From the Division of Nutritional Sciences and the
Institute for Cellular and Molecular Biology, The University of
Texas, Austin, Texas, 78712 and the § Department of
Biochemistry, Molecular Biology, and Biophysics, University of
Minnesota, Minneapolis, Minnesota 55455
Received for publication, January 17, 2001, and in revised form, March 19, 2001
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ABSTRACT |
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Refeeding carbohydrate to fasted rats induces the
transcription of genes encoding enzymes of fatty acid biosynthesis,
e.g. fatty-acid synthase (FAS). Part of this
transcriptional induction is mediated by insulin. An insulin
response element has been described for the fatty-acid synthase gene
region of The consumption of a high carbohydrate diet increases the hepatic
production of malonyl-CoA and its subsequent utilization for de
novo fatty acid biosynthesis (1). In addition to being the
substrate for fatty acid biosynthesis, malonyl-CoA governs the rate of
fatty acid oxidation by virtue of its ability to function as a negative
metabolite effector of carnitine palmitoyltransferase (2). The
concentration of malonyl-CoA is determined by its rate of synthesis by
acetly-CoA carboxylase and its rate of utilization by fatty-acid
synthase. Carbohydrate ingestion causes a coordinate induction in the
expression of both hepatic enzymes (3-10). In the case of fatty-acid
synthase the amount of protein is largely determined by mRNA
abundance for fatty-acid synthase, and this in turn is determined by
the rate of fatty-acid synthase gene transcription (5-10). The
carbohydrate induction of hepatic fatty-acid synthase gene
transcription requires both insulin and glucocorticoids (5, 8, 10-12).
On the other hand, fatty-acid synthase expression is suppressed by
polyunsaturated fatty acids and sterols (9, 12-14) and by the
administration of glucagon and growth hormone (15, 16). The
transcriptional response of the hepatic fatty-acid synthase gene to
carbohydrate reportedly involves a glucose response region located
within the first intron (17) as well as insulin and cAMP response
sequences located between Despite the fact that the proximal promoter region of the fatty-acid
synthase gene contains numerous cis-acting elements that interact with
transcription factors that appear to be regulated by insulin and
glucose, the 2-3-fold enhancement of fatty-acid synthase promoter
activity attributable to the insulin and glucose response elements is
relatively weak in comparison with the 20-30-fold induction in hepatic
fatty-acid synthase gene transcription observed when fasted rats are
refed carbohydrate or when diabetic rats are administered insulin
(5-6, 10). The inability of the proximal promoter regions to yield
in vivo rates of fatty-acid synthase gene transcription
indicated that the fatty-acid synthase gene contained unidentified
enhancer sequences that were essential to the nutritional regulation of
hepatic fatty-acid synthase gene transcription. In an earlier report we
determined that the distal region of the fatty-acid synthase gene
between Primary Hepatocyte Culture and Transfection--
Primary
hepatocytes were isolated from male Harlan Sprague-Dawley rats
(180-220 g) using the collagenase perfusion method (30). After a 4-6
h attachment period, hepatocytes were transfected using Lipofectin
reagent (Life Technologies, Inc.) in Waymouth's medium
containing 5.5 mM glucose, 0.1 µM
dexamethasone, and 1 µM insulin for 12-14 h. After
transfection cells were incubated for 48 h in Waymouth's
medium containing either 5.5 or 27.5 mM glucose,
with or without 0.1 µM dexamethasone and 1 µM insulin. Cells were harvested, and the chloramphenicol
acetyltransferase or luciferase reporter activity was quantified.
Plasmid Constructs--
The parent plasmid In Vivo DNase I Footprinting Analysis--
Nuclei from rat liver
were isolated by the method of Jump et al. (31) and stored
at In Vitro DNase I Footprinting Analysis--
Hepatic nuclear
protein extracts for use in in vitro footprinting and
electromobility shift assays were prepared from male Harlan
Sprague-Dawley rats that had been fasted for 48 h or fasted for
48 h and refed a high carbohydrate fat-free diet for 6 h (33, 25). The Electrophoretic Mobility Shift Assay--
The following
single-stranded oligonucleotides containing the candidate fatty-acid
synthase ChoRE were synthesized for use in gel shift assay:
FAS-ChoRE ( Identification of a Glucose Response Region within the Distal
Promoter of the Fatty-acid Synthase Gene--
Nuclei isolated from the
livers of rats that had been fasted for 48 h and refed a high
glucose diet for 6 h displayed greater sensitivity to DNase I
cleavage than did nuclei from fasted rats. Three DNase hypersensitivity
sites were identified in the 5'-flanking regions of the fatty-acid
synthase gene: (a) The Distal Glucose Enhancer Region of the Fatty-acid Synthase Gene
Contains a ChoRE--
Functional analyses indicated that the sequences
between
Given the high homology between the ChoRE in the L-type
pyruvate kinase and S14 genes, and the sequences located
between The ChoRE of the Fatty-acid Synthase Gene Binds a Carbohydrate
Response Factor and Imparts Carbohydrate Responsiveness--
The ChoRE
of the rat L-type pyruvate kinase and S14 genes
binds a novel hepatic carbohydrate responsive transcription factor (ChoRF) (25, 29). Electrophoretic mobility shift assays using a
partially enriched ChoRF fraction prepared from the hepatic nuclei of
fed rats revealed that the ChoRE of the rat fatty-acid synthase gene
binds the ChoRF (Fig. 5, lane
1). Protein binding activity for the fatty-acid synthase ChoRE was
equal to or even greater than for the ChoRE of L-type
pyruvate kinase or two S14 ChoRE variants that support a
glucose response (Fig. 5, lanes 1-3). In addition to
binding the novel ChoRF, the ChoRE sequence of the fatty-acid synthase
gene also interacts with USF, a transcription factor possibly involved
with the carbohydrate/insulin induction of fatty-acid synthase and
L-type pyruvate kinase (19, 34). The similarity of the
ChoRE of fatty-acid synthase to that of the L-type pyruvate
kinase gene was reinforced by the observation that the fatty-acid
synthase ChoRE effectively competed for the binding of the ChoRF to the
ChoRE of the L-type pyruvate kinase gene (Fig.
6, lanes 1, 4, and
5). The competitive action of the fatty-acid synthase ChoRE
was comparable with that observed for the ChoRE of the S14
gene (Fig. 6, lanes 4-7). On the other hand, the sterol
response element and the hepatic nuclear factor-4 response element were
ineffective as competitors for ChoRF binding (Fig. 6, lanes
8-11).
Finally, in addition to binding a unique ChoRF, the ChoRE of the
fatty-acid synthase gene was found to impart carbohydrate responsiveness to a basal L-type pyruvate kinase promoter
that is otherwise unresponsive to glucose (Table
I). Specifically, when a tandem repeat of
the fatty-acid synthase ChoRE was inserted into the L-type
pyruvate kinase promoter-reporter construct ( The cis-acting elements responsible for the carbohydrate/insulin
induction of fatty-acid synthase gene transcription have been
attributed to sequences within the proximal promoter region (i.e. One approach for identifying gene regions that may play an important
role in gene transcription is to manipulate nutritional status and
examine changes in chromatin structure (27). Using this approach we
found that refeeding glucose to 48-h fasted rats induced changes in
chromatin structure within three regions of the hepatic fatty-acid
synthase gene: (a) The above observations suggested to us that perhaps the proximal
promoter region imparted insulin control to the fatty-acid synthase
promoter, while the distal sequences between The glucose-responsive element within the distal region of the
fatty-acid synthase gene appears to reside within the area of The type of transcription factor that interacts with the ChoRE of the
fatty-acid synthase gene and functions as the target for the glucose
signal is unknown, but the 5'-CACGTG sequence is a consensus
binding sequence for basic/helix-loop-helix/leucine zipper
transcription factors (28). Hasegawa et al. (41)
recently reported that a "glucose response element-binding protein"
interacted with the CACGTG motifs located in the ChoRE of rat
L-type pyruvate kinase, and in the insulin response element
( In summary, we have shown that glucose regulation of fatty-acid
synthase transcription is mediated at least in part by a distal enhancer located over 7,000 bp upstream from the transcriptional start
site. Within this enhancer a specific ChoRE binds the ChoRF and confers
glucose regulation. The ChoRF appears to work in conjunction with
SREBP-1c, and possibly USF-1 and -2, to impart glucose and insulin
responsiveness to the fatty-acid synthase promoter. Hence, regulation
of fatty-acid synthase expression in response to carbohydrate requires
both insulin and glucose signaling pathways working synergistically to
provide the overall dramatic effects observed by carbohydrate diet.
Recently, a similar situation has been found for the S14 gene (25). It is not unreasonable to speculate that many other lipogenic enzyme genes regulated by these two effectors may utilize a
similar dual regulatory pathway for glucose and insulin stimulation.
600 to +65, but the 2-3-fold increase in fatty-acid
synthase promoter activity attributable to this region is small
compared with the 20-30-fold induction in fatty-acid synthase gene
transcription observed in fasted rats refed carbohydrate. We have
previously reported that the fatty-acid synthase gene region between
7382 and
6970 was essential for achieving high in vivo
rates of gene transcription. The studies of the current report
demonstrate that the region of
7382 to
6970 of the fatty-acid
synthase gene contains a carbohydrate response element
(CHO-REFAS) with a palindrome sequence
(CATGTGn5GGCGTG) that is nearly identical to
the CHO-RE of the L-type pyruvate kinase and
S14 genes. The glucose responsiveness imparted by
CHO-REFAS was independent of insulin. Moreover,
CHO-REFAS conferred glucose responsiveness to a
heterologous promoter (i.e. L-type pyruvate kinase). Electrophoretic mobility shift assays demonstrated that CHO-REFAS readily bound a unique hepatic ChoRF and that
CHO-REFAS competed with the CHO-RE of the
L-type pyruvate kinase and S14 genes for ChoRF
binding. In vivo footprinting revealed that fasting reduced
and refeeding increased ChoRF binding to CHO-REFAS. Thus, carbohydrate responsiveness of rat liver fatty-acid synthase appears to
require both insulin and glucose signaling pathways. More importantly, a unique hepatic ChoRF has now been shown to recognize glucose responsive sequences that are common to three different genes: fatty-acid synthase, L-type pyruvate kinase, and
S14.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
444/
278,
278/
131, and
120/
50 (15,
18-26). Functional studies have revealed that the sequence between
70 and
57 imparts insulin responsiveness to the fatty-acid synthase
promoter (19), while the region of
99 to
92 may contain the cAMP
target (15). Transcription factors that interact with the insulin
response region include
USF-1,1 USF-2, SREBP-1, NF-Y,
and Sp1 (19-26). Moreover, the hepatic content of SREBP-1 and the DNA
binding activity of Sp1 are increased by insulin and glucose ingestion
and decreased by fasting (20, 26). In addition, the binding of SREBP-1
to its DNA recognition sequence within the insulin response region of
fatty-acid synthase may enhance the binding of Sp1 to its response
element at
80, which may in turn enhance the interaction of NF-Y with
its recognition sequence at
90 (14, 24).
7382 and
6970 contained elements that imparted to the
fatty-acid synthase promoter a rate of transcriptional activity that
was comparable with that observed in fasted-refed rats (27). Sequence
analysis revealed that this region contained a 17-base pair segment
that was homologous to the ChoRE of the L-type pyruvate
kinase and S14 genes (28). This observation led us to
hypothesize that the 17-base pair segment within the distal enhancer
region of the fatty-acid synthase gene is a functional ChoRE and that
this candidate ChoRE may bind the recently identified ChoRF (25,
29).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
250FAS.PGL2 was
described previously (14). The fatty-acid synthase gene 5'-deletion
series were inserted in a unique BamHI site of the
250FAS.PGL2 to generate
7382FAS.LUC,
7242FAS.LUC, and
7150FAS.LUC (27). The 2×FASChoRE.PK construct was created by
inserting into the HindIII site of PK(
96)LUC (25) two
copies of an oligonucleotide containing the fatty-acid synthase sequence from
7218 to
7191 flanked on each side by a
HindIII site. To prepare the
7382/
6970FAS.PK construct,
the fragment
7382/
6970 was excised from a previously described
construct (27) with HindIII and inserted into
PK(
96)LUC.
80 °C until use. The digestion of nuclei with DNase I was
performed with slight modifications of the method described previously
(27). DNA (200 µg) was incubated with 0.25 unit of DNase I at
30 °C for 10 min in a total volume of 400 µl. The digestion with
proteinase K was performed at 37 °C overnight without RNase A
digestion. The in vivo footprinting method was based on that
of Mueller and Wold (32). The sequences of primer sets are:
5'-CCTCACTATCTGCACCTCCC-3', 5'-TGGCATCTCTGGGCTGCTGTACC-3', and
5'-GGCATCTCTGGGCTGCTGTACCTTAGC-3' for the reverse reaction. The
linker sequence was identical to that used by Mueller and Wold (32).
After extracting DNA, the first primer extension was performed in the
following condition: 5 µg of cleaved genomic DNA, 5 pmol of primer,
40 mM NaCl, 10 mM Tris-Cl (pH 8.9), 5 mM Mg2SO4, 0.01% gelatin, 0.2 mM dNTPs, and 0.5 unit of Vent DNA polymerase (New England
Biolab) in a total volume of 30 µl. The linker ligation was performed
at 14 °C overnight after mixing the following: 30 µl of the first
primer extension mix, 100 pmol of linker, 3 units of T4 DNA ligase and
its buffer (Life Technologies, Inc.) in a final volume of 75 µl. DNA
precipitated from the ligation reaction with ethanol was dissolved in
20 µl of H2O. Polymerase chain reaction
amplification was performed in the following reaction mix: 3 µl of
the ligated DNA, 0.3 µM concentration each of the second gene-specific primer and the linker primer, 200 µM
concentration each of dNTPs, 1.75 mM
MgCl2, 1.2 units of Taq DNA polymerase and its
buffer (Promega) in the total volume of 23 µl. The temperature program used was 30 cycles of 95 °C for 20 s followed by
75 °C for 2 min. The third gene-specific primer (1.7 pmol/sample)
was end-labeled using T4 polynucleotide kinase (United State
Biochemical) and [
-32P]ATP (PerkinElmer Life
Sciences, 6,000 Ci/mmol). The polymerase chain
reaction-amplified DNA was labeled in the following reaction mixture: 5 µl of the polymerase chain reaction reaction mix,
40 nM concentration of the end-labeled primer, 100 nM concentration each of fresh dNTPs, 0.25 unit of
Vent and its buffer (New England Biolab) in final volume of 25 µl.
The reaction was repeated twice at 77 °C for 10 min after denaturing
at 95 °C for 1 min. The labeled DNA was separated by denaturing
polyacrylamide gel electrophoresis using a gel with 6% polyacrylamide,
42% urea, 0.8 mm thick and 40 cm long. The DNA fragments were
visualized by autoradiography after drying the gel.
7282/
6970 fragment of the fatty-acid synthase gene was
released as a NcoI/HpaII fragment. The antisense
strand was labeled by filling in the NcoI site using Klenow
fragment in the presence of [
-32P]dCTP. Labeled DNA
(~50,000 cpm) was incubated on ice for 10 min with 1 µg of
poly(dI-dC) and various amounts of nuclear protein extracts in a final
volume of 50 µl (25 mM Tris-HCl (pH 8), 50 mM
KCl, 6.25 mM MgCl2, 0.5 mM EDTA,
10% glycerol, 0.5 mM dithiothreitol). Subsequent to the
addition of 50 µl of 5 mM CaCl2 and 10 mM MgCl2, the binding reaction was treated at
room temperature (22 °C) with 1 unit of RQ1 RNase-free DNase I
(Promega). The reaction was stopped after 1 min by addition of 90 µl
of 83 mM EDTA-EGTA and 5 µl of proteinase K (10 µg/µl). After a 30-min incubation at 42 °C the reaction was
ethanol-precipitated and the precipitate resuspended in 80% formamide.
The DNA fragments were separated by electrophoresis using a 6%
polyacrylamide, 7 M urea sequencing gel.
7221/
7190),
5'-CTTCCTGCATGTGCCACAGGCGTGTCACCCTC-3' and
3'-GAAGGACGTACACGGTGTCCGCACAGTGGGAG-5'. Complementary strands were annealed by combining equal amounts of each
oligonucleotide, heating to 95 °C for 5 min, and cooling to
25 °C. The annealed oligonucleotide was end-labeled with
[
-32P]ATP using T4 polynucleotide kinase and incubated
on ice with nuclear protein extract for 30 min in 10 mM
HEPES (pH 7.9), 75 mM KCl, 1 mM EDTA, 5 mM MgCl2, 5 mM dithiothreitol, and
10% glycerol. A typical reaction contained 50,000 cpm of labeled
oligonucleotide (0.5-1 ng). For the experiments shown in Figs. 4 and
5, electrophoretic mobility shift assay was performed as
described previously (29) using a fraction of liver nuclear
extract enriched for ChoRF binding by heparin-Sepharose and salmon
sperm DNA-Sepharose chromatography. After incubation samples were
subjected to electrophoresis on a 5% nondenaturing polyacrylamide gel
in a Tris-glycine buffer system. DNA-protein complexes were visualized
by autoradiography after drying the gel.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7382 to
6970; (b)
600 to
400; and (c)
100 to +50 (27). Although the proximal sites (b and c) correspond to the regions that
are involved with the gene's response to insulin (18, 19, 22), these
regions do not appear to impart glucose responsiveness to the
fatty-acid synthase promoter (Fig. 1,
note
7150 and
250). However, transfection-reporter analyses using
primary cultures of rat hepatocytes revealed that constructs that
included the distal region of
7382 to
6970 with the proximal
insulin elements yielded 7-10-fold more promoter activity than did
constructs containing only the insulin response region of the proximal
promoter (27). More importantly, the region between
7382 and
6970
imparted glucose responsiveness to the fatty-acid synthase promoter
(Fig. 1). Specifically, hepatocytes that had been transfected with a
luciferase reporter construct that linked the distal enhancer sequences
of
7382 to
6970 with the proximal insulin response element of the
fatty-acid synthase promoter expressed 2-fold more luciferase when the
medium contained 27.5 mM glucose than when it contained
only 5.5 mM glucose. More importantly this response was
independent of insulin. Deleting the sequences between
7382 and
7242 had no effect on the glucose stimulation of fatty-acid synthase
promoter activity. However, deleting the region between
7242 and
7150 completely eliminated the glucose induction of the fatty-acid
synthase promoter.
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Fig. 1.
Glucose induces rat fatty-acid synthase
promoter activity. Rat primary hepatocytes were transfected with
luciferase reporter constructs containing 5'-flanking deletions of the
rat fatty-acid synthase gene. Cells were cultured in 5.5 (open
bars) or 27.5 mM glucose (solid bars) for
48 h in absence (A) or presence (B) of 1 µM insulin and 0.1 µM dexamethasone. The
data are expressed as -fold increase in luciferase activity over 5.5 mM glucose. The data represent the means ± S.E. of
three to four independent experiments with three replicate
transfections per experiment. *, p < 0.05.
7242 and
7150 of the fatty-acid synthase gene contained a
glucose response element (Fig. 1). In vitro footprinting
using nuclear protein extracts from fed rats revealed that the sequence
between
7240 and
7120 contained six sites that were protected from
DNase I cleavage (Fig. 2). The protected
site between
7214 and
7190 (sites IV and V) contains the palindrome
5'-CATGTG(n5)GGCGTG-3' that has a high degree of
sequence similarity with the ChoRE of the S14 and
L-type pyruvate kinase genes (Fig.
3) (28). The protected site between
7150 and
7125 (site I) contains a CCAAT box, which electrophoretic
mobility shift and supershift assays revealed bind the transcription
factor, C/EBP
(data not shown). In addition, this protected area
also contains a TGGA(n6)GCCAA sequence that may
be a recognition sequence for NF-1. The two footprints between
7180
and
7170 (sites II and III) and the footprint between
7235 and
7220 (site VI) do not appear to contain sequences that correspond to
any known nuclear transcription factors.
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Fig. 2.
In vitro footprint of the
fatty-acid synthase gene between 7240 and
7120. The fragments
of the fatty-acid synthase gene between
7190 and
7120
(A) and
7240 and
7180 (B) were subjected to
in vitro DNase I footprinting as described under
"Experimental Procedures." The radiolabeled probes were incubated
with DNase I plus 0 (lanes N), 10, and 50 µg of liver
nuclear proteins extract (lanes 1 and
2). The DNase I protected regions are marked by the
brackets at the right and are described in
C.
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Fig. 3.
The fatty-acid synthase ChoRE sequence
similarity with the ChoRE sequence of L-type pyruvate kinase and
S14 genes.
7214 and
7190 of the fatty-acid synthase gene (Fig. 3), we
hypothesized that nuclear protein binding to this site would change
with fasting and fasting-refeeding. In vivo footprint
analysis of the fatty-acid synthase gene between
7350 and
7130
revealed that refeeding fasted rats a high glucose diet resulted in
protection of the candidate ChoRE site from DNase I cleavage (Fig.
4, sites 2 and 3).
Fasting reinstated DNase I cleavage within the ChoRE (Fig. 4). Fasting
also resulted in protein binding at two sites: a,
7210 to
7190 and b,
7170 to
7160 (Fig. 4). Interestingly, site a appears to involve sequences that are components of
the ChoRE, and site b was equivalent to site 4 of
the fed. Site 1 in the fed animals remains to be identified,
but site 5 corresponds to the C/EBP
site (Fig. 4).
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Fig. 4.
In vivo DNase I footprint analysis
of the rat liver fatty-acid synthase gene region of 7350/
7130.
Nuclei isolated from liver of fasted and fasted-refed rats were
digested with DNase I as described under "Experimental Procedures."
The figure represents the DNase I footprint/hyper sites of the coding
strand from
7350 to
7130. The end points of protected regions shown
in boxes were identified by chemical sequencing
(G/A and C/T lanes). Only regions
showing clear appearance/disappearance of bands were considered. Areas
affected by fasting are denoted by a and b
(solid boxes); sequences affected by refeeding are labeled
as 1, 2, 3, 4, and
5 (open boxes). Sequence 1 binds an unknown
factor(s); a and 2 and 3 sequences
represent the ChoRE; sequence b and 4 bind
unknown factors; and sequence 5 is the C/EBP
binding site.
*, denotes hypersensitivity sites.
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Fig. 5.
The fatty-acid synthase ChoRE ( 7214/
7198)
binds ChoRF. Electrophoretic mobility shift assays employed
the following radiolabeled oligonucleotides: fatty-acid synthase
(
7214/
7198) (lane 1), rat L-type pyruvate
kinase ChoRE (
171/
142) (lane 2), rat S14
ChoRE (
1448/
1422) (lane 3), "mut 3/5" ChoRE
(lane 4), and "mut 3-6" ChoRE (lane 5). The
"mut 3/5" ChoRE is derived from the rat S14 ChoRE (34)
and the "mut 3-6" ChoRE is derived from the mouse S14
ChoRE (29). Each oligonucleotide was labeled to approximately the same
specific activity and used with partially enriched ChoRF fraction from
rat liver (29). Arrows indicate the ChoRF and the USF
complexes.
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Fig. 6.
The fatty-acid synthase ChoRE competes for
the binding of the ChoRF to the L-type pyruvate kinase ChoRE.
Competition experiments were conducted with the L-type
pyruvate kinase (L-PK) ChoRE as the radiolabeled
probe and cold ChoRE from the fatty-acid synthase (FAS) and
the rat S14 genes; a consensus sterol response element
(SRE-1) (43) or the hepatic nuclear factor 4 (HNF4) binding site from the L-type pyruvate
kinase promoter ( 146/
124). The competing oligonucleotides were
added as 25× and 50× molar excess to the binding reaction.
96/+12) PK.LUC (lacking
the PK ChoRE sequence), luciferase expression was increased nearly
5-fold; and when ligated to (
40/+12) PK.LUC, luciferase activity was
increased 30-fold (Table I). The greater glucose induction with the
(
40/+12) PK.LUC construct was due to the fact that the
40 has lower
promoter activity than does the
96 L-type pyruvate kinase
promoter when hepatocytes are maintained in a low glucose media.
Finally, the enhancer activity of the fatty-acid synthase ChoRE was
very comparable with that of the S14 ChoRE (Table I).
The ChoRE of fatty-acid synthase confers glucose responsiveness
7382/
4606m FAS construct contains a mutation in the CHO-RE site as
indicated by bold characters TCTAGA CTACA GGCGTG
(wild type CATGTG CCACA GGCGTG). The data are representative of two to
three independent experiments with three replicate transfection per
experiment.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
644 to
50) of the gene (18-22). Of particular
interest has been the sequence between
68 and
57. This area
contains an E-box binding site for USF-1 and -2 and a binding site for SREBP-1c that involves sequences on either side of the E-box (14, 23).
Disruption of either the USF-1 or USF-2 genes reduced the hepatic
abundance of fatty-acid synthase mRNA to a level that was only
10-15% of that found in wild type mice (35). Moreover, an early
report by Sul and co-workers (19) indicated that the DNA binding
activity and protein abundance of USF-1 was reduced by fasting and
increased by carbohydrate refeeding. However, subsequent studies have
clearly demonstrated that the abundance of hepatic USFs is not altered
by nutritional manipulations (7). In contrast to USF, the hepatic
abundance of SREBP-1c was rapidly induced by refeeding fasted rats a
high glucose diet, and by administering insulin to diabetic rats, or by
treating rat hepatocytes in primary culture with insulin (36-38). This
induction of hepatic SREBP-1c expression was paralleled by an increase
in hepatic fatty-acid synthase gene transcription (20). The importance
of SREBP-1c to fatty-acid synthase gene transcription was further
suggested by the observation that disrupting the binding site for
SREBP-1c without affecting the E-box site for USF eliminated the
insulin/glucose activation of the fatty-acid synthase promoter (24).
Thus, the nuclear abundance of SREBP-1c and the binding of SREBP-1c to
its recognition sequences within the area of
68 to
57 appear to be
important determinants for hepatic fatty-acid synthase promoter activity. However, disruption of the SREBP-1 gene only partially prevented the increase in hepatic fatty-acid synthase mRNA that results from feeding fasted rats a high glucose diet (27). This suggested that nutritional regulation of fatty-acid synthase gene transcription required factors other than SREBP-1c. Using transgenic mice to characterize 5'-flanking sequences between
2100 and +67, Sul
and co-workers (19, 21) recently discovered that a USF binding site at
332 and an SREBP-1 binding site at
150 of the fatty-acid synthase
gene were essential for insulin and carbohydrate regulation of the
gene. Moreover, the investigators proposed that the region between
444 and +67 is sufficient to replicate the in vivo insulin
and carbohydrate induction of fatty-acid synthase gene transcription.
However, a close examination of their data reveals that the
transcription of the native fatty-acid synthase gene is ~2-3-fold
greater than that observed for the reporter gene. Thus elements
critical to the in vivo regulation of fatty-acid synthase
gene transcription appear to be located outside of the proximal
promoter region.
7300 and
6900, (b)
600
to
400, and (c)
150 to +50 (27). Surprisingly, the
region between +283 and +303, which reportedly contains a glucose
response element (17), did not display a DNase I cleavage site (27). Consistent with this observation we were also unable to demonstrate that the +283 to +303 region imparted glucose responsiveness to the
fatty-acid synthase
promoter.2 The two DNase I
hypersensitivity sites between
600 and +50 corresponded to the
regions within the fatty-acid synthase promoter, which contain insulin
response sequences (18-22). Moreover, insulin treatment of hepatocytes
transfected with constructs containing this region of the proximal
promoter region increased fatty-acid synthase promoter activity
2-3-fold, but the proximal promoter region failed to confer glucose
responsiveness to the fatty-acid synthase promoter (Fig. 1). On the
other hand, when the distal region of
7382 to
6970 was linked to
the proximal sequences of the fatty-acid synthase gene, fatty-acid
synthase promoter activity was increased 3-4-fold (27), and the
transcriptional activity of the promoter was comparable with the rate
of gene transcription observed in rats fed a high glucose diet (5-9).
Interestingly, the 3-4-fold enhancement of fatty-acid synthase
promoter activity contributed by the sequences between
7382 and
6970 was approximately that amount required for the proximal promoter
region of
444 to +67 to achieve in vivo rates of
fatty-acid synthase gene transcription (19, 21, 27).
7382 and
6970
conferred carbohydrate stimulation to the promoter. Consequently the
sequences between
7382 and
6970 were examined more closely for
their role in the glucose induction of fatty-acid synthase gene
transcription. Functional mapping studies indicated that the
7382 to
6970 region contained glucose-responsive sequences. More importantly,
the glucose enhancement of fatty-acid synthase promoter activity
appeared to be independent of insulin (Fig. 1). The ability of glucose
to enhance fatty-acid synthase promoter activity by an
insulin-independent mechanism is consistent with early observations
showing that the feeding of fructose to diabetic rats induced hepatic
fatty-acid synthase gene expression (39, 40).
7242 to
7150 (Fig. 1). Sequence analysis revealed that this 100-nucleotide region contains candidate recognition sites for several transcription factors, including C/EBP
and NF-1, but the
sequence located between
7240 and
7190 was of particular interest,
because it possesses characteristics that are similar to the ChoRE of
the L-type pyruvate kinase and S14 genes. Like the ChoRE of the L-type pyruvate kinase and S14
genes, the E-box motif of the ChoRE for fatty-acid synthase exists as
palindromic sequences that are separated by a 5-bp spacer (Fig. 3). In
the case of the fatty-acid synthase ChoRE, one motif contains a five out of six nucleotide match and the other displays a four out of six
nucleotide match with the ChoRE of the other two genes. Mutation
analysis of the rat S14 and L-type pyruvate
kinase genes indicates that the first 4 bp (CACG) of the E-box are
critical to conferring glucose responsiveness, while mutations in the
fifth and sixth positions did not disrupt the glucose response (41). Spacing between the E-boxes is critical to the function of the ChoRE,
because shortening the spacer to 4 bp resulted in a loss of the glucose
response, and lengthening the spacing sequence to 6 bp blunted the
glucose response (28).
71 to
50) of the fatty-acid synthase gene. However, this unknown
protein interacted weakly with the ChoRE of the rat S14
gene and did not interact at all with comparable motifs in the
acetyl-CoA carboxylase and citrate lyase genes. Moreover the insulin
response element between
71 and
50 is not a glucose response
sequence for the fatty-acid synthase gene (Fig. 1). In contrast we
found that the unique ChoRF of rat liver nuclei (25, 29) readily
interacted with the ChoRE L-type pyruvate kinase,
S14, and fatty-acid synthase genes (Fig. 6). While the
identity of ChoRF is unclear, it has now been shown to recognize
specific glucose-responsive sequences common to three genes:
L-type pyruvate kinase, S14, and fatty-acid
synthase. It also recognizes variant versions of these elements, such
as the mut3/5 oligonucleotide (Fig. 6), that retain glucose
responsiveness but have greatly reduced binding affinity for USF. On
the other hand, mutations of these naturally occurring ChoREs that fail to support a response to glucose do not bind ChoRF. Likewise, ChoRF
does not bind to a consensus sterol response element or the adenovirus
major late promoter USF binding site. Based on these correlations,
ChoRF appears to be a strong candidate for mediating the effects of
glucose on lipogenic gene expression. At present the identity of ChoRF
is unknown, but it does not appear that ChoRF is identical to SREBP-1c
or USF. The migration of the ChoRF complex is significantly slower than
the migration of either of these factors. In addition, antibodies to
USF or SREBP-1 fail to inhibit binding of ChoRF to the ChoRE (25). We
thus propose that ChoRF is a novel hepatic factor, most likely of the
basic/helix-loop-helix family, that is regulated by changes in glucose metabolism.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK 53872 (to S. D. C.), DK 26919 (to H. C. T.), and DK 09723 (to M. T. N.); by the Universidad Nacional Autonoma De Mexico (to M. T.-G.); and by the sponsors of the M. M. Love Chair in Nutritional, Cellular, and Molecular Sciences at The University of Texas at Austin (to S. D. C.).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: 115 Gearing, The University of Texas at Austin, Austin, TX 78712. E-mail: stevedclarke@mail.utexas.edu.
Published, JBC Papers in Press, March 28, 2001, DOI 10.1074/jbc.M100461200
2 C. Rufo, M. Teran-Garcia, M. T. Nakamura, and S. D. Clarke, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
USF-1 and -2, upstream stimulatory factors 1 and 2;
SREBP, sterol regulatory element
binding protein;
NF-Y, nuclear factor Y;
Sp1, stimulatory protein 1;
ChoRF, carbohydrate response factor, ChoRE, carbohydrate response
element;
c/EBP, CAAT enhancer-binding protein
;
LUC, luciferase;
S14, spot 14;
NF-1, nuclear factor 1;
FAS, fatty-acid
synthase.
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