(Received for publication, July 13, 1995)
From the
We have defined a 105-base pair tissue-restricted promoter for the cholesteryl ester transfer protein (CETP) gene that contains a nuclear hormone receptor response element essential for transcriptional activity. DNaseI protection and electrophoretic mobility shift assays showed specific binding of nuclear extracts from HepG2 (hepatic) and Caco-2 (intestinal) cells (expressing cell types) to 3 sites (designated A (-26 to -57), B (-59 to -87), and C (-93 to -118)) within the 105-base pair minimal promoter element between -138 and -33. Mutagenesis studies indicated that the function of the promoter was dependent upon synergistic interactions between transcription factors bound to these sites. Mutation of site C reduced transcription by 50 and 80%, respectively, in HepG2 and Caco-2 cells, and electrophoretic mobility shift assays showed that nuclear hormone receptors, including ARP-1 and its homologue Ear-3/COUP-TF, were occupants of site C in both of these cell types. Overexpression of ARP-1 or Ear-3/COUP-TF with CETP promoter/chloramphenicol acetyltransferase gene reporter plasmids repressed transcriptional activity of the CETP promoter containing sequences up to -300, but activated transcription in the context of larger constructs containing sequences up to -636. Thus ARP-1 may assume a dichotomous role as both a transcriptional repressor and a transcriptional activator dependent on the promoter context. In addition, the architecture of the CETP gene promoter suggests that its expression is under the control of multiple transcriptional signaling pathways mediated by inducible transcription factors as well as nuclear hormone receptors.
Epidemiologic studies have shown that plasma high density
lipoprotein (HDL) ()cholesterol levels are an independent
risk factor for coronary heart disease(1) . One hypothesis that
explains the protective effect of HDL is its role in reverse
cholesterol transport(2) , the net transport of cholesterol
from peripheral tissues to the liver for excretion. The reverse
cholesterol transport process requires CETP, a 74-kDa protein that
mediates the heteroexchange of cholesteryl ester from HDL for
triglyceride in LDL and very low density lipoprotein(3) . HDL
cholesteryl ester, which is derived from free cholesterol taken up from
the peripheral tissues, thus can be directed to the liver (via LDL and
its receptor) for removal from the body. Low plasma CETP activity
appears to be protective against coronary heart disease(4) , as
exemplified in homozygous CETP deficiency, which results in reduced
LDL, increased HDL, absence of coronary heart disease, and longevity (5) . In primates, LDL cholesterol levels and the development
of coronary atherosclerosis is strongly correlated with plasma CETP
activity(6) , while in populations, CETP plasma activity is
inversely correlated with plasma HDL levels, which in turn are strongly
and inversely associated with the development of coronary heart disease (7) .
In humans, CETP mRNA is expressed predominantly in the liver, spleen, and adipose tissue with lower levels of RNA seen in small intestine, adrenal, kidneys, and heart(8, 9) . Macrophages and smooth muscle cells also possess CETP mRNA and activity as do a variety of transformed cells lines, for example HepG2 (hepatic) and Caco-2 (intestinal) cells. CETP gene expression and CETP activity in plasma is affected by hormones(10) , drugs(11) , and diet(6, 12, 13) . However, little is known about the specific regulatory pathways that affect transcription of the CETP gene directly. The data from transgenic mice bearing the human CETP gene strongly suggest that its expression is largely if not exclusively under the control of promoter sequences contained within 3.2 kilobases of its 5`-flanking region(14) . Only one study to date has examined transcriptional regulation of the CETP gene in liver cells(15) . A DNA region upstream of the CETP gene was shown to bind CCAAT/enhancer-binding protein, and CETP gene transcription in liver cells was activated by CCAAT/enhancer-binding protein overexpression.
In this report, we define a tissue-restricted promoter for CETP gene expression that contains three DNA-protein binding regions including a nuclear hormone receptor response element (NHRRE) required for transcription. Nuclear hormone receptors and their ligands have been implicated in a multitude of processes governing cellular metabolism, development, and differentiation (16, 17) and exist in the regulatory regions of several genes involved in the synthesis and metabolism of lipoproteins, specifically the apolipoprotein AI, AII, CIII, and B genes(18, 19, 20) . Thus these factors may be capable of both coordinate regulation of certain genes involved in lipid metabolism as well as a diversity of transcriptional responses. The ``orphan'' nuclear receptors (so-called because no ligand for them has been identified) ARP-1 and Ear-3/COUP-TF bind and repress transcriptional activity of the CETP minimal promoter, but activate transcription in the context of larger constructs. Furthermore, the binding of these and other nuclear hormone receptors to the CETP NHRRE raise the intriguing possibility that as of yet undiscovered signal transduction pathways may be involved in the expression of this gene.
Figure 1:
Transcriptional
regulation of CETP promoter/CAT fusion constructs in expressing and
non-expressing cell types. A restriction map of the
-4700/+27 CETP promoter fragment is shown at the top
left. The indicated plasmid constructs were transfected into
HepG2, Caco-2, COS-1, Sol8, HeLa, and NIH 3T3 cells. The right
panel represents the CAT activity of each CETP promoter/CAT
construct in various cell types expressed as an average of at least 5 separate experiments. The CAT enzymatic activity is
expressed as the percent conversion of
[C]chloramphenicol to its acetylated form and is
relative to the activity of the -33CETP.CAT construct (activity
= 1). X, XbaI; P, PstI; Ac, AccI; H, HindIII; Av, AvaI. 5`-Nucleotide positions are indicated relative to the
transcriptional start site, +1.
Figure 2: DNaseI footprinting analysis of the proximal 5` promoter region of the CETP gene. Autoradiogram showing DNaseI protection patterns of the -138 to -5 CETP gene promoter (upper strand) in the absence of nuclear extracts (-NE) and presence of HepG2 or HeLa nuclear extracts. Protected regions are indicated by boxes along the autoradiogram, and nucleotide positions relative to the transcriptional start site (+1) for each site are shown. Nucleotide sequence of the -138 to -5 region is presented below. A protection pattern similar to HepG2 NE was obtained using Caco-2 nuclear extracts (data not shown).
To characterize DNA-protein
interactions in the CETP gene promoter, synthetic oligonucleotides
corresponding to sites A, B, and C or oligos containing random
mutations in these sites (Amut, Bmut, Cmut) were used in
electrophoretic mobility shift assays (EMSA, see ``Experimental
Procedures'') as indicated in Fig. 3and 4. Incubation of
labeled oligoA with HepG2 (Fig. 3) or Caco-2 (data not shown)
nuclear extracts resulted in the formation of two bands (1 and
2), whereas only the slower migrating band (
1) was formed
with HeLa nuclear extracts (Fig. 3). Both bands were
specifically competed by an excess of unlabeled oligoA but not by an
unrelated oligo, demonstrating sequence specificity. Site A contains a
consensus binding site for Sp1; therefore an oligo containing an Sp1
binding site (29) was used as a competitor in these
experiments. Complex
1 was specifically competed by an excess of
the Sp1 oligo and supershifted by an anti-Sp1 antibody without
affecting complex
2. Thus the ubiquitous factor Sp1 binds to site
A in all cell types studied and is not present in complex
2.
Figure 3:
Binding of Sp1 and other HepG2 nuclear
proteins to CETP site A. Autoradiogram of EMSA showing binding of HeLa
and HepG2 nuclear extracts to end-labeled wild-type CETP site A oligo (lanes 1 and 2), in the presence of 100-fold molar
excess of unlabeled site A (lane 3), 100-fold molar excess of
an unlabeled unrelated oligo (lane 4), or 100-fold molar
excess of an unlabeled Sp1 consensus oligo (lane 5). HepG2
nuclear extract and radiolabeled oligo A were also incubated in the
presence of preimmune sera (lane 6), of an anti-Sp1 antibody (lane 7), or in the presence of an anti-HNF4 antibody (lane 8). Lanes 9, 10, and 11 show
EMSA using HepG2 NE and labeled oligo A mutant 1 (lane 9),
oligo A mutant 2 (lane 10), and oligo A mutant 3 (lane
11), respectively. The sequence of the probes used in these assays
are shown below the autoradiogram. The sequence GGGCGG (boldface) represents the consensus binding sequence for Sp1; underlined bases represent introduced mutations. All
oligonucleotides were engineered with 5`-GATC (BamHI
compatible) overhangs for cloning purposes. 1 indicates the slower
migrating and
2 indicates the faster migrating complex. The arrow shows the supershift complex in lane 7. Results
similar to HepG2 nuclear extract were obtained using Caco-2 nuclear
extract. S, specific; NS, nonspecific; ab,
antibody; PI, preimmune sera.
In
cells that express the CETP gene, site A maybe occupied by protein(s)
in addition to Sp1. To determine the binding requirements for these
proteins, a series of point mutations were introduced into site A and
the resulting oligos were used as probes (Fig. 3). The results
showed that the presence of an intact Sp1 site is sufficient for the
formation of complex 1, while the presence of intact sequences
within and upstream of the Sp1 site are necessary for complex
2
formation. Neither oligo Amut1, which contained mutations within and
upstream of the Sp1 site, nor oligo Amut2, which contained a mutation
in the Sp1 site only, formed a complex. However, with oligo Amut3,
containing mutations upstream of the Sp1 site, an intense band was
formed with the mobility of complex
1 that contained Sp1. Thus in vivo, the occupation of this site may be determined by
competition of Sp1 with another factor(s) that requires the Sp1 site
for binding.
Complexes formed between oligos corresponding to sites B and C and nuclear extracts from HepG2 and Caco-2 cells were also specifically competed with an excess of unlabeled oligo B and C, respectively (Fig. 4); only one specific complex was observed in each case. These complexes were not competed by 100-fold molar excess of oligos Bmut and Cmut respectively; furthermore, neither oligo Bmut nor Cmut formed complexes when used as probes (data not shown). No complex was seen when HeLa nuclear extracts were incubated with a probe to site B, and a weak but specific band was observed when HeLa nuclear extracts were incubated with a probe to site C. Thus the data from EMSA are consistent with DNaseI analysis showing tissue-specific binding activities for sites A, B, and C.
Figure 4: Specific binding of HepG2 nuclear proteins to CETP sites B and C. Autoradiograms of EMSA showing binding of HepG2 or HeLa nuclear extracts to CETP site B (left panel) and site C (right panel). Complexes formed with HepG2 nuclear extracts and sites B and C were competed with 100-fold molar excess of oligo B or C, respectively (second lane of each gel), but not with 100-fold molar excess of an unlabeled unrelated oligo (third lane of each gel) or an excess of an unlabeled mutant oligo of site B (fourth lane, left panel) or a mutant oligo of site C (fourth lane, right panel). HeLa nuclear extract did not form any complex with site B (last lane, left panel) but produced weak binding with site C (last lane, right panel). The arrows represent the location of the specific complexes. The sequence of the oligonucleotides used are as follows (upper strand only): B, 5`-GATCAGGAAGACCCTGCTGCCCGGAAGAGCCTC-3`; Bmut, 5`-GATCAGGAAGACCCTGCTGCCCGTATGAGCCTC-3`; C, 5`-GATCGCTGGGCAGGAAGGAGGTGAATCTCTGGGGCCAGGAAGACC-3`; Cmut, 5`-GATCGCTGGGCAGGAAGGATGTGAATCTCTGGGGCCAGGAAGACC-3`. The oligonucleotides in boldface italic in the sequence of site Bmut and site Cmut indicate oligonucleotide substitutions. The oligonucleotides underlined indicate the homology to NHR consensus binding sequence. All oligos contained an additional 5`-GATC overhang used for cloning purposes. Similar results were obtained using Caco-2 nuclear extract (data not shown). S, specific; NS, nonspecific; mut, mutant oligonucleotide.
To examine the functional relevance of sites A, B, and C in CETP gene transcription, mutations corresponding to Amut, Bmut, and Cmut were tested in transcriptional activation assays (Fig. 5). Mutations of site A (Amut1, Amut2, or Amut3) in -138CETP.CAT all resulted in a 60% decrease of the transcriptional activity compared with the wild-type -138CETP.CAT. Interestingly, transcriptional activity of the construct containing Amut3, which retains Sp1 binding, was decreased, suggesting that the binding of Sp1 to site A is not sufficient for maximal transcription. Mutated sites B and C in the context of -138CETP.CAT resulted both in a 60% decrease of the CAT activity, respectively. Furthermore, transfection of constructs containing the individual sites A, B, or C ligated to the TATA box resulted in 10% of the activity of -138CETP.CAT (data not shown). To determine whether sites A, B, and C were required for maximal transcriptional activity in other expressing cell lines, we transfected CETP promoter/CAT constructs in COS-1 cells. The data showed that mutation of sites A or B reduced transcriptional activity by 65%, but mutation of site C reduced transcription by only 30%. Thus it appeared that occupation of site C was less critical for CETP gene expression in COS-1 than in Caco-2 and HepG2 cells. Furthermore, in EMSA with COS-1 nuclear extracts, site C formed a specific complex of differing mobility than that with HepG2 or Caco-2 nuclear (data not shown). Thus binding of tissue-specific proteins to sites A, B, and C is required for maximal transcriptional activity of the CETP gene promoter in liver and intestine, and this activity is the result of synergistic interactions of proteins bound to these sites.
Figure 5: Transcriptional activity of mutated CETP promoter/CAT reporter constructs. Wild-type and mutant constructs containing the CETP promoter region between -138 and +27 fused to the CAT gene were transfected into HepG2 and Caco-2 cells. The wild-type construct is indicated at the top left; all other constructs contain the mutant version of site A, B, or C as indicated. The sequence of these mutant sites are described in Fig. 3and 4. CAT activity is reported as described in the legend to Fig. 1.
Figure 6: ARP-1 binding on CETP site C in liver cells. Left panel, autoradiogram of EMSA using radiolabeled oligo C incubated with HepG2 nuclear extract in the absence of competitor (lane 1), in the presence of 100-fold molar excess of unlabeled oligo C (lane 2), of 100-fold molar excess of unlabeled apoAI site A (lane 3), of 100-fold molar excess of an unlabeled unrelated oligo (lane 4), of an antibody specific for ARP-1 (lane 5), or in the presence of preimmune sera (lane 6). Right panel, EMSA of oligo C incubated with whole cell extracts from COS-1 cells transfected with pMT2 (lane 1) or pMT2-ARP-1 (lanes 2-6) in the presence of 100-fold molar excess of unlabeled oligo C (lane 3), of 100-fold molar excess of and unlabeled unrelated oligo (lane 4), of an anti-ARP-1 antibody (lane 5), or in the presence of preimmune sera (lane 6). The arrows indicate the specific complexes. Results similar to HepG2 nuclear extracts were obtained using Caco-2 nuclear extract (data not shown). S, specific; NS, nonspecific; ab, antibody; PI, preimmune sera.
Figure 7:
Binding of nuclear hormone receptors to
CETP site C. Recombinant nuclear hormone receptors were overexpressed
in COS-1 cells and whole cell extract were prepared and incubated with
labeled site C for EMSA. CETP site C was incubated with
pMT2-Ear-3/COS-1 (lane 1), pMT2-ARP-1/COS-1 (lane 2),
pMT2-RXR/COS-1 (lane 3), pMT2-TR/COS-1 (lane 4),
pMT2-RXR
/pMT2-TR/COS-1 (lane 5), pMT2-HNF4/COS-1 (lane 6), and pMT2/COS-1 (lane 7). About 1 µl of
these extracts were typically used (average concentration of extracts:
3-8 mg of protein/ml) in a final reaction volume of 20 µl
(see ``Experimental Procedures''). All complexes formed with
site C were found to be specific (data not
shown).
Figure 8: Effects of overexpression of ARP-1 and Ear-3/COUP-TF on CETP gene transcription in liver cells. Wild-type and mutant CETP constructs as well as a construct containing two copies of the NHRRE from the apoAI gene were transfected with 5 µg of pMT2 and ARP-1 or Ear-3/COUP-TF expression vector (pMT2-ARP-1 or pMT2-Ear-3) as indicated. The CAT activities are relative to the activity of the -33CETP.CAT construct without ARP-1. The CAT activity was measured as described in the legend to Fig. 1. Similar results were obtained in Caco-2 cells (data not shown).
To determine whether the effects of ARP-1 and Ear-3/COUP-TF were mediated by site C, a construct containing a mutation of site C, which prevents ARP-1 binding, -138CmutCETP.CAT, was used (for sequence of Cmut see the legend to Fig. 4). The basal expression of this construct was reduced significantly in both cell types, and the effects of ARP-1 and Ear-3/COUP-TF were completely eliminated. These results show that site C is necessary for maximal CETP gene expression and that site C, in the context of the minimal CETP promoter, is necessary for repression by ARP-1 and Ear-3/COUP-TF.
It has been previously shown that proper tissue-specific expression of the human CETP transgene in a mouse required not more that 3.2 kilobases of its 5`-flanking sequences(14) . In this report, we define a highly active, tissue-restricted promoter of the CETP gene that resides immediately upstream of its transcriptional start site. The promoter is modular and is composed of three protein binding activities that function synergistically to produce full transcriptional activity and tissue specificity of expression. One of these binding regions is recognized by several members of the nuclear hormone receptor family of transcription factors, raising the possibility that the transcription of the CETP gene is under the control of hormonal signal transduction pathways. The orphan receptors ARP-1 and/or Ear-3/COUP-TF are occupants of the CETP promoter in vivo and repressed the activity of the minimal CETP promoter in vitro but activated transcription in the context of larger promoter segments. Thus these factors and possibly other members of the nuclear hormone receptor family likely play an important role in controlling CETP gene expression.
The PEA-3 transcription factor binding motif 5`-GGAAG-3` (41) is repeated twice in site B, and mutagenesis of one of these sequences is sufficient to eliminate binding, raising the possibility that PEA-3 participates in the regulation of the CETP gene promoter. PEA-3 belongs to a class of several transcription factors whose activity is regulated by oncogene expression, 12-O-tetradecanoylphorbol-13-acetate, or serum(41, 42, 43) . These observations together with those for site A raise the intriguing possibility that the CETP gene is regulated by multiple synergistically acting signal transduction pathways mediated by inducible transcription factors binding to these sites.
Overexpression of ARP-1 and Ear-3/COUP-TF repress transcription from
-300CETP.CAT and -138CETP.CAT, but activate transcription
from constructs containing additional 5`-flanking sequences upstream of
the CETP gene. Transcriptional repression of the shorter constructs by
ARP-1 was surprising since a promoter construct containing a mutation
in site C, which does not allow binding of ARP-1 or Ear-3/COUP-TF
(-138CmutCETP.CAT, see figure 5) had reduced transcription activity by
60%, a result that strongly suggested that the occupant of site C was
an activator of transcription. If ARP-1 were the only occupant of site
C, then mutation of site C would, by preventing the binding of ARP-1
and resultant transcriptional repression, yield higher levels of
transcriptional activity than that of the wild-type promoter construct.
We suggest that site C is occupied by a heterogeneous group of proteins
in HepG2 and Caco-2 cell nuclear extracts. This hypothesis is supported
by several lines of evidence: 1) competition of the site C-protein
complex in EMSA with oligonucleotides which bind nuclear hormone
receptors but not ARP-1 results in the reduction but not elimination of
the binding complex, 2) the transcriptional activity of the
-138Cmut CETP.CAT construct is lower that the wild type in CV-1
cells, a cell line that does not contain ARP-1 and Ear-3/COUP-TF, and 3) HeLa cell nuclear extracts that contain a large amount of
ARP-1 and Ear-3/COUP-TF (18, 19) showed no protection
of site C in DNaseI analysis (Fig. 2). Thus HeLa cells may
contain a factor(s) that prevents binding of ARP-1 to site C or, more
likely, contain factors in limiting quantities that are essential for
ARP-1 complex formation with site C. Since nuclear hormone receptors
bind their cognate DNA elements as dimers(6) , and since it has
been shown that ARP-1 can form heterodimers with several nuclear
hormone receptors known to be resent in liver and intestine (e.g. RXR
)(45) , it is likely that the true occupant of
site C is a heteromeric complex of which only part is ARP-1 or
Ear-3/COUP-TF.
How does ARP-1 repress CETP gene transcription? The foregoing discussion presupposes that ARP-1 acts directly on the CETP promoter through site C. Evidence supporting this mechanism rather than the interaction of ARP-1 with proteins off the DNA (as in, for example, squelching) comes from the observation that the -138CmutCETP.CAT construct is unaffected by co-expression (overexpression) of ARP-1. If overexpression of ARP-1 recruited proteins away from sites A or B, then it would be expected that transcriptional activity of the mutant construct would be reduced. It has been shown by others that ARP-1 does not interact with the basic transcriptional proteins(40) . Thus it appears that ARP-1 acts directly through site C. Under conditions of ARP-1 excess, which would favor homodimeric complexes, transcriptional repression is observed, but under physiologic conditions that may permit heteromeric complex formation between ARP-1 and other nuclear hormone receptors, activation is seen. These observations would be consistent with the notion that transcriptional repression mediated by ARP-1 homodimers is actually an intermediate state between different states of transcriptional activation(40) . Furthermore, while it may be that overexpression of transcription factors may produce cellular conditions that are not physiologic, the fact that ARP-1's effects are site-specific and result in both activation under certain promoter conditions and repression under others argues that we are observing the true transcriptional response of this gene to the local concentration of ARP-1.
The up-regulation of transcription
by the region between -636 and -300 in the presence of
ARP-1 or Ear-3/COUPTF suggests that these factors bound to site C are
capable of multiple interactions with other proteins in the CETP
promoter region. In the context of -300CETP.CAT or
-138CETP.CAT constructs, transcriptional repression occurs most
likely due to displacement of an activator or activator complex from
site C. This hypothesis is supported by the finding that a single
downstream regulatory element (site C) in the context of a minimal
promoter containing only the TATA box is transcriptionally active and
is repressed by ARP-1 in cotransfection experiments. The
addition of sequences from -300 to -636 decreases basal
transcriptional activity, suggesting that this region or its cognate
binding proteins interact with proteins in the -138 to -33
region and decrease their capacity to activate transcription. The same
interaction may take place in the context of ARP-1 or Ear-3/COUP-TF
overexpression (their tendency to repress is restrained, if not
antagonized by the upstream sequences between -300 and
-636). It is of interest that we have identified two consensus
binding sites for nuclear hormone receptors in this DNA region that
also bind ARP-1 and Ear-3/COUP-TF.
The role of these
sequences in CETP gene transcription in response to these factors is
currently under investigation.
It is interesting to note that many genes involved in cholesterol and lipid homeostasis which are expressed in liver and intestine are regulated by ARP-1 and Ear-3/COUP-TF(18, 19, 20) , suggesting that these genes may be coordinately regulated by these factors in these tissues. In most cases, the transcription of these genes is repressed by ARP-1 or Ear-3/COUP-TF. However, in the case of the CETP gene, up-regulation of transcription is observed when these factors are overexpressed. An antithetical relationship between levels of CETP and apoAI appear to determine plasma HDL levels; conceivably then, the finding of CETP gene up-regulation by ARP-1 and concomitant down-regulation of the apoAI gene may suggest a deterministic role for cellular levels of ARP-1 in HDL synthesis and metabolism.
In summary, we have shown that transcription of the CETP gene is under the control of a promoter element that potentially responds to diverse signaling events. In particular, the finding that orphan nuclear receptors bind to and affect the function of the promoter suggests that this gene may respond to as of yet unidentified ligands. In addition, the effects of ARP-1 on transcriptional activity from this gene promoter may provide a model system in which to study the mechanism for the conversion of a transcriptional repressor into a transcriptional activator.