of the manufacturer (Stratagene). The isolation of
additional cDNAs was carried out by standard methods using the
cDNA identified by Southwestern screening as a probe.
DNA Sequencing and Expression of the Cloned cDNAs by in Vitro Transcription and Translation-- The nucleotide sequence of the cDNA clones were determined on both the sense and antisense strands by the dideoxynucleotide chain-termination method using modified T7 DNA polymerase (Sequenase; United States Biochemicals). In vitro transcription of the isolated clones was carried out with 2 µg of linearized pBluescript plasmid using an mRNA capping kit (Stratagene). In vitro translation was performed with a commercially available rabbit reticulocyte lysate system according to the instructions of the manufacturer (Promega), and when needed, [35S]methionine was added to the translation mixture.
RNA Isolation and Northern Blot Analysis--
Total RNA was
extracted from cultured cells and from limbs and hearts of adult mice
or embryos isolated at 12, 14, and 16 days postcoitum (dpc) by the
guanidine isothiocyanate method (20). Mouse multiple tissue Northern
blot was purchased by CLONTECH. Fifteen micrograms
of total RNA were fractionated by electrophoresis on denaturing agarose
gel, transferred to nylon membranes, and hybridyzed as described
previously (6). A 2.3-kb (kilobase) BamHI fragment
containing the almost entire coding region was isolated from the A22
cDNA and used as a probe. As a control of the amount of RNA loaded
per lane and to check differentiation of myogenic cells in cultures,
filters were rehybridized with a chicken actin cDNA (6) and/or
with a human glyceraldehyde-3-phosphate dehydrogenase (GAPD) cDNA
(ATCC 5701)
Generation of Polyclonal Antibodies and Immunoblot Analysis-- DNA fragments encoding different portions of the ZF22 protein were subcloned into the bacterial expression vector pGEX-2T (Pharmacia). A 344-bp BstXI-AccI fragment and a 884-bp ClaI-BamHI fragment were isolated from the A22 cDNA, whereas a 2-kb EcoRV-BamHI fragment was excised from the A21 cDNA encoding the truncated ZF21 polypeptide. The three glutathione S-transferase-ZF22 fusion proteins, which consisted of the amino-terminal region from amino acid 26 to amino acid 111, the zinc finger region from amino acid 76 to amino acid 319 and the carboxyl-terminal region from amino acid 430 to amino acid 740, respectively, were overexpressed in Escherichia coli and affinity purified by binding to glutathione-linked Sepharose beads (21). Rabbit anti-ZF22 polyclonal antisera were raised against the purified fusion proteins, and antibodies were immunopurified on columns containing the respective glutathione S-transferase-fusion protein used as immunogen according to established procedures (22). For immunoblot analysis, nuclear proteins (5 µg), prepared as described in the following section, or total cell lysates (30 µg) obtained by extraction in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycolate, 0.1% SDS, 1 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml aprotinin, 1 µg/ml leupeptin, 0.5 µg/ml pepstatin, were resolved by electrophoresis on an SDS-7% polyacrylamide gel and electroblotted to a nitrocellulose membrane (Hybond-C, Amersham Corp.). The membrane was incubated with affinity purified anti-ZF22 antibodies (0.5 µg/ml) and then with a secondary antibody conjugated to horseradish peroxidase (Pel-Freez) (1:5000).The antigen-antibody complexes were visualized by enhanced chemiluminescence (ECL kit, Amersham Corp.).
Preparation of Nuclear Extracts and EMSAs--
Nuclear extracts
were prepared from cultured cells and embryonic muscle tissue (limbs
isolated from 12-dpc mouse embryos), as described previously (15).
Preparation of nuclear extracts from skeletal muscle tissue isolated
from adult mice was according to Zahradadka et al. (23). The
following double-stranded oligonucleotides were used as probes
and competitors in electrophoretic mobility shift assays (EMSAs):
BEE-1w (5-AGCTGTTCTGAGTGGGGGAGGGGGCTGCGCCTGC-3
), wild-type consensus
(15); BEE-1m (5
-AGCTGTTCTGAGTGGGACTCTAGGCTGCGCCTGC-3
), mutated consensus (15); Sp1 site (5
-ATTCGATCGGGGCGGGGCGAGC-3
), canonical Sp1 binding site (24); E-boxL
(5
-TTTAACCCAGACATGTGGCTGCCCC-3
), left E-box from muscle
creatine kinase enhancer (25).
Construction of Reporter Expression Vectors and Plasmids
Expressing Wild-type ZF22 and Its Deletion Mutants--
Details about
the construction of the enolase-chloramphenicol acetyltransferase
gene (CAT) expression vectors used in this study have been published
(15). Reporter constructs containing multiple copies of the BEE-1
binding site or a mutated consensus site upstream of the
enolase
promoter, termed pB3-BEE-1w4X and pB3-BEE-1m4X, were obtained by
blunt-end ligation into the HindIII site of the pB3-CAT of
BEE-1w or BEE-1m double-stranded oligonucleotides (four copies),
respectively. Orientation and proper insertion of the four BEE-1
consensus sites was determined by polymerase chain reaction (PCR)
analyses with appropriate primers and confirmed by nucleotide
sequencing. A NarI-BamHI fragment containing the four wild-type or mutated consensus sites and the
enolase promoter was isolated from the above described constructs and used to replace the homologous fragment present in the pB3SV-CAT plasmid to generate pB3SV-BEE-1w4X and pB3SV-BEE-1m4X, respectively. For cotransfection experiments, ZF21/22 expression vectors encoding the short or the long
form of the factor, were generated by inserting a 2.3-kb BamHI fragment, isolated from the A21 cDNA, and a 2.5-kb
NciI fragment, isolated from the A22 cDNA, into the
BamHI and EcoRV sites, respectively, of the
cytomegalovirus promoter-directed expression vector pCDNAI
(Invitrogen). The resulting constructs are indicated as pCDNAI-ZF21
and pCDNAI-ZF22. Plasmids expressing fusion polypeptides of ZF22
and the yeast GAL4 DNA-binding domain were obtained by inserting into
the EcoRI site of the expression vector pSG424 (30) DNA
fragments encoding different portions of ZF22. Briefly, an
EcoRI site with the same reading frame as the
EcoRI site in pSG424 was introduced at the 5
end of the
ZF22 coding region, using the following pair of oligonucleotide
primers: forward, 5
-CCGGAATTCAACATTGACGACAAACTGGA-3
(oligomer A); reverse, 5
- ATCATGGGATATCATATCCTG-3
(oligomer B). The
221-bp EcoRI-EcoRV DNA fragment obtained by PCR
was used to replace the EcoRI-EcoRV fragment of
the A22 cDNA, which contains the 5
-untranslated sequence, and the
full-length coding sequence was then isolated by restriction with
EcoRI and XhoI (the latter is the 3
-cloning
site) and used to generate GAL4-ZF22 (2-794) (see Fig. 9). Constructs
c, d, f, and g (shown in Fig. 9B), which encode
carboxyl-terminal deletion mutants, were generated from the plasmid
containing the full-length coding region by excision of DNA fragments
up to the SacI, EcoRI, AccI, and
EcoRV sites, respectively. Construct e, GAl4-ZF22 (2-184), was obtained by PCR using oligomer A as forward primer and as reverse
primer, oligomer C (5
-CCGGAATTCATAGTTCGTTCTAAAGGCAG-3
), which contains an EcoRI cloning site. Similarly, construct
k, Gal4-ZF22 (136-184), was obtained by PCR using oligomers D
(5
-CCGGAATTCGAGCCAGTAGACTTACAGAA-3
) and C as forward and
reverse primers, respectively. To generate construct j, GAL4-ZF22
(444-794), which encodes the carboxyl-half portion of the protein, the
ClaI-XhoI DNA fragment was first subcloned into
pBluescript/ET-3a (Novagen) to add supplementary cloning sites in frame
with the pSG424 polylinker. Finally, constructs h and i were obtained
from construct j by excision of DNA fragments up to the
EcoRI and SacI sites, respectively (see Fig.
1A for a partial restriction map). Nucleotide sequence of
ZF22 DNA fragments generated by PCR was confirmed by DNA sequencing,
whereas proper expression and stability of the GAL4-ZF22 fusion
proteins was tested by immunoblot analysis on extracts of transiently
transfected COS 7 cells, using a rabbit antiserum against the
DNA-binding domain of GAL4 (Santa Cruz Biotechnology) (data not shown).
In the case of cotransfection experiments with the various GAL4-ZF22 mutants, the reporter construct used was pG5TKCAT. This construct was
obtained by inserting a HindIII-XbaI fragment,
excised from the pG5E1bTATACAT plasmid and containing five GAL4-binding
sites (31), upstream of the thymidine kinase gene (TK)
promoter in plasmid pBL2CAT (32).
Cell Culture, Transfection, and CAT Assays--
C2C12 myogenic
cells (33) were cultured as proliferating myoblasts in a growth
factor-rich medium consisting of Dulbecco's modified Eagle's medium
(Life Technologies, Inc.) supplemented with 20% fetal calf serum (Life
Technologies, Inc.). Differentiation into myotubes was induced by
exposure of subconfluent cultures to a medium containing 5% horse
serum (Life Technologies, Inc.) and 5 µg/ml of insulin (Sigma) for
48-72 h. Embryonic and fetal myoblasts were cultured from limbs of
mouse embryos (11 dpc) and fetuses (16 dpc) as described previously (6,
34). PAF human fibroblasts, CV1 and COS7 monkey kidney cells, and
C3H10T1/2 mouse fibroblasts from laboratory stocks were maintained in
Dulbecco's modified Eagle's medium with 10% fetal calf serum. Cells
were transfected by the calcium phosphate method (35) as described previously (15). Briefly, 10 µg of recombinant CAT plasmid, 2 µg of
the -galactosidase expression plasmid pON1 (36) (used to monitor
transfection efficiency), and 0.5-5 µg of BERF-1 expression vectors
(either pCDNAI-ZF22/21 or GAL4-ZF22 constructs) were used to
transfect 3-4 × 105 cells in a 5.8-cm-diameter
culture dish. Cells were harvested 48 h later and subjected to
-galactosidase and CAT assays as described previously (15). All
transfections were performed on multiple sets of cultures with at least
two different DNA preparations for each plasmid.
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RESULTS |
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Cloning of BEE-1-binding Proteins: Isolation of cDNAs Encoding
the Zinc Finger Polypeptides ZF21 and ZF22--
The BEE-1 binding site
is a G-rich element required to support muscle-specific expression of
the enolase gene together with an adjacent MEF-2 site. For
isolation of BEE-1 binding factors, an embryonic muscle cDNA
expression library was screened by the Southwestern method using a
concatenated BEE-1 probe. One single clone (A21) that displayed
sequence-specific binding to the BEE-1 probe was isolated (Fig.
1A). The nucleotide sequence
of the cDNA insert revealed the presence of an open reading frame
(ORF) of 960 bp, potentially encoding a polypeptide of 320 amino acid
residues with a calculated molecular mass of 35 kDa (Fig. 1B,
ZF21). Additional clones were isolated by screening the cDNA
library with 5
and 3
fragments of the cDNA originally identified
by Southwestern screening. One cDNA (A22) was sequenced and
appeared to be identical to the one encoding ZF21 except for the
presence of a longer 5
-untranslated region and a one-nucleotide
deletion. This deletion would result in a frameshift (Fig.
1A) and the creation of an ORF of 2382 bp encoding a
794-amino acid polypeptide with a predicted molecular mass of 89 kDa
(Fig. 1B, ZF22). The same deletion was found in six
independent clones, suggesting that the originally isolated cDNA
encoding ZF21 might be representative of a rare message or be the
result of a reverse-transcriptase error that occurred during the
preparation of the library.
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Identification of ZF22 as the Major BEE-1-binding Protein in C2C12 Nuclear Extracts-- To confirm the predicted ORF of both A21 and A22 cDNAs and to check the ability of the encoded polypeptides to bind the BEE-1 element, the pBluescript plasmids carrying the two cDNAs were in vitro transcribed and translated. The major product of the ZF22-encoding plasmid was a polypeptide migrating with an apparent molecular mass of about 110-120 kDa, whereas two major polypeptides of about 50 and 43 kDa were detected in the translation mixture programmed with the ZF21-encoding mRNA, likely resulting from the utilization of more than one translation start site (data not shown). Consistent with results previously reported for BFCOL1 (38), both ZF22 and ZF21 migrate in SDS-polyacrylamide gel electrophoresis more slowly than expected from their predicted molecular masses (89 and 35 kDa, respectively). In gel shift experiments, using as a probe the same oligonucleotide utilized in the Southwestern screening, the in vitro-translated ZF22 gave rise to a major DNA-protein complex comigrating with the major endogenous binding activity detected in C2C12 nuclear extracts (Fig. 2A, compare lanes 1 and 2), whereas two faster migrating DNA-protein complexes were resolved with ZF21 (Fig. 2A, lane 5). This result was consistent with the heterogeneity of the translation products. The specificity of the binding was confirmed by addition of a molar excess of the oligonucleotide containing either a wild-type BEE-1 consensus or a mutated consensus site (Fig. 2A, lanes 3 and 6 and lanes 4 and 7, respectively). These results indicated that both ZF22 and ZF21 contain a DNA-binding domain, presumably the four zinc fingers, and suggested that ZF22 corresponds to the predominant BEE-1-binding protein detected in nuclear extracts by EMSA.
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The Zinc Finger Factor Expression Is Down-regulated During
Myogenesis--
To investigate both the pattern of expression of the
ZF22 mRNA in various tissues and the relationship between its
expression and that of the enolase, a Northern blot analysis of
RNAs from different sources was performed. Three major transcripts
corresponding to 3.4, 4.1, and 7.6 kb were observed in all of the
tissues and cell cultures analyzed (Figs.
3 and 4),
but other, less abundant messages larger than 7.6 were also detected,
as previously reported for ht
and ZBP-89 (18, 37). These multiple
bands may be explained by the existence of additional mouse cDNAs
containing 3
-untranslated sequences of different length as result of
the utilization of different polyadenylation sites (data not shown).
The amount of ZF22 transcripts increased from day 12 to day 14 of mouse
embryonic limb development, followed by a remarkable decrease from day
14 to day 16 (Fig. 3A, lanes 1-3) and by a further decrease
in limb skeletal muscle of newborn and adult mice (Fig. 3A, lanes
4 and 5); similarly, a lower level of expression was
observed in adult cardiac muscle tissue when compared with earlier
stages (Fig. 3A, lanes 6-8). RNA was also isolated from
primary cultures of embryonic (11 dpc) and fetal (16 dpc) myotubes (6,
34). Fig. 3B shows that ZF22 transcripts were present in a
relatively large amount in embryonic myotubes, whereas they were barely
detectable in fetal myotubes. ZF22 expression both in vivo
(during muscle development) and in myogenic primary cultures inversely
correlates with
enolase expression (6). Analysis of mRNA from
adult tissues showed that the ZF22 message was ubiquitously expressed; however, the relative amount varied greatly among tissues,
i.e. the message was more abundant in brain and liver than
in skeletal muscle and heart (Fig. 3C). Northern blot
analysis of RNA extracted from C2C12 myogenic cells at various times
during differentiation induced by withdrawal of growth factors (Fig.
4A) revealed that following differentiation of myoblasts to
multinucleated myotubes, the level of ZF22 transcripts slightly
decreased (compare lanes 2-4 with lane 1);
however, the degree of such down-regulation was much less significant
than in vivo during muscle histogenesis (Fig. 3,
A and B). Total protein lysates were prepared
from C2C12 cells maintained for the same lengths of time in
differentiation medium and analyzed by Western blot (Fig.
4B). All three anti-ZF22 antibodies recognized two closely
migrating polypeptides, with apparent molecular masses of about 110 and
120 kDa, clearly resolved when electrophoresis was carried out on
longer gels (see Fig. 6). Both polypeptides appeared to be slightly
more abundant in myoblasts than in myotubes. The specificity of the
antibodies was confirmed by use of preimmune sera as a negative
control; furthermore, the apparent molecular weight of the two
endogenous polypeptides was consistent with the size of the ZF22
protein obtained by in vitro transcription-translation. The
two proteins may be the products of alternatively spliced messages or
result from posttranslation modifications of a single product.
Immunofluorescence analysis (not shown) confirmed that ZF22 protein is
ubiquitously expressed in the mesoderm of mouse embryos but is no
longer detectable in mesoderm of mouse fetuses.
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The DNA Binding Activity of the Zinc Finger Factor Is Diminished in
Adult Skeletal Muscle Tissue Where Novel BEE-1 Binding Activities Are
Detected--
The discrepancy observed between ZF22 expression in
muscle tissues and C2C12 myogenic cells prompted us to investigate the presence of BEE-1 binding activity in skeletal muscle tissues. Nuclear
extracts were prepared from embryonic muscle, using as a source limbs
of 12-day mouse embryos, and from skeletal muscle of adult mice; both
extracts were used in EMSAs (Fig. 5).
Strikingly, although the BEE-1-ZF22 complex was easily detected in
nuclear extract from embryonic muscle (Fig. 5A, ME, lane 2),
it was barely detectable in nuclear extract prepared from adult muscle
(Fig. 5A, MA, lane 3). The DNA-ZF22 complex obtained with
nuclear extract from embryonic muscle was not distinguishable from the
one obtained with C2C12 nuclear extract (Fig. 5A, compare
lanes 1 and 2), as confirmed by the use of
anti-ZF22 antibody (data not shown), whereas it was replaced by one
major faster migrating complex and several minor complexes in adult
muscle nuclear extract (Fig. 5, A and B, MA, lane
3). The newly detected complexes are specific because they were
drastically reduced by addition of an excess of unlabeled probe
containing a wild-type BEE-1 consensus site and were not affected by a
molar excess of a mutated consensus site (Fig. 5B, lanes 2 and 3). Furthermore, the adult muscle-specific complexes were unaffected by addition of all three anti-ZF22 sera and antibody against MNF (data not shown), a factor reported to bind a CACCC-box within the muscle-specific enhancer of the myoglobin gene (27), indicating that the proteins in the complexes are not related to both
factors. An independent assessment of the quality of the extracts is
provided in Fig. 6, C and
D, which show no significant difference in Sp1 and E-box
site binding proteins between nuclear extracts of embryonic and adult
muscle; the binding activity detected with the left E-box from the
enhancer of the muscle creatine kinase gene was slightly higher in
adult muscle than in embryonic tissue (Fig. 6D, lanes 1 and
2). Consistent with the results obtained by EMSA, a Western
blot analysis of the same nuclear extracts confirmed that ZF22
polypeptides were present at a relative comparable level in extracts of
C2C12 myotubes and embryonic skeletal muscle but were not detectable in
a equal amount of nuclear extract prepared from adult muscle (Fig. 6,
lanes 2-5). As observed in total lysates, ZF22 polypeptides
are slightly more abundant in the nuclear extract of C2C12 myoblasts
than in that of myotubes (Fig. 6, lanes 2 and 3).
The proteins displayed the same apparent molecular weight in nuclear
extract both from murine myogenic cells and muscle tissue, whereas two
polypeptides with slightly different mobilities were detected in the
extract of a human fibroblast cell line used as a control (Fig. 6,
lane 1). The apparent molecular weight of the human proteins
detected with the anti-ZF22 antibodies is consistent with a ORF much
larger than the one reported for ht, supporting the data by Hasegawa
et al. (38) on the isolation of a human cDNA clone with
a continuous ORF and in agreement with a recent report on the
expression of the factor in human cell lines and tissues (42).
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Overexpression of the Zinc Finger Factor Results in Repression of
Both Basal and Activated Transcription--
To address the question of
the regulatory role exerted by the zinc finger factor on enolase
gene expression, both ZF22 and ZF21 proteins were overexpressed in
transfection assays with CAT reporter plasmids either carrying the
enolase promoter and the entire first intron (nucleotides
172 to
+706) with the muscle-specific enhancer in its wild-type location or
bearing the minimal enhancer sequence (nucleotides +532 to +611)
upstream of the promoter (Fig. 7A, pB10-CAT and pB3-5
PCR1,
respectively). In both cases, expression of ZF22 and ZF21 resulted in a
consistent and comparable reduction of the CAT activity relative to the
activity detected in cells transfected with the parental, insertless
expression vector (Fig. 7B). The results shown in Fig. 7
were obtained in transiently transfected C2C12 myotubes, but a similar
degree of repression was observed in CH310T1/2 and CV1 cells (data not
shown). To further investigate the mechanisms involved in the observed
transcriptional repression, different concentrations of the ZF22
expression plasmid were cotransfected with reporter constructs in which
CAT gene expression is driven by the
enolase promoter with multiple
copies of a wild-type or a mutated ZF22 binding site (Fig.
8A, pB3-BEE-1w4X and
pB3-BEE-1m4X) and similar constructs carrying the promiscuous SV40
enhancer downstream of the CAT transcription unit (Fig. 8A, pB3SV-BEE-1w4X and pB3SV-BEE-1m4X). Fig. 8B shows that a
dose-dependent transcriptional repression was observed in
all cases; ZF22 was able to repress transcription from the reporter
plasmids carrying four mutated consensus sites but was quantitatively
less repressive on these plasmids than on the reporter plasmids
containing four wild-type binding sites. When lower amounts of the ZF22
expression plasmid were transfected (Fig. 8B, 0.5 µg)
repression was greater when the reporter plasmid contained the
wild-type binding sites, and this difference was more significant when
constructs containing the strong SV40 enhancer were used as reporters
(Fig. 8B, compare a, b, c, and d).
Reporter constructs carrying heterologous promoters, (TK
promoter or SV40 early promoter) behaved similarly, suggesting that the
observed activity does not depend upon the promoter used (data not
shown), although all are TATA-box containing promoters. The results of
these transfection experiments indicated that both ZF22 and ZF21 exert
a transcriptional repression activity on basal as well as activated
transcription and suggested that the activity may reside in the
amino-terminal half of the protein. The factor clearly repressed
transcription more efficiently when it was targeted to the promoter,
although the protein may be able to repress transcription by a
mechanism that does not require DNA binding, such as interaction with a
component of the basic transcriptional machinery.
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ZF22 Contains a Transferable Repression Domain and a Positive
Regulatory Domain--
Because the BEE-1 element was identified as a
positive regulatory element controlling enolase expression, we
decided to conduct experiments to further substantiate the
transcriptional repression activity of the isolated BEE-1 binding
factor. The full coding region of ZF22 was connected in-frame to the
DNA-binding domain of yeast activator GAL4 (amino acids 1-147) to
generate GAL4-ZF22 (2-794), and different amounts of the construct
were cotransfected with a reporter containing the CAT gene under the control of the TK promoter and five copies of the GAL4
binding site. Fig. 9A shows that repression
by ZF22 is concentration-dependent, and no transcriptional
activation was observed in a range of transfected expression vector
from 5 to 4000 ng. These results were obtained by transfection in C2C12
myogenic cells, but comparable CAT activities were detected in CV1
cells (data not shown).
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DISCUSSION |
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The aim of this work was the identification of factors binding to
the G-rich element (AGTGGGGGAGGGGGCTGCG, termed BEE-1) that are
required, together with an adjacent MEF-2 site, to regulate tissue-specific and differentiation-induced expression of the enolase gene in skeletal muscle cells. One clone was isolated by
screening of a phage expression library and used to isolate additional
clones that resulted to encode a Kruppel-like zinc finger factor
homologous to a human DNA-binding protein reported to bind a CACCC
sequence (18). Northern blot analyses showed that the protein is
ubiquitously expressed, but interestingly, expression decreases in
limbs and hearts of mouse embryos during development; this
down-regulation temporally correlates with the appearance of the
secondary muscle fibers (45, 46) and up-regulation of both
enolase
proteins and transcripts (6, 11). Consistent with these results, in
skeletal muscle nuclear extracts from adult mice, the binding activity
due to the zinc finger factor was dramatically reduced, whereas novel
binding activities were observed. On the contrary, in nuclear extracts
from C2C12 myotubes, where relatively abundant levels of mRNAs and
proteins are still present, a strong binding activity is detectable and
presumably it does not allow detection of weaker binding activities.
Cotransfection of the zinc finger factor expression vector with the
native
enolase promoter/enhancer-CAT fusion gene resulted in a
reproducible repression of the CAT activity; therefore, we named the
factor BERF-1 for
enolase repressor factor 1. The previously
identified human factor, ht
, has been reported to exert a weak
activation on the T cell receptor promoter (18), whereas in the recent
report on the isolation of the rat homologue, ZBP-89, it was shown that the factor represses basal promoter activity and inhibits induction by
EGF of the gastrin promoter (37); finally, the recently isolated amino
terminus shorter form, BFCOL1, has been reported not to activate
transcription and to repress the mouse pro-a2(I) collagen promoter only
at high concentrations (38). Furthermore, based on the identity of the
binding site (15) and on the molecular weight and the biochemical
features of the purified proteins, we think that another, previously
described human factor, H4TF1 (47, 48), which binds the histone H4
CTCCC-box, might be related to BERF-1. This hypothesis cannot be proven
since neither antibodies nor cDNAs encoding H4TF1 are available;
however, the factor has been described as a positive regulator of
histone H4 expression. Recently, it has been proposed that H4TF1 might
bind a sequence within the distal enhancer of the human vimentin gene
that acts as a positive regulatory element (49). This discrepancy among the activities may be due to differences in the reporters and the cell
lines used, and in the case of BFCOL1, it might also be related to the
structural difference observed; alternatively, different forms of the
factor may exert different activities, as suggested by the
identification of two polypeptides that differ slightly in their
apparent molecular weight in both mouse and human cells.
In transfection assays using BERF-1 expression vectors and CAT reporter
constructs containing the BEE-1 element, we observed a
dose-dependent repression of the reporter gene activity,
but at the same time BERF-1 was able, though at a lower extent, to inhibit promoters lacking its binding site or bearing multiple copies
of a mutated consensus site. One possible explanation for these
observations is that BERF-1 is a bona fide repressor factor capable of
inhibiting basal as well as activated transcription, according to the
repression mechanisms proposed for the eukaryotic gene transcriptional
regulation (50, 51). Alternatively, the overexpression of BERF-1
results in "squelching" (52), as has been suggested for BFCOL1
(38). Several lines of evidence indicate that our data are consistent
with a repression mechanism rather than with a phenomenon of
squelching: (i) transcriptional activation was never observed under any
experimental conditions we used, regardless the reporter, the cell
type, and the concentration of the transfected BERF-1 expression
vector; (ii) the amount of the expression plasmid required for
repression is within the range that is normally used to measure
transcriptional activity in transient transfection assays (53); (iii)
the BERF-1 binding activity in vitro is reduced in skeletal
muscle as differentiation proceeds, correlating well with up-regulation
of the enolase gene; and (iv) using GAL4 fusion polypeptides, it
has been shown that the factor contains a transferable repression
domain (54). The BERF-1 repression domain does not contain alanine-,
glutamine-, or proline-rich sequences, which are considered typical
features of repression motifs present in suppressor factors like
Kruppel and WT1 (55, 56). Recently, the repertoire of the primary amino
acid sequences within such domains has expanded as more transcription
repressors are characterized (reviewed in Ref. 54). Several reports
have indicated high charge as a common features among repression motifs (57-59), and the BERF-1 repression region, which contains a highly basic domain, may fall into this category. The identification of a
putative activation domain in the serine-rich carboxyl-terminal region
of BERF-1, a feature shared with BFCOL1 (38), leaves open the
possibility that the factor or variant forms may behave as activator
depending upon the promoter or the cell context.
The BEE-1 element was originally identified as a positive
cis-regulatory element that functionally cooperates with the
neighboring MEF-2 binding site in conferring muscle-specific
transcription to the enolase gene. Although none of the
substitution mutations in the BEE-1 element that we tested inhibited
the binding of the repressor factor that we describe in this report and
simultaneously result in an increase in the reporter activity in
transfected C2C12 myotubes,2
we propose that at early stage of differentiation activity of the
enolase enhancer is repressed by the abundant presence of BERF-1, which
precludes a potential activator from binding to the BEE-1 site or to an
overlapping site. During muscle differentiation, the ratio of positive
to negative regulatory binding activities changes in favor of the
activator due to the developmental down-regulation of BERF-1 and/or by
the increasing of different binding activities, as we observed in EMSA
experiments with nuclear extracts from adult muscle. The proposed model
implies that BERF-1 exerts its activity through competition for binding
site occupancy, which is considered a passive transcriptional
repression mechanism, but on the other hand, the factor possesses an
intrinsic repressing activity that inhibits transcription initiation
directly, and this is a mode of action of the active transcriptional
repressors (50). However, as more studies contribute to unravelling
repression mechanisms, this classification has become loose; for
example, it has been recently reported that the human Cut homeodomain
protein represses gene expression by both mechanisms: active repression and competition for binding site occupancy (60).
A candidate for the putative positive regulator competing for binding
to the BEE-1 site might be Sp1, as has been proposed for the gastrin
gERE element, which has been shown to bind both Sp1 and the rat
homologue of BERF-1 (37). Although this possibility cannot be entirely
excluded, our data indicate that different factors are probably
involved. In nuclear extract from adult muscle where BERF-1 binding
activity was dramatically reduced, we did not observe a consequent
increasing of Sp1 binding activity but rather the appearance of novel
DNA-protein complexes. These results are consistent with preliminary
Southwestern analyses that indicate the presence of at least one
muscle-enriched polypeptide that binds in a sequence-specific manner to
the BEE-1 element.2 Further studies are in progress to
identify the positive regulator(s) acting on the BEE-1 element and
clarify the molecular mechanisms controlling the developmentally
regulated expression of the enolase gene in skeletal muscle.
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ACKNOWLEDGEMENTS |
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We thank H. Schoeler for kindly providing anti-Sp1 and anti-Sp3 antibodies and R. Bassel-Duby for anti-MNF serum. We thank Stefano Ferrari and Hans Schoeler for critically reading of manuscript and providing helpful suggestions.
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FOOTNOTES |
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* This work was supported in part by Telethon-Italia (projects 416 and 943 to A. G.) and MURST to S. F. and G. C. We are indebted to the UILDM-Palermo and the Fondazione Telethon for the excellent work in administering the Telethon grants.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X98096 (A22 cDNA sequence), L04282 (human ht), U30381 (rat ZBP-89), and U80078 (mouse BFCOL1).
§ Supported by a fellowship under the Telethon Grant.
** To whom correspondence should be addressed: Istituto di Biologia dello Sviluppo del Consiglio Nazionale delle Ricerche, 90146 Palermo, Italy. Tel.: 39-91-6809-518; Fax: 39-91-6809-548; E-mail: Giallong{at}mbox.unipa.it.
1
The abbreviations used are: BEE-1, enolase
element 1; BERF-1,
-enolase repressor factor 1; dpc, day(s)
postcoitum; kb, kilobase; bp, base pair; GAPD,
glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic
mobility shift assay; CAT, chloramphenicol acetyltransferase gene; PCR,
polymerase chain reaction; TK, thymidine kinase; ORF, open
reading frame.
2 R. Passantino, unpublished data.
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REFERENCES |
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