© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
SREBP-1 Binds to Multiple Sites and Transactivates the Human ApoA-II Promoter In Vitro
SREBP-1 Mutants Defective in DNA Binding or Transcriptional Activation Repress ApoA-II Promoter Activity
From the Section of Molecular Genetics, Cardiovascular Institute, Departments of Biochemistry and Medicine, Boston University Medical Center, Boston, Mass.
Correspondence to Vassilis I. Zannis, Section of Molecular Genetics, Cardiovascular Institute, Department of Medicine, Boston University Medical Center, 700 Albany St, W-509, Boston, MA 02118-2394.
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Abstract—Screening of an expression human liver cDNA library resulted in the isolation of several cDNA clones homologous to sterol regulatory element-binding protein-1 (SREBP-1) that recognize the regulatory element AIIAB and AIIK of the human apoA-II promoter. DNaseI footprinting of the apoA-II promoter using SREBP-1 (1 to 460) expressed in bacteria identified 5 overall protected regions designated AIIAB (-64 to -48), AIICD (-178 to -154), AIIDE (-352 to -332), AIIHI (-594 to -574), and AIIK (-760 to -743). These regions contain inverted E-box palindromic or direct repeat motifs and bind SREBP-1 with different affinities. Transient cotransfection experiments in HepG2 cells showed that SREBP-1 transactivated the -911/29 apoA-II promoter 3.5-fold as well as truncated apoA-II promoter segments that contain 1, 2, 3, or 4 SREBP binding sites. Mutagenesis analysis showed that transactivation by SREBP was mainly affected by mutations in element AIIAB. Despite the strong transactivation of the apoA-II promoter by SREBP-1 we could not demonstrate significant changes on the endogenous apoA-II mRNA levels of HepG2 cells after cotransfection with SREBP-1 or in the presence or absence of cholesterol and 25-OH-cholesterol. An SREBP-1 mutant lacking the amino-terminal activation domain bound normally to its cognate sites and repressed the apoA-II promoter activity. Repression was also caused by specific amino acid substitutions of Leu, Val, or Gly for Lys359, which affected DNA binding. Repression by the DNA binding-deficient mutants was abolished by deletion of the amino-terminal activation domain (1 to 90) of SREBP-1. Overall, the findings suggest that the wild-type SREBP-1 can bind and transactivate efficiently the apoA-II promoter in cell culture. SREBP-1 mutants lacking the activation domain bind to their cognate sites and directly repress the apoA-II promoter whereas mutants defective in DNA binding indirectly repress the apoA-II promoter activity, possibly by a squelching mechanism.
Key Words: sterol regulatory element-binding protein-1 • apoA-II • transcriptional regulation • basic helix-loop-helix zipper factors • upstream regulatory factor
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Introduction |
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Sterol regulatory element-binding protein 1 (SREBP-1) was initially identified as the transcription factor that recognizes the sterol regulatory element-1 (SRE-1) of the LDL receptor promoter.1 2 Two isoforms of SREBP-1, 1a and 1c, have been described along with a homologous protein designated SREBP-2.3 4 Although SREBP-1 and SREBP-2 belong to the family of basic helix-loop-helix zipper (bHLHZip) proteins, they are much larger than other bHLHZip proteins, having 1147 and 1141 amino acids, respectively, and can recognize direct (CACCCCAC) as well as E-box type (CANNTG) palindromic repeats.3 4 5 SREBP family members possess 2 transmembrane domains that anchor them to the endoplasmic reticulum.6 7 8 Their transcriptional activation domain is highly acidic and is located at the amino terminus.7 On sterol depletion, the protein is cleaved by 2 proteases to produce the active part of the molecule of molecular weight (Mr) 68 kDa that translocates to the nucleus and activates the cholesterol-responsive genes.6 8 9 10
The SREBP-1 protein was also cloned independently by screening of an adipocyte expression library with E-box elements as probes.11 The protein was named ADD1 for adipocyte differentiation-dependent protein 1 on the basis of its induction after differentiation of the 3T3L1 preadipocytes. It has been proposed, on the basis of the high levels of expression of SREBP-1 in liver and fat tissues, that SREBP family members may regulate not only cholesterol-responsive genes1 2 but also genes involved in fatty acid biosynthesis and metabolism in adipocytes.12 13 14 15 After the cloning of SREBP-1 and SREBP-2, other genes have been shown to be regulated by the same factors. Many of them are involved in cholesterol and fatty acid metabolism.9 13 14 15
Analysis of sterol-resistant Chinese hamster ovary (CHO) cell lines have provided strong evidence for the physiological significance of SREBPs in the negative-feedback loop that regulates the expression of the LDL receptor. These cell lines have a frameshift mutation that produces a truncated SREBP-2 protein lacking the transmembrane domain.16 This truncated SREBP-2 form constitutively activates the LDL receptor and 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) synthase genes.
Human apoA-II is a major protein component of HDL and is synthesized by the liver and to a much lesser extent by the intestine.17 18 Genetic studies in mice have pointed out that apoA-II may alter the antiatherogenic functions of apoA-I and may thus contribute to the pathogenesis of atherosclerosis.19 20 The transcription of the human apoA-II gene is controlled by a complex array of distal and proximal regulatory elements, designated AIIA to AIIN, which have been divided in 3 functional regions. The distal region (-911/-614) consists of elements AII-I to AIIN and displays an enhancer-type activity with heterologous promoters in HepG2 cells.21 22 Important for the overall promoter activity is also the regulatory element AIIAB located in the proximal region.
Recently, we have also characterized several previously described as well as new transcription factors that bind to these elements.21 An important regulatory role is exerted by the factor CIIIB123 that binds to the regulatory elements AIIAB, AIIK, and AIIL. Simultaneous nucleotide substitutions that prevented the binding of CIIIB1 to elements AIIAB, AIIK, and AIIL reduced the hepatic and intestinal transcription to 6% to 7% of control.24 25 The current study shows that SREBP-1 binds to previously described as well as new regulatory elements and transactivates the human apoA-II promoter. Two of the binding sites of SREBP-1 on elements AIIAB and AIIK overlap with the binding site of CIIIB1, which was shown recently to correspond to the bHLHZip upstream stimulatory factor (USF).26 In addition, use of SREBP-1 mutants defective in DNA binding or transcriptional activation has provided new insights in the activation of the apoA-II promoter by SREBP-1.
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Methods |
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Materials
The Klenow fragment of the DNA polymerase I, restriction enzymes, T4 ligase, T4 polynucleotide kinase, and Vent polymerase were purchased from New England Biolabs. [
32P]ATP (5000
Ci/mmol), [
32P]dCTP, and
[32P]dGTP (3000 Ci/mmol) were purchased from
New England Nuclear. DNaseI was from Worthington. Anti-FLAG monoclonal
antibody was from Kodak, IBI. The secondary antibody was from Santa
Cruz. O-nitrophenyl-1
D-galacto-ß-pyranoside (ONPG),
cholesterol, and 25-OH-cholesterol were from
Sigma. Chloramphenicol acetyltransferase (CAT) ELISA kit was from
Boehringer Mannheim. The RNase protection kit and the ß-actin
probe were purchased from Ambion. Sequenase 2.0 and the ECL system were
from Amersham. Reagents for automated DNA synthesis were purchased from
Applied Biosystems, Inc. Bactotryptone and bacto yeast extract were
purchased from Difco. Double-stranded poly(dI-dC) was purchased from
Pharmacia LKB Biotechnology, Inc. Acrylamide, SDS, urea,
and Tris were purchased from International Biotechnologies, Inc.
Bacterial XL-1 Blue cells were purchased from Stratagene.
Preparation of Concatenated Probes and Screening of Human Liver
cDNA Library
Ten micrograms each of the sense and the antisense strands of a
single-stranded oligonucleotide of AIIAB having 3 G or
C, respectively, at their 5' and 3' ends (Table 1
) were
phosphorylated for 2 hours at 37°C using
T4 polynucleotide kinase. Ten
micrograms each of the sense and antisense strands of the
single-stranded oligonucleotides AIIAB (Table 1)
were annealed and purified by phenol extraction and ethanol
precipitation. The annealed oligonucleotides were
ligated with T4 DNA ligase to produce
concatenated probes that were labeled by nick translation. Plating and
screening of the human liver cDNA library was performed as
described.27 Briefly, the pellet from 20 mL of overnight
culture of Escherichia coli
(Y1090(r-)) was dissolved in 8 mL of ice-cold
10 mmol/L MgSO4, and cells from 0.2 mL of
the overnight culture were infected with 0.1 mL of 1x
dilution
buffer containing 10 000 plaque-forming units (pfu) of gt11, human
liver cDNA library, Clontech. They were incubated at 37°C for 15
minutes, mixed with 6 mL of top agarose (45°C), and plated on
150-mm-diameter prewarmed plates (42°C). After 3 to 4 hours' growth
at 42°C, the plates were covered with dried filters that had been
soaked in 10 mmol/L
isopropyl-ß-D-galactopyranoside (IPTG) and
incubated overnight at 37°C. The next day, the filters were lifted
and air-dried. Proteins bound to the filters were denatured by
treatment with denaturing solution containing 5 mol/L guanidine
hydrochloride (GnHCl) and renatured by sequential dilution of
the solution followed by 5 minutes' incubation of the filters at
4°C. Next the filters were incubated in blocking solution (Blotto)
containing 5% nonfat dry milk and were washed with washing solution
(20 mmol/L HEPES, pH 7.6, 50 mmol/L KCl, 5 mmol/L
MgCl2, 1 mmol/L EDTA, 0.25% nonfat dry
milk, 8% glycerol, 1 mmol/L DTT, pH 7.6, 0.1% Triton X-100) to
remove excess milk. Hybridization solution was added and the filters
were hybridized overnight at 4°C. The filters were washed the
following day for 10 minutes, air-dried, and autoradiographed as
described.27 Candidate clones underwent secondary and
tertiary screening to isolate pure clones.
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To isolate DNA from pure phage clones,
Y1090(r-) cells were infected with phage
particles at a multiplicity of infection of 8:1 to ensure confluent
lysis and were plated on a 150 mmol/L plate (Luria
broth-agarose). After 4 to 5 hours at 42°C, the cell lysate
was extracted in 10 mL of sodium magnesium buffer (0.1 mol/L
NaCl, 0.01 mol/L MgSO4, 0.05 mol/L Tris-Cl pH 7.5
and 2% gelatin) by incubation at 4°C overnight. The SM buffer was
transferred to a 15-mL tube and centrifuged for 10 minutes at
6000 rpm at 4°C to remove debris. The supernatant was transferred to
a clean tube and centrifuged for 90 minutes at 25 000 rpm at
4°C using an SW41 rotor (Beckman Co). The pellet was
resuspended in 400 µL of deionized H2O,
50 µL of 10% SDS, 40 µL of 0.5 mol/L EDTA, and 25 µL of
proteinase K (20 mg/mL), and the mixture was incubated at 65°C for 10
minutes. The DNA was then purified by chloroform extraction and ethanol
precipitation, and stored at -80°C. The DNA was digested with
EcoRI, and the insert was subcloned into pBluescript SK
vector (Stratagene). Partial DNA sequence was obtained from both ends
using the Sequenase 2.0 kit (Amersham). The sequences obtained were
submitted for homology comparison against the sequences deposited in
Genbank.
Plasmid Constructions
ApoA-II Promoter Mutants
To remove the middle elements AIIC to AIIF from the apoA-II
promoter, the -911 to -616 and -67 to 29 regions were amplified
separately by PCR using the -911/29 apoA-II pUCSH CAT plasmid
as a template.25 The region of the apoA-II enhancer (-911
to -616) was amplified with the 5-rev-26 and AII3ApaI primers (Table 1
). The proximal region of the apoA-II promoter was amplified
with the primers AIIAB and 3-CAT-26. The primers AII3ApaI and AIIAB
contain an ApaI site. The PCR products were
digested with XbaI and ApaI (-911 to -616) and
with ApaI and XhoI (-67 to 29). The parental
pUCSH CAT vector23 was also digested with
XbaI and XhoI. The 2 PCR fragments and the pUCSH
CAT vector were ligated in a triple ligation to produce apoA-II
promoter with deletion of the region -616 to -67 (AIIAB wild-type
AIIK wild-type). The mutation of the AIIK element was produced by
amplification of the apoA-II enhancer with 2 sets of overlapping
primers (5-rev-26 and AIIK3) and (AIIK4 and AII3ApaI). An aliquot
containing 1% of each of the PCR products was mixed and amplified
with the external 5-rev-26 and AII3ApaI primers. The mutant enhancer
was digested with XbaI and ApaI. The AIIAB
element was mutated by amplification of the wild-type apoA-II promoter
with the primers AIIABM7 and 3-CAT-26. The PCR product was digested
with ApaI and XhoI. The mutant apoA-II enhancer
was ligated to the wild-type proximal (-67 to 29) region and the pUCSH
CAT vector to produce the AIIAB wild-type AIIK mutant construct.
Alternatively the mutant apoA-II enhancer was ligated to the mutated
-67 to 29 region and the pUCSH CAT vector to produce the AIIAB mutant
AIIK mutant construct. Finally, the wild-type apoA-II enhancer was
ligated to the mutated -67 to 29 region and the pUCSH CAT vector to
produce the AIIAB mutant AIIK wild-type construct. The final constructs
and the mutations in the elements AIIAB and/or AIIK were verified by
DNA sequencing.
Wild-type and Mutant SREBP-1 Expression Plasmids
The cDNA for the SREBP-1a was kindly provided by Dr M. Brown
(University of Texas Health Science Center).4 The flagged
versions of SREBP-1 (1–460) were constructed by PCR-mediated ligation.
Briefly, 2 regions of SREBP-1 cDNA (-132 to 1 and 1 to 1548) were
amplified separately with 2 sets of primers (SREBP-1A, SREFLAGR) and
(SREFLAGF, SREBP-1R) (Table 1). The primers SREFLAGF and
SREFLAGR contained overlapping portions of the FLAG peptide
sequence (DYKDDDDK). In the next PCR, an aliquot containing 2% of the
2 amplification products were mixed together and amplified with the
outside primers (SREBP-1A and SREBP-1R; Table 1) to produce the
final product. These 2 primers contained restriction sites for the
BamHI and EcoRI enzymes that were used to
subclone the construct into the corresponding sites of the pcDNAI.Amp
vector (Invitrogen). The final protein contains the FLAG peptide
followed by 460 amino acids of SREBP-1. The SREBP-1 deletion mutant was
constructed using the same strategy but different sets of overlapping
primers (SRE90FLG and SREFLAGR). Both primers contain the overlapping
FLAG sequence; however, the SRE90 FLAG corresponds to the cDNA region
encoding amino acids 90 to 94 (Table 1). For the construction of
the point mutants in the DNA-binding domain of SREBP-1, 2 overlapping
oligonucleotides were used that contained 2 degenerate
bases corresponding to the amino acid Lys359 (SREKI-F2 and SREKI-R2).
After DNA amplification, this degeneracy resulted in the
production of 4 different mutated cDNAs that had the Lys359
mutated to Arg, Val, Gly, or Ile. All the mutant cDNA were confirmed by
restriction enzyme digestion and DNA sequencing of the mutated
regions.
Expression of SREBP-1 Protein in BL21(DE3) Cells
The region 167 to 1548 of the SREBP-1 cDNA, which encodes a
protein of 460 amino acids, was amplified and subcloned into the
NdeI and EcoRI sites of the bacterial vector
pAED4 (pET3a derivative). The construct was used to transform E
coli BL21 (DE3). This strain expresses T7 RNA polymerase under the
control of lac promoter. An overnight culture of bacteria was diluted
80-fold in 4 mL of Luria broth medium containing 100 µg/mL ampicillin
and incubated for approximately 1.5 hours.
Isopropyl-ß-D-thiogalactopyranoside (IPTG) was then added to a final
concentration of 1 mmol/L, and the culture was maintained for an
additional 3 hours. Next the bacteria were spun for 5 minutes at 5000
rpm, and the pellet was dissolved in 0.5 mL of denaturation buffer
(20 mmol/L HEPES, pH 7.6, 50 mmol/L KCl, 5 mmol/L
MgCl2,1 mmol/L EDTA, 8% glycerol, 1
mmol/L DTT, 6 mol/L GnHCl, pH 7.6) supplemented with protease
inhibitors (1 mmol/L aprotinin, 1 mmol/L
benzamidine, 1 mmol/L leupeptin, 1 mmol/L PMSF). The
bacterial lysates were rotated on a platform for 30 minutes at 4°C
and centrifuged for 5 minutes in a microfuge, and the
supernatant was dialyzed twice for 1 hour against an excess of the
denaturation buffer without GnHCl and containing 1 mmol/L PMSF.
After dialysis, the extracts were centrifuged for 5 minutes at
4°C in a microfuge to remove precipitated material, and the
supernatant containing SREBP-1 was stored at -80°C.
DNaseI Footprinting Assays
The -911/29, -614/29, and -440/29 apoA-II pUCSH CAT
constructs25 were amplified with external primers
(5-rev-26 and 3-CAT-26; Table 1). The primer 5-rev-26 was
end-labeled with T4 polynucleotide
kinase. These PCR products were used to footprint the distal (AIIK,
AIIHI, and AIIDE) SREBP-1–binding sites. To footprint the proximal
(AIIAB and AIICD) sites, the -911/29 apoA-II pUCSH CAT25
was digested with XhoI, end-labeled with
T4 polynucleotide kinase, and then
digested with SalI. To footprint SREBP sites on the apoC-III
promoter, the -890/24 apoC-III pUCSH CAT23 was
digested with XhoI, end-labeled with
T4 polynucleotide kinase, and then
digested with XbaI. All the labeled promoter fragments were
separated on 5% polyacrylamide gel electrophoresis (PAGE) and
purified by electroelution. Footprinting was performed with 4-µL
extracts of bacteria expressing SREBP-1 as
described.23 The position of the binding sites of
SREBP-1 in the apoA-II and apoC-III promoters were determined by
comparing the SREBP-1 footprints with the G+A ladder produced by
chemical cleavage of the same DNA fragment.
Gel Electrophoretic Mobility Shift Assays
This analysis was performed with 1-µL extracts of
bacteria expressing SREBP-1 as described.23 The final
probe concentration in all reactions was 2.5 nmol/L (50 000 cpm). The
competitors were used at 10- to 250-fold excess in relation to the
32P labeled probe.
Transient Transfection Assays
Human hepatoma HepG2 cells and monkey kidney COS-1 cells were
routinely grown in Dulbecco's modified Eagle's medium (DMEM)
containing 10% FBS. HepG2 cells were plated in 30-mm dishes at a
density of 5x105/plate in DMEM supplemented with
10% FBS. They were transfected by calcium phosphate coprecipitation
method with a total of 6 µg of DNA (3 µg of reporter CAT plasmid, 1
µg of cytomegalovirus (CMV) ß-galactosidase (ß-gal), and
appropriate amounts [0.2 to 2 µg] of SREBP-1). COS-1 cells were
transfected with 4 µg of hepatic nuclear factor 4 (HNF-4) or the
FLAG-SREBP-1 (1–460) and used for the production of whole cell
extracts. Forty-eight hours after transfection, cells were lysed with
lysis buffer (Boehringer Mannheim) for 30 minutes at room
temperature. The lysates were collected and used for CAT and ß-gal
assays. All experiments were done in duplicate and repeated at least 3
times.
The relative amounts of the CAT enzyme in the cells was determined by sandwich ELISA assays using the CAT ELISA kit (Boehringer Mannheim). The method uses 96-well plates coated with anti-CAT antibody.28 The lysate was added for 1 hour and incubated at 37°C. After 5 washes with the wash solution, anti-CAT antibody labeled with digoxigenin was added to the wells, and the mixture was incubated for an additional hour. The antibody was then removed by 5 washes, and the secondary antibody conjugated with horseradish peroxidase was added for 1 hour. After the incubation, the samples were washed 5 times and the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) substrate was added to the wells. The absorbance of the solution was then measured at 410 nm.28 The ß-gal activity of the extracts was used to normalize the efficiency of transfection.
SDS-PAGE and Western Blotting Analysis
Protein extracts were analyzed on standard (10%
resolving, 4% stacking) mini Protean gels (Bio-Rad) in Tris glycine
buffer (0.025 mmol/L Tris, 0.2 mol/L glycine, 0.1% SDS). After
electrophoresis, the proteins were either stained with Coomassie
brilliant blue for direct visualization or transferred to
nitrocellulose filters for 1 hour at 100 V in transfer buffer
(0.025 mmol/L Tris, 0.2 mol/L glycine, 20% methanol). The bound
proteins, carrying the FLAG epitope at the amino terminus (DYKDDDDK),
were detected using monoclonal anti-FLAG M2 antibody (Kodak, IBI).
Briefly, the membranes were blocked with 5% nonfat dried milk in
50 mmol/L Tris HCl, pH 7.4, and 150 mmol/L NaCl for 30
minutes at room temperature. The primary antibody was then added to a
final concentration of 10 µg/mL for 30 minutes. After the incubation,
the membranes were washed briefly twice with the same solution without
milk. The secondary antibody conjugated with horseradish peroxidase
(HRP) (Santa Cruz) was added in dilution 1:1000 in blocking solution,
and the membranes were incubated for 30 minutes. Before detection, the
membranes were washed 3 times in the same solution without milk for 5
minutes each time, and the proteins were detected with the enhanced
chemiluminescence (ECL) system (Amersham).
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Results |
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Human ApoA-II Promoter Contains 5 Binding Sites for SREBP-1
Screening of 106 plaques of a human liver cDNA library (Clontech) for transcription factors that recognize the regulatory element AIIAB of the human apoA-II promoter resulted in the isolation of 4 cDNA clones homologous to SREBP-1. Limited sequencing of the 4 clones showed identity with the 447 to 546, 545 to 609, 819 to 848, and 2565 to 2624 nucleotide sequences of SREBP-1 on the basis of the Genbank sequence information.4
The existence of an SREBP-1–binding site on the human apoA-II promoter
prompted us to investigate further the role of SREBP-1 on the
transcriptional regulation of the human apoA-II gene. It was shown
previously that the amino terminal segment of SREBP-1, extending to
amino acids 410, 476, or 513, translocates directly into the
nucleus.7 This bypasses the requirement for
sterol-regulated proteolytic cleavage of the intact SREBP-1 molecule to
generate the transcriptionally active amino-terminal
form.8 Initially a 1.55-kb segment of SREBP-1 cDNA
encoding for the first 460 amino acids was expressed in E
coli BL21(DE3) strain, and bacterial extracts containing SREBP-1
were prepared. With these bacterial extracts, DNaseI footprinting
showed that SREBP-1 binds to 5 distinct sites in the apoA-II promoter.
Two of these sites, AIIAB (-64 to -48) and AIIK (-760 to -743),
were previously identified as binding sites for rat liver nuclear
proteins.25 The remaining 3 sites, designated AIICD
(-178 to -154), AIIDE (-352 to -332), and AIIHI (-594 to -574),
are unique binding sites for SREBP-1 and are located between the
previously defined footprints C and D, D and E, and H and I,
respectively (Figure 1A through 1D and 1F).
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DNaseI footprinting analysis was also performed with the human
apoC-III promoter, because the library screening also showed that
SREBP-1 recognizes the regulatory element CIIIB (data not shown). This
element has been shown to contain a hormone response element (HRE) that
binds a variety of nuclear hormone receptors, including
HNF-4.29 HNF-4 expressed in COS-1 cells and SREBP-1
(1–460) expressed in bacterial cells were used for this purpose. This
analysis showed that the 2 factors bind to overlapping sites
within the CIIIB element (Figure 1E and 1G). The binding site of
SREBP-1 coincides with the binding site of the factor CIIIB1, which has
been previously characterized by us30 and was shown
recently to correspond to the transcription factor
USF.26
Comparison of the SREBP-1–Binding Sites Found in the ApoA-II and
ApoC-III Promoters to Previously Reported SREBP-Binding Sites
Alignment of SREBP-1–binding sites from the apoA-II and apoC-III
promoters identified by DNaseI footprinting, previously reported
consensus SREBP-binding sites, and the original SRE found on the
LDL receptor is shown in Table 2. The
SREBP-1–binding sites on the apoA-II promoter contain either an
inverted E-box type palindromic repeat of CANNTG, or a sequence highly
homologous to the direct repeat ATCACCCCAC.4 This direct
repeat is found in the SRE-1 element of the LDL receptor, which is
recognized only by SREBP.
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The binding of SREBP-1 to the regulatory elements identified by DNaseI
footprinting was also tested by gel electrophoretic mobility shift
assays (EMSA). For this purpose, double-stranded
oligonucleotides corresponding to the protected regions
defined by DNaseI footprinting were synthesized and used as probes in
EMSA (Table 3). Extracts of bacteria
transformed with the empty expression vector were used as a negative
control. This analysis showed that SREBP-1 expressed in
bacteria can bind to all 5 elements of the apoA-II promoter. The
binding is competed out by excess of unlabeled
oligonucleotide, indicating that the binding is
specific. No binding was observed in lanes containing extract from
bacteria from cells transformed with the empty vector pAED4 (Figure 2A).
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The relative affinity of the 5 SREBP-1–binding sites on the
apoA-II and apoC-III promoters were compared by extensive competition
assays using the SRE-1 element from the LDL promoter as a probe and
competing with oligonucleotides containing
SREBP-1–binding sites from the apoA-II and apoC-III promoters and the
original SRE-1 found in the LDL receptor promoter. The concentrations
of the competitor oligonucleotides used were 10- to
250-fold molar excess over the probe. Figure 2B shows that the
unlabeled SRE-1 and the AIIAB elements of the LDLR and apoA-II
promoters, respectively, compete with comparable efficiency for binding
to the labeled SRE-1 probe. The order of competition of the probes for
the binding of SREBP-1 to the element SRE-1 is approximately
SRE-1~AIIAB>AIIK>AIICD~AIIDE~AIIHI>CIIIB.
SREBP-1 (1–460) Transactivates the Human ApoA-II
Promoter
The -911/29 apoA-II region is a strong promoter in cells of
hepatic origin, and its activity is approximately 30% of the activity
of CMV promoter (H.-Y. Kan and V.I. Zannis, unpublished data,
1997). To evaluate the effect of SREBP-1 on the activity of the
human apoA-II promoter, plasmids expressing the amino-terminal portion
of SREBP-1 cDNA encoding the amino acids 1 to 460, along with different
apoA-II promoter constructs, were used in the cotransfection
experiments of HepG2 cells. This analysis showed that SREBP-1
transactivated 3.5-fold the human apoA-II promoter (Figure 3A). The transactivation reaches a
plateau at 
200 ng of SREBP-1 expression plasmid, suggesting either
that saturation of the apoA-II promoter occurs or that another factor
may become limiting. This 3.5-fold transactivation is significant given
the very strong activity of the apoA-II promoter in HepG2 cells. A
mutated (-614/29) apoA-II promoter, which lacks the apoA-II enhancer
region and contains the regulatory elements AII-I to AIIN, has
approximately 1% of the activity of the -911/29 apoA-II promoter in
HepG2 cells.25 This promoter is transactivated
40-fold by 5 00 ng of SREBP-1 expression plasmid. The activity of
this truncated promoter reaches approximately 40% of the wild-type
promoter activity in the presence of SREBP-1 (Figure 3A).
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The truncated apoA-II promoter segments that contain only 1
(AII-80/29), 2 (AII-230/29), 3 (AII-440/29) or 4 (AII-614/29)
SREBP-1–binding sites were also tested for transactivation by SREBP-1
(Figure 3B and 3C). The transactivation of these truncated
apoA-II promoter segments initially increases with the number of
SREBP-1–binding sites present and ranges from 10-fold for the
AII-80/29 construct, which has 1 SREBP-1–binding site, to
approximately 40-fold for the AII-440/29 and AII-614/29 promoter
constructs, which have 3 and 4 SREBP-1–binding sites, respectively
(Figure 3B). These truncated promoter constructs provide a
useful set of reporter plasmids to explore further the structure and
functions of SREBP-1 family members.
Previous mutagenesis analysis indicated that elements AIIAB and
AIIK are very important for promoter activity in HepG2 cells. These
elements are also SREBP-1–binding sites. Point mutations or deletions
of these elements reduced the promoter activity to 10% to 20% of the
control in HepG2 cells.21 24 To test the importance of
these elements in the SREBP-1–mediated transactivation, 4 new apoA-II
promoter constructs were made carrying an internal deletion that
removed elements AIIC to AIIH and linked the enhancer region (AII-I to
AIIN) to element AIIAB. One construct has both elements intact (AB
wild-type K wild-type). Two constructs carry mutations either in
element AIIAB (AB mutant K wild-type) or in element AIIK (AB wild-type
K mutant), and 1 construct carries mutations in both elements AIIAB and
AIIK (AB mutant K mutant). The mutations were designed to eliminate the
binding of SREBP-1 to this site and test the contribution of the
individual sites in SREBP-1 mediated transactivation as well as
possible synergism between SREBP-1 molecules bound to the proximal and
the distal site. The same mutations, designed to abolish the binding of
SREBP-1, were introduced into elements AIIAB and AIIK, and the mutant
oligonucleotides were used as probes or competitors in
DNA binding assays (Table 3). In agreement with previous
findings,25 transient cotransfection assays using these
constructs showed that the internal deletion of the region (-616 to
-67) does not decrease the apoA-II promoter activity in HepG2 cells,
but rather increases the activity of the mutant promoter 2-fold (Figure 4A and 4B). The introduction of mutations
in elements AIIAB and AIIK progressively decreased the activity of the
mutant apoA-II promoter constructs. Although the promoter activity of
the constructs carrying mutations in elements AIIAB or AIIK was
comparable with that of the wild-type promoter, the extent of
transactivation was not affected by mutations in element AIIK but was
reduced significantly by mutations in element AIIAB (Figure 4A and 4B).
A small increase in transactivation observed with
the construct carrying a double mutation may represent residual
weak binding to the mutated site AIIK (Figure 5). The effect of the mutations on DNA
binding was tested by DNA binding gel electrophoresis and competition
assays. The competition experiments showed that the mutated
oligonucleotides (AIIAB mutant, AIIABM1, and AIIK
mutant) (Table 3) have decreased ability to compete for the
binding of SREBP-1 to the wild-type oligonucleotides.
The direct binding assays demonstrated that the mutations severely
affect the ability of SREBP-1 to bind to the mutated elements AIIAB and
AIIK (Figure 5).
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SREBP-1 Mutants Defective in DNA Binding or Transcriptional
Activation Repress the ApoA-II Promoter Activity
Early cotransfection experiments with an SREBP-1 (1-460) form
produced by PCR amplification of the SREBP-1 cDNA resulted in a
dose-dependent repression of the apoA-II promoter
activity.31 Sequence analysis of this construct
revealed that it contained a substitution of Ile for Lys359, introduced
during the amplification and cloning of the SREBP-1. Close examination
of the position of this amino acid across the members of the family of
bHLHZip binding proteins showed that it is located at the beginning of
helix 2 of the bHLHZip domain and it is highly conserved among the
members of the family (Figure 6D). To
elucidate the mechanisms of this repression, we introduced additional
mutations into the same position, changing the Lys359 to either Gly,
Val, or Arg. To monitor the expression of these mutant SREBP-1 (1–460)
forms, the flagged version of different SREBP-1 mutants were cloned in
mammalian and bacterial vectors. Immunoblot
analysis was used to assess their expression after transient
transfection of COS-1 and transformation of bacterial BL21(DE3) cells.
This analysis showed that SREBP-1 mutants were expressed
efficiently in COS-1 as well as in bacterial cells and have the
expected molecular mass (Figure 6A through 6C). The relative
location of the site of mutagenesis of SREBP-1 is shown in Figure 6C.
|
Transient cotransfection assays showed that SREBP-1 mutants carrying
substitution of Ile, Gly, or Val for Lys359 decreased the -911/29
apoA-II promoter activity, reaching 50% decrease at 500 ng of SREBP-1
(1-460) expression plasmid, as compared with 3.5-fold activation
observed with the wild-type SREBP-1 (1-460). On the other hand, a
mutant SREBP-1 (1-460) form carrying a substitution of Arg for Lys359
transactivated 2.5-fold the -911/29 apoA-II promoter (Figure 7A). Qualitatively similar results were
obtained when the SREBP-1 mutants were used in cotransfection
experiments with the -911/29 AB wild-type K mutant apoA-II promoter.
The activity of this promoter was reduced to approximately 50% to 60%
by the mutants carrying substitution of Ile, Gly, or Val for Lys359 and
was increased 2-fold by substitution of Arg for Lys359 (Figure 7B). These SREBP-1 (1-460) mutants were also used in
cotransfection with the apoA-II (-614/29) promoter constructs. This
analysis showed that the mutants carrying substitutions of Ile,
Val, or Gly for Lys359 are virtually inactive. Only the mutant carrying
a substitution of Arg for Lys359 displayed some transactivation ability
(Figure 7C). It is important to note that, in the context of the
wild-type apoA-II (-911 to 29) promoter, the mutant carrying a
substitution of Arg for Lys359 functions much better compared with the
truncated apoA-II (-614 to 29) promoter (Figure 7A through 7C).
This suggests that interactions of the SREBP-1 mutant carrying a
substitution of Arg for Lys359 with the factors that bind to the
apoA-II enhancer region are important for transactivation of the
-911/29 apoA-II promoter by SREBP-1. When the enhancer region that
contains regulatory elements AII-I to AIIN is deleted, protein-protein
interactions between the mutant SREBP-1 and other factors bound to the
enhancer are lost. As a result, transactivation by the mutant protein
is severely compromised.
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The first 90 amino acids of SREBP-1 contain the transcriptional
activation domain of the protein, which is highly acidic in character.
Amino-terminal deletion mutants of hamster SREBP-1 behave as dominant
negative repressors.7 To explain the repression of the
apoA-II promoter activity by mutants defective in the DNA-binding
domain of SREBP-1 (1-460), 2 additional SREBP-1 variants were
generated. One variant lacks the first 90 amino-terminal residues
(SREBP-1 
90). The second variant lacks the first 90 amino-terminal
residues and carries an additional substitution of Ile for Lys359 in
its DNA-binding domain (SREBP-1 90K359I). Both mutants showed
similar levels of expression to the wild-type SREBP-1 (1–460) (Figure 6A and 6B). Cotransfection of the SREBP-1 90 mutant along
with the wild-type apoA-II (-911 to 29) promoter decreased the
promoter activity to 17% of control (Figure 7D); the double
mutant with deletion of the amino-terminal activation domain and
substitution of Ile for Lys359 in the DNA-binding domain (SREBP-1
90K359I) was unable to activate or repress the apoA-II
promoter activity (Figure 7D).
In contrast, the SREBP-1 mutant that carries a substitution of Ile for
Lys359 and has an intact activation domain (SREBP-1 K359I) caused a
dose-dependent decrease in the -911/+29 apoA-II promoter activity,
which reached 20% of the control value at 2 µg of SREBP-1 (Figure 7E).
Transactivation of the wild-type apoA-II promoter by SREBP-1 (1-460)
correlated with the ability of the SREBP-1 mutant proteins expressed in
bacteria to bind to the elements AIIAB and SRE-1 in gel EMSAs. Similar
amounts of the SREBP-1 mutant proteins were used in these assays (as
assessed by immunoblotting of the bacterial extracts;
Figure 6B). This analysis showed that substitution of
Ile, Val, or Gly for Lys359 decreased the binding affinity of SREBP-1
mutant proteins for the SRE-1 and AIIAB elements (Figure 8A and 8C). The most severe effect was
observed with the mutant carrying the substitution of Gly for Lys 359,
which did not bind to either element AIIAB or SRE-1 (Figure 8A and 8C). As shown in Figure 7A through 7C, all these mutants
repressed the apoA-II promoter activity. The SREBP-1 mutant carrying
the substitution of Arg for Lys359 showed the best relative binding
among the mutants, in agreement with the transactivation data of the
Figure 7A through 7C.
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Similar analysis showed that the SREBP-1 carrying a
deletion at the mutant amino-terminal SREBP-1 90 could bind
efficiently to its cognate sites on element AIIAB and SRE-1 (Figure 8B and 8D) and could repress the apoA-II promoter activity
(Figure 7D). In contrast, the double mutant (SREBP-1 90K359I)
displayed weak DNA binding (Figure 8B and 8D) and had no effect
on the apoA-II promoter activity (Figure 7D and 7E). The
findings suggest that repression by the DNA binding-deficient mutants
requires the activation domain of SREBP-1. One unexpected observation
from the gel EMSAs was that both deletion mutants SREBP-1 90 and
SREBP-1 90 K359I displayed a slower migration in the
polyacrylamide gel than the wild-type protein (Figure 8B and 8D). The cause of this anomalous migration is not known. We
speculate that this may be the result of a large change in the
structure or charge of the protein caused by the deletion of the acidic
amino terminal domain.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present in vitro studies show that the wild-type SREBP-1 binds to 5 sites and transactivates strongly the apoA-II in HepG2 cells whereas SREBP-1 mutants defective in their DNA-binding domain repress the apoA-II promoter activity. A potential role of SREBP-1 in apoA-II gene regulation in vivo was not established.
In Vivo Role of SREBP-1 in Transactivation of
Cholesterol-Responsible Genes
In vivo studies in hamsters indicated that SREBP-1 decreases under
conditions of cholesterol deprivation and
indicates that SREBP-1 may be responsible for maintenance of
the basal activity of the cholesterol-responsive
genes.32 Furthermore, expression of the SREBP-1a
isoform in transgenic mice caused a massive accumulation of
triglycerides and cholesterol in the liver,
with elevations in many of the enzymes involved in
cholesterol and fatty acid synthesis but did not alter
levels of apoA-I, apoB, and apoE mRNA.33 These data are
consistent with cotransfection experiments, which showed that
SREBP-1 was unable to transactivate the human apoA-I and apoB
promoters (V.I. Zannis, unpublished data, 1996). The apoA-II
mRNA levels were not examined in these animals.33
In the present study RNase protection experiments on HepG2 cell cultures grown in lipoprotein-deficient serum or in the presence or absence of cholesterol and 25-OH-cholesterol showed small relative increase in the apoA-II mRNA/glycerol-3 phosphate dehydrogenase mRNA in cholesterol-depleted cells compared with cholesterol-overloaded cells. However, no difference was observed in the apoA-II mRNA/ß-actin mRNA ratio (data not shown). Thus, despite the multiplicity of SREBP-1 sites on the apoA-II promoter and the in vitro data presented below, apoA-II may behave differently from the prototype cholesterol-responsive genes that are regulated by SREBP.
It is possible that the apoA-II protein, which participates in the transport of cholesterol and other lipids throughout the body, is expressed constitutively most of the time. However the present in vitro data can neither exclude nor support the possibility that cholesterol availability may influence moderately the expression of this gene via SREBP-1 to adjust apoA-II levels to different physiological demands.
SREBP-1 Binds to Multiple Sites and Transactivates the
Human ApoA-II Promoter Activity in HepG2 Cells
A total of 5 binding sites for SREBP-1 were found in the apoA-II
promoter by DNaseI footprinting using SREBP-1 expressed in bacteria.
The reason for the presence of 5 binding sites is not clear, but given
the strong activity of the apoA-II promoter in HepG2 cells the multiple
sites may be needed to enable SREBP-1 to compete effectively with other
factors that can occupy the same sites for the activation of the
apoA-II promoter. It has been shown that SREBP-1 recognizes palindromic
E-box type CANNTG repeats, which can also be recognized by other
members of the bHLHZip family of transcription factors.5
SREBP-1 can also recognize a direct CACCCCAC repeat, exemplified by the
SRE-1 element of the human LDL receptor.1 It was found
that an atypical tyrosine residue present in the bHLHZip domain of
SREBP-1 contributes to the dual DNA-binding specificity.5
The human apoA-II promoter contains both types of SREBP-1–binding
sites. The AIICD and AIIDE elements contain a direct repeat, and the
AIIAB, AIIHI, and AIIK elements contain a palindromic repeat (Table 2). DNA binding competition assays showed that the SRE-1 element
of the LDL receptor promoter and the AIIAB element from the apoA-II
promoter have the highest relative affinity among the elements tested.
The lower affinity of the other binding sites does not necessarily
exclude them from a potential role in the transactivation of the
apoA-II promoter. Several lower-affinity binding sites may bring about
high-level synergistic activation of a promoter. As discussed below,
the E-box binding sites in the apoA-II promoter (AIIAB and AIIK) are
also occupied by other E-box binding proteins,24 26 and
SREBP-1 would have to compete with them for binding to these elements.
On the other hand, the lower-affinity direct repeats AIICD and AIIDE
are unique binding sites for SREBP. Thus, despite the lower relative
affinity, the binding of SREBP-1 to several sites may bring about
synergistic transactivation of the apoA-II promoter.
The approximately 3.5-fold increase in promoter activity by SREBP-1 is significant given the strong activity of this promoter in HepG2 cells. Furthermore, truncated promoter segments that lack the enhancer region (AIII to AIIN) and have low activity can be transactivated up to 40-fold with 0.5 µg of SREBP-1. Transactivation depends largely on the number of SREBP-1–binding sites. These findings suggest that, in the context of the entire -911/29 apoA-II promoter, other factors besides SREBP-1 may become limiting and this is the reason that transactivation reaches a plateau at 3.5-fold in the full-length -911/29 apoA-II promoter.
The SREBP-1–binding site in the apoC-III promoter (CIIIB) overlaps
completely with the binding site of CIIIB1/USF26 30 34 35 36 37 38
and partially with the binding site of nuclear receptors. The binding
of both transcription factors to this element may be mutually exclusive
(Figure 1E). In addition, CIIIB element is a weak binding site
for SREBP-1 and this may be the reason that cotransfection experiments
with SREBP-1 (1-460) did not significantly affect the apoC-III promoter
activity (data not shown).
SREBP-1–Binding Sites on Elements AIIAB and AIIK Overlap With
Binding Sites of Transcription Factor CIIIB1/USF
Of the 5 binding sites identified, the AIIAB and AIIK are of
special interest because they have been shown previously to bind
another nuclear factor, designated CIIIB1.30 Mutations in
sites AIIAB or AIIK that prevented the binding of CIIIB1 to these sites
decreased the promoter activity in HepG2 cells to approximately 60%
and 20% of control, respectively. The binding of SREBP-1 to elements
AIIAB and AIIK overlaps completely with the binding site of factor
CIIIB1, a heat-stable protein of 41 kDa that has been purified to
homogeneity.30 It was shown recently that several
properties of CIIIB1 correspond to the properties of USF1/USF2
heterodimers, 2 related and previously described bHLHZip transcription
factors.26 34 35 36 37 38 USF-mediated transcriptional activation
involves interactions with TFIID,35 TFII-I,39
and TATA box-binding protein–associated factor
TAFII 5540 and requires positive
cofactors.41 The binding site of CIIIB1 on element CIIIB
contains an E-box type motif CACCTG on the antisense strand between
nucleotides -71 and -76. Nucleotide
substitutions within this sequence abolished the binding of the
purified factor.30 Purified CIIIB1 protein also protects
the region -65 to -48 of the apoA-II promoter that contains an
identical E-box type CACCTG motif on the sense strand between
nucleotides -59 and -54.30 CIIIB1 also binds
with high affinity to the regulatory elements AIIK and AIIL of the
apoA-II gene.24 Both the in vitro mutagenesis of the
CIIIB1/USF binding sites and cotransfection experiments suggest that
CIIIB1/USF is an activator of the apoA-II promoter
activity.24 26 Although CIIIB1/USF and SREBP-1 occupy
identical sites on elements AIIAB and AIIK on the apoA-II promoter, our
findings suggest that the mechanism of transactivation of the apoA-II
promoter by these factors may be different. In the former case, binding
of the CIIIB1/USF to element AIIK is the most important, whereas in the
latter case, binding of SREBP-1 to element AIIAB is the most important.
A promoter construct containing the element AIIAB and the apoA-II
enhancer displayed a 2-fold increase in activity compared with the
wild-type apoA-II promoter and was transactivated 2-fold by
SREBP-1. It is interesting that although mutation in either element
AIIAB or AIIK decreased the activity of this promoter to 50% of the
control, transactivation was affected only by mutations in element
AIIAB, thus demonstrating the importance of this element for the
SREBP-1–mediated transactivation. Collectively, our findings suggest
that the SREBP-1–binding sites in the apoA-II promoter are functional
and the binding of SREBP-1 to these sites leads to the transactivation
of the apoA-II promoter. In contrast, promoters that contain weak
SREBP-1–binding sites, such as the apoC-III promoter, or promoters
that lack SREBP-1 sites, such as apoA-I, are not transactivated
by SREBP-1 (Zannis VI and Nagaoka, unpublished data, 1996).
Mutations in the DNA-Binding and Activation Domains of SREBP-1
Repress ApoA-II Promoter Activity
Previous mutagenesis in the DNA-binding domain of SREBP-1
that targeted an atypical tyrosine residue showed that this amino acid
is required for the recognition of the direct repeat
motif.5 The mutations reported in this study targeted the
Lys 359 residue, which is located at the beginning of the second helix
of the helix-loop-helix domain (Figure 6D). An unexpected
finding of our early work was that mutation of this residue with
substituted Ile for Lys359 not only decreased the binding of SREBP-1 to
DNA, but also decreased the apoA-II promoter activity to 20% of
control.31 The crystal structures of other bHLHZip
proteins have indicated that the homologous Lys residue is contacting
the backbone of the DNA and is making a hydrogen bond with an Asn
residue that also contacts the backbone of the DNA.42
Thus, we rationalized that this Lys residue might contribute to the
overall affinity of SREBP-1 for DNA but probably might not alter its
binding specificity to direct or palindromic repeats.
Previous studies using SREBP-1 constructs containing mutations in the
DNA-binding domain reduced by 31% to 50% the activity of reporter CAT
constructs driven by 2 copies of repeats 2 and 3 of the LDL receptor
promoter.7 This homopolymeric promoter has relatively low
activity and has to be transactivated by wild-type SREBP-1 to
observe repression by SREBP mutants defective in DNA binding. Therefore
any negative regulatory effect by SREBP-1 mutants might have been
pronounced compared with the apoA-II promoter, which has high activity
in HepG2 cells and is more sensitive to inhibitory than
stimulatory factors. To understand the underlying reasons for the
inhibitory effects of this SREBP-1 mutant, we introduced
additional mutations in SREBP-1 by substituting Gly, Val, or Arg for
Lys359. Two other mutants were also constructed: 1 that carries
deletion of the activation domain (SREBP-1 90) and another which
combined the amino-terminal deletion with the substitution of Ile for
Lys359 (SREBP-1 90K359I). The binding and activation properties of
these SREBP-1 mutants were tested by DNA binding as well as in
cotransfection experiments. As expected, substitutions of Ile, Gly, or
Val for Lys359 significantly decreased the binding of SREBP-1 expressed
in bacteria to their cognate sites on the elements AIIAB and SRE-1
compared with the wild-type SREBP-1. The SREBP-1 mutant carrying a
substitution of Gly for Lys 359 actually lost completely its ability to
bind to the DNA. It is possible that introduction of a glycine residue
at position 359 could have a significant impact on the adjacent
secondary structures of SREBP-1, because glycine lacks a side chain and
has a large rotational freedom. On the other hand, substitution of Arg
for Lys359 had only a slight impact on the binding of SREBP-1 to the
AIIAB and SRE-1 elements. Most of the members of the bHLHZip family
have a conserved Lys residue at this position except Max, which has an
Arg residue (Figure 6D).43 Thus conservation of a
positive charge at position 359 appears to be necessary for efficient
binding of SREBP-1 to DNA. Binding of the other SREBP-1 mutants
containing a deletion of their activation domain also yielded some
interesting information. Unexpectedly, the deletion mutants exhibited a
slower migration in native polyacrylamide gel than their
full-length counterparts. Such anomalous migration is usually
attributed to a large conformational change of the mutated protein. In
the case of SREBP-1, deletion of the amino-terminal domain resulted in
the loss of 16 acidic amino acid residues. Thus the deletion mutant 1
to 90 SREBP-190, which binds as a dimer to DNA, would have 32 fewer
negative charges per complex with DNA than the full-length SREBP-1 and
this difference could explain at least partially the different mobility
of the DNA-protein complex to the cathode. Consistent with this
interpretation is the observation that retardation in the migration of
mutants carrying deletion of amino acids 1 to 90 (SREBP-1 90 and
SREBP-1 90 K359I) is more pronounced on the SRE-1 probe, which is
18-bp long, than on the AIIAB probe, which is 36-bp long (Table 3). Deletion of the activation domain is usually not expected to
affect the binding of a transcription factor. In our experiments, the
deletion mutant SREBP-1 90 showed a somewhat weaker binding to the
AIIAB element compared with the full-length SREBP-1, but it displayed
strong binding to the SRE-1 element. It has been proposed that
positioning of the 2 monomers of SREBP-1 is different on palindromic
and direct repeat elements (AIIAB and SRE-1,
respectively),4 and their dimerization interface may also
be different. This could be responsible for the difference observed in
binding of the SREBP-1 mutants carrying a deletion of residues 1 to 90
to the AIIAB and SRE-1 elements.
The activation properties of the SREBP-1 mutants correlate well with their DNA binding in vitro to the elements AIIAB and SRE-1. The SREBP-1 mutants carrying substitutions of Ile, Gly, or Val for Lys359 have lower binding ability and repress the apoA-II promoter. On the other hand, the mutant having substitution of Arg for Lys359, which retains relatively good binding, is still able to transactivate the apoA-II promoter albeit weaker than the wild-type SREBP-1. Binding of SREBP-1 to DNA and the presence of an intact activation domain are essential for SREBP-1 to transactivate the apoA-II promoter. Thus, consistent with previous findings,7 deletion of the amino-terminal region of SREBP-1, which contains an acidic activation domain, converts the protein from an activator to a repressor. This repression could result from competitive displacement of the positive regulator CIIIB1/USF that binds to the same sites.30 Another possibility would be recruitment of an active repressor by the activation-defective form of SREBP-1.44 45
Insight into the mechanism of this repression was obtained by
comparison of the 2 mutants that carry substitution of Ile for Lys359,
but only 1 of them has the amino-terminal activation domain deleted
(SREBP-1 K359I and SREBP-1 90K359I). This analysis showed
that the full-length mutant with defects in DNA binding repressed the
apoA-II promoter activity in a dose-dependent fashion whereas the
truncated mutant that lacks the activation domain and is defective in
DNA binding did not have any effect. It is possible that the activation
domain of the mutant SREBP-1 K359I, which is defective in DNA binding,
associates with another factor that is important for the activation of
the apoA-II promoter. This association could titrate this putative
factor out of the promoter, thus causing a decrease in the apoA-II
promoter activity. Similar interference has been observed in other
systems as well.46 The identity of the putative titratable
factor remains unknown. However, recent studies have shown that CBP
(cyclicAMP regulatory element-binding protein) associates with the
activation domain of SREBP-1 and enhances its activation
potential.47 CBP also associates with many transcription
factors46 47 48 49 50 51 and possesses histone acetylase activity,
which is thought to cause remodeling of the chromatin
structure.52 53 54
In summary, this study documents that SREBP-1 binds to multiple sites on 2 types of binding motifs in vitro and transactivates different apoA-II promoter segments in HepG2 cells. SREBP-1 mutants defective in their DNA-binding domain repress the apoA-II promoter activity, possibly by a squelching mechanism. We suggest that apoA-II promoter constructs containing multiple SREBP-1–binding motifs may become valuable probes in future studies to assess different activation domains of SREBP-1 and possibly its interactions with potential coactivators or corepressors. Additional studies are required to establish a potential role of SREBP-1 in apoA-II gene regulation in vivo.
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Acknowledgments |
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This work was supported by a grant from the National Institute of Health (HL33952). We thank Drs Eileen Falvey and Helen Dell for editorial corrections and comments, and Anne Plunkett for typing the manuscript, and Dr Michael Brown of the Health Science Center in Texas for providing the SREBP-1 cDNA.
Received February 9, 1998; accepted October 21, 1998.
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