THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 275, No. 17, Issue of April 28, pp. 12497–12502, 2000
Printed in U.S.A.

Transcriptional Regulation of the ATP Citrate-lyase Gene by Sterol
Regulatory Element-binding Proteins*(Received for publication, January 7, 2000, and in revised form, February 2, 2000)

Ryuichiro Sato‡§¶, Akihiro Okamoto§, Jun Inoue§, Wataru Miyamoto§, Yuko Sakai§,
Noriaki Emoto储, Hitoshi Shimano**, and Masatomo Maeda§
From the ‡Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The
University of Tokyo, Tokyo 113-8657, §Laboratory of Biochemistry and Molecular Biology, Graduate School of
Pharmaceutical Sciences, Osaka University, Osaka 565-0871, the 储International Center for Medical Research, Kobe
University School of Medicine, Kobe 650-0017, and the **Department of Metabolic Diseases, Faculty of Medicine, The
University of Tokyo, Tokyo 113-8655, Japan

In an attempt to identify unknown target genes for
SREBP-1, total RNA from a stable Chinese hamster
ovary cell line (CHO-487) expressing a mature form of
human SREBP-1a (amino acids 1– 487) with a LacSwitch
Inducible Mammalian Expression System was subjected
to a polymerase chain reaction subtraction method. Oneof the fragments was found to have 90 and 86% homology
with rat and human ATP citrate-lyase (ACL) cDNA, respectively. When Hep G2 cells are cultured under either
sterol-loaded or -depleted conditions, expression of thegene is induced approximately 2–3-fold by sterol depletion. To investigate the direct effect of SREBP-1a ontranscription, luciferase assays using the promoter ofthe human ACL gene were performed. These deletion
studies indicated that a minimum 160-base pair segment
contains the information required for the transcriptional regulation brought about by enforced expressionof SREBP-1a. Luciferase assays using mutant reporter
genes revealed that SREBP-dependent transcriptional
regulation is mediated by two nearby motifs, the
SREBP-binding site (a TCAGGCTAG sequence) and the
NF-Y-binding site (a CCAAT box). It was confirmed bygel mobility shift assays that recombint SREBP-1a binds
to the sequence. Data from studies with transgenic miceand reporter assays show that the ACL gene promoter isactivated by SREBP-1a more strongly than SREBP-2 incontrast to the HMG CoA synthase and LDL receptor
gene promoters, which exhibit the same preference forthe two factors. Therefore, SREBPs transcriptionallyregulates ACL enzyme activity, which generates the cytosolic acetyl CoA required for both cholesterol andfatty acid synthesis.

Two SREBPs,1 designated SREBP-1 and -2, regulate thetranscription of a number of genes encoding enzymes and proteins involved in cholesterol or fatty acid metabolism (1).
SREBPs are synthesized as 125-kDa membrane-bound precur-* This work was supported by grants from the Ministry of Education,
Scinece, Sports, and Culture of Japan. The costs of publication of thisarticle were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: 1-1-1 Yayoi, Bunkyo,
Tokyo 113-8657, Japan. E-mail: aroysato@mail.ecc.u-tokyo.ac.jp.
1
The abbreviations used are: SREBP, sterol regulatory elementbinding protein; ACL, ATP citrate-lyase; HMG CoA, 3-hydroxy-3-methylglutaryl Coenzyme A; IPTG, isopropyl ␤-D-thiogalactopyranoside;
LDL, low density lipoprotein; PCR, polymerase chain reaction; SRE,
sterol regulatory element; CHO, Chinese hamster ovary; bp, basepair(s).
This paper is available on line at http://www.jbc.org

sors and are activated by releasing the transcriptionally active
NH2-terminal portion in a two step proteolysis (2– 4). Studies
using transgenic mice overexpressing either a mature form of
SREBP-1 or SREBP-2 have revealed that SREBP-1 mainly
regulates lipogenic gene expression and that SREBP-2 regulates cholesterogenic gene expression (5, 6). This characterization is further supported by our previous findings that
SREBP-2 gene expression is controlled by the intracellularcholesterol level and that this transcription is directly regulated by SREBPs through the SREBP-binding site in the promoter region (7).
Unlike SREBP-2, two isoforms of SREBP-1 (SREBP-1a and
SREBP-1c) are produced by alternate promoters (8). SREBP-1a
has a longer and therefore more potent transactivation domain
at the extreme NH2 terminus than SREBP-1c. RNA protection
assays have revealed that the SREBP-1c is more abundant
than SREBP-1a in most mouse and human tissues (9). However,
SREBP-1c has been reported to be weaker than SREBP-1a instimulating the transcription of target genes in in vitro assays
because of its shorter transactivation domain (10, 11).
In the current study we have established a CHO cell linetransiently expressing a more potent transcription factor, human SREBP-1a, to identify unknown target genes for
SREBP-1. In these cells expression of exogenous SREBP-1a isrepressed until an inducer, isopropyl ␤-D-thiogalactopyranoside (IPTG), is added to the medium. When the cells are cultured with a medium containing sterols and IPTG, this systemenables genes induced in response to transient expression of
SREBP-1a to be picked up, independent of the effects of endogenous SREBPs inactivated by sterols in the medium. Using asubtraction PCR method, we have subsequently managed toobtain several gene fragments induced by SREBP-1a. We havecharacterized the 5⬘-flanking region of one of these genes, thehuman ACL gene, and defined the region responsible for SREBPdependent regulation. Furthermore, we have examined with
reporter assays and transgenic mice expressing either
SREBP-1a or SREBP-2 which isoform, SREBP-1 or -2, is preponderantly involved in regulation.
EXPERIMENTAL PROCEDURES

Materials—Hygromycin B, G418, and lipoprotein-deficient serum
were purchased from Sigma. Restriction enzymes were obtained from
New England Biolabs.
Construction of an Expression Plasmid for Human SREBP-1a—To
generate a construct of human SREBP-1a containing amino acids
1– 487, designated pOPI3SREBP1, a 1.5-kilobase pair fragment wasmade by PCR including NotI restriction sites at both ends. The NotINotI PCR-generated fragment was ligated into the pOPI3CAT vector(Stratagene).
Stable Cell Lines—A lac repressor expression plasmid, p3⬘SS (Stratagene), was stably transfected into CHO cells. On day 0, 1.5 ⫻ 105 CHO

12497

12498Transcriptional Regulation of ACL by SREBPs

FIG. 1. The human ACL promoter
sequence (13). The transcription startsite is ⫹1. Three putative SRE sites and a
CCAAT sequence are boxed. The sites
used for preparation of truncated reporter
gene constructs are indicated by arrows.
The mutant sequence in either the SRE
sites or the CCAAT box is shown by italic
letters under the individual original
sequence.

cells were seeded into each 60-mm dish with medium A (Dulbecco’s
modified Eagle’s medium/Ham’s F12 medium, 100 units/ml penicillin,and 100 ␮g/ml streptomycin) supplemented with 7% fetal calf serum.
On day 1, each monolayer was transfected with 3 ␮g of the plasmidusing a calcium phosphate precipitation method. On day 2, the cells
were trypsinized and reseeded into three 100-mm dishes. On day 4, thecells were refed with medium A supplemented with 7% fetal calf serumand hygromycin (400 ␮g/ml). Visible clones were picked up, subcloned,
and maintained in the same medium. One of the clones expressing thelac repressor was established and designated CHO-Lac. The CHO-Lac
cells were further transfected with pOPI3SREBP1 and cultured with amedium containing hygromycin and G418 (500 ␮g/ml) to obtain the
CHO-487 cell line as described above.
PCR Subtraction—On day 0, 7 ⫻ 105 cells (CHO-Lac or CHO-481)
were seeded into 100-mm dishes with medium A supplemented with 7%
fetal calf serum. On day 3, the cells were refed with the same medium
containing 1 ␮g/ml 25-hydroxycholesterol, 10 ␮g/ml cholesterol, and 1
mM IPTG. After 19 h of incubation, the cells were harvested and total
RNA was prepared. Poly(A) RNA was purified with an oligo(dT) column(Amersham Pharmacia Biotech). According to the procedures describedin a previous paper (12), PCR fragments induced by SREBP-1a were
subcloned into a Bluescript vector. To pick up real positive fragments,
Northern blot analyses on CHO-487 were carried out using each PCR
fragment as a probe, and nucleotide sequences for inducible fragments
were determined.
Construction of the Reporter Genes for Luciferase Assays—The luciferase reporter plasmids were constructed by cloning the BglII-HindIII
PCR fragments coding the 5⬘-flanking region of human ACL gene (13)into the same restriction sites of a pGL3 basic vector (Promega). To
generate ACL-300, ACL-251, ACL-174, ACL-151, ACL-131, ACL-94,
ACL-60, and ACL-30, PCR primers were designed to hybridize at thecorresponding position (Fig. 1) and were coupled with the common
downstream primer from nucleotide ⫹29. To disrupt one of three putative SREBP-binding sites within ACL-131 (the sites, SREa, SREb, andSREc), the megaprimers were amplified with the upstream primer withfour to six mutations (GCCCTG 3 GAATTC at the SREa, AGGCTA 3
GAATTC at the SREb, TCGCCG 3 GAATTC at the SREc) and thecommon downstream primer, and further extended in the 2nd PCR as
described previously (14). The expression plasmids, pSREBP1(1– 487)
and pSREBP2(1– 481), and reporter plasmids, pHMG S containing a
0.5-kilobase human HMG CoA synthase promoter and pLDLR containing a 1.5-kilobase human LDL receptor promoter, have been described
previously (15, 16).
Tissue Cultures and Cell Transfection—HEK 293 cells were cultured,and cell transfection, luciferase, and ␤-galactosidase assays were performed as described previously (14 –16).
Northern Blot Analysis for Stable CHO Cells and Hep G2 Cells—
Total RNA from CHO-Lac and CHO-487 cells were prepared as described above. Hep G2 cells were set up on day 0 (1.8 ⫻ 106 cells/
100-mm dish) in medium B (Dulbecco’s modified Eagle’s medium, 100
units/ml penicillin, and 100 ␮g/ml streptomycin) supplemented with 7%
fetal calf serum. On day 1, the medium was removed, and the cells werethen washed with phosphate-buffered saline and refed with medium B
containing 5% lipoprotein-deficient serum supplemented either with 1
␮g/ml 25-hydroxycholesterol plus 10 ␮g/ml cholesterol or with a 50 ␮Mconcentration of a HMG CoA reductase inhibitor (pravastatin) plus 50
␮M sodium mevalonate. After 48 h of culture, total RNA was extracted
and fractionated on formaldehyde/agarose gels and then transferred to
Nylon membranes (Roche Molecular Biochemicals). Riboprobes wereprepared using CHO ACL cDNA, human S17 cDNA (from 1 to 370 bp)(17) and human HMG CoA synthase (from 576 to 867 bp) (18) with adigoxigenin RNA labeling kit (Roche Molecular Biochemicals). Hybridization signals were quantified with AttoPhos (Amersham Pharmacia
Biotech) using a FluorImager 595 (Molecular Dynamics).
Gel Mobility Shift Assay—A double-stranded DNA fragment corresponding to nucleotides ⫺118 to ⫺102 of the ACL gene was 3⬘ endlabeled with a digoxigenin-11-ddUTP using a digoxigenin gel shift kit(Roche Molecular Biochemicals). The digoxigenin-labeled probe wasincubated with 50 ␮g of recombinant SREBP-1a in binding buffer. The
reaction conditions have been described previously (15). In competition
assays, an excess amount of an unlabeled 17-bp fragment containing
SREb in the human ACL promoter or the mutant SREb was added priorto addition of the labeled probe. Recombinant SREBP-1a (1– 487) containing six consecutive histidines were expressed using a pET28 vector(Novagen) in Escherichia coli and purified by nickel-nitrilotriacetic acidagarose affinity chromatography (Qiagen).
Transgenic Mice Study—Transgenic mice were as described previously (19, 20). Animals of each group (n ⫽ 3) were fed a high protein/low
carbohydrate diet for 2 weeks and fasted overnight prior to sacrifice. An
equal aliquot of liver total RNA was pooled (15 ␮g) and subjected to
Northern blot analysis using cDNA probes for mouse HMG CoA Synthase, LDL receptor and ACL (21, 22). The resulting bands were quantified by exposure of the filters to BAS2000 with BAStation software(Fuji Photo Film, Co., Ltd, Japan), and the results were normalized tothe signal generated from 36B4 mRNA.
RESULTS

Expression of ACL mRNA Is Highly Up-regulated by
SREBP-1a in CHO-487 Cells—For the establishment of CHO487, an expression plasmid containing human SREBP-1a (1–487) under the control of the Rous sarcoma virus promoter andlac operator was introduced into CHO cells constitutively expressing the Lac repressor (CHO-Lac). After 19 h of incubationof the cells with 1 mM IPTG, we examined the SREBP-1a
inducibility by RNase protection assays. In CHO-487 cells
SREBP-1a mRNA was slightly detectable even in the absenceof IPTG but was increased approximately 10-fold by IPTG
(data not shown). To carry out the subtraction PCR, we prepared total RNA from CHO-487 and CHO-Lac cells cultured
with sterols plus 1 mM IPTG. It should be noted that sterols
decrease the amount of the endogenous nuclear form ofTranscriptional Regulation of ACL by SREBPs
SREBPs in both cell lines and that IPTG increases exogenous
SREBP-1a (1– 487) in CHO-487 cells. We obtained several PCR
fragments induced by SREBP-1a including fatty acid synthase,
HMG CoA synthase, and stearoyl CoA desaturase-2 cDNA. Oneof these fragments was found to have 90 and 86% homology
with rat and human ACL cDNA, respectively (data not shown),suggesting that this fragment encodes CHO ACL. Fig. 2 showsthat expression of the mRNA for HMG CoA synthase wascompletely down-regulated in CHO-Lac cells and was inducedin CHO-487 cells only by exogenous SREBP-1a. Expression of
ACL mRNA was approximately 7-fold higher in CHO-487 cells.
Unlike HMG CoA synthase, expression of ACL mRNA was notcompletely suppressed even in the presence of the high concentration of sterols.

FIG. 2. Northern blot analysis for the ACL and HMG CoA synthase gene in CHO-Lac and CHO-487 cells. CHO-Lac (A) and CHO487 (B) cells were cultured with 1 ␮g/ml of 25-hydroxycholesterol, 10
␮g/ml of cholesterol, and 1 mM IPTG for 19 h. 20-␮g total RNA samples
were fractionated on 1% agarose gel, transferred to nylon membrane,
and hybridized with a digoxigenin-labeled riboprobe for ACL, HMG
CoA synthase, or S17. The fold change in ACL mRNA, relative to thatin CHO-Lac cells, was calculated after correction for loading differenceswith S17. Signals were quantified with a FluorImager 595. In three
separate experiments the same relative mRNA levels were obtained.

FIG. 3. ACL mRNA expression is regulated by cellular cholesterol levels. Hep G2 cells were cultured with medium containing 5%
lipoprotein-deficient serum supplemented with either 1 ␮g/ml of 25hydroxycholesterol plus 10 ␮g/ml of cholesterol (sterol-loaded conditions) or 50 ␮M of a HMG CoA reductase inhibitor, pravastatin plus 50
␮M of sodium mevalonate (sterol-depleted conditions) for 48 h. Northernblot analysis was carried out as described in the legend to Fig. 2.

FIG. 4. Regulation of ACL promoter-luciferase reporter genes by
SREBP-1a(1– 487). HEK 293 cells weretransfected with one of human ACL promoter reporter genes (200 ng), a plasmid
encoding ␤-galactosidase (100 ng), and an
expression plasmid (10 ng), pSREBP1(1–487) for 4 h. The cells were incubated for
48 h and then lysed, and enzyme activities were determined. The ratio of luciferase activity in relative light units is divided by the ␤-galactosidase activity (U,units) to give a normalized luciferase
value (relative light units/unit). The values given are the averages of data from
more than three experiments performedin triplicate.

12499

Expression of ACL mRNA Is Regulated by Sterols in Hep G2
Cells—To determine whether expression of ACL mRNA is regulated by the intracellular cholesterol level, human hepatoma
cells, i.e. Hep G2 cells, were cultured under either sterol-depleted or -loaded conditions, and Northern blot analysis was
performed. Under the sterol-depleted conditions, expression of
ACL mRNA was up-regulated 2–3-fold (Fig. 3), indicating thatthe induction is not restricted in CHO-487 cells. Taken together, it is likely that the expression of the ACL gene iscontrolled by the intracellular cholesterol level through theaction of SREBPs.
SREBP-1a Directly Regulates the Transcription of Human
ACL Gene—To determine whether SREBP-1a directly regulates the transcription of ACL gene, we carried out luciferase
assays using reporter genes including various deletion versionsof the human ACL 5⬘-flanking region. HEK 293 cells were
cotransfected with one of the reporter genes and either an
expression plasmid for human SREBP-1a (1– 487), pSREBP1(1– 487), or an empty expression vector. Because a previous
report demonstrated that all the minimal response elements
for the ACL gene expression are localized within the human
ACL promoter ⫺300-bp region (13), we focused on the promoter
activity of the first 300 bp 5⬘ to the transcription start site. Fig.
4 clearly shows that enforced expression of SREBP-1a enhances the luciferase activities from 5- to 15-fold as long as thereporter gene contained the segment from ⫺131 to ⫹29 bp (thetranscription start site is position ⫹1). We also obtained thesame results when the cells were cultured under either steroldepleted or -loaded conditions (data not shown). Although it
was found that expression of ACL mRNA in transgenic miceoverexpressing either SREBP-1 or SREBP-2 was accelerated, it
has been remained unclear whether this elevation is a direct
effect of SREBPs action. This is, therefore, the first evidence
demonstrating that SREBP-1 directly regulates the transcription of the ACL gene.
The Elements Responsible for the SREBP-mediated Transcriptional Regulation of the ACL Gene—To identify the sequence motifs in the region (from ⫺131 to ⫹29 bp) responsible
for the SREBP-mediated transcriptional regulation of the ACL
gene, we further carried out luciferase assays using various
mutant versions of reporter genes. We found three putative
SREBP-binding sites in the region (SREa, SREb, and SREc in
Fig. 1) and mutated each of them to generate mutant versionsof reporter genes. Furthermore, we mutated a CCAAT se-

12500Transcriptional Regulation of ACL by SREBPs

FIG. 5. Effect of the mutation of the
SRE or the NF-Y binding site on theexpression of reporter gene. HEK 293
cells were transfected and cultured as described in the legend to Fig. 4. The foldactivation (luciferase activity with
SREBP-1 versus without SREBP-1) isshown. The luciferase activities obtainedby the reporter genes were in the range of
400 –1000 relative light units/unit. The
values given are the averages of data from
more than three experiments performedin triplicate.

quence, which is recognized by a ubiquitous transcription factor, NF-Y, to determine whether NF-Y is involved in the transcriptional regulation of the human ACL gene as well as
farnesyl diphosphate synthase, HMG CoA synthase, squalene
synthase, and SREBP-2 genes (14, 15, 23). Mutation of either
the SREb or the CCAAT sequence resulted in a significant
suppression of the SREBP-dependent induction of luciferase
activities, whereas a more than 10-fold induction of luciferase
activity by SREBP-1a was still observed even after the mutation of either the SREa or the SREc segment (Fig. 5). These
results demonstrate that both the SREb and CCAAT sequence
are important for transcription regulation.
SREBP-1a Binds to the SREb Site—To confirm that the
SREb sequence is recognized by SREBP-1a, gel mobility shift
assays were performed with recombinant SREBP-1(1– 487). As
shown in Fig. 6, a single-shifted DNA-protein complex was
observed in the presence of recombinant SREBP-1a (lane 2).
The band almost completely disappeared in the presence of anexcess amount of an unlabeled wild-type probe but not a mutant probe (lanes 3 and 4). These results clearly show that
SREBP-1a is capable of binding the TCAGGCTAG sequence in
the ACL promoter.
ACL Gene Expression Is Preferentially Induced by
SREBP1a—To assess the induction of ACL gene expression by
SREBP-2, the luciferase assays were performed using an expression plasmid, pSREBP2(1– 481). Fig. 7 shows that
SREBP-2 is capable of stimulating the luciferase activities in adose-dependent manner but that its effect is much less potent
than that of SREBP-1a. Because we could not rule out thepossibility that the amounts of expressed SREBP-1a and -2
proteins are not equal in each dose of transfected DNA, theinduction of luciferase activities driven by either the human
HMG CoA synthase or the human LDL receptor gene promoter
was also investigated. The induction of both HMG CoA synthase and LDL receptor gene expression by SREBP-1a isslightly higher but almost comparable with that by SREBP-2.
These results indicate that SREBP-1a is a more potent activator of ACL gene expression.
Expression of mRNA for the ACL, HMG CoA Synthase, andLDL Receptor Gene in the Livers of Wild-type and Transgenic
Mice—It has been reported that expression of ACL mRNA in
the livers of SREBP-1a transgenic (TgBP-1a) and SREBP-2
transgenic (TgBP-2) mice is enhanced (6). In the current experiment, expression of mRNA for the ACL, HMG CoA synthase, and LDL receptor genes in the livers of wild-type andtransgenic mice were carefully investigated after 2-week feedings of a high protein/low carbohydrate diet followed by over-

FIG. 6. SREBP-1 binds to the TCAGGCTAG sequence in the
ACL promoter. Double-stranded DNA fragment corresponding to nucleotides ⫺128 to ⫺112 was 3⬘ end-labeled with a digoxigenin-11ddUTP. In lanes 1 and 2, the reaction mixture was incubated without orwith recombinant SREBP-1a(1– 487). In competition assays, a 1000-foldmolar excess of an unlabeled 17-bp fragment (SREb, a wild-type fragment; SREb KO, a mutant fragment with GAATTC instead of AGGCTA
in the SREb) was added prior to addition of the labeled probe (third andfourth lanes).

night fasting to induce the transgene and to minimize theeffects of endogenous SREBPs. The levels of mRNA for the
HMG CoA synthase and LDL receptor gene were almost thesame in the TgBP-1 and 2 mice, whereas ACL gene expression
was tremendously stimulated only in the TgBP-1a mice (Fig.
8). These findings are entirely consistent with the results ofreporter assays in Fig. 7.
DISCUSSION

The current assay system using a cell line inducibly expressing nuclear SREBP-1a (CHO-487) enabled us to identify a newtarget gene for SREBP-1a, the ACL gene. The transient expression system driven by IPTG is critical for seeing the effects oftranscription factors that are usually activated only in the
short term. Western blot analyses reveal that the level of
SREBP-1a protein induced by IPTG is within the physiologicalrange in CHO-487 cells (data not shown). In addition, at least
three known sterol-responsive genes were identified by the
PCR subtraction method in these cells, suggesting that thesystem functions correctly and that ACL is also one of thephysiological targets of SREBPs. Furthermore, the fact thatACL gene expression is also stimulated in Hep G2 cells when
the cells are cultured under the sterol-depleted conditions (Fig.
3) supports this.
When the 5⬘-flanking region of the rat ACL gene (24) isTranscriptional Regulation of ACL by SREBPs

12501

FIG. 7. Differential sensitivity of
the ACL, HMG CoA synthase, andLDL receptor promoters to overexpressed SREBP-1a or SREBP-2. HEK
293 cells were transfected with one ofreporter genes (ACL-131, pHMG S, andpLDLE; 200 ng), a plasmid encoding
␤-galactosidase (100 ng), and an indicated amount of expression plasmid,pSREBP1a(1– 487) or pSREBP2(1– 481),
for 4 h. The cells were incubated for 48 hand then lysed, and enzyme activities
were determined. The fold activation (luciferase activity in the presence of
SREBP-1a or SREBP-2 versus in the absence) is shown. The values given are theaverages of data from three experiments
performed in triplicate.

FIG. 8. Quantification of mRNA levels of ACL, HMG CoA synthase, and LDL receptor in the livers from wild-type (WT),TgBP-1a, and TgBP-2 mice as measured by Northern blot analysis. The values are fold changes versus corresponding wild-type signals that are arbitrarily set at 1. Animals of each group (n ⫽ 3) were fed
a high protein/low carbohydrate diet for 2 weeks and fasted overnightprior to sacrifice. An equal aliquot of liver total RNA was pooled (15 ␮g)and subjected to Northern blot analysis using a 32P-labeled cDNA probe
for mouse HMG CoA synthase, LDL receptor, and ACL. The resulting
bands were quantified by exposure of the filters to BAS2000 with
BAStation software, and the results were normalized to the signal
generated from 36B4 mRNA (21, 22).

compared with that of the human gene, both the SREb sequence and the CCAAT box with the 13-bp spacing are highly
conserved. The current study shows that ACL gene expression
is enhanced by SREBP-1 in CHO cells and the livers of transgenic mice as well as human Hep G2 cells. Taken together, it islikely that transcription of ACL gene is regulated by a combination of SREBPs and NF-Y across species. Several genes,
including SREBP-2, farnesyl pyrophosphate synthase, squalene synthase, and HMG CoA synthase have been shown to be
regulated by the same combination of transcription factors (14,15, 23). The distances between the two binding sites for SREBP
and NF-Y in the promoter of these genes are all in the 15–21-bp
spacing range that has been shown to give the highest induc-tion of transcription by SREBP-2 in in vitro reporter assays(14). It may be possible that SREBP-2 requires this 15–21-bp
spacing for the regulation of these genes involved in cholesterolsynthesis but that SREBP-1 has a shorter necessary or ideal
spacing for the induction of genes involved in fatty acid metabolism, including ACL and glycerol-3-phosphate acyltransferasewith their 13- and 9-bp spacing, respectively. Interestingly, theSREb sequence (TCAGGCTAG) in the human ACL gene resembles the SRE motif (TCAGCCTAG) observed in the mouseglycerol-3-phosphate acyltransferase gene most closely among
known SRE sequences (25).
ACL as well as acetyl CoA carboxylase and fatty acid synthase has been demonstrated to be induced during lipogenesis
in the liver and belongs to a class of lipogenic enzymes. Recent
studies with SREBP-1 knockout mice demonstrated that
SREBP-1 plays a crucial role in nutritional induction of lipogenic enzymes including ACL (21, 22). In the absence of
SREBP-1, the ACL mRNA level was profoundly suppressed
despite concomitant activation of SREBP-2. This suggests that
SREBP-1 is a major physiological regulator for ACL in theliver. It is consistent with our obtained result that SREBP-1
rather than SREBP-2 predominantly stimulates ACL gene expression in the transfection studies. However, residual ACL
mRNA level was still found in the livers of SREBP-1 knockout
mice that had been refed after fasting. In contrast, in a fasted
state with both SREBP-1 and -2 suppressed, ACL mRNA wascompletely absent irrespective of the presence of SREBP-1,
suggesting some significant contribution of SREBP-2 to ACL
gene transcription in a nonfasted state. Because ACL is acytosolic enzyme that generates and provides acetyl CoA for
both cholesterol and fatty acid synthesis de novo, it might bereasonable that ACL gene transcription can be maintained in asterol-regulatory fashion under the control of SREBP-2, eventhough the main regulation of ACL is mediated by SREBP-1 ina lipogenic regulatory manner. When CHO-Lac cells were cultured with sterols, there was a marked decline in HMG CoA
synthase mRNA, but a much smaller decline in ACL mRNA
(Fig. 2). This finding also support the concept that basal transcription of the ACL gene is driven primarily by factors otherthan SREBPs, whereas the HMG CoA synthase gene is more
dependent on SREBPs, presumably on SREBP-2 in particular.
It has been reported that insulin activates expression ofmRNA for ACL as well as other lipogenic enzymes. Recently,
several investigations have demonstrated that SREBP-1c expression is transcriptionally stimulated by insulin (26, 27) andthat the insulin-dependent hepatic expression of lipogenesisrelated genes including fatty acid synthase, acetyl CoA carboxylase, S14, and L-pyruvate kinase is mediated by SREBP-1c

12502

Transcriptional Regulation of ACL by SREBPs(28). It is therefore probable that hepatic ACL gene expressionis under the control of SREBP-1c, a predominant form in theliver and mediator of insulin action. Intensive analyses of the
rat ACL promoter sequence revealed that the region from ⫺104
to ⫺20 of the gene is responsible for regulation because of
insulin (29, 30). However, this region does not contain thecorresponding SREb site required for sterol-mediated regulation. It therefore remains unclear whether the SREBP action
through the SREb motif might be involved in the insulin-dependent stimulation of transcription. In our recent study wefound that a putative insulin response element in the humanmicrosomal triglyceride transfer protein gene promoter contains a functional SRE sequence (16). Because both insulin and
SREBPs negatively regulate microsomal triglyceride transfer
protein gene expression, SREBP-1c, which is believed to respond to insulin, might partly mediate insulin action underphysiological conditions. Further work is required to elucidate
the mechanism by which ACL gene expression is regulated by
the insulin cascade and the SREBPs.
In this study we have demonstrated that ACL is a new target
gene for SREBP-1 using CHO-487 cells transiently expressing
an active form of SREBP-1a. It has been reported that SREBPs
activate the transcription of a number of genes encoding enzymes involved in cholesterol and fatty acid synthesis. It isnoteworthy that the SREBPs, especially SREBP-1, can further
regulate these two pathways by governing the synthesis of an
initial common substrate, acetyl CoA, by a regulation of ACL
gene expression.
Acknowledgment—We thank Dr. Kevin Boru for reviewing themanuscript.
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