From the Department of Biochemistry, School of Medicine, West Virginia University, Morgantown, West Virginia 26506
Received for publication, July 5, 2000, and in revised form, October 6, 2000
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ABSTRACT |
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Triiodothyronine (T3) stimulates a 7-fold
increase in transcription of the acetyl-CoA carboxylase- When the intake of dietary carbohydrate exceeds the immediate
energy needs of the animal, excess carbohydrate is converted to
triacylglycerols, which can be used for energy during periods of
fasting. One of the enzymes that plays a pivotal role in mediating this
response is acetyl-CoA carboxylase
(ACC).1 ACC catalyzes the
ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA,
which is the donor of all but two ( The concentration of ACC ACC Thyroid hormone action is initiated by the binding of T3 to nuclear
receptors. Nuclear T3 receptors (TRs) are members of a superfamily of
ligand-dependent transcription factors that include the
receptors for steroid hormones, vitamin A derivatives, vitamin D3,
oxysterols, prostanoids, and a large family of receptor-like proteins
with unknown ligands (orphan receptors) (12, 13). TRs bind to DNA
sequences denoted as T3 response elements (T3REs). A wide diversity of
T3RE structures have been reported in T3-responsive genes (14). In
general, T3REs consist of multiple copies of a hexameric sequence
related to a consensus RGGWMA arranged as inverted repeats, everted
repeats, direct repeats, or extended single copies of the hexamer. The
nucleotide sequence of the hexameric half-sites and flanking DNA and
the spacing of the half-sites influence the binding of TR and its
interactions with other proteins (15-18). Consequently, the structure
of the T3RE is an important factor influencing the transcriptional
activity of TR. TRs can bind T3REs as monomers, homodimers, or
heterodimers with retinoid X receptor (RXR) (13, 14). TR/RXR
heterodimers are thought to be the principal species of TR bound to
T3REs in vivo, since this complex binds DNA with higher
affinity and modulates transcription more effectively than TR monomers
and TR homodimers (19-21). In addition to TR and RXR, other members of
the nuclear hormone receptor family can regulate transcription by
binding to T3REs. For example, the TRs bound to T3REs exhibit two regulatory activities. In the absence of
T3, TRs repress transcription (23-26). The addition of T3 causes a
derepression of transcription and, in some instances, a further
activation of transcription above that observed in the absence of TR
(23). This dual regulatory activity of TR arises, in part, from the
ability of TR to recruit auxiliary regulatory proteins referred to as
corepressors and coactivators. In the absence of T3, TR binds to
corepressors such as silencing mediator for retinoic and thyroid
hormone receptors (27) and nuclear receptor corepressor (28). The
presence of T3 causes the release of corepressors and the subsequent
association of TR with coactivators (29, 30). Examples of coactivators
of TR include CREB-binding protein (CBP) (31, 32), steroid
receptor coactivator-1 (33), CBP-interacting protein (34, 35), and
p300/CBP associated factor (36). Corepressors and coactivators
may mediate the transcriptional effects of TR by directly interacting
with the basal transcriptional machinery, by modulating interactions
between TR and the basal transcriptional machinery, and by modifying
chromatin structure (13, 29, 37).
In the present report, we have investigated the mechanism by which T3
regulates ACC Reporter Plasmids--
An 18-kilobase pair ACC
The cDNAs for human RXR Cell Culture and Transient Transfection--
Primary cultures of
chick embryo hepatocytes were prepared as described previously (42) and
maintained in serum-free Waymouth's medium MD705/1 containing 50 nM insulin (gift from Lilly) and corticosterone (1 µM). Chick embryo hepatocytes were incubated on 60-mm
Petri dishes (Fisher) at 40 °C in a humidified atmosphere of 5%
CO2 and 95% air. Cells were transfected 6 h after
plating, using 20 µg of Lipofectin (Life Technologies), 2.5 µg of
p[ACC Gel Mobility Shift Analysis--
Twenty hours after being placed
into culture, chick embryo hepatocytes were incubated in Waymouth's
medium containing insulin and corticosterone with or without T3 for the
times indicated in the figure legends. Cells were harvested, and
nuclear extracts were prepared as described (46) except that the
protease inhibitors, leupeptin (0.25 µg/ml), benzamidine (10 mM), aprotinin (8 µg/ml), and phenylmethylsulfonyl
fluoride (0.5 mM) were added to the extraction buffer at
the indicated concentrations. Chicken TR Identification of Sequences That Confer T3 Regulation on ACC
Analysis of 3'-deletions of ACC
To obtain additional data indicating the presence of a T3RE in the
5'-flanking region of ACC
Analysis of the sequences between Characterization of Nuclear Proteins That Bind the ACC
Antibody supershift experiments were performed to identify the proteins
that interacted with ACC
The ability of the ACC
Results from DNA binding analyses suggest that complex 1 and complex 2 mediate the enhancer activity of ACC
The major T3RE mediating the T3 regulation of the chicken malic enzyme
gene is a DR4-type element located between
Previous work from our laboratory has shown that in hepatocytes
incubated in the presence of insulin, the stimulation of ACC Effects of Expression of Exogenous LXR, RXR, and TR on ACC
We also investigated the effects of overexpression of TR Previous studies analyzing the functional properties of TRs
in vivo and in vitro have shown that the
unliganded form of TR represses transcription and that the binding of
T3 to TR reverses this effect and, in some instances, stimulates
transcription above that observed in the absence of TR (13, 23-26).
Accordingly, several native and artificial T3REs have been shown to
confer T3 responsiveness by repressing transcription in the absence of T3 and activating transcription in the presence of T3 (53-56). In the
present report, we have identified a T3RE (ACC Results from DNA binding (Figs. 4, 5, and 7), competition (Fig. 6), and
transient transfection (Fig. 8) analyses suggest that the
T3-independent enhancer activity of ACC LXRs were initially identified as orphan receptors by screening
libraries for homologues of nuclear hormone receptors and were
subsequently shown to be bound and activated by naturally occurring
oxysterols at physiological concentrations (47-49, 57). LXR/RXR
binding sites have been identified in the genes for cholesterol 7 Previous work from our laboratory has shown that a relatively long time
(24 h) is required to reach maximal rates of ACC In contrast to ACC In theory, the ability of a T3RE to bind TR and repress transcription
in the absence of T3 serves to amplify the activating effects of T3 on
transcription. Indeed, the robust T3-induced stimulation of
transcription conferred by ME-T3RE2 in chick embryo hepatocytes
(>270-fold) (53) is consistent with this hypothesis. What is the
physiological significance of the enhancer activity of ACC In summary, we have characterized a T3RE in the ACC (ACC
)
gene in chick embryo hepatocytes. Here, we characterized an ACC
T3
response element (ACC
-T3RE) with unique functional and protein
binding properties. ACC
-T3RE activated transcription both in the
absence and presence of T3, with a greater activation observed in the
presence of T3. In nuclear extracts from hepatocytes incubated in the
absence of T3, ACC
-T3RE bound protein complexes (complexes 1 and 2)
containing the liver X receptor (LXR) and the retinoid X receptor
(RXR). In nuclear extracts from hepatocytes incubated in the presence of T3 for 24 h, ACC
-T3RE bound a different set of complexes. One complex contained LXR and RXR (complex 3) and another contained the
nuclear T3 receptor (TR) and RXR (complex 4). Mutations of ACC
-T3RE
that inhibited the binding of complexes 1 and 2 decreased transcriptional activation in the absence of T3, and mutations of
ACC
-T3RE that inhibited the binding of complexes 3 and 4 decreased transcriptional activation in the presence of T3. The stimulation of
ACC
transcription caused by T3 was closely associated with changes
in the binding of complexes 1-4 to ACC
-T3RE. These data suggest
that T3 regulates ACC
transcription by a novel mechanism involving
changes in the composition of nuclear receptor complexes bound to
ACC
-T3RE. We propose that complexes containing LXR/RXR ensure a
basal level of ACC
expression for the synthesis of structural lipids
in cell membranes and that complexes containing LXR/RXR and TR/RXR
mediate the stimulation of ACC
expression caused by T3.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) of the carbon atoms for
the synthesis of long-chain fatty acids. This reaction is the
pace-setting step of the fatty acid synthesis pathway (1, 2). There are
two ACC isoforms that are encoded by distinct genes. ACC
(260 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
(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 (3).
in liver is subject to nutritional and
hormonal regulation. For example, in starved animals, the concentration
of hepatic ACC
is low; feeding a high carbohydrate, low fat diet
stimulates an 8-20-fold increase in the amount of the enzyme (4-7).
The effects of nutritional manipulation on ACC
concentration are
mediated primarily by changes in the rate of transcription of the
ACC
gene (8). Diet-induced changes in ACC
transcription are
mimicked in primary cultures of chick embryo hepatocytes by
manipulating the concentration of hormones and nutrients in the culture
medium. The addition of 3,5,3'-triiodothyronine (T3) to the culture
medium stimulates a 7-fold increase in ACC
transcription (9).
Interestingly, a relatively long time (24 h) is required to achieve
maximal rates of ACC
transcription after the addition of T3,
suggesting that the accumulation of a rate-limiting intermediate is
involved in mediating this response. The molecular mechanism by which
T3 regulates ACC
transcription remains to be determined.
transcription is initiated from two promoters, resulting in
mRNAs with heterogeneity in the 5'-untranslated region (2). The
more upstream promoter (promoter 1) flanks exon 1, while the more
downstream promoter (promoter 2) flanks exon 2. In livers of rats (10,
11) and chickens,2 the
increase in total ACC
mRNA abundance caused by the consumption of a high carbohydrate, low fat diet is mediated by alterations in the
activities of promoter 1 and promoter 2, with the latter promoter
playing a quantitatively greater role in mediating this response.
Alterations in promoter 2 activity are also primarily responsible for
the T3-induced stimulation in total ACC
mRNA abundance in chick
embryo hepatocytes.2
and
isoforms of the liver X
receptor (LXR) heterodimerize with RXR on an artificial T3RE composed
of directly repeated half-sites separated by a 4-bp spacer (22).
Binding of LXR/RXR to this T3RE causes a stimulation of transcription
in the absence and presence of ligands for LXR and RXR. Evidence that
LXR/RXR heterodimers modulate transcription through T3REs of native
genes is presently lacking.
transcription in hepatocytes. A strongly active T3RE
in the 5'-flanking region of ACC
promoter 2 has been characterized.
An interesting feature of this ACC
T3RE that distinguishes it from
T3REs of other T3-responsive genes is that it activates transcription
both in the absence and presence of T3. In addition, this ACC
T3RE
binds multiple protein complexes in hepatic nuclear extracts, and
several of these complexes contain LXR/RXR heterodimers. We also show
that the T3-induced stimulation of ACC
transcription in hepatocytes
is closely associated with dramatic changes in the binding of LXR/RXR
complexes and TR/RXR complexes to the ACC
T3RE. From these data, we
propose that T3 regulates ACC
transcription by a novel mechanism
involving changes in the binding of nuclear receptor complexes to a
T3RE.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
genomic clone
was obtained by screening a
chicken genomic library
(CLONTECH) with a polymerase chain
reaction-generated DNA fragment extending from
1500 to
855 bp
relative to the start site of transcription of chicken ACC
promoter
2. The sequence of the primers used to generate the
1500 to
855 bp
fragment was derived from the published ACC
promoter 2 sequence
(38). ACC
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. To construct
p[ACC
4900/+274]CAT (HindIII/XhoI),
p[ACC
2054/+274]CAT (BamHI/XhoI),
p[ACC
854/+274]CAT (PstI/XhoI),
p[ACC
391/+274]CAT (BsmI/XhoI),
p[ACC
212/+274]CAT (MscI/XhoI),
p[ACC
136/+274]CAT (EagI/XhoI), and
p[ACC
94/+274]CAT (BstEII/XhoI), ACC
restriction fragments indicated in brackets were inserted upstream of
the chloramphenicol acetyltransferase (CAT) gene in KSCAT (39). To
construct p[ACC
108/+274]CAT, p[ACC
84/+274]CAT,
p[ACC
59/+274]CAT, p[ACC
41/+274]CAT, and p[ACC
30/+274]CAT,
ACC
fragments were amplified by polymerase chain reaction and
inserted upstream of the CAT gene in KSCAT. pBLCAT2 (pTKCAT) was
obtained from B. Luckow and G. Schutz (German Cancer Research Center)
(40). The cryptic activator protein-1 site located 5' of the multiple
cloning site in pBLCAT2 (41) was removed by excising the
NdeI/HindIII fragment from this plasmid followed
by religation. Fragments of the ACC
promoter/regulatory region were
inserted into SphI and SalI site 5' of the herpes
simplex virus thymidine kinase (TK) promoter in pTKCAT to form
ACC/TKCAT constructs. p[ACC
212/
82]TKCAT, p[ACC
171/
82]TKCAT, and p[ACC
212/
108]TKCAT were constructed by first amplifying the
indicated ACC
fragments using polymerase chain reaction and then
subcloning them into pTKCAT. p[ACC
136/
82]TKCAT,
p[ACC
108/
82]TKCAT and pTKCAT constructs containing mutations in
the
108 to
82 fragment were made by inserting annealed
complementary synthetic oligonucleotides into pTKCAT. Structures of
reporter plasmids were confirmed by restriction enzyme mapping and
nucleotide sequence analyses.
and chicken TR
were provided by R. Evans (Salk Institute) and H. Samuels (New York University), respectively. D. Mangelsdorf (University of Texas Southwestern Medical
Center) provided the cDNAs for human LXR
and LXR
. Expression plasmids for RXR
, TR
, LXR
, and LXR
were developed by
subcloning the cDNAs for these receptors into pSV-SPORT1 (Life
Technologies, Inc.).
4900/+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, chick embryo hepatocytes
were harvested, and cell extracts were prepared as described by Baillie
et al. (43). CAT activity (44) and protein (45) were assayed
by the indicated methods. All DNAs used in transfection experiments
were purified using the Qiagen endotoxin-free kit.
, human LXR
, human
LXR
, and human RXR
were translated in vitro using the TNT SP6 coupled reticulocyte lysate system (Promega). To assess the
relative efficiency of synthesis of the different receptor proteins,
incorporation of [35S]methionine into receptor proteins
was measured in parallel reactions. 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. Binding reactions were carried out in 20 µl containing 18 mM HEPES, pH 7.9, 90 mM KCl,
0.18 mM EDTA, 0.45 mM dithiothreitol, 18%
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 10 µg of nuclear extract or 2 µl of in
vitro translated proteins. The reaction was carried out on ice for
60 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 subject 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 at 0 °C prior to the addition of the oligonucleotide
probe. Monoclonal antibody that recognizes the
,
, and
forms
of RXR was generously provided by P. Chambon (Strasbourg, France).
Polyclonal antibodies that recognized chicken ovalbumin upstream
promoter-transcription factor I (N19), the
and
forms of chicken
TR (FL-408), and the
and
forms of LXR (C19) were purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The synthetic
oligonucleotides that were used as probes or competitors in gel
mobility shift assays are listed in Figs. 3A and
6C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Promoter 2--
Results from RNase protection analyses indicated that
the T3-induced stimulation of ACC
expression in chick embryo
hepatocytes is mediated primarily by changes in the activity of
promoter 2 of the ACC
gene.2 Transient transfection
experiments were performed to identify the T3RE(s) mediating this
regulation. In our initial experiments, chick embryo hepatocytes were
transfected with a series DNA constructs containing 5'-deletions of
ACC
promoter 2 linked to the CAT gene. In hepatocytes transfected
with the longest construct, p[ACC
4900/+274]CAT, T3 caused a
4.2-fold increase in CAT activity (Fig.
1). Deletion of ACC-
sequences to
2054,
854,
391, and
212 bp had no effect on T3 responsiveness.
Deletion of ACC
sequences from
136 to
108 bp caused a 2.1-fold
increase T3 responsiveness, suggesting that the region between
136
and
108 bp contains a sequence that inhibits T3 responsiveness.
Deletion of ACC
sequences from
108 to
94 bp caused a marked
decrease in promoter activity in the absence and presence of T3.
Because the extent of the decrease in promoter activity was greater in
cells incubated in the presence of T3, T3 responsiveness was decreased
by 83%. Further deletion of ACC
sequence to a 5'-end points of
84,
59, and
41 bp had no effect on residual T3 responsiveness
(about a 2-fold increase in CAT activity). Further deletion of ACC
sequence to a 5'-end point of
30 bp abolished promoter activity in
the absence and presence of T3 (data not shown). These data suggest
that the region between
108 and
94 bp overlaps with a T3RE or an
accessory element that augments T3 responsiveness. In addition, another
T3RE of weaker activity is located downstream of
41 bp.
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Fig. 1.
Effects of deletions of the 5'-flanking
region of ACC promoter 2 on transcriptional
activity in the absence and presence of T3. Chick embryo
hepatocytes were transiently transfected with p[ACC
4900/+274]CAT or
equimolar amounts of other plasmids as described under "Experimental
Procedures." After transfection, cells were treated with or without
T3 for 48 h. Cells were then harvested, extracts were prepared,
and CAT assays were performed. Left, the constructs used in
these experiments. The number at the left of each
construct is the 5'-end of ACC
DNA in nucleotides relative to the
transcription initiation site of promoter 2. The 3'-end of each
construct was +274 bp. Right, CAT activity of cells
transfected with p[ACC
212/+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 the CAT activity for
hepatocytes treated with T3 (+T3) by that for hepatocytes
not treated with T3 (
T3). The -fold responses were
calculated for individual experiments and then averaged. The results
are the means ± S.E. of six experiments. CAT activity of
T3-treated hepatocytes transfected with p[ACC
212/+274]CAT was
320 ± 42% conversion/h/mg of protein. a, the -fold
stimulation by T3 for p[ACC
108/+274]CAT is significantly higher
than that of any other construct (p < 0.05).
DNA in the context of
p[ACC
94/+274]CAT indicated the presence of a T3RE or T3RE accessory element between +179 and +151
bp.3 However, this T3
regulatory sequence was not detected in 3'-deletions containing the T3
regulatory sequence between
212 and
94 bp. This observation
suggests that sequences upstream of the transcription start site
mediate most, if not all, of the T3 regulation of transcription initiated from ACC
promoter 2.
promoter 2, chick embryo hepatocytes 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 chick hepatocytes (Fig.
2). Appending an ACC
DNA fragment from
212 to
82 bp to the TK promoter caused a 3.6-fold increase in T3
responsiveness. To further define the location of the T3RE in the
212
to
82 bp fragment, a series of deletions of p[ACC
212/
82]TKCAT
were tested for their ability to confer T3 responsiveness. Deletion of
the 5'-end to
171 bp caused a 36% increase in T3 responsiveness. Further deletion to
136 bp had no effect on T3 responsiveness. When
the 5'-end was deleted to
108 bp, T3 responsiveness increased from
5.7- to 10.2-fold. This observation is consistent with data from
5'-deletion analysis in the context of p[ACC
4900/+274]CAT (Fig. 1),
suggesting the presence of a T3 inhibitory element between
136 and
108 bp. When the 3'-end of the
212 to
82 fragment was deleted to
108 bp, T3 responsiveness was abolished. Thus, the sequence between
108 and
82 bp contains a strongly active T3RE. Interestingly, this
T3RE confers T3 regulation by stimulating promoter activity both in the
absence and presence of T3, with a greater stimulation observed in the
presence of T3. These data support the results from 5'-deletion
analysis in the context of p[ACC
4900/+274]CAT (Fig. 1) indicating
the presence of a T3-dependent and T3-independent enhancer
element between
108 and
94 bp.
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Fig. 2.
A strongly active T3RE is located between
108 and
82 bp of the ACC
gene.
Fragments of the ACC-
gene were linked to the minimal TK promoter in
pTKCAT. Hepatocytes 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, constructs used
in these experiments. Numbers indicate the 5' and 3'
boundaries of ACC
DNA relative to the transcription initiation site
of promoter 2. Right, CAT activity in cells transfected with
p[ACC
108/
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 four experiments. The CAT activity of
extracts from T3-treated hepatocytes transfected with
p[ACC
108/
82]TKCAT was 251 ± 36% conversion/h/mg of
protein. Significant differences between means within a column
(p < 0.05) are as follows. a,
versus pTKCAT; b, versus any other
construct.
108 and
82 bp revealed the
presence of four hexameric half-sites (Fig.
3A). Two of these half-sites
(sites 2 and 3) conform perfectly to the consensus T3RE half-site
sequence, RGGWMA. The other two half-sites (sites 1 and 4) are
degenerate copies of the consensus half-site sequence. Half-sites 1 and
3 form an imperfect direct repeat with a 4-bp spacer (DR4) on the
coding strand, and sites 2 and 4 form an imperfect DR4 on the noncoding
strand. These putative DR4 elements partially overlap with each other.
Other combinations of half-sites could form inverted or everted repeat
structures. To investigate which of the putative T3RE half-sites were
involved in mediating T3 regulation, hepatocytes were transfected with
constructs that contained mutations of individual half-sites in the
context of p[ACC
108/
82]TKCAT. Mutating the first 3 bp of
half-site 1 (mut 1) had no effect on T3 responsiveness (Fig.
3B). In contrast, mutating the first 3 bp of half-site 2 (mut 2) or half-site 4 (mut 4) abolished T3 responsiveness. This effect
was mediated by a decrease in promoter activity in the absence and
presence of T3, with a greater effect in the presence of T3. The first 3 bp of half-site 3 also comprise part of the spacer separating half-sites 2 and 4. Mutating these 3 bp (mut 3) caused a 72% decrease in T3 responsiveness. This effect was mediated by a decrease in promoter activity in the presence of T3. These data demonstrate that T3
regulation conferred by the
108 to
82 bp fragment is mediated by
two half-sites (sites 2 and 4) arranged as direct repeats on the
noncoding strand. Both of these half-sites are required for the
T3-dependent and T3-independent transcriptional activation
functions of this T3RE. The T3-dependent enhancer function is also modulated by sequences in the spacer separating the two half-sites.
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Fig. 3.
Delineation of the sequences in the 108 to
82 bp ACC
fragment that confer
transcriptional activation in the absence and presence of T3.
A, native and mutant sequences of putative half-sites
located between
108 and
82 bp of the ACC
gene.
Underlined and overlined sequences in the native
108 to
82 bp fragment are motifs that are similar to the consensus
T3RE half-site (top). The half-sites are numbered 1-4. The
arrows indicate the orientation of the half-sites.
Half-sites 2 and 3 conform perfectly to the consensus T3RE half-site
sequence, whereas half-sites 1 and 4 are degenerate copies of the
consensus sequence. Mutations were introduced into individual
half-sites in the context of p[ACC
108/
82]TKCAT. mut 1 to mut 4 refer to the number of the half-site that was mutated in each
construct. Mutated sequences are boxed. B, CAT
activity of hepatocytes transfected with mutant reporter constructs.
Hepatocytes were transiently transfected as described in the legend of
Fig. 1 and under "Experimental Procedures." After transfection,
cells were treated with or without T3 for 48 h. CAT activity in
cells transfected with p[ACC
108/
82]TKCAT and treated with T3 was
set at 100, and the other activities were adjusted proportionately. The
results are the means ± S.E. of five experiments. The CAT
activity of extracts from T3-treated hepatocytes transfected with
p[ACC
108/
82]TKCAT was 238 ± 26% conversion/h/mg of
protein. Significant differences between means within a column
(p < 0.05) are as follows. a,
versus p[ACC
108/
82]TKCAT treated without T3;
b, versus p[ACC
108/
82]TKCAT treated with
T3; c, versus p[ACC
108/
82]TKCAT.
T3RE
between
108 and
82 bp--
Gel mobility shift analyses were
conducted to assess the binding of hepatic nuclear proteins to the T3RE
between
108 and
82 bp. We will refer to this T3RE as ACC
-T3RE.
Nuclear extracts were prepared from hepatocytes incubated in the
absence and presence of T3 for 24 h. Incubation of nuclear
extracts with a 32P-labeled DNA probe containing
ACC
-T3RE resulted in the formation of four protein-DNA complexes
designated 1-4 in the order of increasing mobility (Fig.
4, A and B). The
abundance of complex 1 and complex 2 was markedly increased in nuclear
extracts from hepatocytes incubated in the absence of T3 relative to
nuclear extracts from hepatocytes incubated in the presence of T3.
Conversely, the abundance of complex 3 and complex 4 was markedly
elevated in nuclear extracts from hepatocytes incubated in the presence
of T3 compared with nuclear extracts from hepatocytes incubated in the
absence of T3. A 100-fold molar excess of unlabeled ACC
-T3RE
competed for the binding of complexes 1-4, suggesting that the binding
of these protein-DNA complexes was specific (Fig. 4A).
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Fig. 4.
ACC -T3RE binds
hepatic protein complexes containing LXR/RXR and TR/RXR. Gel
mobility shift assays were performed using nuclear extracts prepared
from hepatocytes incubated with or without T3 for 24 h. A
double-stranded DNA fragment corresponding to
108 to
82 bp of the
ACC
gene (ACC
-T3RE) was labeled with [
-32P]dCTP
using the Klenow fragment of E. coli DNA polymerase. The
radiolabeled probe was incubated with 10 µg of nuclear protein as
described under "Experimental Procedures." DNA and DNA-protein
complexes were resolved on 6% nondenaturing polyacrylamide gels.
A, nuclear extracts were incubated with antibodies against
RXR and TR prior to the addition of the probe. Competition analysis was
performed by mixing the labeled probe with a 20- and 100-fold molar
excess of unlabeled ACC
-T3RE prior to the addition of nuclear
extract. B, nuclear extracts were incubated with antibodies
against COUP-TFI and LXR prior to the addition of the probe. Positions
of the specific protein-DNA complexes (arrows), nonspecific
complexes (asterisk), and supershifted complexes
(SS) are indicated. These results are representative of four
experiments employing independent preparations of nuclear extract.
NS, normal serum.
-T3RE. Antibodies directed against
,
,
and
isoforms of RXR completely disrupted the formation of complex 3 and complex 4 (Fig. 4A). This result was more readily observed in nuclear extracts from T3-treated hepatocytes in which the
abundance of complex 3 and complex 4 was elevated. RXR antibodies partially disrupted the formation of complex 1 and complex 2 in nuclear
extracts from hepatocytes incubated without T3. The disruption of
complexes 1, 2, 3, and 4 by RXR antibodies was associated with the
appearance of a supershifted complex of high intensity. These results
suggest that most of the protein-DNA complexes that interact with
ACC
-T3RE contain RXR or a protein highly related to RXR. Antibodies
directed against the
and
isoforms of TR completely disrupted
the formation of complex 4 but had no effect on the formation of
complexes 1-3. The disruption of band 4 by TR antibody was associated
with the appearance of a supershifted complex. Thus, complex 4 appears
to contain TR/RXR heterodimers. Other proteins that can heterodimerize
with RXR on DR4 elements include the
and
isoforms of LXR (22,
47). LXR
and LXR
are expressed abundantly in liver (47-49).
Preincubation of nuclear extracts with antibodies directed against
LXR
and LXR
partially disrupted the formation of complexes 1 and
2 in nuclear extracts from hepatocytes incubated without T3 and
disrupted the formation of complex 3 in nuclear extracts from
hepatocytes incubated with T3 (Fig. 4B). Thus, complexes
1-3 appear to contain LXR/RXR heterodimers. The orphan receptor,
chicken ovalbumin upstream promoter-transcription factor (COUP-TF),
also has been reported to bind T3REs (50, 51). Antibodies against
COUP-TFI had no effect on protein binding to ACC
-T3RE.
-T3RE to bind heterodimers containing LXR/RXR
and TR/RXR was confirmed by gel mobility shift experiments employing
in vitro synthesized receptors. Incubation of in
vitro translated LXR
, LXR
, RXR
, or TR
with the
ACC
-T3RE probe resulted in little or no DNA binding activity (Fig.
5A). Inclusion of RXR
in
the binding reactions with LXR
, LXR
, and TR
stimulated the formation of high affinity protein-DNA complexes containing
LXR
/RXR
, LXR
/RXR
, and TR
/RXR
, respectively.
Competition analyses indicated that the affinities of LXR
/RXR
,
LXR
/RXR
, and TR
/RXR
for ACC
-T3RE were similar (Fig.
5B).
View larger version (37K):
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Fig. 5.
ACC -T3RE binds
LXR
, LXR
, and
TR
as heterodimers with
RXR
. Gel mobility shift assays were
performed as described under "Experimental Procedures" using
in vitro synthesized nuclear receptors and
32P-labeled ACC
-T3RE (
108 to
82 bp) as the probe.
A, equimolar amounts of in vitro synthesized
LXR
, LXR
, or TR
were incubated with the radiolabeled probe in
the absence or presence of RXR
as indicated. In lanes
8-10, receptor preparations were incubated with antibodies
against LXR or RXR prior to the addition of the probe. Positions of
heterodimeric, homodimeric, monomeric, and supershifted receptor
complexes are indicated by arrows. Nonspecific complexes are
indicated by an asterisk. B, the radiolabeled
ACC
-T3RE probe was incubated with a 3-, 6-, 20-, and 100-fold molar
excess of unlabeled ACC
-T3RE or ME-T3RE2 prior to the addition of
in vitro synthesized LXR
/RXR
, LXR
/RXR
, and
TR
/RXR
. The sequence of ME-T3RE2 is shown in Fig.
6C.
-T3RE in the absence of T3 and
that complex 3 and complex 4 mediate the increase in enhancer activity
of ACC
-T3RE caused by the addition of T3. To obtain further data
supporting this hypothesis, competition experiments were performed
using unlabeled DNA fragments containing native or mutant forms of
ACC
-T3RE. These ACC
-T3RE fragments were the same sequences
assayed in the transfection experiments described in Fig. 3. Unlabeled
ACC
-T3RE, ACC
-T3RE mut 1, and ACC
-T3RE mut 3 were more
effective than ACC
-T3RE mut 2 and ACC
-T3RE mut 4 in competing for
the binding of complexes 1 and 2 in nuclear extracts from hepatocytes
incubated in the absence of T3 and complex 3 in nuclear extracts from
hepatocytes incubated in the presence of T3 (Fig.
6A). Thus, ACC
-T3RE mutants
that abolished enhancer activity in the absence and presence of T3
(i.e. ACC
-T3RE mut 2 and ACC
-T3RE mut 4) bound
proteins in complex 1, complex 2, and complex 3 with reduced affinity.
These findings are consistent with a role of complexes 1 and 2 in
mediating the enhancer activity of ACC
-T3RE in the absence of T3 and
a role of complex 3 in mediating enhancer activity in the presence of
T3. The competition profile for complex 4 was different from that of
complexes 1-3. In nuclear extracts from hepatocytes incubated with T3,
ACC
-T3RE and ACC
-T3RE mut 1 were more effective than ACC
-T3RE
mut 2 and ACC
-T3RE mut 3 in competing for complex 4 binding
activity. Thus, the inhibition of T3-induced transcriptional activity
caused by ACC
-T3RE mut 2 and ACC
-T3RE mut 3 is associated with a
decrease in the binding of complex 4. However, ACC
-T3RE mut 1 and
ACC
-T3RE mut 4 exhibited a similar ability to compete for the
binding of complex 4 despite the fact that these mutations caused
markedly different effects on T3-induced transcriptional activity.
Thus, the elimination of T3-dependent enhancer activity
caused by mutation of the more upstream half-site of the ACC
-T3RE
(i.e. ACC
-T3RE mut 4) is not associated with changes in
the binding of TR/RXR. Previous studies have shown that variations in
the structure of the upstream half-site of DR4-type T3REs can
profoundly alter T3-dependent enhancer activity without
causing changes in TR/RXR binding affinity (16). The mechanism
mediating the alteration in T3-dependent enhancer activity
involves changes in the conformation of TR/RXR heterodimers bound to
DNA (52). This mechanism may explain the effect of ACC
-T3RE mut
4 on T3-dependent enhancer activity.
View larger version (55K):
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Fig. 6.
The ability of native and mutant T3REs to
compete for the binding of hepatic nuclear proteins to
ACC -T3RE. Gel mobility shift experiments
were performed as described under "Experimental Procedures" using
32P-labeled ACC
-T3RE as a probe and nuclear extracts
prepared from hepatocytes incubated with or without T3 for 24 h.
Nuclear extracts were incubated with the ACC
-T3RE probe in the
presence of different concentrations of unlabeled competitor DNAs. Each
reaction contained 10 µg of nuclear protein. DNA and DNA-protein
complexes were resolved on 6% nondenaturing polyacrylamide gels.
Specific protein-DNA complexes are indicated by arrows.
Bands 1 and 2 and bands
3 and 4 are the predominant protein-DNA complexes
observed with nuclear extracts from hepatocytes incubated in the
absence of T3 and presence of T3, respectively. A,
competition analysis with native and mutant forms of ACC
-T3RE. The
sequences of the ACC
-T3RE competitors are shown in Fig.
3A. Unlabeled competitor DNAs (6-, 20-, or 40-fold molar
excess) were mixed with the radiolabeled probe prior to the addition of
nuclear extract. B, competition analysis with the major T3RE
of the chicken malic enzyme gene (ME-T3RE2). Unlabeled ME-T3RE2 and
ACC
-T3RE (3-, 6-, 20-, or 100-fold molar excess) were mixed with the
radiolabeled probe prior to the addition of nuclear extract.
C, comparison of the sequence of ME-T3RE2 with that of
ACC
-T3RE. The arrows indicate the position and
orientation of the half-sites.
T3 NE, nuclear
extracts from hepatocytes treated without T3; +T3
NE, nuclear extracts from hepatocytes treated with T3. These
data are representative of four experiments employing independent
preparations of nuclear extract.
3883 and
3858 bp and is
referred to as ME-T3RE2 (39, 53). In contrast to the transcriptional
enhancer function of ACC
-T3RE in chick embryo hepatocytes incubated
in the absence of T3 (Figs. 1, 2, and 3B), ME-T3RE2
functions as a strong repressor of transcription in chick embryo
hepatocytes incubated under the same experimental conditions (53). Both
ACC
-T3RE and ME-T3RE2 activate transcription in hepatocytes
incubated in the presence of T3. The different activities of
ACC
-T3RE and ME-T3RE2 in hepatocytes incubated in the absence of T3
may be due to differences in the binding affinities of these T3REs for
complexes 1 and 2. To investigate this possibility, we examined the
ability of different concentrations of unlabeled ME-T3RE2 to compete
for protein binding to 32P-labeled ACC
-T3RE. A 100-fold
molar excess of ME-T3RE2 was not effective in competing for the binding
of complex 1 and complex 2 in nuclear extracts from hepatocytes
incubated without T3 (Fig. 6B). The inability of ME-T3RE2 to
bind the proteins in complex 1 and complex 2 is consistent with these
complexes functioning as activators of transcription in the absence of
T3. To investigate whether common factors were involved in mediating
the T3-dependent enhancer activities of ACC
-T3RE and
ME-T3RE2, competition experiments were performed using nuclear extracts
from hepatocytes incubated in the presence of T3. ME-T3RE2 was
effective in competing for the binding of complex 4 but was not
effective in competing for complex 3. This observation suggests that
the proteins comprising complex 4 (i.e. TR/RXR heterodimers)
mediate the T3-dependent enhancer activities of both
ACC
-T3RE and ME-T3RE2. The inability of ME-T3RE2 to bind complexes
containing LXR/RXR heterodimers (i.e. complexes 1-3) was
confirmed by experiments demonstrating that ME-T3RE2 was not effective
in competing for binding of in vitro synthesized
LXR
/RXR
(Fig. 5B).
transcription by T3 occurs in two phases (Fig.
7A) (9). Incubating hepatocytes with T3 for 1 h causes a small increase (1.9-fold) in
ACC
transcription. Between 1 and 5 h of T3 treatment, there is
no change in the rate of ACC
transcription. After 5 h of T3 treatment, the rate of ACC
transcription increases again. A maximal rate of ACC
transcription is reached after 24 h of T3 treatment and is about 7 times the transcription rate observed before the addition of T3. To investigate the temporal relationship between ACC
transcription and the binding of nuclear proteins to ACC
-T3RE, the
time course of the effects of T3 on protein binding to ACC
-T3RE was
determined. Nuclear extracts from hepatocytes incubated with or without
T3 for different times were subjected to gel mobility shift analysis
using ACC
-T3RE as a probe. As observed earlier in Fig. 4,
ACC
-T3RE primarily bound complexes 1 and 2 in nuclear extracts from
hepatocytes incubated in the absence of T3 (Fig. 7B).
Incubation of hepatocytes with T3 for 1 and 5 h had no effect on
the pattern of protein binding to ACC
-T3RE. Between 5 and 24 h
of T3 treatment, the binding of complexes 3 and 4 increased and the
binding of complexes 1 and 2 decreased. The pattern of protein binding
to ACC
-T3RE at 48 h of T3 treatment was similar to that at
24 h of T3 treatment (data not shown). Thus, T3-induced alterations in protein binding to ACC
-T3RE are closely correlated with the large increase in ACC
transcription between 5 and 24 h
of hormone treatment. This observation provides further evidence that
alterations in the binding of complexes 1-4 are involved mediating the
T3-induced increase in ACC
transcription.
View larger version (25K):
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Fig. 7.
Time course for T3-induced changes in the
binding of hepatic nuclear proteins to
ACC -T3RE. A, previously
published data for the time course of the T3-induced increase in ACC
transcription. Nuclear run-on assays were performed using nuclei
isolated from hepatocytes incubated in the absence or presence of T3
for the indicated time periods. These data were taken from Hillgartner
et al. (9). B, time course for T3-induced changes
in protein binding to ACC
-T3RE. Gel mobility shift assays were
performed using nuclear extracts prepared from hepatocytes incubated in
the absence or presence of T3 for the indicated time periods. Nuclear
extracts (10 µg of protein) were incubated with
32P-labeled ACC
-T3RE as described under "Experimental
Procedures." DNA and DNA-protein complexes were resolved on 6%
nondenaturing polyacrylamide gels. Specific protein-DNA complexes are
indicated by arrows. Nonspecific complexes are indicated by
an asterisk. These data are representative of four
experiments employing independent preparations of nuclear
extract.
-T3RE
Function--
Results of DNA binding analyses (Figs. 4 and 5) suggest
that complexes containing LXR/RXR heterodimers mediate the
transcriptional enhancer activity of ACC
-T3RE in hepatocytes
incubated in the absence of T3. To obtain additional data supporting
this hypothesis, we determined the effects of expression of exogenous
LXR
, LXR
, RXR
, LXR
plus RXR
, or LXR
plus RXR
on
transcription directed by the ACC
-T3RE. Chick embryo hepatocytes
were transiently transfected with p[ACC
108/
82]TKCAT and
expression plasmids containing or lacking the genes for LXR
, LXR
,
or RXR
. Overexpression of RXR
alone had no effect on
p[ACC
108/
82]TKCAT activity in hepatocytes incubated in the
absence or presence of T3 (Fig. 8). In
contrast, overexpression of LXR
or LXR
alone or in combination
with RXR
stimulated a 2.5-3-fold increase in
p[ACC
108/
82]TKCAT activity in the absence of T3; overexpression
of these receptors had no effect on p[ACC
108/
82]TKCAT activity in
the presence of T3. When hepatocytes were transfected with a reporter
plasmid lacking the ACC
-T3RE (pTKCAT), overexpression of LXR
,
LXR
, RXR
, LXR
plus RXR
, or LXR
plus RXR
had no effect
on promoter activity in the absence or presence of T3 (data not shown).
These data are consistent with a role of LXR/RXR heterodimers in
mediating the enhancer activity of ACC
-T3RE in the absence of
T3.
View larger version (15K):
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Fig. 8.
Effects of expression of exogenous
LXR , LXR
,
RXR
, and TR
on
transcription directed by ACC
-T3RE. Chick
embryo hepatocytes were transiently transfected with
p[ACC
108/
82]TKCAT (1 µg/plate) and pSV-SPORT1-based expression
plasmids (0.1 µg/plate) expressing the genes for LXR
, LXR
,
RXR
, and TR
. In transfections containing two receptor expression
plasmids, 0.1 µg of each plasmid was transfected per plate. Empty
expression plasmid (pSV-SPORT1) was added to bring the total amount of
expression plasmid transfected per plate to 0.2 µg. Control
transfections contained 0.2 µg/plate of empty expression plasmid.
After transfection, cells were treated with or without T3 for 48 h. CAT activity in cells transfected with p[ACC
108/
82]TKCAT plus
empty expression plasmid (control) and treated with T3 was set at 100, and the other activities were adjusted proportionately. The results are
the means ± S.E. of three experiments. The CAT activity of
extracts from T3-treated hepatocytes transfected with
p[ACC
108/
82]TKCAT and empty expression plasmid was 214 ± 33% conversion/h/mg of protein. *, mean is significantly different
(p < 0.05) from that of cells transfected with
p[ACC
108/
82]TKCAT plus empty expression plasmid (control) and
incubated without T3.
on
p[ACC
108/
82]TKCAT activity in chick embryo hepatocytes. In contrast to the results for LXR
and LXR
, overexpression of TR
and TR
plus RXR
caused an 88 and 91% decrease in
p[ACC
108/
82]TKCAT activity, respectively, in hepatocytes
incubated in the absence of T3; overexpression of these receptors had
no effect on p[ACC
108/
82]TKCAT activity in hepatocytes incubated
in the presence of T3 (Fig. 8). These data provide further evidence
that TR/RXR heterodimers are not involved in mediating the enhancer
activity of ACC
-T3RE in the absence of T3.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-T3RE) with different
functional properties. ACC
-T3RE confers T3 regulation on ACC
promoter 2 in chick embryo hepatocytes by stimulating transcription
both in the absence and presence of T3, with a greater stimulation
observed in the presence of T3. These transcriptional effects of
ACC
-T3RE were observed in the presence of endogenous cellular
proteins; overexpression of TR or other proteins was not required to
elicit a T3 response from transfected genes in chick embryo
hepatocytes. To our knowledge, this is the first report describing a
T3RE that functions as a T3-independent enhancer of transcription
during physiological conditions.
-T3RE is mediated by protein
complexes containing LXR/RXR heterodimers (complexes 1 and 2) and that
the increase in ACC
-T3RE enhancer activity caused by T3 treatment is
mediated by protein complexes containing TR/RXR heterodimers (complex
4) and LXR/RXR heterodimers (complex 3). This is the first time that
LXR/RXR heterodimers have been shown to play a role in mediating the
transcriptional activity of a physiologically relevant T3RE. The
different LXR/RXR complexes that bind ACC
-T3RE in hepatocytes may
contain LXR and/or RXR of different sizes. The binding of coregulatory
proteins to LXR/RXR heterodimers may also account for the different
LXR/RXR complexes.
-hydroxylase (58), cholesterol ester transfer protein (59), and
ABC1 (60, 61). These binding sites resemble DR4 elements and confer
transcriptional activation in both the absence and presence of
oxysterols, with a greater activation observed in the presence of
oxysterols. There are no reports that the LXR/RXR binding sites in the
genes for cholesterol 7
-hydroxylase, cholesterol ester transfer
protein, and ABC1 confer T3 regulation of transcription. LXR has been
proposed to play a key role in the regulation of cholesterol excretion
in animals, as cholesterol 7
-hydroxylase, cholesterol ester transfer
protein, and ABC1 are proteins involved in the regulation of reverse
cholesterol transport. Conclusive evidence supporting a role of LXR
in the regulation of cholesterol catabolism has come from the
characterization of the LXR
knockout mice. LXR
ablation causes an
accumulation of cholesterol esters in liver as a result of an inability
of cholesterol 7
-hydroxylase to be induced by dietary cholesterol
(62). Studies with LXR
knockout mice also indicate that LXR
regulates expression of genes involved in fatty acid metabolism. For
example, ablation of LXR
in mice causes a marked decrease in the
expression of fatty acid synthase and stearoyl-CoA desaturase in liver
(62); the molecular mechanisms mediating these effects are not known. The results of the present study demonstrating that LXR binds and
activates the ACC
gene in hepatocytes provide additional support for
a role of LXR in the regulation of lipogenic enzyme expression.
transcription after
the addition of T3 and that most of the increase in transcription
occurs between 5 and 24 h of hormone treatment (9). Comparison of
the time course of T3-induced changes in ACC
transcription with the
time course of T3-induced changes in ACC
-T3RE binding activity
indicates that the increase in ACC
transcription between 5 and
24 h of T3 treatment is closely associated with an increase in the
binding of complexes 3 and 4 and a decrease in the binding of complexes
1 and 2 (Fig. 7). This observation suggests that regulation of ACC
transcription by T3 is mediated by novel mechanism involving
alterations in the composition of nuclear receptor complexes bound to
ACC
-T3RE. We propose the following model for the regulation of
ACC
transcription by T3. The small increase in transcription
observed within 1 h of T3 addition is mediated by a ligand-induced
stimulation of the derepression/activation function of TR in complex 4. This initial stimulation of ACC
transcription by T3 is limited by
the low binding activity of complex 4 to the ACC
-T3RE relative to
that of complex 1 and complex 2. The large increase in ACC
transcription between 5 and 24 h of T3 treatment is mediated by
the increase in binding of complex 3 and complex 4 and the decrease in
binding of complex 1 and complex 2. In this model, complex 3 and
T3-bound complex 4 are more potent activators of transcription than
complex 1 and complex 2. The relatively long time (>5 h) required to
detect T3-induced changes in protein binding to ACC
-T3RE suggests
that alterations in the synthesis of a regulatory protein are involved
in mediating this effect. While the identity of this regulatory protein
is not known, it does not appear to be TR or RXR, since expression of
TR
, TR
, RXR
, and RXR
is not affected by T3 treatment in
chick embryo hepatocytes (63). Data from Western blot analyses indicate
that the concentration of LXR
and LXR
in chick embryo hepatocytes is also not affected by T3 treatment.3 We speculate that T3
regulates the expression of nuclear receptor accessory proteins that,
in turn, modulate protein binding to ACC
-T3RE. Such accessory
proteins may regulate ACC
-T3RE binding activity by physically
interacting with complexes 1, 2, 3, and/or 4 or by altering the
phosphorylation state of these protein complexes.
-T3RE, the major T3RE of the malic enzyme gene,
ME-T3RE2, functions as a potent repressor of transcription in the
absence of T3 (53). If complex 1 and complex 2 mediate the
T3-independent enhancer activity of ACC
-T3RE in hepatocytes, then
what are the factors that mediate the T3-independent repressor activity
of ME-T3RE2? In previous work, we have shown that ME-T3RE2 binds to
four complexes in nuclear extracts from chick embryo hepatocytes
incubated without T3 (63). In contrast to the protein binding profile
for ACC
-T3RE, all four complexes that interact with ME-T3RE2 contain
TR, and three of the four complexes contain RXR. Thus, the
T3-independent repressor activity of ME-T3RE2 is likely to be mediated
by one or more complexes containing unliganded TR. This supposition is
consistent with previous studies demonstrating that unliganded TR
functions as a repressor of transcription (26). Interestingly, in chick
embryo hepatocytes incubated in the absence of T3, the transcription
rate of the malic enzyme gene as determined by nuclear run-on analysis
is 30% of that observed for the ACC
gene.4 This difference in
transcription rate between malic enzyme and ACC
is likely to be
mediated, at least in part, by the contrasting T3-independent
activities of ME-T3RE2 and ACC
-T3RE.
-T3RE in
the absence T3? In addition to its role in energy homeostasis, ACC
is required for the synthesis of structural lipids in cell membranes.
Consequently, a basal level of ACC
expression is observed under
conditions when the enzyme is not induced by hormonal and nutritional
factors. We propose that the ACC
-T3RE also has a dual physiological
role. In addition to mediating the hormonal regulation of ACC
expression, ACC
-T3RE ensures a basal level of ACC
expression for
obligatory cellular processes. The latter function of the ACC
-T3RE
is mediated by the binding of LXR/RXR complexes. ACC
-T3RE may also
contribute to the basal expression of ACC
in extrahepatic tissues,
as LXR
is expressed in a wide variety of tissues (48, 49).
gene with unique
functional and protein binding properties. In addition, we have
developed data suggesting that the delayed actions of T3 on ACC
transcription are mediated by a novel mechanism involving alterations
in the composition of nuclear receptor complexes bound to the
ACC
-T3RE. The observation that the ACC
-T3RE co-localizes with a
prominent DNase I-hypersensitive site in chromatin from livers of
intact chickens supports a role for this cis-acting sequence in
mediating the regulation of ACC
transcription in vivo.2 Future experimentation will be directed toward
defining the mechanism by which T3 modulates the binding of nuclear
receptor complexes to the ACC
-T3RE in hepatocytes.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. D. Mangelsdorf (LXR
and LXR
), R. Evans (RXR
), and H. Samuels (TR
) for providing
the indicated cDNAs. We thank Dr. P. Chambon for providing RXR antiserum.
![]() |
FOOTNOTES |
---|
* This work was supported by Cooperative State Research Service/United States Department of Agriculture Grant 980-3711.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.
To whom correspondence should be addressed: Dept. of Biochemistry,
P.O. Box 9142, West Virginia University, Morgantown, WV 26506-9142. Tel.: 304-293-7751; Fax: 304-293-6846; E-mail: fbhillgartner@ hsc.wvu.edu.
Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M005894200
2 L. Yin, Y. Zhang, T. Charron, and F. B. Hillgartner, manuscript in preparation.
3 Y. Zhang and F. B. Hillgartner, unpublished results.
4 F. B. Hillgartner and T. Charron, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ACC, acetyl-CoA carboxylase; bp, base pair(s); COUP-TF, chicken ovalbumin upstream promoter transcription factor; CAT, chloramphenicol acetyltransferase; DR4, half-sites arranged as direct repeats with a 4-bp spacer; LXR, liver X receptor; RXR, retinoid X receptor; TK, thymidine kinase; T3, 3,5,3'-triiodothyronine; TR, nuclear T3 receptor; T3RE, T3 response element; CBP, CREB-binding protein.
![]() |
REFERENCES |
---|
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