From the
The B creatine kinase gene is regulated by an array of positive
and negative cis-elements in the 5`-flanking DNA that function in both
muscle and nonmuscle cells. In C
Creatine kinase (CK)
Like other multigene families
expressed in muscle the CK genes undergo an isoenzyme switch during
development(13, 14, 15, 16) . B CK is
expressed in immature proliferating myogenic cells (myoblasts). Muscle
differentiation is characterized by down-regulation of the B gene,
which is switched off during myogenesis, and induction of M CK, which
becomes the major isoform present in both cardiac and skeletal muscle
(17, 18). With the use of M- and B- specific cDNA probes we showed that
coordinate up-regulation of M and B mRNA occurs in the early stages of
myogenesis in C
We showed that the human B CK gene is
regulated by an array of positive and negative cis-elements in the
5`-flanking DNA that function in both muscle and nonmuscle cells and
that sequences between -92 and +80 confer expression to
chimeric plasmids that resembles that of the endogenous B CK gene in
C
The creatine kinase gene family represents an interesting
model of coordinate regulation of isoproteins that can be studied in
convenient cell culture systems. A great deal of attention has been
given to elucidating the molecular mechanisms that regulate the M CK
gene. The regulatory elements that are essential for expression of this
gene have been characterized by transfection experiments in cell
culture (33-35), direct gene injection into heart and skeletal
muscle(36, 37) and in transgenic animals(38) .
The M CK gene is regulated by a tissue-specific enhancer that contains
two MEF-1 motifs or E-boxes (CANNTG) that bind myogenic factors (MyoD,
myogenin, Myf-5, and MRF-4) and an MEF-2 motif that binds a MADS box
transcription factor(39) . In contrast, regulatory mechanisms
that control genes that are turned off during myogenesis have received
less attention. The down-regulation of
The regulation of
the B CK gene has been studied in a number of nonmuscle systems. A
61-base pair sequence between -98 and -37 that contains
both the CCAAT and TATA sequences is important for efficient in
vitro transcription from a minimal B CK promoter (43-46).
The results of transfection experiments have identified 5`-upstream
sequence elements and sequences within the first (untranslated) exon
that are important for expression in HeLa cells (47) and neuroblastoma
cells(48) . The B CK gene promoter has strong sequence
similarity to the adenovirus E2E gene promoter and like the E2E gene is
regulated by the viral activator Ela. This finding may be of
significance for the metabolic energy-requiring events that take place
after oncogenic activation(49) . In osteoblastic cells
transcriptional up-regulation of the gene during differentiation is
associated with the formation of a nuclear protein-DNA complex that
binds a cis-element between +1 and +228 of the
gene(5) . In U937 cells the highly conserved 3`-NTR of B CK mRNA
is important for translational regulation of B CK protein(50) .
We present evidence that sequences within the first exon are
important for regulation of expression of the B CK gene in transfected
myogenic cells in culture. This sequence does not contain recognition
sites for any previously described transcription factor (Genetics
Computer Group (GCG) transcription factor recognition sites release
6.5). The presence of regulatory sequences within the first exon of a
gene is an unusual finding but is not unique to the B CK gene.
Important regulatory sequences within the first exon have been
described in the human tissue-type plasminogen activator
gene(51) , the skeletal troponin I gene(52) , and a human
nonmuscle myosin heavy chain gene(53) . Interestingly, none of
the exonic regulatory sequences important for expression of other genes
in muscle cells share significant identity with the sequence we
describe.
Protein phosphorylation is an important control mechanism
for regulation of the myogenic developmental program. Both MyoD and
myogenin are phosphoproteins(54, 55) . Phosphatase
treatment of MyoD attenuates specific binding of MyoD-E12 heterodimers
to the M CK gene enhancer(56) . Fibroblast growth factor
inhibits myogenesis by inactivating myogenic helix-loop-helix proteins
by phosphorylating a site in the DNA binding domain of
myogenin(54) . Treatment of C
We thank Kimberly Goodwin, Kimberly Hawker, and Nancy
Brada for technical assistance, Kelly Hall for secretarial assistance,
and Michael Ritchie for contributions to the early stage of this
project.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
C
myogenic
cells M and B creatine kinase mRNAs are coordinately up-regulated in
the early stages of myogenesis and then undergo distinct regulatory
programs. The B creatine kinase gene is down-regulated in the late
stages of myogenesis as M creatine kinase becomes the predominant
species in mature myotubes. Sequences between -92 and +80 of
the B creatine kinase gene confer a regulated pattern of expression to
chimeric plasmids that closely resembles the time course of expression
of the endogenous B creatine kinase gene in C
C
cells undergoing differentiation. We show that sequences within
the first exon of the B creatine kinase gene are important for the
developmental regulation of the gene in C
C
cells and that these sequences bind a nuclear protein that shows
a similar tissue-specific distribution and developmentally regulated
expression to that of the endogenous B creatine kinase gene.
(
)catalyzes the
reversible phosphorylation of ADP and creatine and is important in the
regulation and maintenance of cellular energy metabolism. The M and B
creatine kinase genes encode highly homologous M and B subunit proteins
that associate in the cytoplasm to form three dimeric cytoplasmic
isoenzymes (MM, MB, and BB). B CK is expressed in many tissues, is
regulated by steroid hormones(1, 2) , and undergoes
developmental regulation in the lens of the eye(3) , in
cartilage(4) , and in osteoblastic cells in culture(5) .
B CK is a marker for certain histologic types of lung cancer (6) and for brain damage(7, 8) . B-containing
isoenzymes (MB and BB) increase in skeletal muscle during chronic
exercise training (9) and in the myocardium in response to
hypertrophy, acute myocardial ischemia, and heart failure (10, 11, 12) in adaptation to conditions of
decreased energy reserve(10) .
C
cells (19) and in the
developing heart (20) prior to down-regulation of B mRNA during
the final stages of differentiation as M mRNA becomes the predominant
species. This fetal pattern of expression of the CK genes is
recapitulated in heart in response to acute pressure
overload(21) . Interestingly, the developmental expression of B
CK mRNA is very similar to that of cardiac actin mRNA, which is also
up-regulated in the early stages of differentiation of
C
C
cells and down-regulated as it is replaced
by skeletal actin mRNA in the late stages of myogenesis(22) .
Thus, the pattern of expression of certain fetal isoforms such as B CK
and cardiac actin may represent an evolutionarily conserved
developmental program in muscle. This program is different from that of
other fetal isoforms such as
and
actin, which are expressed
in myoblasts and are down-regulated during all subsequent stages of
myogenesis(23) .
C
cells undergoing
differentiation(19) . We now show that sequences within the
first exon of the B CK gene are important for the developmental
regulation of the gene in C
C
cells and that
these sequences bind a nuclear protein that may play a role in the
developmentally regulated expression of the B CK gene.
Preparation of BCKCAT and BCKneo Reporter
Plasmids
The plasmid BCKCAT92 (Fig. 1A) that
contains 92 base pairs of 5`-flanking DNA, the first exon
(untranslated), and the first 12 base pairs of the first intron of the
human B CK gene inserted in the HindIII site of pSVOCAT was
described previously(19) . To determine whether sequences within
the first exon and first intron are important for expression of
BCKCAT92 we prepared BCKCAT92del (+3 to +80), a construct
that contains the first 92 base pairs of upstream DNA and the cap site (Fig. 1B). This construct was prepared by synthesizing
oligonucleotides representing the sense and antisense strands of the
sequence from -92 to +2 designed to reconstitute HindIII-compatible ends after annealing to facilitate
subcloning into the HindIII site of pSVOCAT. We also prepared
a 3`-deletion series of constructs with identical 5`-ends containing
sequences from -92 to +2 and different 3`-ends designed to
delete select sequences from the first exon and intron of the B CK gene (Fig. 1, C-F). The constructs were prepared by
ligating annealed double-stranded oligonucleotides representing the
desired sequences into the HindIII site of
pSVOCAT(24) . All plasmids were sequenced in both strands to
ensure a single copy of the desired insert was present in the correct
orientation(25) .
Figure 1:
Identification of a regulatory element
within the first exon of the human B CK gene. A, the plasmid
BCKCAT92 was described previously (19). The hatchedbox represents sequences contained in the first exon. B-F, 3`-deletion series through the region +3 to
+80 prepared as described under ``Materials and
Methods'' and drawn to scale. G, the plasmid MCKCAT2620
(not drawn to scale) that contains the human M CK gene enhancer was
described previously (30). CC
cells were
transfected and harvested as myoblasts or myotubes, and extracts were
assayed for [
C]chloramphenicol conversion.
Induction represents CAT activity (mean ± S.D.) present
in extracts of cells transfected with chimeric plasmids (26.5% ±
9.4% conversion [
C]chloramphenicol for the most
active construct) relative to pSVOCAT (3.1% ± 1.8% conversion
[
C]chloramphenicol). N, total number of
dishes of cells transfected with each construct. At least two
preparations of each plasmid and different batches of
C
C
cells were used for transfection
experiments.
Because the neomycin resistance (neo) gene encodes a very stable transcript (26) we
prepared BCKneo reporter plasmids to facilitate detection of
transcripts in transiently transfected cells. BCKCAT92 and
BCKCAT92del(+3 to +80) were digested completely with BamHI and then partially with HindIII to release the
chloramphenicol acetyltransferase (CAT) gene from the plasmid. The neo gene obtained by digesting pSVneo with BamHI and HindIII was ligated to BamHI/HindIII-digested BCKCAT92 and BCKCAT92del
(+3 to +80) resulting in the constructs BCKneo92 and
BCKneo92del (+3 to +80).
Cell Culture
The murine skeletal muscle cell line
CC
(ATCC #CRL1772) was maintained in an
atmosphere of 8% CO
, 92% air in growth medium
(Dulbecco's modified Eagle's medium) supplemented with 20%
fetal calf serum, penicillin (50 µg/ml), and streptomycin (50
µg/ml)). Differentiation medium was Dulbecco's modified
Eagle's medium supplemented with 10% horse serum and antibiotics.
Cell Transfection and Chloramphenicol Acetyltransferase
Assays
CC
cells were plated 24 h before
transfection at a density of 3.5
10
cells/60-mm
dish in 3 ml of growth medium. Transfections were performed by the
calcium phosphate coprecipitation method(19) . Precipitates
contained a total of 20 µg of DNA comprised of 15 µg of test
plasmid and 5 µg of pMSV
gal as an internal standard to correct
for transfection efficiency. After a 4-h incubation with the
precipitate, cells were subjected to a 3-min glycerol shock and
harvested 24-48 h later. Other dishes of cells were fed with 3 ml
of differentiation medium after the transfected cells had become fully
confluent and harvested 60 h later as fully differentiated myotubes.
Cell extracts were prepared, and assays for
-galactosidase and
chloramphenicol acetyltransferase were performed as described
previously(19) . The amount of extract used for chloramphenicol
acetyltransferase assays was based on the results of the
-galactosidase assay and contained 25-100 µg of protein.
The assays were terminated after 60 min.
Northern Blot Hybridization
CC
cells plated as described above were transfected with 15 µg
of either BCKneo92 or BCKneo92del(+3 to +80) and 5 µg of
pCMVCAT as an internal standard to control for transfection efficiency.
The cells were harvested 28 h after transfection, and
Poly(A)
mRNA was purified directly from transfected
cells with the use of a Micro-FastTrack mRNA isolation kit
(Invitrogen). Northern blots were prepared with 7 µg of
Poly(A)
mRNA and Nytran membranes (Schleicher and
Schuell) as recommended by the supplier. The probes were a 1321-base
pair HindIII/SmaI fragment of the neo gene
and a 550-base pair HindIII/NcoI fragment of the CAT
gene radiolabeled with [
P]dCTP (Amersham Corp.)
as described(19) . Autoradiograms prepared with Kodak XAR film
and Cronex intensifying screens were analyzed with an LKB Ultroscan XL
laser densitometer.
Preparation of Nuclear Extracts
Extracts were
prepared from CC
cells and tissues by a
modification of the method described by Heberlein et
al.(27) . The protein concentration was determined by the
method of Bradford(28) .
Preparation of Probes for Gel Mobility Shift
Assays
A DNA fragment comprising the first exon and first 13
base pairs of the first intron of the B CK gene was used as the
template in a polymerase chain reaction with primers designed to
include bases +1 to 19, the reverse complement of +64 to
+80, and with [P]dCTP at a final
concentration of 0.32 µM(29) .
DNA Gel Mobility Shift Assays
Gel mobility shift
assays were performed as described previously (30) with 1.0
10
dpm of double-stranded
P-labeled
probe and 10 µg of nuclear protein extract.
Phosphatase Treatment of Nuclear Extract
Nuclear
extract was incubated with 19.2 milliunits of potato acid phosphatase
(Sigma) or with buffer (50 mM Tris HCl, pH = 9.3, 1.0
mM MgCl, 0.1 mM ZnCl
, and 1.0
mM spermidine) and 2.0 units of calf intestinal alkaline
phosphatase (Promega) for 30 min at 37 °C. After phosphatase
treatment
P-body-labeled probe was added to the extract,
and gel mobility shift assays were performed.
Transfection Experiments
We showed that
sequences within -92 to +80 of the B CK gene confer a
regulated pattern of expression to CAT reporter plasmids in
CC
cells that resembles the time course of
expression of B CK mRNA(19) . The plasmid BCKCAT92 (Fig. 1A) showed peak expression 24-48 h after
transfection and was not expressed above background in fully
differentiated myotubes. In nonmyogenic cells that express B CK (HeLa
cells, Hep G2 cells, and NIH3T3 cells) BCKCAT92 expression was above
background 48-108 h after transfection(19) . These results
show that the decrease in expression of BCKCAT92 in
C
C
cells is due to differentiation and does
not simply reflect the time course of expression of a plasmid in a
transient transfection experiment. To determine the importance of
exonic and intronic sequences for expression of BCKCAT92 in
C
C
cells we prepared a construct in which the
exonic and intronic sequences were deleted (BCKCAT92del (+3 to
+80), Fig. 1B). When compared to the expression of
BCKCAT92, which was approximately 5-fold above background in myoblasts,
deletion of exon I and intron I sequences from BCKCAT92 resulted in a
plasmid that was not expressed above background in either myoblasts or
myotubes (Fig. 1B). These results were unexpected and
showed that sequences from +2 to +80 were critical for
expression of BCKCAT92. Accordingly, to determine the sequences from
+2 to +80 that regulate expression of BCKCAT92 we prepared a
3`-deletion series (Fig. 1, C-F). Deletion of
sequences from +26 to +80 had no effect on expression of the
resultant plasmid (Fig. 1, C-D). Although
expression of the plasmid BCKCAT92del (+26 to +80) was higher
than that of BCKCAT92 (Fig. 1, A and D) the
difference was not statistically significant (Student's t test). However, deletion of sequences from +18 to +25
resulted in a construct that was not expressed above background in
myoblasts or myotubes (Fig. 1, E and F). In
contrast, the plasmid MCKCAT2620 that contains the human M CK gene
enhancer (30) was inactive in myoblasts and was expressed
19-fold above background in fully differentiated myotubes (Fig. 1G). These results show that sequences from
+18 to +25 are important for expression of BCKCAT chimeric
plasmids in C
C
myoblasts.
Exon I Regulates Expression of Chimeric Plasmids at the
Level of mRNA Accumulation
BCKCAT mRNA transcripts derived from
the constructs shown in Fig. 1, A-F are different.
This suggests expression may vary with translational efficiency of
BCKCAT mRNAs. Accordingly, we sought to determine whether the observed
difference in CAT activity in cells transfected with these constructs
correlated with CAT mRNA levels. We performed Northern blot analysis of
mRNA extracted from CC
cells transfected with
BCKCAT92 and BCKCAT92del(+3 to +80). Because CAT mRNA was not
detectable with this technique we prepared constructs in which the CAT
gene was replaced with the neo gene, which encodes a more
stable transcript. C
C
cells were transfected
with BCKneo92 and BCKneo92del(+3 to +80), and neo mRNA transcripts were analyzed by Northern blot hybridization. The
steady-state level of neo mRNA directed by BCKneo92 was 9-fold
greater than that of BCKneo92del(+3 to +80) when normalized
to CAT mRNA encoded by the internal standard pCMVCAT (Fig. 2).
These results show that sequences within B CK exon I modulate
expression of chimeric plasmids at the level of mRNA accumulation.
Effects at the level of transcription as well as mRNA processing and
stability are all formal possibilities.
Figure 2:
B
CK exon I affects BCKneo mRNA accumulation in transfected cells. RNA
extracted from cells transfected with the plasmid BCKneo92del(+3
to +80) (lane1) or BCKneo92 (lane2) and pCMVCAT as an internal standard was analyzed by
Northern blot hybridization with P-labeled cDNA probes
derived from the CAT gene and the neo gene as described under
``Materials and Methods.''
Gel Mobility Shift Assays
To characterize the
trans-acting factors that interact with sequences within the first exon
of the B CK gene we prepared nuclear extracts from
CC
myoblasts and myotubes at select
developmental stages and performed gel mobility shift assays. We were
particularly interested in factors that are expressed in myoblasts and
are down-regulated with differentiation that could be important
mediators of B CK gene expression. A representative result from two
independent preparations of extracts is shown in Fig. 3. Three
nuclear protein-DNA complexes are depicted with arrows.
Complex 1 exhibited marked down-regulation with differentiation.
Complex 2 was not consistently detected in different preparations of
extract tested, and complex 3 was not regulated with differentiation.
These results identify a nuclear protein complex that is expressed in
myoblasts and is down-regulated with differentiation resembling the
expression of B CK in myogenic cells in culture.
Figure 3:
Gel mobility shift assays with B CK exon I
probe and nuclear extracts from CC
cells at
select developmental stages. Nuclear extract was prepared from
C
C
myoblasts (lane2), and
from C
C
myotubes after 24 h (lane3), 48 h (lane4), and 120 h (lane5) in differentiation medium for gel mobility shift
assays with
P-labeled probe (B CK +1 to +80).
Uncomplexed probe is shown in lane1.
To determine
whether the DNA-protein complexes shown in Fig. 3represented the
specific interaction of nuclear proteins with sequences within B CK
exon I shown to be important for expression of chimeric plasmids in
transfected cells, additional gel mobility shift experiments were
performed with competitor DNA (Fig. 4). Unlabeled DNA including
sequences from +1 to +25 was added to the gel mobility shift
reaction in molar excess to P-labeled exon I probe. The
cold DNA competed with the probe for binding to nuclear protein present
in complex 1 but not complex 3 (Fig. 4, lanes
1-4). In comparison, unlabeled DNA that included sequences
from +1 to +17 did not compete with the probe for binding to
complex 1 (Fig. 4, lanes 5-7). These results show
that sequences from +18 to +25 that are critical for
expression of chimeric plasmids in transfected cells also represent the
binding site for complex 1.
Figure 4:
Gel mobility shift assays with the B CK
probe and competitor DNA. P-labeled probe was incubated
with nuclear extract prepared from C
C
myoblasts. Lane1 represents a reaction without
competitor DNA. In lanes2-4 unlabeled DNA
containing sequences from +1 to +25 was added to the reaction
in 45-, 90-, and 175-fold molar excess as a competitor. In lanes5-7 unlabeled DNA containing sequences from +1
to +17 was added to the reaction in 90-, 185-, and 375-fold molar
excess as a competitor.
We used gel mobility shift assays to
evaluate the expression of protein 1 in select tissues. We prepared
nuclear extracts from brain, a tissue in which B CK is expressed, and
from heart and skeletal muscle, tissues in which the CK genes undergo
developmental regulation(20, 31) . The results showed
that expression of protein 1 was abundant in brain (Fig. 5, laneBR) and expressed at a lower level in heart, a
tissue in which the MB isoenzyme of CK is found and in which B CK mRNA
is present (Fig. 5, laneH). Mature skeletal
muscle, which expresses only trace amounts of B mRNA, did not show
detectable amounts of protein 1 (Fig. 5, laneM). These results show that the expression of protein 1
in the tissues evaluated correlated well with that of B CK mRNA. In
contrast, band 3 was detected in all tissues. In skeletal muscle and
heart, B CK mRNA is down-regulated with development. To determine
whether the developmental regulation of protein 1 in heart and skeletal
muscle resembled that of B CK mRNA, we prepared nuclear protein
extracts from tissues obtained from 1-day-old neonatal mice, a stage at
which B CK mRNA expression is abundant relative to that of mature
tissue(20) . The results of gel mobility shift assays showed
that protein 1 was abundant in both neonatal tissues and was
down-regulated with development, correlating well with the
developmental regulation of B CK mRNA in these tissues (Fig. 6).
In contrast, band 3 was not developmentally regulated.
Figure 5:
Expression of band 1 protein in tissues. P-labeled B CK probe was incubated with nuclear extract
prepared from adult mouse skeletal muscle (M), heart (H), and brain (BR) and subjected to a standard gel
mobility shift assay.
Figure 6:
Developmental expression of band 1 protein
in tissues. P-labeled B CK probe was incubated with
nuclear extract prepared from heart and skeletal muscle from day 1
neonatal (N) and adult (A) mice and evaluated in a
standard gel mobility shift assay.
Role of Phosphorylation in Activation of Band 1
Protein
Developmental down-regulation of band 1 protein could be
due to either a decrease in synthesis or a post-translational
modification of the protein resulting in lower affinity for the target
DNA. Because phosphorylation has been shown to modulate the DNA binding
activity of many transcription factors(32) , we determined the
effect of treatment of extracts with phosphatases on binding of the
probe by band 1 protein. Treatment of extract with either enzyme
resulted in marked inhibition of formation of complex 1 (Fig. 7).
These results suggest that phosphorylation of band 1 protein may be an
important post-transcriptional mechanism that mediates affinity for the
target DNA.
Figure 7:
Effect of phosphatase treatment of nuclear
extract on the formation of complex 1. Nuclear extract was prepared
from CC
myoblasts as described under
``Materials and Methods.'' A, the extract was either
untreated (lane1), treated with acid phosphatase (lane2), or incubated with phosphatase buffer
without enzyme (lane3) before the addition of
P-labeled B CK probe and the gel mobility shift assay. B, the extract was untreated (lane1),
treated with acid phosphatase that was boiled for 15 min (lane2), or treated with acid phosphatase (lane3) prior to the addition of
P-labeled probe
and the gel mobility shift assay. C, the extract was incubated
with phosphatase buffer (lane1) or buffer and
alkaline phosphatase (lane2) prior to the addition
of
P-labeled probe and the gel mobility shift
assay.
SDS-Polyacrylamide Gel Electrophoresis Analysis of the
DNA Binding Protein
To determine the molecular weight of protein
1 we performed a gel mobility shift assay with the B CK probe and
nuclear protein extract from CC
myoblasts.
Band 1 was excised from a wet gel after a brief exposure to Kodak XAR
film to facilitate localization of the band in the gel. The protein was
separated from the gel with the use of electroelution, analyzed by
electrophoresis in a 7.5% SDS-polyacrylamide gel, and detected with the
silver reagent (Bio-Rad). A single protein of approximate molecular
weight 150 kDa (n = 6 gels) was seen consistently (Fig. 8). This band was not seen when free DNA probe was
separated from a band shift gel by electroelution, subjected to
SDS-polyacrylamide gel electrophoresis, and stained with silver
reagent. Autoradiography of the gel showed that the band represented
protein, and not protein complexed to [
P]DNA
probe.
Figure 8:
A
single protein is contained in DNA-protein complex 1. A standard gel
mobility shift assay was performed with P-B CK probe and
nuclear protein extract from C
C
myoblasts. The
position of DNA-protein complex 1 on the wet gel was identified by
autoradiography. The band was excised, and the protein was separated
from the gel with the use of electroelution. The protein was evaluated
by electrophoresis in a 7.5% SDS-polyacrylamide gel that was stained
with the silver reagent (Bio-Rad). The protein is depicted with an arrow. The migration of
C-protein standards
(Amersham Corp.) is shown to the left of the figure.
-actin mRNA during
myogenesis is controlled at the level of transcription by conserved
sequences in the 3`-nontranslated region of the gene(40) . The
cardiac
-actin gene is regulated by the interaction of MyoD1, the
serum response factor, and Sp1 with promoter elements(41) . The
helix-loop-helix protein Id is expressed in C
C
myoblasts and is down-regulated with differentiation(42) .
Id associates specifically with MyoD and attenuates its ability to
trans-activate muscle-specific genes such as the M CK gene. There is no
evidence that Id or other helix-loop-helix proteins play a direct or
indirect role in the regulation of the B CK gene.
C
myoblasts with the protein phosphatase inhibitor okadaic acid
inhibits skeletal muscle cell differentiation and MyoD expression and
induces expression of the id gene (57). We describe a nuclear
protein that binds specifically to sequence elements that are important
for expression of B CK chimeric constructs in C
C
myoblasts. Evidence that the protein we describe is important for
regulation of the B CK gene includes specific binding to sequences
within the first exon that confer regulated expression to the CAT gene
in transfected C
C
cells, expression in tissues
that correlates with that of B CK mRNA and protein, and developmental
expression in myogenic cells and in heart and skeletal muscle that
resembles that of B CK mRNA. Our results suggest that expression of B
CK in cells and tissues may be regulated through a mechanism that
alters phosphorylation of this DNA binding protein. Further
investigation may provide insight into these mechanisms that mediate
the developmental down-regulation of genes during myogenic development.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.