From the Department of Molecular Biology and Biochemistry, University of California, Irvine California 92697-3900
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ABSTRACT |
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3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
synthase, a key regulatory enzyme in the pathway for endogenous
cholesterol synthesis, is a target for negative feedback regulation by
cholesterol. When cellular sterol levels are low, the sterol regulatory
element-binding proteins (SREBPs) are released from the endoplasmic
reticulum membrane, allowing them to translocate to the nucleus and
activate SREBP target genes. However, in all SREBP-regulated promoters studied to date, additional co-regulatory transcription factors are
required for sterol-regulated activation of transcription. We have
previously shown that, in addition to SREBPs, NF-Y/CBF is required for
sterol-regulated transcription of HMG-CoA synthase. This heterotrimeric
transcription factor has recently been shown to function as a
co-regulator in several other SREBP-regulated promoters, as well. In
addition to cis-acting sites for both SREBP and NF-Y/CBF, the sterol
regulatory region of the synthase promoter also contains a consensus
cAMP response element (CRE), an element that binds members of the
CREB/ATF family of transcription factors. Here, we show that this
consensus CRE is essential for sterol-regulated transcription of the
synthase promoter. Using in vitro binding assays, we also
demonstrate that CREB binds to this CRE, and mutations within the CRE
that result in a loss of CREB binding also result in a loss of
sterol-regulated transcription. We further show that efficient
activation of the synthase promoter in Drosophila SL2 cells
requires the simultaneous expression of all three factors: SREBPs,
NF-Y/CBF, and CREB. To date this is the first promoter shown to require
CREB for efficient sterol-regulated transcription, and to require two
different co-regulatory factors in addition to SREBPs for maximal activation.
3-Hydroxy-3-methylglutaryl coenzyme A
(HMG-CoA)1 synthase is a key
rate-limiting enzyme of cholesterol biosynthesis, converting acetoacetyl-CoA and acetyl-CoA into HMG-CoA. In order to achieve cellular cholesterol homeostasis, this and other genes of cholesterol metabolism are regulated by classical feedback repression: they are
up-regulated when sterol levels fall and down-regulated when sterol
levels rise (1-5). This control is exerted primarily at the
transcriptional level through the action of the sterol regulatory element-binding proteins (SREBPs), a unique subfamily of basic helix-loop-helix zipper (bHLHZip) proteins. SREBPs are expressed as
125-kDa precursor proteins that are anchored to the endoplasmic reticulum and nuclear membranes. When cellular sterol levels fall, the
precursor is released from the membrane by a two-step proteolytic mechanism, allowing the mature, transcriptionally active SREBPs to
translocate to the nucleus (6). Once inside the nucleus, SREBPs
activate cholesterogenic target genes through binding to sterol
regulatory elements (SREs) present in their promoters. SREBPs also
activate key genes of fatty acid metabolism (7-9) thus, they are
central transcription factors of mammalian lipid metabolism.
SREBPs alone, however, are inefficient transcriptional activators, and
in all SREBP-regulated promoters studied to date, additional transcription factors are necessary for maximal activation in response
to sterol deprivation. Interestingly, the required co-regulator is not
the same for all SREBP-regulated promoters. For example, in the case of
the farnesyl diphosphate synthase promoter, the required co-regulator
is NF-Y/CBF (10), whereas in the LDL receptor promoter, it is Sp1
(11).
The region of the HMG-CoA synthase promoter that is necessary for
sterol-regulated transcription is contained within an approximate 100-base pair region, which includes two consensus SREs, a CCAAT box,
and a consensus cAMP response element (CRE) (12) (see Fig. 1). Previous
studies have demonstrated that both SREs, termed SRE I 5' and SRE I 3',
as well as the inverted CCAAT box, are essential for sterol-regulated
transcription. These elements recruit SREBP and NF-Y/CBF, respectively,
to the promoter under conditions of sterol deprivation (10, 12).
However, the potential involvement of the CRE has not previously been evaluated.
CREs were originally identified as cis-acting elements that confer
transcriptional activation in response to elevated cAMP levels
(13-15). Using the somatostatin promoter, it was shown that this
responsiveness was mediated through activation and recruitment of the
basic leucine zipper containing transcription factor termed cAMP
response element-binding protein (CREB) (16). CREB is phosphorylated in
response to elevated cAMP, and this allows it to interact efficiently with the transcriptional co-activator protein called CREB-binding protein to stimulate transcription of cAMP target genes (17, 18).
Subsequently, numerous related CRE-binding proteins have been
identified and cloned (19) and together they comprise the CREB/ATF
family of transcription factors. Individual members of this family bind
to CREs present in numerous eukaryotic promoters, and activate
transcription in response to various cellular signals (20-22).
In the current studies, we investigated the role of the putative CRE in
sterol-regulated transcription of the HMG-CoA synthase gene. In a
thorough mutational analysis of the promoter regulatory region, we
noted that single point mutations that alter the consensus CRE resulted
in a complete loss of sterol-regulated transcription. Using in
vitro binding assays, we show that CREB binds to the wild type
consensus CRE of the synthase promoter, and mutations within the CRE
that abolish sterol-regulated transcription completely disrupt CREB
binding to this element. Furthermore, using a Drosophila tissue culture cell line that does not express several mammalian transcription factors, we show that maximal activation of the synthase
promoter requires the simultaneous expression of SREBP, NF-Y/CBF, and
CREB. To date, this is the first case in which SREBP has been shown to
require two different co-regulatory DNA-binding proteins to activate a
promoter in response to sterol deprivation. Additionally, this is the
first report demonstrating that a member of the CREB/ATF family can
function together with the SREBPs to mediate sterol-regulated transcription.
Cells and Media--
All of the cell culture lines used
here have been described before (11). All cell culture materials were
purchased from Life Technologies Inc. Lipoprotein-deficient serum was
prepared by ultracentrifugation of newborn bovine serum as described
previously (23). Cholesterol and 25-OH cholesterol were obtained from
Steraloids Inc., and stock solutions were dissolved in absolute ethanol.
Plasmids--
pGL2 basic was purchased from Promega Inc. and was
used as the source of the luciferase reporter gene in all constructs.
Standard techniques were used in all cloning procedures (24).
Construction of the plasmid pSynSRE has been described elsewhere (12).
PSynTLuc is the same as the plasmid "TATA only" described
previously (11). The point mutations were constructed using the
"Altered Sites" mutagenesis kit (Promega Inc.) according to the
manufacturer's protocol. All mutants were confirmed by sequencing.
pCMV2 Cell Culture and Transient Transfection Assays--
CV-1 cells
were cultured at 37 °C and 7% CO2 in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum (normal) and were plated at 125,000 cells/60-mm dish on day 0. On day 1, cells
were refed normal media and transfected by the calcium phosphate co-precipitation method as described (11). Precipitates contained 20 µg each of the luciferase reporter test plasmid and the non-sterol regulated pCMV2
Drosophila SL2 cells were cultured at 25 °C in Shields
and Sang Drosophila media (Sigma) containing 10%
heat-inactivated fetal bovine serum. On day 0, cells were seeded at
1 × 106 cells/60-mm dish. On day 1, the cells were
transfected by a standard calcium phosphate co-precipitation method
(25). On day 3, cells were harvested, extracts were prepared by the
freeze-thaw method, and assays were performed as described below.
Enzyme Assays--
The luciferase activities were measured in an
Analytical Luminescence monolight model 2010 luminometer with a
luciferin reagent from Promega Inc. Gel Mobility Shift Assay--
A 30-base pair double-stranded
oligonucleotide probe corresponding to bases Expression and Purification of Fusion Proteins--
The
expression and purification of amino acids 1-490 of SREBP1a has been
described previously (11). The glutathione S-transferase (GST)-CREB expression construct was made by excising the CREB coding
sequence from the plasmid pPacCREB using HindIII and
BamHI. The resulting fragment was then inserted in-frame
with the GST coding region in the pGex2T plasmid. GST-CREB was
expressed in E. coli strain DH5 Protein-Protein Interaction Assay--
The fusion proteins were
expressed in E. coli strain DH5 Mutations within the Consensus CRE Abolish Sterol-regulated
Transcription of HMG-CoA Synthase--
Previous studies have
demonstrated that a 100-base pair region of the HMG-CoA synthase
promoter is necessary and sufficient for efficient sterol-regulated
transcription. Within this region, two binding sites for SREBP and one
binding site for NF-Y/CBF were shown to be critical (12). To determine
if there are other critical elements within this 100-bp interval, we
introduced a series of single point mutations into the region and fused
the resulting mutant promoters to a luciferase reporter gene. Each construct was then transiently transfected into CV-1 cells and assayed
for sterol-regulated transcription by culturing the transfected cells
either under induced (
Mutations in the 5'-half of the SRE-I 5' resulted in a decrease in
sterol-regulated transcription due to a loss in promoter activity under
induced conditions, as expected. However, mutations in the 3'-half of
the SRE-I 5'did not result in a dramatic decrease in sterol regulation.
In the case of Sterol Defective Mutants That Alter the CRE Element Decrease
Binding by CREB and Not SREBPs--
The mutations at
Using the wild type synthase probe, a shifted complex was observed in
the presence of either SREBP-1a or CREB (Fig. 3, A and B, lanes 2 and 3). However, using probes
corresponding to the single mutations in the consensus CRE at bases
SREBP-1a bound to the wild type probe (Fig. 3B, lanes 2 and
3) and this was significantly decreased when a critical base
in the SRE element was mutated (Fig. 3B, lanes 5 and
6). More importantly, SREBP bound efficiently to the probes
containing either CRE mutation (Fig. 3B, lanes 8-9 and
11-12).
Therefore, the mutations within the CRE of the HMG-CoA synthase
promoter decrease the binding of CREB (and possibly other related
family members), but not SREBP-1a. Taken together, the transfection
results and the DNA binding assays suggest that the recruitment of
CREB, or another related protein, is critical for sterol-regulated
transcription of the HMG-CoA synthase promoter.
Maximal Activation of the HMG-CoA Synthase Promoter Requires SREBP,
NF-Y/CBF, and CREB--
To determine which transcription factors are
simultaneously required for maximal activation of mammalian promoters,
it is possible to take advantage of a Drosophila tissue
culture cell line, SL2. This cell line does not express functional
homologues for several mammalian proteins, including the transcription
factors Sp1 (25) and
NF-Y/CBF.2 Therefore, it is a
useful cell-based assay system for the analysis of transcription factor
requirements for promoter activation because it provides a negative
background for such studies. For example, this assay system was used to
directly show that SREBP and Sp1 function as co-activators of the LDL
receptor promoter (11).
To directly evaluate the ability of SREBP, NF-Y/CBF, and CREB/ATF, or
various combinations thereof, to activate the HMG-CoA synthase
promoter, we used the SL2 assay system (Fig.
4). The wild type synthase
promoter-reporter construct was co-transfected along with increasing
amounts of a Drosophila expression construct for human CREB
(Fig. 4A). CREB, either alone or together with expression
constructs for all three subunits of NF-Y/CBF, was unable to activate
the promoter. Expression of SREBP-1a and the NF-Y/CBF plasmids together
resulted in only a very slight activation of the synthase promoter in
the absence of CREB. However, addition of increasing amounts of CREB
along with constant levels of both SREBP-1a and NF-Y/CBF resulted in a
dramatic increase in promoter activity. Inclusion of expression
constructs for all three subunits of NF-Y/CBF was required for this
activation.2
The effect of CREB was specific and dependent on the consensus CRE, as
there was no activation observed for the CRE mutant promoter-reporter
construct even when all three proteins were co-expressed (Fig.
4B). The results in Fig. 4C show that the
CREB/ATF family member ATF-2 also functions with SREBP and NF-Y/CBF to activate transcription of the HMG-CoA synthase promoter, although the
level of activation was not as high as that observed in the presence of
CREB. This may be due to inherent differences in activation potential
between the two proteins, or simply due to differences in expression
levels of CREB and ATF-2 from the pPac expression vectors in SL2 cells.
Again, this effect was specific, since the LDL receptor
promoter-reporter construct, which does not contain NF-Y/CBF or CREB
sites, was unaffected (Fig. 4D). These results support the
hypothesis that efficient transcriptional activation of the HMG-CoA
synthase promoter requires SREBP, NF-Y/CBF, and CREB (or other members
of the CREB/ATF family) to be present simultaneously.
SREBP Directly Interacts with CREB in Vitro--
How do SREBP and
CREB function, together with NF-Y/CBF, to achieve sterol-regulated
transcription? In an attempt to address this issue, we evaluated
whether SREBP-1a and CREB could interact with each other in solution in
the absence of DNA. CREB was expressed as a GST fusion protein, and
immobilized on glutathione-agarose beads which was then incubated with
purified, recombinant SREBP-1a. SREBP-1a was either loaded directly on
an SDS-PAGE gel (Fig. 5, lane
1) or material eluted from GST-agarose beads after incubation with
either the recombinant GST protein (Fig. 5, lane 2) or with the recombinant GST-CREB fusion protein (Fig. 5, lane 3).
The SDS-PAGE gel was then analyzed by immunoblotting with an antibody directed against SREBP-1. Recombinant SREBP-1a bound specifically to
the GST-CREB fusion protein. Thus, SREBP-1a and CREB are capable of
interacting with each other in solution in the absence of a DNA
fragment containing adjacent binding sites for the two proteins.
In response to cellular sterol deprivation, the HMG-CoA synthase
gene is activated at the transcriptional level primarily through the
action of SREBPs. However, in all SREBP target promoters studied to
date, including the HMG-CoA synthase promoter, recruitment of
additional, nonspecific transcriptional activators to the promoter is
required to achieve this activation (10-12, 26, 27). The HMG-CoA
synthase promoter has a complex array of cis-acting regulatory elements, each with the potential for involvement in sterol-regulated transcription. In the current studies, we show that the consensus CRE,
located within the minimal regulatory region of the promoter, is
essential for sterol-regulated transcription. In a previous report from
our laboratory, it was demonstrated that this element mediated
transcriptional activation of the synthase promoter in response to
phorbol esters and the AP-1 transcription factor (28). Here, we have
demonstrated that this element recruits CREB to the promoter, and
mutations that abolish CREB binding also result in a loss of
sterol-regulated transcription (Figs. 2 and 3). Although other
cholesterogenic promoters contain consensus CREs, to date this is the
first promoter shown to require a CREB/ATF factor for the regulation of
transcription in response to cellular sterol levels.
We have previously shown that the two SREs and the CCAAT box are
required for sterol-regulated transcription (12) of the HMG-CoA
synthase promoter. However, as demonstrated here, these elements are
necessary but not sufficient for sterol-regulated transcription. This
is based on two observations: 1) single point mutations in the CRE
result in a loss of sterol-regulated transcription, even in the
presence of the intact SREs and CCAAT box (Fig. 2); and 2) in a
Drosophila SL2 assay system, maximal transcriptional activation is achieved only in the presence of all three proteins, SREBP, NF-Y/CBF, and CREB (Fig. 4A). This observation is
further unique in that the HMG-CoA synthase promoter is the first in
which three factors, SREBP and two different general factors, are
necessary for efficient transcriptional activation in response to
sterol deprivation.
CREB is a member of a large family of basic leucine zipper proteins
called the CREB/ATF family that consists of at least 10 distinct
members (22, 29). CREB, the first member to be cloned, was originally
identified based on its ability to bind to a cis-acting element termed
the CRE, which was known to confer cAMP responsiveness on corresponding
promoters (16). Subsequently, CREB has been suggested to activate
transcription in response to various other cellular signaling agents,
as well, including insulin, growth factors, and calcium (30-32). In
some cases, CREB alone is not sufficient, and additional transcription
factors are required to achieve activation. For example, in the PEPCK
promoter, the CRE functions together with a CCAAT box to achieve
cAMP-mediated transcriptional activation (14), whereas in the T-cell
receptor The functional differences among the various CREB/ATF proteins are not
completely clear at present. Although they are all highly similar in
their basic and leucine zipper regions and they bind the same
cis-acting consensus sequence, the affinities of the different homo-
and heterodimeric combinations vary for different CREs (36, 37).
Furthermore, they do not all respond to the same cellular signals. For
example, ATF-2 mediates activation by the adenovirus E1a protein (38),
whereas both CREB and ATF-1 mediate activation in response to elevated
cAMP (18, 39). We tested the ability of ATF-2 to substitute for CREB in
activation of the HMG-CoA synthase promoter. Although in an in
vitro binding assay ATF-2 was able to bind to the CRE with the
same specificity as we observed for
CREB,3 it was unable to
activate the HMG-CoA synthase promoter to the same level as that
achieved with CREB (Fig. 4). It is possible that this result is due to
a difference in expression levels of ATF-2 and CREB in SL2 cells, but
this is unlikely since in similar experiments ATF-2 was more effective
than CREB in activating transcription from the HMG-CoA reductase
promoter.2 Interestingly, in the cAMP-independent cell
type-specific expression of the somatostatin promoter mentioned above
(34), ATF-2 was also unable to substitute for CREB. As more is
understood about the functions of the various CREB/ATF proteins, the
reason for this difference will become clearer.
In almost all eukaryotic promoters, multiple DNA binding transcription
factors must assemble on the promoter, together forming a
transcriptionally active complex. In many cases, this complex is
composed of a combination of regulatory-specific transcriptional activators such as SREBPs, together with more ubiquitous, generic, transcription factors. The functions of the individual components can
include enhanced recruitment of other required factors (either by
direct physical interaction or by alteration of DNA structure of nearby
cis-acting elements), presentation of distinct and complimentary activation domains, and/or distinct interactions with essential components of the basal, RNA polymerase II transcription machinery. We
previously reported that SREBP physically interacts with NF-Y/CBF (12),
and another report demonstrated that NF-Y/CBF enhanced the binding of
SREBP to the SRE in the promoter for farnesyl diphosphate synthase,
another enzyme of cholesterol biosynthesis (40). Here we have shown
that SREBP-1a physically interacts with CREB (Fig. 5). Interestingly,
mutations that introduce insertions between the SRE I 3' and the SRE I
5'/CRE are defective for sterol-regulated transcription in transient
transfection assays.3 The reason for this is unclear, as
yet, but may reflect the importance of a requirement for this
protein-protein interaction between SREBP and CREB when they are
recruited to the promoter. Further experiments are required to
determine the mechanistic significance of this interaction.
How does the SREBP family of transcription factors mediate the
regulation of numerous genes involved in distinct but related metabolic
processes such as fatty acid metabolism, cholesterol synthesis, and
cholesterol uptake? Promoter specific effects of the different SREBP
isoforms is likely to provide part of the basis for regulatory
specificity (41). Additionally, the differences in promoter
architecture result in a requirement for unique combinatorial arrangements of transcription factors that are required for activation of the various SREBP target genes. This is also likely to provide part
of a mechanism by which coordinate, yet specific, regulation is
achieved. The involvement of CREB in activation of the HMG-CoA synthase
gene, for example, may add a further level of complexity to its
regulation, relating the responsiveness to sterol deprivation with
other cellular regulatory events.
INTRODUCTION
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Abstract
Introduction
References
MATERIALS AND METHODS
-Gal contains the cytomegalovirus early promoter linked to the
Escherichia coli
-galactosidase gene (11). The plasmid
pPacSp1 was obtained from Al Courey (UCLA), and contains the
Drosophila actin 5C promoter upstream of the coding sequence
for human Sp1 (25). This plasmid was used to construct all pPac
plasmids used here. pPacSREBP1a encodes amino acids 1-490 of the
full-length protein and was described previously (11). The plasmids
pPacCREB, pPacATF-2, pPacNF-YA, pPacNF-YB, and pPacCBF-C (also called
NFY-C) were constructed by polymerase chain reaction amplification of
the corresponding coding sequences from RSVCREB (a kind gift of Dr.
Marc Montminy, Joslin Diabetes Center, Boston, MA), pGexATF-2 (a kind
gift of Dr. Michael Green, University of Massachusetts, Worcester, MA), PET 3b-NF-YA, PET 3b-NF-YB (kind gifts of Dr. Robert Mantovani, Universita Degli Studi Di Milano, Italy), or pCite-CBF-C (a kind gift
of Dr. Sankar Maity, M.D. Anderson Cancer Center, Houston, TX),
respectively, using oligonucleotides containing restriction sites for
BamHI and XhoI. The resulting fragments were
inserted into the pPac vector after excision of the Sp1 coding sequence.
-Gal plasmid. Salmon sperm DNA was added to bring the total DNA to 60 µg/2 ml. An equal volume (0.5 ml) was added to
each of four dishes, for a total of 15 µg of DNA/60-mm dish. Twelve
to 16 h later, the cells were washed three times with
phosphate-buffered saline (PBS), refed either induced (Dulbecco's
modified Eagle's medium + 10% lipoprotein-depleted serum) or
suppressed media (same as induced media but also containing 10 µg/ml
cholesterol and 1 µg/ml 25-OH cholesterol), and returned to the
incubator. On day 3, cells were harvested by scraping in
phosphate-buffered saline and duplicate dishes were pooled prior to
preparation of soluble protein extracts by 3 freeze-thaw cycles.
-Galactosidase assays were
performed by a standard colorimetric procedure with
2-nitrophenyl-
-D-galactopyranoside as substrate (24).
The normalized luciferase activity was obtained by dividing the
luciferase activity in relative light units by the
-galactosidase
activity (OD 420/h).
284 to
259 of the wild
type HMG-CoA synthase promoter was end labeled with
[
-32P]ATP and T4 polynucleotide kinase (Pharmacia
Biotech Inc.). The mutant probes (CRE Mutant A, CRE mutant B, SRE
mutant) are exactly the same except for the mutation
265G
T,
264T
G, or
272A
C, respectively. The labeled probes were used
in an electrophoretic mobility shift assay as described previously (11)
with proteins prepared as described below.
F'. After harvesting the
200-ml cultures, pellets were resuspended in 4 ml of ice-cold sucrose
solution (10 mM Tris, pH 8, 20% sucrose, 1 mM
dithiothreitol), incubated on ice for 15 min in the presence of
lysozyme (50 mg/ml stock), then incubated on ice for 10 min in the
presence of EDTA (final concentration of 1 mM). Sarkosyl
(10% stock solution) was added to a final concentration of 0.05% and
the samples were sonicated 3 times for 10 s each. Cell debris was
pelleted by centrifugation. The supernatant was then incubated with 1:1
slurry of glutathione-agarose (Sigma) on a rotating wheel at 4 °C
for 3 h, followed by extensive washing with Wash Buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 0.1% Triton X-100, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin/pepstatin, 10%
glycerol). Bound proteins were eluted with 5 mM glutathione.
F'. Cultures (100 ml) were
grown to OD600 of 0.6 to 0.8, induced by adding
isopropyl-1-thio-
-D-galactopyranoside to final
concentration of 1 mM, and grown for an additional 3 h
at 37 °C. Cells were then harvested by centrifugation. The cell
pellets were resuspended in NETN (100 mM NaCl, 20 mM Tris, pH 8, 1 mM EDTA, 0.5% Nonidet P-40),
lysed by sonication, and the cell debris was removed by ultracentrifugation. Glycerol was added to the supernatant to a final
concentration of 10%, and extracts were stored in aliquots at
70 °C. The expression of the fusion protein was verified by SDS-PAGE analysis followed by staining with Coomassie Blue, as well as
by an immunoblotting protocol with an anti-GST antibody and horseradish
peroxidase-conjugated anti-rabbit IgG (Sigma). To 10 µl of
glutathione-agarose beads, 100 µl of the indicated crude GST fusion
protein extract, and 300 µl of buffer (Z'0.1, 50 mM Hepes
pH 7.5, 2 mM MgCl2, 100 mM KCl,
10% (v/v) glycerol, 0.1% (v/v) Nonidet P-40) + 0.5% non-fat dry milk + 5 mM dithiothreitol) were added followed by an incubation
on a rotating wheel at 4 °C for 2 h. The bound GST fusion
protein was pelleted in a microcentrifuge and nonspecifically bound
proteins were removed by washing the pellets 3 times with 1 ml of
buffer. Then, 5 µl of purified SREBP-1a was added to the bound
proteins, along with 300 µl of buffer, and incubated on a rotating
wheel for an additional 2 h at 4 °C. The reactions were again
washed 3 times with 1 ml of buffer, and specifically bound proteins
were pelleted, resuspended in sample buffer, and analyzed by SDS-PAGE
followed by Western blot analysis using the ECL kit (Pierce) and an
anti-SREBP-1 primary antibody (IGG2A4).
RESULTS
sterols) or suppressed (+sterols) conditions.
The results for constructs containing mutations within the region
277
to
260, relative to the transcription start site, are shown (Fig.
2A), and the ratio of reporter gene activity under induced
to suppressed conditions is reported as fold regulation (Fig.
2B). SynSRE is the wild type promoter-reporter construct, and SynTLuc is a control promoter-reporter construct containing only
the HMG-CoA synthase minimal TATA box.
268T
G, this was due to an increase in activity
under both induced and suppressed conditions. The reason for this is
not clear. In the promoter constructs containing mutations
corresponding to
268T
A,
267A
C, and
266C
A, an increase in
sterol regulation was observed due to a decrease in the level of
promoter activity under both induced and suppressed conditions. This
effect may be due to disruption of binding of a CREB/ATF protein at the
overlapping CRE, which normally contributes to both the induced and the
suppressed activity (in this case equivalent to the basal activity) of
the promoter. This possibility is further supported by the dramatic
loss in sterol regulation for the mutants
264T
G and
265G
T,
both of which mutate critical bases within the consensus CRE, while
leaving the consensus SRE intact. Taken as a whole, these results
indicate that a CREB/ATF family member is involved in sterol-regulated
transcription of the HMG-CoA synthase promoter.
264 and
265
discussed above are adjacent to the consensus SRE element but are
contained within the putative CRE. To determine if these mutations were
defective for sterol regulation due to a loss in binding of either CREB
or SREBP-1a, recombinant versions of each protein were used in an
electrophoretic mobility shift assay with either the wild type or
selective mutant HMG-CoA synthase probes (Fig. 3).
264 or
265 (Mutant A or B, see Fig.
1) that were defective for
sterol-regulated transcription (Fig. 2),
the CREB-DNA complex was not formed (Fig.
3A, lanes 8-9 and
11-12). Using a probe with a single mutation in the SRE I
5', but outside of the CRE, resulted in a decrease in the shifted complex observed in the presence of CREB (Fig. 3A, lanes 5 and 6), indicating bases outside the concensus CRE are
important in recruiting CREB to the promoter. Similar results were
observed when we used a recombinant version of the CREB family member
ATF-3 (data not shown).
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Fig. 1.
The HMG-CoA synthase promoter. A
schematic of the HMG-CoA synthase promoter from position 324 to
225
linked to
28 to +38, relative to the mRNA start site, is shown.
The relative positions of the SRE I 5' and 3', the CCAAT box, and the
CRE, which bind SREBP, NF-Y/CBF, and CREB/ATF proteins, respectively,
are indicated. "Mutant A" and "Mutant B" refer to the single
base substitutions analyzed in the experiments of Figs. 3 and 4.
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Fig. 2.
Sterol-regulated transcription of
single base mutants of the HMG-CoA synthase promoter. CV-1 cells
were transiently transfected with a luciferase reporter construct
containing either the wild type HMG-CoA synthase promoter
(pSynSRE) or the same construct but containing the indicated
individual base substitution mutation. pSynTLuc is a control luciferase
reporter construct containing the synthase TATA box only. As a control,
a CMV2- -galactosidase construct was co-transfected with each
luciferase construct. After transfection, the cells were cultured in
medium containing 10% lipoprotein-depleted serum in the absence
(induced) or presence (suppressed) of 10 µg/ml
cholesterol and 1 µg/ml 25-hydroxycholesterol. The average of the
normalized luciferase reporter activity under induced and suppressed
conditions for three individual experiments is shown in panel
A. The data from panel A is represented as fold
regulation in panel B by dividing the activity under induced
conditions by the activity under suppressed conditions. The error
bars are presented on the graph in panel B.
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Fig. 3.
CREB binds to the CRE present in
the wild type HMG-CoA synthase promoter. 30-base pair
double-stranded oligonucleotide probes corresponding to the wild
type synthase promoter (lanes 1-3), a single point mutation in the SRE
(lanes 4-6), or individual mutations in the consensus CRE
(CRE mutant A (lanes 7-9) or B (lanes 10-12),
see Fig. 1) were 32P-end labeled and used in gel mobility
shift assays, as described under "Materials and Methods." In
A, the probes were incubated either in the absence ( ) or
presence of 1 µl (+) or 5 µl
(+) of affinity purified
GST-CREB. In B, the probes were incubated either in the
absence (
) or presence of 1 µl (+) or 5 µl
(+) of affinity purified
His-tagged SREBP-1a. In both A and B the
arrow denotes the position of the migration of the
protein-DNA complex.
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Fig. 4.
Transcriptional activation of HMG-CoA
synthase requires the simultaneous expression of SREBP, NF-Y, and
CREB. Drosophila SL2 cells were transiently transfected
with a luciferase reporter construct containing the wild type HMG-CoA
synthase promoter (A and C), synthase MutA
(B), or the LDL receptor promoter (D) and
increasing amounts of a pPac expression vector for CREB (A
and B) or ATF-2 (C and D). As
indicated, 50 ng of a pPac expression vector for SREBP-1a, NF-Y/CBF (50 ng each of pPacNF-YA, pPacNF-YB, pPacCBF-C), or both were
co-transfected. In all experiments, a pPac -galactosidase plasmid
was transfected as a control.
, CREB or ATF-2 only;
, +SREBP;
, +NF-Y;
, +SREBP-1a/NF-Y.
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Fig. 5.
SREBP-1a physically interacts with CREB in
solution. GST (lane 2) or GST-CREB (lane 3)
was bound to glutathione-agarose beads, then incubated with purified,
His-tagged SREBP-1a, followed by extensive washing. The specifically
bound proteins were separated on SDS-PAGE, then subjected to Western
blot analysis using antibody specific for SREBP-1a. Lane 1 is from purified SREBP-1a loaded as a control.
DISCUSSION
gene, the T-cell specific factor TCF-1
must function
together with a CRE-binding protein and the ETS-1 protein to achieve
activation (33). Another example is the cell specific expression of the somatostatin promoter. Using the cell line Tu6, it was shown that this
cell-restricted expression requires the concerted action of an islet
cell-specific LIM family protein Isl-1, CREB (34), and a NF-Y/CBF-like
protein (35).
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ACKNOWLEDGEMENTS |
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We thank Drs. Marc Montminy, Michael Green, Al Courey, Sankar Maity, and Robert Mantovani for generously sharing plasmids used in this study. We also thank Shawn Millinder and Lynn Yieh for contributions to the early stages of this work, and current members of our laboratory for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL48044 and American Heart Association Grant 96008190.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by National Institutes of Health Predoctoral Training
Grant GM07311.
§ Established Investigator of the American Heart Association. To whom correspondence and reprint requests should be addressed: Dept. of Molecular Biology and Biochemistry, University of California, Irvine, 19172 Jamboree Road, Irvine, CA 92697-3900. Tel.: 949-824-2979; Fax: 949-824-8551; E-mail: tfosborn{at}uci.edu.
2 M. M. Magaña and T. F. Osborne, manuscript in preparation.
3 K. A. Dooley and T. F. Osborne unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; SREBP, sterol regulatory element-binding protein; SRE, sterol regulatory element; CRE, cAMP response element; CREB, cAMP response element-binding protein; ATF, activating transcription factor; LDL, low density lipoprotein; NF-Y, nuclear factor-Y; CBF, CAAT binding factor; 25-OH cholesterol, 25-hydroxycholesterol; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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