From the Department of Biochemistry and Molecular Biology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799
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ABSTRACT |
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Transcription of the human squalene synthase (HSS) gene is regulated by variations in the level of cellular cholesterol. Three regulatory elements in the HSS promoter region are known to be involved in the regulation: 1) a modified sterol regulatory element (SRE) 1 (HSS-SRE-1), 2) an inverted SRE-3 (Inv-SRE-3), 3) an inverted Y box (Inv-Y-Box).
We report here the regulatory role of distinct cis-elements in the HSS
promoter by using mutants of an HSS-luciferase promoter reporter. The
activity of a wild-type promoter reporter transiently transfected into
HepG-2 cells is increased by sterol depletion of the cells or by
coexpression of mature forms of the SRE-binding proteins (SREBP) 1a and
SREBP-2. Differential activation by SREBP-1a and SREBP-2 of the
reporter gene mutated at various regions of the promoter is observed.
Mutation of either the HSS-SRE-1 or the Inv-SRE-3 sequence diminished
the activation by SREBP-1a and by sterol depletion but did not affect
the activation by SREBP-2. Simultaneous mutations of both of these
sequences almost completely abolished activation of the promoter by
SREBP-1a or by sterol depletion, but activation by SREBP-2 was retained
at 70%. Mutation of the Inv-Y-Box sequence element decreased the
activity of the promoter by 50% or more, and if mutated together with
both SREs, the activation was almost completely abolished. Mutation of
any single GC box of the two located at 40 to
57 did not affect activity, whereas simultaneous mutation of the two decreased activation by SREBP-2 by 60%, by lipid depletion by 20%, and had no effect on
the activation by SREBP-1a. A Y box motif at
159 to
166 and an
SRE-like sequence element (SRE-1(8/10)) at position
101 to
108 are
also involved in the sterol regulation. These results indicate that the
complex sterol-mediated transcriptional regulation of the HSS gene is
due to the presence of multiple copies of diverse cis elements in the
HSS promoter. The differential activation of the HSS promoter may point
to specific role of the SREBPs in cholesterogenesis.
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INTRODUCTION |
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Squalene synthase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21), which catalyzes the formation of squalene by condensation of two molecules of farnesyl diphosphate, is the first committed enzyme in cholesterol biosynthesis. Its expression, like that of several other key enzymes in the pathway such as 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 3-hydroxy-3-methylglutaryl-coenzyme A synthase, and farnesyl diphosphate synthase, is highly regulated by cellular cholesterol homeostasis (1-4). The 5'-flanking regions of the genes encoding these enzymes contain either a 10-bp1 sequence designated sterol regulatory element (SRE)-1 or a related sequence (5-8). Other sterol-sensitive genes such as the low density lipoprotein receptor, fatty acid synthase, and acetyl-coenzyme A carboxylase (ACC) also contain SRE elements in the promoter region (9-13).
Two structurally related protein transcription factors designated sterol regulatory element binding protein (SREBP) 1 and 2 activate transcription by binding to the SREs (7, 14). The precursor forms of the SREBPs are comprised of three segments: 1) an N-terminal region containing both a basic-helix-loop-helix-leucine zipper DNA binding segment and an acidic transcription activation domain, 2) a middle domain that includes two membrane spanning regions, and 3) a C-terminal region (16). Under sterol depletion conditions, the N-terminal segments are cleaved in two separate steps, thereby releasing the active forms of the SREBPs from the endoplasmic reticulum. They are then free to migrate to the nucleus and affect gene transcription (17-19). Mutations transcribing shorter forms of the SREBP-2 protein, which include the N-terminal active domain, are not subject to this regulation and produce sterol-resistant phenotypes (20).
Studies in cultured cells indicate that the two SREBPs are coordinatively regulated. No differences between them were observed in the activation of target genes (7, 14). However, in hamster liver, the SREBPs are expressed and activated independently. When liver cholesterol is depleted by dietary manipulations, expression and proteolytic activation of SREBP-1 is unaffected, whereas the transcription and activation of SREBP-2 is induced (21).
Electromobility shift studies indicate that the binding of the SREBPs to the SREs is enhanced in the presence of a general transcription factor. The promoters of the low density lipoprotein receptor, fatty acid synthase, and ACC contain GC box sequences, and the transcription factor Sp1 that binds to this motif can fulfill this function (10, 12, 13, 22, 23). The binding of SREBP-1a to the promoter of farnesyl diphosphate synthase is enhanced by the general transcription factor NF-Y, which binds to CCAAT box sequences (8, 24, 26). An NF-Y binding motif is also present in the promoter of 3-hydroxy-3-methylglutaryl-coenzyme A synthase and is likely to participate in the sterol-mediated regulation of this gene (27).
We have previously demonstrated that the human squalene synthase (HSS) gene is transcriptionally regulated in the HepG-2 human hepatoma cell line (28). We have also identified a 69-bp sequence in the HSS promoter, located 131 nucleotides 5' to the transcription start site, that is necessary for the sterol-mediated regulation of an HSS-luciferase reporter gene (pHSS1kb-Luc) (29). Sequence analysis of the 5'-flanking sequences of the HSS gene suggests that the sterol-mediated regulation of this gene is a complex mechanism. Four CCAAT boxes, three SRE-related motifs, and two GC boxes were identified there. Other sterol-regulated genes usually contain a single type of an SRE element and one of the two cis-elements that binds either NF-Y or Sp1. However, the presence of multiple copies of diverse elements in the HSS promoter strongly suggests that the regulation of this gene relies on multiple forms of interaction between the different transcription factors.
We have shown that adipocyte determination- and differentiation dependent factor 1/SREBP-1c, the rat homologue of SREBP-1c (30), binds to a sequence motif similar to the SRE-1 element in the low density lipoprotein receptor promoter, which we designated HSS-SRE-1. The regulatory effect of adipocyte determination- and differentiation-dependent factor 1/SREBP-1c on HSS promoter activity was verified by its activation of the pHSS1kb-Luc reporter in HepG-2 cells, indicating that the HSS-SRE-1 element in the HSS promoter may be functional (25, 29). An SRE-3 element previously identified in the promoter of the farnesyl diphosphate synthase gene (8) is present in an inverted orientation (Inv-SRE-3) 32 nucleotides 3' to the HSS-SRE-1 sequence. In addition, an SRE motif with an 8-nucleotide sequence identity to the 10-bp SRE-1 element, designated SRE-1(8/10), is located 29 nucleotides 3' to the Inv-SRE-3 element. These latter two SRE sequences were shown in electromobility shift assays to selectively bind SREBP-2 but not SREBP-1 (25).
The CCAAT motif is known to bind the general transcription factor NF-Y. Two such sequences are found 17 and 49 nucleotides 5' to the HSS-SRE-1 element. An additional inverted CCAAT sequence called the Y box is located between the HSS-SRE-1 and the Inv-SRE-3 elements (17 nucleotides 3' to the HSS-SRE-1 sequence), and another form of CCAAT sequence that was previously shown to be essential in the transcription of the farnesyl diphosphate gene (8) is present in an inverted orientation (Inv-Y-Box) between the Inv-SRE-3 and the SRE-1(8/10) elements (9 nucleotides 3' to the Inv-SRE-3 element). The binding of NF-Y to the Inv-Y-Box was verified by gel mobility shift assays (25, 29). It is of interest that the orientations of the SRE-3 and the Y box element in the HSS promoter are both inverted compared with those found in farnesyl diphosphate synthase. Their relative position in the promoter is reversed as well. In farnesyl diphosphate synthase, the Y box is 5' to the SRE-3 sequence and they are separated by a 21-bp spacer. In HSS, the Inv-SRE-3 is 5' to the Inv-Y-Box with a 9-bp separation. In both genes, the two elements are arranged sequentially to allow orientation in the same direction. This arrangement may be required for the interaction between an SREBP and NF-Y.
The detailed regulation of the HSS gene and the relative importance of the multiple motifs is not known. Single transversal mutations of DNA regions covering most of the promoter failed to completely eliminate the sterol-mediated regulation of an HSS-luciferase reporter. However, significant loss of activation was observed when the HSS-SRE-1, Inv-SRE-3, or the Inv-Y-Box sequences were mutated. Multiple mutations showed that the contribution of these motifs in the activation of the promoter is additive (25).
The purpose of the present study is to determine how the multiple motifs in the HSS promoter are involved in sterol regulation of the HSS gene. Using combinations of mutations in the HSS promoter, we have characterized elements involved in the regulation. Using these mutant promoters, we were able to identify sequences that differentially interact with SREBP-1a and SREBP-2 in the activation. These differences are, so far, unique to the HSS gene and may elucidate the specific physiological role of the SREBPs in sterol production.
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EXPERIMENTAL PROCEDURES |
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Materials and General Methods--
DNA sequencing was performed
using Sequenase 7-Diaza-dGTP DNA sequencing kit (Amersham Pharmacia
Biotech). Restriction enzymes and modification enzymes were purchased
from New England Biolabs. [-32P]dCTP was obtained from
NEN Life Science Products. Lipofectin reagent was purchased from Life
Technologies, Inc.
Cell Culture and Transient Transfection--
Human hepatoma
HepG-2 cells were maintained in 35-mm plates in a minimal essential
medium (Life Technologies, Inc.) supplemented with 10% fetal bovine
serum, 1 mM glutamine, 1 mM pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C and 5% CO2. Transient transfection of HepG-2 cells was conducted
using the Lipofectin reagent transfection procedure according to the manufacturer's manual and as described previously (29). Each transfection included 5 µg of HSS promoter-luciferase reporter DNA
and 4 µg of DNA of pCMV--galactosidase (pCMV-
GAL) expression vector for calculation of transfection efficiency. Where indicated, various amounts of the pCMV-CSA10 and pCMV-CS2 were also included in
the transfection mixture for the expression of SREBP-1a and SREBP-2. On
the day after transfection, the cells were treated for 24 h with
either suppressing medium, sterol(+), or inducing medium, sterol(
).
Sterol(+) contained 1 µg/ml 25-hydroxycholesterol and 10 µg/ml
cholesterol in 10% lipid-depleted serum; sterol(
) contained the same
medium as sterol(+), except that the sterols were replaced with 5 µg/ml lovastatin. The procedures for cell harvesting, preparation of
cell extracts, and the assays for luciferase activity and
-galactosidase activity have been described previously (29).
Relative luciferase activity is expressed as the ratio of luciferase
activity in relative light units to
-galactosidase activity (in
A574).
Generation of HSS Promoter-Luciferase Reporter
Constructs--
The construct used for the generation of the
replacement mutations is a pBluescript phagemid containing the 1 kb
5'-flanking region of the HSS gene (pHSS1kb-BS, +73 to 934) (29).
First, single strand DNA was prepared from pHSS1kb-BS using the
Sculptor in vitro Mutagenesis kit (Amersham) with minor
modifications. Briefly, TG1 host cells were transformed with pHSS1kb-BS
to obtain fresh transformants. On the following day, one colony was
selected and grown in 2× yeast tryptone medium for 3 h. VCS-M13
helper phage (Stratagene) was then added to the culture, and the
incubation was continued for 4 h. The cells were then lysed, and
the resulting single strand DNA was purified by PEG 8000 precipitation
followed by phenol extraction and ethanol precipitation.
Oligonucleotide-directed mutagenesis was performed as described
previously (25). To overcome the high GC content in the mutation site,
annealing and extension of mutation primers were performed at a
relatively higher temperature. Therefore, the following procedure was
employed. Single strand DNA and mutation primer were denatured at
94 °C for 5 min and slowly cooled to 25 °C to allow annealing.
The annealed primer was then extended in a buffer containing 2 mM dithiothreitol, 6 mM MgCl2, 0.2 mM each of dNTP, and 5 units of Vent DNA polymerase in a
total volume of 100 µl. The reaction was kept at 0 °C for 5 min,
incubated at room temperature for 10 min, 55 °C for 30 min, and
finally, 70 °C for 2 h. Four hundred units of T4 DNA ligase was
then added, and the reaction was incubated at 37 °C for 1 h to
allow for extension and ligation. The resulting DNA was used to
transform XL-1Blue competent cells. The mutated colonies were selected
by colony hybridization and restriction enzyme digestion using the
unique site introduced into mutation primers and verified by DNA
sequencing. The mutated plasmids were used for the subsequent subcloning of the mutated 1-kbp HSS insert into HindIII and
BamHI sites of a pXP1 luciferase reporter gene vector as
described previously for the native promoter (29). The resulting
mutations in pXP1 constructs were confirmed again by DNA sequencing,
and the plasmid DNAs were prepared for transient transfections. The
mutated HSS promoter regions, designated m1 to m8, are shown in Fig. 1.
The sequences of the oligonucleotides used for the mutation of the different sections of the HSS promoter are shown below, with the mutated sequences in bold:
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RESULTS |
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The functional relationship between previously identified
cis-elements in the HSS promoter, a promoter that is involved in the
sterol-mediated regulation, was investigated by mutational analysis.
Fig. 1 shows the nucleotide sequence +1
to 200 of the HSS promoter and seven cis-elements that were
previously identified as potentially important in the transcription of
the HSS gene. This sequence was reported to be sufficient in conferring
the sterol-mediated regulation of the promoter for the HSS gene (25). Two GC boxes are present in position
40 to
57 gapped by six nucleotides. Two other sequences of interest that may potentially bind
general transcription factors are Inv-Y-Box (
121 to
128) and a Y
box (
159 to
161). We have also examined three other cis-elements
with sequences of, or related to, SREs: an SRE-1(8/10) (
101 to
108), an Inv-SRE-3 (
138 to
147), and an HSS-SRE-1 sequence (
180
to
189) (25). Since DNA gel retardation studies have indicated
differences between the SREBPs in the binding to some of the above
sequences (25), in the present study we have used mutational analysis
to determine the functional significance of these elements in the
activation of the HSS promoter by SREBP-1a and SREBP-2.
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We have constructed a series of reporter genes containing mutations in the DNA elements of interest. The sequences in pHSS1kb-Luc, which were modified by transversal replacement mutations, are indicated (m1 to m8) in Fig. 1. In addition, multiple combinations of these mutations in pHSS1kb-Luc were used for the study of the activation by the SREBPs. These studies were done in HepG-2 cells cotransfected with the mutated reporter constructs and an expression construct for one of the SREBPs. Transfected cells were grown in the presence of cholesterol and 25-hydroxycholesterol (suppression media). Promoter activation is given as -fold induction and was calculated from the ratio of luciferase activity in cell extracts transfected with one of the SREBPs to cells without it as described under "Experimental Procedures." In each experiment, the activation of the mutated HSS promoter by the SREBPs was compared with its activation by lipid depletion in cells grown without sterols and in the presence of lovastatin (inducing media) and to the activation of the native pHSS1kb-Luc promoter in cells exposed to inducing media.
The different mutants can be divided into three main groups. The first group consists of constructs having mutations of the HSS-SRE-1 and Inv-SRE-3 elements, which are the two sequences reported to be important in the sterol-mediated regulation of other genes, and the SRE-1(8/10) element. These studies were designed to evaluate the importance of these cis-acting elements in the activation of the promoter by the two SREBPs. The results of these studies are depicted in Fig. 2. As was reported earlier (25), SREBP-2 was somewhat more effective in the activation of the wild-type promoter in pHSS1kb-Luc (Fig. 2a). Mutation of the HSS-SRE-1 sequence lowered the activation of the resulting promoter in pHSSm1-Luc by SREBP-1a or by inducing media but did not affect at all the activation by SREBP-2. At 1 µg of DNA/well, the activation by SREBP-1a was about 60% that of the activation obtained by SREBP-2 (Fig. 2b). Similar results were obtained if instead, the Inv-SRE-3 sequence element was replaced (pHSSm2-Luc construct). Again, significantly lower activation of this promoter by SREBP-1a (about 45% that of SREBP-2 at 1 µg of DNA/well) or by inducing media was observed, but the activation by SREBP-2 was not affected in comparison to the wild-type promoter (Fig. 2c). The most pronounced difference between the activation by SREBP-2 and SREBP-1a was observed using a promoter mutated in both the HSS-SRE-1 and the Inv-SRE-3 elements (pHSSm12-Luc). Although there is a small lowering (20% at 1 µg of DNA/well) in the activation by SREBP-2, the activation by SREBP-1a or by inducing media is almost completely diminished (Fig. 2d). In our earlier report a replacement mutation of the SRE-1(8/10) element by itself had a relatively small lowering effect in the activation of the HSS promoter (less than 30%) in transfected cells exposed to inducing medium (25). However, since the regulation of the HSS promoter may be an additive phenomenon contributed by the multiple regulatory sequences, it is necessary to study this sequence after eliminating other sequences that may possibly contribute to the regulation. Thus, we have decided to reexamine the regulatory contribution of this sequence in a construct in which both the HSS-SRE-1 and the Inv-SRE-3 elements were replaced. As expected, mutation of all the three SRE elements almost completely eliminated activation by lipid depletion or by SREBP-1a. However, unlike pHSSm12-Luc, this additional mutation (pHSSm124-Luc) almost completely abolished the activation by SREBP-2 as well (compare Fig. 2, d and e).
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The second group of experiments was designed to assess the importance of the Inv-Y-Box sequence in the activation of the HSS promoter by SREBPs or by sterol depletion. The actual sequence mutated is indicated as m3 in Fig. 1. The activation of a reporter gene containing this mutation (pHSSm3-Luc) is shown in Fig. 3a. There was a significantly lower activation by the SREBPs or by inducing media. Although SREBP-2 can activate the promoter to approximately 5-fold, the activation by SREBP-1a or by inducing media was only 3-fold. A similar effect was observed using a reporter construct containing, in addition, a mutation of the SRE-1 element (pHSSm13-Luc, Fig. 3b) or the SRE-3 element (pHSSm23-Luc, Fig. 3c). Although in each instance promoter activity is diminished, the activations most affected by these mutations were those induced by SREBP-1a or exposure of the cells to sterol depletion, whereas activation by SREBP-2 was approximately doubled. The most pronounced effect on activation was observed when all the three sequences above were mutated in the promoter (pHSSm123-Luc). The activity of this mutant HSS promoter was almost completely diminished regardless of the activation method (Fig. 3d).
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The third group of mutations was studied to determine the involvement
of the two GC box sequences in the HSS promoter. In earlier work we
reported that mutation of sequences containing the most 5' GC box
element (52 to
57) did not affect the sterol-mediated regulation of
this promoter (25). Therefore, we investigated the regulatory
relationship between the two GC boxes and the three SREs located 5' to
them. For this purpose we have first used a construct in which the two
most 5' SREs were mutated away (pHSSm12-Luc) and in it, sequentially
replaced the m6 sequences containing the 3' GC box element
(pHSSm126-Luc), the m7 sequences containing the 5' GC box element
(pHSSm127-Luc), and the m5 region that includes the two GC boxes
(pHSSm125-Luc). Mutation of a single element in a construct lacking
these SREs had minimal or no effect on the activation by the two SREBPs
or by sterols. In each mutant, the activation by SREBP-2 was mostly
retained (about 7- and 10-fold at 1 µg of DNA/well), whereas
activation by SREBP-1a or by sterol depletion was low (only 2-3-fold
at 1 µg of DNA/well) (Figs. 4, a and b). However, if both GC boxes were
replaced, the activation by SREBP-2 was substantially lowered as well
(3-fold at 1 µg of DNA/well), although it was still twice as high as
the activation by SREBP-1a. (Fig. 4c). Interestingly, a
mutant promoter in which only the two GC boxes were replaced
(pHSSm5-Luc) retained its activation by SREBP-1a or by sterol
depletion, but this time the activation by SREBP-2 was significantly
lower (about 4.5-fold at 1 µg of DNA/well) (Fig. 4d).
Mutation of the two GC boxes together with the SRE-1(8/10) caused
nondiscriminatory loss of 50% activation by the SREBPs and by lipid
depletion (pHSSm45-Luc in Fig. 4e), whereas mutation of the
Y box in addition to the two GC box sequences (pHSSm58-Luc) had a
pronounced lowering of activation by lipid depletion but little effect
on the activation by the two SREBPs (compare Fig. 4, d and
f). The mutation of the Y box by itself did not affect the
activation by SREBP-1a. However, activation by lipid depletion was
lowered by 31% and, although low concentrations of SREBP-2 were
effective in the activation, further activation using amounts of
SREBP-2 above 100 ng of DNA/well was not achieved (Fig. 4g).
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To illustrate the differences in activation of the various mutant HSS promoters by the two SREBPs, we have calculated the -fold differences expressed as the ratio of the -fold inductions (SREBP-2/SREBP-1a). To avoid nonspecific activation by overexpression of the SREBPs, these ratios were obtained in experiments employing DNA concentrations of constructs expressing SREBPs that cause 50% maximal activation of the wild-type promoter by sterol depletion (89 ng of DNA/well for SREBP-1a and 46 ng of DNA/well for SREBP-2). The highest relative activations were observed for promoters that lack the HSS-SRE-1 and the Inv-SRE-3 sequences but still include at least a single GC box sequence (Fig. 5). The only constructs displaying negative ratios are those in which the two GC box sequences were mutated (pHSSm5-Luc and pHSSm58-Luc).
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DISCUSSION |
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We have reported that the expression of HSS mRNA in HepG-2
cells is highly regulated by sterols (28). To study this
sterol-mediated regulation, we have cloned and analyzed the promoter of
the HSS gene (25, 29). In the present study we illustrate the regulated expression of a series of HSS promoter-reporter gene mutants by sterols
and SREBPs. This regulation involves multiple cis-acting elements that
are known to utilize the general transcription factors NF-Y and Sp1 and
specific sterol regulatory sequences that bind SREBPs. These multiple
cis-elements are located within 150 nucleotides (40 to
189) of the
HSS promoter.
The concentration-dependent activation of the wild-type HSS promoter by SREBP-1a and SREBP-2 shows a similar profile (Fig. 1a). It is apparent that the HSS-SRE-1 and the Inv-SRE-3 elements are important in the regulation of the promoter by SREBP-1a. The presence of these two DNA sequences enables an additive effect in the activation by SREBP-1a, since elimination of any single one decreases the activation by approximately 50% (Figs. 1, b and c), whereas the mutation of both resulted in almost complete loss of activation by this transcription factor (Fig. 1d). From these figures it is also apparent that the regulation by sterol depletion resembles the activation by SREBP-1a. These two sequences are needed to obtain maximal regulation by sterols. Contrary to the loss of promoter activation by SREBP-1a, the activation by SREBP-2 is minimally affected by these mutations. Mutation of any one of these SREs has no apparent effect on the activation of the mutant promoter, and mutational modification of both shows limited effect (Fig. 1, a-d). This regulatory difference between the two SREBPs was not demonstrated before, to the best of our knowledge, with any other sterol-regulated genes and may indicate the unique characteristics of HSS as the first committed enzyme in the cholesterol biosynthetic pathway.
In an earlier study, it was reported that a replacement mutation of the SRE-1(8/10) element by itself had a relatively small effect in lowering of the activation of the HSS promoter (less than 30%) by inducing medium. However, this sequence was shown to interact with SREBP-2 in a gel mobility shift assay (25). Since the sterol-dependent regulation of the HSS promoter is affected by several regulatory sequences acting in concert, the relative contribution of a single regulatory sequence to the overall response may be obscured. Therefore, to assess the importance of any element it is necessary to study this sequence after eliminating other sequences that may contribute to the regulation (e.g. HSS-SRE-1 and the Inv-SRE-3). As expected, mutation of all the three SRE elements almost completely eliminated the activation by SREBP-2, indicating possible interaction between the SRE-1(8/10) sequence and SREBP-2 (compare Fig. 2, d and e). This evidence does not eliminate a possible interaction of the SREBP-1a with the SRE-1(8/10) element, since the activation of the pHSSm12-Luc promoter by SREBP-1a is already low. In fact, some evidence indicates that the involvement of SRE-1(8/10) in the activation may be nondiscriminatory for the SREBPs, since in a mutant promoter that lacks the two GC boxes, mutation of the SRE-1(8/10) sequence affects primarily the activation by SREBP-1a (compare Fig. 4, d with e).
The Inv-Y-Box cis-sequence element (121 to
128) is also important
in the transcriptional activation of the HSS promoter. This sequence
motif was shown earlier to be essential for the regulation by sterols
in several sterol-regulated genes. For instance, the binding of
SREBP-1a to the SRE-3 element in farnesyl diphosphate synthase is
enhanced by the binding of NF-Y to the CCAAT box (8, 24, 26). Efficient
sterol-mediated regulation of the gene for SREBP-2 was also shown to
require the presence of SRE-1 and NF-Y elements (15). A general scheme
for the interaction between sterol regulatory proteins and general
transcription factors in the sterol-mediated regulation was suggested
in which the binding of a sterol regulatory transcription factor to its
sequence element is enhanced by the binding of a general protein
transcription factor to a nearby sequence (10, 15, 26). Similar to the mechanism suggested for these genes, the Inv-NF-Y-Box is also shown
here to be important in the efficient activation of the HSS promoter.
Its mutation affects both the activation by sterol depletion or by the
SREBPs. Mutation of this sequence by itself (Fig. 3a) or
together with either the HSS-SRE-1 or the Inv-SRE-3 (Fig. 3,
b and c) reduced the activation dramatically.
Mutation of both SREs together with a mutation of the Inv-Y-Box totally abolished activation by sterol depletion, and only residual activation by high levels of the SREBPs was retained (Fig. 3d). If the
general scheme for sterol mediated-regulation involving an SREBP
interaction with a general transcription factor (such as NF-Y) is valid
for HSS as well, then the limited activation observed in Fig. 3,
a-c must be due to a non-sterol-specific cis-element in the
promoter, possibly the Y box.
As indicated above, the complex regulatory mechanism of HSS is apparent by the high remaining activation by SREBP-2 of a mutant promoter lacking both the HSS-SRE-1 and the Inv-SRE-3 elements (Fig. 2d). This indicates the existence of a remaining SREBP-2 recognition sequence. To completely abolish the SREBP-induced or sterol-mediated activation of the HSS promoter, mutation of the Inv-Y-Box is necessary as well. This triple mutation exists in pHSSm123-Luc. In this mutated promoter, the activation by SREBP-2 was completely abolished (compare Figs. 2d with Fig. 3d).
At the 3' end of the 150-bp sequence containing the multiple cis-elements there are two adjacent GC box elements (Fig. 1). Originally, we assumed that these GC box sequences were of no significance to either the transcription or to the sterol-mediated regulation of the HSS promoter, since mutation of each alone did not affect the activation by sterol depletion (data not shown). In fact, mutation of both elements together resulted in only a marginal decrease of the activation by sterol depletion and had no effect on the activation by SREBP-1a. However, further examination indicated that simultaneous mutation of these two elements significantly decreased the activation by SREBP-2 (Fig. 4d). To eliminate the possible activation by endogenous SREBP-1a that may be produced in the cells despite the added sterols, we have taken advantage of the observation that mutation of the two 5' SREs differentially inhibited the activation by SREBP-1a but not the activation by SREBP-2 (Fig. 2d). Accordingly, we have used constructs that lack these two SREs to study the significance of the two GC box sequences in the specific activation by SREBP-2. These studies show redundancy in the functionality of the two GC boxes. The presence of either one of the GC box sequences is sufficient to allow activation by SREBP-2 (compare Fig. 4, a and b, to Fig. 2d). However, simultaneous mutation of the promoter region m5, which contains the two GC box sequences, and the two 5' SREs significantly lowered the activation by SREBP-2 (Fig. 4c). It can be concluded that if, as proposed above, there is an SREBP-2 binding sequence other than the two 5' SREs, it may affect activation of the HSS promoter by interacting with an Sp1 transcription factor that can bind to either of these cis-elements. Additional multiple mutation studies are needed to indicate the involvement of the SRE-1(8/10) together with the two GC boxes in the activation. The effect observed here is in agreement with the reported interaction between SREBPs and Sp1 in the activation of the promoters for the low density lipoprotein receptor, fatty acid synthase, and ACC (10-13, 22, 23). However, in contrast to HSS, no specificity between the SREBPs was reported for these genes.
The pronounced differential activation of mutant HSS promoters by the two SREBPs is most apparent in Fig. 5. Since we could not determine the amounts of the SREBPs expressed in the cells by transfection, we have used DNA constructs expressing SREBPs at concentrations that yielded 50% of the activation caused by sterol depletion of the wild-type HSS promoter. This, we assume, assures that the SREBPs are not overexpressed in transfected cells above physiological levels, and therefore, the differential effect of the SREBPs on the mutant promoters can be determined. It is apparent that under these conditions the activation by SREBP-1a was affected to a higher extent than by SREBP-2. This differential effect is most pronounced in promoters in which the two 5' SREs are mutated. The differential effect is reversed when the two GC box elements are eliminated, which provides additional evidence that the two SREBPs may not interact interchangeably with regulatory sequences at the HSS promoter. It is interesting that when, in addition to the mutation of the two GC boxes, the Y box is also mutated, the relative activation by SREBP-2 is further reduced (compare pHSSm5-Luc with pHSSm58-Luc in Fig. 5). A possible explanation is that the Y box is involved in enhancing a specific interaction of SREBP-2 with one of the SREs. This possibility is supported by the difference in the activation of a Y box mutant (pHSSm8-Luc) by the two SREBPs. Interestingly, at low levels of the SREBPs, they are both as effective in the activation. However, at concentrations above 100 ng of DNA/well, unlike for SREBP-1a, further activation by SREBP-2 was not achieved (Fig. 4g). This profile of activation supports possible interaction of the SREBP-2 with multiple SRE sequences at the promoter. Those with relatively high affinity for SREBP-2 were not affected by the mutation of the Y box, and thus, low concentrations of SREBP-2 are effective. However, the general transcription factor that binds to the Y box may affect, primarily, the interaction of the SREBP-2 with a low affinity element(s), which regulates transcription only at relatively high levels of SREBP-2 (hence the relatively low decrease in activation by lipid depletion). In this model, the SRE(s) with the low affinity is SREBP-2-specific and did not bind SREBP-1a.
The data presented suggest that in most mutants the regulation by sterols in HepG2 cells is similar to the activation by SREBP-1a and not to the activation by SREBP-2. The exception to this are mutants in the Y box in which the sterol regulation parallels the activation by the latter (see Figs. 4, f and g).
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ACKNOWLEDGEMENTS |
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We thank Dr. Steven K. Nordeen of the University of Colorado for the luciferase pXP1 construct and Dr. Timothy F. Osborne of the University of California, Irvine for providing the plasmids encoding the SREBPs. We also thank Dr. Mark A. Roseman for useful editing support.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL48540 and HL50628.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.
To whom correspondence should be addressed: Tel.: 301-295-3550;
Fax: 301-295-3512; E-mail: shaike{at}usuhsb.usuhs.mil.
1
The abbreviations used are: bp, base pair; SRE,
sterol regulatory element; SREBP, SRE-binding protein; HSS, human
squalene synthase; Inv-SRE-3, inverted SRE-3; Inv-Y-Box, inverted Y
box; CMV, cytomegalovirus; pCMV-GAL, pCMV-
-galactosidase; ACC,
acetyl-coenzyme A carboxylase; kbp, kilobase pair(s).
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REFERENCES |
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