©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Regulation of the Cholesteryl Ester Transfer Protein Gene by the Orphan Nuclear Hormone Receptor Apolipoprotein AI Regulatory Protein-1 (*)

(Received for publication, July 13, 1995)

François Gaudet Geoffrey S. Ginsburg (§)

From the Laboratory of Molecular and Cellular Cardiology, Department of Cardiology, Children's Hospital, Boston, Massachusetts 02115, the Cardiovascular Division, Beth Israel Hospital, Boston, Massachusetts 02215, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Epidemiologic studies have shown that plasma high density lipoprotein (HDL) (^1)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.


EXPERIMENTAL PROCEDURES

Plasmid Construction

A 387-base pair PCR product was obtained from the published sequence of the CETP promoter (21) using the 5`-primer 5`-GGGAAGCTTTGTCTTTTTCTCATAGTCATTGTATTT-3` and the 3`-primer (3`-alpha primer) 5`-GGGAAGCTTGGTTATCAGGCAGTGGTGTGTAAGTGG-3`. This PCR product was digested with HindIII and inserted in the HindIII site of pUC9.CAT to generate -300CETP.CAT. The -138CETP.CAT was constructed by digesting the -300CETP.CAT with XbaI (XbaI site at position -138) and BamHI (BamHI site 3` to the CAT gene) and inserting the resulting 1.8-kilobase fragment into pUC19.CAT digested with XbaI and BamHI. The -33CETP.CAT plasmid was generated by PCR amplification of the -33 to +27 from -138CETP.CAT using the 5`-primer 5`-GGGAAGCTTGGATCCACATACATATACGGGCTCCAGGCTGAA-3` and the 3`-alpha primer. The resulting fragment was digested with HindIII and cloned in the HindIII site of pUC9.CAT. To generate longer promoter regions, the 387-base pair PCR fragment was used as a probe to screen a Charon 40-based human chromosome 16 genomic library (ATCC 57765), and two overlapping clones were isolated. A XbaI-XbaI fragment containing sequences from -138 to -4700 was isolated and inserted in the XbaI site of -138CETP.CAT at position -138 to generate the -4700CETP.CAT construct. Restriction digest of the -4700 construct with PstI and AccI and religation of the vectors resulted in constructs -806CETP.CAT and -636CETP.CAT, respectively. For the site-directed mutant constructs, a first round of two PCR reactions was done. The first reaction involved the -138 XbaI primer (5`-primer; 5`-GGGAGGCTTTCTAGAGGAGGCCGCAGGGG-3`) and the lower strand of the mutant double-stranded oligo containing the mutations to be introduced (3`-primer); the second reaction involved the upper strand of the mutant double-stranded oligo corresponding to the same site of interest (5`-primer) and the CETP 3`-alpha primer. The PCR products were gel purified, mixed together, hybridized, and used as templates for a second round of PCR involving the -138 XbaI primer (5`-primer) and the CETP 3`-alpha primer. The resulting PCR product was digested with HindIII and cloned in the HindIII site of pUC9.CAT. The presence of mutations was confirmed by sequencing.

Cell Culture and Transfections

HepG2, Caco-2, COS-1, Sol8, HeLa cells, and NIH 3T3 were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Sigma), penicillin, and streptomycin under 5% CO(2) at 37 °C. Caco-2 cells were seeded at 2.5 times 10 cells/100-mm dish the day before transfection. All other cell types were seeded at 2.0 times 10 cells/100-mm dish the day before transfection. In general, 20 µg of reporter construct along with 2 µg of pCMV-beta-gal fusion plasmid were transfected into cultured cells by the calcium phosphate coprecipitation method(22) . For -4700CETP.CAT (a 9-kilobase plasmid), the amount of plasmid added for the transfection was corrected for the number of moles of lower molecular weight reporter constructs. For cotransfection experiments with recombinant transcription factors, 10 µg of CAT reporter construct was used with 5 µg of pMT2 or 5 µg of pMT2-ARP-1 or Ear-3/COUP-TF and shocked with 15% glycerol for 2 min the next day. Cells were harvested 48 h after the glycerol shock, resuspended in TE (10 mM Tris, 1 mM EDTA) and disrupted by three successive freeze-thaw cycles. The cell debris were pelleted, and the beta-galactosidase activity was determined from the supernatant as described previously(23) . The CAT assay was performed as described previously (23) and was normalized with beta-galactosidase activity to account for transfection efficiency. All CAT activities were within the linear range of the assay. Data reported were averaged from at least five independent experiments.

Nuclear Extracts

HepG2 and Caco-2 nuclear extracts were prepared from 20 confluent 150-mm dishes by the method described by Dignam (24) except that buffers A and C were supplemented with 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml of both pepstatin A and leupeptin (Sigma). Additionally, buffer C contained NaCl at a final concentration of 0.5 M, and buffer D was replaced by a similar buffer, buffer G (20 mM HEPES, pH 7.8, 0.1 M KCl, 0.2 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20% glycerol). Aliquots of nuclear extracts were snap frozen on dry ice and stored in liquid nitrogen. The protein concentration was determined by the Lowry assay (25) and was from 3 to 8 mg/ml.

Whole Cell Extracts

To make whole cell extracts, COS-1 cells were maintained in Dulbecco's modified Eagle's medium, 10% heat-inactivated fetal bovine serum and were transfected with pMT2 or pMT2-TF (TF, cDNA for ARP-1, Ear-3, HNF4, RXRalpha, or TR) by the DEAE-dextran procedure as described previously(26) . After 48 h, the cells were harvested and resuspended in buffer G (20 mM HEPES, pH 7.8, 0.1 M KCl, 0.2 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20% glycerol). Cell extracts were produced by three freeze-thaw cycles, snap frozen on dry ice, and stored at -70 °C.

DNase I Footprinting Analysis

Nuclear extracts were prepared from HepG2, Caco-2, and HeLa cells. The DNA probes were prepared by 5`-end-labeling of a 165-base pair XbaI-HindIII fragment from -138 to +27 of the CETP promoter region. The assay was performed as described previously (27) except that 1 times buffer Z was used in the assay (10 times Z buffer: 25 mM HEPES, pH 7.5, 100 mM MgCl(2), 1 mM freshly added dithiothreitol, 10 µM ZnSO(4), 20% (v/v) glycerol, 0.1% (v/v) Nonidet P-40).

Oligonucleotides

Oligonucleotides were synthesized on an abi 394 DNA/RNA synthesizer, deblocked at 55 °C for 8 h, and purified by polyacrylamide gel electrophoresis(28) . Complementary oligonucleotides containing 5`-GATC overhanging ends were annealed and used for EMSA and cloning. Some of these oligos were also used for PCR.

Electrophoretic Mobility Shift Assays

HepG2, Caco-2 and HeLa cell nuclear extracts or ARP-1, Ear-3/COUP-TF, RXRalpha, TR, or HNF4 proteins produced from COS-1 cell extracts were incubated with indicated P-labeled double-stranded oligo probes and run on native polyacrylamide gel electrophoresis in buffer Z as described previously(27) . For antibody EMSA experiments, a 1:10 dilution of antiserum to Sp1, HNF4, or ARP-1 were added to the mixtures prior to incubation with the probes for 30 min at room temperature.


RESULTS

Definition of a Promoter Region Responsible for the Tissue-specific Expression of the CETP Gene

The regulatory transcriptional elements for the CETP gene were localized by transfecting a series of CETP promoter/reporter fusion plasmids into HepG2, Caco-2, COS-1, and Sol8 cells (cells derived from liver, intestine, kidney, and muscle, respectively), all of which synthesize CETP mRNA: -4700CETP.CAT, -806CETP.CAT, -636CETP.CAT, -300CETP.CAT, -138CETP.CAT, and -33CETP.CAT. Low levels of CAT gene expression were observed in all cell lines with constructs containing sequences from -4700 to -636, relative to the transcriptional site, +1 (Fig. 1). However, in expressing cells, -300CETP.CAT and -138CETP.CAT had 20- and 70-fold greater activity, respectively, than -33CETP.CAT, a construct that includes CETP promoter sequences up to the TATA box. In HeLa and NIH 3T3 cells, which do not express CETP, levels of CAT activity near background were observed with all constructs. These data indicate that the cis-elements between -138 and -33 are both necessary and sufficient to promote high levels of CETP gene transcription in cell types that express endogenous CETP.


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 [^14C]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.



The -138 to -33 Region Contains Three Tissue-specific DNA-Protein Binding Sites Whose Cognate Proteins Function Synergistically to Activate Transcription

The binding of nuclear proteins to the active CETP gene promoter (-138 to -33) was investigated by DNaseI footprinting analysis. These assays revealed three protected regions (designated A, B, and C) between -138 and -33 using HepG2 and Caco-2 nuclear extracts (Fig. 2). In HeLa cells, which do not express CETP, site A, but not B and C, was bound.


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 (alpha1 and alpha2), whereas only the slower migrating band (alpha1) 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 alpha1 was specifically competed by an excess of the Sp1 oligo and supershifted by an anti-Sp1 antibody without affecting complex alpha2. Thus the ubiquitous factor Sp1 binds to site A in all cell types studied and is not present in complex alpha2.


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. alpha1 indicates the slower migrating and alpha2 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 alpha1, while the presence of intact sequences within and upstream of the Sp1 site are necessary for complex alpha2 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 alpha1 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.



The Complex Formed with Site C Contains the Orphan Nuclear Hormone Receptor, Apolipoprotein AI Regulatory Protein-1 (ARP-1)

We noted that site C contains a consensus sequence recognized by members of the nuclear hormone receptor family of transcription factors. We therefore tested the hypothesis that this site could bind nuclear hormone receptors. The complex between site C and HepG2 or Caco-2 nuclear extracts was competed with an oligo containing a NHRRE from the apoAI gene that binds several different nuclear hormone receptors(19, 30, 31) . This element competed with site C for protein binding and suggested that nuclear hormone receptors were bound to CETP site C (Fig. 6). Moreover, a point mutation in the consensus binding site for nuclear hormone receptors in site C eliminated binding (5`-GATCGCTGGGCAGGAAGGA(GT)GTGAATCTCTGGGGCCAGGAAGACC-3`; the nuclear hormone receptor binding site is shown in boldface, and the mutated base is underlined). ARP-1, produced in COS-1 cells by forced expression was specifically bound to this site and showed a mobility similar to the specific complexes formed with HepG2 or Caco-2 nuclear extracts (Fig. 6). To verify whether ARP-1 was present in the complex formed with HepG2 or Caco-2 nuclear extracts, the assay was performed with an antibody specific for ARP-1, which also recognizes other members of the COUP-transcription factor (COUP-TF) family including Ear-3/COUP-TF(32) . Binding was eliminated with the antibody but not in the control experiment with the preimmune sera. Thus, ARP-1, and possibly other members of the COUP-TF family, bind to CETP site C and may be a major component of the proteins bound to site C in liver and intestinal cells. We tested the ability of other nuclear hormone receptors to bind to site C. Specific binding to site C was observed for Ear-3/COUP-TF, RXRalpha, and a heterodimer of RXRalpha and thyroid hormone receptor, while binding was not seen with thyroid hormone receptor alone or HNF-4 (Fig. 7). These results show that CETP site C contains a selective nuclear hormone receptor DNA-binding site and suggest that the regulation of this gene may be under the control of hormonal or intracrine signals.


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-RXRalpha/COS-1 (lane 3), pMT2-TR/COS-1 (lane 4), pMT2-RXRalpha/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).



The CETP Gene Promoter Is Modulated by ARP-1 and Ear-3/COUP-TF

To determine the effect of ARP-1 and Ear-3/COUP-TF on CETP gene transcription, the cDNAs for these nuclear hormone receptors were cotransfected into HepG2 or Caco-2 cells with reporter constructs containing varying lengths of the CETP gene promoter region. In addition, a construct containing two copies of the apoAI NHRRE, (denoted as [A][A] -41AI.CAT), whose activity has been shown to be repressed by ARP-1 (31) was used as a control. Overexpression of ARP-1 or Ear-3/COUP-TF repressed the -138CETP.CAT construct in both cell lines (Fig. 8) but did not affect the activity of the -33CETP.CAT in HepG2 and Caco-2 cells, suggesting that the effect of ARP-1 was mediated through the -138 to -33 DNA region.


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.

Activation of the CETP Gene by ARP-1 and Ear-3/COUP-TF

We evaluated the functional consequences of ARP-1 and Ear-3/COUP-TF on CETP gene transcription in the context of the reporter constructs -806CETP.CAT and -636CETP.CAT. These studies were undertaken because of the presence of two consensus binding sequences for nuclear hormone receptors located in the region between -636 and -300. In the context of the construct -806CETP.CAT, which has low basal transcriptional activity, both ARP-1 and Ear-3/COUP-TF induced transcription 3-fold in both HepG2 and Caco-2 cells. Deletion of nucleotides -806 to -636 did not affect induction by ARP-1 or Ear-3/COUP-TF, while deletion of nucleotides -636 to -300 abolished induction and resulted in repression as shown above. Thus ARP-1 or Ear-3/COUP-TF bound to site C appear to interact synergistically with other factors upstream of -300 to activate transcription and, depending on the promoter context, transmit signals to activate or repress transcription.


DISCUSSION

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.

Multiple Signal Transduction Pathways May Regulate the CETP Gene through Three DNA-Protein Binding Sites Located in its Minimal Promoter

Site A contains overlapping DNA protein recognition regions for the zinc-finger proteins Sp1 (5`-GGGCGG-3`) and Egr-1 (5`-GCGGGGGCG-3`) (27) as well as the consensus binding sequence for AP-2 (5`-GGGCTGGG-3`)(33) . AP-2 mediates enhanced transcription by both the protein kinase C and protein kinase A pathways(34) . Egr-1 is an early growth response gene that is induced by mitogenic stimulation, cell growth, and differentiation(35) . Overlapping binding motifs for Sp1 and Egr-1 have been observed in several gene promoters(36) , and competition between these factors for overlapping motifs has been reported to play a role in the regulation of transcriptional activity in the promoters of the adenosine deaminase, platelet-derived growth factor A-chain, and colony stimulating factor-1 genes(37, 38, 39) . Thus the local Sp1 concentration may affect the binding of complex alpha2 proteins and their cooperative interaction with B and C proteins to activate transcription. Interestingly, Egr-1 may play a role in the transcriptional regulation of the apoAI gene whose product is coupled with CETP in the efflux of cholesterol from tissues via HDL(40) . Thus the potential exists for coordinate regulation of these two genes (CETP and apoAI) by this class of transcription factors.

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.

Regulation of CETP Gene Transcription by Nuclear Hormone Receptors

Site C binds members of the nuclear hormone superfamily of transcription factors. The binding and function of these transcription factors in CETP gene transcription is highly relevant since 1) we have previously shown that ARP-1 is present in Caco-2 and HepG2 cells using EMSA supershift experiments with antibodies that specifically recognize the ARP-1 family of transcription factors (19) and 2) we have shown that ARP-1 is present in rat intestinal epithelial cells and liver parenchymal cells by in situ hybridization(18) . Sequence analysis of site C suggested that the binding motif for ARP-1 and Ear-3/COUP-TF is an imperfect direct repeat of the hexanucleotide sequence 5`-TG(C)G(A)CCT(C)-3` separated by a spacer of seven nucleotides consistent with binding of dimeric complexes. Mutagenesis analysis of site C has confirmed the requirement of these two half-sites for complex formation with ARP-1 and Ear-3/COUP-TF. (^2)Indeed the binding of ARP-1 and Ear-3/COUP-TF has been recently reported to bind to elements having a seven-nucleotide spacer between consensus half-sites(44) .

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,^2 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. RXRalpha)(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.^2 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.^2 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Parke-Davis American Heart Association Clinician Scientist Award and the Department of Medicine, Beth Israel Hospital. To whom correspondence should be addressed: Cardiovascular Division, Beth Israel Hospital, 330 Brookline Ave., Boston MA 02215. Tel.: 617-667-4110; Fax: 617-667-4833; ginsburg@phenix.tch.harvard.edu.

(^1)
The abbreviations used are: HDL, high density lipoprotein; CETP, cholesteryl ester transfer protein; LDL, low density lipoprotein; NHRRE, nuclear hormone receptor response element; EMSA, electrophoretic mobility shift assay; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; TF, transcription factor.

(^2)
F. Gaudet and G. S. Ginsburg, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. Vikas Sukhatme and Roger Breitbart for thoughtful comments on this work and Emily-Flynn MacIntosh for preparation of the figures. We are grateful for the support of F. G. from Dr. Andrew M. Grant and the Department of Biochemistry, Sherbrooke University, Québec, Canada during a portion of this work.


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