From the Department of Biochemistry and Molecular
Biology, the Institute of Genetic Science, Yonsei University College of
Medicine, 134 Shinchon-dong Seodaemun-gu, Seoul, 120-752, Korea, the
¶ Department of Molecular Biology, Kyung-Hee University College of
Medicine, Seoul, 130-050, Korea, and the
Department of
Biochemistry, Kwandong University College of Medicine,
Kangnung, 210-701, Korea
Received for publication, August 3, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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The 280-kDa Acetyl-CoA carboxylase
(ACC)1 is a key enzyme in
fatty acid biosynthesis catalyzing the transfer of a carboxyl group to
acetyl-CoA to form malonyl-CoA. Malonyl-CoA serves as the active donor
of two carbon atoms in fatty acid synthesis and strongly inhibits fatty
acid Mammals have two major isoforms of acetyl-CoA carboxylase. The 265-kDa
isoform, designated as ACC Compared with ACC Because there are several E-boxes on the ACC Cloning of ACC Primer Extension--
An oligonucleotide
(5'-CAGGAAAAGGTCAGACAGACAGGA-3'), complementary to the region +37 to
+56 from the ATG codon of ACC RT-PCR--
Total RNAs were extracted from Alexander cells,
HepG2 cells, and human skeletal muscle, using the TRIzol (Life
Technologies, Inc.) according to the manufacturer's instructions.
First-strand cDNAs were synthesized from 2 µg of total RNA in 20 µl of reaction volume using SuperScript II reverse transcriptase
(Life Technologies, Inc.). Each reverse transcription mixture (0.5 µl) was used as the template for amplifying ACC Construction of Transfection Plasmids--
The PII Cell Culture and Transient Transfection Assay--
The
established cell lines, obtained from American Type Culture Collection
(Rockville, MD), were cultured in the recommended media (H9C2 in
DMEM/F12; Alexander in MEM; HeLa in MEM; NIH3T3 in Dulbecco's modified
Eagle's medium) supplemented with 10% fetal calf serum, 100 units/ml
penicillin G sodium, 100 µg/ml streptomycin sulfate at 37 °C under
5% CO2, 95% air. All cell culture materials were
purchased from Life Technologies, Inc. For the transient transfection
assay, cells were plated at a density of 2 × 105
cells/35-mm dish. On the next day, transfection was performed with 0.4 µg of the indicated pPII Yeast One-hybrid Screening--
A human skeletal muscle cDNA
library was screened employing the Matchmaker one-hybrid system
(CLONTECH). All procedures were performed according
to the manufacturer's instructions. For the construction of a
target-reporter yeast strain, 3 copies of the 20-bp fragment
corresponding to nucleotides from Preparation of Recombinant MyoD, E47, Myf4, and
Myf6--
Recombinant MyoD and E47 were expressed in E. coli SG13009[pREP4] containing pQT-MyoD or pQT-E47, kindly
provided by Stephan F. Konieczny. Expression plasmids of pET-Myf6 and
pGST-Myf4 were prepared by inserting Myf6 or Myf4 cDNA into
BamHI/HindIII sites of pET-21a (Novagene) or
EcoRI/XhoI sites of pGEX4T3 (Amersham Pharmacia
Biotech), respectively. Myf6 and GST-Myf4 were prepared in E. coli BL21(DE3) and E. coli DH5 Electrophoretic Mobility Shift Assay--
The EMSA probes used
are as follows (sequence of sense oligomers are presented here); E1
(
Each sense oligonucleotide was labeled with polynucleotide kinase
(Takara) using [ Cloning of Human ACC
To determine the transcription initiation site of ACC
We tested the promoter activities of the 5'-flanking fragments of those
two 5'-UTRs. The PII ACC
The multiple E-boxes picked up by the computer-based analysis suggested
MRFs might act on the PII
To identify the MyoD-responsive regions in this promoter, we
constructed a group of deletion reporters containing the PII MyoD Acts on the E-boxes Located in Region A--
MyoD functions
as a heterodimer with E-proteins such as E12 and E47 (21). Although
MyoD/MyoD homodimers can bind to E-boxes in vitro,
the MyoD/E-protein heterodimers recognize E-boxes on DNA strands
in vivo (26, 27). There exist three E-boxes in the Region B Has Multiple cis-Elements Crucial for Basal Transcription
and MyoD Responsiveness--
To investigate the
cis-elements for the MyoD responsiveness located around Probe Pr5 (+4/+29) Is Preferentially Bound by MyoD/MyoD
Homodimer--
The transient transfection assay of the M(GC) mutant
and the EMSA with Pr5 (Fig. 6) indicate the presence of a
MyoD-responsive element, that is not a canonical E-box, in the region
around +4 to +29. To clarify the precise core sequence for
MyoD-binding, competitive EMSA was performed using mutant Pr5
oligonucleotides harboring substitutions (Fig.
7A). Competitors containing
mutations from +17 to +24 could not compete effectively against Pr5,
suggesting that the sequence of GCCTGTCA (+17 to +24) is important for
MyoD binding.
From the previous EMSA (Fig. 6C), MyoD seems to bind to this
element more avidly than to E-box 4. We compared this novel
cis-element with the E-box in vitro by EMSA. As
shown at Fig. 7B, E47 had strong affinity to Pr3 (lanes 1 and 2) and very poor affinity to Pr5 (lane 7 and
8), while MyoD showed much higher affinity to Pr5 than to
Pr3 (lanes 3, 4, 9, and 10). Heterodimerization of E47 and MyoD did not affect the high affinity of E47 to Pr3 (lanes 5 and 6), while it did significantly
decrease complex formation to Pr5 in comparison with MyoD homodimer
(lane 11 and 12). These results suggested that
the novel cis-element in Pr5 prefers the MyoD homodimer to
E47 homodimer or MyoD/E47 heterodimer. Moreover, these results
corresponded to the in vivo results of transient transfection assays using artificial promoter-reporter constructs, which contained the E-box clustering region of PII MRFs Bind to the Novel cis-Element in Vivo--
To isolate the
trans-acting factors acting on region B, we screened a human skeletal
muscle cDNA library using the yeast one-hybrid system. Three bait
constructs were generated by insertion of three copies of the
nucleotide sequence corresponding to the regions of
We tested whether MyoD and Myfs induce transcriptional activation on
the promoters containing this novel cis-element. pPII In this study, we have introduced the muscle-type promoter of
ACC The trans-acting factors involved in the PII Between regions A and B ( In region B, the proximal MyoD-responsive region, a well conserved
E-box 4 (caGCtg) and a novel cis-element containing a
GCCTGTCA sequence (Fig. 7) were identified as responsible for the MyoD responsiveness. This novel MyoD-binding site is very different from
E-boxes where MRF binding is mediated by dimerization with E-proteins.
The presence of E47 weakened the MyoD binding to this element in
vitro, and coexpression of E12 diminished the MyoD-mediated stimulation on p6×(+8/+27)-tk-luc in vivo (Fig. 7). Those
inhibitory effects of E-proteins seem to result from the dimerization
of MyoD with E-proteins. In other words, MyoD binding to this site is
not mediated by heterodimerization with E-proteins such as E47 and E12.
This novel sequence, which was avidly bound by homodimeric MyoD, seems
to be bound by other MRF family factors. Using the yeast one-hybrid
system, we found that other myogenic transcription factors such as Myf4
and Myf6 are recruited by this novel cis-element. They all
are bHLH proteins, responsible for muscle differentiation, bind to
E-boxes, and heterodimerize with E-proteins (23, 28-32). Interaction
of these Myfs with this site was successfully demonstrated in
vitro and in vivo by EMSA and transient transfection
assays (Fig. 8). Taken together, the novel cis-clement
containing GCCTGTCA sequence functions as a general binding site for
bHLH myogenic transcription factors without E-protein mediation.
Region B is a very proximal regulatory region and is thus, thought to
contain basic elements responsible for the initiation of transcription
through interactions with RNA polymerase II and general initiator
factors. The TGAAA at A previous study reported that ACC-isoform of acetyl-CoA
carboxylase (ACC
) is predominantly expressed in heart and skeletal
muscle, whereas the 265-kDa
-isoform (ACC
) is the major ACC in
lipogenic tissues. The ACC
promoter showed myoblast-specific
promoter activity and was strongly induced by MyoD in NIH3T3 cells.
Serial deletions of the promoter revealed that MyoD acts on the E-boxes
located at positions
498 to
403 and on the proximal region
including the 5'-untranslated region. Destruction of the E-boxes
at positions
498 to
403 by site-directed mutagenesis resulted in a
significant decrease of MyoD responsiveness. The "TGAAA" at
32 to
28 and the region around the transcription start site play important roles in basal transcription, probably as a TATA box and an Inr element, respectively. Mutations of another E-box at
14 to
9 and a
"GCCTGTCA" sequence at +17 to +24 drastically decreased the MyoD
responsiveness. The novel cis-element GCCTGTCA was
preferentially bound by MyoD homodimer in EMSA and conferred MyoD
responsiveness to a luciferase reporter, which was repressed by the
overexpression of E12. This finding is unique since activation via
E-boxes is mediated by heterodimers of MyoD and E-proteins. We screened
a human skeletal muscle cDNA library to isolate clones expressing proteins that bind to the region around the GCCTGTCA (+8 to +27) sequence, and isolated Myf4 and Myf6 cDNAs. Electrophoretic
mobility shift assay showed that recombinant Myf4 and Myf6 bind to this novel cis-element. Moreover, transient expression of Myf6
induced significant activation on the ACC
promoter or an artificial
promoter harboring this novel cis-element. These findings
suggest that muscle regulatory factors, such as MyoD, Myf4, and Myf6,
contribute to the muscle-specific expression of ACC
via
E-boxes and the novel cis-element GCCTGTCA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-oxidation by acting as a potent inhibitor of carnitine palmitoyltransferase I (1, 2). Carnitine palmitoyltransferase I
resides on the surface of the mitochondrial membrane and generates palmitoylcarnitine from palmitoyl-CoA. This step is critical for fatty
acid
-oxidation because cytosolic fatty acyl-CoA cannot translocate
into the mitochondria where fatty acid
-oxidation occurs.
Palmitoylcarnitine is transported into mitochondra by a carrier system
and then reconverted into palmitoyl-CoA by carnitine palmitoyltransferase II. This shuttle system is the first step of fatty
acid
-oxidation, which supplies tissues with energy.
, is highly expressed in liver and adipose
tissues, and the 280-kDa isoform, designated as ACC
, is
predominantly expressed in heart and skeletal muscle (3-5). Human
ACC
and ACC
have about 80% amino acid sequence homology and both
produce malonyl-CoA. They are, however, encoded by separate genes,
mapped to chromosome 17q21 and 12q23.1, respectively, and show distinct
tissue distribution and nutritional regulation (6-8). The presence of
the
-isoform in tissues that do not actively synthesize fatty acid
de novo has not been clearly explained. It has been
suggested that ACC
may play a role in mitochondrial fatty acid
-oxidation in muscle tissues. Its product malonyl-CoA might be a
critical regulatory tool for mitochondrial fatty acid uptake and
metabolism (9, 10).
, little is known about the regulation of ACC
.
To study the transcriptional regulation of ACC
we cloned the 17-kb
human ACC
promoter and analyzed its cis-elements and trans-acting factors. Transcriptional regulation of
muscle-specific genes is commonly studied by investigating the effects
of muscle-specific and stage-specific transcription factors (11-14).
One of the sequence motifs characterized as a critical regulatory
component in muscle gene expression is the E-box (CANNTG) (15-17).
Muscle-specific genes, such as muscle creatine kinase, myosin light
chain, and myogenin genes, have multiple E-boxes in their enhancers or
promoters that act cooperatively to regulate gene transcription
(18-20). E-boxes are binding sites for ubiquitously expressed bHLH
family proteins, known as E-proteins. In muscle cells, E-proteins
dimerize with muscle-related bHLH transcription factors, called muscle regulatory factors (MRFs), enabling the MRFs to act on muscle-specific regulatory elements (21, 22). The four muscle-specific MRFs, MyoD,
myogenin, Myf5, and MRF4, have been characterized in terms of their
significance in development through stage-specific utilization of those
factors (23-25).
promoter, we expected
that the muscle-specific expression of ACC
might be explained by the
responsiveness of its promoter to MRFs. In this study, we report the
fact that ACC
is transcriptionally activated by MyoD through the
E-boxes on its promoter. We also report a novel cis-acting
element which serves as an MRF-binding site and is critically required
for the MyoD-mediated ACC
promoter activation. Elaboration upon the
transcriptional regulation of ACC
in the context of
myogenesis-related factors might provide further understanding of the
muscle-specific expression of ACC
.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Promoter--
Previous isolation of the ACC
cDNA from a human skeletal muscle cDNA library (7) yielded two
cDNA clones with different 5'-untranslated regions (5'-UTR) of 300 and 42 bp, respectively.2 In
the presence of the sense primer
(5'-ATGTAACCCTGAATGCACGGTGGGGAGGACAT-3') of the 300 bp 5'-UTR and the
antisense primer (5'-TCACTGGGGATGCAGCCACCAGCTCCATT-3') of the 42-bp
5'-UTR, PCR using human genomic DNA as a template produced a 14.5-kb
DNA fragment spanning these two 5'-UTRs. The PCR reaction was performed
using ExpandTM Long Template PCR system (Roche Molecular
Biochemicals). The PCR was set at 94 °C for 10 s for
denaturation, at 60 °C for 30 s for annealing, and at 68 °C
for 8 min for extension and repeated for 30 cycles. After the final
cycle, the elongation was carried out at 68 °C for 7 min. The
amplified DNA fragment was subcloned into pGEM4Z (Promega) and its
restriction map was determined (Fig. 1A). The 5'-flanking
region of the 300-bp 5'-UTR was obtained by inverse PCR. Ten µg of
human genomic DNA was digested with 50 units of EcoRI and
followed by phenol/chloroform extraction and ethanol precipitation. The
precipitated DNA was self-ligated in the volume of 1 ml using T4 DNA
ligase (100 units) at 16 °C for 14 h. Self-ligated DNA was
precipitated by ethanol after phenol/chloroform extraction, and was
dissolved in 20 µl of TE buffer (pH 8.0). The flanking sequence of
300 bp 5'-UTR was amplified by PCR using 5' antisense primer
(5'-CCTGGAGCACTTAACCTTAACCTTCA-3'), and 3' sense primer
(5'-TAAGCAGCAAGCAGGCTTAG-3'), and 0.5 µg of self-ligated DNA. The
amplified DNA was subcloned into pGEM4Z and sequenced.
was labeled with
[
-32P]ATP by polynucleotide kinase. The labeled
oligonucleotide (105 cpm) was mixed with 1 µg of human
skeletal muscle poly(A)+ RNA (CLONTECH)
in 10 µl of 25 mM PIPES (pH 6.8), 400 mM
NaCl, 1 mM EDTA. The mixture was incubated at 70 °C for
3 min, then slowly cooled down to 37 °C and incubated overnight at
37 °C. Annealed primer/RNA was precipitated by adding 25 µl of
ethanol and used for extension reaction. This primer was extended with 200 units of SuperScript II reverse transcriptase (Life Technologies, Inc.) at 42 °C for 1 h under the buffer conditions the
manufacturer recommended. After phenol/chloroform (1:1, v/v) extraction
and ethanol precipitation, the size of primer extension product was determined by 8% denaturing polyacrylamide gel electrophoresis. The
nucleotide numbering of the ACC
promoter was based on the transcription start site proved by primer extension analysis. To
confirm that the primer extension product contains the 5'-UTR of the
ACC
cDNA, the primer extension product was isolated from the
denaturing polyacrylamide gel and used as a template in following PCR.
Primers used in the PCR were the 5' sense primer corresponding to the
region
72 to
53, or
55 to
34 from the ATG codon and the 3'
antisense primer used in primer extension analysis. The size of the PCR
products was determined on an 8% native polyacrylamide gel.
cDNA. The PCR
primers for the 5' terminal region of ACC
transcript, spanning exon
1b and exon 2, were the sense primer corresponding to the region
72
to
53 from the ATG codon and the antisense primer same as that used in the primer extension analysis. The ACC
cDNA corresponding to
the region +6001 to +7000 from the ATG codon and
glyceraldehyde-3-phosphate dehydrogenase cDNA were amplified using
following primers: ACC
sense, 5'-TCCAACAACCAGCTGGGTGGCGTTC-3';
ACC
antisense, 5'-CCAGCATCCGGCCGGGTGTGTCATG-3'; glyceraldehyde-3-phosphate dehydrogenase sense,
5'-ACCACAGTCCATGCCATCAC-3'; and glyceraldehyde-3-phosphate
dehydrogenase antisense, 5'-GGAGACCTTCTGCTCAGTCGACG-3'. The sizes of
the PCR products were determined on an 8% native polyacrylamide gel or
on 1.0% agarose gels.
promoter-reporter construct of pPII
1317 was generated by
subcloning the 3' 1.4-kb XbaI fragment (
1317 to +65) of
the 14.5-kb PCR product into SmaI site of pGL3-Basic
(Promega). The constructs designated as pPII
1090, pPII
569,
pPII
349, and pPII
203 were produced by self-ligation of
pPII
1317 after digestion with KpnI,
KpnI/ApaI, KpnI/DraI, and
KpnI/HindIII, respectively. The constructs of
pPII
800, pPII
93, pPII
38, and pPII
+7 were generated by
insertion of the DNA fragments amplified by PCR into SmaI
site of pGL3-Basic. The construct of pPII
1317/+17 was produced by
ligation of the 4.5-kb NarI-flushed/XbaI fragment
of pPII
-1317 and 1.7-kb SmaI/XbaI luciferase
gene of pGL3-Basic. The construct, pPII
1317/+3 was prepared by
subcloning the DNA fragment from
1317 to +3 of PII
amplified by
PCR into SmaI site of pGL3-Basic. Mutant constructs were
generated employing QuikChangeTM site-directed mutagenesis
kit (Stratagen) using the mutagenic oligonucleotides. Every mutated
sequence is explained in the legends to Figs. 5 and 6, and the
sequences of all mutant constructs were confirmed by DNA sequencing.
The construction of the pE-box-tk-luc and the p6×(+8/+27)-tk-luc was
performed by inserting
569 to
204 of PII
and six copies of the
fragment from +8 to +27 of PII
, respectively, into the upstream of
the tk promoter of ptk-luc, a tk-promoter-luciferase reporter construct.
-luciferase constructs, 0.2 µg of the
pCMV
-galactosidase plasmid (CLONTECH) and the
indicated amounts of pcDNA3 (Invitrogen), pcMyoD, or pcE12. The
pcMyoD and pcE12 plasmids were kindly provided by Stephan F. Konieczny
(Purdue University) and express MyoD and E12, respectively, under the control of the cytomegalovirus IE promoter. Transfection was
performed using LipofectAMINE Plus transfection reagent (Life
Technologies, Inc.) for 3 h according to the manufacturer's
instructions. After 2 days, cells were washed with phosphate-buffered
saline (Life Technologies, Inc.) and lysed in 200 µl of reporter
lysis buffer (Promega). Luciferase activities were measured using the
Luciferase Assay System (Promega) and normalized by the
-galactosidase activities to correct for the transfection
efficiency. All plasmid DNA for transfections was purified by Qiagen
Plasmid Midi kit (Qiagen Inc.)
42/
23,
10/+10, and +8/+27 was
inserted into the SacI site of pHisi-1 and pLacZi. The
resulting plasmids were linearized and transformed into
Saccharomyces cerevisiae strain YM4271 by the lithium
acetate method, and the transformants were selected on SD-His,Ura solid
medium for integration of the target-reporter construct into the
HIS3 locus. To screen the clones expressing the proteins
that bind to the target sequences in vivo, a human skeletal
muscle cDNA library constructed in pGAD10 was introduced into the
above transformed YM4271. pGAD10, the expression vector of the
Matchmaker yeast two-hybrid system produces the inserted gene product
as a C terminus of GAL4 activation domain-fusion protein. Clones were
selected on SD-His,Ura,Leu solid medium containing 40 mM
3-amino-1,2,4-triazole (Sigma). Plasmids isolated from the selected
yeast clones were transformed into the Escherichia coli strain DH5
and subjected to sequencing for identification.
, respectively. The
bacteria freshly transformed with each expression vector were grown to mid-log phase and proteins were induced for 4 h with 1 mM isopropyl-1-thio-
-D-galactopyranoside. The bacteria were harvested by centrifugation and disrupted by sonication. The recombinant proteins containing histidine-tag were
purified to homogeneity by Ni-NTA-agarose (Qiagen) chromatography. GST-Myf4 was purified by glutathione-Sepharose 4B (Amersham Pharmacia Biotech) chromatography according to the protocol provided by the
manufacturer. The purity and the concentration of the recombinant proteins were verified by SDS-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining.
418/
394), 5'-CCCTTGGAACATCTGTCGATGCTG-3'; E2 (
435/
412),
5'-GGCGTCTGACAGATGAACCCCTTG-3'; E3 (
512/
489), 5'-CTGTGTGTCCAGCTGGCCATTCGT-3'; Pr1 (
43/
15)
5'-CAGCCTCCCGCTGAAAGGTGACACTCTGCC-3'; Pr2 (
33/
15),
5'-CTGAAAGGTGACACTCTGCC-3'; Pr3 (
24/+4),
5'-GACACTCTGCCAGCTGGGTTCCCTTAGT-3'; Pr4 (
10/+10),
5'-TGGGTTCCCTTAGTCACCCT-3'; Pr5 (+4/+29),
5'-TCACCCTGTGGGCGCCTGTCAGCCTC-3'. Pr5 mutants used for
competition assay are shown at Fig. 7A.
-32P]ATP as substrate and annealed
with 5-fold excess of its complementary, unlabeled oligonucleotide by
heating at 90 °C for 5 min and allowing it to slowly cool to room
temperature in 0.1 M Tris (pH 8.0), 50 mM NaCl.
The annealed probe was purified by spun column chromatography and
probes of 100,000 cpm were used for each reaction. Recombinant MyoD,
GST-Myf4, Myf6, or MyoD/E47 heterodimer were incubated with the
prepared probes in a final volume of 20 µl containing 10 mM HEPES (pH 7.9), 75 mM KCl, 1 mM
EDTA, 5 mM dithiothreitol, 5 mM MgCl2, 10% glycerol, 1 µg of poly(dI-dC) (Amersham
Pharmacia Biotech), and 0.5% fetal calf serum. After 20 min incubation
at room temperature, samples were subjected to electrophoresis on 5%
polyacrylamide gel in 1 × TBE (45 mM Tris, 45 mM boric acid, 1 mM EDTA) at 250 V for ~1 h
at room temperature. For the competition assays, competitors prepared
by annealing the unlabeled sense and antisense oligonucleotides were
added at ~50-200-fold molar excess for each competition assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Promoter--
Two ACC
cDNA clones
containing different 5'-UTRs of 300 and 42 bp, respectively, were
previously isolated from a human skeletal muscle cDNA library (7).
A sense primer based on the 300-bp 5'-UTR and an antisense primer based
on the 42-bp 5'-UTR were used to amplify a 14.5-kb product. To clone
the upstream sequence of the 300-bp 5'-UTR, inverse PCR was performed
using circular genomic DNA self-ligated after EcoRI
digestion as template. The resulting 3.5-kb DNA fragment contained
2.3-kb upstream and 1.2-kb downstream sequences of the 300-bp 5'-UTR.
The cloning strategy used to obtain the 17-kb 5'-flanking sequence and
its restriction map are shown in Fig.
1A. The regions of 300- and
42-bp 5'-UTRs were named as exon 1a and exon 1b, respectively, and
their 5'-flanking sequences were designated as PI
and PII
.
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Fig. 1.
Cloning of the upstream 17-kb region of
ACC gene and determination of its
transcription initiation site. A, describes the cloning
strategy used to obtain the 17-kb 5'-flanking region of the ACC
gene
and the restriction sites of the cloned fragment. The regions of two
5'-UTRs (300 and 42 bp described in text) are indicated as exon
1a and exon 1b, and their 5'-flanking regions are
designated as PI
and PII
, respectively. The ATG start codon in
exon 2 is illustrated. B, shows the primer extension product
resolved on an 8% denaturing polyacrylamide gel (a, lane
5). A dideoxy sequence ladder (a, lanes 1, 2, 3, and
4) is shown for size determination of the primer extension
product. The primer extension product (132 bp) was eluted from gel
slice and used for the nested PCR to amplify the region
55 to +56
(b, lane 1) or
72 to +56 (b, lane 2) from the
ATG codon. The size of nested PCR products was measured on a 8% native
polyacrylamide gel in 1 × TBE by comparing with the
X174
DNA/HaeIII marker (b, lane 3)
, a
32P-labeled primer complementary to the region +37 to +56
from the ATG codon of ACC
cDNA was extended using human skeletal
muscle poly(A)+ RNA as template. The products were analyzed
on a sequencing gel and a single 132-bp fragment was observed (Fig.
1B, a). Because the primer-extension gave only one product
smaller than the 300-bp 5'-UTR, we performed PCRs to confirm whether it
contained the sequence of the 42-bp 5'-UTR. The 132-bp fragment was
isolated from the sequencing gel slice and used as a template, and two 5' sense primers corresponding to the 42-bp 5'-UTR and one common 3'
antisense primer of the previous primer extension, were used. Those
primers amplified two PCR products of our expecting sizes, 111 bp
(
55/+56) and 128 bp (
72/+56), respectively. This result suggests
that the ACC
mRNA in muscle originates from PII
(Fig. 1B, b). The first transcribed nucleotide of PII
,
determined by the primer extension, was designated as +1 for numbering
the nucleotides in the ACC
gene.
region from
1317 to +65 showed high promoter
activity in H9C2 myoblast cells, while the 2.3-kb PI
region upstream
of the 300-bp 5'-UTR did not have transcription activity at all in
various cell lines. Computer-based analysis (MatInspector V2.2 based on
Transfac 4.0) of PII
revealed several transcription factor-binding
sites including multiple E-boxes (Fig.
2). Therefore, in this study we
investigated the cis-elements and trans-acting
factors involved in the transcription regulation of PII
promoter.
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Fig. 2.
Sequence analysis of the 1317 to +65
fragment of the PII
promoter. The E-boxes
and other putative cis-elements recognized by computer-based
analysis (MatInspector V2.2 based on Transfac 4.0) are boxed
on the sequence of the PII
promoter (GenBankTM accession
number AF268379). An arrow indicates the transcription start
site.
Promoter Was Highly Active in Myoblast Cells and Was
Strongly Activated by MyoD--
According to previous reports of the
tissue-specific expression of ACC
(6, 7, 26), we tested the promoter
activity of the
1317 to +65 fragment in various cell lines. A
transiently transfected luciferase reporter construct (pPII
-1317)
showed the highest promoter activity in H9C2, a rat cardiomyoblast cell line. However, its promoter activity was very low or hardly detectable in Alexander, a human hepatoma cell line, in HeLa, a human cervical carcinoma cell line, or in NIH3T3, a mouse embryonic fibroblast cell
line (Fig. 3A). The extremely
low activity of pPII
1317 in Alexander cells was rather intriguing,
because ACC
expression has been observed in human liver and HepG2,
another human hepatoma cell line (6, 26). The inactiveness of this
promoter in Alexander cells suggested that liver might employ another
promoter for ACC
expression. To explore whether a different promoter
from PII
drives ACC
expression in liver, we examined if the
ACC
mRNA in liver cell lines has different 5'-UTR from that of
muscle tissue. We prepared cDNAs from total RNA of Alexander cells,
and HepG2 cells, and human skeletal muscle tissue to perform RT-PCR.
Only the muscle cDNA generated the 128-bp PCR product containing
5'-UTR (exon 1b) sequences, while those three cDNAs produced a 1-kb
product for the C terminus coding region (Fig. 3B). Both
muscle and liver definitely express the ACC
mRNAs but the
mRNAs in those two tissues have different 5'-UTRs. These results
strongly imply the presence of liver-type ACC
promoter, and we
concluded that PII
is the muscle-type ACC
promoter.
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Fig. 3.
Muscle-specific promoter activity of
PII and its MyoD responsiveness in various
cell lines. A, pPII
-1317 was transiently transfected
into H9C2, NIH3T3, Alexander, and HeLa cells. The basal activity and
the MyoD stimulated activity of the reporter in each tested cell line
are presented as white bars and black bars,
respectively. Reporter (0.4 µg) and pCMV
-galactosidase (0.2 µg)
were transfected into each cell line with pcMyoD or pcDNA3 (0.4 µg each). The reporter activities are shown as the relative
luciferase activity normalized by the
-galactosidase activity. The
data represents the mean ± S.D. of three independent experiments
performed in triplicates. B, RT-PCR to compare the ACC
transcripts of Alexander cells (A), HepG2 cells
(H), and human muscle tissue (S). The ACC
cDNA fragments, containing the sequence of exon 1b and exon 2 (a), or +6001 to +7000 from the ATG codon (b), or
glyceraldehyde-3-phosphate dehydrogenase cDNA as an internal
control for the quality of cDNAs (c) were PCR-amplified
and the product sizes were measured on a 8% polyacrylamide gel
(a) or 1% agarose gel (b and c). For
the size marker (M), the 25-bp DNA ladder or 1-kb DNA ladder (Life
Technologies, Inc.) was used.
(Fig. 2). Overexpression of MyoD, a
typical MRF, indeed activated the transiently transfected ACC
promoter effectively in every cell line except in HeLa cells (Fig.
3A). Its relative activation by MyoD in H9C2 cells is lower than in NIH3T3 cells or Alexander cells due to high basal transcription activity in H9C2 cells, probably caused by the endogenous myogenic transcription factors. We therefore used NIH3T3 cells for further experiments instead of myoblast cells to exclude the effect of endogenous MyoD and other muscle related factors.
promoter serially deleted from the 5' or 3' end of the
1317 to +65
fragment (Fig. 4). The deletion of
569
to
349 and
38 to
19, among the 5' deletion constructs, most
drastically decreased the MyoD responsiveness. Deletion of the 220 bp
from
569 to
349, containing a cluster of three E-boxes, decreased
the MyoD responsiveness by about 50%. The reduced responsiveness was
recovered in further deletions up to
39, suggesting the presence of
inhibitory elements for MyoD responsiveness in the region from
340 to
39. The deletion from
93 to
39, where Sp1 can bind, most
outstandingly enhanced the MyoD responsiveness. Although we cannot
reveal the prescise mechanisms involved in this inhibitory action in
the present study, the Sp1-binding site in this inhibitory region is
considered to be noteworthy since Sp1 expression markedly decreases
during myogenesis (36, 37). Deletion from
38 to
19 resulted in a
significant decrease in basal transcription as well as the decrease in
MyoD responsiveness, and the further deletion to +7 completely
abolished MyoD responsiveness of PII
. Deletions of the 5'-UTR from
pPII
1317 also significantly diminished the MyoD responsiveness and
the basal transcription level. The regions of
569 to
349 and
39 to +65, which play important roles in MyoD responsiveness and/or basal
transcription, are hence designated as regions A and B, respectively
(Fig. 4).
View larger version (17K):
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Fig. 4.
MyoD-responsive regions of
PII have been narrowed down to two main
regions by transient transfection analysis of serially deleted
reporters. Schematic diagram of the PII
serial deletion
reporter constructs and their MyoD responsiveness are presented. The
two putative regions on which MyoD acts, designated as region
A and region B, are presented at the bottom.
Reporter (0.4 µg) and pCMV
-galactosidase (0.2 µg) were
transfected into NIH3T3 cells with pcMyoD or pcDNA3 (0.4 µg
each). The reporter activities are shown as the relative luciferase
activity normalized by the
-galactosidase activity. The data
represents the mean ± S.D. of four independent experiments
performed in triplicate.
569 to
349 region designated as E-box 1, E-box 2, and E-box 3 (Fig.
5A). E-box 1 and 2 are
juxtaposed with intervening 12 nucleotides, and E-box 3 is located
about 60 bp upstream from E-box 2. Interestingly, strikingly similar
distributions of three E-boxes are found in various muscle-specific
promoters and enhancers, such as the myosin light chain enhancer,
muscle creatine kinase promoter, and acetylcholine receptor
and
subunit enhancers (26). We presumed the cluster of three E-boxes on the
ACC
promoter might contribute to its MyoD responsiveness and
examined their role in MyoD responsiveness by introducing a
site-specific mutation (TG
AA) in each (Fig. 5B). Each
single E-box mutation such as M(E1), M(E2), and M(E3) reduced the MyoD
responsiveness to different extents. Mutation on E-box 1 most
significantly decreased the responsiveness. Interestingly, the
influences of the E-box destructions were neither synergistic nor
additive. Double mutant (M(E1+2)) or triple mutant (M(E1+2+3)) showed a
similar extent of MyoD responsiveness to that of the single mutant
M(E1). To confirm the MyoD binding to these E-boxes in
vitro, we performed EMSA with recombinant MyoD and recombinant E47
(Fig. 5, C and D). The probe containing E-box 1 (E1) was well shifted by MyoD, E47, or the mixture of MyoD and E47
(MyoD/E47). The probes containing E-box 2 (E2) or E-box 3 (E3) were not
effectively bound by MyoD, but were shifted by E47 or MyoD/E47. All
three probes were bound by MyoD/E47, even though the intensity of their
shifted bands was significantly different from one another, E1 showed
the strongest band shift, E3 the next, and E2 the weakest. To determine
whether the differences result from differences in their binding
affinity, we performed competition assays (Fig. 5E). The
complex formation between E1 and MyoD/E47 was effectively competed by
cold E1, the self-competitor, moderately by cold E3, and slightly by
excess amount of cold E2. Thus, E-box 1 has the strongest affinity to
MyoD/E47 among the three E-boxes in region A.
View larger version (45K):
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Fig. 5.
MyoD acts on the three E-boxes of region
A. A shows the sequences of the E-boxes in region A and
the location of the probes E1, E2, and E3 used in D and
E. B, single, double or triple E-box mutants
(mE1, mE2, mE3, mE1+2, and mE1+2+3), schematically shown at the
left side, were prepared by site-directed mutagenesis
substituting TG of CANNTG with AA. Wild type (pPII 569) or
E-box-mutant reporters (0.4 µg) and pCMV
-galactosidase (0.2 µg)
were transfected into NIH3T3 cells with pcMyoD or pcDNA3 (0.4 µg
each). The reporter activities are shown as the relative luciferase
activity normalized by the
-galactosidase activity. The data
represents the mean ± S.D. of three independent experiments
performed in triplicate. C, SDS-PAGE of the recombinant E47
and MyoD. Those proteins are bacterially expressed and purified for
EMSA. M is the molecular weight standard. D, EMSA
of the E-boxes with recombinant MyoD and/or E47.
32P-Labeled probes (E1, E2, and E3) were incubated with 300 ng of MyoD (lanes 1, 4, and 7) or 30 ng of E47
(lanes 2, 5, and 8) or 150 ng/15 ng of MyoD/E47
mixture (lanes 3, 6, and 9). E,
competition assays. The E1 band shifted by MyoD/E47 (150 ng/15 ng) was
competed by 50-200-fold molar excess amount of unlabeled competitors,
such as E1 (lanes 2-4), E2 (lanes 8-10), and E3
(lanes 5-7).
38
to +65 (region B), we first introduced a mutation on the single E-box
(
14 to
9), designated as E-box 4, of pPII
93 (Fig.
6A). The mutation of TG to AA
(M(E4)) in E-box 4 reduced the MyoD responsiveness of pPII
93 by
about 50% (Fig. 6B). Because the destruction of the sole
E-box in pPII
93 did not abolish its entire MyoD responsiveness, we
presumed there might exist other important sites for MyoD action in
this short region, and prepared several mutant reporters to identify
those motifs. We substituted the first and second TG sequences with AA,
and designated the resulting mutants as M(
32) and M(
25) according
to their locations. Mutation of the
32/
31 TG drastically decreased
the MyoD responsiveness and the basal transcription by 74 and 50%, respectively, whereas mutation of the
25/
24 TG showed no
significant change on the basal promoter activity and the stimulation
by MyoD (Fig. 6, A and B). The TGAAA sequence
from
32 to
28 appears to play a role of TATA box in the PII
promoter because it is located in the typical TATA box position and its
mutation significantly affects the basal transcription. Next, the
transcription start site (CCTT+1AG) and a
palindromic hexanucleotide (GGCGCC) located at +14 to +19 were
substituted with AAGCTT. Those mutants, designated as M(start) and
M(GC), showed remarkably decreased MyoD responsiveness by 89 and 85%,
respectively. M(start) also showed significantly decreased basal
transcription by over 80% while M(GC) did not show decrease in basal
transcription, suggesting that the transcription start region might
serve as an Inr element. To discern the cis-elements for
MyoD from those for the general transcription complex, we examined
where MyoD can bind in this region by EMSA using the probes shown in
Fig. 6A. Probe Pr3 containing E-box 4 and probe Pr5
containing the GGCGCC palindrome could form a complex with recombinant
MyoD, whereas the other probes could not (Fig. 6C).
View larger version (28K):
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Fig. 6.
Region B has several putative
cis-elements for the basal transcription and the MyoD
responsiveness. A, DNA sequences from 43 to +30 is
shown to describe the five mutations introduced in region B and the
five probes used in B and C. B, the
MyoD-stimulated activities of the five mutant reporters were measured
by transient transfection with co-transfection of pcMyoD into NIH3T3
cells. The basal activity of each tested mutant is shown in the
inset. Reporter (0.4 µg) and pCMV
-galactosidase (0.2 µg) were transfected into NIH3T3 cells with pcMyoD or pcDNA3 (0.4 µg each). The reporter activities are shown as the relative
luciferase activity normalized by the
-galactosidase activity. The
data represents the mean ± S.D. of five independent experiments
performed in triplicate. C, probes (Pr1 to Pr5) presented at
the bottom of A were subjected to an EMSA to test
the binding of MyoD (150 ng).
View larger version (53K):
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Fig. 7.
Pr5 contains a novel
cis-element for MyoD binding whose characteristics are
different from those of the E-box. A, the novel
cis-element has GCCTGTCA as its core motif. The Pr5 band
shifted by MyoD (100 ng) was competed by mutant cold oligonucleotides
shown in the right panel. Eight mutant oligonucleotides
(lanes 3-8) and one wild type (lane 2)
oligonucleotide were added to the binding reaction in the 200-fold
molar excess. B, comparison of E-box 4 and the novel
cis-element in vitro. 32P-Labeled Pr3
(lanes 1-6) and Pr5 (lanes 7-12) were incubated
with 50 or 100 ng of E47 (lanes 1, 2 and 7, 8),
100 or 200 ng of MyoD (lanes 3, 4 and 9, 10), and
50/25 or 100/50 ng of the MyoD/E47 mixture (lanes 5, 6 and
11, 12). C, comparison of the E-box 4 and the
novel cis-element in vivo. The E-box clustered
region ( 569 to
204) or 6 copies of the +8 to +27 sequence were
inserted upstream of the minimal tk promoter of ptk-luc construct as
described under "Materials and Methods." Each reporter (0.4 µg)
with pCMV
-galactosidase (0.2 µg) was co-transfected with
pcDNA3 (0.4 µg, white bar), pcE12/pcDNA3 (0.1/0.3
µg, light gray bar), pcMyoD/pcDNA3 (0.1/0.3 µg,
black bar), or pcE12/pcMyoD/pcDNA3 (0.1/0.1/0.2 µg,
dark gray bar) into NIH3T3 cells. The control reporter
(ptk-luc) was transfected in the same way to check the effect of MyoD
or E12 on the minimal tk promoter. The reporter activities are shown as
the relative luciferase activity normalized by the
-galactosidase
activity. The data represents the mean ± S.D. of five independent
experiments performed in triplicate.
(
569 to
204) (pE-box-tk-luc) or six copies of +8 to +27 sequence
(p6×(+8/+27)-tk-luc) upstream of tk minimal promoter (Fig.
7C). The tk minimal promoter itself was not affected by
overexpression of E12 or MyoD. E12 alone did not stimulate
pE-box-tk-luc or p6×(+8/+27)-tk-luc, while MyoD-overexpression
enhanced the transcription of both reporter constructs. Coexpression of
E12 with MyoD synergistically activated the transcription in
pE-box-tk-luc compared with the transcription driven by MyoD alone.
However, coexpression of E12 significantly decreased the transcription
of p6×(+8/+27))-tk-luc stimulated by MyoD. Taken together, this novel
cis-element is more apt to respond to the MyoD homodimer
rather than to the E-protein/MyoD heterodimers and therefore is
distinct from E-boxes.
42 to
23,
10
to +10, and +8 to +27 upstream of the HIS3 gene. We isolated
the clones expressing the GAL4 activation domain fused to DNA-binding
proteins, which bind to the bait sequences, and thus activate the
HIS3 gene expression allowing selection in
histidine-depleted media. We could not obtain any positive clones as
for the
42 to
23 or
10 to +10 regions, while several positive
clones were isolated for the +8 to +27 region. Sequencing of those
isolated clones revealed that one of them contained the full-length
Myf4 cDNA and the other seven clones contained various lengths of
Myf6 cDNA. These data suggest that MRFs bind to the +8 to +27
region in vivo, where the E-box consensus sequence does not
exist. To confirm their binding ability to this DNA sequence in
vitro by EMSA, we prepared recombinant Myf4, Myf6, and MyoD expressed in bacteria (Fig. 8B). MyoD,
Myf4, and Myf6 could bind to this region in vitro. Those
bindings were effectively competed by a 200-fold molar excess of the
unlabeled wild type Pr5 (Fig. 8A, lanes 3, 6, and
9), while the addition of excess cold mPr5.4, an
oligonucleotide containing GGCGCC
AAGCTT mutation, did not decrease
complex formation (Fig. 8A, lanes 4, 7, and
10).
View larger version (29K):
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Fig. 8.
The novel cis-element
containing GCCTGTCA sequence shows MRFs responsiveness.
A, EMSA of Pr5 with 100 ng of MyoD (lanes 2-4),
GST-Myf4 (lanes 5-7), or Myf6 (lanes 8-10).
Both wild type (lanes 3, 6, and 9) and mutant
(mPr5.4) unlabeled double-stranded oligonucleotides (lanes 4, 7, and 10) were used as competitors. B,
SDS-PAGE showing the purified recombinant proteins used in the EMSA.
Lanes 1-4, the molecular weight size markers (Genepia,
Korea), MyoD, GST-Myf4, and Myf6, respectively. C, the
reporter constructs (0.4 µg each), such as ptk-luc,
p6×(+8/+27)-tk-luc, and pPII 93, were transfected into NIH3T3
cells with pcMyoD, pcMyf6, or pcDNA3 (0.4 µg each), and pCMV
-galactosidase (0.2 µg) was co-transfected. Fold increase of the
MyoD- or Myf6-mediated induction compared with the basal activity of
each reporter is shown above the bars. The
reporter activities are shown as the relative luciferase activity
normalized by the
-galactosidase activity. The data represents the
means ± S.D. of five independent experiments performed in
triplicate.
93 and p6×(+8/+27)-tk-luc were greatly activated by MyoD or Myf6, while
tk promoter itself was not activated by MyoD or Myf6 (Fig. 8C). All together these in vivo and in
vitro data, we conclude that the GCCTGTCA motif truly is a novel
cis-element for MRF transcription factors.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and characterized the cis-elements and
trans-acting factors related. Moreover, we are suggesting
the presence of the liver-type ACC
promoter. The PII
promoter in
the present study was cloned according to the information of 5'-UTR
sequence of ACC
cDNA originating the from skeletal muscle.
However, because ACC
is expressed not only in skeletal muscle but
also in liver, another promoter different from the PII
seems to
induce the ACC
expression in liver. The following evidence supports
this suggestion. First, the PII
promoter was highly active in
myoblast but not in the hepatoma cell line. Second, the sequences of
exon 1b transcribed by the PII
exists only in the ACC
mRNA of
skeletal muscle and not in that of hepatoma cell lines such as
Alexander and HepG2, while the sequences for the C terminus region of
ACC
could be detected in both mRNAs from skeletal muscle and
hepatoma cell lines. Conclusively, ACC
expression might employ
another promoter in liver, and the PII
is its muscle-specific promoter.
promoter are
identified as MRF family members, such as MyoD, myogenine/Myf4, and
MRF4/Myf6. The myogenic regulatory factor-mediated transcription in
this promoter depends on four E-boxes and one novel
cis-element immediately downstream from the transcription
start site, and at least two cis-elements which
are not fully characterized but appear to be important for basal
transcription. Among the four E-boxes, three of them were located in a
distal region (
498 to
403), and one resides in a very proximal
region (
14 to
9). The distal E-box clustered region (region A) is
responsible for up to 50% of the MyoD responsiveness of this promoter,
and the proximal region (region B), harboring the fourth E-box and a
novel downstream cis-element, is responsible for the other
50% of MyoD responsiveness. E-box 1 of the clustered three E-boxes is
thought to be most critical for the MyoD responsiveness of region A,
although E-boxes 2 and 3 are also required for full activity. This
distribution of triple E-boxes and the finding that, while all are
necessary, one of the three is more crucial, has been reported by
Wentworth et al. (26) in the myosin light chain enhancer. In
addition, the E-box 1 (caTCtg) has never been reported as an
MRF-binding site as far we know, while E-box 3, the less critical MyoD
responsive site (caGCtg) has been repeatedly reported as a binding
motif for MyoD/E-protein heterodimers in several muscle gene promoters (29, 30). E-box 2 (caGAtg) is reported to exist in the promoter of
mouse acetylcholine receptor
subunit (35), but its weak binding
affinity for MyoD/E47 do not allow ignoring the possibility that other
transcription factors bind to this region and help the action of
neighboring MyoD.
349 to
39), unidentified repressor
elements that might control the MyoD responsiveness seem to exist,
because the deletion of this region enhanced the MyoD responsiveness. The enhancement was most prominent when the region containing an
Sp1-binding site was excised out. According to our unpublished EMSA
data2 using the nuclear extracts of the H9C2 myoblasts and
its differentiated myotubes, Sp1 binding to the consensus motif (
70
to
65) is severely decreased in muotubes. This result corresponds to
the fact that myogenesis accompanies Sp1 decrease (36, 37). The Sp1
binding to this region might suppress the MyoD responsiveness in
myoblasts and down-regulation of Sp1 during differentiation to myotube
would be required for complete activation of the ACC
gene by MyoD.
32 region appears to play a role similar to a
"TATA" element since it is located at the general TATA locus and
the mutation at this region dramatically reduced the basal
transcription. The previously reported consensus Inr sequence
(YYA+1N(T/A)YY) of metazoan (33, 34) is similar to
the start region (CTT+1AGTC) of this ACC
promoter
and disruption of this site decreased the basal transcription
significantly. These findings suggest that this region might function
as the Inr element of basal transcription.
expression is not observed in
myoblast cells which express high levels of MRFs and is observed only
after the differentiation of myoblast cells into myotubes (7). However,
the promoter activity of ACC
was very high in H9C2 myoblasts in our
transient transfection assay (Fig. 3). According to our unpublished
data,2 the ACC
promoter, stably transfected into H9C2
myoblast cells, shows very high basal activity and was not further
enhanced by differentiation into myotubes (data not shown). These
discrepancies between transfection assays and endogenous gene
expression suggest that MRFs are required for maintnaining high level
expression of ACC in muscle cells, and another switching system exist
to turn on the expression of this muscle-specific gene. Further studies on the switching system might provide a general understanding for
stage-specific turning on and off of muscle-specific genes. We
now introduce the first report on the regulation mechanism for the
muscle-type ACC
promoter and also suggest the possible presence of
the liver-type ACC
promoter.
![]() |
ACKNOWLEDGEMENTS |
---|
We appreciate Dr. Stephan F. Konieczny for providing pcMyoD, pcE12, pQT-MyoD, and pQT-E47. We thank Dr. Ki-Han Kim for considerate advice.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Yonsei University College of Medicine for Research Instructors Research Grant 1998-01 and financial support of the Korean Research Foundation for the program year of 1998.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 sequences of PI and PII
have
been submitted to the GenBankTM/EBI Data Bank
with accession numbers AF268378 and AF268379, respectively.
§ Recipient of a scholarship from the Brain Korea 21 Project For Medical Science, Ministry of Education, South Korea.
** To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry and Molecular Biology, Institute of Genetic Science, Yonsei University College of Medicine, 134 Shinchon-dong Seodaemun-gu, Seoul 120-752, Korea. Tel.: 82-2-361-5186; Fax: 82-2-312-5041; E-mail: kyungsup59@yumc.yonsei.ac.kr.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M007002200
2 J-J. Lee, Y-A. Moon, J-H. Ha, D-J. Yoon, Y-H. Ahn, and K-S. Kim, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ACC, acetyl-CoA carboxylase; MRF, muscle regulatory factor; bHLH, basic helix-loop-helix; 5'-UTR, 5'-untranslated region; EMSA, electrophoretic mobility shift assay; PCR, polymerase chain reaction; kb, kilobase pair(s); bp, base pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; PIPES, 1,4-piperazinediethanesulfonic acid.
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