Transcriptional activities of nuclear SREBP-1a, -1c,and -2 to different target promoters of lipogenic andcholesterogenic genes
Michiyo Amemiya-Kudo,* Hitoshi Shimano,1,§ Alyssa H. Hasty,* Naoya Yahagi,*
Tomohiro Yoshikawa,* Takashi Matsuzaka,§ Hiroaki Okazaki,* Yoshiaki Tamura,* Yoko Iizuka,*
Ken Ohashi,* Jun-ichi Osuga,* Kenji Harada,* Takanari Gotoda,* Ryuichiro Sato,†
Satoshi Kimura,* Shun Ishibashi,* and Nobuhiro Yamada§
Department of Metabolic Diseases,* Faculty of Medicine, and Department of Applied Biological Chemistry,†
Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan; and Department ofInternal Medicine,§ Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan

Abstract Recent studies on the in vivo roles of the sterol
regulatory element binding protein (SREBP) family indicate that SREBP-2 is more specific to cholesterogenic gene
expression whereas SREBP-1 targets lipogenic genes. To define the molecular mechanism involved in this differential
regulation, luciferase-reporter gene assays were performed
in HepG2 cells to compare the transactivities of nuclear
SREBP-1a, -1c, and -2 on a battery of SREBP-target promoters containing sterol regulatory element (SRE), SRE-like, orE-box sequences. The results show first that cholesterogenic
genes containing classic SREs in their promoters arestrongly and efficiently activated by both SREBP-1a and
SREBP-2, but not by SREBP-1c. Second, an E-box containing reporter gene is much less efficiently activated by
SREBP-1a and -1c, and SREBP-2 was inactive in spite of its
ability to bind to the E-box. Third, promoters of lipogenic
enzymes containing variations of SRE (SRE-like sequences)are strongly activated by SREBP-1a, and only modestly andequally by both SREBP-1c and -2. Finally, substitution of theunique tyrosine residue within the basic helix-loop-helix(bHLH) portion of nuclear SREBPs with arginine, the conserved residue found in all other bHLH proteins, abolishes
the transactivity of all SREBPs for SRE, and conversely results in markedly increased activity of SREBP-1 but not activity of SREBP-2 for E-boxes. These data demonstrate
the different specificity and affinity of nuclear SREBP-1 and-2 for different target DNAs, explaining a part of the mechanism behind the differential in vivo regulation of cholesterogenic and lipogenic enzymes by SREBP-1 and -2, respectively.—Amemiya-Kudo, M., H. Shimano, A. H. Hasty, N.
Yahagi, T. Yoshikawa, T. Matsuzaka, H. Okazaki, Y. Tamura,Y. Iizuka, K. Ohashi, J-i. Osuga, K. Harada, T. Gotoda, R.
Sato, S. Kimura, S. Ishibashi, and N. Yamada. Transcriptional activities of nuclear SREBP-1a, -1c, and -2 to different

Manuscript received 6 December 2001 and in revised form 26 March 2002.
DOI 10.1194/jlr.M100417-JLR200

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target promoters of lipogenic and cholesterogenic genes. J.
Lipid Res. 2002. 43: 1220–1235.
Supplementary key words lipogenesis • cholesterol • triglycerides •fatty acids • transcription factors

The liver synthesizes both cholesterol and fatty acids;
however, their biosynthetic pathways are under distinct
and separate regulation (1). In contrast to cholesterol synthesis, which is tightly regulated by a feedback system tomaintain cellular cholesterol levels, fatty acid synthesis isdriven primarily by the availability of carbohydrates. Despite these different patterns of regulation, recent evidence suggests that both biosynthetic pathways can becontrolled by a common family of transcription factors
designated sterol regulatory element binding proteins(SREBPs) (2, 3). SREBPs belong to a large class oftranscription factors containing basic helix loop helix(bHLH)-Zip domains. Unlike other members of this class,
SREBPs are synthesized as membrane-bound precursors
that require cleavage by a two-step proteolytic process inorder to release their amino-terminal bHLH-Zip-containing domain into the nucleus to bind to a specific DNA sequence, sterol regulatory element (SRE), and activate
their target genes in a sterol-regulated manner (2). Another unique feature of this family is their DNA binding
specificity. One family member, SREBP-1c, also named

Abbreviations: ACL, ATP citrate lyase; bHLH, basic helix loop helix; FAS, fatty acid synthase; FPP, farnesyl diphosphate; G6PD, glucose6-phosphate dehydrogenase; GK, glucokinase; ME, malic enzyme; PK,pyruvate kinase; S14, spot 14; SRE, sterol regulatory element; SREBP,
sterol regulatory element binding protein.
1 To whom correspondence should be addressed.
e-mail: shimano-tky@umin.ac.jp
Copyright © 2002 by Lipid Research, Inc.
This article is available online at http://www.jlr.org

ADD1, was cloned independently as a protein that binds
to E-boxes, universal cis-elements for bHLH proteins, andpresumably promotes adipocyte differentiation (4). Therefore, ADD1 has dual binding specificity to both classic palindromic E-boxes and nonpalindromic SREs (5). This
unique binding specificity is attributed to the tyrosine residue in the basic region, which is unique to the SREBP
family, whereas other common bHLH proteins have arginine at this position (5).
Although the list of SREBP target genes is expanding,they are functionally categorized into two groups. The
first group includes cholesterol biosynthetic genes suchas HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, squalene synthase, and SREBP-2 itself, as well as the LDL receptor gene, each of which contain the classic SRE sequence (ATCACCCCAC) or its
modified form SRE3 (CTCACACGAG), and adjacent cofactor (NF-Y or Sp1) sites in their promoters (6–9). The
second group represents lipogenic enzyme genes thathave been known to be nutritionally regulated at a transcriptional level (10, 11). They include acetyl-CoA carboxylase, fatty acid synthase (FAS), stearoyl CoA desaturase 1
and 2, glycerol-3-phosphate acyltransferase, diazepambinding inhibitor/acyl-CoA-binding protein, and spot 14(S14), for which promoter analyses revealed that SREBP
binding and activation sites in their promoters seem tohave more diverse consensus sequences than the classic
SRE, and are tentatively designated SRE-like sequences(12–17). Some lipogenic enzyme genes such as S14 (16),
FAS, liver-type pyruvate kinase (PK), and glucokinase(GK) contain E-boxes or E-box like sequences in theirpromoters that could confer carbohydrate, glucose, or insulin response sensitivity (18, 19), and could also be potential SREBP-targets due to the dual binding specificityof SREBPs to these sites (5).
To date, three SREBPs have been identified; SREBP-1aand SREBP-1c are produced from a single gene throughthe use of alternate promoters, and SREBP-2 is transcribed from a separate gene. All actively growing
cultured cells studied to date produce predominantly
SREBP-1a and SREBP-2, whereas most organs, including liver, from adult animals predominantly synthesize
SREBP-1c and SREBP-2 (20). All three SREBPs are capable of activating each of the known target genes, althoughwith differing efficiencies. SREBP-1c is weaker than
SREBP-1a and SREBP-2 due to its shorter transactivation
domain (21, 22).
To gain insight into the distinct roles of each SREBP isoform in vivo, transgenic mice that overexpress truncated,
active nuclear forms of human SREBP-1a, -1c, or -2 inliver, were produced and characterized (21, 23, 24). The
different SREBP-overexpressing transgenic animals showed
different patterns of increase in hepatic synthesis and accumulation of cholesterol and/or fatty acids. These datasuggest that the SREBP-1 isoforms are more selective in
activating fatty acid biosynthetic genes, while SREBP-2 ismore specific for controlling cholesterol biosynthesis.
From other lines of evidence, the physiological role ofhepatic SREBP-1c as a nutritional regulator of lipogenic

enzymes has been implicated by the distinct regulation of
SREBP-1c and -2 in different nutritional conditions. The
amounts of SREBP-1c protein and mRNA, but not thoseof SREBP-2, dramatically increase when mice are placedon a high carbohydrate diet or after refeeding fasted micein a similar manner to changes in lipogenic enzyme
mRNAs (25, 26). Furthermore, dietary polyunsaturated
fatty acids, known to inhibit hepatic lipogenic enzyme expression, decreased the amount of nuclear SREBP-1c protein without affecting nuclear SREBP-2 protein (27–29);
therefore, SREBP-1c seems to be a mediator of this polyunsaturated fatty acid regulation (16, 27). Finally, using
survivors of SREBP-1 deficient mice, it was confirmed that
SREBP-1 is responsible for nutritional induction of hepatic lipogenic enzymes whereas SREBP-2 controls cholesterol biosynthesis (30). All these in vivo data establisheddistinct roles of SREBP-1c and -2 in hepatic lipogenesis
and cholesterogenesis, respectively. Whereas SREBP-2 exerts sterol regulation through cleavage of the membranebound precursor protein to liberate the active nuclear
form into the nucleus, SREBP-1 controls lipogenic enzymes by self-regulating its own transcription level. However, their precise molecular mechanisms are not fully understood. Present studies were performed to test the
hypothesis that the molecular differences in transcriptional activities of nuclear SREBP isoforms can explain the
distinct specificities in activation of cholesterol biosynthetic and lipogenic genes in vivo. Luciferase reporter
gene assays in HepG2 cells were performed to comparetranscriptional activities of SREBP wild-type isoforms andtheir YR mutant versions (substitution of tyrosine for arginine) for a whole battery of promoters of cholesterogenic
and lipogenic genes. The results clearly demonstrated
that SREBP-1a, -1c, and -2 showed differing DNA binding
specificities to SRE, SRE-like, and E-box sequences, with
SREBP-1a being the strongest activator for all of these targets. However, hepatic major isoforms SREBP-1c and -2
showed similar activities for lipogenic enzyme genes.
Therefore, to account for distinct in vivo roles of SREBP-1
for lipogenesis, there must be a linked but distinct nutritional and metabolic regulation in the supply of nuclear
forms of SREBP-1 and SREBP-2.

EXPERIMENTAL PROCEDURES
Materials
Standard molecular biology techniques were used. We obtained cholesterol and 25-hydroxycholesterol from Sigma, N-Acetyl-Leu-Leu-norleucinal-CHO (ALLN, calpain inhibitor I) from
Calbiochem, Redivue [-32P]dCTP (6,000 Ci/mmol) from Amersham Pharmacia, and restriction enzymes from New England
Biolabs. Plasmid DNAs for transfection were prepared with EndoFree Plasmid Maxi kits (Qiagen).

Construction of expression, luciferase-reporter plasmids
Cytomegalovirus (CMV) promoter expression plasmids containing cDNA encoding human nuclear form of SREBP-1a and-1c (pCMV-SREBP-1a and -1c) were prepared as described (21).

Amemiya-Kudo et al. DNA specificity of nuclear SREBP-1a, -1c, and -2

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Human truncated SREBP-2 expression plasmid (pCMV-SREBP2) was prepared by inserting the same cDNA fragment used forthe transgene construct of TgSREBP-2 (24) into the SmaI site ofpCMV7. The tyrosine residue in the basic region of each SREBP
expression plasmid (at amino acid 335 for SREBP-1a, 321 for
SREBP-1c, and 342 for SREBP-2) was altered to an arginine residue by site-directed mutagenesis using a commercial kit (Transformer, Invitrogen). These YR mutated versions were designated
pCMV-SREBP-1aM, pCMV-SREBP-1cM, and pCMV-SREBP-2M.
Luciferase gene reporter plasmids for artificial multimerized SRE promoter, human LDL receptor, HMG-CoA synthase,farnesyl diphosphate synthase, and fatty acid synthase gene promoters (SRE-Luc, pLDLR-Luc, pHMGCoASyn-Luc, pFPP-Luc,and pFAS-Luc, respectively) were prepared as previously described (21, 31). Luciferase reporter genes for promoters ofmouse ATP citrate lyase (ACL), rat malic enzyme (ME), rat glucose-6-phosphate dehydrogenase (G6PD), rat liver-type pyruvatekinase (PK), rat spot 14 (S14), and rat GK genes (pACL-Luc,
pME-Luc, pG6PD-Luc, pPK-Luc, pS14-Luc, and pGK-Luc) wereconstructed as follows. Each promoter was obtained by PCR
using Pfu DNA polymerase (Stratagene), and rat or mouse
genomic DNA as a template. The primers used were: rat ACL
5 primer, AGCCCCTGAAGCGATCAGGCCACA (300), 3 primer,
GGATCTCTCCAGCCGCCTGCAGCTG (15) (32); rat ME 5
primer, GAATTCGCATAGCCCAGAAGCTATAGCTGT (783), 3
primer, CGGGAGTGCGGCGGGCGGCGGCCGTGCGAC (7),(33); rat G6PD 5 primer, GTCGGCCAGAAGTGAAGAGGGCAGGAGCAG (926), 3 primer, TTTAGTTGCCGCTGCCAAACACGTTCACAG (58) (34); rat PK 5 primer, GACAGGCCAAAGGGGATCCAGCAGCATG (197), 3 primer, ACGTTGCTTACCTGCTGTGTCTGTGGGTCT (12) (35); rat S14, 5
primer CCTTCTAACTGGTTGAGCAGCTG (250), 3 primer,
AACTCAGAGACCAGCAAACTGC (12) (36); and rat GK 5
primer CCCACGAGGATCCCCCACTG (318), 3 primer CTTCCGCACTAACGGGCCTGA (11) (37).
A restriction site, KpnI and Hind III (or XhoI), was added toeach 5 primer and 3 primer, respectively. The PCR products
were digested with KpnI and Hind III (or XhoI), and were inserted
into the respective sites of pGL2-Basic Vector (Promega). For pEbox-Luc, six copies of carbohydrate response element at position(1448 to 1422) of the rat S14 gene (GCCAGTTCTCACGTGGTGGCCCTGTGC) (18) were inserted into the SmaI site ofpGL2-Promoter Vector (Promega) in which the SV40 early promoter was added to the pGL2-Basic Vector. Other enhancer constructs were also produced by inserting the following oligonucleotides into SmaI/Nhe I sites of pGL2-Promoter Vector. SRESp1:
gctagcaaaatcaccccactgcaaactcctccccctgcgctagc; SRE-likeNFY (7bp): gctagcaaaatcgcctgatgcaaactattggctgcgctagc; E-box: cccgggaaatcacgtgatgcgctagc; E-boxSp1:cccgggaaatcacgtgatgcaaactcctccccctgcgctagc; E-boxNFY: cccgggaaatcacgtgatgcaaactattggctgcgctagc; SRE-like  NF-Y enhancer region from S14 promoter was
produced by PCR using the following primers and S14-Luc as atemplate: 5 primer: aaacccgggtttgtccctgggtag and 3 primer:
AAAGCTAGCTTGTTTTGAGCCAATCCC. PCR product was inserted into SmaI/Nhe I sites of pGL2-Promoter Vector.

tive control, the same amount of basic plasmid CMV7 (38) wastransfected in place of expression plasmids. Five hours after
transfection, the cells were incubated in the same medium supplemented with cholesterol (10 g/ml) and 25-hydroxycholesterol (1 g/ml) to suppress endogenous SREBP activity for an
additional 16 h prior to harvest. The amount of luciferase activity
in transfectants was measured and normalized to the amount of
-galactosidase activity as measured by standard kits (Promega).

Gel mobility shift assay
Gel mobility shift assays were performed as previously described (19). DNA used as probes were as follows: SRE probe, thesequence from SRE and Sp1 sites of human LDL receptor genepromoter and used for construction of pSRE-Luc; E-box probe,
the sequence from the S14 gene enhancer region used forconstruction of pE-box-Luc (refer to construction of expression,luciferase-reporter plasmids); DNA fragment A (GGCCCGCCCCTCACCCGTCGGTGCCCAGGTC) and B (TCGCCACCCCCTCTCGCCACCCACGCCCGCCCC) from the rat malic enzyme
gene promoter (34) as indicated in Fig. 7. The DNA probes werelabeled by fill-in reaction with Klenow enzyme and [-32P]dCTP
after annealing the oligomers, and were purified on G50 Sephadex
columns. The labeled DNA was incubated with a recombinant
protein (100 ng) of human nuclear SREBP-1a, -2, or nuclear extracts (1 g) from cells transfected with each CMV-SREBP expression plasmid as described in transfection and luciferase studies in a mixture containing 10 mM Tris-HCl, pH 7.6, 50 mM KCl,
0.05 mM EDTA, 2.5 mM MgCl2, 8.5% glycerol, 1 mM dithiothreitol, 0.5 g/ml poly(dI-dC), 0.1% Triton X-100, and 1 mg/ml
nonfat milk for 30 min on ice. The DNA-protein complexes wereresolved on a 4.6% polyacrylamide gel.

Immunoblot analysis
Some sets of HepG2 cells for transfection studies were incubated for the last 2 h in medium supplemented with ALLN (5g/ml). The nuclear extracts were prepared and subjected toimmunoblot analysis using anti-human SREBP-1 or SREBP-2 antibody as the primary antibody as previously described (21).

Northern blot analysis of SREBPs in livers fromtransgenic miceTransgenic mice overexpressing human SREBP-1a, -1c, and -2
under the control of the rat phosphoenolpyruvate carboxykinase
promoter were established as described (21, 23, 24). Homozygous mice for Tg-SREBP-1c and -2 were obtained by mating hemizygotes. Wild type littermates of hemizygous Tg-SREBP-1a mice
were used as controls. Three male animals from each group were
fed high protein/low carbohydrate diet for 2 weeks to induce
transgene expression. The animals were fasted 12 h prior to sacrifice. Total liver RNAs were prepared using TRIZOL reagent(GIBCO BRL, Life Technologies). Northern blot analyses were
performed with the indicated cDNA probes as previously described (23).

Transfections and luciferase assays
Human hepatoma HepG2 cells were cultured in DMEM containing 25 mM glucose, 100 U/ml penicillin, and 100 g/ml
streptomycin sulfate supplemented with 10% FCS. On day 0,
cells were plated on a 12 well plate at 4.5  104 cells/well. On
day 2 each expression plasmid (indicated amount or 0.5 g),each luciferase reporter plasmid (0.5 g), and an SV--galactosidase reference plasmid (pSV--gal, Promega, 0.5 g) wastransfected into cells using SuperFect Transfection Reagent(QIAGEN) according to the manufacturer’s protocol. As a nega-

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RESULTS
Distinct in vivo activation of cholesterogenic andlipogenic genes by SREBPs in livers of transgenic mice
Gene expression of different cholesterogenic and lipogenic enzymes in the livers of transgenic mice overexpressing nuclear forms of SREBP-1a, -1c, and -2 was estimated by Northern blot analysis in the same set ofexperiments for direct comparison. Overexpressed transgene products were mature active forms that automatically enter the nucleus and activate the target genes irrespective of the cleavage processing of precursor proteins.
The SREBP-1a, -1c, -2 transgenic lines were prepared sothat the protein amounts of the transgene products in theliver nuclei were comparable (24). Therefore, transcriptional activities of the three isoforms for a target gene canbe compared by mRNA levels of the gene in transgenic
and wild type livers. As shown in Fig. 1, the patterns of induction of mRNAs by SREBP isoforms as compared with

Fig. 1. Hepatic mRNA levels of cholesterogenic and lipogenic enzymes in nuclear SREBP-1a, -1c, and -2 transgenic mice as measured
by Northern blot analysis. Transgenic mice overexpressing humannuclear SREBP-1a, -1c, and -2 under the phosphoenolpyruvate carboxykinase promoter were constructed as described (23, 24). Experimental animals were homozygous for SREBP-1c and -2 andhemizygous for SREBP-1a, to attain comparable levels of transgene
products. Controls were wild-type littermates of SREBP-1a transgenic mice. Three male animals from each group were fed a highprotein/low carbohydrate diet for 2 weeks and fasted 12 h prior tosacrifice. Aliquots of total RNA prepared from livers were pooled(15 g) and subjected to Northern blot analysis with indicatedcDNA probes as previously described (24).

wild type were very similar for low density lipoprotein receptor (LDLR), HMG-CoA synthase, and farnesyl diphosphate (FPP) synthase genes. SREBP-1a and -2 were equallystrong for activation of these cholesterogenic genes
whereas SREBP-1c was essentially inactive. For lipogenic
enzyme genes, SREBP-1a profoundly activated most lipogenic genes tested here (S14, FAS, ACL, ME, and G6PD).
Although to a lesser extent than SREBP-1a, SREBP-1c and-2 overexpression induced mRNA levels of these genes tosimilar extents. Among glycolytic genes, liver-type pyruvate kinase (PK) was modestly activated while only a slightactivation of glucokinase (GK) was detected by overexpression of SREBP-1a. Although it has been suggested that
SREBP-1c could be a mediator of hepatic gene induction
by insulin, overexpression of SREBP-1c in our transgenic
mice did not induce PK or GK gene expression in theliver.
Luciferase assays for cholesterogenic and lipogenic
gene promoters
To further elucidate the molecular mechanism of different activities of SREBP isoforms for cholesterogenic
and lipogenic enzymes, we set up luciferase reporter geneassays for different gene promoters and compared transcriptional activities of nuclear SREBP-1a, -1c, and -2 forthem. Figure 2 shows a schematic representation of thepromoter constructs of the genes to be tested for the luciferase reporter gene assays. The promoters of cholesterogenic genes (LDLR, HMG-CoA synthase, and FPP synthase genes) have been extensively studied and shown tocontain one or two functional classic SRE or SRE3 sites
with adjacent cofactor binding sites (Sp1 or NF-Y), which
are indispensable for SREBP activation (9, 39–41). In contrast, lipogenic enzyme gene promoters contain variable
regulatory sequences for SREBP. They can be roughly separated into two groups: SRE-like containing promotersand E-box containing promoters. The former group includes FAS, ACL, ME, and S14. These genes contain regulatory sequences that resemble a classic SRE. In contrast,
the latter groups are E-box containing promoters, which
include S14, PK, GK, and FAS. Some of their E-box sequences have been reported to be responsible for nutritional regulation acting as carbohydrate, glucose, or insulin response elements (18, 19). Previous data fromSREBP-1 knockout mice indicate that SREBP-1 could besomehow involved in nutritional regulation of these enzymes, possibly through these E-boxes. Because of the low
stringency in defining putative SRE-like sequences, some
genes, such as FAS and S14, can be included in bothgroups. The complexity of the FAS promoter will be discussed later. Although the exact binding sites of SREBPs
in ME and G6PD promoters have yet to be identified by
precise promoter analysis, their expressions have beenshown to be controlled by SREBPs in studies with transgenic and knockout mice (30, 42). Putative elements responsible for the effect of SREBPs in these promoters areillustrated in Fig. 2. The ACL promoter contains an SRElike sequence with a close NF-Y site that is responsible for
SREBP activation (43).

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genes and to give a clue as to functional and conformational distinction of SREBPs.
Prior to performing luciferase assays, the expression
level of each SREBP nuclear form was estimated by an immunoblot analysis. Nuclear proteins were prepared fromthe same set of experiments as used for luciferase reporter
assays. As shown in Fig. 3B, expression plasmids for
SREBP-1a, -1c, -1aM, and -1cM produced similar amountsof the truncated proteins in the nuclear extracts of thetransfected cells. The amounts of SREBP-2 and -2M werealso similar. Although direct comparison of protein
amounts expressed from SREBP-1 and -2 constructs is difficult, mRNA levels were similar and comparable (datanot shown). Therefore, luciferase assays in the same set ofexperiments can afford quantitative results for relativetranscriptional activities among different constructs.

Fig. 2. Schematic representation of promoters of cholesterogenic
and lipogenic enzyme genes used for luciferase reporter gene(Luc) assays in the current study. Cholesterogenic enzyme genepromoters contain one or two classic SREs accompanied by cofactor binding sites (NF-Y or Sp1). Lipogenic enzyme promoters areclassified into two groups: SRE-like containing promoters andE-box containing promoters. SREBP binding sites for pyruvate kinase (PK), glucokinase (GK), malic enzyme (ME), and glucose-6-phosphate dehydrogenase (G6PD) gene promoters have not been identified, and thus binding of SREBPs to E-boxes or SRE-likesequences in those genes is putative. Figure is not to scale. FPP, farnesyl diphosphate synthase; S14, spot 14; PK, pyruvate kinase; GK,
glucokinase; FAS, fatty acid synthase; ACL, ATP citrate lyase; ME,malic enzyme; G6PD, glucose-6-phosphate dehydrogenase.

Expression vectors encoding nuclear SREBP-1a, -1c, or-2 under the CMV promoter were constructed and thetranscriptional activities compared for different promoterluciferase constructs in human hepatoma HepG2 cells.
We also set up a mutant version (M) for each SREBP expression vector in which an arginine residue was substituted for the tyrosine residue that is conserved in the basic
region in the SREBP family. This tyrosine residue renders
ADD1, the rat homolog for SREBP-1c, unique in terms ofdual DNA binding specificity to SREs as well as to E-boxes(5). In contrast, other bHLH members have a conserved
arginine at this position, and have binding affinity only toE-boxes, and not to SRE. The YR mutation of SREBPs waspredicted to alter their binding specificity to their target
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Cholesterogenic enzyme gene promoters
The transcriptional activity of each SREBP expression
plasmid for SRE was estimated using an optimum artificial
SRE-reporter gene (44). In this SRE-Luc construct, threecopies of a classic SRE (ATCACCCCAC)-Sp1 site, formerly
designated repeats 2 and 3 in the analysis of the promoterof LDL receptor gene, were connected to a TATA box(44) (Fig. 2). After transfection, HepG2 cells were incubated in a medium containing FCS supplemented withcholesterol and 25OH cholesterol to suppress endogenous SREBP activities. As shown in Fig. 3A, we estimate
the transcriptional activity of each SREBP isoform in arange between 0.1 ng and 500 ng of transfected expression plasmid DNA. The maximal effect was observed at 10
ng and, at higher concentrations, the activity was decreased, showing relatively high affinity and low saturation
kinetics between a classic SRE and SREBPs. However, at allconcentrations, relative activities among SREBP isoforms
were similar. In our expression experiments, SREBP-1a isslightly stronger than SREBP-2, although the two isoforms
were reported to have similar transcriptional activities
with different protocols and cells (22). In contrast,
SREBP-1c was 10-fold weaker and essentially inactive. Consistent with the previous report (5), the YR mutated
SREBP-1c in our construct lost its luciferase activity completely (Fig. 3B). Predictably, no transcriptional activity
was found in the mutated SREBP-1a because its bHLH domain is identical to that of mutated SREBP-1c. Furthermore, when we tried this mutation analysis for SREBP-2,
arginine substitution almost completely abolished SREBP-2
activity for SRE as well. Therefore, the importance of thistyrosine residue for activation of the classic SRE is universal among all SREBP isoforms.
We chose LDL receptor, HMG-CoA synthase, and FPP
synthase genes to estimate the effects of wild type and mutant SREBPs on the activities of native cholesterol-related
gene promoters (Fig. 4). LDL receptor and HMG-CoA
synthase promoters contain one copy and two copies of
SRE (9, 39), respectively, while FPP synthase is regulated
by a similar, but distinct sequence designated SRE3 (Fig.
2) (6). In HepG2 cells, SREBP-1a and -2 activated all threegene promoters with similar efficiency while SREBP-1c

Fig. 3. Comparison of transcriptional activities of nuclear SREBP-1a (closed circle), -1c (closed square), -2(open triangle)(A and C), and respective YR mutants (B and D) on SRE (A and B) and E-box (C and D) as
measured by luciferase reporter gene assay in HepG2 cells. An artificial SRE-luciferease reporter plasmid,pSRE-Luc contains three sets of classic SRE and Sp1 site from LDL receptor promoter (refer to Fig. 2). An
E-box enhancer luciferase reporter gene (pE-box-Luc) was constructed by integrating six copies of the carbohydrate response element of rat S14 gene (1448 to –1422 bp) containing two E-boxes into the pGL2-Promoter vector (refer to Fig. 2). The HepG2 cells were transfected with the indicated DNA amount of CMV
promoter-expression plasmid, pSRE-Luc or pE-box-Luc (0.5 g), and pSV--gal (0.5 g) as reference plasmid using SuperFect (Promega). Expression plasmids used were pCMV-SREBP-1a, pCMV-SREBP-1c, andpCMV-SREBP-2 encoding nuclear active forms of human SREBP-1a, -1c, and -2, respectively, under the CMV
promoter, and YR mutated versions of the wild-type SREBP constructs (pCMV-SREBP-1aM, pCMV-SREBP1cM, and pCMV-SREBP-2M) in which arginine was substituted for tyrosine residue in the basic region. A
CMV7 empty vector (Cont) was used as a negative control. After transfection, the cells were incubated for 16 h
in DMEM supplemented with 10% FCS, 1 g/ml 25-hydroxycholesterol, and 10 g/ml cholesterol to suppress endogenous SREBP activity. Luciferase activity was measured and normalized to  -galactosidase activity. The values are expressed as ratios of the value from control (Cont; open column). Each point represents
the mean SE of three independent transfections. Inset in B: Immunoblot analysis of SREBP proteins expressed in transfected cells used for luciferase reporter assays. Another set of the cells for transfection wasused to verify that the amounts of expressed SREBPs were comparable. Nuclear extracts were prepared andsubjected to immunoblot analysis as previously described (15 g)(21). Membranes were incubated with antihuman SREBP-1 or human SREBP-2 as primary antibody. ECL was used for visualization of the bands.

activation was very weak. These data are essentially consistent with the results from SRE-Luc as well as mRNA levels in transgenic studies (Fig. 1). The YR mutation in anyof the SREBP constructs abolishes the activity of the LDL
receptor promoter, as observed in SRE-Luc. In contrast,
the YR mutation reduced the promoter activities of HMGCoA and FPP synthase genes only 2- to 3-fold. The residual activities could be due to the abilities of the mutant

SREBPs to weakly bind to modified SREs or other sequences in these promoters.
Lipogenic enzyme gene promoters
E-box-Luciferase reporter. It was previously reported that
CACGTG-type E-box was preferentially picked up in theselection of the ADD1 binding motif from random oligonucleotides (5). This E-box is also present in the carbohy-

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Fig. 4. Comparison of transcriptional activities of nuclear SREBP1a, -1c, -2, and respective YR mutants on LDL receptor, HMG-CoA
synthase, and FPP synthase gene promoters as measured by luciferase reporter gene assay in HepG2 cells. The HepG2 cells weretransfected with the CMV promoter-expression plasmid as indicated below (1 g), either pLDLR-Luc, pHMGSyn-Luc, or pFPPSyn-Luc as a reporter gene (0.5 g, refer to Fig. 2), and pSV-gal (0.5g) as reference plasmid using SuperFect (Promega).
Expression plasmids used were pCMV-SREBP-1a, -1c, -2, -1aM, -1cM,-2M, and CMV7 (an empty plasmid as a control). After transfection,
the cells were incubated for 16 h in DMEM supplemented with
10% FCS, 1 g/ml 25-hydroxycholesterol, and 10 g/ml cholesterol to suppress endogenous SREBP activity. Luciferase activity wasmeasured and normalized to  -galactosidase activity. The values
are expressed as ratios of the value from control (Cont; open column). Each bar represents the mean
SE of four independenttransfections.

drate response element of the S14 gene, whose expression
in the liver has been known to be nutritionally regulated
similarly to other lipogenic enzymes (18). The E-box is located 1.6 kb upstream of the transcription start site anddoes not have any promoter activity alone in preliminary
experiments (data not shown), suggesting its potential enhancer activity. An enhancer reporter gene was constructed in which six copies of this carbohydrate response
element were inserted into a luciferase gene reporter,
which contains the SV40 promoter (pGL2-Promoter Vector)(Fig. 2). This reporter gene contains essentially only
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Journal of Lipid Research Volume 43, 2002

E-boxes fused to an intrinsic promoter and is designated
E-box-Luciferase reporter (E-box-Luc) as a tester to estimate enhancer activities of each SREBP isoform on a representative E-box. As shown in Fig. 3, dose-activation
curves of SREBPs for E-box-Luc were markedly distinct
from those for SRE-Luc. SREBP-1a and -1c exhibited asimilar activity for E-box in a dose-dependent manner(Fig. 3C). It is noteworthy that significant activation of
E-box-Luc requires 10–100 ng of transfected SREBP expression plasmid DNA, which was higher than the DNA
concentration for maximal activity for SRE. This suggeststhat SREBP-1 is more efficient for activation of SRE thanE-boxes. In contrast, SREBP-2 had essentially no activity
for E-box-Luc. Very interestingly, the YR mutation increased enhancer activities of SREBP-1a and -1c markedly,
but did not restore any activity of SREBP-2 (Fig. 3D).
Based upon these results, we evaluated the dual binding
specificity of each SREBP isoform for SRE and E-box (Fig.
5). This dual binding specificity was originally found for
SREBP-1c/ADD1 (5). SREBP-1a shares the same bHLH
protein with SREBP-1c and should have the same binding
specificity. We performed the gel shift assays using the nuclear extracts derived from SREBP transfected cells (Fig.
5A). DNA containing an SRE and an Sp1 site, the same sequence as used for SRE-Luc and LDL-Luc, was labeled as
an SRE probe. As shown in left panel of Fig. 5A, nuclear
extracts from control cells in which endogenous SREBPs
were suppressed by addition of 25-OH cholesterol andcholesterol in the medium, exhibited shifted bands unrelated to SREBPs, presumably due to an abundant presence of Sp1 family in the cells, since these bands disappeared after the addition of excess unlabeled DNA
containing only Sp1 binding site. Nuclear extracts fromthe cells transfected with the SREBP-1a expression plasmid showed a new shifted band by SREBP-1a. Its specificity of SREBP-1 binding was confirmed by a supershift after
addition of the specific antibody. In the middle panel, we
compared SREBP-1a, YR mutant SREBP-1a (1aM),
SREBP-2, and YR mutant SREBP-2 (2M) in binding to thisSRE probe. As expected, SREBP-1aM did not cause the gel
shift of the SRE. Similarly, SREBP-2 bound, but SREBP-2M
did not bind, to the SRE. These data are consistent with
their transcriptional activities for the SRE-containing promoter (SRE-Luc) as estimated by luciferase assays (Fig. 3).
Therefore, the importance of the tyrosine residue in thebasic region for binding to SRE (5) was verified for both
SREBP-1a and -2. We examined binding of SREBPs to the
E-box using a probe from E-box-Luc (right panel of Fig.
5A). We found that both SREBP-1a and -1aM bound to the
E-box, which was consistent with the luciferase results. Unexpectedly, both SREBP-2 and -2M also bound to the
E-box. This is a discrepancy with the results from luciferase assay that SREBP-2 was essentially inactive and
SREBP-2M was completely inactive for the E-box containing promoter (Fig. 3C, D). Ability of SREBP-1a and -2 tobind to the E-box was reconfirmed using recombinant
proteins (Fig. 5B). Competitive studies with excess SRE
and E-box DNAs suggested that SREBP-2 appears to havea higher binding affinity for the SRE than for the E-box,

Fig. 5. Gel mobility shift assay for binding of native and YR mutant SREBPs expressed to SRE and E-box. A:
Double stranded DNA fragments containing SRE-Sp1 site (derived from the human LDLR gene promoter,
refer to Fig. 2) and E-box (derived from a carbohydrate responsive element of rat S14, refer to Fig. 2) werelabeled with [-32P] dCTP. These labeled SRE and E-box probes were incubated with nuclear extracts fromthe cells transfected with pCMV-SREBP-1a, -2, -1aM, -2M, and CMV (an empty plasmid as control) and wererun on polyacrylamide gels. Endogenous SREBPs were suppressed by pre-incubation of the cells with 25OH
cholesterol and cholesterol. All the nuclear extracts including control exhibited shifted bands unrelated to
SREBPs. These non-SREBP shifted bands were due to Sp1 binding to the SRE probe and to USF1 binding to
the E-box probe (data not shown). In the left panel, specificity of SREBP-1a binding to the SRE probe (indicated by arrow) was confirmed by a supershift after addition of SREBP-1 antibody. The specificities of otherbindings of SREBPs to the SRE and E-box (indicated by arrows) were confirmed by supershifts after additionof respective antibodies (data not shown). B: Gel mobility shift assay for binding of recombinant SREBP-1
and -2 to SRE and E-box. Labeled SRE and E-box probes were also incubated with recombinant nuclear
SREBP-1 and -2 and subjected to gel mobility shift assay. Specificity of each protein binding to SRE and E-box
was confirmed by supershift by addition of the respective specific antibody. In competitive assays, a 1,000-foldmolar excess of indicated unlabeled probes was added prior to addition of the labeled probe.

Amemiya-Kudo et al. DNA specificity of nuclear SREBP-1a, -1c, and -2

1227

relative to SREBP-1. This suggests that binding of SREBP-2
to the promoter might not be sufficient for activation ofits downstream genes.

Pyruvate kinase and glucokinase gene promoters
The liver-type pyruvate kinase gene promoter contains
a similar carbohydrate (glucose) response element to thatof the S14 gene; two tandem E-box like sequences designated the L4 box (18). PK-Luc, containing this region,
was tested as an SREBP target. Preliminary data show thatthe luciferase construct fused to 200 bp PK promoter containing this region was activated 2- to 3-fold by changing
from low glucose (2.75 mM) to high glucose medium (25mM) for 16 h for confirmation of this construct as a glucose response element. In addition, overexpression of
USF1 (CMV-USF1), which is known to activate both PK
and GK promoters, increased PK-Luc activity 3.10.3fold (three independent experiments). In contrast to thecarbohydrate response element of S14 (E-box-Luc), thePK promoter did not significantly respond to SREBP overexpression (relative fold changes vs. control vector are:
SREBP-1a, 1.1 0.2; SREBP-1c, 1.6 0.3; SREBP-2, 1.2
0.3; SREBP-1aM, 1.6 0.3; SREBP-1cM, 1.4 0.1; SREBP2M, 0.6 0.1; mean SE from six independent transfections). As shown in Fig. 1, hepatic PK mRNA was modestly
up regulated by SREBP-1a overexpression in the transgenic mice. It is not currently known whether this effect of
SREBP-1a is direct or indirect; however, it can be concluded that the effect of SREBP on PK expression in vivo
is not mediated through the carbohydrate response element of the PK gene. In our previous report, SREBP-1 disruption did not severely impair the high-sucrose induction of PK (30). Taken together, these results indicate that
SREBP-1 is not significantly involved in the physiologicalregulation of PK. Another glycolytic and lipogenic enzyme, GK, was reported to have an insulin response element in its proximal promoter region. There is a
CACGTG-type E-box close to this region. Consistent with
the in vivo data (Fig. 1), GK-Luc was not significantly activated by any of the SREBP constructs (relative foldchanges vs. control vector are: SREBP-1a, 1.0
0.1;
SREBP-1c, 1.8
0.2; SREBP-2, 0.6
0.05; SREBP-1aM,
1.4
0.2; SREBP-1cM, 0.8
0.1; SREBP-2M, 0.3
0.1;
mean
SE from three independent transfections)whereas USF1 expression plasmid markedly induced the
GK promoter (162). These data suggest that SREBPs
are not strong activators for GK, which is contrary to a previous report that adenoviral overexpression of SREBP-1c/
ADD1 activates GK expression (45).
S14 proximal gene promoter
Recently the element responsible for SREBP-1c binding
and activation of the S14 gene promoter was identified in
the known polyunsaturated fatty acid suppressive region(16). This element (distinct from the carbohydrate response element used for E-box-Luc) is located at 140 bp
upstream of the transcription start site. The sequence
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Journal of Lipid Research Volume 43, 2002

(TCGCCTGAT) exhibited a slight similarity to both E-box
and SRE. The S14 promoter-luciferase containing this sequence with adjacent NF-Y site was estimated for SREBP
activation (Fig. 6A). The S14 promoter was strongly activated by SREBP-1a, and by SREBP-1c and -2 with less efficiency at any concentration of the transfected DNA. The
induction of hepatic S14 mRNA levels in transgenic mice
(Fig. 1) was less marked than that in the luciferase assays.
It is presumably because other transcription factors andhormones such as USFs and thyroid hormones regulate
the long native promoter of S14 gene. However, this promoter along with the enhancer region (Fig. 3C) shouldplay a role in nutritional induction of hepatic S14 gene
expression (31). Interestingly, the YR mutation abolishedthe SREBP-2 activation of the S14 promoter Luc, but did
not affect the SREBP-1 activation, suggesting that the molecular mechanism for SREBP activation of the S14 promoter could be different between SREBP-1 and -2. YR mutated SREBP-1a and -1c showed dose-dependent activationin all concentrations in contrast to native SREBPs whose
peak activities were at 100 ng.

FAS gene promoter
The fatty acid synthase promoter is a unique case where
two perfect SRE sites flank an E-box, also recognized as an
insulin or carbohydrate response element (refer to Fig.
2)(19, 46). As depicted in Fig. 6B, wild-type SREBP1a had
the strongest activity for the FAS promoter among SREBP
isoforms. The highest activation was observed at 100 ng ofDNA. SREBP-1c and -2 showed lower levels of activation
than SREBP-1a, but similar dose-dependent patterns. The
YR mutation significantly impaired the transcriptional activities for SREBP-1a and -1c at 100 ng and lower concentrations, but the activation by YR mutant SREBP-1a and-1c was dose-dependent and resulted in the same level ofactivation of FAS-Luc as respective native SREBP-1a and-1c at 500 ng. This is consistent with a previous report that
YR mutated ADD1 still retained its binding to and transactivation of the FAS gene promoter in CAT assays (25). In
contrast, the mutation caused SREBP-2 to lose its activity
to FAS promoter, showing a discrepancy in the effect of YR
mutation between SREBP-1 and -2.

ACL gene promoter
ATP citrate lyase catalyzes the reaction for the translocation of acetyl-CoA from mitochondria to cytosol and iscritical for biosynthesis of both cholesterol and fatty acidsin the cytosol. ACL is regulated in a lipogenic fashion andcontrolled by SREBP-1c in the liver (31). Recently, an
SREBP binding and activation site in the rat and human
ACL promoters was identified (43). It is an SRE-like sequence (TCAGGCTAG) accompanied by an NF-Y site.
Mouse ACL promoter (300 bp), that contains the same
SREBP binding site and a region responsible for insulin
and polyunsaturated fatty acid effects, was fused to theluciferase reporter gene (ACL-Luc) (32, 47) (Fig. 2). As
shown in Fig. 6C, SREBP-1a profoundly activated theFig. 6. Comparison of transcriptional activities of nuclear SREBP-1a (closed circle), -1c (closed square), -2(open triangle), and respective YR mutants on the S14 (A), FAS (B), and ACL (C) gene promoters measured
by luciferase reporter gene assay in HepG2 cells. Reporter plasmids p S14 Luc, pFAS Luc, and pACL Luc
were constructed by fusing rat S14 proximal gene, rat FAS gene promoter, andmouse ACL gene promoter topGL2-Basic Vector (refer to Fig. 2). The HepG2 cells were transfected with the indicated amount of CMV
promoter-expression plasmid, p S14 Luc, pFAS Luc, or pACL Luc as a reporter gene (0.5 g), and pSV--gal(0.5g) as reference plasmid using SuperFect (Promega). Expression plasmids used were pCMV-SREBP-1a,-1c, -2, -1aM, -1cM, -2M, and CMV7 (an empty plasmid as control). The cells were treated and luciferase activity was corrected for by  -galactosidase activity measured as described in the legend of Fig. 4. The values
were expressed as ratios of the value from control (Cont; open column). Each point represents the mean
SE of three independent transfections.

ACL promoter with peak activity at 100 ng. Activation of
ACL by SREBP-2 and SREBP-1c was lower, but significant. The YR mutation abolished the transactivity of eachisoform.

Malic enzyme gene promoter
In the reported rat malic enzyme promoter sequence,no known authentic SRE site was found (33). However,there are several SRE half sites that have been shown to be

Amemiya-Kudo et al. DNA specificity of nuclear SREBP-1a, -1c, and -2

1229

Fig. 7. Comparison of transcriptional activities of nuclear SREBP-1a(closed circle), -1c (closed square), -2 (open triangle), and respective YR
mutants (dashed lines) on the malic enzyme gene promoter measured
by luciferase reporter gene assay in HepG2 cells (A) and gel mobility
shift assay for binding of SREBP-1 to the malic enzyme gene promoter(B). A: The HepG2 cells were transfected with the indicated amount of
CMV promoter-expression plasmid, pME Luc (rat malic enzyme genepromoter fused to pGL2-Basic Vector, refer to Fig. 2) as a reporter gene(0.5 g), and pSV--gal (0.5g) as reference plasmid using SuperFect(Promega). Expression plasmids used were pCMV-SREBP-1a, -1c, -2,-1aM, -1cM, -2M, and CMV7 (an empty plasmid as control). The cells
were treated and luciferase activity was corrected for by  -galactosidase
activity measured as described in the legend of Fig. 4. The values wereexpressed as ratios of the value from control (Cont; open column). Eachpoint represents the mean SE of three independent transfections. B:
Double stranded DNA fragments containing two palindromic SRE halfsites with an adjacent Sp1 site (Fragment A) and containing one SRE
half site with an Sp1 site (fragment B) in the rat malic enzyme gene werelabeled with [-32P]dCTP and were used as probes. The labeled fragment A and B were incubated with a recombinant nuclear SREBP-1 protein. Specificity of SREBP-1 binding to the fragment A was confirmed bysupershifts after addition of SREBP-1 antibody. In competitive assays, a
1,000-fold molar excess of unlabeled fragment A or B was added prior toaddition of the labeled probe.

involved in the binding of SREBP to the SRE-like regionof the acetyl-CoA carboxylase promoter (Fig. 2)(46). Notably, at 228 bp, there is a cluster of SRE half sites(TCACCC) consisting of a region of two palindromic SRE
half sites with an adjacent Sp1 site (fragment A in Fig. 7)
and a region of a single SRE half site with an Sp1 site(fragment B in Fig. 7), which are potential SREBP binding sites. An 880 bp malic enzyme promoter-luciferase reporter gene was constructed and used in transfection
studies with SREBP expression plasmids. As shown in Fig.
7A, SREBP-1c has activity equal to that of SREBP-1a andhigher than that of SREBP-2. The fold activation by eachisoform was relatively small, but dose-dependent. The YR
mutation in each SREBP completely abolished this activity
for the ME promoter. To show a direct binding of SREBP-1
to the malic enzyme gene promoter, we performed a gel
shift assay (Fig. 7B). Both fragment A and B probes were
shifted after the addition of an SREBP-1 recombinant pro1230

Journal of Lipid Research Volume 43, 2002

tein, and specificity of its binding was validated by the disappearance of both bands in the presence of either excess
unlabeled fragment A or B, and supershifts in the presence of SREBP-1 antibody. These indicate that SREBP-1
binds to both regions.

G6PD gene promoter
The reported sequence of the G6PD promoter (approximately 0.8 kb) contains E-box, SRE, or SRE-likesites without neighboring Sp1 or NF-Y sites (Fig. 2) (34).
The G6PD promoter was fused to luciferase (G6PDLuc)and was used to assess activation by SREBPs. As shown in
Fig. 8, G6PD-Luc was activated 20-fold by SREBP-1a at
100 ng and was only slightly (3–5 fold) activated by
SREBP-1c and -2. These activations were slightly decreased by the YR mutation, the physiological significance of which is marginal.

Fig. 8. Comparison of transcriptional activities of nuclear SREBP-1a(closed circle), -1c (closed square), -2 (open triangle), and respective
YR mutants on the G6PD gene promoter measured by luciferase reporter gene assay in HepG2 cells. The HepG2 cells were transfectedwith the indicated amount of CMV promoter-expression plasmid,
pG6PD Luc (rat G6PD gene promoter fused to pGL2-Basic Vector,
refer to Fig. 2) as a reporter gene (0.5 g), and pSV--gal (0.5g) as
reference plasmid using SuperFect (Promega). Expression plasmids
used were pCMV-SREBP-1a, -1c, -2, -1aM, -1cM, -2M, and CMV7 (anempty plasmid as control). The cells were treated and luciferase activity was corrected for by -galactosidase activity measured as described in the legend of Fig. 4. The values were expressed as ratios ofthe value from control (Cont; open column). Each point represents
the mean SE of three independent transfections.

Representative SRE, SRE-like, and E-box
enhancer constructs
To explore the general features on different specificities of SREBP isoforms for SRE, SRE-like and E-box containing promoters estimated in the current study, we
made another set of enhancer constructs containing representative SRE, SRE-like, or E-box motif. As shown in Fig.
9A, we selectively chose SRE from LDLR promoter, SRElike sequence from S14 promoter, and an E-box that waspreviously reported as the cis-element that had most preferential affinity for ADD1/SREBP-1c. Fig. 9B compares activation of these constructs by native and mutant SREBPs.
The data were essentially similar to results from respective
native promoters, demonstrating that SRE, SRE-like, andE-box in the SREBP target promoters could essentiallyrepresent all SREBP activations. Furthermore, it was
found that the distance between SRE-like and NF-Y site
was very important since SREBP activation was abolishedby shortening the spacing length from the original 26 bp
to 7 bp. The importance of spacing between SRE and NF-Y
site has been reported for SRE and NF-Y site (48). A single
copy of E-box is a very weak enhancer for SREBP activation, but strong for mutant SREBPs. Interestingly, mutant
SREBP activation of E-box does not change in the presence or absence of neighboring NF-Y or Sp1 site. All thesedata suggest that SRE, SRE-like, and E-box have different
features of SREBP activation in terms of SREBP isoform
difference and requirement for cofactors.

DISCUSSION
The current studies clearly demonstrate different specificities of the three nuclear SREBP isoforms to different

cholesterogenic and lipogenic enzyme gene promoters.
Overall, the relative transcriptional activities of the SREBP
isoforms for different genes in luciferase assays were essentially similar to the extents of fold increases in corresponding mRNA levels observed in livers of SREBP transgenic mice. This indicates consistency of overexpression
tests for SREBPs between liver and HepG2 cells. It also
supports the evidence that SREBPs are directly involved in
activation of those gene promoters.
The most striking observation was found with the
E-box-Luc construct. As demonstrated by gel shift assays
in Fig. 5, SREBP-1 and -2 and their YR mutated versions
could all bind to this E-box, demonstrating that the dual
binding specificity for both SREs and E-boxes is true of
not only SREBP-1c/ADD1, which was originally described(5), but also of SREBP-1a and SREBP-2. However, very interestingly, the luciferase assays demonstrated that SREBP-2
was essentially inactive and YR mutated SREBP-2 was completely null, whereas SREBP-1 could weakly activate E-boxLuc. SREBP-2 could bind to E-box, but could not activate
its downstream gene unlike SREBP-1.
A recent paper by T. F. Osborne and colleagues elegantly
showed that binding of SREBPs to SRE induces recruitment of cofactors Sp1 and NF-Y to adjacent sites to result inacetylation of histone H3 (49). It could be speculated that
SREBPs require interaction with cofactors after binding totarget DNAs to activate the downstream gene, but SREBP-2,
unlike SREBP-1, might have an impaired interaction withthese cofactors on the E-box containing promoter. This
would be an intriguing example demonstrating that DNA
binding of a transcription factor is a necessary, but not a
sufficient condition for transactivation.
SREBP-1 can activate some other E-box or E-box-like sequences: CATGTG contained in the FAS promoter and
CGCCTG in the S14 promoter, whereas it is essentially inactive for degenerated E-boxes in the PK promoter(CACGGG and CCCGTG). Therefore, there is some preference for SREBP-1 among E-box-like sequences. Although SREBP-1 seems to prefer a complete c-myc E-box,
CACGTG, for binding (5), this sequence alone is notenough for promoter activation by SREBP-1 because thissequence was also found in the GK promoter, which
SREBP-1 could not significantly activate (Fig. 2). It was reported that overexpression of SREBP-1c/ADD1 by adenoviral vector activated GK expression. We speculated that
this GK induction could be indirect effect of SREBP-1.
The YR mutation makes SREBP-1a and -1c superactive tothe E-box-Luc while mutated SREBP-2 remained inactive.
Although SREBP-1 and not SREBP-2 is functionally capable of activating E-boxes, still arginine appears to be preferable to tyrosine in the basic region, raising a question of
its physiological action on E-box containing promoters.
SRE-containing promoters absolutely require NF-Y or Sp1
as cofactors for SREBP activation (9, 40, 50, 51). In contrast, the current study suggested that these factors do nothighly contribute to activation of E-box-Luc by native andmutant SREBP-1 (Fig. 9) and, thus, the mechanisms for
SREBP activation of E-box and SRE should be distinct.
Furthermore, the different dose-dependency for SRE-Luc

Amemiya-Kudo et al. DNA specificity of nuclear SREBP-1a, -1c, and -21231

Fig. 9. Comparison of transcriptional activities of nuclear SREBP-1a, -1c, -2, and respective YR mutants on enhancer constructs containing
representative SREBP binding sites (SRE, SRE-like, and E-box motifs) as measured by luciferase reporter gene assay in HepG2 cells . A: Construction of six enhancer luciferase reporters containing SRE (from LDLR promoter), SRE-like (from S14 promoter), and E-box (ATCACGTGAT) with or without Sp1 site or NF-Y site is shown. B: The HepG2 cells were transfected with the CMV promoter-expression plasmid, enhancer luciferase construct as indicated below (0.5 g), and pSV--gal (0.5g) as reference plasmid using SuperFect (Promega).
Expression plasmids used were pCMV-SREBP-1a, -1c, -2, -1aM, -1cM, -2M, and CMV7 (an empty plasmid as a control). After transfection, thecells were incubated for 16 h in DMEM supplemented with 10% FCS, 1 g/ml 25-hydroxycholesterol, and 10 g/ml cholesterol to suppress
endogenous SREBP activity. Luciferase activity was measured and normalized to -galactosidase activity. The values are expressed as ratios ofthe value from control (Cont; open column). Each bar represents the mean SE of four independent transfections.

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Journal of Lipid Research Volume 43, 2002

and E-box-Luc (Fig. 3) suggests that SREBP-1 has muchhigher affinity and more efficient activation for SRE thanE-box. Taken together with the fact that SREBPs did not
activate E-box containing GK and PK promoters, physiological relevance of SREBP to E-box containing promotersis currently unknown. A more precise base-by-base mutational analysis is needed to clarify the effects of SREBP-1
and -2 on different E-box sequences and to determine aphysiological role of SREBP-1 in E-box containing genepromoters.
In contrast, for lipogenic enzyme genes containing
SRE-like sequences, all the SREBP isoforms significantly
activated their transcription. This indicates that any formof SREBP can stimulate expression of most lipogenic
genes as long as the mature protein is targeted to the nucleus. In light of relative activities among the isoforms,
SREBP-1a activation is extraordinarily strong. SREBP-1acontains a longer transactivation domain and thus has
stronger transactivity than SREBP-1c. This trend is true ofmost lipogenic gene promoters with the exception of ME,
upon which SREBP-1a and -1c activate similarly. In spite ofa similar length amino-terminal transactivation domain, lipogenic enzyme gene promoters prefer nuclear SREBP-1a
to nuclear SREBP-2 in terms of molecular specificity, although the difference is not so clear as that for E-boxes.
The effect of the YR mutation on SRE-like containing promoters was partial, which places the features of SRE-likesequences between classic SREs and E-boxes. The relativetranscriptional activities of nuclear SREBP-1a, -1c, and -2
for SRE-, SRE-like, and E-box containing promoters are
summarized in Fig. 10.
The FAS promoter activity by each SREBP expression
construct implicates something intriguing. The DNA
binding site of FAS promoter for SREBPs is complicatedto interpret. Two SREBP binding sites are connected in apalindromic fashion and both sites are important for sterol regulation (13). Meanwhile, the junction of these
SREs composes an E-box, which has been shown to be responsible for the carbohydrate response of the FAS gene(Fig. 2). SREBP-1a was the strongest activator and the YR
mutation did not affect the FAS promoter activity. In lightof the presence of an E-box, it is reasonable that the YR
mutation retains the binding of SREBP to this region, as
observed in SREBP-1a and -1c, although relatively highamount of SREBPs was required. Meanwhile, SREBP-2
that is more specific to sterol regulation and is now known
to have no activity for E-boxes (Fig. 3), was sensitive to the
YR mutation for binding to this region (Fig. 5). This sensitive and fine-tuned activation property of the FAS promoter with SREBP-1 and -2 could explain why FAS is regulated in both sterol and lipogenic fashions. Another
upstream SRE (Fig. 2) has been reported to be involved inin vivo nutritional regulation of FAS gene expression by
SREBP (52).
In spite of an incredibly wide range of SREBP-1 control
over all lipogenic genes, the extent of regulation of E-box
containing lipogenic genes such as PK and GK by SREBPs
seems considerably limited. In the current study, SREBP-1
is shown not to be involved in well-defined carbohydrate

Fig. 10. Schematic representation of relative transcriptional activities of SREBP family to different DNA targets. Target sequences include classic SRE, which is usually found in the promoters of cholesterogenic enzyme and LDL receptor genes, various SRE-likesequences, and E-box (-like) sequences in lipogenic enzyme genes.
Arrows and their boldness show specificities and transcriptional activities. nSREBP, nuclear SREBP.

or insulin responsive element in the PK and GK promoters. Consistently, in the recent studies with SREBP-1
knockout mice, although induction of most of lipogenic
enzymes was severely impaired in the liver, some lipogenic
enzymes such as PK retained lipogenic induction (30).
The residual transcriptional activities for lipogenic enzymes in SREBP-1 knockout mice were more prominent
in adipose tissues. All these data indicate that there must
be other lipogenic regulators that control those lipogenic
enzymes both in liver and adipose tissue. Furthermore,
the marked superphysiological activity of YR mutated
SREBP-1 for CACGTG-type E-box also prompts us to
speculate the presence of an unknown bHLH proteinthat is structurally similar to SREBP-1 but has an arginine
residue in the basic region, and thus controls E-box typelipogenic genes. This protein would not activate SRE,
but activate E-box containing lipogenic enzyme genes
and could participate in physiological regulation ofthese genes. Upstream stimulatory factors (USF), whichhave been shown to bind to PK, GK, S14, and FAS, could
be involved; however, it does not appear that USF alone
can account for this activity as the amount of nuclear
USF did not change in these lipogenic conditions. Investigation for this residual activity could shed some lighton the understanding of transcriptional factor(s) responsible for the carbohydrate response, which cannot
be explained for by SREBP-1 and USF.
The current data may have different in vivo physiological relevance, as the major isoforms of SREBP in the liver
are SREBP-1c and SREBP-2 (20). While cholesterogenic
genes are activated by SREBP-2 and not by SREBP-1c,
these two factors are almost equally active for lipogenic
genes. However, our recent data from SREBP-1 knockout
mice confirmed that SREBP-1c dominates hepatic nutritional regulation of lipogenic enzyme genes while SREBP-2
specifically regulates cholesterol biosynthesis (30).
Taken together, it can be speculated that different methods of nuclear SREBP-1c and -2 supply are required for
distinct nutritional and metabolic regulation of cholesterol and fatty acid biosynthesis. One possibility is that
SREBP-1c controls hepatic lipogenesis by changing thebulk amount supply of nuclear SREBP-1c, overwhelming

Amemiya-Kudo et al. DNA specificity of nuclear SREBP-1a, -1c, and -21233

nuclear SREBP-2, rather than by their molecular specificities to the promoters of lipogenic genes. It seems that in
the regular fed state, SREBP-1c is much more abundant in
the liver nuclei than is SREBP-2 and maintains basal lipogenesis. In lipogenic conditions such as in a refed state after fasting or placement on a high carbohydrate diet, mature SREBP-1c protein further accumulates in the nuclei
and activates lipogenic genes; however, it is not strong
enough for activation of cholesterogenic genes (27, 30).
In contrast, SREBP-2 is not regulated by these nutritionalchanges (26, 30). This would make SREBP-1c a key transcriptional regulator for nutritional regulation of lipogenic enzymes in the liver. Another possibility is that nuclear SREBP-2 might have higher affinity to SRE than to
SRE-like sequences, causing it to preferentially bind toand activate cholesterogenic gene promoters rather thanlipogenic genes. In a cholesterol-depleted state, the reciprocal regulation of SREBP-1c and -2 is observed in livers
from mice treated with lovastatin and Colestipol (53). Nuclear SREBP-2 is robustly increased whereas nuclear
SREBP-1 is moderately suppressed. These changes resultin marked induction of all cholesterogenic genes whilesome lipogenic genes such as FAS and ACL are concomitantly induced. Drs. Goldstein and Brown have extensively
analyzed the mechanism for the release of nuclear
SREBP-2 upon sterol regulation. It has been shown to becompletely due to the activity of cleaving the SREBP2-SCAP complex by site 1 protease with its translocationfrom rER to Golgi (2, 3, 54). In contrast, the mechanismof SREBP-1 activation in liver is currently unknown. Although SREBP-1c also requires SCAP for cleavage, its
overall regulation seems to be at both cleavage and transcriptional levels. Recently, LXR has been identified as thedominant activator for SREBP-1c gene promoter, suggesting a link between lipogenesis and oxysterol regulation(55–57). Future studies are needed to elucidate the mechanism of transcriptional regulation of SREBP-1c (58).

The authors thank Drs. Joseph L. Goldstein and Michael S.
Brown for continuous support of the project. This study was
supported by the Promotion of Fundamental Studies in Health
Science of the Organization for Pharmaceutical Safety and Research (OPSR) and, in part, by a grant from the Kamakura
Women’s University.

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