(Received for publication, April 20, 1995)
From the
We have cloned and characterized the 5`-flanking region of the gene encoding human squalene synthase. We report here the promoter activity of successively 5`-truncated sections of a 1 kilobase of this region by fusing it to the coding region of a luciferase reporter gene. DNA segments of 200 base pairs (bp) 5` to the transcription start site, as determined by primer extension analysis, show a strong promoter effect on the expression of the luciferase chimeric gene and a high response to the presence of sterols when transiently transfected into the human hepatoma cell line HepG2 or to the hamster-derived CHO-K1 cells. An approximately 50-fold induction of luciferase activity, in the absence of sterols, was observed in transiently transfected HepG2 cells for fusion constructs containing sections of 200, 459, and 934 bp of the putative human squalene synthase promoter. Loss of promoter activity and response to sterols was localized to a 69-bp section located 131 nucleotides 5` to the transcription start site. Sequence analysis of this region showed that it contained a sterol regulatory element 1 (SRE-1) previously identified in other sterol regulated genes (Smith, J. R., Osborne, T. F., Brown, M. S., Goldstein, J. L., and Gil, G.(1988). J. Biol. Chem. 263, 18480-18487) and two potential NF-1 binding sites. Additional CCAAT box, SRE-1 element, and two Sp1 sites were identified 3` to this section. Sequences within this 69-bp DNA, including the SRE-1 cis-acting element, show strong binding to the purified nuclear transcription factor ADD1 (Tonzonoz, P., Kim, J. B., Graves, R. A., and Spiegelman B. M.(1993) Mol. Cell Biol. 13, 4753-4759) by mobility shift assay and footprinting analyses.
Squalene synthase (farnesyl-diphosphate:farnesyl-diphosphate
farnesyltransferase, EC 2.5.1.21) is the first enzyme specific to the
cholesterol biosynthetic pathway. The activity of rat hepatic squalene
synthase is regulated by dietary cholesterol and by the dietary
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) ()reductase
inhibitors, lovastatin, or fluvastatin(1, 2) . The
activity of human squalene synthase (HSS) and the level of its mRNA are
regulated by sterols in the human hepatoma cell line HepG2.
Sterol-mediated regulation has been localized to a 10-base pair (bp) element in the 5`-flanking region of other sterol-regulated genes. This 10-bp sterol regulatory element 1 (SRE-1) mediates increased transcription of the genes encoding HMG-CoA synthase and the low density lipoprotein (LDL) receptor in sterol-depleted cells, and its activity is inhibited by sterols(3, 4) . Proteins that bind to the SRE-1 of the LDL receptor (SREBPs) were purified by DNA affinity chromatography from nuclear extracts of HeLa cells. A cDNA for SREBP-1 was isolated from adipocyte cDNA library(5) . This cDNA, designated ADD1, activated transcription of a reporter gene containing an ``E-box'' sequence present in the promoter of fatty acid synthase in transfected NIH 3T3 cells. Cloned SREBP cDNA contain two major classes of proteins, SREBP-1 (5) and SREBP-2(8) . Three different cDNAs for SREBP-1 were isolated, suggesting multiple forms of the mRNA and perhaps different proteins as well. The physiological significance of these subclasses is unclear(6) . Different SREBP-1 proteins may have specific physiological roles because mRNAs for the various isoforms are differentially regulated by sterol depletion in HepG2 cells(8) .
Proteolytic cleavage of the C-terminal membrane-associated domain of the nascent SREBP-1 (125 kDa) forms its nuclear form (68 kDa). This proteolytic maturation was proposed to be accomplished by a sterol-inhibited protease. The calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) induced the mRNA for HMG-CoA synthase and was proposed to inhibit the degradation of the mature SREBP-1(9) .
In other sterol-regulated genes, the SRE-1 is not involved in sterol-mediated transcriptional regulation. Although the promoter region of farnesyl diphosphate synthase contains multiple forms of the SRE-1 element, these elements are not involved in the sterol-mediated transcriptional regulation(10) . Similarly, the promoter of the hamster HMG-CoA reductase contains unique sites for sterol regulation. Red 25, a nuclear hamster liver protein, binds to this regulatory region but did not bind to the sterol regulatory regions of the LDL receptor and HMG-CoA synthase promoters(11) .
In this report we characterize the 5` region of the HSS gene. The promoter activity and the sterol-mediated regulation of this DNA were assessed by fusing 5`-flanking DNA to a luciferase reporter gene and transfecting it into HepG2 cells and Chinese hamster ovary (CHO-K1) cells. A 69 bp DNA sequence confers transcriptional competence and sterol regulation. ADD1 binds to this 69 bp sequence in two places, one of which contains an SRE-1 element.
Standard recombinant technology procedures were used(12) . DNA sequencing was performed by the dideoxy chain-termination method (13) employing Sequenase 7-Diaza-dGTP DNA sequencing kit (U. S. Biochemical Corp.).
Three probes
were used in the electrophoretic mobility shift assay. One is a 156-bp
DNA fragment generated by digesting the 1-kb promoter of human
squalene synthase with NheI and BanI (nucleotides
-91 to -246). The two other probes are synthetic
oligonucleotides. One of them is a DNA containing a wild-type SRE-1
sequence existing in the HSS promoter (HSS-SRE-1), and the other has
the same sequence except that four bases were mutated (nucleotides
-183, -184, -186, and -187) and introduced into
the SRE-1 site (HSS-SRE-1-mut). The complementary oligonucleotides were
reanealed and the probes were P-end-labeled by Klenow
fill-in reaction as described above. The sequences of the two synthetic
DNA probes are shown below with boldface letters indicating the mutated
bases: HSS-SRE-1, 5`-TAGAGTGTTATCACGCCAGTCTCCTT-3` and
3`-TCACAATAGTGCGGTCAGAGGAAGG-5`; HSS-SRE-1-mut,
5`-TAGAGTGTTATCTAGGAAGTCTCCTT-3` and
3`-TCACAATAGATCCTTCAGAGGAAGG-5`.
Figure 2:
Schematic representation of the HSS
promoter and the sequencing strategy of the 5`-flanking region of the
HSS gene. A 2-kb DNA section of a PstI partially digested
fixII HSS genomic clone was subcloned into pBluescript KS vector
and sequenced. The positions of the primers, in relation to the 1.5-kb
5` end of this clone used for the sequencing, are shown as arrows on topwith the descending order representing
the sequence of the reactions. The relative positions of the
endonuclease restriction sites used for the preparation of different
chimeric luciferase reporter genes are indicated below. The
type and positions of various putative transcription and regulatory
elements found within the 300 nucleotides flanking the transcription
start site are indicated in marked boxes in the bottom of the scheme. The sequences of the two SRE-1-like elements are boxed.
A 1-kb SmaI restriction fragment of pHSS2kbBS was isolated and subcloned into an EcoRV site of pBluescript to give the correctly oriented insert (pHSS1kb-BS) in this construct for the further preparation of the luciferase expression plasmid in the pXP1 vector (repeatedly, insertion into the SmaI site of pBluescript resulted in the reversed orientation of the insert). pHSS1kb-BS was digested with HindIII and BamHI, and the 1-kb DNA fragment was ligated to the same restriction sites in pXP1, a luciferase vector (S. K. Nordeen, University of Colorado, (15) ), to form pHSS1kb-Luc. pHSS1kb-BS was digested with PstI to remove the 5` 477-bp of the HSS insert and religated to form pHSS532-BS. A HindIII-BamHI fragment was removed from pHSS532-BS and ligated to same sites in pXP1 vector to form pHSS532-Luc. pHSS273-BS was prepared from pHSS1kb-BS by an XbaI digestion followed by self religation. The corresponding pHSS273-Luc plasmid was prepared from the latter by inserting an HindIII-SacI 273-bp DNA fragment from pHSS273-BS into an HindIII-SstI digestion product of pXP1. The next chimeric gene pHSS204-Luc was prepared by inserting a 204-bp HindIII-MscI DNA fragment of pHSS532BS into a HindIII-SmaI-digested pXP1 vector. To prepare the smallest chimeric reporter gene, pHSS164-Luc, we first digested the pHSS1kb-BS DNA with an NheI. The linear plasmid was blunt-ended with Klenow. pHSS164-BS was prepared by a SmaI digestion of the linearized DNA following by religation. The corresponding pHSS164-Luc reporter gene was prepared from the latter by inserting a HindIII-BamHI-164-bp DNA fragment from pHSS164-BS into an HindIII-BamHI-digested pXP1 vector. pHSS1kbRev-BS containing a reversely oriented 1-kb HSS insert (relative to pHSS1kb-BS) was prepared by inserting the SmaI, HSS 1 kb, digestion product from pHSS2kbBS into the SmaI site of the BlueScript vector. The corresponding pHSS1kbRev-Luc reporter gene was prepared from the latter by inserting a HindIII-BamHI 1-kb DNA fragment from pHSS1kbRev-BS into an HindIII-BamHI-digested pXP1 vector. All luciferase reporter genes except pHSS1kbRev-Luc had the same 3` ends in a HindIII site of pXP1 and varying 5` ends. All plasmids were verified by sequencing.
Figure 1: Nucleotide sequence of the HSS promoter. The sequence shown contains 1487 nucleotides 5` to the transcription start site of the longest mRNA transcript and 96 nucleotides 5` to the ATG initiation codon, which is at the 3` end of the sequence. The cytidine at the transcription start site is designated +1. Putative transcriptional and regulatory elements are doubleunderlined and identified above the sequence. Endonuclease restriction sites, used for the preparation of various chimeric luciferase reporter genes, are underlined and are also identified above the sequence. The SmaI site at position +73 is the 3` end of the HSS inserts in common to all fusion luciferase reporter genes.
Figure 3:
Determination of the human squalene
synthase transcription initiation site. A schematic diagram of the
procedure (bottom) and an autoradiogram (top) are
shown. The 20-nucleotide primer 5`-GCGAAACTGCGACTGGTCTG-3` (H10),
located 164 nucleotides 3` to the AUG translation initiator, was
5`-P-labeled using T4-polynucleotide kinase and
[
-
P]ATP. 50 fmol of the
P-labeled primer (6
10
cpm) and 3
µg of poly(A
) RNA were used for the extension
reaction. After hybridization for 45 min at 60 °C, the reaction was
carried out at 45 °C for 60 min with RNA reverse transcriptase (100
units) and 2.5 mM of dNTPs in 40 µl of reaction volume.
The radioactive extension product was analyzed on 5% sequencing gel. An
M13 single-stranded DNA sequencing product is represented by lanesG, A, T, and C and is used for
size determination. The two largest
P-labeled extension
products (EP) were of 262 and 261 nucleotides. The
transcription initiation site, therefore, is calculated to be at either
98 or 97 nucleotides 3` to the adenosine of the AUG
initiator.
Figure 4:
Promoter activity and response to sterols
of fusion genes containing successively 5` truncated HSS promoter.
Varying lengths promoter fragments of the human squalene synthase gene
used for the construction of pHSS1kb, -532, -273,
-204, -164, and -1kbRev-Luc are represented by a solid and hatchedbar. The endonuclease
restriction sites used for the construction of this plasmids are
indicated above and on each side of the HSS DNA fragments. The
HSS transcription initiation site, indicated by an arrow, is
numbered +1, and all other sites are relatively numbered. The hatchedbar represents a section of the
5`-untranslated region in the first exon of the HSS gene and is linked
at its 3` end (SmaI site at position +73) to the coding
region of a luciferase reporter gene. The HSS DNA in pHSS1kbRev-Luc is
the same as the DNA in pHSS1kb-Luc but inserted in an opposite
orientation. Activities were measured in transfected HepG2 cells, which
were also co-transfected with a pCMV-GAL plasmid for the
expression of nonregulated
-galactosidase activity in the presence
and absence of sterols. Relative luciferase activities were determined
as a ratio of RLU to the activity of
-galactosidase and were
normalized to 100 for pHSS1kb-Luc in the absence of sterols. In the
absence of sterols, cells were incubated in 10% LPDS containing media
in the presence of 5 µg/ml lovastatin. In the presence of sterols,
the cells were incubated in the same LPDS media in the presence of 5
µg/ml 25-hydroxycholesterol and 15 µg/ml cholesterol, according
to the protocol described under ``Materials and
Methods.''
The expression of the different chimeric
HSS-luciferase constructs was tested in transiently transfected cells.
We chose to examine both hepatic-derived and fibroblast cell lines in
order to test the possibility that the transcriptional regulation
characteristics of the HSS promoter might display cell type specificity
and, therefore, might exhibit a different transcriptional regulation of
the luciferase reporter fusion genes in these two cell lines. Thus, we
introduced the various chimeric constructs by transfection into both
the human hepatoma cell line HepG2 and the hamster-derived CHO-K1
fibroblast cell line. For normalization of the activity in the
different transfectant cells, we co-transfected the cells with
pCMV-GAL, a non-sterol-regulated
-galactosidase expression
vector, and the results were calculated as the ratio of the luciferase
to the
-galactosidase activities.
In HepG2 cells, the highest
expression of luciferase, relative to the normalizing
-galactosidase activity, was obtained with the pHSS1kb-Luc fusion
gene. This expression resulted when the transfected cells were treated
with LPDS and lovastatin in the growth media. High luciferase
expression was also observed in cells transfected with pHSS532-Luc and
pHSS273-Luc. However, further reduction in the size of the synthase
promoter resulted in a substantially lower expression of the luciferase
activity. Accordingly, pHSS204-Luc and pHSS164-Luc showed relatively
low reporter activity (Fig. 5). As expected, reversal of the
orientation of the HSS promoter resulted in complete loss of luciferase
activity in HepG2 cells (see pHSS1kbRev-Luc in Fig. 5). With slight, although reproducible, differences, a
similar pattern was observed for the expression of these constructs in
CHO cells (Fig. 5). For further studies of the regulation of the
synthase promoter, we chose to use the HepG2 cells.
Figure 5:
Comparison of promoter activities of
fusion genes containing varying lengths of the HSS promoter expressed
in CHO-K1 and HepG2 cells. CHO-K1 and HepG2 cells were transfected with
one of the chimeric genes pHSS1kb, -532, -273, -204,
-164, and -1kbRev-Luc, containing varying length promoter
fragments of the human squalene synthase fused to a luciferase reporter
gene and with a pCMV-GAL plasmid for the expression of
nonregulated
-galactosidase activity. The cells were maintained in
LPDS containing media in the presence of 5 µg/ml
25-hydroxycholesterol and 15 µg/ml cholesterol, according to the
protocol described under ``Materials and Methods.'' Relative
luciferase activities were determined as a ratio of RLU to the activity
of
-galactosidase and was normalized to 100 for pHSS1kb-Luc in
both cell lines. The relative activity of pHSS1kb-Luc in HepG2 was 1.35
times higher then in CHO-K1 cells.
When transfected HepG2 cells were treated with cholesterol and 25-hydroxycholesterol in LPDS-containing medium (fully suppressed conditions) or with lovastatin in LPDS medium (fully induced conditions), a definite regulation was observed. A 47.6-fold increase in luciferase activity was observed between fully induced and fully suppressed conditions in extracts of pHSS1kb-Luc transfected cells treated for 24 h (see Fig. 4). Thus, the 934-bp 5`-flanking region of the HSS promoter contained sequence elements that conferred sterol regulation. A similar regulatory response was observed for the pHSS532-Luc and pHSS273-Luc constructs. A 42.9- and a 51.3-fold increase between fully induced and fully suppressed conditions in HepG2 cells was observed for these two constructs, respectively (Fig. 4). Since in fully suppressed conditions the luciferase activity in all of the above three constructs is marginal, it is assumed that it may affect the accuracy of this ratio. Nonetheless, it reflects the pronounced effect sterols have on the regulation of these reporter constructs.
The relative synthase
promoter activities in pHSS1kb-Luc, pHSS531-Luc, and pHSS276-Luc
constructs under fully induced conditions are very similar. Repeatedly,
pHSS1kb-Luc produced a somewhat higher luciferase response than the
latter two (see Fig. 6). Since the amounts of the three chimeric
DNA constructs as well as the amount of the DNA of the normalizing
pCMV-GAL were kept constant in all transfections, the molar DNA
equivalents driving the three luciferase expressions are not equal and
are higher in the cells containing the smaller constructs. But even
with this consideration, the luciferase expression in the smaller
vectors is still considerable. Insertion of the 1-kb 5`-flanking
sequence in a reverse orientation into the pXP1 expression vector
completely failed to induce luciferase activity. The resulting
pHSS1kbRev-Luc construct showed background levels of luminescence
either under fully suppressed or fully induced conditions (Fig. 4).
Figure 6:
Expression of luciferase reporter gene,
containing varying lengths of the HSS promoter, in HepG2 cells
maintained in different sterol-containing media. HepG2 cells were
transfected with one of the chimeric genes pHSS1kb, -532,
-273, -204, -164, and -1kbRev-Luc, containing
varying lengths promoter fragments of the human squalene synthase fused
to a luciferase reporter gene, and with a pCMV-GAL plasmid for the
expression of nonregulated
-galactosidase activity. The cells were
exposed to one of the following treatment media: (a) medium
containing 10% (v/v) FBS; (b) media containing 10% (v/v) LPDS
(LPDS media) supplemented with 15 µg/ml cholesterol; (c)
LPDS media supplemented with 5 µg/ml lovastatin; (d) LPDS
media supplemented with 5 µg/ml 25-hydroxycholesterol; (e)
LPDS media supplemented with 15 µg/ml cholesterol, and 5 µg/ml
25-hydroxycholesterol. Cholesterol and 25-hydroxycholesterol were added
in a 5-µl ethanol solution. After incubation for 24 h with the
appropriate treatment media, the cells were harvested, and the cell
extracts were assayed for luciferase and
-galactosidase
activities. Relative luminescence are expressed as a ratio of
luciferase activities, in RLU, to the activities of
-galactosidase (A
).
Truncation of the 5` HSS flanking sequences at the MscI site to produce a 204 bp insert almost completely abolished its promoter activity. Only 6.8% of the luciferase activity remained, and a mere 3.4-fold increase in activity was observed for the pHSS204-Luc between fully induced and fully suppressed conditions. Similar results were also obtained for the smaller pHSS164-Luc construct.
Cholesterol is a much weaker regulator then 25-hydroxycholesterol. Suppression of luciferase activity with 15 µg/ml cholesterol supplementation to the LPDS-containing media resulted in a decrease of activity to 17.9% of fully induced conditions, whereas the addition of 5 µg/ml 25-hydroxycholesterol instead resulted in 3.5% remaining activity in cells transfected with pHSS1kb-Luc. A similar, albeit not identical ratio was observed for the cells grown in 10% FBS (see Fig. 6). Similar ratios were also observed in cells transfected with pHSS532-Luc and pHSS273-Luc. It is interesting to note that the shortest promoter in pHSS164-Luc failed completely to respond to the presence of cholesterol, but its relative activity did decrease somewhat in the presence of 25-hydroxycholesterol (Fig. 6).
Figure 7:
DNase I footprinting of human squalene
synthase promotor. A, footprinting of coding strand of a DNA
fragment corresponding to -91 to -459 of the gene. B, footprinting of noncoding strand of a probe (+73 to -246). Both probes were end-labeled with
[-
P]dCTP as described under
``Experimental Procedures.'' The probe was incubated with or
without ADD1 (1 or 2 µg as indicated), followed by DNase I
digestion and analysis on a 6% sequencing gel. Lane1 is the chemical cleavage of the probe at A+G residues, which
served as a sequence marker. Lane2 is the probe
digested with DNase I in the absence of ADD1. Lanes3 and 4 correspond to the DNase I-digested probe in the
present of 1 and 2 µg of ADD1. The sequence position of the probe
is shown to the left of the gel. The protected region is
indicated on the right side of the gel, and SRE-1 sequence is
marked by a bracket.
Figure 8:
Binding of ADD1 to human squalene synthase
promoter. A probe, corresponding to nucleotides -91 to -246
of the human squalene synthase promoter and containing the SRE-1
sequence 5`-ATCACGCCAG-3`, was generated by the digestion of the HSS
promoter with NheI and BanI. This probe was
end-labeled with [-
P]dCTP. The labeled
probe was incubated with various amounts of ADD1, as indicated on the top of the gel, under the condition described under
``Experimental Procedures.'' Separation of the free and bound
probes was done by electrophoresis on a 4% native polyacrylamide gel.
The retarded band, which corresponds to the ADD1-bound probe and the
free probe band is indicated by arrows.
Figure 9:
Binding of ADD1 to HSS-SRE-1 and
HSS-SRE-1-mut. Two P-labeled DNA probes, which include
either a wild-type SRE-1 sequence found in HSS promoter (HSS-SRE-1) or
the same sequence except with four bases mutated at nucleotides
-183, -184, -186, and -187 (HSS-SRE-1-mut) were
incubated with ADD1 in a competition assay as described under
``Materials and Methods.'' Lanes1-3 show the binding of 1 ng of labeled HSS-SRE-1 to increasing
concentrations of ADD1. Lane1, unbound HSS-SRE-1
probe; lanes2 and 3, HSS-SRE-1 probe
incubated with 0.5 and 1.0 µg of ADD1, respectively. Lanes4-6 show competition assays of 1 ng of
labeled HSS-SRE-1 with increasing concentrations of the same unlabeled
probe. The molar excess of the unlabeled competing probe for each lane
is as follows: lane4, 100; lane5,
1,000; lane6, 10,000. Lanes7-9 show the binding of 1 ng of labeled HSS-SRE-1-mut probe to
increasing concentrations of ADD1. Lane7, unbound
HSS-SRE-1-mut probe; lanes8 and 9,
HSS-SRE-1-mut probe with 0.5 and 1.0 µg of ADD1, respectively. The
binding products were analyzed on 4% native polyacrylamide
gel.
Squalene synthase was shown to be a highly regulated enzyme in mammals(1, 2, 14, 20) . The sterol-mediated regulation of HSS in HepG2 cells was shown to be primarily transcriptional(14) . Therefore, to determine whether the 5`-flanking region of the HSS gene contained sterol regulatory elements, we prepared chimeric fusion constructs containing various lengths of the 5`-flanking HSS sequences fused to a luciferase reporter gene. These fusion constructs were introduced to human hepatoma (HepG2) and nonhepatic CHO-K1 cell lines by transfection for transient expression.
Current studies show that the 5`-flanking region of the HSS gene confers a strong promoter activity in the fusion luciferase genes. The 1-kb, 532-bp, and 273-bp 5`-flanking sequences of the HSS gene have comparatively similar promoter function activity. However, deletion of 69 bp at the 5` end of the pHSS273-Luc construct almost completely diminished the promoter activity of the resulting pHSS204-Luc chimeric gene.
The transient expressions of pHSS1kb-Luc, pHSSXP532-Luc, and pHSS273-Luc are found to be highly responsive to the presence of sterols in both CHO (data not shown) and HepG2 cells (see Fig. 6). The sterol-mediated response of the luciferase activity in transiently transfected HepG2 and CHO cells exceeded the reported responses of similar CAT constructs containing the 5`-flanking regions of the genes for farnesyl diphosphate synthase in transiently transfected HepG2 cells(10) ; that of the LDL receptor and HMG-CoA synthase in transiently transfected CV-1 cells(4) ; and stably transfected CHO cells with HMG-CoA reductase or HMG-CoA synthase CAT fusion genes(21, 22) .
Since there is no significant difference between pHSS532-Luc (containing HSS nucleotides +73 to -459) and pHSS273-Luc (with nucleotides +73 to -200) in their promoter activity and the sterol-mediated response, we can assume that the two distal CCAAT sequences at -210 and at -243 are marginally important, if at all, to the synthase promoter activity and response to sterols. The pHSS204-Luc chimeric gene (nucleotides +73 to -131) retained little of the promoter activity (Fig. 5). Thus, we have to assume that essential cis-acting element(s), necessary for the transcription and the sterol-mediated regulation, are present in the 69-bp sequence between nucleotides -131 and -200. There are some recognizable potential cis-acting transcription elements located within this sequence. Starting at nucleotide -187, there is a 7:8 base pair match of the octamer cis-acting element SER-1, which was shown to confer response to sterols in several genes involved in cholesterol homeostasis(23, 24) . This cis-acting element is preceded by AT at its 5` end, which was also shown to be essential, in conjunction with the SRE-1 sequence, for sterol-mediated regulation in the promoter of the LDL receptor(4) . At nucleotide -163, the sequence ATTGG is a reverse CCAAT box in the opposite strand. This sequence was shown to be recognized by a family of CTF/NF-1 cellular binding proteins that are known to be involved in transcription initiation (25, 26, 27, 28) . Finally, the MscI restriction site at nucleotide -128 used for the preparation of pHSS204-Luc disrupted a cis- acting TGGCCAAT sequence, which is also a binding sequence for a CTF/NF-1 initiation site. Thus, elimination of any one of these three sequences may explain the loss of promoter activity and the response to sterols observed for pHSS204-Luc.
The TGGCCAAT sequence is located 15 bp 5` to the octamer CACCCCAC, an SRE-1 consensus sequence element at nucleotide -108. However, this element lacks the AT sequence at its 5` end, which is essential for the binding and activity of the SREBP-1 transcription factor. The pHSS164-Luc construct was designed to eliminate this SRE-1 element. Since most of the promoter activity was lost by the deletion of sequences 5` to this SRE-1 element, there is no way of assessing, based on the present data, its importance as a regulatory element of the promoter for HSS. An indication that this SRE-1 sequence may not be important for sterol-mediated regulation comes from the observation that this sequence does not show footprinting in the presence of ADD1. Future experiments, involving mutational substitution and single nucleotide mutagenesis should elucidate the involvement and the functional interrelationship of the different regulatory elements.
The 5`-flanking region of the HSS gene also contains two adjacent Sp1 transcription elements with the sequence GGGCGG at the core of each. These two elements are present at nucleotides -57 to -40 and are located approximately 50 nucleotides 3` to the consensus SRE-1 sequence. Sp1 sites were shown to be promoter-specific transcription activation sequences of RNA polymerase II(29) . These two elements are present in pHSS164-Luc and apparently are not sufficient to confer by themselves any significant promoter activity.
The promoter for HSS does not contain a TATA box 5` to the transcription initiation site. In this respect, the HSS gene is similar to the HMG-CoA reductase gene and different than other highly regulated genes in the cholesterol biosynthetic pathway, such as the genes for HMG-CoA synthase, farnesyl diphosphate synthase, and the LDL receptor (10, 24, 30, 31) .
There are multiple forms of the trans-acting sterol regulatory element binding protein (SREBP) factors, which bind to the SRE-1 elements of the promoters of the LDL receptor and the HMG-CoA synthase genes. The two major forms are the SREBP-1 (6) and SREBP-2 (7) . However, the existence of different cDNAs for SREBP-1 may indicate the cellular existence of different forms of this protein(6) . The processing and translocation of the SREBP-1 from the membrane to the nucleus was shown to be initiated by a proteolytic process. It was also reported that its further degradation by the calpain protease inhibitor ALLN increased the level of the mRNA for HMG-CoA synthase (9) . In early experiments, we failed to observe an increase in the level of HSS mRNA when HepG2 cells were exposed to ALLN (data not shown). Having an HSS-luciferase reporter construct enabled the determination of the effect of ALLN on the transcription of this chimeric gene. Again, we failed to observe an increase in luminescence in response to ALLN in HepG2 cells treated with either 10% serum or LPDS. This observation is in agreement with the recent report that the inhibition of SREBP-1 degradation by ALLN treatment of HepG2 cells increased the mRNA levels for the LDL receptor but not for squalene synthase(8) .
The lack of response to ALLN and the indication that ADD1 interact with a promoter element that contains an SRE-1 sequence as shown by the gel mobility shift ( Fig. 8and Fig. 9) and the footprinting (Fig. 7) assays brings up the interesting possibility that ADD1, which is considered to be the rat homologue for SREBP-1(5, 32) , may actually have a different mode of maturation. To test that, future studies will have to be done in cultured cells with transfected ADD1 precursor.
The footprinting assay indicates that purified ADD1 interacts with two sequences at the HSS promoter. One at nucleotides -165 to -191, which contains the SRE-1 element, and another at nucleotides -149 to -131. This later promoter DNA sequence does not contain an SRE-1 element nor does it contain the E-box motif CANNTG that was used in the oligonucleotide screening procedure for the isolation of the ADD1 protein(5) . Thus, if this sequence is involved in the binding of ADD1 and the regulation of its promoter activity, the essential DNA motif for its binding will have to be elucidated, in future studies, by mutagenesis methodology. However, the results available clearly indicate the binding of the DNA sequence containing the SRE-1 element to ADD1 ( Fig. 8and Fig. 9). As shown, mutation of this sequence in nucleotides that were previously shown to be essential for the binding of the LDL receptor promoter to SREBP-1 (4, 6) also abolished the binding of the HSS SRE-1 sequence to ADD1.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U18994[GenBank].