(Received for publication, June 16, 1994; and in revised form, September 1, 1994 )
From the
Regulation of the low density lipoprotein (LDL) receptor promoter by cholesterol requires a well defined sterol regulatory site and an adjacent binding site for the universal transcription factor Sp1. These elements are located in repeats 2 and 3 of the wild type promoter, respectively. The experiments reported here demonstrate that Sp1 participates in sterol regulation of the LDL receptor in an orientation-specific fashion. We present data which suggest that sterol regulatory element-binding protein (SREBP) increases the binding of Sp1 to the adjacent repeat 3 sequence. We also demonstrate that SREBP and Sp1 synergistically activate expression from the LDL receptor promoter inside the cell by co-transfecting expression vectors encoding each protein into Drosophila tissue culture cells that are devoid of endogenous Sp1. In addition, other transcription factor sites were unable to substitute for Sp1 in sterol regulation when placed next to the SREBP-binding site. These studies together with recent data from others provide the basis of a working model for sterol regulation of the LDL receptor promoter. The presence of Sp1 sites in several other regulated promoters suggests that this universal transcription factor has been recruited to participate in many regulatory responses possibly by a similar mechanism.
Cellular sterol balance is efficiently maintained by a feedback
mechanism. When cellular sterol levels are low genes involved in
cholesterol synthesis and uptake are activated(1, 2) .
When sufficient cholesterol is amassed they are efficiently shut off.
The protein involved in cholesterol uptake is a cell surface receptor
that rapidly internalizes cholesterol-rich low density lipoprotein
(LDL) ()particles from outside the cell. Transcription from
the LDL receptor gene is efficiently regulated by cholesterol, and its
simple promoter is composed of three related sequence elements referred
to as repeats 1-3 located upstream of a TATA box-like
element(3, 4, 5) . The repeats display a
12/16 base pair match with each other.
Repeats 1 and 3 bind the universal transcription factor Sp1. However, repeat 2 harbors a special element that is directly responsible for regulation by sterols, and it does not bind Sp1. The sterol regulatory element (SRE-1) within repeat 2 was identified by mutational studies as a 10-base pair contiguous sequence that binds a family of basic helix-loop-helix zipper proteins called the sterol regulatory element binding proteins or SREBPs(5, 6, 7, 8) . The SRE-1 functions as a conditionally positive element activating expression only when sterol levels are low. It cannot function efficiently by itself even when present in multiple copies ((8) and this study). In the native LDL receptor promoter the adjacent binding site for transcription factor Sp1 located in repeat 3 is also required(4) .
Transcription factor Sp1 was first identified as a trans-acting sequence-specific DNA-binding protein required for expression of the SV40 virus early promoter(9) . Soon thereafter, Sp1 sites were identified in several other viral and cellular promoters. Because of its widespread occurrence Sp1 is probably involved in activation of transcription from a wide variety of promoters(10) . This basic function of Sp1 underscores its fundamental importance in gene transcription. Many of the promoters with Sp1 sites contain other elements that specify regulated expression of the encoded gene. These specific regulatory elements may function by influencing the activity of Sp1 bound at a neighboring site. Since normal sterol regulation requires both a special sterol regulatory element (SRE-1) and an adjacent Sp1-binding site, the LDL receptor promoter provides a system to study how Sp1 is involved in regulated transcription.
The experiments reported here were designed to address the role of the Sp1 site of repeat 3 in sterol regulation. We show that sterol regulation is lost when the sequence of repeat 3 is inverted relative to repeat 2. We also present data which suggest that SREBP binding at repeat 2 stimulates Sp1 binding at repeat 3. Furthermore, when sites for other known transcription factors were substituted in place of repeat 3 normal sterol regulation was not observed. These data provide the basis for a working model of how SREBP and Sp1 function together to modulate cellular sterol levels through regulation of the LDL receptor.
The low affinity Sp1 site from distal signal 1 from the herpes virus thymidine kinase gene (14) (CTAG-CGCCGCCCCGACTGC) and binding sites for transcription factor nuclear factor-1 (15) (CTAG-CTGGCATGCTGCCAAT), the octamer family (16) (CTAG-CCGAATGCAAATCACT), and AP-1 (17) (CTAG-CTTGATGAGTCAGCCG) were inserted next to repeat 2 exactly as described for repeat 3 above. The identity of all of the plasmids used in this study was confirmed by DNA sequence analysis.
pCMV2 -Gal was obtained from K. Normington from the Department
of Molecular Genetics at the University of Texas Southwestern Medical
School and contains the cytomegalovirus early promoter linked to the Escherichia coli
-galactosidase gene. Some of the
oligonucleotides used here were purchased from Operon Technologies, and others were synthesized by Andy Craddock in the core facility
of the Molecular Biology and Biochemistry Department at the University
of California, Irvine.
Drosophila SL2 cells were
obtained from Dr. Al Courey (UCLA) and cultured at 25 °C in Shields
and Sang Drosophila media (Sigma) containing 10%
heat-inactivated fetal bovine serum. They were seeded at 1
10
cells/60-mm dish and transfected by a standard calcium
phosphate co-precipitation method(18) . Plasmid pPACSp1
containing the Sp1 coding sequence under the control of the Drosophila Actin 5C promoter was also obtained from Dr. A.
Courey. The SREBP-1 protein used in this study represents amino acids
1-490 of the full-length protein. The corresponding region of the
mRNA was cloned from HepG2 cell cDNA by polymerase chain reaction using
oligonucleotides designed from the published sequence(6) . We
inserted the corresponding DNA fragment into the pPAC expression
vector.
Figure 1:
Sterol
regulation of LDL receptor promoter-luciferase fusion plasmids. The
diagram at the bottom depicts the relevant features of the plasmids
used in the study. indicates the normal orientation of the
repeat 3 sequence, and
indicates the reverse orientation. A, the indicated plasmids were transfected into CV-1 cells and
analyzed for sterol regulation as described in the text. The data
represent the ratio of luciferase activity (relative light units) for
the indicated test plasmid to the
-galactosidase activity (OD 420
nM/h) expressed from the control cytomegalovirus promoter. The
average of at least five independent experiments for each plasmid
performed in duplicate is presented. I stands for induced and
represents the activity measured from cells cultured in the presence of
lipoprotein depleted serum. S stands for suppressed and
represents the activity measured from cells cultured in the presence of
lipoprotein-depleted serum plus 10 µg/ml of cholesterol and 1
µg/ml of 25-OH cholesterol. The induced level for the plasmid with
repeat 3 in the native orientation was set at 100%, and all values are
plotted relative to this value.
Figure 3: DNase I footprint analyses with both Sp1 and SREBP. Purified Sp1 or SREBP was used in a footprint reaction essentially as described in the legend to Fig. 2A and under ``Materials and Methods.'' The indicated amount of each protein was added alone or together with the DNA probe as indicated, and reactions were processed by the standard footprinting procedure. A, analysis with repeat 3 in the native orientation. B, analysis with repeat 3 in the reverse orientation. All symbols and notations are the same as in Fig. 2.
Figure 2:
DNase footprint analyses of repeat 3
containing plasmids from Fig. 1. A, DNA probes P end-labeled on the bottom strand were prepared from each
plasmid containing repeat 3, and 10 fmol were used in each binding
reaction with the indicated amount of purified Sp1 protein. Two and 10
µl correspond to 6 and 30 ng of protein. DNase I footprinting was
performed as described under ``Materials and Methods.'' A
picture from an autoradiogram of a 7% denaturing polyacrylamide gel is
shown. The lane marked G represents an aliquot of the
probe treated by the guanine-specific cleavage reaction used in Maxam
and Gilbert DNA sequencing(52) . The positions of each copy of
repeat 2 and the location and orientation of repeat 3 are indicated.
The vertical arrowpointing down indicates the wild
type orientation. The horizontal arrow points to a prominent
DNase I hyper-cleavage site observed upon binding of Sp1 to repeat 3 in
the inverse orientation. B, DNA probes
P
end-labeled on the top strand were prepared from each plasmid
containing repeat 3 and 10
10
mol were used
in each binding reaction with the indicated amount of purified Sp1
protein. Other symbols and notations are identical to Fig. 2A. Note that one copy of repeat 2 was
electrophoresed off the bottom of the gel.
Figure 4:
SREBP increases Sp1 binding to repeat 3.
Purified Sp1 and SREBP were used in a standard gel shift analysis as
described under ``Materials and Methods.'' A, 0.5 ng
of the P end-labeled probe containing one copy each of
repeats 2 and 3 was incubated in the absence of any protein (lane
6) with 10 ng of SREBP alone (lane 1), with increasing
amounts of Sp1 alone (lanes 7-10) or with both proteins
together (lanes 2-5) prior to electrophoresis as
indicated at the top of the figure. The proteins present in each
resolved complex are indicated by arrows at the side of the
figure. B, the position of the Sp1 complex was located in the
dried gel by alignment with an autoradiogram, and it was excised, mixed
with aqueous scintillation mixture, and the amount of radioactivity was
determined by liquid scintillation counting. A background of 70
counts/min was obtained from a gel slice from the corresponding
position of the probe only sample (lane 6), and this was
subtracted from all values. C, 0.25 ng of the
P
end-labeled probe containing one copy each of repeats 2 and 3 was
incubated in the absence of any protein (lane 1) with 50 ng of
SREBP alone (lane 6), with increasing amounts of Sp1 alone (lanes 2-5), or with both proteins together (lanes
7-10) prior to electrophoresis and all symbols and notations
are as in Fig. 4A. The conditions for electrophoresis
are described under ``Materials and Methods.'' The small
amount of upper complex in lane 6 containing only SREBP
migrating slightly slower than the SREBP-Sp1 co-complex is due to
protein aggregation that occurs at high
concentrations.
To study the role of the LDL receptor repeat 3 in sterol
regulation, we prepared a series of luciferase fusion plasmids based on
the diagram at the bottom of Fig. 1. A TATA box fragment
containing sequence from -28 to +39 from the promoter for
HMG-CoA synthase was fused to the luciferase coding sequence as
described under ``Materials and Methods.'' We inserted three
copies of the repeat 2 sequence from the human LDL receptor promoter
into this TATA only construct. All three copies were directed in the
same orientation that is present in the wild type promoter. Next, we
inserted a single copy of repeat 3 in both possible orientations. Each
of these plasmids was transfected into CV-1 cells that were
subsequently cultured for 24 h in the presence or absence of regulatory
sterols, and aliquots of total cell lysates were used to measure
luciferase activity. We included an internal control plasmid, pCMV2
-Gal, which encodes
-galactosidase driven by the powerful and
non-cholesterol-regulated human cytomegalovirus early promoter. All of
the values presented here represent a ratio of luciferase to
-Gal
to correct for nonspecific variations in the transfection and assay
procedures. The TATA only containing plasmid displayed very low levels
of activity (Fig. 1A, lanes 7 and 8)
and insertion of the three copies of repeat 2 activated expression only
slightly and it was not induced upon sterol deprivation (lanes 5 and 6). This is consistent with previous experiments and
clearly demonstrates that repeat 2 cannot activate high levels of
sterol-regulated expression on its own even when present in multiple
copies(8) .
When one copy of repeat 3 from the LDL receptor was placed immediately adjacent to the repeat 2 insertion in the same relative orientation as is found in the wild type promoter, expression was enhanced significantly, and it was 4-fold higher in cells cultured in serum depleted of lipoproteins (lanes 1 and 2). This 4-fold induction by sterol deprivation is similar to the magnitude observed with the entire wild type promoter(4, 5) . When the orientation of repeat 3 was reversed the level of activity in the presence of regulatory sterols was similar to the wild type orientation indicating that basal activation afforded by Sp1 at repeat 3 is independent of orientation (compare lanes 2 and 4). However, activation upon removal of serum lipoproteins is abolished (compare lanes 1 and 3). Therefore, only the activation of the promoter upon sterol depletion is affected by the inversion of repeat 3.
The above observations indicate that Sp1 has
a dual role in transcription of the LDL receptor promoter. First, it
provides a basal level of activation that is independent of
orientation. Second, it has a role in sterol regulation which is
critically dependent on the orientation of the recognition site. This
suggests that Sp1 binds to repeat 3 in an asymmetric fashion. To
evaluate this more directly, we analyzed the binding of purified Sp1 to
the plasmids discussed above. We prepared footprinting probes labeled
with P on the bottom DNA strand from the plasmid with
repeat 3 in the native (Fig. 2A, lanes
1-5) or inverted orientation (lanes 6-10). An
aliquot of each probe was incubated with purified Sp1 followed by a
standard DNase I footprinting experiment. Fig. 2A shows
that Sp1 bound to repeat 3 in both plasmids; however, there was an
important distinction. The footprint with repeat 3 in the inverted
orientation resulted in a DNase I hyper-cleavage site between repeats 2
and 3 (compare lanes 3 and 4 with lanes 8 and 9 and note the horizontal arrow). This is
outside of the inverted repeat 3 sequence and is, therefore, not due to
a DNA strand-specific difference. The reason for this distinction is
the directional manner in which Sp1 binds to its site and could be due
either to a specific alteration in the DNA structure or to a more
favorable microenvironment for cleavage by DNase I that is induced by
Sp1.
If this hypersensitive cleavage site truly was a consequence of
the asymmetric interaction of Sp1 with its recognition site then an
identical hyper-cleavage site should be induced on the complementary
DNA strand in the other plasmid on the opposite side of the repeat 3
insertion. Therefore, we prepared probes P labeled on the
opposite DNA strand and repeated the DNase I footprinting reactions.
The results displayed in Fig. 2B clearly show that an
identical hyper-cleavage site is generated (note the horizontal
arrow in Fig. 2B and compare lanes 2 and 3 with lanes 6 and 7).
To study potential SREBP-Sp1 interactions at the LDL receptor promoter, DNA probes from plasmids containing repeat 3 in both orientations were used for DNase I footprinting analyses with both proteins (Fig. 3, A and B). SREBP-1 bound to all three copies of the repeat 2 sequence as expected when added alone, and a strong hyper-cleavage site was induced at the 5` boundary of each binding site (lane 8 of both A and B). Titrations of Sp1 were performed alone (lanes 2-4) and in the presence of a saturating amount of recombinant SREBP (lanes 5-7). The footprint regions for each separate protein directly abut each other and may partially overlap (compare lane 4 with lane 8). When both proteins are added together, Sp1 bound to repeat 3 made the adjacent site protected by SREBP more accessible to DNase I. SREBP is partially displaced to allow access to the DNase I, but it is still bound since the hyper-cleavage site at the 5` side is still visible. This effect of Sp1 on SREBP was independent of the orientation of repeat 3 (compare lanes 5 and 8 of both A and B). Since the two binding sites are immediately adjacent to one another and their footprint regions may slightly overlap, this result is probably due to steric interference by Sp1. The close proximity suggests that direct communication by the DNA-bound proteins may be essential to the synergistic activation of transcription observed when cellular sterol levels fall.
From the experiments presented above there appeared to
be an interaction between the DNA bound Sp1 and SREBP. Additionally, in
some of our footprint experiments SREBP seemed to increase the binding
of Sp1 to repeat 3. ()To study this further, we performed
gel shift experiments with a DNA probe containing one copy each of
repeats 2 and 3 (Fig. 4A). When either SREBP (lane
1) or Sp1 (lanes 7-10) were added alone each bound
to the probe and produced a stable gel-shifted complex. The SREBP
complex migrated slightly slower than the Sp1 complex through the gel.
When a constant level of SREBP was incubated with Sp1 in increasing
amounts, the Sp1 binding activity was significantly enhanced (lanes
2-5). This effect was quantified by cutting the complexes
from the gel and determining the amount of radioactive DNA present by
liquid scintillation spectrometry (Fig. 4B). From the
picture of the gel (Fig. 4A) and the graph (Fig. 4B), it is clear that Sp1 binding is
significantly enhanced by SREBP. This effect requires that the
SREBP-binding site and repeat 3 are on the same DNA molecule.
The enhanced binding of Sp1 resulted in a dramatic increase in
the Sp1 only protein
DNA complex, and little of the complex
containing both proteins was observed. The dual complex does appear at
higher levels of Sp1, but the amount of the complex varied slightly
between experiments and it was not significantly increased when the
concentration of Sp1 was increased over a range where the amount of the
Sp1 only complex was greatly stimulated (lanes 3-5).
This suggests that the SREBP-DNA interaction was destabilized by Sp1,
and the association is unstable to the gel electrophoresis conditions.
The data of Fig. 3which shows that Sp1 partially displaced the
adjacently bound SREBP in the footprint reaction supports this notion.
This would be similar to an observation concerning the HTLV Tax
protein. Tax has been reported to stimulate DNA binding of a
co-regulatory protein without the formation of a stable ternary complex
under standard gel-shift conditions. However, alteration of the
conditions for native gel electrophoresis made it possible to detect
substantial amounts of the stable ternary complex (22) .
Therefore, we sought to validate our interpretation of the experiment of Fig. 4A by altering conditions of the gel-shift experiments to stabilize the ternary complex between Sp1, SREBP, and the DNA. By increasing the concentration of SREBP 5-fold and altering the gel-shift conditions (see ``Materials and Methods''), we were able to detect substantial amounts of the stimulated ternary complex (Fig. 4C). Once again, the SREBP significantly enhanced Sp1 binding (compare lanes 2-5 with lanes 6-10).
All of the experiments to date (including those reported here) document that repeat 3 is essential to sterol regulation. Since Sp1 binds to this site in vitro it was assumed that Sp1 was the regulatory protein inside the cell. However, repeat 3 is not a classic GC box containing Sp1 site and considering that several other proteins have been shown to bind to Sp1-like sites in vitro(23, 24, 25) , it was not clear if Sp1 was the protein involved in synergistic activation of the LDL receptor promoter along with SREBP inside the cell. To evaluate this we performed co-transfection studies in Drosophila SL2 cells. This line has been used previously to analyze Sp1 function since the cells are devoid of endogenous Sp1 yet retain the necessary accessory proteins required to support an Sp1 activation response(18) . The SREBP-1 coding sequence from amino acid 1-490, which contains all the information required for transcriptional activation(6, 26) , was inserted into the Drosophila pPAC expression vector (18) and transfected into SL2 cells in the presence and absence of pPAC Sp1 and various luciferase reporter plasmids (Fig. 5). We used the plasmid from Fig. 1containing three copies of repeat 2 linked to repeat 3 in the native orientation (lanes 1-4), the wild type LDL receptor promoter (lanes 5-8), and as a control we used a plasmid containing the SV40 early promoter which contains six Sp1 sites and no high affinity binding sites for SREBP (lanes 9-12). The SV40 promoter did not respond to SREBP co-transfection alone (lane 11) but was dramatically activated by Sp1 (lane 10). The addition of SREBP along with Sp1 resulted in less than a 2-fold further increase from the SV40 promoter (compare lanes 10 and 12). The wild type LDL receptor and the synthetic repeat 2-repeat 3 promoters are weakly activated by SREBP or Sp1 alone (lanes 2, 3, 6, and 7), and co-transfection with both Sp1 and SREBP resulted in synergistic activation (lanes 4 and 8). These results strongly support the model that SREBP and Sp1 work in concert to activate the LDL receptor promoter.
Figure 5: Synergistic activation by SREBP and Sp1 in Drosophila SL2 cells. Drosophila SL2 cells were transfected by a standard calcium phosphate co-precipitation protocol. Three luciferase reporter plasmids were used. They correspond to the synthetic promoter containing three copies of repeat 2 linked to repeat 3 in the native orientation (see Fig. 1, lanes 1-4), the wild type human LDL receptor promoter (lanes 5-8), and the SV40 early promoter containing plasmid called pGL2-promoter(9, 10, 11, 12) . 2 µg of each reporter was transfected alone or with 25 ng of each expression vector singly or in combination as indicated at the bottom of the figure. The data are from a typical experiment, and each one has been repeated at least three times with nearly identical results. The data are presented as Fold Activation where the value of luciferase activity normalized to total cell protein for the reporter alone is set at 1.0. The ratio of luciferase relative light units/microgram of total cell protein for the samples with co-transfection of both SREBP and Sp1 were as follows: lane 4, 61,447; lane 8, 52,565; lane 12, 403,409.
The data presented so far indicate that the orientation of the Sp1 site in the repeat 3 element is crucial for normal regulation of the LDL receptor promoter and that SREBP increases the binding of Sp1 to the repeat 3 site. Since Sp1 is a universal transcription factor that activates expression of several other promoters, we next asked whether other transcription factor-binding sites could substitute for Sp1. A series of reporter plasmids containing binding sites for other ubiquitous transcription factors in place of the repeat 3 Sp1 site were made and analyzed as described above (Fig. 6). When a low affinity Sp1 site from the herpes virus type 1 thymidine kinase gene (TK) was inserted in place of repeat 3 a low level of expression was observed in the presence of regulatory sterols that was induced upon sterol depletion (compare lanes 3 and 4). This would be expected if the resulting transcriptional activity is dependent on the affinity of Sp1 for its site. Binding sites for either nuclear factor 1 or the octamer family of regulators did not functionally replace repeat 3 (lanes 5, 6, 9, and 10); however, insertion of a binding site for AP-1 did partially restore regulation (lanes 7 and 8). This is potentially significant since there is an AP-1-binding site adjacent to an SREBP-binding site in the sterol-regulated HMG-CoA synthase promoter (see ``Discussion''). Since none of these other sites fully substitute for repeat 3 it is likely that Sp1 is specifically required for normal sterol regulation of the LDL receptor promoter.
Figure 6: Other transcription factor sites cannot substitute for repeat 3 for regulation by sterols. The binding site for a low affinity Sp1 site from the herpes virus thymidine kinase gene (distal element 1) or consensus DNA-binding sites for the indicated transcription factors were placed next to the three copies of repeat 2 exactly as described for repeat 3. The sequence for each site is presented under ``Materials and Methods.'' These plasmids were transfected into CV-1 cells and analyzed as described under ``Materials and Methods'' and the legend to Fig. 1.
The sterol regulatory site located in repeat 2 of the LDL
receptor promoter cannot activate expression on its own ( Fig. 1and (8) ), and we have shown here that the
trans-acting protein which binds to this element, SREBP, is a weak
activator by itself (Fig. 5). The Sp1 site contained in repeat 3
can drive basal expression, but it is not subject to sterol
regulation(7, 8) . In the native LDL receptor
promoter, repeats 2 and 3 are located next to each other, and efficient
sterol regulation requires both of
them(3, 4, 7, 8) . The simple
activation afforded by Sp1 under sterol suppressing conditions was
10-fold higher than the minimal activity observed from the
plasmids containing only the TATA box or three copies of the LDL repeat
2 in addition to the TATA box (Fig. 1). This basal level of
expression afforded by Sp1 was not orientation dependent (Fig. 1, lanes 2 and 4). The significant
difference between the two plasmids containing three copies of repeat 2
plus a single copy of repeat 3 in each possible orientation was evident
only when the cells were cultured in medium that had been depleted of
serum lipoproteins. The plasmid containing repeat 3 in the native
orientation was efficiently induced; however, when repeat 3 was
inverted induction was abolished. These experiments demonstrate that
Sp1 has a dual role in the LDL receptor promoter. It provides a basal
activation required for a low suppressed level of promoter activity,
and it also participates in sterol regulation of the LDL receptor in an
orientation-specific fashion. Its role in sterol regulation requires
that it communicate with SREBP bound to the adjacent repeat 2 sequence.
The data presented here suggest that SREBP stimulates the activity of Sp1 bound at repeat 3 by direct interaction on the DNA. A directional model for Sp1 binding to its recognition site has been proposed based on extensive chemical footprinting, analysis of both DNA and protein mutagenesis studies, and comparisons between the three Sp1 DNA-binding zinc fingers and the DNA-binding domain of a related protein whose crystal structure has been solved when bound with DNA(18, 27, 28) . This model predicts that the long amino-terminal portion of Sp1 would be oriented toward repeat 2 when the repeat 3 Sp1 site is in the wild type orientation(27, 28) . Additionally, the SRE-1 has been proposed to be a direct repeat element that interacts in a directional fashion with an SREBP(5, 6, 29) . The simultaneous binding of both SREBP and Sp1 would align specific domains from each protein to facilitate productive protein-protein interactions. It is potentially interesting that Sp1 contains glutamine-rich regions in its amino-terminal domain which are involved in multimerization of Sp1 protomers and synergistic activation (30, 31) . In addition, one of the recently characterized SREBPs also contains a glutamine-rich domain(32) . It is possible that Sp1 and an SREBP interact through these domains.
The footprinting experiment of Fig. 3suggests that Sp1 and SREBP communicate with each other
when simultaneously bound to DNA. Sp1 alters the SREBP binding such
that the protected region is more accessible to DNase cleavage. When
SREBP was prebound to the DNA the subsequent addition of Sp1 resulted
in the same partial displacement observed when both proteins were added
simultaneously. This further supports the idea that Sp1 can
bind and displace SREBP. The gel shift data of Fig. 4indicates
that SREBP facilitates the binding of Sp1 to the adjacent repeat 3
site. At low concentrations of SREBP, the stimulated complex
co-migrates with the complex containing only Sp1. Therefore, it is
likely that after Sp1 binds, the SREBP-DNA interaction is not stable,
and it falls off the DNA during electrophoresis. This interpretation is
supported by the partial displacement of SREBP by Sp1 observed in the
footprint experiment of Fig. 3. In addition, other reports have
documented that predicted ternary complexes appeared not to be stable
to native electrophoresis (22, 33, 34) . By
altering the gel-shift conditions, one group was able to stabilize the
complex containing both proteins(22) . Therefore, we altered
our standard conditions and increased the ratio of SREBP/Sp1 in the
binding reaction (see ``Materials and Methods''). With these
modifications we did detect substantial amounts of a stimulated complex
containing both proteins (Fig. 4C).
Taken together,
the data presented here coupled with recent data from the Brown and
Goldstein group (26) suggest a model for how SREBP and Sp1
function together to provide tight regulated expression of the LDL
receptor. When cellular sterol levels fall, SREBP is processed from its
120-kDa membrane form to a soluble 65 kDa mature form which is
translocated to the nucleus. The nuclear SREBP then binds to repeat 2
and increases the binding of Sp1 to the adjacent repeat 3 sequence.
After Sp1 binds to its site, transcription is stimulated by DNA-bound
Sp1 and SREBP. With Sp1 bound adjacently, the SREBP-DNA interaction
would be relatively unstable. However, under conditions of chronic low
cellular sterol levels SREBP would accumulate in the nucleus and a
sustained high level of LDL receptor expression would be sustained.
When cellular sterol levels rise again due to high LDL receptor
activity and lipoprotein-cholesterol uptake, the flux of SREBP into the
nucleus would be curtailed. This would result in dissociation of
DNA-bound SREBP at the LDL receptor promoter followed by a rapid
decline in its nuclear concentration due to degradation of the free
SREBP. The net result would be a significant decline in LDL receptor
mRNA synthesis. This model is consistent with recent data that SREBP is
synthesized constitutively and is translocated to the nucleus following
sterol regulated proteolysis from its extranuclear membrane location (26) . In further support of this model, the same investigators
reported that nuclear SREBP-1 is rapidly degraded by a calpain-like
protease (26) . This mechanism would at least partially explain
the synergistic activation of transcription afforded by both SREBP and
Sp1 together. In addition, this quickly reversible response (in the
absence of a continual influx of SREBP into the nucleus) would provide
a clever mechanism for the cell to quickly alter LDL receptor
expression in response to subtle changes in cellular sterol levels. A
rapid and inherently transient response is essential to an effective
metabolic regulatory mechanism.
The co-transfection studies in SL2 cells (Fig. 5) directly demonstrate that SREBP is a weak transcriptional activator by itself, and it cooperates with Sp1 to achieve high levels of expression from the LDL receptor promoter inside the cell. Since other proteins bind to GC-rich Sp1-like sites in vitro(23, 24, 25, 35) it was important to show that Sp1 and SREBP could specifically work together in vivo. It is interesting to note that the wild type promoter was more dramatically activated than our synthetic construct. This is likely due to the additional Sp1-binding site located in repeat 1 which is required for efficient expression of the native LDL receptor promoter(4) .
We do not believe that enhanced binding of Sp1 by SREBP can explain the synergistic activation alone. When bound together, SREBP and Sp1 are likely to interact more efficiently with the basic transcriptional apparatus than a single Sp1 or SREBP site alone. The amino-terminal portion of SREBP is acidic in character and has been recently identified as a potential transactivation domain(36) . Acidic transactivation domains have been identified in several other transcriptional regulatory proteins(37, 38) . Interestingly, Sp1 belongs to a separate class of regulatory proteins that contain activation domains rich in glutamine residues(18, 38) . It is likely that each class of activator interacts with distinct co-activator molecules to facilitate transcription(39) . The concerted action of both Sp1 and SREBP may result in synergistic activation by coupling two different co-activator pathways together(31, 39, 40) .
Other transcription
factors did not substitute for the Sp1 site in sterol regulation.
However, as shown in Fig. 6, the insertion of an AP-1 site
resulted in partial activation when cells were cultured in the absence
of serum sterols. This is potentially significant because there is an
AP-1 site adjacent to an SREBP-binding site in the sterol-regulated
HMG-CoA synthase gene promoter (13) . ()Interestingly, c-Jun which is one of the proteins that
constitutes the heterodimeric AP-1 factor also contains a
glutamine-rich activation domain (41) that could interact with
adjacently bound SREBP. It is possible that AP-1 participates in sterol
regulation of HMG-CoA synthase similar to Sp1 in the LDL receptor
promoter.
It is noteworthy that in previous studies the sequence encompassing repeats 2 and 3 was shown to function efficiently for sterol regulation in either orientation. In every case the relative orientation of the two repeats was not altered(3, 5, 7) . This strengthens the argument that the two repeats function together in a modular fashion to activate transcription when cellular sterol levels are low.
The involvement of two adjacent direct repeat elements in a regulatory response is reminiscent of studies on the nuclear receptor family of transcriptional activators. The relative orientation and spacing of the recognition half-sites determines the composition of receptor homo- or heterodimer and the biological response mediated by each site. Changing either the orientation or spacing between each repeat can dramatically alter the binding selectivity and ultimate regulatory response (42, 43, 44) .
The current studies have
identified an essential role for Sp1 in regulation of intracellular
cholesterol metabolism. Recent studies have shown that Sp1 is involved
in synergistic transcriptional activation from the HIV promoter along
with NFB in response to phorbol esters and cytokines(45) .
As in the LDL receptor this interaction is orientation dependent and
Sp1 could not be substituted for by other transcription factor sites.
The HIV response to phorbol esters and cytokines should be quick and
reversible, similar to LDL receptor regulation by cellular sterols.
Interestingly, these authors show that the DNA-binding p65 subunit of
NF
B does not form a stable gel shift complex along with Sp1.
Indeed, from their data ( Fig. 2of (45) ) it appears
that formation of the DNA
protein complex containing only p65 is
stimulated by Sp1.
It is also interesting to note that both SREBP
and NFB are maintained in a latent extranuclear form before they
are activated to enter the nucleus to stimulate transcription. When
cellular sterol levels fall, SREBP is released from its location in the
endoplasmic reticulum membrane by proteolysis, and the amino-terminal
fragment enters the nucleus and activates LDL receptor
expression(26) . NF
B is sequestered in the cytosol by an
inhibitory protein, IkB, that prevents it from entering the nucleus.
Upon activation, NF
B is released and enters the nucleus to
activate the appropriate target genes(46, 47) .
One
other recent report (48) has demonstrated that Sp1 acts
together with an adjacent C/EBP site to activate expression of a
rat cytochrome P-450 gene during liver development. This response would
be predicted to result in stable activation since it defines a
developmental response, and in this case the authors were able to
demonstrate a stable gel-shifted complex containing both proteins.
The available data indicate that Sp1 can participate in regulation by diverse biological signals in subtly different ways. The presence of Sp1 sites in several other promoters regulated by distinct cellular signals suggests that Sp1 may be involved in regulated expression of many other promoters(10, 38, 49, 50, 51) .