Available online at www.sciencedirect.com

Biochemical and Biophysical Research Communications 368 (2008) 261–266
www.elsevier.com/locate/ybbrc

Mouse Elovl-6 promoter is an SREBP target
Shin Kumadaki a, Takashi Matsuzaka a,b, Toyonori Kato a, Naoya Yahagi a,
Takashi Yamamoto a, Sumiyo Okada a, Kazuto Kobayashi a, Akimitsu Takahashi a,
Shigeru Yatoh a, Hiroaki Suzuki a, Nobuhiro Yamada a, Hitoshi Shimano a,b,*a

Department of Internal Medicine (Endocrinology and Metabolism), Graduate School of Comprehensive Human Sciences,
University of Tsukuda, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japanb
Center for Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Received 9 January 2008
Available online 28 January 2008

Abstract
Elovl-6, a long fatty acid elongase, contributes to de novo synthesis of fatty acids and regulates hepatic insulin sensitivity. Hepatic
regulation of Elovl-6 gene expression in various nutritional conditions suggested that, like other lipogenic enzyme genes, Elovl-6 is atarget of SREBP-1, a transcription factor governing fatty acid synthesis. Supportively, adenoviral RNAi knockdown of SREBP-1 inmouse liver suppressed Elovl-6 mRNA and fatty acid synthase levels. Therefore, we analyzed mouse Elovl-6 gene promoter to determine
its role as an SREBP-1 target. Luciferase reporter assays of 1.4-kb 50 ﬂanking region of mouse Elovl-6 gene in HepG2 cells demonstrated
that nuclear SREBPs activated the Elovl-6 promoter, highlighting two SREBP binding sites: proximal SRE-1 and distal SRE-2. EMSA
indicated that SRE-1 had higher aﬃnity than SRE-2 for SREBP. ChIP assays conﬁrmed in vivo binding of hepatic nuclear SREBP-1c
protein. These data demonstrated that Elovl-6 is regulated directly and primarily by SREBP-1c.
Ó 2008 Elsevier Inc. All rights reserved.
Keywords: Fatty acids; Elongase; Sterol regulatory element-binding protein; Lipogenic enzymes; Transcription factor

Sterol regulatory element-binding proteins (SREBPs)
are members of the basic helix–loop–helix-leucine zipper
family of transcription factors [1–3] that regulate biosynthesis of both cholesterol and fatty acids. SREBP is initiallybound to the rough endoplasmic reticulum membrane, andis 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 bind to speciﬁc sterol response elements (SREs) in the promoters of target genes. Three isoforms of SREBP, SREBP-1a and -1c (also known as
ADD1), and SREBP-2 have been characterized [4–6].
*

Corresponding author. Address: Department of Internal Medicine(Endocrinology and Metabolism), Graduate School of Comprehensive
Human Sciences, University of Tsukuda, 1-1-1 Tennodai, Tsukuba,
Ibaraki 305-8575, Japan. Fax: +81 29 853 3174.
E-mail address: shimano-tky@umin.ac.jp (H. Shimano).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2008.01.075

SREBP-2 plays a crucial role in the regulation of cholesterol biosynthesis, whereas SREBP-1 mainly controls thegene expression of lipogenic enzymes [7,8]. Nuclear
SREBP-2 has high aﬃnity for classic SREs, which are usually found in the promoters of cholesterogenic genes andthe LDL receptor gene. Nuclear SREBP-1 also has a broadbinding capacity for SRE-like sequences, including E-boxes
that are occasionally found in the promoters of lipogenic
genes [9].
Elovl-6 (also known as LCE/FACE) is a lipogenic
enzyme and was identiﬁed as an SREBP-1 target by microarray analysis of SREBP-1 transgenic mice. Elovl-6 exhibits fatty acyl-CoA elongase activity speciﬁc for long chains(C12–C16 saturated and monosaturated fatty acids) and isimportant for tissue fatty acid composition. Recently, analysis of Elovl-6-deﬁcient mice demonstrated that hepatic
Elovl-6 plays a crucial role in obesity-induced insulin
resistance [10–12].

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S. Kumadaki et al. / Biochemical and Biophysical Research Communications 368 (2008) 261–266

Hepatic expression of Elovl-6 is regulated by nutritional
factors and liver X receptor (LXR), both of which areimportant regulators of SREBP-1c. Moreover, the nutritional regulation of hepatic Elovl-6 is signiﬁcantly suppressed in SREBP-1-deﬁcient mice [11]. These data
indicate that Elovl-6 is regulated by SREBP-1 in mouse
liver. Although one potential SRE:SREBP binding site in
the Elovl-6 promoter was mentioned on the basis ofsequence alignment analysis [10], the mouse Elovl-6 promoter has not been fully analyzed and has yet to be conﬁrmed as a direct target of SREBPs. In this study, adetailed promoter analysis of mouse Elovl-6 gene was performed. Our results conﬁrmed that the Elovl-6 promoter is
an SREBP target. Two SREBP binding sites were found
and were required for maximal activation by SREBP-1,
but the downstream SRE was more crucial than the
upstream SRE.

Materials and methods
Materials. T0901317 and eicosapentaenoic acid (EPA) sodium salt
were obtained from Sigma Chemicals. Redivue [a-32P] dCTP was purchased from GE Healthcare Bio-Sciences KK. Restriction enzymes wereobtained from Takara Bio Inc. and plasmid DNAs for transfection wereprepared with the QIAGEN Plasmid Midi Kit (Qiagen).
Animal experiments. All animal studies were approved by the Animal
Care Committee of the University of Tsukuba. Male C57BL/6J mice werepurchased from Clea Japan. The mice were housed in colony cages,
maintained on a 12-h light/dark cycle, and given free access to water and astandard chow diet (MF, Oriental Yeast) or a high-fat/high-sucrose diet[13]. After adenovirus injection, the mice were sacriﬁced in the light phasein a 4 h-fasted state. Tissues were isolated immediately, weighed, andstored in liquid nitrogen.
Preparation of recombinant adenovirus. SREBP-1-speciﬁc RNA interference (RNAi) constructs were subcloned into U6 entry vector (Invitrogen) using the SREBP-1 coding sequences 50 -tagagcgagcgttgaactgtatt-30 ,and a recombinant adenoviral plasmid was generated by homologous
recombination with pAd promoterless vector (Invitrogen). We produced
recombinant adenovirus in 293 cells and puriﬁed them by CsCl gradientcentrifugation as described previously [14,15].
Total RNA preparation and Northern blotting. Total RNA was isolated
from mouse livers and H2.35 cells using Sepazol RNA I Super reagent(Nacalai Tesque Inc.). Northern blot analysis was performed using a 32Plabeled probe as described previously [11,12].
Expression plasmids and reporter plasmids. All expression vectors havebeen described previously [16,17]. The reporter plasmid Elovl-6-1399-Luc
was engineered to contain a fragment of the mouse Elovl-6 promoter from
1399 to ±0 bp cloned into the MluI/XhoI sites of the pGL3 basic vector(Promega) containing the coding sequences of ﬁreﬂy luciferase cDNA.
Other constructs were produced by PCR using this construct as a DNA
template, and the PCR products were inserted into the pGL3 basic vector.
The primers used in PCR were as follows: 50 -primer Elovl-6-473-Luc, 50 -cttacgcgtagggtgctgcgggat-30 ; Elovl-6-273-Luc, 50 -cttacgcgtccaagaggtggcgaa
tg-30 ; Elovl-6-173-Luc, 50 -cttacgcgtaacaaaactatcccccagggc-30 ; Elovl-6-103Luc, 50 -ccgacgcgt- cccgatcgcacgaggggga-30 ; and Elovl-6-88-Luc, 50 -cttacgc
gtgggaggagatttccctgtaga-30 , 30 -primer 50 -gggctcgagaccgtaggaggaaatgaatg30 . The site-directed mutagenesis constructs, SRE-1(m) and SRE-2(m)Luc, were produced by PCR with the following primers: SRE-1(m)-50 , 50 gggttaattttcccgatataaagagggggaggagatttc-30 ; SRE-1(m)-30 , 50 -gaaatctcctccc
cctctttatatcgggaaaattaaccc-30 ; SRE-2(m)-50 , 50 -catttatttgcctggagttctacg-gac
agagaacctggc-30 ; and SRE-2(m)-30 , 50 -gccaggttctctgtccgtagaactccaggcaaa
taaatg-30 . The double-mutagenesis construct SRE-1 2(m)-Luc was produced by ligation of SRE-1(m) and SRE-2(m) PCR products digested with

HindIII. Ligation was performed using the Quick Ligation Kit (NewEngland Biolabs Inc.).
Cell cultures. HepG2 cells were cultured in minimum essential medium
Eagle (Sigma) supplemented with 1 mM sodium pyruvate (GIBCO), 1%
non-essential amino acids (GIBCO), 10% FBS, and 1% penicillin–streptomycin (Sigma). For luciferase assay, HepG2 cells were seeded in 24-wellplates at a density of 2.5  105 cells/well. H2.35 cells were cultured in
Dulbecco’s modiﬁed Eagle’s medium supplemented with 200 nM
dexamethasone (Sigma), 4% FCS, and 1% penicillin–streptomycin. For
chromatin immunoprecipitation assay (ChIP), H2.35 cells were seeded in
15-cm plates and the indicated chemicals were added to the conﬂuent cells.
Transfection and luciferase assays. Each expression plasmid (200 ng),luciferase reporter plasmid (200 ng), and pSV40-Renilla plasmid (50 ng)
was cotransfected into HepG2 cells using the FuGENE6 reagent (Roche
Applied Science) according to the manufacturer’s instructions. After 24 hof transfection, cells were washed with phosphate buﬀer saline (PBS) andharvested. Luciferase assays were performed according to the manufacturer’s instructions (Promega), and luciferase activity was quantiﬁed using
the ARVO SX 1420 multilabel counter (Perkin Elmer Life Sciences). As
the internal standard, SV40 Renilla luciferase control vector was also
cotransfected to normalize for transfection eﬃciency.
Electrophoretic mobility shift assay. Electrophoretic mobility shift
assay (EMSA) was performed as described previously [17,18]. In brief, arecombinant SREBP-1a protein was produced using the TNT T7 quickcoupled transcription/translation system (Promega). The reaction mixture
was incubated with in vitro synthetic protein lysate and then analyzed on
5% polyacrylamide gels in 0.25  TBE at room temperature. The SREBP1 antibody (anti-SREBP-1, sc-8984; Santa Cruz) was used to conﬁrm
speciﬁc binding. The wild-type (wt) and mutant (mut) probes for EMSA
were as follows: Elovl-6 SRE-1 (wt), gttaattttcccgatcgcacgagggggaggac;
Elovl-6 SRE-1 (mut), gttaattttcccgatATAaAgagggggaggac; Elovl-6 SRE-2(wt), gcctggagttctcatgccagagaacctggc; and Elovl-6 SRE-2 (mut),
gcctggagttctACGgAcagagaacctggc. Each probe contains the indicatedSRE (underlined). Mutations in these SREs are shown in capital letters.
ChIP assay. ChIP assays were performed as previously described withsome modiﬁcations [19]. In brief, we immunoprecipitated the complexes ofgenomic DNA and SREBP-1 with antibodies against SREBP-1 (rabbit
polyclonal; No. 931 for mouse SREBP-1 [20]) from H2.35 cells. The
extracted genomic DNA was then subjected to PCR ampliﬁcation. To
amplify the Elovl-6 promoter region containing SRE-1 and SRE-2, thefollowing primer sets were used: for SRE-1, 50 -ccagtccaccaaccaccttc-30(sense), and 50 -aatgaatgccttgcggtctt-30 (antisense); and for SRE-2, 50 -cggg
atcgaggtagtgagga-30 (sense), and 50 -gcctggcctttgaatgaagt-30 (antisense).

Results
To evaluate the short-term eﬀect of SREBP-1c deletion
on Elovl-6 gene expression, normal C57BL6 mice were
infected with adenoviruses containing SREBP-1 RNAi.
Northern blot analysis revealed that knockdown of
SREBP-1 was complete and was accompanied by a strong
suppression of fatty acid synthase levels, a representative
SREBP-1 target gene. Elovl-6 gene expression was also
severely inhibited, indicating that Elovl-6 could be a potential target of SREBP-1 (Fig. 1A). Subsequently, the mouse
Elovl-6 promoter was tested as an SREBP target. The 1.4kb 50 region upstream of the mouse Elovl-6 gene wascloned into a luciferase vector and used for luciferase
reporter assay after the transfection of expression plasmids
for various transcription factors into cells. The Elovl-6 promoter was strongly activated by coexpression of SREBP1a, -1c, and -2. The reporter was weakly activated by
HNF-4a and Sp-1, but not LXR, RXR, and PPARs(Fig. 1B).

S. Kumadaki et al. / Biochemical and Biophysical Research Communications 368 (2008) 261–266

B
Chow

HF/HS

LacZi LacZi BP1i
SREBP-1
FAS

70
60
50
40
30
20
10
0

pc
D
N
A
3
S R . 1(EB +)SR P-1EB a
P
S R - 1c
EB
PLX 2
R
α
R
X
R
H α
N
FH 1β
N
F4α
Sp
-1
PP PPAAR Rαα+Fe
P n o.
PP PARAR γγ+
P
Fo io.
x
Fo o-1xo-1
3A

Elovl-6

Relative Luciferase Activity

A

263

36B4

Fig. 1. Regulation of Elovl-6 promoter by SREBP-1. (A) Northern blot analysis of 36B4, SREBP-1, FAS, and Elovl-6 gene expression in the liver of
SREBP-1 knockdown mice. Knockdown of hepatic SREBP-1 was performed by adenoviral expression of RNA interference (RNAi). Seven-week-old male
C57BL/6J mice were fed a standard chow or high-fat/high-sucrose (HF/HS) diet for 15 weeks, followed by tail vein injection with adenovirus encoding
RNAi targeting SREBP-1 (BP1i) or LacZ (LacZi) sequence (adenoviral dose of 1.0  1011 viral particles per mouse). After 6 days of HF/HS feeding, themice were sacriﬁced in a 4-h-fasted state. (B) Activation of the Elovl-6 promoter by SREBPs. The mouse Elovl-6 promoter region (1399 bp) was fused to aluciferase reporter gene (Elovl-6-1399-Luc). HepG2 cells were cotransfected with Elovl-6-1399-Luc as a reporter gene, pSV40-Renilla as a reference, andthe indicated expression plasmids or control vector (pcDNA3.1). At the same time, 100 lM fenoﬁbrate (Sigma) or 10 lM pioglitazone (Cayman Chemical)
was added to the indicated cells. The relative Luc activity was expressed as a fold change to that of the control vector (pcDNA3.1). The luciferase activity
was normalized to Renilla luciferase activity and the assays were performed in triplicate. Results are expressed as means ± SEM. Feno, fenoﬁbrate; Pio,pioglitazone.

As shown in Fig. 2A, the mouse Elovl-6 promoter
sequence contains two putative SREs as potential SREBP
binding sites, SRE-1 and SRE-2, and an E-box. Sequential
deletion studies indicated that SRE-1 was mainly responsi-

A

Mouse Elovl-6 Promoter
-1399

B

ble for SREBP activation (Fig. 2B), whereas SRE-2 contributed weakly and E-box did not contribute at all.
EMSA indicated that SREBP-1a protein binds strongly
to SRE-1, but weakly to SRE-2 (Fig. 3A). The introduction

Elovl6-1399-Luc
Elovl6-473-Luc
Elovl6-273-Luc
Elovl6-173-Luc
Elovl6-103-Luc

Transcription
±0

SRE-2

E-box

SRE-1

TCATGCCAG
SRE-2-398 - -390

GAGGTC
E-box-147 - -142

TCGCACGAG
SRE-1-98 - -90

SRE-2

E-box

SRE-1

SRE-2

E-box

SRE-1

E-box

SRE-1

E-box

SRE-1
SRE-1

Elovl6-88-Luc

pcDNA3.1
SREBP-1apGL3-Basic
0

5

10

15

20

25

30

35

Relative Luciferase Activity(Fold Activation of Control)

Fig. 2. Requirement of both SRE-1 and SRE-2 regions for SREBP-1a activation of mouse Elovl-6 promoter. (A) Schematic representation of mouse
Elovl-6 promoter with the sequences and locations of potential SREBP binding sites (SRE-2, E-box, and SRE-1 as novel potential sites). (B) Deletion
studies in SREBP-1a activation of the mouse Elovl-6 promoter. The schematic representation of mouse Elovl-6 promoter luciferase deletion constructs
used in this study is shown. Various reporter gene plasmids (Elovl-6-1399, -473, -273, -173, -103, and -88-bp-Luc) were cotransfected into HepG2 cells with
pSV40-Renilla as a reference and SREBP-1a expression plasmid or pcDNA3.1. The relative Luc activity was expressed as a fold change to that of thecontrol vector (pcDNA3.1). The luciferase activity was normalized to Renilla luciferase activity, and the assays were performed in triplicate. Results are
expressed as means ± SEM.

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S. Kumadaki et al. / Biochemical and Biophysical Research Communications 368 (2008) 261–266

A

LDLR
SRE

Probe
SREBP-1a-Elovl-6
SRE-2(wt)

Elovl-6
SRE-2(mut)Elovl-6
SRE-1(wt)

Elovl-6
SRE-1(mut)

-

-

-

-

BP1a
NS

Elovl-6
SRE-2(wt)

B ProbeAnti-BP1
SREBP-1aCompetitor

-

+

+

+

+

LDLR SRE-2
wt mutElovl-6
SRE-1(wt)
+

+

+
+

-

+

SRE-1

+

+

+

LDLR SRE-1
wt mut

+

+

+
+

SRE-2

BP1a
NS

Fig. 3. SREBP-1a binding to SRE-1 and SRE-2 in mouse Elovl-6 promoter. (A and B) EMSA with radiolabeled probes for the authentic human LDL
receptor SRE (LDLR SRE) and Elovl-6 SREs (wild-type (wt) and mutant (mut) as indicated in ‘‘Materials and methods”). In vitro translated SREBP-1aproteins were incubated with these labeled probes. After incubation, the binding reaction mixtures were subjected to EMSA on a nondenaturing
polyacrylamide gel as described in ‘‘Materials and methods”. (A) Various amounts of in vitro translated products were incubated with probes. (B) The
competition was performed using unlabeled probes as competitors in 250- or 1000-fold molar excess. Speciﬁc binding was conﬁrmed using a polyclonal
antibody against SREBP-1 for competition. The bound SREBP-1a/DNA complex (BP1a) and a non-speciﬁc shifted complex (NS) are shown in Fig. 3.

of mutations into the sites SRE-1 and SRE-2 cancelled thebinding. The signal was abolished by the addition of cold
authentic SRE DNA probe from the LDL receptor promoter, and supershifted by the addition of SREBP-1 antibody, conﬁrming the speciﬁcity of SREBP bindings to
SRE-1 and SRE-2 (Fig. 3B). Mutual competition between
SRE-1 and SRE-2 in EMSA demonstrated that SRE-1 has
higher aﬃnity for SREBP than SRE-2. This supports datafrom the deletion studies, which show that SRE-1 is more
important for SREBP activation. Results from EMSA
and the deletion studies were further supported by mutation analysis (Fig. 4A). The promoter construct containing
both SRE-1 and SRE-2 was activated by SREBP. Mutation of SRE-1 and/or SRE-2 decreased this activation to
the extents predicted by the deletion studies and EMSA.
Finally, direct binding of SREBP-1 to SRE-1 and SRE-2
in vivo was estimated by ChIP assay. First, SREBP-1c in
H2.35 cells was activated by T0901317, an LXR agonist,and suppressed by EPA (Fig. 4B). Concomitantly, Elovl6 expression was activated by T0901317 and suppressedby EPA. ChIP assay of nuclei from these sets of treated
H2.35 cells conﬁrmed direct binding of SREBP-1 to
SRE-1 in vivo (Fig. 4C). The signal from SRE-1 was consistently changed by T0901317 and EPA in an SREBP-1regulated manner. In contrast, the positive signal showing
direct binding to SRE-2 could not be detected.

Discussion
The present study clearly demonstrates that Elovl-6 is an
SREBP target. As previously reported, expression of Elovl6 was regulated in an SREBP-dependent manner in transgenic and knockout mice [11]. Nutritional regulation of
Elovl-6 expression, such as fasting–refeeding, high-fat diet,polyunsaturated fatty acids, or in ob/ob mice, as well asactivation by LXR could be mediated through SREBP1c. Our data conﬁrmed that SREBP-regulated Elovl-6
expression is mediated through the direct binding of
SREBP to the Elovl-6 promoter. In luciferase reporter
assays, SREBP-1a and SREBP-2 equally activated the
Elovl-6 promoter, whereas SREBP-1c was less active. Thispattern of transactivation, depending upon SREBP isoforms, was similarly observed in many typical SREBP target gene promoters [9]. Elovl-6 was a crucial step in thefatty acid synthesis pathway, suggesting that SREBP-1c isthe major transcription factor for the physiological regulation of Elovl-6.
A series of promoter analyses demonstrated that there
are two SREBP binding sites: SRE-1 and SRE-2. Althoughboth could potentially contribute to the activation of the
Elovl-6 promoter, proximal SRE-1 having higher aﬃnity
for SREBP is more important for SREBP activation. ChIP
assay of our experimental settings demonstrated that

S. Kumadaki et al. / Biochemical and Biophysical Research Communications 368 (2008) 261–266

A265

pGL3-Basic
Elovl6-88-Luc
SRE-1

Elovl6-103-Luc
Elovl6-473-Luc(wt)

SRE-2

SRE-1

SRE-2(m)

SRE-2

SRE-1

SRE-1(m)

SRE-2

SRE-1

SRE-1,2(m)

SRE-2

SRE-1

pcDNA3.1
SREBP-1a

0

5

10

15

20

25

30

35

40

Relative Luciferase Activity(Fold Activation of Control)

B

V

C

T090 EPA

Input

Mw V T090 EPA

SREBP-1

Ab-SREBP1

IgG

V T090 EPA V T090 EPA Mw

Elovl-6

SRE-1 primer(187 bps)

300bp
200bp
100bp

36B4

SRE-2 primer(183 bps)

300bp
200bp
100bp

Fig. 4. SRE-1 as a novel SREBP binding site is stronger than SRE-2 in exogenous and endogenous SREBP-1 activation of Elovl-6 promoter, (A) Eﬀects
of mutation in SRE-1 and/or SRE-2 on SREBP-1a activation of Elovl-6 promoter. Native (Elovl-6-473, -103, and -88-bp-Luc), mutant-SRE-1 (SRE1(m)), mutant-SRE-2 (SRE-2(m)), or combined mutant- SRE-1 and 2 (SRE-1, 2(m)) reporter gene plasmids were cotransfected into HepG2 cells with
pSV40-Renilla and SREBP-1a expression plasmid or pcDNA3.1. These mutant plasmids have the same mutations in the indicated SREs as the EMSA
probes. The relative Luc activity was expressed as a fold change to that of the control vector (pcDNA3.1). The luciferase activity was normalized to Renillaluciferase activity and the assays were performed in triplicate. Results are expressed as means ± SEM. (B) Northern blot analysis of 36B4, SREBP-1, andElovl-6 gene expression in H2.35 cells. Conﬂuent cells were incubated with T0901317 (1 lM) or EPA (50 lM) for 24 h. V, vehicle. T090, T0901317. (C)Chromatins prepared from H2.35 cells (under the same conditions as described in A) were subjected to chromatin immunoprecipitation assay with an antimouse SREBP-1 antibody and rabbit IgG as the negative control. Immunoprecipitate samples were subjected to PCR using primers to amplify the SRE-1or SRE-2-containing region of the mouse Elovl-6 promoter. Mw, molecular weight.

SREBP bound only to SRE-1 in vivo. Therefore, theseresults indicate that SRE-1 is the primary binding site of
SREBP in the Elovl-6 promoter.
Regulatory mechanism(s) other than SREBPs may also
exist. To elucidate such mechanisms, further studies on
Elovl-6 regulation using regions far upstream or intronsof this gene are required. However, presently it can be concluded that SREBPs are the primary transcription factors
for the proximal promoter region of mouse Elovl-6 gene.
Acknowledgments
This work was supported by Grants-in-Aid from the
Ministry of Science, Education, Culture, and Technologyof Japan.
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