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INTRODUCTION |
In livers of birds and mammals, consumption of carbohydrate in
excess of the immediate energy requirements of the animal increases the
expression of enzymes involved in the conversion of carbohydrate to
triacylglycerols (1). These enzymes include those in glycolysis, such
as glucokinase and L-pyruvate kinase; those in fatty acid synthesis, such as acetyl-CoA carboxylase-
(ACC)1 and fatty acid
synthase; those in NADPH production, such as malic enzyme; and those in
triacylglycerol synthesis, such as glycerol-3-phosphate acyltransferase. For most of the lipogenic enzymes studied, the induction of enzyme levels by dietary carbohydrate is mediated primarily by changes in gene transcription (1).
Several signaling pathways are involved in mediating the stimulatory
effects of dietary carbohydrate on lipogenic gene transcription in
liver. Increased insulin secretion and glucose metabolism are two
important signals mediating this response (1). Another factor signaling
changes in carbohydrate status is the active form of thyroid hormone,
3,5,3'-triiodothyronine (T3). Ingestion of a high carbohydrate meal
stimulates a rapid increase in the secretion of thyroxine from the
thyroid gland and the conversion of thyroxine to T3 in extrathyroidal
tissues (2, 3). The resulting increase in T3 concentration in liver
activates the transcription of the genes for ACC (4), fatty acid
synthase (5), malic enzyme (6), and Spot 14 (S14) (7). Ingestion of carbohydrate also increases the levels of nuclear T3 receptors (TRs)
in liver (8). This phenomenon may also contribute to the stimulation of
lipogenic gene transcription by dietary carbohydrate. A role of thyroid
hormones in mediating the nutritional regulation of lipogenic enzyme
expression is supported by the observation that hypothyroidism in high
carbohydrate-fed animals causes a marked reduction in expression of
ACC, fatty acid synthase, malic enzyme, and S14 in liver
(9-11).
ACC catalyzes the pace-setting step of the fatty acid synthesis pathway
(1). There are two ACC isoforms that are encoded by distinct genes.
ACC
(280 kDa) is the major isoform observed in heart and skeletal
muscle, where it is thought to function primarily in the regulation of
-oxidation of fatty acids (12). ACC (265 kDa) is the principal
isoform expressed in tissues that exhibit high rates of fatty acid
synthesis such as liver, adipose tissue, and mammary gland. ACC
expression is induced by nutritional and hormonal factors that promote
high rates of fatty acid synthesis. For example, feeding previously
starved chickens a high carbohydrate, low fat diet stimulates an
11-fold increase in transcription of the ACC gene in liver (13).
This effect is partially reproduced in primary cultures of chick embryo
hepatocytes (CEH) by manipulating the concentration of T3 in the
culture medium. The addition of T3 stimulates a 7-fold increase in
ACC transcription in CEH (4).
The ACC gene is transcribed from two promoters, resulting in
mRNAs with heterogeneity in their 5'-untranslated region (14). These ACC promoters are designated promoter 1 and promoter 2. The
increase in total ACC mRNA abundance caused by high carbohydrate feeding in chickens and by T3 in CEH is mediated by alterations in the
activities of both promoter 1 and promoter 2, with the latter promoter
playing a quantitatively greater role in mediating these responses
(15). Additional studies have shown that the stimulatory effect of T3
on promoter 2 activity is mediated by a T3 response element (T3RE) with
unique functional and protein binding properties (16). This T3RE
enhances ACC promoter activity both in the absence and presence of
T3, with a greater stimulation observed in the presence of T3. The
results of DNA binding analyses with nuclear extracts from CEH suggest
that the T3-independent enhancer activity of the ACC T3RE is
mediated by the binding of protein complexes containing LXR·RXR
heterodimers and that the increase in enhancer activity caused by T3
treatment is mediated by the binding of a different set of protein
complexes. One of these complexes contains TR·RXR heterodimers, and
another contains LXR·RXR heterodimers. Based on these observations,
we have hypothesized that the ACC T3RE not only mediates T3
regulation of ACC transcription but also ensures a basal level of
ACC expression for the synthesis of structural lipids in cell membranes.
In addition to regulation by nutrients and hormones, ACC
transcription is controlled by tissue- or cell-specific factors. For
example, ingestion of carbohydrate has little or no effect on ACC
transcription in heart, kidney, brain, and skeletal muscle (13). Cell
type-dependent differences in the regulation of ACC are
also observed in cells in culture. T3 regulation of ACC
transcription in chick embryo fibroblasts (CEF) is markedly diminished
compared with that in CEH (17). The mechanisms responsible for cell
type-dependent differences in the regulation of ACC
transcription remain to be determined.
Studies analyzing the regulation of other T3-responsive genes have
shown that optimal T3 regulation of transcription is dependent not only
on the presence of a T3RE but also the presence of accessory elements
that bind proteins that are distinct from nuclear hormone receptors
(18-23). In isolation, these accessory elements are devoid of
T3-responsive activity; they act by enhancing the degree of T3
responsiveness initiated by the T3RE. Some T3 accessory elements bind
proteins that are differentially expressed in different cell types and
thus play a key role in conferring cell type-dependent differences in T3 responsiveness (18, 22, 23). There is little
information on the mechanism by which T3 accessory elements modulate T3
regulation of transcription.
In the present study, we have identified an accessory element that
enhances T3 responsiveness of ACC promoter 2 in CEH. This element is
located 5 bp downstream of the ACC T3RE and binds sterol regulatory
element-binding protein-1 (SREBP-1). We have developed data suggesting
that SREBP-1 enhances T3 regulation of ACC transcription by directly
interacting with TR·RXR heterodimers bound to the ACC T3RE. In
addition, we provide evidence that alterations in SREBP expression play
a role in mediating cell type-dependent differences in T3
regulation of ACC transcription.
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EXPERIMENTAL PROCEDURES |
Plasmids--
DNA fragments used to construct reporter plasmids
were named by designating the 5' and 3' ends of each fragment relative
to the transcription start site of promoter 2 of the ACC gene.
p[ACC
108/+274]CAT has been described previously (16). This
construct contains ACC sequences between 108 and +274 bp ligated
upstream of the CAT gene in KSCAT (24). A block mutation of the SRE-1
between 79 and 72 bp was introduced into p[ACC108/+274]CAT
using a polymerase chain reaction-based strategy (25). pBLCAT2 (pTKCAT) was obtained from B. Luckow and G. Schutz (German Cancer Research Center) (26). The cryptic activator protein-1 site located 5' of the
multiple cloning site in pBLCAT2 (27) was removed by excising the
NdeI/HindIII fragment from this plasmid followed by religation. p[ACC108/82]TKCAT, p[ACC108/66]TKCAT,
p[ACC-84/66]TKCAT, and pTKCAT constructs containing mutations in
the 108 to 66 bp fragment were made by first annealing
complementary synthetic oligonucleotides containing ACC sequences
and then inserting them into SphI and SalI site
5' of the thymidine kinase (TK) promoter in pTKCAT.
A full-length cDNA for chicken SREBP-1 was obtained by screening a
chicken liver cDNA library (Stratagene) using a human SREBP-1 cDNA probe (nucleotides 721-1103 relative to the start site of translation) and by 5'-RACE.2
The N-terminal amino acid sequence of this chicken SREBP-1
(GenBankTM accession number AY029224) more closely
resembles the 1a isoform than the 1c isoform described for mammalian
species (28). The data from RNase protection analyses indicate that
other forms of SREBP-1 containing variations in the N terminus are not
expressed in chicken cells. An expression plasmid encoding the mature
form of chicken SREBP-1 was developed by subcloning a SREBP-1 cDNA fragment encoding amino acids 1-464 into pSV-SPORT1 (Invitrogen) to
form pSV-SPORT1-SREBP-1 (1-464). N-terminal and C-terminal truncations
of the mature form of SREBP-1 were generated by PCR. PCR products
encoding SREBP-1 polypeptides (78-464, 108-464, 238-464, 1-266,
1-340, 1-389, and 300-464) were subcloned into pSV-SPORT1. To
generate a plasmid that expresses a fusion protein containing glutathione S-transferase (GST) linked to the mature form of
chicken SREBP-1, a SREBP-1 cDNA fragment encoding amino acids
1-464 was subcloned into pGEX-2T (Amersham Biosciences). The
structures of all reporter plasmids and expression plasmids were
confirmed by restriction enzyme mapping and nucleotide sequence analyses.
pGEM-3Zf()-based plasmids that express full-length chicken TR
(1-408) and N-terminal and C-terminal deletion derivatives of chicken
TR (1-118, 1-157, 21-408, 51-408, and 120-408) have been
previously described (19). The construction of a plasmid that expresses
a fusion protein containing GST linked to full-length TR
(pGEX-2T-TR (1-408)) has been described (19). The cDNAs for human RXR and human LXR were provided by R. Evans (Salk Institute) and D. Mangelsdorf (University of Texas Southwestern Medical Center), respectively. Expression plasmids for RXR and LXR were developed by subcloning the cDNAs for these receptors into pSV-SPORT1.
Cell Culture and Transient Transfection--
Primary cultures of
CEH were prepared as previously described (29) and maintained in
serum-free Waymouth's medium MD705/1 containing 50 nM
insulin (gift from Eli Lilly Corp.) and 1 µM corticosterone. CEH were incubated on 60-mm Petri dishes (Fisher) at
40 °C in a humidified atmosphere of 5% CO2 and 95%
air. The cells were transfected 6 h after plating, using 20 µg
of Lipofectin (Invitrogen), 1.3 µg of p[ACC108/+274]CAT, or an
equimolar amount of another reporter plasmid and pBluescript KS(+) to
bring the total amount of transfected DNA to 3.0 µg/plate. At 18 h of incubation, the transfection medium was replaced with fresh medium
with or without T3 (1.5 µM). At 66 h of incubation,
CEH were harvested, and the cell extracts were prepared as described by
Baillie et al. (30). CAT activity (31) and protein (32) were
assayed by the indicated methods. All of the DNAs used in transfection experiments were purified using the Qiagen endotoxin-free kit.
CEF were obtained from SPAFAS, Inc. (Norwich, CT) and were routinely
cultured in DMEM/M199 (Dulbecco's modified Eagle's medium (25 mM glucose) with medium 199 (Invitrogen) in a 1:1 (v/v)
ratio, containing 10,000 units/liter penicillin G, 10 mg/liter
streptomycin sulfate, and 25 µg/liter amphotericin B supplemented
with 5% fetal bovine serum). These cells were transfected with
p[ACC108/+274]CAT or p[ACC108/+274]CAT containing a block
mutation of the SRE-1 using the calcium phosphate method (33). Briefly,
the cells were seeded on T-75 flasks and grown in DMEM/M199 containing
5% fetal bovine serum until 70% confluent. Twenty-four hours before transfection, the medium was changed to DMEM/M199 containing 50 nM insulin and 1 µM corticosterone. This
medium was used throughout the experiment. The cells were transfected
with 15 µg of reporter plasmid. Exposure to the calcium phosphate/DNA
precipitate was for 16 h. Following transfection, the cells were
trypsinized and distributed to 60-mm tissue culture plates. T3 was
added to the medium at this time. After 48 h of incubation, the
cells were harvested, and the extracts were prepared for CAT and
protein assays.
Preparation of Membrane and Nuclear Extracts--
All of the
procedures were carried out at 4 °C. To prevent proteolysis, a
mixture of protease inhibitors (CompleteTM; Roche Molecular
Biochemicals) was included in all the buffers. The nuclear extracts
were prepared from CEH and CEF by a modification of the method
described by Dignam et al. (34). Briefly, CEH from four
100-mm plates or CEF from twelve 100-mm plates were pooled and
centrifuged at 1000 × g for 5 min at 4 °C. The
resulting cell pellet was homogenized in buffer 1 (10 mM
Hepes, pH 7.9, 10 mM KCl, 1.5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol) using a 20 strokes in a Dounce
homogenizer. The homogenate was centrifuged at 1100 × g for 10 min, and the resulting nuclear pellet was washed
once in buffer 1. The nuclear pellet was resuspended in Buffer 2 (20 mM Hepes, pH 7.9, 420 mM NaCl, 25% (v/v)
glycerol, 1.5 mM MgCl2, 1 mM EDTA,
1 mM EGTA, 1 mM dithiothreitol). This suspension was rotated for 30 min and then centrifuged 15,000 × g for 30 min. The resulting supernatant was designated as
the nuclear extract fraction. The membrane extract fraction was
prepared by centrifuging the supernatant of the original 1100 × g spin for 1 h at 100,000 × g. The
resulting membrane pellet was dissolved in Buffer 3 (10 mM
Tris, pH 6.8, 100 mM NaCl, 1% (w/v) SDS, 1 mM
EDTA, 1 mM EGTA, 1 mM dithiothreitol). The
protein content of the nuclear and membrane extracts was determined as
described (32).
Gel Mobility Shift Analysis--
Chicken TR, human RXR,
and chicken SREBP-1 were translated in vitro using the TNT
SP6-coupled reticulocyte lysate system (Promega). Double-stranded
oligonucleotides were prepared by combining equal amounts of the
complementary single-stranded DNA in a solution containing 10 mM Tris, pH 8.0, 1 mM EDTA followed by heating
to 90 °C for 2 min and then cooling to room temperature. The
annealed oligonucleotides were labeled by filling in overhanging 5'
ends using the Klenow fragment of Escherichia
coli DNA polymerase in the presence of
[-32P]dCTP and/or [-32P]dGTP. The
binding reactions were carried out in 20 µl of 20 mM
Tris, pH 7.9, 100 mM KCl, 5 mM
MgCl2, 1 mM EDTA, 1 mM
dithiothreitol, 5% glycerol (v/v), 0.3 mg/ml bovine serum albumin, and
2 µg of poly(dI·dC). A typical reaction contained 20,000 cpm (0.1 ng) of labeled DNA and 5 µl of in vitro translated
proteins and was performed at 37 °C for 30 min. DNA and DNA-protein
complexes were resolved on 6% nondenaturing polyacrylamide gels at
4 °C in 0.5× TBE (45 mM Tris, pH 8.3, 45 mM
boric acid, 1 mM EDTA). Following electrophoresis, the gels
were dried and subjected to storage phosphor autoradiography. For
competition experiments, unlabeled competitor DNA was mixed with
radiolabeled oligomer prior to the addition of nuclear extract. For
antibody supershift experiments, nuclear extracts were incubated with
antibodies for 30 min prior to addition of the oligonucleotide probe. A
mouse monoclonal antibody against SREBP-1 (IgG-2A4) was obtained from
the American Type Tissue Collection (Manassas, VA). P. Chambon
(Strasbourg, France) kindly provided a monoclonal antibody against RXR.
A polyclonal antibody that recognizes chicken TR (FL-408) was purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The synthetic oligonucleotides that were used as probes in gel mobility shift assays
are listed in Fig. 1A and in the legend to Fig. 6.
Western Blot Analysis--
The proteins in nuclear extract and
membrane fractions were subjected to electrophoresis in 8%
SDS-polyacrylamide gels and then transferred to polyvinylidene
difluoride membranes (Amersham Biosciences) using an electroblotting
apparatus (Owl Scientific). Immunoblot analyses were performed as
described in the Western blotting protocol from Santa Cruz
Biotechnology. Briefly, the blots were blocked in Blotto (5% nonfat
dry milk, 10 mM Tris-HCl, pH 8.0, 150 mM NaCl)
at 4 °C overnight and then incubated with monoclonal antibody
against SREBP-1 (IgG-2A4) or SREBP-2 (IgG-1D2) diluted to 1 µg/ml in
Blotto containing 0.01% (v/v) Tween 20. The results of gel mobility
shift experiments employing in vitro translated proteins or
chicken liver nuclear extracts indicated that IgG-2A4 and IgG-1D2
reacted with the chicken forms of SREBP-1 and SREBP-2, respectively.
After incubation with primary antibody for 1 h at room
temperature, the blots were washed in TBS (10 mM Tris-HCl,
pH 8.0, 150 mM NaCl) containing 0.01% Tween 20. Next, the
blots were incubated with a donkey anti-mouse IgG conjugated to
horseradish peroxidase (Jackson ImmunoResearch) diluted 1:10,000 in
Blotto, 0.01% Tween 20 for 1 h at room temperature. After washing with TBS, 0.01% Tween 20, antibody-protein complexes on blots were
detected using enhanced chemiluminescence (Amersham Biosciences). Chemiluminescence on the blots was visualized using a FluorChem 8000 imager (Alpha Innotech Corporation), and signals for the precursor and
mature forms of SREBP-1 and SREBP-2 were quantified using FluorChem
V200 software.
RNase Protection Assay--
cDNA fragments for chicken
SREBP-1, SREBP-2, and -actin were subcloned into the
HindIII and BamHI sites of pBluescript
KS+ (Stratagene). The SREBP-1 cDNA fragment (300 bp)
contained sequences between nucleotides 829 and 1128 relative to the
start site of translation. The SREBP-2 cDNA fragment (460 bp) was
isolated using reverse transcriptase-PCR. The primers used to generate
this fragment were based on a SREBP-2 mRNA sequence reported in the
GenBankTM data base (accession number AJ310769). The 5'
boundary of this SREBP-2 fragment was defined by the primer sequence,
5'-agcagggcaaccataagcTGA-3', and the 3' boundary was defined by the
primer sequence, 5'-CAGCCAAACCATCCACCTGT-3'. The -actin cDNA
fragment (256 bp) contained sequences between nucleotides 4 and 259 relative to the start site of translation. Each subclone was linearized
with BamHI, and antisense RNA was transcribed with
[-32P]CTP (specific activity, 3000 Ci/mmol) using
bacteriophage T3 RNA polymerase (Promega). Labeled RNAs were purified
by polyacrylamide gel electrophoresis. RNA was extracted from CEH and
CEF by the guanidinium thiocyanate/phenol/chloroform method (35). RNase protection assays were performed using the RPA II kit (Ambion). Total
RNA (20 µg) was hybridized to 4 × 104 cpm of
32P-labeled RNA at 45 °C for 16 h. The sample was
then digested with a mixture of RNase A and RNase T. Protected
fragments were separated on 8 M urea, 5% polyacrylamide
gels. The gels were dried and subjected to storage phosphor
autoradiography. The images were quantified using ImageQuaNT software
by Molecular Dynamics.
Protein-Protein Interaction--
GST and GST fusion proteins
were expressed in E. coli (BL21, pLysS) and purified using
standard techniques (36). Briefly, the bacteria were transformed with
pGEX-2T or recombinant pGEX-2T plasmids expressing GST fusion proteins.
Overnight bacterial cultures in ampicillin (250 µg/ml) were diluted
1:100 into 250 ml of Luria broth and grown at 37 °C to an
A600 of 1.0 before induction with 1 mM isopropyl-1-thio--D-galactopyranoside for
60 min. The cells were pelleted and resuspended in 5 ml of buffer A (25 mM Hepes, pH 7.9, 50 mM KCl, 6% (v/v)
glycerol, 5 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol, 0.05% Triton X-100). The cells were
lysed on ice by sonication and centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant was mixed for
1 h at 4 °C on a rotator with 0.5 to 1 ml of 50%
glutathione-Sepharose beads (Amersham Biosciences) that were preswollen
in buffer A. After absorption, the beads were collected by
centrifugation at 4 °C and washed three times with 1 ml of buffer A. The fusion proteins coupled to the glutathione-Sepharose beads were
stored at 4 °C as 50% (v/v) slurry in buffer A. The concentrations
and sizes of GST and GST fusion proteins were estimated by SDS-PAGE,
using a known quantity of molecular weight standards.
L-[35S]Cysteine- or
L-[35S]methionine-labeled proteins were
prepared by using TNT reticulocyte lysates (Promega). Approximately 2.5 × 104 to 5 × 104 cpm of
35S-labeled protein was incubated with 300 ng of GST fusion
protein immobilized on glutathione-Sepharose beads in 300 µl of
buffer A for 1 h at 4 °C on a rotator. Where indicated 1 µM T3, 9-cis-retinoic acid, or
22(R)-hydroxycholesterol was included in the binding reaction mixture. In experiments examining the effects of DNA binding
on the interaction between TR and SREBP-1 (see Fig. 6), oligonucleotides (30 ng) containing the ACC T3RE and/or ACC SRE-1
were included in the binding reaction. The beads were collected by
centrifugation at 4 °C and washed three times with 1 ml of buffer A. The bound proteins were eluted with SDS gel loading buffer and analyzed
by SDS-PAGE followed by storage phosphor autoradiography. 35S-Labeled proteins were analyzed by electrophoresis and
storage phosphor autoradiography to ensure that similar levels of input radioactivity of the labeled protein were used in the GST binding assays.
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RESULTS |
A SRE-1 Located Immediately Downstream of a Strongly Active T3RE in
the ACC Gene Augments T3 Regulation of Transcription in a Cell
Type-specific Manner--
Previous studies with native thyroid
hormone-responsive genes have shown that optimal T3 regulation of
transcription requires the presence of one or more accessory
factor-binding sites in addition to a T3RE (18-23). These accessory
elements are usually located within 100 bp of the T3RE. In previous
work analyzing the T3 regulation of ACC promoter 2 in CEH,
5'-deletion analyses failed to detect a positive accessory element
upstream of the strongly active T3RE between 101 and 86 bp (16).
Deletion analyses also failed to detect TR accessory sequences between +31 and +274 bp. We investigated the possibility that sequences immediately downstream of the ACC T3RE contained a TR accessory element. Sequence comparison analysis of the region between 86 and
+31 bp revealed the presence of a 10-bp element (80 and 71 bp) that
perfectly matched the SRE-1 identified in the rat, hamster, and frog
genes for the low density lipoprotein receptor (37). Transient
transfection experiments were performed to determine the effects of the
SRE-1 on T3 regulation of ACC promoter 2 in CEH. In hepatocytes
transfected with a reporter construct containing ACC sequences
between 108 and +274 linked to the CAT gene, T3 stimulated a
14.3-fold increase in CAT activity (Fig.
1B). Mutation of sequences
between 79 and 72 bp (SRE-1 mut) in the context of the 108 to
+274 ACC fragment caused a 53% decrease in T3 responsiveness. This
effect was mediated by a reduction in promoter activity in the presence
of T3. These data indicate that T3 regulation of ACC transcription
in hepatocytes is enhanced by the presence of a SRE-1 located 5 bp
downstream of a strongly active T3RE in the proximal region of ACC
promoter 2.
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Fig. 1.
A SRE-1 between 80 and 71 bp of the
ACC gene enhances T3 regulation of
ACC transcription in CEH but not in CEF.
A, sequence of the chicken ACC gene between 108 and
66 bp. The hexameric half-sites comprising the T3RE are indicated by
arrows. The SRE-1 is boxed. The sequence of a
block mutation of the SRE-1 (SRE-1 mut) is shown underneath
the SRE-1. B and C, p[ACC108/+274]CAT or a
construct containing a block mutation of the SRE-1 (SRE-1
mut) in the context of p[ACC108/+274]CAT was transiently
transfected into CEH and CEF as described under "Experimental
Procedures." After transfection, the cells were treated with or
without T3 for 48 h. The cells were then harvested, extracts were
prepared, and CAT assays were performed. Left panels, the
constructs used in these experiments. The numbers indicate
the 5' and 3' boundaries of ACC DNA in nucleotides relative to the
transcription initiation site of promoter 2. Right panels,
CAT activity of cells transfected with p[ACC108/+274]CAT and
treated with T3 was set at 100, and the other activities were adjusted
proportionately. The fold stimulation by T3 was calculated by dividing
CAT activity for cells treated with T3 (+T3) by that for
cells not treated with T3 (T3). The fold responses were
calculated for individual experiments and then averaged. The results
are the means ± S.E. of five experiments. The results of
experiments performed with CEH and CEF are shown in B and
C, respectively. The asterisk in B
indicates that the fold stimulation by T3 was significantly lower than
that of cells transfected with p[ACC108/+274]CAT
(p < 0.05).
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T3 regulation of the ACC gene in nonhepatic cells such as CEF is
markedly diminished relative to that of CEH (17). Previous studies with
other T3-responsive genes have shown that TR accessory proteins play an
important role in mediating cell type-dependent differences
in T3 regulation of transcription (18-23). Transient transfection
experiments were performed with CEF to determine whether the SRE-1
between 80 and 71 bp is involved in mediating differences in T3
responsiveness of the ACC gene between CEH and CEF. In CEF
transfected with p[ACC108/+274]CAT, the T3-induced stimulation in
CAT activity (2.4-fold) was substantially lower than that observed in
CEH (14.3-fold) (Fig. 1, B and C). Mutation of
the SRE-1 in the context of p[ACC108/+274]CAT had no effect on T3
responsiveness in CEF (Fig. 1C). These data contrast with those for CEH, demonstrating that mutation of the SRE-1 causes a marked
reduction in T3 responsiveness (Fig. 1B). The decreased ability of the SRE-1 to stimulate T3 responsiveness in CEF relative to
CEH suggests that this element plays a role in conferring cell type-dependent differences in T3 regulation of ACC
transcription. Other sequences within the 108 to +274 fragment also
contribute to differences in T3 responsiveness between CEH and CEF,
because mutation of the SRE-1 did not eliminate differences in T3
responsiveness between CEH and CEF.
We next asked whether the functional interaction between the ACC
SRE-1 and ACC T3RE in CEH required the presence of additional cis-acting sequences. To address this question, we
determined whether the ACC SRE-1 could function alone to enhance T3
regulation conferred by the ACC T3RE. CEH were transfected with
constructs containing ACC DNA fragments linked to the minimal
promoter of the herpes simplex virus TK gene. The TK promoter alone was
unresponsive to T3 in CEH (Fig. 2).
Appending a DNA fragment containing the ACC T3RE (108 to 82 bp)
to TK-CAT caused a 9.6-fold increase in T3 responsiveness (compare fold
stimulation by T3 of p[ACC108/82]TKCAT with that of TKCAT). A
substantially greater increase in T3 responsiveness was observed
(20-fold) when a DNA fragment containing both the T3RE and SRE-1 (108
to 66 bp) was linked to TKCAT. The increase in T3 responsiveness
caused by the SRE-1 was due to an increase in promoter activity in the
presence of T3. This increase in T3 responsiveness was not due to
changes in the spacing between the T3RE and the TK promoter, because T3
responsiveness of a construct containing a block mutation of the SRE-1
(SRE-1 mut) in the context of p[ACC108/66]TKCAT was similar to
that of p[ACC108/82]TKCAT. These data indicate that the
stimulation of T3-induced ACC transcription by the SRE-1 is mediated
by direct interactions between the SRE-1 and T3RE. Appending a DNA
fragment containing the SRE-1 alone (84 to 66 bp) to TKCAT had no
effect on promoter activity in the absence or presence of T3. This
finding indicates that the SRE-1 requires the presence of the T3RE to
be transcriptionally active. This conclusion is consistent with results
from previous 5'-deletion analyses in the context of
p[ACC-4900/+274]CAT demonstrating that deletion of ACC sequences
from 84 bp to 59 bp has no effect on ACC transcription in the
absence or presence of T3 (16).
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Fig. 2.
The ACC SRE-1 alone
enhances T3 regulation directed by the ACC
T3RE. Fragments of the ACC gene containing the T3RE
and/or SRE-1 were linked to the minimal TK promoter in TKCAT. CEH were
transiently transfected with these constructs and treated with or
without T3 as described in the legend of Fig. 1 and under
"Experimental Procedures." Left panel, constructs used
in these experiments. The numbers indicate the 5' and 3'
boundaries of ACC DNA relative to the transcription initiation site
of promoter 2. The ACC sequence between 108 and 66 bp is shown
in Fig. 1A. Right panel, CAT activity in CEH
transfected with p[ACC108/82]TKCAT and treated with T3 was set at
100, and the other activities were adjusted proportionately. The fold
stimulation by T3 was calculated as described in the legend to Fig. 1.
The results are the means ± S.E. of six experiments. The
asterisk indicates that the fold stimulation by T3 for
p[ACC108/66]TKCAT was significantly higher than any other
construct (p < 0.05).
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SREBP-1 Physically Interacts with TR·RXR Heterodimers--
SREs
bind a class of basic helix-loop-helix-leucine zipper (bHLH-Zip)
factors referred to as sterol regulatory element-binding proteins
(SREBPs) (38). SREBPs are synthesized as 125-kDa precursor proteins
that are anchored to the endoplasmic reticulum. To become transcriptionally active, precursor SREBP must be translocated to the
Golgi, where it encounters two proteases that act in sequence to
release the N-terminal segment of SREBP referred to as mature SREBP
(39). Mature SREBP is the protein that enters the nucleus and binds the
SRE of target genes. In chickens and mammals, SREBPs are expressed from
two distinct genes designated as SREBP-1 and SREBP-2 (38, 40).
Previous studies employing transgenic animals indicate that
SREBP-1 is more effective than SREBP-2 in modulating triacylglycerol synthesis, whereas SREBP-2 is more effective than SREBP-1 in modulating cholesterol synthesis (41, 42).
DR-4-type (half-sites arranged as direct repeats with a 4-bp
spacer) T3REs bind TR·RXR heterodimers in a specific spatial arrangement in which RXR contacts the more 5' half-site and TR contacts
the more 3' half-site (43). If TR·RXR directly interacts with mature
SREBP on the ACC gene, then flipping the orientation of the T3RE may
alter the ability of the SRE-1 to enhance T3 regulation of
transcription. To investigate this possibility, CEH were transfected with reporter constructs containing the ACC T3RE in the native or
flipped orientation. In CEH transfected with p[ACC108/66]TKCAT containing the T3RE in the native orientation, mutation of the SRE-1
inhibited T3 responsiveness by 56% (Fig.
3). In contrast, mutation of the SRE-1
had no effect on T3 responsiveness when the orientation of the T3RE in
p[ACC108/66]TKCAT was flipped (compare fold stimulation by T3 of
p[ACC108/66]TKCAT containing T3RE flip with that of
p[ACC108/66]TKCAT containing T3RE flip and SRE-1 mut). These data
suggest that SREBP interacts with TR·RXR in an
orientation-dependent manner to enhance T3 regulation of the ACC gene.
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Fig. 3.
The ability of the ACC
SRE-1 to enhance T3 responsiveness requires the presence of the
ACC T3RE in its native orientation.
Fragments of the ACC gene containing the T3RE in the native or
flipped orientation were linked to the minimal TK promoter in TKCAT.
CEH were transiently transfected with these constructs and treated with
or without T3 as described in the legend of Fig. 1 and under
"Experimental Procedures." Left panel, constructs used
in these experiments. The numbers indicate the 5' and 3'
boundaries of ACC DNA relative to the transcription initiation site
of promoter 2. The orientation of the half-sites comprising the T3RE
are indicated by the arrows. The ACC sequence between
108 and 66 bp is shown in Fig. 1A. Right
panel, CAT activity in CEH transfected with
p[ACC108/66]TKCAT and treated with T3 was set at 100, and the
other activities were adjusted proportionately. The fold stimulation by
T3 was calculated as described in the legend to Fig. 1. The results are
the means ± S.E. of five experiments. The asterisk
indicates that the fold stimulation by T3 for p[ACC108/66]TKCAT
(T3RE in the native orientation) was significantly higher than
that of p[ACC108/66 SRE-1mut]TKCAT (T3RE in the native
orientation) (p < 0.05).
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The close proximity of the SRE-1 and T3RE on the ACC gene coupled
with functional data indicating a direct and orientation-specific interaction between these regulatory elements (Figs. 1-3) led us to
hypothesize that mature SREBP physically interacted with TR·RXR heterodimers to augment T3 regulation of ACC transcription. To investigate this hypothesis, we determined using a pull-down assay whether the mature form of chicken SREBP-1 physically interacted with
TR and RXR. In our initial experiments, we tested the ability of
in vitro synthesized TR and RXR to bind a bacterially
expressed fusion protein containing GST linked to the mature form of
SREBP-1. 35S-Labeled TR bound GST-SREBP-1, and the
presence of T3 enhanced this interaction (Fig.
4). Inclusion of unlabeled RXR in the binding reaction had no effect on the interaction of
35S-labeled TR with GST-SREBP-1 (data not shown). No
interaction was observed between 35S-labeled TR and GST.
In contrast to the results for TR, little or no interaction was
observed between 35S-labeled RXR and GST-SREBP-1 (Fig.
4). The lack of interaction between RXR and SREBP-1 was confirmed by
pull-down experiments employing GST-RXR as the bait and
35S-labeled SREBP-1 synthesized in vitro (data
not shown). We also investigated whether SREBP-1 interacted with
LXR, because LXR·RXR complexes bind the ACC T3RE in CEH and are
postulated to play a role in mediating the T3-induced increase in
ACC promoter activity (16). No interaction was observed between
35S-labeled LXR and GST-SREBP-1 (Fig. 4). These data
indicate that SREBP-1 selectively interacts with TR.
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Fig. 4.
The mature form of SREBP-1 specifically binds
TR in a T3-regulated manner. Bacterially
produced GST or GST linked to the mature form of chicken SREBP-1
(GST-SREBP-1) was immobilized on glutathione-Sepharose
beads. These preparations were then incubated with in vitro
translated and 35S-labeled TR, RXR, or LXR as
described under "Experimental Procedures." The incubations were
performed in the absence or the presence of ligand for TR (1 µM T3), RXR (1 µM
9-cis-retinoic acid), or LXR (1 µM
22(R)-hydroxycholesterol) as indicated. After the matrix was
extensively washed, the labeled proteins retained on the beads were
eluted, resolved by SDS-PAGE, and visualized by storage phosphor
autoradiography together with 10% of the total radiolabeled receptor
input used in each binding reaction. This experiment was repeated three
times with similar results.
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We next set out to identify the motifs in TR that interacted with
SREBP-1. Pull-down experiments were performed using GST-SREBP-1 as the
bait and various truncations of TR labeled with 35S
in vitro. Deletion of the first 20 or 50 amino acids from
the N terminus of TR had little or no effect on the binding of TR to GST-SREBP-1 (Fig. 5A). When
deletion of the N terminus of TR was extended to amino acid 120, binding of TR to GST-SREBP-1 was abolished. To further analyze the
interaction between TR and SREBP-1, TR polypeptides containing
amino acids 1-157, amino acids 1-118, and amino acids 51-157 were
tested for their ability to interact with GST-SREBP-1. A strong
interaction was observed between GST-SREBP-1 and a TR polypeptide
containing amino acids 1-157. Weaker interactions were observed
between GST-SREBP-1 and TR polypeptides containing amino acids
1-118 and amino acids 51-157. Collectively, these data suggest that a
TR region containing the DNA-binding domain plus flanking sequences
(amino acids 21-157) is required for optimal binding to SREBP-1.
Interestingly, interactions between GST-SREBP-1 and TR truncations
lacking the ligand-binding domain were not enhanced by the presence of
T3. This observation is consistent with the scenario that T3 binding to
the ligand-binding domain of TR causes a conformational change that
enhances the ability of the N-terminal region of TR to interact with
SREBP-1.
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Fig. 5.
A region of TR
containing the DNA-binding domain plus flanking sequences is
required for optimal binding to SREBP-1, and a region of SREBP-1
containing the bHLH-Zip domain is required for optimal binding to
TR. Wild-type and truncated forms of TR or mature
SREBP-1 were translated in vitro in the presence of
L-[35S]cysteine- or
L-[35S]methionine. 35S-Labeled
proteins were then incubated with GST, GST-SREBP-1, or GST-TR bound
to glutathione-Sepharose beads as described in the legend of Fig. 4 and
under "Experimental Procedures." The incubations were performed in
the absence and presence of 1 µM T3. After the matrix was
extensively washed, the labeled proteins retained on the beads were
eluted, resolved by SDS-PAGE, and visualized by storage phosphor
autoradiography together with 10% of the total radiolabeled protein
input used in each binding reaction. A, results of
experiments using GST-SREBP-1 as the bait protein and full-length and
truncated versions of 35S-labeled TR as test proteins. A
schematic representation of chicken TR is shown in the upper
panel. The DNA-binding domain (DBD) and ligand-binding
domain (LBD) are indicated. Embedded within the LBD of TR
is a heptad repeat region involved in dimerization. A ligand-induced
change in the conformation of the LBD is responsible for providing an
interface for interaction with coactivator proteins. B,
results of experiments using GST-TR as the bait protein and
full-length and truncated versions of mature SREBP-1 as test proteins.
A schematic representation of the mature form of chicken SREBP-1 is
shown in the upper panel. A region of SREBP-1 enriched in
acidic amino acids (amino acids 1-51) mediates interactions with
coactivator proteins (55, 56). The bHLH-Zip motif between amino acids
300 and 370 is required for DNA binding (38). The region between amino
acids 51 and 213 is enriched in proline and serine residues. These
experiments were repeated twice with similar results.
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To map the motifs in SREBP-1 that interacted with TR, pull-down
analyses were performed using a GST fusion protein containing full-length TR as a bait and various truncations of mature SREBP-1 labeled with 35S in vitro. Consistent with the
results of experiments employing GST-SREBP-1 as the bait protein, the
full-length, mature form of SREBP-1 (amino acids 1-464) interacted
with GST-TR (Fig. 5B). N-terminal deletions of SREBP-1 to
amino acid 78, 108, 238, or 300 had little or no effect on the binding
SREBP-1 to GST-TR. A C-terminal deletion of SREBP-1 to amino acid
389 also had no effect on the binding of SREBP-1 to GST-TR. In
contrast, C-terminal deletions of SREBP-1 to amino acids 349 and 266 abolished the binding of SREBP-1 to GST-TR. These data indicate that
a SREBP-1 region containing the bHLH-Zip domain (amino acids 300-389)
is required for binding to TR. In contrast to results of experiments analyzing interactions between GST-SREBP-1 and 35S-labeled
TR, interactions between GST-TR and 35S-labeled
SREBP-1 proteins were not affected by the presence of T3. The latter
observation suggests that appending GST to the N terminus of TR
blocks ligand-induced conformational changes that facilitate
interactions between TR and SREBP-1. A similar observation has been
reported for interactions between TR and the homeodomain protein,
PBX1 (19).
The TR region between amino acids 21-157 and the SREBP-1 region
between amino acids 300 and 389 overlap with motifs mediating DNA
binding activity. This prompted us to examine whether the binding of
TR and SREBP-1 to their DNA response elements altered the
interaction between these proteins. Pull-down assays were carried out
with GST-SREBP-1 and in vitro synthesized TR and RXR
in the absence or presence of oligonucleotides containing the ACC
SRE-1 alone (T3REmut-SRE-1), the ACC T3RE alone (T3RE-SRE-1mut), or
both the ACC T3RE and ACC SRE-1 (T3RE-SRE-1). None of the oligonucleotides had an effect on the interaction between GST-SREBP-1 and TR in the absence and presence of T3 (Fig.
6). These results suggest that DNA does
not modulate the interaction between SREBP-1 and TR.
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Fig. 6.
The interaction between TR
and SREBP-1 is not altered by the binding of these proteins to
DNA. GST or GST-SREBP-1 immobilized on glutathione-Sepharose beads
was incubated with in vitro translated and
35S-labeled TR and an equimolar concentration of
unlabeled RXR in the absence and presence of T3. Oligonucleotides
(30 ng) containing the ACC T3RE and/or SRE-1 were included in some
incubations as indicated. After the matrix was extensively washed, the
labeled proteins retained on the beads were eluted, resolved by
SDS-PAGE, and visualized by storage phosphor autoradiography together
with 10% of the total radiolabeled protein input used in each binding
reaction. T3RE-SRE-1 (ACC sequences between 108 and
66 bp) contains both the ACC T3RE and SRE-1.
T3RE-SRE-1mut contains a mutation of the SRE-1 in the
context of the 108 to 66 bp ACC fragment.
T3REmut-SRE-1 contains a mutation of the T3RE in the context
of the 108 to 66 bp ACC fragment. The sequence of T3RE-SRE-1 and
T3RE-SRE-1mut is shown in Fig. 1A. The sequence of
T3REmut-SRE-1 is
5'-AGGTGGTTGAATGGAGGTAAAGACTCGCATCACACCACCGCGG-3'
(the mutated bases are underlined). Additional experimental
details are described in the legend of Fig. 4 and under "Experimental
Procedures." This experiment was repeated twice with similar
results.
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SREBP-1 Forms a Tetrameric Complex with TR·RXR Heterodimers
on a ACC DNA Fragment Containing Sequences between 108 and 66
bp--
The finding that SREBP-1 physically interacted with
TR·RXR when both complexes were bound to DNA raised the
possibility that SREBP-1 enhanced T3-induced ACC transcription by
forming a tetrameric complex with TR·RXR on the ACC gene. To
obtain evidence supporting this proposal, gel mobility shift
experiments were performed using in vitro translated TR,
RXR, and mature SREBP-1 and an ACC probe containing the T3RE and
SRE-1 (T3RE-SRE-1, 108 to 66 bp). Incubation of the T3RE-SRE-1
probe with TR, RXR, and SREBP-1 resulted in the formation of
three complexes (Fig. 7A). The
results of supershift analyses with antibodies against TR, RXR,
and SREBP-1 indicated that the top band contained a
SREBP-1·TR·RXR complex, the middle band contained
SREBP-1 homodimers, and the bottom band contained
TR·RXR heterodimers. To determine whether the top band
contained a SREBP-1/TR·RXR trimer or a
SREBP-1·SREBP-1/TR·RXR tetramer, gel mobility shift
experiments were performed with ACC probes (108 to 66 bp)
containing mutations in either the upstream half-site or downstream
half-site of the ACC SRE-1. Because binding of SREBP dimers to DNA
requires the presence of both SRE-1 half-sites (37), mutation of either
ACC SRE-1 half-site would likely prevent the binding of a tetrameric
complex. Mutation of either SRE-1 half-site in the 108 to 66 bp ACC
probe abolished the binding of the middle and top
bands (data not shown). This observation suggests that the
top band is a SREBP-1·SREBP-1/TR·RXR tetrameric complex.
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Fig. 7.
SREBP-1 forms a tetrameric complex with
TR·RXR heterodimers
on a DNA probe containing ACC sequences
between 108 and 66 bp. Gel mobility shift assay were performed
as described under "Experimental Procedures" using
32P-labeled DNA probes and in vitro synthesized
TR, RXR, and mature SREBP-1. A, a DNA probe containing
ACC sequences between 108 and 66 bp (T3RE-SRE-1) was
incubated with TR and RXR (1:1 molar ratio) in the presence of
mature SREBP-1. In some incubations, in vitro translated
proteins were incubated with antibodies against TR, RXR, SREBP-1, or
COUP-TF prior to the addition of the probe. B, the
T3RE-SRE-1 probe was incubated with increasing amounts of TR and
RXR (1:1 molar ratio, 0-2 µl) in the presence of a fixed level of
mature SREBP-1 (0 or 2 µl). Unprogrammed lysate was added to bring
the total amount of lysate to 4 µl/reaction. T3 (1 µM)
was added to binding reactions as indicated. This experiment was
repeated three times with similar results. C, experiments
were performed using a ACC probe (108 to 66 bp) containing a
mutation of the T3RE (T3REmut-SRE-1). The incubation
conditions were the same as those described for B. This
experiment was repeated twice with similar results. D,
ACC DNA fragments (108 to 66 bp) containing the T3RE in the
native orientation (T3RE-SRE-1) or the flipped orientation
(T3RE Flip-SRE-1) were labeled with 32P to the
same specific activity. These ACC probes were incubated with
increasing amounts of mature SREBP-1 (0-2 µl) in the presence of a
fixed level of TR·RXR (0 or 2 µl). Unprogrammed lysate was
added to bring the total amount of lysate to 4 µl/reaction. Similar
amounts of radiolabeled T3RE-SRE-1 and T3RE Flip-SRE-1 were used in the
binding reactions. This experiment was repeated twice with similar
results. The positions of the tetrameric, heterodimeric, and
homodimeric protein complexes are indicated by arrows.
Nonspecific complexes are indicated by the asterisks. The
sequence of the ACC probes are shown in Fig. 1A and in
the legend to Fig. 6.
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In Fig. 7B, the T3RE-SRE-1 probe was incubated with
increasing amounts of TR·RXR in the presence of a fixed
concentration of SREBP-1. Total SREBP-1 binding activity (signal of the
SREBP-1·SREBP-1/TR·RXR complex plus the SREBP-1 homodimeric
complex) was higher in the presence of TR·RXR than in the
absence of TR·RXR. The extent of the increase in total SREBP-1
binding activity caused by TR·RXR was greater in the presence
of T3 than in the absence of T3. In contrast, total TR·RXR
binding activity (signal of the SREBP-1·SREBP-1/TR·RXR complex plus the TR·RXR heterodimeric complex) was not affected by the presence of SREBP-1. These data indicate that SREBP-1 can form a
tetrameric complex with TR·RXR on the ACC gene and that the
formation of this complex enhances the DNA binding activity of SREBP-1
but has no effect on the DNA binding activity of TR·RXR.
To determine whether tetrameric complex formation on the T3RE-SRE-1
probe and the concomitant increase in SREBP-1 DNA binding activity
required the binding of TR·RXR to the T3RE, gel mobility shift
experiments were performed using a probe containing a mutation of the
T3RE in the context of the 108 to 66 bp ACC fragment (T3REmut-SRE-1). Incubation of this probe with in vitro
synthesized SREBP-1 resulted in the formation of a single protein-DNA
complex containing SREBP-1 homodimers (Fig. 7B). The
addition of increasing amounts of TR·RXR to the binding
reaction had no effect on the pattern or intensity of protein binding.
These data indicate that tetrameric complex formation on the 108 to
66 bp ACC fragment requires the binding of TR·RXR to the
T3RE. The results of experiments employing a probe containing a block
mutation of the SRE-1 in the context of the 108 to 66 bp ACC
fragment (T3RE-SRE-1mut) indicate that tetrameric complex formation
also requires the binding of SREBP-1 to the SRE-1 (data not shown).
The data in Fig. 3 demonstrated that the stimulation of
T3-induced transcription by the ACC SRE-1 was dependent on the
orientation of the ACC T3RE. The effect of T3RE orientation on SRE-1
activity may be mediated by changes in the ability of SREBP to form a
tetrameric complex with TR·RXR on DNA. To investigate this
possibility, gel mobility shift experiments were performed using
in vitro synthesized proteins and ACC probes containing
the T3RE in the native and flipped orientation. ACC fragments (108
to 66 bp) containing the T3RE in the native orientation (T3RE-SRE-1)
or flipped orientation (T3RE Flip-SRE-1) were labeled with
32P to the same specific activity. Equal amounts of each
probe were incubated with a fixed concentration of TR·RXR in
the presence of T3 and increasing concentrations of SREBP-1. At each
concentration of SREBP-1, the abundance of tetrameric complexes
(SREBP-1·SREBP-1/TR·RXR) was higher on the T3RE-SRE-1 probe
than on the T3RE Flip-SRE-1 probe (Fig. 7C). This
observation provides support for a role of tetrameric complex formation
in mediating the stimulatory effects of the SRE-1 on T3-induced ACC transcription.
The TR·RXR-induced increase in SREBP-1 binding to the ACC
SRE-1 (Fig. 7) may be due to an increase in the affinity of SREBP-1 for
the SRE-1 and/or a decrease in the dissociation rate of SREBP-1 from
the SRE-1. To determine whether TR·RXR altered the dissociation rate of SREBP-1 from the SRE-1, protein complexes prebound to the
labeled T3RE-SRE-1 probe were incubated for various times with a
1,000-fold molar excess of an unlabeled competitor DNA containing the
ACC SRE-1 alone (T3REmut-SRE-1). The dissociation rate of SREBP-1 in
the SREBP-1·SREBP-1/TR·RXR tetrameric complex was decreased
by 61% compared with the dissociation rate of the SREBP-1 homodimeric
complex (Fig. 8A). These data
indicate that tetrameric complex formation stabilizes the binding of
SREBP-1 to the ACC SRE-1. This phenomenon accounts for at least part of the TR·RXR-induced increase in SREBP-1 binding to the
T3RE-SRE-1 probe.
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Fig. 8.
The formation of the
SREBP-1·SREBP-1/TR·RXR
tetrameric complex stabilizes the binding of SREBP-1 to the
ACC SRE-1. In vitro
synthesized TR, RXR, and mature SREBP-1 were bound to
32P-labeled T3RE-SRE-1 as described under "Experimental
Procedures." The binding mixture was then incubated with a 1,000-fold
molar excess of unlabeled T3REmut-SRE-1 (A) or T3RE-SRE-1mut
(B). At various time points after the addition of competitor
DNA, the aliquots of the binding mixture were subjected to
nondenaturing gel electrophoresis. Left panel, data from a
representative experiment. Right panel, plot of log protein
binding activity versus time. The signals for
protein-DNA complexes containing SREBP-1·SREBP-1,
TR·RXR, or SREBP-1·SREBP-1/TR·RXR were
quantitated using a PhosphorImager. The protein binding
activities at 0 min were set at 100, and the activities at other time
points were adjusted proportionately. The values are the means ± S.E. of three experiments. The positive standard error bar |
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Transcription in Hepatocytes*
TOP
ABSTRACT
INTRODUCTION
