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Human Molecular Genetics Advance Access originally published online on July 27, 2005
Human Molecular Genetics 2005 14(18):2607-2618; doi:10.1093/hmg/ddi291
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Functional interaction between –629C/A, –971G/A and –1337C/T polymorphisms in the CETP gene is a major determinant of promoter activity and plasma CETP concentration in the REGRESS Study

Eric Frisdal1, Anke H.E.M. Klerkx2, Wilfried Le Goff1, Michael W.T. Tanck3, Jean-Pierre Lagarde1, J. Wouter Jukema4,5, John J.P. Kastelein2, M. John Chapman1 and Maryse Guerin1,*

1Institut National de la Santé et de la Recherche Médicale, INSERM Unité 551, Dyslipoproteinemia and Atherosclerosis, Paris Cedex, France, 2Department of Vascular Medicine and 3Department of Clinical Epidemiology and Biostatistics, AMC, Amsterdam, The Netherlands, 4Department of Cardiology, LUMC, Leiden and 5Interuniversity Cardiology Institute of the Netherlands, Utrecht, The Netherlands

* To whom correspondence should be addressed at: INSERM Unité 551, Hôpital de la Pitié, Pavillon Benjamin Delessert, 83, boulevard de l'Hôpital, 75651 Paris Cedex 13, France. Tel: +33 142177977; Fax: +33 145828198; Email: mguerin{at}chups.jussieu.fr

Received June 2, 2005; Revised July 12, 2005; Accepted July 20, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
Cholesteryl ester transfer protein (CETP) plays a key role in the determination of high-density lipoprotein (HDL) levels via its action on intravascular HDL metabolism. The TaqIB polymorphism of the CETP gene is associated with plasma CETP and high-density lipoprotein cholesterol (HDL-C) levels and with premature coronary artery disease. Such associations appear to result from linkage disequilibrium between TaqIB and other functional polymorphisms. To date, only one functional promoter variant, which may explain the effects of TaqIB, has been identified at position –629 in the CETP gene. Here we describe a C/T polymorphism located at position –1337 in the human CETP gene (C allele frequency: 0.684), which is significantly associated with plasma HDL-C and CETP levels (P=0.0001 and P<0.0001, respectively). Transient transfection of a reporter gene construct containing the CETP promoter from –1707/+28 in liver cells (HepG2) revealed that the –1337T allele was expressed to a significantly lower degree (–34%, P<0.0001) than the –1337C allele. In addition, we clearly demonstrated that the –971G/A polymorphism is functional and that its functionality is intimately linked to the presence of the –1337 site. In vitro evaluation of potential interaction between –1337C/T and other functional variants of the CETP gene (–971G/A and –629C/A) demonstrated that these three functional CETP promoter polymorphisms can interact together to determine the overall activity of the CETP gene and thus contribute significantly to variation in plasma CETP mass concentration.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
The cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters from anti-atherogenic high-density lipoprotein (HDL) to pro-atherogenic apoB-containing lipoprotein particles including VLDL, VLDL remnants, IDL and LDL with hetero-exchange of triglycerides. CETP therefore plays a key role in the reverse cholesterol transport pathway and in the intravascular remodeling and recycling of both atherogenic and anti-atherogenic lipoprotein particles. Hypercholesterolemia, mixed hyperlipidemia, type 2 diabetes and metabolic syndrome involve dyslipidemic states which are associated with elevated circulating concentrations of atherogenic lipoprotein particles, thereby favoring enhanced arterial cholesterol deposition and accelerated atherogenesis. These dyslipidemic states are equally associated not only with elevated concentrations and activity of CETP in plasma (1Go,2Go), but also with low plasma high-density lipoprotein cholesterol (HDL-C) and apoAI levels (3Go,4Go). In contrast, CETP deficiency, which represents the most frequent cause of hyperalphalipoproteinemia in Asian populations, is associated with elevated plasma levels of HDL-C which may attain plasma concentrations 3–6-fold above the normal range (5Go).

Several polymorphisms have been identified in the human CETP gene. Most correspond to single nucleotide polymorphisms and are located in the promoter region of the gene or in introns or exons as well as in the 3' flanking region (6Go). Polymorphisms in the CETP gene constitute key genotypic markers of the relationship between the CETP gene and its metabolic and clinical impacts (7Go). The TaqIB polymorphism is the most extensively studied variant and is located in the first intron of the human CETP gene (8Go); this polymorphism accounted for 6% of the variance in plasma CETP levels (9Go). Subjects displaying the B1 allele display higher levels of CETP and lower levels of HDL-C when compared either with heterozygous or with homozygous subjects for the B2 allele (10Go,11Go). The effects of TaqIB on these plasma parameters are gender-dependent; furthermore, they have been reported to be influenced by alcohol consumption, body mass index and insulin levels (8Go,12Go). The TaqIB polymorphism is associated with coronary artery disease (CAD) risk (11Go,13Go). Indeed, the TaqIB polymorphism was associated with the progression of coronary atherosclerosis by computer-assisted quantitative angiography in the Regression Growth Evaluation Statin Study (REGRESS) (14Go).

Owing to its location, it is assumed that the TaqIB polymorphism does not represent a functional regulatory site, but rather that it is a marker for another functional sites. In addition, its effects on plasma CETP and HDL-cholesterol levels are independent (15Go,16Go), suggesting that the TaqIB polymorphism is a marker for at least two functional variants. The search for such variants led to the identification of the polymorphism located at position –629 in the upstream region of the CETP gene promoter, which modulate the transcriptional activity of the CETP gene in vitro (17Go). The –629C/A polymorphism is significantly associated with plasma CETP and HDL-cholesterol levels and constitutes a strong candidate to explain, at least in part, the effects of the TaqIB polymorphism. Moreover, the –629 CETP promoter polymorphism exerts independent effects on CETP concentration and HDL-C levels (9Go). A further single nucleotide polymorphism located at position –971G/A in the human CETP gene promoter has been identified which is significantly associated with both plasma CETP concentration and HDL-C level. However, transient transfection experiments using a construct containing 1054 bp of the CETP promoter region failed to demonstrate the functionality of the –971G/A polymorphism (18Go). In addition, it has been demonstrated that the –971G/A polymorphism interacts with both the functional –629C/A site and the TaqIB polymorphism in regulating plasma HDL-C levels, but not with plasma CETP concentration. In contrast, Klerkx et al. (9Go) revealed a strong interactive effect between the –971G/A and the –629C/A polymorphisms on plasma CETP concentration. Taken together, these observations strongly suggest the existence of at least one additional functional polymorphism within the human CETP promoter.

In this study, we identified the –1337C/T polymorphism as a functional variant located in the human CETP gene promoter. This polymorphism is associated with plasma CETP and HDL-C levels and accounts for at least 7% of the determination of plasma CETP levels. In addition, transient transfection experiments in HepG2 cells clearly demonstrated that the –971G/A polymorphism is functional and that its functionality requires the distal region (up to 1054 bp) of the human CETP gene promoter. Finally, we demonstrate that the three functional CETP polymorphisms, i.e., –1337C/T, –971G/A and –629C/A, can interact together to determine the overall activity of the CETP gene promoter and thus contribute significantly to variation in plasma CETP mass concentration.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
Localization and frequencies
In this study, the region from –500 to –2000 bp of the human CETP gene promoter has been analyzed by polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP) and sequencing. A total of five polymorphisms located at positions –1932, –1674, –1337, –875 and –827 were identified and genotyped in 230–269 subjects of the REGRESS population. The distribution of the genotypes and the relative allele frequencies is shown in Table 1. The frequency distributions of genotypes were consistent with those expected from Hardy–Weinberg equilibrium. Allele frequencies of –1674T/C, –1337C/T, –875C/T and –827C/T polymorphisms were in good agreement with those previously obtained with an independent population (19Go). Moreover, the –875C/T represented a CETP promoter variant with an 8.2% allele frequency for the –875T allele among Caucasians. The –875C/T polymorphism was not in significant linkage disequilibrium (LD) with –2708G/A, –1932T/C and –1674T/C variants. All other promoter polymorphisms analyzed in this study were in strong LD with one another and with previously described polymorphisms in the human CETP gene promoter (Table 2) (9Go,18Go,19Go).


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  		Table 1. Genotype distribution, allele frequencies and association of five CETP gene promoter polymorphisms with plasma CETP concentration and HDL-C levels
 

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  		Table 2. Pairwise LD coefficient between CETP gene promoter polymorphisms in the REGRESS population
 
Effects on plasma CETP mass and HDL-cholesterol levels
Table 1 shows plasma CETP concentrations and HDL-C levels as a function of the –1932T/C, –1674T/C, –1337C/T, –875C/T and –827C/T CETP promoter polymorphisms. Among the five polymorphisms genotyped in 230–272 subjects of the REGRESS population, only –1337T/C was significantly associated with plasma CETP levels (P=0.014). Thus, genotyping of the –1337T/C polymorphism was completed using the entire population available in the REGRESS bank (n=534). In the whole population studied, allele frequencies were 0.684 and 0.316 for the C and T alleles, respectively. As shown in Table 3, the –1337T/C polymorphism was significantly associated with the increased plasma concentration of CETP (P<0.0001) and lower HDL-C concentrations (P=0.0001). No significant association was observed between the –1337T/C polymorphism and triglycerides, plasma total cholesterol or LDL-cholesterol levels. Single polymorphism genotype analysis showed that the –1337 CETP promoter variant explained approximately 7% of the variation in plasma CETP concentration and 3.4% of the variation in HDL-C.


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  		Table 3. Plasma lipid levels (mmol/l) and CETP mass (mg/l) in 534 individuals of the REGRESS population according to –1337C/T polymorphism
 
To investigate whether the effects of the –1337 were independent or due to the LD between –1337 and the previously analyzed –2708, –971 and –629 polymorphisms that were proposed to represent putative functional sites, a linear regression using a backward selection procedure was performed (9Go). Backward analysis of the effects on CETP concentration revealed that of these four polymorphisms, both –1337 and –629 remained in the model with P-values of 0.017 and 0.024, respectively, which together explained 10.1% of the variation in CETP concentration (Table 4). This indicates the independent effects of these promoter polymorphisms on plasma CETP concentrations. For HDL-C, only the –629 polymorphism remained in the model, which accounted for 6.1% of the variation in HDL-C.


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  		Table 4. CETP polymorphisms in the final backward selection regression model for plasma lipid and CETP levels
 
Functionality of –1337C/T polymorphism
To determine whether the –1337C/T polymorphism might influence the activity of the human CETP gene promoter, transient transfections of HepG2 cells were performed with a luciferase reporter gene. First, we analyzed the effects of variation at the –1337 site in a 1707-bp fragment of the CETP promoter with either C or T at position –1337 in a construct with G at position –971 and C at –629. As shown in Figure 1 by constructs C/G/C and T/G/C, luciferase activity of the plasmid containing the T allele was 34% lower than the C allele and this was highly significant (P<0.0001). This finding is consistent with the lowest plasma CETP levels observed in subjects displaying the TT genotype. Indeed, plasma CETP levels were significantly reduced (–22%; P<0.0001) in patients with the TT genotype as compared with those displaying the CC genotype (Table 3).



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  		Figure 1. Expression studies of CETP promoter constructs carrying potential haplotypes for the –1337C/T, –971G/A and –629C/A polymorphisms. Modulation of the activity of the –1337 site as a function of the –971 and –629 haplotypes. 0 indicates the most frequent allele and 1 indicates the less frequent allele. CETP promoter activity is expressed as fold increase of RLU relative to pGL3b. Values are mean±SD of five independent experiments each corresponding to at least six point determinations. Statistical analyses were performed by ANOVA Newman–Keuls post-test:**P<0.0001 and *P<0.02.

 
Determination of the binding of nuclear proteins by electrophoretic mobility shift assay
Electrophoretic mobility shift assays (EMSAs) were carried out to determine whether DNA–protein interaction occurs in the CETP promoter region containing the –1337C/T polymorphism and whether such interaction may account for the differential transcriptional activity of the two –1337 forms (Fig. 2). A DNA–protein interaction was observed with both the –1337C and –1337T probes (Fig. 2A and B, lane 1). The formation of this major DNA–protein complex was partially competed by a molar excess of unlabeled –1337C probe (Fig. 2A, lanes 3 and 4) or of unlabeled –1337T probe (Fig. 2B, lanes 3 and 4). In contrast, the DNA–protein complex was not removed in the presence of an excess of non-specific competitor (Fig. 2A and B, lane 2). Moreover, a molecular excess of unlabeled –1337C or –1337T probe represented equivalent competitors of retarded complexes formed with the other radiolabeled probe, –1337T (Fig. 2A, lanes 5 and 6) or –1337C (Fig. 2B, lanes 5 and 6), respectively.



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  		Figure 2. Analysis of binding by EMSA with nuclear extracts from HepG2 cells. An amount of 0.125 pmol of radiolabeled probes with either C (A) or T (B) at position –1337 was incubated with 5.2 µg of nuclear extracts (lane 1), in the presence of a molar excess (100x) of unlabeled non-specific competitor oligonucleotide (lane 2), –1337C oligonucleotides (lanes 3 and 4 in A and lanes 5 and 6 in B), –1337T oligonucleotide (lanes 5 and 6 in A and lanes 3 and 4 in B), consensus Sp1 oligonucleotide (lane 7) and in the presence of antibodies specific to human Sp1 (lane 8) or Sp3 (lane 9). An amount of 0.125 pmol of each end-radiolabeled probe corresponding to the Sp1 consensus oligonucleotide (C) was incubated with 5.2 µg of nuclear extracts in the absence of competitor (lane 1), in the presence of a molar excess of consensus Sp1 oligonucleotide (lane 2), in the presence of antibodies specific to human Sp1 (lane 3) or Sp3 (lane 4), 1337C oligonucleotide (lane 5) and –1337T oligonucleotide (lane 6).

 
Analysis of the CETP promoter fragment surrounding the –1337C/T polymorphism (5'-CCCTcGGTC-3') revealed that this promoter region displayed similarities with a consensus sequence for transcriptional factors of the Sp family (5'-(G/T)GGGCGGPuPuPy-3'). In addition, the specific DNA–protein complex formed with either the 1337C or the 1337T probe displayed a molecular weight between 100 and 150 kDa (Fig. 2A and B). This observation is consistent with the expected molecular weight of the Sp1 transcription factor (109 kDa). Moreover, the use of an Sp1 probe as a competitor significantly reduced the abundance of the DNA–protein complex formed with either the 1337C or the 1337T probe (Fig. 2A and B, lane 7). Incubation of nuclear extracts from HepG2 cells in the presence of antibodies against Sp1 or Sp3 abolished the formation of the specific DNA–protein complex with the 1337C probe (Fig. 2A, lanes 8 and 9), whereas the complex formed with the 1337T probe was significantly decreased (Fig. 2B, lanes 8 and 9). Unfortunately, no supershift could be detected in any case. However, a major and specific DNA–protein complex of similar size was also obtained with a radiolabeled probe specific for the consensus sequence of the Sp-transcription factor family Sp1 probe (Fig. 2C). In addition, the DNA–protein complex formed with the Sp1 probe was completely competed by a molar excess of unlabeled –1337C (Fig. 2C, lane 5) or –1337T (Fig. 2C, lane 6) probe. Taken together, these results indicate the implication of nuclear factors Sp1 and Sp3 in the formation of the specific complex observed with either the 1337C or the 1337T probe.

Effects of haplotype at the –1337, –971 and –629 sites on in vitro activity of the CETP promoter
To evaluate potential interactions between the three polymorphisms located in the human CETP gene promoter, which were significantly associated with plasma CETP mass, i.e., –1337C/T, –971G/A and –629C/A, we performed transient transfection experiments using a set of vectors corresponding to the eight potential haplotypes. We observed that the haplotype carrying all three of the less frequent alleles (T/A/A) accounted for only 20% (P<0.0001) of promoter activity of the haplotype carrying the more frequent alleles for the three polymorphisms (C/G/C) (Fig. 3A). Interestingly, haplotypes carrying only one mutated site (C/A/C, C/G/A and T/G/C) or two mutated sites (T/G/A, C/A/A and T/A/C) displayed significant lower promoter activity (–30% and –50%, respectively) than the wild-type haplotype (C/G/C). This observation was consistent with in vivo results showing that patients homozygous for the 1337T/971A/629A haplotype displayed a significant lower (–23%, P<0.009) plasma CETP mass as compared with patients homozygous for the 1337C/971G/629C haplotype (1.55±0.35 and 2.01±0.52 µg/ml, respectively) (Fig. 3B). In vitro promoter activity correlates (r=0.8) with plasma CETP concentration, indicating that variation in plasma CETP levels in patients from the REGRESS population is linked to the participation of these three functional polymorphisms –1337C/T, –971G/A and –629C/A in overall CETP expression (Fig. 4). However, this correlation did not reach statistical significance (P=0.109). Moreover, the less frequent allele for each polymorphism tested displayed a significantly lower in vitro promoter activity than the more frequent allele, thereby indicating that these three polymorphisms (i.e. –1337C/T, –971G/A and –629C/A) are functional (Figs 1,5 and 6). The functionality of the –629C/A variant has been previously described by Dachet et al. (17Go), who observed a significantly lower promoter activity for the –629A allele than the –629C allele. Interestingly and in contrast to our previous study (18Go), the –971G/A polymorphism appeared to be functional in our four pairs of constructs with a reduced promoter activity for the –971A allele as compared with the –971G allele (Fig. 5). Moreover, using four pairs of constructs in which each carried a distinct haplotype, the reduction in CETP promoter activity observed with –1337T versus the –1337C allele ranged from –14 to –58% (Fig. 1). Similar variations were observed with both the –971G/A (–25 to –65%; Fig. 5) and –629C/A (–15 to –48%; Fig. 6) polymorphisms. These results clearly indicate that the three functional variants presently studied interact together to determine the overall in vitro CETP gene promoter activity. The –971A allele enhances the inhibitory action of the –1337T allele on CETP gene promoter activity, whereas the –971G allele can reduce the effect of the –1337T allele through interaction with the –629A allele. In addition, the –1337T allele partially abolished the repressive effect of the –629A allele through an interaction with the –971G allele. The –1337T allele increased the effect of the –971A allele on CETP promoter activity. In addition, the presence of the –629A allele potentiated the effect of the –1337T allele on the –971G/A polymorphism.



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  		Figure 3. (A) CETP promoter activity as a function of the number of mutated sites. 0 indicates the most frequent allele and 1 indicates the less frequent allele. §P<0.0001 significantly different from other constructs. (B) Plasma CETP concentration in corresponding CETP promoter haplotype. Haplotypes displaying a frequency less than 1% were excluded. The –1337C/–971G/–629C haplotype was significantly different from the –1337T/–971A/–629A haplotype (P<0.009).

 


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  		Figure 4. Relationship between in vitro CETP promoter activity and plasma CETP concentration in patients from the REGRESS population. Coefficient of correlation was calculated using the non-parametric spearman test. Haplotypes displaying a frequency less than 1% were excluded from the analysis. n represents the number of subjects for each haplotype.

 


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  		Figure 5. Expression studies of CETP promoter constructs carrying potential haplotypes for the –1337C/T, –971G/A and –629C/A polymorphisms. Modulation of the activity of the –971 site as a function of the –1337 and –629 haplotypes. 0 indicates the most frequent allele and 1 indicates the less frequent allele. CETP promoter activity is expressed as fold increase of RLU relative to pGL3b. Values are mean±SD of five independent experiments each corresponding to at least six point determinations. Statistical analyses were performed by ANOVA Newman–Keuls post-test: **P<0.0001 and *P<0.02.

 


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  		Figure 6. Expression studies of CETP promoter constructs carrying potential haplotypes for the –1337C/T, –971G/A and –629C/A polymorphisms. Modulation of the activity of the –629 site as a function of the –1337 and –971 haplotypes. 0 indicates the most frequent allele and 1 indicates the less frequent allele. CETP promoter activity is expressed as fold increase of RLU relative to pGL3b. Values are mean±SD of five independent experiments each corresponding to at least six point determinations. Statistical analyses were performed by ANOVA Newman–Keuls post-test: **P<0.0001 and *P<0.02.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIAL AND METHODS
 REFERENCES
 
We demonstrate the functionality of the –1337C/T and –971G/A polymorphisms located in the human CETP promoter. The functionality of the –971G/A variant is intimately linked to the presence of the –1337 site. Equally, our data indicate that these two polymorphisms, newly identified as functional, can interact together and with the –629C/A variant to modulate CETP gene expression and thus contribute to variation in plasma CETP mass. We propose that this interactive effect is associated with a common transcription factor. Indeed our findings reveal that the nuclear factors Sp1 and Sp3 are implicated in the formation of the specific DNA–protein complex formed at the –1337 site. The functionality of the –629C/A polymorphism is associated with specific binding of Sp1 and Sp3 to the –629A allele (17Go). We therefore propose a putative molecular mechanism underlying the interactive effect of the –1337C/T, –971G/A and –629C/A polymorphisms on CETP gene expression which involves not only the Sp1 and Sp3 transcription factors, but also three-dimensional DNA conformation.

Five polymorphisms located in the human CETP gene promoter have presently been identified and genotyped to determine their potential impact on plasma CETP concentration and HDL-cholesterol levels. Among them, only the –1337C/T variant appeared to be associated with both plasma CETP and HDL-C concentrations. Interestingly, all these five polymorphisms were identified and genotyped earlier by Lu et al. (19Go) in a population of elderly Japanese men. By contrast, the allele frequencies of –1932T/C polymorphism (0.716/0.284) observed in this study in a Caucasian population are quite distinct from those reported by Lu et al. (19Go) in a Japanese population (0.54/0.46). Allele frequencies of polymorphisms (–1674T/C, –1337T/C, –875C/T and –827C/T) observed in this study are, however, in good agreement with those previously reported by Lu et al. (19Go). It is likely that the above discrepancy concerning the –1932T/C polymorphism may result from the distinct origin of the two populations analyzed. Despite the fact that Lu et al. (19Go) genotyped the –1337C/T polymorphism, no data on the potential association between this polymorphism and plasma CETP concentration and HDL-C levels was presented.

Recently, it has been shown that the human CETP gene promoter region from position –1012 to position –1398 induces a marked activation (+50%) of CETP promoter activity (20Go). A binding site for the orphan nuclear receptor CYP7A promoter binding factor (CPF) at promoter position –1042 bp, which transactivates CETP promoter activity, has been identified (20Go). This site accounts for only half of the activation mediated by the –1012/–1398 promoter region in transient transfection experiments in HepG2 cells, suggesting the presence of additional positive regulatory element(s) in this region. Therefore, the functional –1337C/T polymorphism, which is located in this region, may potentially account for the remaining activation of the CETP promoter. DNA constructs used by Le Goff et al. (20Go) displayed a C, a G and a C in positions –1337, –971 and –629, respectively, and thus correspond to the C/G/C construct of the present study. Using such a construct, we observed that the promoter activity of the –1337T allele was 34% lower than that of the C allele (T/G/C versus C/G/C, see Fig. 1). This observation is consistent with the implication of the –1337C/T polymorphism in the activation of the CETP promoter.

In contrast to our previous study (18Go), we now demonstrate the in vitro functionality of the –971G/A polymorphism. Interestingly, using extensive genotype and haplotype analyses, Klerkx et al. (9Go) proposed that variation located at positions –2708 and –971 in the CETP promoter may represent putative functional sites acting in concert with the –629C/A polymorphism in the determination of plasma CETP concentration in an independent cumulative and interactive manner, respectively. Transient transfection of HepG2 cells revealed that the –2708G/A polymorphism alone did not modulate the transcriptional activity of the human CETP gene promoter (data not shown). The DNA constructs used in this study to evaluate the in vitro functionality of the –971G/A variant contained the proximal region of the human CETP gene promoter up to –1707 bp, whereas the functionality of the –971G/A polymorphism was previously evaluated using DNA constructs containing a shorter fragment (1054 pb) of the proximal promoter region (18Go). The fact that transient transfections using DNA constructs containing 1054 bp of the CETP promoter region failed to reveal the in vitro functionality of the –971G/A polymorphism, whereas similar experiments using DNA constructs containing 1707 pb clearly demonstrated the functionality of this site, thereby suggest that the CETP promoter region from –1054 to –1707 bp is involved in the functionality of the –971G/A polymorphism. In addition, it is important to note that the –1337 site was absent in the DNA construct containing 1054 pb of the CETP promoter region. Moreover, transient transfection experiments using DNA constructs containing 1707 bp of the CETP promoter, each carrying a different haplotype for the –1337C/T, –971G/A and –629C/A polymorphisms, revealed an interaction between both the –1337 and –971 sites. Considered together, these observations strongly support the concept that the presence of the distal CETP promoter region, and in particular the –1337 site, is required for the functionality of the –971G/A polymorphism.

Until now, the –629C/A polymorphism has been the sole functional polymorphism described in the CETP gene promoter (17Go). Indeed, the authors observed that the –629A allele displayed a significantly lower (–25%) promoter activity than the –629C allele. DNA constructs used in the previous study contained only 777 bp of the proximal CETP promoter region, and thus both the –1337 and –971 sites were absent. Interestingly, in this study using four couples of constructs each carrying a distinct haplotype for the –1337C/T and –971G/A polymorphisms, we observed that the –629A allele displayed a reduced promoter activity in the range from 15 to 48% (Fig. 6). These observations clearly demonstrate the interaction between the three functional polymorphisms on CETP gene expression. In a recent study, Thompson et al. (21Go) have shown that the functionality of the –629 site is modulated by another variant located at position –38 in the CETP promoter. Indeed, the difference in transcriptional activity observed between the two –629 alleles is reduced to only 10% when the less common –38A allele is present, whereas this difference reached 25% in the presence of the –38G allele. This newly characterized –38G/A polymorphism appears to be specific to African Americans and occurs at very low frequency among Caucasians with a 0.17% allele frequency for the 38A allele (21Go,22Go).

In this study, we observed that a specific DNA–protein complex was formed with both the –1337C and –1337T probes, however no clear difference between the two alleles was observed. Interestingly, similar observations were reported by Le Goff et al. (18Go) for the –971G/A polymorphism. In addition, the DNA–protein complex formed with either the 1337C or 1337T probes involved nuclear factors Sp1 and Sp3. Unfortunately, results obtained from the EMSA analysis do not allow us to propose a molecular mechanism able to explain the in vitro functionality of both the –1337 and –971 sites. However, we observed that the two presently studied functional variants (–1337C/T and –971G/A) interact together to modulate in vitro CETP promoter activity. We hypothesize that a specific three-dimensional conformation of promoter DNA is required for DNA–protein interaction involving these two polymorphic sites. Indeed, Zhang and Dufau (23Go,24Go) described a model in which the Sp1-4(I) binding site is the primary site for binding of Sp1/Sp3 proteins which subsequently recruit HDAC1/HDAC2/s/mSin3A/RbAp48 complexes to silence expression of the CG/LH gene. More recently, it has been shown that Sp1/Sp3/Sp4 and ‘Sp-like’ proteins are involved in recruiting HDAC complexes to the proximal promoter, thus preventing chromatin remodeling and resulting in transcriptional repression of the gene (25Go). It has been previously demonstrated that the functionality of the –629C/A polymorphism is associated with the binding of Sp1 and Sp3 transcription factors (17Go). Indeed, the 629A allele, but not the 629C allele, corresponds to an Sp1/Sp3 binding site acting as a repressor of promoter activity. As we observed significant interactions between the –629C/A polymorphism and the other polymorphic sites, –1337C/T and –971G/A, we hypothesize that a common transcription factor may be involved in such a modulation. Therefore, we propose that the Sp1 and Sp3 transcription factors represent good candidates to explain the observed interactions between the three polymorphisms on CETP promoter activity.

The results presented here extend significantly those reported by Klerkx et al. (9Go) and indicate the presence of at least two functional polymorphisms upstream of –629C/A. Indeed, both the –1337C/T and –971G/A polymorphisms in the CETP promoter are functional and interact together and with the –629C/A variant to determine plasma CETP levels. Our study clearly demonstrates that patients homozygous for –1337C/–971G/–629C haplotype are associated with elevated plasma CETP levels resulting from enhanced promoter activity as compared with patients homozygous for the 1337T/–971A/–629A haplotype. As elevated plasma CETP levels are associated with a high CAD risk, the goal of our future studies will be to determine whether patients with the C/G/C haplotype are more susceptible to atherosclerosis than patients with the T/A/A haplotype.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
REFERENCES
 
Study population
The design of the REGRESS was described earlier (26Go). Male patients were recruited from the Dutch Caucasian population. Subjects were eligible if they were less than 70 years old and if they presented obstruction of at least 50% of a major coronary artery as assessed by quantitative coronary angiography. Plasma total cholesterol levels were between 4 and 8 mmol/l (1.55 and 3.10 g/l) and plasma triglyceride concentrations were less than 4 mmol/l (3.50 g/l).

Analysis of plasma lipid parameters
Data for lipid, lipoprotein and apolipoprotein levels in the REGRESS cohort have already been reported (14Go). Plasma CETP concentration was determined using a two-antibody sandwich immunoassay as previously described (27Go).

Identification of gene polymorphisms by SSCP and genotyping
A 1.5-kb length of the human CETP gene promoter region between –2000 and –500 pb (GenBank accession no AF027656) was divided into overlapping fragments of approximately 300 bp. SSCP was performed using DNA from 50 individuals as previously described (18Go). DNA from individuals corresponding to different SSCP patterns was reamplified and sequenced using the T7 sequenase version 2.0 kit (Amersham). Genotyping was performed by restriction fragment length polymorphism (RLFP) or by direct sequencing of PCR products using the DNA Big DyeTM (Terminator Cycle Sequencing kit (ABI Prism, Applera) and a Perkin Elmer 377 DNA sequencer. Table 5 presents the PCR primers, the restriction enzymes used to detect the identified polymorphisms and the length of the resulting fragments. DNA amplification was performed as follows: DNA (50 or 100 ng) was added in a 20-µl mixture containing 16 pmol of each primers, 200 µM of each dNTPs, 4.5 mM MgCl2 and 0.5 Unit Taq DNA polymerase (Qiagen). The PCR reaction was carried out on a Peltier Thermal Cycler (PTC-220-Dyad) and comprised a 5-min denaturation at 94°C followed by 30 cycles of a 30-s denaturation at 94°C, 30-s annealing at 56°C, and a final 1-min extension at 72°C. After 30 cycles, an ultimate 7-min extension at 72°C was performed. PCR products (20 µl) were digested for 3 h with the appropriate enzyme (RsaI: 5 U, StyI: 10 U, FokI: 4 U and MscI: 3 U) at 37°C according to the manufacturer's instructions. The restriction fragments were separated by gel electrophoresis on a 1.5% agarose gel containing ethidium bromide.


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  		Table 5. Single-strand conformation polymorphism primers designed for analysis of the CETP gene promoter region (lower mismatch in primer to suppress a restriction site) and methods for RFLP screening of the CETP gene promoter (except for genotyping of –1674T/C polymorphism determined by sequencing)
 
DNA constructs
The DNA construct p1707 used in this study was previously described in detail by Le Goff et al. (20Go). Briefly, a 1707-pb fragment corresponding to the region from +36 to –1707 of the CETP promoter was cloned between the SacI and BsrGI sites of the pGL3 basic luciferase expression vector (Promega). The p1707 construct displayed a C in position –1337 (p1337C). A single point mutation was introduced at the position –1337 using the GeneEditorTM in vitro Site-Directed Mutagenesis System kit (Promega) in order to generate the p1337T. The oligonucleotide used to create the mutation was 5'-GGGCACCCTtGGTCATTGC-3' with the lowercase letter indicating the mutation site. Three polymorphisms in the human CETP gene promoter have been identified in positions –629, –631 (17Go) and –971 (18Go). Both the p1337C and p1337T constructs displayed a C in positions –629 and –631 and a G in position –971. To analyze the effect of haplotypes at positions –1337, –971 and –629 on the in vitro activity of the CETP promoter, the p1337C (C/G/C: 1337C/971G/629C) and p1337T (T/G/C: 1337T/971G/629C) constructs were derived to generate six additional vectors: C/G/A, C/A/C, C/A/A and T/G/A, T/A/C, T/A/A, respectively. An additional single point mutation was introduced at position –971 and/or –629 using the QuickChange Site-Directed Mutagenesis kit (Stratagene). Oligonucleotides used to create the mutation at positions –971 and –629 were 3'-GGAGCAGGTGCAAACTCaGGACTAGGGCAGGACCC-5' and 3'-CTCAGAGGCTGTATACCCaCCCAGAGTATTTTTATG-5', respectively. The integrity of inserts and the presence of mutations were verified by sequencing using the DNA Big DyeTM (Terminator Cycle Sequencing kit (ABI Prism, Applera) and a Perkin Elmer 377 DNA sequencer. The pGL3 basic vector (pGL3b) without an insert was used as a negative expression control.

Cell culture and transient transfection experiments
The human hepatocellular carcinoma cell line HepG2 was obtained from American Type Culture collection and maintained in culture in 5% CO2 at 37°C in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Invitrogen), 2 mM L-glutamine and 50 µg/ml gentamycin. Cells were seeded on 6-well plates at 2.5x106 cells per well. After 48-h incubation, 3 µg of each CETP promoter construct was cotransfected with 0.25 µg of a ß-galactosidase expression vector (pCMV.Sport-ß gal, Invitrogen) using the Lipofectine Liposomal reagent (Invitrogen) according to the manufacturer's instructions. Twenty-four hours after transfection, the medium was replaced by fresh medium and the cells were incubated for an additional period of 16 h. Cells were harvested with 150 µl of Cell Culture Lysis Reagent (Promega). The lysate was centrifuged for 2 min at 12 000 r.p.m. in order to remove the cellular fragments. Luciferase activity was measured on the supernatant using the Luciferase Assay System kit (Promega) in a 1420 VICTOR Multilabel counter (Perkin Elmer), and ß-galactosidase activity was measured using the ß-galactosidase Enzyme Assay System kit (Promega). Transcriptional activity was expressed in relative luciferase units (RLUs) after normalization for ß-galactosidase activity. Data were average from five independent experiments each corresponding to at least six point determinations.

Electrophoretic mobility shift assay
HepG2 nuclear extracts were prepared from confluent 75-cm2 flasks as previously described by Dignam et al. (28Go) and stored at –80°C before use. The protein concentration of nuclear extracts was determined by the bicinchoninic acid (BCA, Pierce) protein assay. The EMSA was performed using 19-bp synthetic oligonucleotides with either C or T at position –1337 (–1337C: 5'-GGGCACCCTcGGTCATTG-3' –1337T: 5'-GGGCACCCTtGGTCATTGC-3'). Oligonucleotides were annealed with their respective complementary strand at 100°C for 2 min in a solution containing 670-mM Tris–HCl (pH 7.5), 130-mM MgCl2, 13-mM EDTA, 13 mM spermidine and 67-mM DTT.

Double-strand probes (12.5 pmol) were radiolabeled with 20 µCi (6.7 pmol) of [{gamma}32P] ATP (3000 Ci/mmol, Amersham) by T4 polynucleotide kinase (Invitrogen) at 37°C for 30 min. Radiolabeled double-strand probes (0.125 pmol) were incubated for 20 min at 4°C in a final volume of 20 µl containing 10-mM Tris–HCl (pH 7.5), 65-mM NaCl, 4.3-mM MgCl2, 0.5-mM EDTA, 1-mM DTT, 5% glycerol, 2-µg poly(dI-dC), 2.3-mM spermidine, 1-µg BSA and 6 µg of nuclear extracts. In experiments that required the presence of unlabeled competitor (50- and 100-fold excess), this latter was added to the mixture before the addition of the radiolabeled probe. When indicated, 1 µg of rabbit affinity-purified polyclonal antibody raised against Sp1 or Sp3 (Santa Cruz Biotechnology) was incubated with nuclear extracts for 30 min at 0°C before addition of radiolabeled probe. After incubation, samples were loaded onto a 5% acrylamide gel (acrylamide/bis-acrylamide, 29 : 1). Electrophoresis was performed at room temperature at 200 V for 3 h, and the gels were transferred onto 3MM paper (Whatman), dried and exposed to Hyperfilm MP (Amersham) at –20°C overnight. Double-strand Sp1 consensus oligonucleotides (5'-ATTCGATCGGGGCGGGGCGAGC-3') were purchased from Promega. The non-specific competitor double strand had the following sequence: 5'-GCAGATCACTTGAGGTCAGG-3'.

Statistical analysis
Allele frequencies were calculated from the genotype counts. The observed genotype counts were compared with those expected under Hardy–Weinberg equilibrium with a {chi}2-test with one degree of freedom. The presence of LD between pairs of polymorphisms was tested using a likelihood ratio test (29Go) and the extent of disequilibrium was expressed in terms of D'=D/Dmax or D/Dmin (30Go). Single and multiple CETP polymorphism genotype effects were tested by analysis of variance and a backward regression analysis, respectively. Haplotype effects were estimated using a method described by Tanck et al. (31Go). Throughout, P-values <0.05 were interpreted as significant.

In transient transfection experiments, results were expressed as means±SD and statistical significance was determined by ANOVA Newman–Keuls post-test.


   ACKNOWLEDGEMENTS
 
INSERM provided generous support for these studies. The authors thank Dr. Thierry Huby for stimulating discussion. J. Wouter Jukema is an established investigator of the Netherlands Heart Foundation (2001D032).

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
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