THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 41, pp. 34577–34589, October 14, 2005
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Sterol Regulatory Element-binding Proteins Activate
Insulin Gene Promoter Directly and Indirectly through
Synergy with BETA2/E47*
Received for publication, June 21, 2005 Published, JBC Papers in Press, July 29, 2005, DOI 10.1074/jbc.M506718200

Michiyo Amemiya-Kudo‡, Junko Oka‡, Tomohiro Ide§, Takashi Matsuzaka§, Hirohito Sone§, Tomohiro Yoshikawa¶,
Naoya Yahagi¶, Shun Ishibashi¶, Jun-ichi Osuga¶, Nobuhiro Yamada§, Toshio Murase‡, and Hitoshi Shimano§1
From the ‡Okinaka Memorial Institute for Medical Research, Toranomon Hospital, Toranomon 2-2-2, Minato-ku, Tokyo 105-8470,
Japan, ¶Department of Diabetes and Metabolic Diseases, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan,and §Department of Internal Medicine (Endocrinology and Metabolism), Graduate School of Comprehensive Human Sciences andCenter for Tsukuba Advanced Research Alliance, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8575, Japan
Insulin gene expression is regulated by pancreatic ␤ cell-specificfactors, PDX-1 and BETA2/E47. Here we have demonstrated thatthe insulin promoter is a novel target for SREBPs established as
lipid-synthetic transcription factors. Promoter analyses of rat insulin I gene in non-␤ cells revealed that nuclear SREBP-1c activatesthe insulin promoter through three novel SREBP-binding sites(SREs), two of which overlap with E-boxes, binding sites for BETA2/
E47. SREBP-1c activation of the insulin promoter was markedly
enhanced by co-expression of BETA2/E47. This synergistic activation by SREBP-1c/BETA2/E47 was not mediated through SREs but
through the E-boxes on which BETA2/E47 physically interacts with
SREBP-1c, suggesting a novel function of SREBP as a co-activator.
These two cis-DNA regions, E1 and E2, with an appropriate distanceseparating them, were mandatory for the synergism, which implicates formation of SREBP-1c䡠BETA2䡠E47 complex in a DNA looping structure for efficient recruitment of CREB-binding protein/p300. However, in the presence of PDX1, the synergistic action of
SREBP-1c with BETA2/E47 was canceled. SREBP-1c-mediatedactivation of the insulin promoter and expression became overt in ␤cell lines and isolated islets when endogenous PDX-1 expression
was low. This cryptic SREBP-1c action might play a compensatoryrole in insulin expression in diabetes with ␤ cell lipotoxicity.
Insulin gene expression is restricted to pancreatic ␤ cells and is
induced by glucose (1). Extensive studies on the insulin promoter have
unveiled ␤ cell-specific transcription factors responsible for glucoseinducible transcription, such as PDX-1, BETA2/E47, and MafA, andhave located their respective binding sites, A3, E1, and C1 (2–7). It has
been well documented that PDX-1 plays a major physiological role inglucose-regulated expression of the insulin gene (2, 8, 9) in addition toits important role in the development and differentiation of pancreatic
islets. BETA2, a ␤ cell-specific basic helix-loop-helix (bHLH)2 protein is* This work was supported by grants-in-aid from the Ministry of Science, Education,
Culture, and Technology of Japan. The costs of publication of this article weredefrayed in part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
1
To whom correspondence should be addressed: Dept. of Internal Medicine, Graduate
School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba Ibaraki, Japan 305-8575. Tel.: 81-29-853-3053; Fax: 81-29-853-3174; E-mail:shimano-tky@umin.ac.jp.
2
The abbreviations used are: bHLH, basic helix-loop-helix; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; HEK, human embryonic kidney;
Luc, luciferase; SREBP, sterol regulatory element-binding protein; CMV, cytomegalovirus; GST, glutathione S-transferase; ChIP, chromatin immunoprecipitation; ␤-gal,
␤-galactosidase; TA, transactivation domain; HIT, hamster insulinoma tumor cells.

OCTOBER 14, 2005 • VOLUME 280 • NUMBER 41

also an important regulator of the insulin gene (3) and of pancreaticdevelopment (10). The pancreas of BETA2-null mice fail to develop
mature islets, and animals become diabetic (10). BETA2 heterodimerizes with E47, a ubiquitous bHLH (3), the gene disruption of whichcauses no apparent phenotype in ␤ cells (11). The rat insulin promoter
I, the most studied insulin gene promoter, contains E2- and A3/4-boxes.
BETA2/E47 binds to E2-box and PDX-1 binds to A3/4. PDX-1, BETA2/
E47, and HMGI(Y) physically interact (12) and show synergistic activation of insulin gene expression by facilitating recruitment of coactivator
CBP/p300 (13, 14).
Sterol regulatory element-binding proteins (SREBPs) are members of
the basic helix-loop-helix leucine zipper family of transcription factorsthat regulate fatty acid and cholesterol synthesis (reviewed in Refs. 15
and 16). SREBPs are initially bound to the rough endoplasmic reticulum
membrane. By virtue of the SREBP cleavage-activating protein/Insig
system, SREBP is cleaved in a sterol-dependent manner to liberate theamino-terminal portion containing a basic helix-loop-helix leucine zipper domain (nuclear SREBP), which enters the nucleus where it can bindto specific sterol response elements (SREs) in the promoters of target
genes. Three isoforms of SREBP, -1a, -1c, and -2, are known. Although
SREBP-2 plays a crucial role in the regulation of cholesterol synthesis,
SREBP-1c controls the gene expression of lipogenic enzymes (reviewed
in Refs. 17 and 18). Nuclear SREBP-2 has a high affinity with classic
sterol regulatory elements, which are usually found in the promoters ofcholesterogenic genes and the LDL receptor gene. Nuclear SREBP-1
also has broad binding capacity to SRE-like sequences, includingE-boxes that are occasionally found in the promoters of lipogenic
genes (19).
SREBP-1c has been well established as controlling the nutritional
regulation of lipogenic genes in the liver (20). However, the role of
SREBP-1c in pancreatic ␤ cells is yet to be elucidated. Because an excess
of fatty acids or triglycerides could impair the functions of ␤ cells, a
potential pathophysiologic consequence of diabetes, often referred to as
lipotoxicity, estimation of SREBP-1c in ␤ cell function is an important
issue to be addressed. It has been reported that insulin-resistant obese
animals have a high expression of SREBP-1c in ␤ cells (21). A ␤ cell line
in which nuclear SREBP-1c is overexpressed showed impaired insulin
secretion (22–26). Overall, these data support the potential involvementof SREBP-1c in the lipotoxicity theory of ␤ cells. However, the effect of
SREBP on insulin gene expression, especially in the context with interaction with ␤ cell-specific transcription factors, has never been investigated. In the current study, we performed promoter analysis on rat
insulin promoter I to investigate the potential effects of SREBPs on
insulin gene expression in both non-␤ and ␤ cell lines.

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Activation of Insulin Promoter by SREBPs
Transfection and Luciferase Assays—HEK293 cells (3.5 ⫻ 104 cells/well),
HepG2 cells (4.2 ⫻ 104 cells/well), and HIT cells (3.5 ⫻ 105 cells/
Standard molecular biology techniques were used. We obtained cholesterol and 25-hydroxycholesterol from Sigma, Redivue [␣-32P]dCTP well) were plated on 12-well plates. Each expression plasmid (0 – 0.5(6000 Ci/mmol) from Amersham Biosciences, and restriction enzymes ␮g), luciferase reporter plasmid (0.5 ␮g), and pSV-␤-gal (0.5 ␮g) werereagent (Qiagen) forfrom Takara Bio Inc. Plasmid DNAs for transfection were prepared with co-transfected using SuperFect transfection
TM
HEK293
and
HepG2
cells
and
using
JetPEI-Man(Polyplus-transfecEndoFree Plasmid Midi kits (Qiagen).
tion)
for
HIT
cells.
The
total
amount
of
DNA
in
each
transfection was
Expression Plasmids—Expression vectors encoding rat PDX-1, rat␮g/well.
The
luciferase
activityintransfectantswasadjustedto
1.5–2.0
BETA2, and rat E47 were generated by PCR amplification and insertion
measuredbyMicroLumat
Plus(Berthold)andnormalized
tothe␤-gaof the cDNAs into the cytomegalovirus (CMV)-driven vector (pCMV7)(27). The following primers were used: PDX-1-(1–284), 5⬘-primer 5⬘- lactosidase activity as measured by standard kit (Promega).
Gel Mobility Shift Assays—Gel shift assays were performed as previATGAACAGTGAGGAGCAGT-3⬘, 3⬘-primer 5⬘-TCACCGGGGTTously described (30). Recombinant SREBPs (SREBP-1c and -2) (Fig. 1E)
CCTGCGG-3⬘; BETA2-(1–357), 5⬘-primer 5⬘-ATGACCAAATCATand nuclear extracts from HEK293 cells transfected with pCMVACAGCGAGAG-3⬘, 3⬘-primer 5⬘-CTAATCGTGAAAGATGGCATSREBP-1c, pCMV-SREBP-1cM, or pCMV-⌬TA-SREBP-1c-Myc (Fig.
3⬘; E47-(1– 647), 5⬘-primer 5⬘-ATGAACCAGTCTCAGAGAATG-3⬘,
6B) were used. The DNA probes prepared by annealing both strands
3⬘-primer 5⬘-TCACAGGTGCCCGGCTGGGTTGT-3⬘; BETA2⌬ADwere as follows: SRE1/E1-box, 5⬘-TCAGCCCCTCTCGCCATCTGC2-(1–299), 5⬘-CTAAAAGGCATAATTTTTTTCAAACTC-3⬘; BETACTACCTA-3⬘; SRE2, 5⬘-GAACTGCTTCATCAGGCCATC-3⬘; E22⌬AD1AD2-(1–170), 5⬘-CTACTGTACAAAGGAGACGAG-3⬘; BETbox, 5⬘-GGCCCATCTGGCCCCTTGTTAAC-3⬘; and SRE2/E2-box,
A2⌬bHLH-(169 –357), 5⬘-GTACAGACACTCTG-3⬘; E47⌬AD15⬘-GGACTGCTTCATCAGGCCATCTGGCCCCTTGTTAATAAT(178 – 647), 5⬘-CCTGGTCTTCCTTCCTCG-3⬘; E47⌬AD1AD2CTAATTACCCTAGGTCTAAC-3⬘.
(510 – 647), 5⬘-GAAGACAAGAAGGACCTG-3⬘; E47⌬bHLHThe 32P-labeled probe and proteins were incubated in 15 ␮l of bind(1–537), 5⬘-TCACAGGTCCTTCTCCTC-3⬘. CMV expression plasmids
ing buffer (10 mM Tris-HCl, pH 7.6, 50 mM KCl, 0.05 mM EDTA, 2.5 mM
encoding human nuclear SREBPs (pCMV-SREBP-1a, -1c, and -2) (28),
MgCl2, 8.5% glycerol, 1 mM dithiothreitol, 0.5 mg/ml poly(dI-dC), 0.1%
Tyr3 Arg mutated versions (pCMV-SREBP-1aM, -1cM, and -2M) (19)
Triton X-100, and 1 mg/ml nonfat milk) for 30 min on ice. Myc or
and pCMV-⌬TA-SREBP-1c, which lacks the amino-terminal transactiSREBP antibodies were preincubated with proteins for 60 min before
vation domain (1–90) of SREBP-1c (20), were previously described.
adding the labeled probes. The DNA䡠protein complex was resolved on a
Reporter Plasmids—The reporter plasmid Ins715-Luc contains a
4.6% polyacrylamide gel. Gels were dried and exposed to the Bioimaging
fragment of the rat insulin I gene promoter from ⫺715 to ⫹31 bp cloned
Analyzer System station software (Fuji Photo Film).
into the SmaI site of pGL2 basic vector (Promega) containing the coding
In Vitro Protein-Protein Interaction Assay—[35S]Methionine-labeledsequences of firefly luciferase cDNA. Other constructs were produced
proteins, luciferase, SREBP-1c, ⌬TA-SREBP1c, BETA2, and E47 wereby PCR using this construct as a DNA template, and the PCR products prepared using the in vitro translation transcription system (Promega).
were inserted into pGL2 basic vector. The primers used for PCR were as Labeled protein (3 ␮l) was mixed with 1 ␮l of Myc antibody bound to 5
follows: 5⬘-primer Ins715-Luc, 5⬘-TCTCAACTCCTTGAAAATAGC- ␮l of protein G-Sepharose beads (Amersham Biosciences) in 200 ␮l of
TACCT-3⬘; Ins646-Luc, 5⬘-GCTGTGCTACTGAGGCCTGATG-3⬘; interaction buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA,
Ins265-Luc, 5⬘-GGTACCTGATTGTGCTGTGAACTGCT-3⬘; Ins118- 0.5% Nonidet P-40, 10 ␮g/ml leupeptin, 1 mM phenylmethylsulfonyl
Luc, 5⬘-GGTACCTCTCGCCATCTGCCTACC-3⬘; Ins104-Luc, 5⬘-GG- fluoride) and incubated for 1 h at 4 °C. The beads were washed three
TACCTACCCCTCCTAGAGCCCTT-3⬘; SRE2 (m)-Luc, 5⬘-GGTACC- times with interaction buffer. The bound proteins were eluted with 30
TGTGAACTGCTTCATAAAGCCATCTGGCCC-3⬘; E2 (m)-Luc, ␮l of sample buffer (1% SDS, 100 mM dithiothreitol, 50 mM Tris-HCl,
5⬘-GGTACCTGTGAACTGCTTCATCAGGCCATCAAGCCCCTTGT- pH 7.5, 0.1% Bromothymol Blue) and heated 95 °C for 5 min. The boundTAAT-3⬘; del SRE2-Luc, 5⬘-GCCATCTGGCCCCTTGTTAAT-3⬘, 3⬘- proteins (15 ␮l) were subjected to SDS-PAGE and autoradiography.
primer 5⬘-AAGCTTGTAGCTGGTCACTTAGGGTT-3⬘. Restriction
GST Binding Assay—GST fusion proteins, GST alone, GSTsites KpnI and HindIII were added to each 5⬘-primer and 3⬘-primer, SREBP-1c (nuclear form-(24 – 460)), GST-SREBP-1c (bHLH-(286 –respectively.
364)) (31) were expressed in Escherichia coli and purified according to
The site-directed mutagenesis constructs, SRE1(m)-Luc and E1(m)- the manufacturer’s recommendations (Promega). [35S]Methionine-laLuc were produced by PCR with the following primers: SRE1 (m)-5⬘, beled E47 (bHLH)-(498-634)) was prepared using the in vitro transla5⬘-GCTAGCCATCTGCCTACCTACCC-3⬘; SRE1 (m)-3⬘, 5⬘-GCTA- tion transcription system (Promega). GST precipitation assays wereGCGGGGCTGAAGCTGTAATT-3⬘; E1 (m)-5⬘, 5⬘-GGTACCAGGC- modified as previously reported (12, 32).
CTACCTACCCCTC-3⬘; E1 (m)-3⬘, 5⬘-AGGCCTATGGCGAGAPreparation of Nuclear Extracts—Nuclear extracts from HEK293,
GGGGCTGAA-3⬘. The constructs lacking the intervening sequences HIT, and INS1 cells were performed as described by Hua et al. (33). Two
between E1 and E2 regions were produced as follows: The fragment was hours prior to cell collection, ALLN (N-acetyl-Leu-Leu-norleucinalamplified by 5⬘-primer, 5⬘-CCCTCTCGCCATCTG-3⬘ and 3⬘-primer, aldehyde, calpain inhibitor I) (Calbiochem) (25 ␮g/␮l) was added to the5⬘-AAGCTTGTAGCTGGTCACTTAGGGTT-3⬘ using Ins265-Luc as cells. After collection, the cells were washed with phosphate-buffereda template and was inserted to the pGL2 basic vector. Restriction sites saline and suspended in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5
NheI and HindIII were added to each primer. 50-bp- and 86-bp-deleted mM MgCl2, 1 mM EDTA, 1 mM EGTA). Pellets were passed through afragments were amplified by 5⬘-primer 5⬘-GGTACCTGATTGTGCT- 26-gauge needle eight times and centrifuged at 1,600 ⫻ g for 5 min. The
GTGAACTGCT-3⬘ and 3⬘-primer, 5⬘-GGGTAATTAGATTAT- supernatant was centrifuged at 55,000 rpm for 20 min, and the memTAA-3⬘ (del 86) or 3⬘-primer, 5⬘-GCGCTCATTGGACGTCA-3⬘ (del brane-containing pellets were suspended in membrane buffer (50 mM
50). These fragments were inserted into the above vector. Restriction Tris-HCl, pH 8.0, 2 mM CaCl2, 80 mM NaCl, 1% Triton X-100). The
sites KpnI and NheI were added to each primer. Rat insulin II gene pellet containing the nuclei was suspended in buffer C (20 mM Hepes,
pH 7.9, 25% glycerol, 0.4 M NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM
promoter InsII251-Luc was previously described (29).

MATERIALS AND METHODS

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Activation of Insulin Promoter by SREBPs

FIGURE 1. Insulin gene promoters are novel
SREBP targets. A, schematic representation of therat insulin I and II genes and human insulin genepromoters with the sequences and location ofnovel SRE elements (SRE1, SRE2, and SRE3) andknown E-box motifs. SRE1 and SRE2 elements partially overlap with the E1- and E2-boxes. SRE3 has
an inverted consensus SRE sequence. B, the comparison of relative rat insulin promoter I activities.
HEK293 cells were co-transfected with Ins715-Luc(0.5 ␮g) as a reporter gene, pSV-␤-gal (0.5 ␮g) as areference, and the indicated amount of CMVdriven expression plasmids PDX-1, BETA2, SREBP1a, and/or empty vector (pCMV7). The firefly luciferase activity normalized to ␤-gal activity (mean ⫾S.E.) was expressed. RLU, relative light unit; OD,
optical density at 415 nm for ␤-galactosidase. C,transcriptional activities of nuclear SREBP-1a, -1c,
-2, and respective Tyr3 Arg mutants on Ins715Luc in HepG2 cells. D, deletion studies in SREBP-1c
activation of the rat insulin I promoter. Shown isthe schematic representation of rat insulin gene I
luciferase deletion constructs used in this study.
Various reporter genes (Ins-715, -646-, -265-, -118-,
and -104-bp-Luc), pSV-␤-gal, control vector(pCMV7), or SREBP-1c expression plasmid (0.5 ␮g)were transfected in HepG2 cells. The relative Luc
activity was expressed as a fold change to that of
the control vector (pCMV7). E, gel shift assay for
SREBP binding to SRE1 and SRE2 of the rat insulin I
gene promoter. Double-stranded DNA fragments
corresponding to the E1 and E2 regions (SRE1/E1box, SRE2, and E2-box probe) were labeled with
[␣-32P]dCTP and incubated in the reaction mixture
with or without recombinant SREBP-1c and
SREBP-2. Specificity of SREBPs binding to the E1 or
E2 region probes was confirmed by a supershift
after the addition of SREBP-1 or SREBP-2 antibodies (Ab) (lanes 4, 5, 10, 15). In competition assays of
the E2 region, a 1000-fold molar excess of an unlabeled SRE2 element or E2-box probe was added(lanes 8, 9, 13, 14).

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Activation of Insulin Promoter by SREBPs

FIGURE 2. Synergistic activation of the insulin
promoter by SREBP-1c with BETA2, E47. A, thecomparison of relative insulin promoter activities(Ins265-Luc) by indicated combinations of expression plasmids (0.125 ␮g each) in HepG2 cells. B andC, in the presence of the indicated expression plasmids (0.125 ␮g each), SREBP-1c (0 – 0.5 ␮g, B) or
BETA2 (0 – 0.125 ␮g, C) was co-transfected with
Ins265-Luc and pSV-␤-gal in HepG2 cells. The relative Luc activity (mean ⫾ S.E.) normalized to ␤-gal
activity was expressed as a fold change to that of
the control vector (pCMV7).

EGTA), rotated at 4 °C for 30 min, and centrifuged at 10,000 ⫻ g for 20
min. The protein from the nuclear extract is in the supernatant. Allbuffers used contain a mixture of protease inhibitors (25 ␮g/␮l ALLN,
10 ␮g/␮l leupeptin, 2.1 ␮g/␮l aprotinin, 5 ␮g/␮l pepstatin, and 5 ␮g/␮lphenylmethylsulfonyl fluoride).
Isolation of Rat Islet and Mouse Pancreas—Pancreatic islets of Langerhans were isolated from 18-h-fasting male Sprague-Dawley rats (8weeks old) and C57Bl6 mice (12 weeks old) by perfusion of the pancreatic duct and in situ collagenase digestion (34). The islets were subsequently purified by Ficoll density gradient and by hand picking. Immediately after isolation, the islets were cultured overnight in RPMI 1640
medium containing 5.5 mM glucose with or without T0901317 (3 ␮M).
Total RNA was prepared using TRIzol reagent (Invitrogen).

34580 JOURNAL OF BIOLOGICAL CHEMISTRY

Immunoblot Analysis—Aliquots of proteins were subjected to 10%
SDS-PAGE transferred to nitrocellulose membrane (Hybond-ECL,
Amersham Biosciences) and incubated with rabbit anti-mouse
SREBP-1 or goat anti-PDX-1 (N-18) antibodies (Santa Cruz Biotechnology). Secondary antibodies were horseradish peroxidase-conjugateddonkey anti-rabbit or donkey anti-goat (Santa Cruz Biotechnology), anddetection of immunoreactive bands was performed using the ECL kit(Amersham Biosciences).
Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays were
performed as previously published with some modification (35, 36). A
total of 3.0 ⫻ 107 INS-1 cells (from two confluent 10-cm plates) was
cross-linked with formaldehyde (final concentration 1%) after washingwith phosphate-buffered saline and incubation for 15 min at room tem-

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Activation of Insulin Promoter by SREBPs

FIGURE 3. Effects of mutations in SREs andE-boxes on activation of the insulin promoter
by SREBP-1c, BETA2/E47, and PDX-1. A, schematic presentation of various luciferase reporters
containing native (Ins265-Luc) and mutant SREs
(SRE1 and SRE2) and E-boxes (E1 and E2) in E1
and E2 regions in the rat insulin I gene. The indicated mutant plasmids were constructed bysite-directed mutagenesis of Ins265-Luc (see“Materials and Methods”). Dots indicate mutant
bases. B, native and various mutant reporter
plasmids were co-transfected into HepG2 cellswith pSV-␤-gal and expression plasmids(BETA2⫹E47, PDX-1⫹BETA2⫹E47, SREBP-1c, andBETA2⫹E47⫹SREBP-1c) (0.125 ␮g each). The activity (mean ⫾ S.E.) normalized to ␤-gal activity was
expressed as a fold change to that of the control
vector (pCMV7).

perature. The reaction was stopped by adding glycine at a final concentration of 0.125 M. After 5 min, the cells were scraped and washed twotimes with cold phosphate-buffered saline with protease inhibitors (10␮g/ml leupeptin, 2 ␮g/ml aprotinin, 12.5 ␮g/ml ALLN, 2.5 ␮g/␮l pepstatin A, and 1 mM phenylmethylsulfonyl fluoride). The pellets were
resuspended in 0.5 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM
Tris-HCl, pH 8.1, plus protease inhibitors) and subjected to sonication
at setting 10 for six 10-s pulses plus setting 30 for five 10-s pulses to sharethe chromatin to 1000-bp fragments. The samples were diluted 4-foldwith immunoprecipitation dilution buffer (0.01% SDS, 1.1% Triton
X-100, 1.2 mM EDTA, 167 mM NaCl, 16.7 mM Tris-HCl, pH 8.0, plus

OCTOBER 14, 2005 • VOLUME 280 • NUMBER 41

protease inhibitors). To reduce the nonspecific binding, the samples
were incubated with 60 ␮l of sonicated salmon sperm DNA/protein
A-Sepharose slurry (Amersham Biosciences) on a rotating platform at
4 °C for 2 h. After centrifugation, the supernatant was incubated with 10
␮l of rabbit anti-mouse SREBP-1 antibody and rabbit normal IgG as anegative control and rotated overnight at 4 °C. 0.9 ⫻ 107 INS-1 cells foreach antibody were used. Immune complexes were collected by adding
20 ␮l of salmon sperm DNA/protein G-Sepharose slurry (Amersham
Biosciences) for 4 h with rotation. Samples were subsequently washedusing 1.0 ml of wash buffer A (0.1% SDS, 1% Triton X-100, 2 mM EDTA,
150 mM NaCl, 20 mM Tris-HCl, pH 8.1), wash buffer B (0.1% SDS, 1%

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Activation of Insulin Promoter by SREBPs

FIGURE 4. Both the E1 and E2 regions with an appropriate distance are crucial for the synergistic activation of rat insulin I promoter. A, deletion studies in the synergistic
activation of the rat insulin I gene promoter in HepG2 cells. The indicated promoter plasmids, pSV-␤-gal, and expression plasmids (PDX-1⫹BETA2⫹E47 and BETA2⫹E47⫹SREBP-1c)(0.125 ␮g each) were transfected. The relative Luc activity (mean ⫾ S.E.) normalized to ␤-gal activity was expressed as a fold change to that of the control vector (pCMV7) (A, B, andC, left). B, schematic representation of the comparison of the rat insulin promoters I and II in the E1 and E2 regions. The synergistic activities of Ins265-luc by the indicated expression
plasmids (0.125 ␮g each) were compared with that of the rat insulin II luciferase promoter InsII-(251)-luc in HepG2 cells. Dots indicate different bases. C, schematic representation of
Ins265-luc and mutant constructs with shorter intervening sequences (64 and 28 bp) between the E1 and E2 regions. Wild type (WT, Ins265-Luc) or deleted reporter plasmids(-50del-Luc, -86del-Luc) were transfected into HepG2 cells with pSV-␤-gal in the presence of expression plasmids (SREBP-1c, PDX-1⫹BETA2⫹E47, or BETA2⫹E47⫹SREBP-1c) (0.125 ␮geach) (left panel). In HIT cells, each reporter plasmid (0.5 ␮g) and pSV-␤-gal were co-transfected (right panel). The activities by ␤ cell-specific endogenous factors were measured.

Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl, pH 8.1),
wash buffer C (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM
EDTA, 10 mM Tris-HCl, pH 8.1), and wash buffer D (1 mM EDTA, 10
mM Tris-HCl, pH 8.0), and eluted by 30-min incubations with 0.5 ml ofelution buffer (1% SDS, 50 mM NaHCO3, 10 mM dithiothreitol). NaCl
was added to the elution at a final concentration of 0.3 M, and thesamples were incubated at 65 °C for 6 h to reverse the formaldehydeinduced cross-linking. DNA and protein were ethanol-precipitatedovernight at ⫺20 °C and dissolved in 100 ␮l of Tris EDTA buffer (pH7.4). Digestion was performed by the addition 25 ␮l of proteinase K
buffer (50 mM Tris-HCl, pH 7.5, 25 mM EDTA, 1.25% SDS) with 2 ␮g/␮l
proteinase K (2 ␮l), and samples were placed at 55 °C for 1 h. Chromatin
DNA was extracted with phenol/CHCl3 followed by ethanol precipitation. Samples were dissolved in 100 ␮l of Tris EDTA buffer (pH 7.4).
3-␮l aliquots were used for PCR analysis. To amplify the rat insulin I
promoter region containing SRE1 and SRE2, the following primer sets
were used: 5⬘-TGCTTCATCAGGCCATCTGG-3⬘ for sense and 5⬘-G-34582 JOURNAL OF BIOLOGICAL CHEMISTRYGTAGGCAGATGGCGAGAGGG-3⬘ for antisense. Primers sets for rat
fatty acid synthase promoter regions containing SRE were used as apositive control: 5⬘-GACGCTCATTGGCCTGG-3⬘ for sense and 5⬘-TCTGGAGGCAGACGACAAG-3⬘ for antisense. The PCR conditions
were 5 min at 94 °C and 30 cycles of 30 s at 94 °C, 30 s at 55 °C, and 1 min
at 72 °C. Following amplification, PCR products were electrophoresedin a 3% agarose gel and visualized by ethidium bromide staining.
Statistics—Statistical significance was assessed with the Student’s ttest for unpaired data.

RESULTS
Identification of Insulin Gene Promoters as SREBP Targets—As schematized in Fig. 1A, pancreatic ␤ cell-specific cis-elements for the insulin
promoter included E-boxes (E1 and E2) as binding sites for bHLH protein heterodimers BETA2/E47, and A-boxes (A1 and A3/4) for a
homeobox PDX-1. In the process of searching for glucose/insulin-responsive elements in promoters of nutritionally regulated genes in rela-

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Activation of Insulin Promoter by SREBPs

FIGURE 5. SREBP protein physically binds to E47
and BETA2. A, in vitro-translated, 35S-labeled luciferase (a– c), E47 (a and b), SREBP-1c (a), and/or
BETA2 (c) protein, and/or unlabeled E47 (c) wereincubated with Myc epitope-tagged BETA2(BETA2-Myc, a), SREBP-1c (SREBP1c-Myc, b and c),or TA-deleted SREBP-1c (⌬TA-BP1c-Myc, b and c).
The mixtures were immunoprecipitated with antiMyc antibody. 10% of the 35S-labeled proteinsused for the assay were resolved by SDS-PAGE as
input. B, in vitro-translated 35S-labeled E47 (bHLH(498 – 634)) was incubated with 1 ␮g of GST alone,
GST-SREBP-1c (nuclear form (24 – 460)), or GSTSREBP-1c (bHLH-(286 –364)) fusion proteins immobilized on glutathione-Sepharose beads, andbound proteins were resolved on 15% gel by SDSPAGE followed by autoradiography. 5% of the 35Slabeled E47 bHLH-(498 – 634) protein was loaded
as input.

tion to SREBP regulation, we found that the rat insulin I gene promoter
possesses three potential binding sites for nuclear SREBPs putatively
designated SRE1, SRE2, and SRE3 (Fig. 1A). Intriguingly, SRE1 and SRE2
were located adjacent to the E1- and E2-boxes with partial overlapping
sequences, respectively. The cluster of A1, E1, and SRE1 was tentatively
designated as the E1 region and the distal cluster of A3/4, E2, and SRE2
as the E2 region (Fig. 1A). SRE3 was located upstream and in a reverse
orientation. These new potential SREBP binding elements are highly

OCTOBER 14, 2005 • VOLUME 280 • NUMBER 41

conserved in rat II insulin and human insulin gene promoters (37).
Luciferase assays in non-␤ HEK293 and HepG2 cells (data not shown)demonstrated that overexpression of the active form (nuclear) of
SREBP-1a markedly and dose-dependently induced the activity of therat insulin I promoter containing these regions (Ins715-Luc) (Fig. 1B).
Strikingly, the activation of Ins715-Luc by nuclear SREBP-1a was evenhigher than by co-expression of PDX-1 and BETA2, both of which are
crucial for ␤ cell-specific and robust expression of the insulin gene (12,JOURNAL OF BIOLOGICAL CHEMISTRY

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Activation of Insulin Promoter by SREBPs
38). Among the nuclear SREBP isoforms (referred to as SREBPs, hereafter), SREBP-1a had the highest activity (Fig. 1C). SREBP-1 has beenshown to bind both SRE and some E-boxes, low stringent consensus
sequences for bHLH proteins (39). Tyr3 Arg mutants of SREBPs in
which the tyrosine residue in the basic region of SREBP are replaced byarginine conserved in regular bHLH proteins have been shown to abolish SREBP binding to SRE elements but retain binding to E-boxes (39).
All SREBP Tyr3 Arg mutants (SREBP-1aM, -1cM, and -2M) completely lost activity, supporting evidence that SREBP activation of theinsulin promoter is mediated through binding to the SRE elements andnot to the E-boxes (Fig. 1C). Sequential deletion studies demonstrated
that SRE1, SRE2, and SRE3 elements all contribute to the SREBP activation of the insulin promoter (Fig. 1D).
To evaluate the direct binding of SREBPs to these putative SRE elements, gel mobility shift assays were performed. Both recombinant
SREBP-1c and recombinant SREBP-2 bind to both SRE1/E1 box (Fig.
1E, left) and SRE2/E2 box (data not shown). The specificity of the binding was confirmed by their supershifts after the addition of SREBP-1c or
SREBP-2 antibodies. The signals from DNA probes specific to SRE2 andE2-box and competition with unlabeled probes indicate that SREBP-1
binds to SRE2 and not to E2-box (Fig. 1E, right). The binding of SREBP
to the SRE3 element was also confirmed, although with a slightly weakersignal than to SRE2 and SRE1 (data not shown). These data demonstrated that SREBPs activate the rat insulin I gene promoter throughbinding to the SRE1, SRE2, and SRE3 elements. SREBPs were also foundto bind to other insulin SREs in rat insulin I and II and human insulin
promoters (data not shown). Taken together, it was concluded that theinsulin gene is a new SREBP target gene.
Synergistic Activation of Insulin Gene by SREBP-1 with ␤ Cell-specific
Factors—Insulin gene expression is highly induced by a tertiary complexof PDX-1䡠BETA2䡠E47 riding over E-box and A-box. Considering theoverlapping location of the SREs and the E-boxes, it is possible that
potential interactions between SREBPs and BETA2/E47 could modify
the insulin promoter activity. The effects of co-expression of SREBP-1c
with these pancreatic ␤ cell-specific factors were evaluated using a luciferase construct containing the E1 and E2 regions (Ins265-Luc) in
HepG2 cells (Fig. 2A). Activation of Ins265-Luc by SREBP-1c wasenhanced by co-expression of BETA2. When both BETA2/E47 wereadded to SREBP-1c, the luciferase activity was further enhanced andwas close to the authentic maximal activation of the insulin promoter by
PDX-1/BETA2/E47 in HepG2 cells, which was reported to be comparable with the intrinsic insulin promoter activity in ␤ cells (38). Fig. 2, B
and C, shows that SREBP-1c and BETA2 dose-dependently contributeto the synergistic activation by either SREBP-1c/BETA2 or SREBP-1c/
BETA2/E47 (Fig. 2C). However, when SREBP-1c was added to theauthentic combination of PDX-1/BETA2/E47, the potent activity wasslightly diminished (Fig. 2B).
To evaluate the importance of individual SREs and E-boxes in synergistic activation by SREBP-1c and BETA2/E47 more precisely, various
mutations in the luciferase constructs were tested in HepG2 cells (Fig.
3). As shown in Fig. 3B, mutation of either the E1- or E2-box completely
abolished PDX-1/BETA2/E47 activation as previously reported (40),reconfirming that E1- and E2-boxes are essential for PDX-1/BETA2/
E47 activation. For activation by SREBP-1c alone, SRE2 considerably
contributed to its activation, as already suggested by the deletion study(Fig. 1D), but SRE1 was found to be most crucial. Synergistic activation
by SREBP-1c/BETA2/E47 was almost eliminated by the disruption ofeither the E1- or E2-box, whereas mutations in SRE1 or SRE2 only
slightly decreased. These data suggested that SREBP activation and
SREBP synergism with BETA2/E47 could be mediated through differ-

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FIGURE 6. Effects of TA deletion (⌬TA-SREBP-1) and Tyr3 Arg mutation (SREBP-1cM)
on the synergistic activation by SREBP-1c/BETA2/E47. A, schematic representation ofwild type of SREBP-1c, ⌬TA-SREBP-1 in which the amino-terminal transactivation domainof SREBP-1c was deleted, and SREBP-1cM in which arginine (R) was substituted for tyrosine (Y) residue conserved in the basic region of DNA binding sites of SREBP. Basic, DNAbinding site; HLH, helix-loop-helix structure. B, left, inhibitory effects of ⌬TA-SREBP-1 and
SREBP-1cM on the activation of Ins265-Luc by SREBP-1c. Ins265-luc, pSV-␤-gal, andpCMV-SREBP-1c (0.125 ␮g) were transfected with the indicated amounts of ⌬TASREBP-1 or SREBP-1cM (0 – 0.5 ␮g) in HepG2 cells. Right, electrophoretic mobility shift
analysis (EMSA) of DNA-binding activities of SREBP-1c, SREBP-1cM, and ⌬TA-SREBP-1.
Each of the SREBP plasmids was transfected into HEK293 cells, and nuclear proteins wereextracted. The SRE2⫹E2-box region was used as a probe. Supershift assay was performed using antibodies against SREBP-1 or Myc. C, effects of ⌬TA-SREBP-1 or SREBP1cM on the activation of Ins265-Luc by BETA2⫹E47 or SREBP-1c⫹BETA2⫹E47. The indicated amounts of ⌬TA-SREBP-1 or SREBP-1cM (0 – 0.5 ␮g) were transfected with Ins265luc and pSV-␤-gal in HepG2 cells. The relative Luc activity (mean ⫾ S.E.) was expressed asa fold change to that of the control vector (pCMV7).

ent cis-elements. Both the E1- and E2-boxes appear to be indispensable
for synergistic activation by SREBP-1c/BETA2/E47 as well as by PDX1/BETA2/E47 activation.
The Synergistic Activation of SREBP-1 and ␤ Cell-specific Factors Is
Mediated through the Inter-region Interaction between E1 and E2
Regions—Effects of sequential deletion of the E1 and E2 regions were
estimated in terms of SREBP-1c/BETA2/E47 activation in HepG2 cells.
The results highlighted the importance of the E2 region, showing that
the E1 region alone does not have any synergistic activation (Fig. 4A).
Meanwhile, from the evaluation of the construct in which distal E2
region was fused to a SV-40 promoter-driven luciferase reporter gene, it
was found that the E2 region alone did not have any enhancer activity(data not shown). All these data indicated that both the E1 and E2

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Activation of Insulin Promoter by SREBPs

FIGURE 7. Mapping of essential domains in BETA2 and E47 for the synergistic activation of insulin promoter. A, schematic domain maps of BETA2 (left) and E47 (right). Bothproteins contain two activation domains AD1 and AD2 and a bHLH domain. The bars indicate regions for the protein-protein interaction. The constructs are as follows: BETA2-(1–357),
BETA2⌬AD2-(1–299), BETA2⌬AD2AD1-(1–170), and BETA2⌬bHLH-(170 –357), E47-(1– 647), E47⌬AD1-(178 – 647), E47⌬AD1AD2-(510 – 647), and E47⌬bHLH-(1–537). B, effects ofnative or truncated BETA2 (0.125 ␮g) on Ins265-Luc activity with PDX-1⫹E47 (0.125 ␮g) or SREBP-1c⫹E47 (0.125 ␮g) in HepG2 cells. Basal activities of native and truncated BETA2
were indicated on the left. C, effects of native or truncated E47 (0.125 ␮g) on Ins265-Luc activity with SREBP-1c (0.125 ␮g), PDX-1⫹BETA2 (0.125 ␮g), or SREBP-1c⫹BETA2 (0.125 ␮g)in HepG2 cells. Basal activities of native and all truncated E47s were 1.0 (data not shown.). The relative Luc activity is expressed as a fold change to that of the control vector (pCMV7).

regions are mandatory for the synergism, implicating that SREBP-1c/
BETA2/E47 as well as PDX-1/BETA2/E47 synergistically activate theinsulin promoter through inter-region (E1 and E2 regions) interaction.
This hypothesis was supported by experiments showing that the synergistic activation was not observed in the rat insulin II promoter, which
does not have an E-box in the E2 region (Fig. 4B). To examine the impactof the length between the two regions on inter-region synergism, theeffects of deletions in the intervening sequence (hypothetical bending
part of the DNA) were evaluated (Fig. 4C). In HepG2 cells, synergistic
activation by PDX-1/BETA2/E47 or SREBP-1c/BETA2/E47 was essentially abolished in both of the constructs with shorter intervening
sequences (-50del, -86del) (Fig. 4C, left). In HIT cells, the hamster insulinoma cell line, deletion of the intervening sequence affected the insulin
promoter activity (Fig. 4C, right). Thus, the synergism required two
separate E1- and E2-boxes with a certain appropriate distance in a bending form presumably to form the complex of DNA and SREBP-1c (orPDX-1)䡠BETA2䡠E47.

OCTOBER 14, 2005 • VOLUME 280 • NUMBER 41

Direct Interaction of SREBP-1c with BETA2/E47 in Vitro—Basedupon the synergistic activation by these bHLH proteins, the physicalinteraction between SREBP-1c, BETA2, and E47 was estimated using
co-immunoprecipitation experiments. As shown in Fig. 5A, labeled invitro-translated E47 protein was co-immunoprecipitated with
SREBP-1c protein, indicating that E47 and SREBP-1c physically interact. The signal of SREBP-1c/E47 was comparable with that of authentic
BETA2/E47 formation (Fig. 5A, a). Meanwhile BETA2/SREBP-1c binding gave a very weak signal (Fig. 5A, a). However, labeled BETA2 could
be immunoprecipitated by SREBP-1c in the concomitant presence ofE47, suggesting the formation of a SREBP-1c䡠BETA2䡠E47 complex (Fig.
5A, c). ⌬TA-SREBP-1c also showed similar results, demonstrating thatinteraction of SREBP-1c with E47 is mediated through the bHLH portion of SREBP-1c and not its transactivation domain (Fig. 5A, b and c).
Pull-down assays using GST fusion proteins of the bHLH portion of
SREBP-1c and E47 also provided similar results, indicating that thebHLH portions of SREBP-1c and E47 directly interact (Fig. 5B). These

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FIGURE 8. Effects of SREBP-1c on rat insulin I
gene activity in ␤ cells. A, a, comparison of theactivation of wild type (Ins265-Luc) and its various
mutation constructs by ␤ cell-specific endogenous factors in HIT cells, the hamster insulinoma
cell line. The structure of constructs were as
described in the legend to Fig. 3A. The delSRE2Luc SRE2 element but contains SRE1, E1-, andE2-boxes. The A3/4-box (5⬘-TAATAATCTAATAC3⬘) was mutated as 5⬘-TCCTCCTCTGCTAG-3⬘. The
reporter plasmid and pSV-␤-gal were transfected
in HIT cells. The relative luminescence versus
Ins265-Luc (set as 1.0) is shown. b, activation of
Ins265-Luc and mutation constructs by SREBP-1coverexpression in HIT cells. The activity (mean ⫾S.E.) was expressed as a fold change to that of thecontrol vector (pCMV7). **, p ⬍ 0.001; *, p ⬍ 0.01
when compared with INS265-Luc (WT) activity
with pCMV7. B, Western blot analysis of endogenous SREBP-1c and PDX-1 in HIT cells induced by
glucose and T0901317. Cells were incubated withor without T0901317 (10 ␮M) for 19 h and further
incubated in 5.5, 10, and 25 mM glucose in medium
for 5 h. Membrane and nuclear proteins wereextracted. C, Ins265-Luc and pSV-␤-gal were transfected in HIT cells in the same condition as Fig. 8B.
Values are given as relative activity to Ins265-Luc
in 10 mM glucose without T0901317 (set as 1.0).
Error bars represent standard error (n ⫽ 12). N.S.,
not significant.

data suggested that SREBP-1c could form the complex with E47 andBETA2 on the insulin promoter.
SREBP-1c Is Involved in Synergistic Activation of Insulin Promoter
without DNA Binding—The mutation analysis on E1 and E2 regions
indicated that synergistic activation of the insulin promoter by SREBP1c/BETA2/E47 require both E1- and E2-boxes, rather than SRE1 and
SRE2. Thus, SREBP-1c involvement in this synergism could not be
mediated through direct DNA binding. We further examined the effectof the SREBP-1c Tyr3 Arg mutant (SREBP-1cM) (Fig. 6A). By electrophoretic mobility shift analysis, SREBP-1cM does not bind to either
SREs or E-boxes of the insulin promoter I (Fig. 6B, right). Consistent
with this, SREBP-1cM, by itself, did not activate the insulin promoter inluciferase assays (Fig. 1C). SREBP-1cM only slightly inhibited the activity of SREBP-1c in a dose-dependent manner (Fig. 6B, left). However,
SREBP-1cM robustly activated the insulin promoter synergistically with
BETA2/E47, although less efficiently than SREBP-1c (Figs. 2C and 6C).
When SREBP-1cM was added to SREBP-1c/BETA2/E47, the activation
by SREBP-1c/BETA2/E47 was further potentiated. Thus, SREBP-1cM
did not bind to the insulin promoter but still exhibited the synergistic
activation similar to SREBP-1c. These data strongly suggested that thesynergistic activation by SREBP-1c and BETA2/E47 on the rat insulin
promoter I is mediated through the direct interaction of SREBP-1c with
BETA2/E47 on the E-boxes and not through DNA binding of SREBP-1c
to SRE elements.
Activation Domains of SREBP-1, BETA2, and E47 Mediate the Synergistic Activation of the Insulin Promoter—Each domain of SREBP-1c,
BETA2, and E47 was estimated in terms of their synergistic activation of
the insulin promoter. ⌬TA-SREBP-1 (⌬TA-BP1) (Fig. 6A) has beenshown to inhibit the transcriptional activity of SREBP-1 in a competitiveway (Fig. 6B) (20, 41). By electrophoretic mobility shift analysis, ⌬TA-

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BP1 binds to SREs of the insulin promoter I (Fig. 6B, right). ⌬TA-BP1, byitself, had no activity for Ins265-Luc (data not shown). ⌬TA-BP1 partially but dose dependently inhibited the activation of Ins265-Luc by
BETA2/E47 or SREBP-1c/BETA2/E47 (Fig. 6C). These data indicatethat the transactivation domain of SREBP-1 is crucial for activation of
the insulin promoter by SREBP-1 alone and is necessary for full synergistic activation with BETA2/E47.
Fig. 7A, left, shows the function structure of BETA2. Activation of
Ins265-Luc by BETA2 alone was essentially abolished by the deletion of
AD2 or bHLH (Fig. 7B). Partial deletion of AD2 (BETA2⌬AD2-(1–299))
and lack of both AD1 and AD2 (BETA2⌬AD2AD1-(1–170)) completely
attenuated the synergistic activation, suggesting that both AD1 andAD2 are essential for the full synergistic activation of SREBP-1c/BETA2/E47 as well as for PDX-1/BETA2/E47, as previously reported (14).
Deletion of bHLH of BETA2 domains (BETA2⌬bHLH-(170 –357)),
which disrupts heterodimerization with E47, caused a marked reduction in activities in SREBP-1c/BETA2/E47 with some residual activity,
reflecting that SREBP-1c alone could bind to the SRE elements andactivate the insulin promoter. These data demonstrated that BETA2
contributes to the synergistic activation of the insulin promoter by
SREBP-1c/BETA2/E47 through AD1 and AD2.
E47, an alternative splicing form of the E2A gene, consists of two
activation domains, AD1 and AD2, and a bHLH domain (Fig. 7A, right)(42). Presumably because of the presence of endogenous E2A products,
E47 expression did not enhance the SREBP-1c effect on Ins265-Luc (Fig.
7C, left). Domain deletion studies indicated that SREBP-1c/BETA2/E47(and also PDX-1/BETA2/E47) activation of Ins265-Luc was markedly
impaired by the deletion of both AD1 and AD2 (E47⌬AD1AD2-(510 –647)) in a dominant negative fashion. Taken together, the activation

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Activation of Insulin Promoter by SREBPs
domains of SREBP-1c, BETA2, and E47 are highly involved in synergistic activation of the insulin promoter by SREBP-1c/BETA2/E47.
Discrepancy in the SREBP Activation of the Insulin Promoter between
␤ Cells and Non-␤ Cells—To determine whether SREBPs contribute toinsulin gene expression in ␤ cells, reporter assays using Ins265-Luc andits mutant constructs were performed in HIT cells. As shown in Fig. 8A,a, both E-box mutants and the A3/4-box mutant completely abolished
the activity, confirming the key roles of PDX-1 or BETA2/E47 in ␤ cells.
In contrast, when SRE2 and SRE1 were deleted and mutated, respectively, the activity of the insulin promoter was only marginally reduced(Fig. 8A, a), suggesting a small, if any, contribution of endogenous
SREBP in the insulin promoter activity in ␤ cells. Overexpression of
SREBP-1c in HIT cells exhibited a complex, bell-shaped activation of
Ins265-Luc with a slight suppression at a higher dose (Fig. 8A, b, left).
Conversely, Ins265-Luc with SRE2 deletion or SRE1 disruption, whichcancelled the direct binding of SREBP-1c to the promoter, caused dosedependent inhibition by SREBP-1c, suggesting the inhibitory effect of
SREBP-1c on endogenous PDX-1/BETA2/E47 (Fig. 8A, b, left). Whenthe E-boxes or A3/4-box were disrupted to cancel the effects of the ␤cell-specific factors, exogenous SREBP-1c robustly activated the promoter presumably by binding to intact SRE1 and SRE2 elements, as
observed in non-␤ cells (Fig. 8A, b, right). These data suggested that,
although SREBP-1c has a potential to activate the insulin promoter, thepresence of ␤ cell-specific factors masked this effect and SREBP-1coverexpression could, rather, inhibit the intrinsic insulin promoter
activity.
To further investigate the physiological relevance of SREBP-1c in ␤
cells, the effects of two conditions known to induce endogenous
SREBP-1c expression (high glucose and treatment with the T0901317
synthetic LXR agonist) on insulin gene expression were tested. Immunoblot analysis on cell membranes (precursor form of SREBP-1c) andnuclear extracts (mature form) demonstrated that incubation with highglucose and/or treatment with T0901317 additively elevated both membrane and nuclear forms of SREBP-1c protein in HIT cells (Fig. 8B).
Endogenous PDX-1 protein content was low at 5.5 mM glucose andinduced at higher concentrations (10 and 25 mM). T0901317 did notchange the PDX-1 protein level. Insulin promoter activity was measured
in these conditions (Fig. 8C). The activity was increased at high glucose
concentrations, presumably reflecting high PDX-1 expression. The
LXR ligand slightly but significantly induced the Ins265-Luc at low glucose concentration with low PDX-1 expression but not at higher glucose concentrations with high PDX-1 expression.
Of more physiological relevance, the direct binding of endogenous
SREBP-1 to the insulin promoter (containing SRE2 and SRE1 elements)
was estimated in vivo by ChIP assays of the nuclei from INS1 cells, therat insulinoma cell line. Both membrane and nuclear SREBP-1 proteins
were highly induced by the addition of the LXR ligand, as previously
observed in HIT cells (Fig. 9A) and liver cells (43). In response to the
induction of nuclear SREBP-1 in both 5.5 and 10 mM glucose in medium
with T0901317, direct binding of SREBP-1 to SRE2 and/or SRE1 in theinsulin promoter was shown by ChIP assay as well as to the fatty acidsynthase promoter, an authentic SREBP-1c target (Fig. 9B). Finally,
insulin gene expression was measured in isolated islets after the
SREBP-1c induction. Isolated rat and mouse islets were incubated at 5.5mM glucose with T0901317 (3 ␮M). With high induction of SREBP-1c
expression, the insulin mRNA level could be increased in both islets(Fig. 9C). These data demonstrated that endogenous SREBP-1c could
directly bind to the insulin promoter and increase insulin gene expression in ␤ cells in accordance with the findings from non-␤ cells.

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FIGURE 9. Endogenous SREBP-1c is induced by T0901317, directly binds to insulin
promoter, and increases insulin gene expression in ␤ cells. A, Western blot analysis of
endogenous SREBP-1c in INS1 cells, the rat insulinoma cell line. Cells were incubatedwith or without T0901317 (1 ␮M) for 19 h and further incubated in 5.5 or 10 mM glucose
in medium for 5 h. Membrane and nuclear proteins were extracted. B, chromatins prepared from INS1 cells (under the same conditions as described for A) were subjected to
the ChIP assay with anti-mouse SREBP-1 antibody and rabbit IgG as a negative control.
Immunoprecipitate samples were subjected to PCR using primers to amplify rat insulin I
gene and rat fatty acid synthase (FAS) gene promoters. Mw, molecular weight. C, Northern blot analysis of insulin gene expression in mouse and rat pancreatic islet treated withor without T0901317. Isolated islets were incubated in 5.5 mM glucose with or without
T0901317 (3 ␮M) for 24 h. Amounts of mRNA for insulin, islet amyloid polypeptide (IAPP),
or SREBP-1c were measured. Total RNA was isolated and subjected to blot hybridization
with the indicated 32P-labeled probe.

DISCUSSION
Identification of Insulin Promoter as an SREBP Target—Our current
studies clearly demonstrated that the insulin gene is a novel SREBPtarget and that SREBP activates the insulin gene promoter, strongly innon-␤ cells and potentially in ␤ cells. The rat insulin I promoter possesses three novel SRE elements to which SREBP homodimers can bindand which contribute to the SREBP-mediated activation. To date,almost all of the known promoters of the SREBP target genes involved inlipid metabolism have a site(s) for co-activators, NF-Y, and/or Sp1 in theneighboring region to recruit CBP/p300 (44 – 46). Because ␤ cell factors
also use CBP/p300 as a co-activator for insulin gene expression (13, 14),
it is plausible that CBP/p300 recruitment is crucial for SREBP-mediatedactivation. Complete loss of the activity by deletion of the transactiva-

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Activation of Insulin Promoter by SREBPs
tion domain of SREBP-1, a known CBP binding site, supports thisnotion.
Synerigistic Activation of SREBP-1 and BETA2/E47—There are a few
base pairs overlapping the SRE and E-box in both the E1 and E2 regions.
Considering space hindrances in the case of SREBP-1c/BETA2/E47 cotransfection, it is impossible that SREBP-1c binds to SRE and BETA2/
E47 binds to E-box in the same region simultaneously. Comparative gel
shift assays suggested that, overall, the affinities of BETA2/E47 for E1and E2-boxes are higher than those of SREBP-1 for SRE1 and SRE2 (datanot shown). Thus, when SREBP-1c and BETA2/E47 are both present,
BETA2/E47 should dominate the E1 and E2 regions. This could partiallyexplain the finding that the SREBP-1c contribution to insulin promoter
activity was small in HIT cells.
As shown by mutation and deletion studies, SREBP-1c/BETA2/E47
synergism requires both the E1 and E2 regions. In particular, E-boxes(and not SREs) in E regions are crucial for the synergistic activation of
SREBP-1c/BETA2/E47. Thus, it is BETA2/E47 that directly binds to theinsulin promoter (E-boxes). As suggested by immunoprecipitation and
SREBP-1cM experiments, SREBP-1c could be committed to this synergism by physically interacting with BETA2/E47 without DNA binding.
This illustrates a dual functionality of SREBP-1c; along with being anestablished transcription factor that binds to SRE, SREBP-1c can also
serve as a co-activator for recruitment of CBP/p300. Each component of
the SREBP-1c䡠BETA2䡠E47 complex has transactivation (TA) domainsthat can bind to CBP/p300. Thus, the complex with multiple TAdomains could efficiently and stably recruit CBP/p300, leading to transcription as high as that seen with PDX-1/BETA2/E47 (13, 14). Transactivation domain deletion studies of SREBP-1c, BETA2, and E47 supported this (Figs. 6 and 7). Furthermore, this synergism could also
involve DNA looping so that two sets of BETA2/E47 binding to E1- andE2-boxes could be bridged by SREBP-1c through protein-protein interaction without DNA binding. In many eukaryotic genes, transcription
factors bind to promoters at sites distant from one another, yet act
synergistically in DNA looping to activate transcription (47, 48). The
length between two regions is also important for DNA looping, as
evidenced in both PDX-1/BETA2/E47 and SREBP-1c/BETA2/E47
synergism (Fig. 4C).
Potential SREBP Contribution to Insulin Gene Regulation in ␤ Cells—
The current data suggested that SREBP-1c could potentially participate
in insulin gene regulation. ChIP assays confirmed that endogenous
SREBP-1c could directly interact with the insulin gene in ␤ cells. In
non-␤ cells, PDX-1/BETA2/E47 could increase insulin promoter activity to the level of what is seen in ␤ cells (38). SREBP-1c/BETA2/E47
caused a similar effect, suggesting that SREBP-1c could functionallyreplace PDX-1. However, when both SREBP-1c and PDX-1 were present, they did not synergistically work (Figs. 2B and 8A). Consistent withcomplex situations, the effect of SREBP-1c expression on the insulin
promoter in ␤ cells was biphasic. The molecular mechanism for thisapparent antagonism between SREBP-1c and PDX-1 is currently underinvestigation.
These data question the physiological relevance of SREBP-1c to theregulation of insulin genes under normal conditions. Supportively, as
shown in Figs. 8C and 9C, SREBP-1c could contribute to insulin promoter activation and gene expression only when the PDX-1 level is low.
Thus, SREBP-1c could have a chance to contribute to the insulin gene
regulation in pathophysiological states where PDX-1 function is compromised, as observed in insulin resistance and diabetes. In insulinresistant animals, SREBP-1c could be increased in the liver and potentially in ␤ cells (21, 49). Hyperglycemia could also increase SREBP-1c
expression in liver (50) and a ␤ cell line (22). Potential involvement of

34588 JOURNAL OF BIOLOGICAL CHEMISTRYSREBP-1c in ␤ cell lipotoxicity leading to impaired insulin secretion was
implicated in the SREBP-1c-inducible INS-1 cell line (22), in adenovirus
SREBP-1c-infected MIN6 cells (23) and rat pancreatic islets (24, 25),and ␤ cell-specific SREBP-1c transgenic mice (26). In these SREBP-1coverexpression systems, insulin gene expression was not increased but
rather suppressed. However, in our hands, under different conditions,
including in ␤ cell lines, ␤ cell-specific SREBP transgenic mice islets, andwild islets incubated with fatty acids, changes in insulin gene expression
were not consistent in contrast to decreased insulin secretion. We
assumed that this discrepancy was because of differences in the expression levels of PDX-1- and/or ␤ cell-specific factors. It could also be
speculated that activation of the insulin promoter by SREBP-1c in ourfindings could be masked in normal conditions but become overt as a compensatory adaptation for impaired insulin action in insulin resistance ordiabetes, especially when associated with decreased PDX-1 expression.
Further studies are needed to clarify the physiological and pathophysiological function of SREBP-1 in insulin gene expression and secretion.
Acknowledgments—We are grateful to Dr. A. H. Hasty for critical reading of
this manuscript. We appreciate Drs. S. Miwa, R. Takemura, K. Ohashi, Y.
Iizuka, Y. Tamura, H. Okazaki, S. Okazaki, Y. Najima, T. Yamamoto, and M.
Sekiya for helpful discussion.

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