From the
Department of Biochemistry and Molecular
Biology, Institute of Genetic Science, Yonsei University College of Medicine,
134 Shinchondong Seodaemungu, Seoul 120-752, Korea,
Brain Korea 21 Project for Medical Science,
Yonsei University, and the ¶Department of
Biochemistry, Institute of Medical Science, Kwandong University College of
Medicine, 522 Naekokdong, Kangnung, Kangwondo 210-701, Korea
Received for publication, January 17, 2003 , and in revised form, April 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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ACC is also expressed in liver and HepG2 cells
(4,
12). Oxidation of fatty acid
also occurs actively in liver, but its regulation is different from that in
skeletal muscle. In liver, fatty acid oxidation is increased during the
fasting period and is almost blocked by food intake, whereas in skeletal
muscle, nutritional status does not change the rate of oxidations of fatty
acids significantly. Interestingly, animals lacking ACC
showed marked
increase in blood ketone bodies by overnight fasting compared with wild type
animals (13). These facts
suggest the possibility that ACC
might also control fatty acid oxidation
in liver and the regulation of its activities might be different in liver and
muscle.
Most of the lipogenic enzymes, including ACC, fatty acid synthase,
stearoyl-CoA desaturase-1, and ATP citrate-lyase, are regulated by dietary
regimen and insulin at the transcription level in liver. These regulations are
known to be mediated by SREBP-1, which is a member of the basic
helix-loop-helix/leucine zipper family of transcription factors
(1418).
SREBP-1 has been identified as two isoforms (SREBP-1a and -1c) derived from a
single gene through the use of alternative transcription start sites and
splicing (19). The precursors
of SREBPs (
125 kDa) are located in endoplasmic reticulum. Upon
activation, SREBPs are released from the membrane by a sequential two-step
cleavage process and translocated into the nucleus as a mature protein
(
68 kDa) (20). SREBP-1c
is a primary regulator in liver and adipose tissues, which mediates the
activation of gene transcription by insulin and food intake
(2123),
whereas SREBP-1a is major isoform in established cell lines.
In the present study, we first demonstrate that ACC gene
transcription driven by promoter II is induced in liver by the intake of
high-carbohydrate diet. Moreover, the nuclear form of SREBP-1 activates
ACC
promoter II by its binding to SREs and food intake
increases the occupancy of SREBP-1c in ACC
promoter II in
liver.
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EXPERIMENTAL PROCEDURES |
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Animals and DietsMale Sprague-Dawley rats weighing 150200 g were used for all experiments. For fasting and refeeding studies, rats were fasted for 48 h and refed with a fat-free high-carbohydrate diet for 0, 12, 24, or 48 h. All experiments were performed at least twice. The fat-free high-carbohydrate diet contained 82% (w/w) carbohydrates (74% starch, 8% sucrose), 18% (w/w) casein, 1% (w/w) vitamin mixture, and 4% (w/w) mineral mixture. All the materials for diet were purchased from Harlan Teklad Co. (Madison, WI).
Cell Culture and Transient TransfectionHepG2 cells were
maintained in minimal essential medium (MEM) supplemented with 10% (v/v) fetal
bovine serum and 100 µg/ml antibiotics/antimycotics at 37 °C in an
8090% humidified atmosphere. Cells were set up for experiments at 1
x 106 cells per well on a 6-well plate, and then incubated
for 1620 h. At 80% confluent state, cells were transfected with the
indicated plasmids using LipofectAMINE PLUS according to the manufacturer's
protocols. Briefly, the plasmid DNA and 4 µl of PLUS reagent were mixed in
100 µl of MEM and then added to 100 µl of MEM containing 2 µl of
LipofectAMINE reagent. The total amount of DNA in each transfection was
adjusted to the same amount by addition of mock vector plasmid. The cells were
washed with phosphate-buffered saline and supplied with serum-free MEM. After
15 min of incubation, the LipofectAMINE/DNA mixtures were added into wells.
The cells were transfected for 3 h with the plasmid, then washed twice with
phosphate-buffered saline and then grown in MEM supplemented with 10% fetal
bovine serum and 100 µg/ml antibiotics/antimycotics. After 48 h, the cells
were harvested and lysed by 200 µl of reporter lysis buffer (Promega), and
cell debris was removed by centrifugation. Luciferase activities were measured
using 10 µl of cell extract and 50 µl of Luciferase assay reagent
(Promega). For -galactosidase assay, the hydrolysis of
o-nitrophenol-
-D-galactopyranoside (Sigma) at 37
°C was measured at 420 nm
(24). Total proteins of
lysates were determined by the Bradford method. Luciferase activities were
normalized by the amount of total proteins because cytomegalovirus
promoter-driven expression of
-galactosidase is suppressed by
overexpression of SREBP-1a.
Drosophila SL2 cells were grown in Schneider's insect media
(Sigma) supplemented with 10% (v/v) fetal bovine serum and 1%
penicillin/streptomycin at 25 °C without supplemental CO2. For
transfection, cells were plated at a density of 5 x 105
cells/35-mm dish and were co-transfected on the next day by the calcium
phosphate coprecipitation method. The promoter-luciferase construct (0.4
µg) of phP-II93/+65 or mSp1 was co-transfected with 0.2 µg
of expression plasmids, such as pPac_SREBP-1a, pPac_SREBP-1c, pPac_Sp1, and/or
pPac mock vector, together with 0.2 µg of pPac_
-galactosidase. For
each dish, 146 µl of 0.25 M CaCl2 containing the DNA
was added dropwise to 146 µl of 140 mM NaCl, 1.5 mM
Na2HPO4, 50 mM HEPES, pH 7.0 and incubated at
room temperature for 15 min. The cells were harvested at 48 h after
transfection and luciferase and
-galactosidase activities of cell
extracts were measured.
Northern Blot Hybridization of mRNATotal RNA was isolated
from liver of rat, which was fasted or refed for the indicated periods, by
TRIzol (Invitrogen) according to the manufacturer's protocol. To remove the
glycogens in each sample, isolated RNA was suspended in distilled water and
precipitated by addition of one-third volume of LiCl buffer (7.5 M
LiCl, 50 mM EDTA). Total RNA isolated from two animals of each
group were pooled. Twenty µg of each sample were denatured with RNA sample
loading buffer (20 mM MOPS, pH 7.0, 2 mM sodium acetate,
1 mM EDTA, 8% (v/v) formaldehyde, 50% (v/v) formamide), and
subjected to electrophoresis in a 0.9% denaturing formaldehyde-agarose gel,
and transferred to Nylon membrane. The 420-bp fragment corresponding to exon 2
of the rat ACC gene was labeled with
[
-32P]dCTP using the Megaprime Labeling Kit (Amersham
Biosciences) and used as a probe. The membranes were hybridized with the probe
for 2 h at 65 °C with Rapid-Hybrid buffer (Amersham Biosciences). After
hybridization, the membrane was washed twice with high salt washing buffer
(0.1% SDS, 2x SSC) at room temperature for 30 min followed by low salt
washing buffer (0.1% SDS, 0.2x SSC) at 65 °C for 15 min. The
membrane was exposed to Kodak BioMax film with intensifying screen at
70 °C.
Western Blot AnalysisRat liver was homogenized in Buffer A
(50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10% (v/v) glycerol,
1% (v/v) Triton X-100, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, 50 mM NaF, 5 mM sodium
pyrophosphate) with a glass homogenizer and sonicated three times for 30 s on
ice. The homogenate was centrifuged to remove the cell debris, and protein
concentration of the soluble fraction was determined with Bradford reagent.
Extracts were separated in 5% SDS-polyacrylamide gels and transferred to
Protran nitrocellulose membranes (Schleicher & Schuell). Immunoblot
analysis was carried out with polyclonal anti-ACC antibody or
horseradish peroxidase-conjugated strepavidin, and specific bands were
visualized using ECL Kit (Amersham Biosciences).
RNase Protection AssaysThe pCRII plasmids containing cDNA for exon 1a (90 bp) or exon 1b (52 bp) extending to part of exon 2 (69 bp), were used as templates for cRNA synthesis. After linearization of each plasmid (1 µg) by HindIII digestion, 32P-labeled cRNA was synthesized by T7 RNA polymerase (Ambion, Austin, TX). Probes were purified by gel elution after 6% polyacrylamide, 6 M urea gel electrophoresis. RNase protection assays with purified probes were performed with RPAIII kit. Total RNA (20 µg) isolated from rat liver was hybridized with probe (1.6 x 105 cpm) in 30 µl of hybridization buffer at 42 °C for 1216 h. The unhybridized RNA was digested by adding 150 µl of the diluted solution (1:100) of RNase A/T1 mixture in RNase I digestion III buffer and incubating at 37 °C for 30 min. Probes protected from RNase were precipitated by addition of 225 µl of RNase inactivation/precipitation III solution and centrifugation for 15 min at 12,000 rpm. Precipitates were washed with 70% ethanol, and then denatured with 4 µl of sequencing gel loading buffer at 95 °C for 3 min and resolved on 6% polyacrylamide, 6 M urea gel. Gels were dried and exposed to Kodak BioMax film at 70 °C with intensifying screens. A sequencing ladder was loaded in the adjacent lane to determine the size of the product.
DNase I Footprinting AssayDNA fragment covering the region from 244 to +51 bp were labeled in one strand by PCR with a primer set of which one is labeled with [32P]. The sequences of the primers for PCR were 5'-ACCTAAGCTTGAGGTCAGGA-3' and 5'-AGCTCCATTCTTGAGTGAGG-3'. Indicated amounts of recombinant SREBP-1 protein, purified as described in Ref. 17, were incubated with 3 x 105 cpm of labeled probe for 20 min on ice under the condition of 10 mM HEPES, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 7% (v/v) glycerol, and 2 µg of poly(dI-dC), and then 50 µl of diluted DNase I solution (0.0020.001 units/µl) was added to the DNA-protein binding reactions. After 2 min of digestion at room temperature, the reaction was stopped by adding 100 µl of stop buffer containing 1% (w/v) SDS, 200 mM NaCl, 20 mM EDTA, pH 8.0, and 0.1 µg/µl glycogen. The DNA was extracted with phenol/chloroform and recovered by ethanol precipitation. The pellets were dissolved in sequencing gel loading buffer and then resolved on denaturing 6% polyacrylamide, 6 M urea gel. The footprints were compared with G + A ladder produced by the chemical cleavage sequencing reaction of the same probe to determine the corresponding nucleotide sequences.
Electrophoretic Mobility Shift Assay (EMSA)The probes
corresponding to nucleotide 93 to 21 of ACC
promoter II were generated by PCR, using 32P-labeled antisense
primer (40 to 21) and unlabeled sense primer (93 to
74). The phP
-II93/+65 and corresponding mutant constructs
were used in PCR as template. Amplified DNA was eluted from the gel after 8%
PAGE. The probe corresponding to 82/53 was made using
PAGE-purified oligonucleotides as followings. Ten picomoles of a
single-stranded oligonucleotide was labeled at the 5' end by incubation
with polynucleotide kinase and 30 µCi of [
-32P]ATP (6000
Ci/mmol) at 37 °C for 90 min. Five molar excess of complementary
oligonucleotide was added to the reaction mixture, and heated to 95 °C for
3 min, followed by cooling down to room temperature. After annealing reaction,
free isotope was removed by passing the reaction mixture through a Sephadex
G-50 spun column. For EMSA, the probes (1 x 105 cpm) were
incubated with purified recombinant SREBP-1 (10 ng) or liver nuclear extract
(10 µg) in a final volume of 20 µl containing 10 mM HEPES, pH
7.6, 75 mM KCl, 1 mM EDTA, 10 mM
dithiothreitol, 10% (v/v) glycerol, 1 µg of poly(dI-dC), and 0.5% bovine
serum albumin. After 20 min of incubation at room temperature, the samples
were resolved on a 4% polyacrylamide gel in 1x TBE (45 mM
Tris, 45 mM boric acid, 1mM EDTA) at 250 V for
1 h
at room temperature. After electrophoresis, the polyacrylamide gel was dried
and exposed to Fuji HR-G30 x-ray film for 3 h at 70 °C with
intensifying screen.
Construction of PlasmidsThe luciferase constructs of human
ACC promoter II were described in Lee et al.
(25). Human ACC
promoter I (770 bp) was amplified with human genomic DNA using sense primer
(5'-TCCACCTTCCCTGTTGCCTGA-3') and phosphorylated antisense primer
(5'-ACCGTGCATTCAGGGTTACA-3') and inserted in SacI and the
SmaI site of pGL3-Basic. Rat ACC
promoter I
(576/+159) and promoter II (95/+65) were amplified by PCR using
rat genomic DNA and the primer sets of
5'-ACTGAGCTCAGCGGCCAGACATG-3'/5'-TTTCAAGCTCCTCTGTGGCT-3'
and
5'-CCGAGTACTGGCCAAGCCCCT-3'/5'-TCACTGGGGACGTGGCCGCCA-3',
respectively. Amplified rat ACC
promoter I and II sequences
were inserted into SacI/SmaI and SmaI sites of
pGL3-Basic, respectively. The expression plasmids, pPac_SREBP-1a and
pPac_SREBP-1c, were constructed by insertion of 1.4 kb of SREBP-1a and
SREBP-1c cDNA into the EcoRV site of the pPacPL vector. The
expression vector pPac_Sp1 was the gift from R. Tjian (University of
California, Berkeley, CA) and the pPacPL was the gift from Carl S. Thummel
(University of Utah).
Chromatin Immunoprecipitation (ChIP) AssayChIP assay
protocol was modified from the description by Duong et al.
(26). Livers from the rats
fasted and refed with high-carbohydrate diets were perfused with DMEM for 5
min at a flow rate of 10 ml/min through the portal vein, then fixed with 5%
formaldehyde in DMEM for 5 min at the same flow rate. Livers were then washed
with DMEM for 5 min, and the fixed liver was excised and washed in cold
phosphate-buffered saline. The weight of liver was measured and stored in
70 °C. Because the weight of liver is markedly changed according to
feeding status, the 0.5% fraction of each liver weight (50100 mg) was
homogenized with 1 ml of buffer A, and pelleted by centrifugation at 12,000
rpm for 5 min at 4 °C. Pellet was resuspended in 800 µl of SDS lysis
buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0). To
shear chromatin, the lysate was sonicated on ice for 3 min with sonicator tip
of 2.5 mm in diameter at 30% amplitude and 0.5 cycle (UP400s, Dr. Hielscher
GmbH, Germany). Samples were centrifuged at 13,000 rpm for 10 min at 4 °C,
and 200 µl of supernatant was divided into aliquots for subsequent 10-fold
dilution in ChIP dilution buffer (0.01% SDS, 1% Triton X-100, 1.2
mM EDTA, pH 8.0, 16.7 mM Tris-HCl, pH 8.0, 167
mM NaCl). To test the amount of input DNA for each sample, 20 µl
of diluted aliquot was saved for further processing in parallel with all other
samples at the reversal of cross-linking step. Each 2 ml of chromatin samples
were precleared with 60 µl of 50% slurry of protein A-agarose (v/v)
(Peptron Co., Daejeon, Korea) containing 200 µg/ml herring sperm DNA
for1hat4 °Con a rotating wheel, after which the beads were pelleted, and
the supernatant was transferred to a new tube. The 15 µg of anti-SREBP-1
IgG or preimmune IgG were added to precleared chromatin sample and incubated
for 1218 h at 4 °C on a rotating wheel. Immune complexes were
collected with 60 µl of 50% slurry of protein A-agarose, 200 µg/ml
herring sperm DNA, while rotating for 3 h at 4 °C, followed by
centrifugation at 1000 rpm for 1 min at 4 °C. The beads were washed for 5
min in low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA,
20 mM Tris-HCl, pH 8.0, 150 mM NaCl), high salt wash
buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM
Tris-HCl, pH 8.0, 500 mM NaCl), LiCl wash buffer (0.25 M
LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10
mM Tris-HCl, pH 8.0), and twice with TE buffer. Chromatin complexes
were eluted from the beads in 30 min with 400 µl of elution buffer (1% SDS,
0.1 M NaHCO3) at room temperature. To reverse the
cross-linking, 200 mM NaCl was added, and the samples were
incubated at 65 °C for 4 h. To digest proteins, samples were incubated at
45 °C for 90 min after the addition of the following: 10 mM
EDTA, 40 mM Tris, pH 6.5, 50 µg/ml proteinase K. Samples were
extracted twice with phenol/chloroform/isoamyl alcohol (25:24:1), and DNA was
precipitated with 20 µg of glycogen and 2 volumes of 100% ethanol. The
pellets were collected by centrifugation for 15 min at 4 °C. Samples were
resuspended in 100 µl of deionized water and stored at 80 °C.
Four microliters of input control or ChIP samples were used as a template in
PCR using the primer sets for ACC promoter I
(5'-TGCCACTCAGTGCCTTGAAGGTTA-3',
5'-TTTCAAGCTCCTCTGTGGCT-3') or ACC
promoter II
(5'-CCGAGTACTGGCCAAGCCCCT-3',
5'-TCACTGGGGACGTGGCCGCCA-3'), respectively. The cycle numbers of
PCR were determined, where the amplifications of target DNA were dependent on
the amounts of samples. PCR products were subjected to electrophoresis in a 2%
agarose gel, and visualized by ethidium bromide staining.
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RESULTS |
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5'-Untranslated Regions for ACC mRNA in
Liver and the Responsiveness of ACC
Promoters to
SREBP-1Human ACC
gene is known to be controlled by
two kinds of promoters: P-I and P-II (Fig.
2B) (11).
To address which of these two promoters directs the expression of the
ACC
gene in liver, we have analyzed the 5'-untranslated
region of ACC
transcripts from rat liver by RNase protection
assays using cRNA probes covering parts of exon 1a (90 nucleotides), or exon
1b (52 nucleotides), which extends to exon 2 (69 nucleotides). Each probe is
designated as probe I and probe II for exon 1a and exon 1b, respectively, as
shown in Fig. 2A. The
lengths of the original probes were shown in reactions without adding RNase
A/T1 mixture (Fig. 2A,
lanes 1 and 8) and the complete digestion of probes in
reactions without adding total RNA (Fig.
2A, lanes 2 and 9). The protected
fragments of both probes from digestion with RNase were markedly increased in
refed rat livers, coinciding with the result of Northern blot analysis of
ACC
mRNA. The protected fragments of probe I appeared around 69
nucleotides in size corresponding to the exon 2 region of the probe
(Fig. 2A, lanes
36), whereas those of probe II showed 2 major bands around 120
nucleotides representing exon 1b-exon 2
(Fig. 2A, lanes
1013). These data suggest that promoter II mainly drives the
expression of ACC
in rat liver and is activated by food intake.
Most lipogenic enzymes, such as fatty acid synthase, ATP citrate-lyase, and
ACC
, were induced in liver by the intake of fat-free high-carbohydrate
diet, and SREBP1c is reported to be a main mediator
(1618).
Thus, we have tested the effects to SREBP-1 on ACC
promoters.
Overexpression of SREBP-1a markedly activated only promoter II of both human
and rat ACC
genes, but not promoter I
(Fig. 2C). These data
suggests that promoter II is the principal promoter responsible for regulated
expression of ACC
by SREBP-1 in rat liver.
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Localization of SREBP-1 Responsive Region in Promoter IITo
define the region mediating SREBP-1 responsiveness, the transient transfection
assays were performed with serial deletion constructs of human
ACC promoter II (Fig.
3). Co-transfection of pcSREBP-1a remarkably increased the
promoter activity. The deletion of the sequences up to nucleotide 93
did not change the SREBP-1 responsiveness of the promoter, even with a gradual
increase in the activity of the promoter. However, deletion of the sequences
up to nucleotide 38 markedly suppressed the promoter activity driven by
SREBP-1 near to the basal activity of the promoter II. The deletion of the
region from 38 to 18, including the TATA-like element
(32/28), almost completely abolished the basal promoter
activity. This data suggests that the region from 93 to 38 plays
an important role in mediating responsiveness of ACC
promoter
II to SREBP-1.
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Identification of SREBP-1 Response Elements in Promoter II of
ACC GeneFor the purpose of identifying the SREBP-1
binding region in the human ACC
promoter II, DNase I
foot-printing assay was performed (Fig.
4). The region from 64 to 49 was protected from
DNase I digestion by recombinant SREBP-1, strongly suggesting the binding of
SREBP-1. The region around the footprinted sequences is highly conserved
between human and rat (Fig.
5A), and two potential elements for SREBP binding in
human sequences, denoted as SRE1 (62/54) and SRE2
(52/44) were predicted by sequence analysis. In rat, SRE1 is
highly conserved and three overlapping consensus sequences were predicted in
SRE2 locus. For EMSA, the probes (93/21), containing wild and
mutated sequences on SRE1 and/or SRE2 denoted in
Fig. 5A, were
generated by PCR with the same 32P-labeled antisense primer
(40/21) and cold sense primer (93/74) using
ACC
promoter-reporter constructs with/without mutations.
Because the specific activities of all probes, including wild and mutant
probes, were the same, the differences in intensities between shifted bands in
EMSA might be caused by differences in the affinities of SREBP-1 to probes.
The probe, containing intact SRE1 and SRE2, formed a complex with SREBP-1,
resulting in the single shifted band (Fig.
5B). The mutation of SRE1 (mSRE1) severely decreased the
SREBP-1 binding, but the mutation of SRE2 (mSRE2) slightly suppressed the
complex formation with SREBP-1. The double mutation of SRE1 and SRE2 (mSRE1 +
2) almost completely inhibited the SREBP-1 binding. These findings indicate
that SREBP-1 has strong affinity to SRE1, and weak to SRE2.
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The region from 73 to 68 (CCCTCC) in rat promoter matches perfectly with CT motif reported as the binding site for Sp1 and Sp3 in a variety genes, such as ATP citrate-lyase, lipoprotein lipase, and low density lipoprotein receptor genes (2729). In human sequence, this region (71/66) is also highly conserved except for the A to T nucleotide substitution. To address the binding of Sp1 to this promoter region, we carried out EMSA with the probe from 82 to 53 of the human sequence. This probe produced one major complex by incubation with rat liver nuclear extract, and the complex was supershifted by antibody against Sp1 (Fig. 5C). The mutation of CCCACC to CCCAAA abolished the Sp1 binding (data not shown). These data suggest that this locus immediately upstream of SRE1 binds Sp1 in both human and rat promoters.
Next, we tested the effect of these mutations on SREBP-1 responsiveness of
promoter II (Fig. 6A).
The stimulation of promoter activity of phP-II(93/+65) by SREBP-1
was inhibited about 88% by the mutation in SRE1 (mSRE1). The mutation in SRE2
also resulted in marked inhibition (63%) of SREBP-1-mediated induction. The
double mutation of SRE1 and SRE2 almost completely suppressed SREBP-1
activation. These data indicated that SRE1 and SRE2 play a critical role in
SREBP-1-mediated induction of ACC
promoter. Interestingly, the
mutation at the Sp1 binding site markedly suppressed basal promoter activity
and SREBP-1-mediated activation (Fig.
6A), even though the deletion construct
(phP-II
38/+65) devoid of this region did not show any decrease of
basal promoter activity (Fig.
3). The reason why the basal activity is suppressed by mutation at
the Sp1 binding site could not be explained by far in the present study. To
confirm the role of the Sp1 binding for SREBP-1 stimulation in
ACC
promoter II, the transient transfection assay in the SL2
insect cell line was performed (Fig.
6B). The overexpression of Sp1 markedly increased
reporter gene expression in the phP-II
93/+65 construct, whereas
the mutation of the Sp1-binding consensus (mSp1) inhibited this stimulation.
The overexpression of SREBP-1a also increased luciferase activities in both
wild and mSp1 constructs, but overexpression of SREBP-1c did not. The
overexpression of Sp1 with SREBP-1a or SREBP-1c showed the synergistic
activation of the reporter gene in the wild type reporter construct, but did
not in the mSp1 mutant construct. These data indicated that Sp1 binding to
71/66 is essential for SREBP-1-mediated stimulation of
ACC
promoter II.
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SREBP-1 Binding in ACC Promoter II Was Increased by
Refeeding High-carbohydrate Diet in VivoFinally, we have performed
ChIP assay to observe whether SREBP-1 occupancy to ACC
gene
promoter II is actually influenced in rat livers by dietary status. The
hepatic chromosomal DNA was cross-linked to the transcription factors binding
to them by perfusion of DMEM containing formaldehyde into the hepatic portal
vein. Antibody specific to SREBP-1 was used to immunoprecipitate the
fragmented chromatin, and then the specific portion of the ACC
gene was amplified by PCR (Fig.
7A). The association of SREBP-1 to promoter I was not
detectable in both the fasted and refed states
(Fig. 7C). In the
refed group, the amounts of amplified promoter II sequence were increased
proportionally to the amounts of chromatin DNA immunoprecipitated by
anti-SREBP-1 until 33 cycles of PCR, whereas the amplifications were not
detected in the fasted group (Fig.
7B). The amounts of input DNAs in all samples, which were
used for immunoprecipitation, were almost the same, referring to similar
patterns of amplification in both fasted and refed groups. These results
indicate that the occupancy on the proximal region of promoter II by SREBP-1
is drastically regulated in vivo by feeding status and this
regulation might mediate the control of ACC
gene expression in
liver by diet.
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DISCUSSION |
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It was previously reported that the human ACC gene has the
two promoters, designated as promoter I and II. Human promoter II responded
well to muscle regulatory factors, such as MyoD, myogenin, and MRF4, which
were proven to control the muscle-specific gene expression
(25,
34). In the present study,
RNase protection assays revealed that promoter II is a principal promoter in
liver and activated by the intake of high-carbohydrate diet.
ACC
promoter II is activated by overexpression of SREBP-1,
which is known to play a critical role in dietary control of lipogenic enzyme
genes
(1418).
However, promoter I does not respond to SREBP-1 at all. DNase I foot-printing
and EMSA revealed the 2 potential SREBP-1-binding sites (SRE1,
62/54 and SRE2, 52/44) and Sp1-binding site (CT
motif, 71/66). SREBP-1 showed strong affinity to SRE1 and the
mutation in SRE1 or SRE2 resulted in 88 and 63% reduction in SREBP-1
activation. The mutations of both SRE1 and SRE2 almost completely suppressed
SREBP-1 activation. These data indicate that both SRE1 and SRE2 play a major
role in SREBP-1 action on ACC
promoter II. The mutation of the
CT motif inhibiting Sp1-binding, suppressed SREBP-1 activation in animal cells
and inhibited the synergistic activation of ACC
promoter II by
Sp1 and SREBP-1 in insect cells. From these data, we assumed that Sp1 binding
to 71/66 is needed by SREBP-1 action at SRE1 and SRE2 for the
activation of the ACC
gene. These results support the reports
of Sp1 and SREBP-1 synergism in the transcription in many other genes
(29,
3537).
All of the in vitro experiments indicate that the region from
71 to 44, containing the binding elements for Sp1 and SREBP-1,
plays an important role in SREBP-1-mediated activation of the
ACC
gene. ChIP assay in vivo showed that the occupancy
of SREBP-1 is induced in ACC
promoter II by the intake of a
high-carbohydrate diet. This result suggests that SREBP-1c binding to
ACC
promoter II is regulated by dietary status and may be
responsible for activation of ACC
gene expression. In the present study,
we first showed that the expression of the ACC
gene is
regulated by nutritional status at the transcriptional level, and SREBP-1c
binding to functional SREs plays the critical role in this control.
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FOOTNOTES |
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|| To whom correspondence and reprint requests should be addressed: Dept. Biochemistry and Molecular Biology, Yonsei University College of Medicine, 134 Shinchondong Seodaemungu, Seoul, 120-752, Korea. Tel.: 82-2-361-5184; Fax: 82-2-312-5041; E-mail: kyungsup59{at}yumc.yonsei.ac.kr.
1 The abbreviations used are: ACC, acetyl-CoA carboxylase; SREBP, sterol
regulatory element-binding protein; EMSA, electrophoretic mobility shift
assay; ChIP, chromatin immunoprecipitation assay; DMEM, Dulbecco's modified
Eagle's medium; MEM, minimal essential medium; MOPS,
4-morpholinepropanesulfonic acid.
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REFERENCES |
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