Functional analysis of the chimpanzee and human apo(a) promoter sequences: identification of sequence variations responsible for elevated transcriptional activity in chimpanzee.

Lp(a) concentrations vary considerably among individuals and are primarily determined by the apo(a) gene locus. We have previously shown that mean plasma Lp(a) levels in the chimpanzee are significantly higher than those observed in humans (Doucet, C., Huby, T., Chapman, J., and Thillet, J. (1994) J. Lipid Res 35, 263-270). To evaluate the possibility that this difference may result from a high level of expression of chimpanzee apo(a), we cloned and sequenced 1.4 kilobase (kb) of the 5'-flanking region of the gene and compared promoter activity to that of its human counterpart. Sequence analysis revealed 98% homology between chimpanzee and human apo(a) 5' sequences; among the differences observed, two involved polymorphic sites associated with Lp(a) levels in humans. The TTTTA repeat located 1.3 kb 5' of the apo(a) gene, present in a variable number of copies (n = 5-12) in humans, is uniquely present as four copies in the chimpanzee sequence. The second position concerns the +93 C>T polymorphism that creates an additional ATG start codon in the human apo(a) gene, thereby impairing translation efficiency. In chimpanzee, this position did not appear polymorphic, and a base difference at position +94 precluded the presence of an additional ATG. In transient transfection assays, the chimpanzee apo(a) promoter exhibited a 5-fold elevation in transcriptional activity as compared with its human counterpart. This marked difference in activity was maintained with either 1.4 kb of 5' sequence or the minimal promoter region -98 to +141 of the human and chimpanzee apo(a) genes. Using point mutational analyses, nucleotides present at positions -3, -2, and +8 (relative to the start site of transcription) were found to be essential for the high transcription efficiency of the chimpanzee apo(a) promoter. High transcriptional activity of the chimpanzee apo(a) gene may therefore represent a key factor in the elevated plasma Lp(a) levels characteristic of this non-human primate.

To evaluate the possibility that this difference may result from a high level of expression of chimpanzee apo(a), we cloned and sequenced 1.4 kilobase (kb) of the 5-flanking region of the gene and compared promoter activity to that of its human counterpart. Sequence analysis revealed 98% homology between chimpanzee and human apo(a) 5 sequences; among the differences observed, two involved polymorphic sites associated with Lp(a) levels in humans. The TTTTA repeat located 1.3 kb 5 of the apo(a) gene, present in a variable number of copies (n ‫؍‬ 5-12) in humans, is uniquely present as four copies in the chimpanzee sequence. The second position concerns the ؉93 C>T polymorphism that creates an additional ATG start codon in the human apo(a) gene, thereby impairing translation efficiency. In chimpanzee, this position did not appear polymorphic, and a base difference at position ؉94 precluded the presence of an additional ATG. In transient transfection assays, the chimpanzee apo(a) promoter exhibited a 5-fold elevation in transcriptional activity as compared with its human counterpart. This marked difference in activity was maintained with either 1.4 kb of 5 sequence or the minimal promoter region ؊98 to ؉141 of the human and chimpanzee apo(a) genes. Using point mutational analyses, nucleotides present at positions ؊3, ؊2, and ؉8 (relative to the start site of transcription) were found to be essential for the high transcription efficiency of the chimpanzee apo(a) promoter. High transcriptional activity of the chimpanzee apo(a) gene may therefore represent a key factor in the elevated plasma Lp(a) levels characteristic of this non-human primate.
The atherothrombogenic lipoprotein(a) (Lp(a) 1 ) consists of an low density lipoprotein-like particle containing an additional glycoprotein, apolipoprotein (a) (apo(a)), which is attached to apo B100 via a disulfide bridge (1,2). Apo(a) and plasminogen cDNA sequences display remarkable homology (3). In humans, apo(a) contains an inactive plasminogen-like protease domain, and two plasminogen-like kringle domains, kringles IV and V. The kringle IV domain is present in multiple tandem copies of variable number. This variation is responsible for an elevated degree of size heterogeneity in the apo(a) protein (4 -6). The plasma concentration of Lp(a) remains quite constant throughout life in a given individual, although considerable variation (up to a 1000-fold) in Lp(a) level is observed between individuals (Ͻ0.001 to Ͼ1 mg/ml). Twin, family, and sib-pair linkage studies have revealed that such intra-individual variability in plasma concentration is under genetic control and almost entirely explained by variations at the apo(a) gene locus (7). Indeed, apo(a) isoform size polymorphism has been shown to account for 30 -70% of the total variability in Lp(a) levels, according to the ethnic origin of the populations examined (7)(8)(9). This effect is thought to result primarily from differential efficacy of post-translational processing of apo(a) isoforms in the hepatic cell (10,11). In addition to the number of KIV repeats, variations in apo(a) gene sequence in the 5Ј-flanking region of the gene may also contribute to variance in plasma Lp(a) levels (12)(13)(14). In this regard, it is notable that genetic studies have revealed that a pentanucleotide (TTTTA) repeat polymorphism and a C/T polymorphism, both located in the 5Ј-region of the apo(a) gene, are associated with Lp(a) levels (15)(16)(17)(18).
In addition to humans, the presence of Lp(a) has been detected only in Old World monkeys (19) and, surprisingly, in the European hedgehog (20). The chimpanzee (Pan troglodytes) is the non-human primate most closely related to humans, and indeed, Lp(a) in this species displays some remarkable characteristics. We observed that the mean plasma levels of Lp(a) and the distribution of apo(a) isoforms are distinct in chimpanzee as compared with those reported in humans, cynomolgus monkey, or baboon (21). Lp(a) concentrations in the chimpanzee are significantly higher than those observed in either a Caucasian population (mean Lp(a) level ϭ 0.61 mg/ml versus 0.18 mg/ml) or in African populations for which the highest mean Lp(a) levels (ϳ0.30 -0.40 mg/ml) have been reported (8,9). As generally observed in humans, an inverse correlation between Lp(a) concentrations and apo(a) isoform sizes was found in the group of chimpanzees that we examined (21). However, apo(a) isoforms of low molecular mass were detected with a greater frequency in monkeys as compared with humans (mean values 665 Ϯ 121 kDa versus 789 Ϯ 114 kDa), which could partly account for the elevated Lp(a) levels observed in the chimpanzee. Nevertheless, chimpanzees generally exhibited superior levels of Lp(a) as compared with humans for apo(a) isoforms of similar size. In view of these characteristics, we hypothesized that sequence differences in the 5Ј-flanking region of the apo(a) gene might account for the elevated expression level of apo(a) in chimpanzee. Consequently, we cloned and sequenced the 5Ј-flanking region of the chimpanzee apo(a) gene. The transcriptional activity of this region was 5-fold greater as compared with that of the corresponding region of the human apo(a) gene. Site-directed mutagenesis revealed that this difference is due to three base substitutions in the very proximal promoter region.

EXPERIMENTAL PROCEDURES
Subjects and Samples-All animals were housed at the Regional Center for Training and Research in Human Reproduction in Gabon. Blood samples were taken from 50 unrelated animals, and genomic DNA was prepared by phenol extraction.
Cloning and Sequencing of the 5Ј-Region of the Chimpanzee apo(a) Gene-The 5Ј-flanking region of the chimpanzee apo(a) gene was amplified by PCR using five different sets of primers to obtain overlapping fragments (Table I, primer sets P1, P3, P4, P5, and LP); these fragments covered 1.4 kb of 5Ј sequence. Primers were designed according to the corresponding region of the human apo(a) gene (12). The conditions of the PCR reaction were as follows: in a 50-l final volume, 100 ng of genomic template DNA were mixed with 20 pmol of each primer, 10 pmol of each dNTP, and 1 unit of Taq polymerase (Stratagene) in a buffer containing 10 mM Tris-HCl and 1.5 mM MgCl 2 at pH 8.8. PCR reactions were carried out using the following program: denaturation at 95°C for 30 s, annealing at the appropriate temperature (Table I) for 30 s, and elongation at 72°C for 30 s. 30 cycles were carried out under these conditions after an initial cycle for which the denaturation step was 5 min at 95°C, in a Techne PHC-3 thermocycler. The PCR products were purified and ligated to an EcoRV-digested cloning vector (pBluescript). The 5Ј-and 3Ј-extremities of PCR clones were sequenced by the dideoxy chain termination method of Sanger (22) with a Sequenase 2.0 kit (USB/Amersham Pharmacia Biotech) using T3 and T7 primers. The sequences were characterized from at least two independently amplified PCR clones.
Screening of TTTTA Repeat Polymorphism-The region starting at Ϫ1410 bp upstream of the ATG codon and comprising the pentanucleotide repeat sequence was amplified by PCR from genomic DNA using REPD and REPR primers ( Table I). The (TTTTA) n repeat was detected by electrophoresis on 10% polyacrylamide gels. 10-l aliquots of the PCR reaction were loaded on the gels and run for 45 min at 30 mA in a Miniprotean II instrument (Bio-Rad, France). The gels were stained with ethidium bromide and photographed under ultraviolet light.
Plasmid Constructs-The 1.4-kb fragment of the chimpanzee apo(a) 5Ј-flanking region was amplified by PCR under the same conditions as mentioned above, except that the elongation time was 2 min. The primers used were MB22 and PCR78 described by Zysow et al. (13) for the amplification of the corresponding 5Ј-region of the human apo(a) gene. Primer PCR78 encompasses the translation initiation start site of the apo(a) gene, but a base pair mismatch transforms the ATG codon into an AGG sequence in the amplified fragment. MluI and BglII sites, present at the 5Ј-end of primer MB22 and PCR78, respectively, were used for directional cloning of the PCR product into MluI/BglII-digested pGL3-basic (Promega), a promoterless luciferase reporter gene vector. The resulting plasmid was designated pCH. The corresponding region of the human apo(a) gene, containing a C at position ϩ93, was equally cloned in the pGL3-basic expressing vector (plasmid pHU). To generate the 5Ј-deletion constructs 5Ј⌬pCH and 5Ј⌬pHU, in which the luciferase gene is driven by the minimal apo(a) promoter sequence (Ϫ98 to ϩ141), plasmids pCH and pHU were digested by MluI (5Ј cloning site, see above) and PvuII (Ϫ98) to completion. Each vector was then separated from the excised apo(a) fragment on agarose gel and religated after blunting the cohesive termini with the Klenow enzyme. All Plasmids used in transfection experiments were purified on AX-100 columns (Macherey-Nagel, France).
Site-directed Mutagenesis-Substitution mutants for positions Ϫ3 and Ϫ2 were prepared from either the pCH or the pHU constructs by using the Gene Editor kit (Promega, France). Synthetic oligonucleotides containing two mismatched bases were used to introduce mutations in the human (CC3 TT) and the chimpanzee (TT3 CC) promoters. Briefly, the mutagenic oligonucleotides (mutTT-2-3: 5Ј-TAATGTTT-GAATTCTGCTGAGCCAG-3Ј or mutCC-2-3: 5Ј-TAATGTTTGAACCCT-GCTGAGTCAG-3Ј) were annealed to heat-denatured plasmids together with a second oligomer, which allows the selection of the mutated vectors with the Gene Editor antibiotic mix. The bound primers were extended and ligated with T4 polymerase and T4 ligase. The mixture was then used to transform the repair-deficient Escherichia coli strain BMH 71-18 mutS. Plasmid DNA prepared from an overnight culture was used to transform JM109 cells. Mutants were then selected on LB plates containing both ampicillin and the Gene Editor antibiotic selection mix. Mutated plasmids were identified by the absence (for the mutated human sequence) or the presence of an EcoRI site (for the mutated chimpanzee sequence). The presence of the mutations was confirmed by sequencing. As described above for pCH and pHU, the mutated constructs were digested with MluI and PvuII to generate 5Ј⌬pCHmut.-3,-2 and 5Ј⌬pHUmut.-3,-2 for which the firefly luciferase cassette is under the control of the first 239 bp of the apo(a) promoter. The same mutagenesis procedure was applied to plasmid 5Ј⌬pHU as described above to simultaneously substitute positions Ϫ3, Ϫ2 (CC3 TT) along with a third position (Ϫ51 G3 C, or Ϫ24 A3 T, or ϩ8 C3 T, or ϩ65 G3 A, or ϩ94 G3 A) by the nucleotides found in the chimpanzee sequence at the respective positions. Mutations at positions Ϫ2, Ϫ3, and ϩ8 were introduced using the mutagenic oligonucleotide mut-2-3ϩ8 (5Ј-TAATGTTTGAATTCTGCTGAGTCAG-3Ј). For the generation of the other mutants, mutTT-2-3 (see above) was used in combination with a second mutagenic oligonucleotide encompassing the third position to mutate (position Ϫ51: 5Ј-ATCTATTGACATTC-CACTCTC-3Ј, position Ϫ24: 5Ј-TATAAGACTCTTTATTCAAGG-3Ј, position ϩ65: 5Ј-GGTTTGTGGATGCGTTTACTC-3Ј, position ϩ94: 5Ј-GT-CAACAACATCCTGGGATTG-3Ј). After a first screening of the mutated constructs by restriction analysis, the sequences of the selected mutants (5Ј⌬pHUmut.-3,-2,-51, 5Ј⌬pHUmut.-3,-2,-24, 5Ј⌬pHUmut.-3,-2,ϩ8, 5Ј⌬pHUmut.-3,-2,ϩ65, and 5Ј⌬pHUmut.-3,-2,ϩ94) were verified by sequencing.
Cell Culture and Transfection Experiments-HepG2 cells were maintained in culture in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, and 2 mM glutamine. For transient transfections, 2 ϫ 10 5 cells were plated into six-well dishes, grown for 24 h, and then incubated with 1 ml of serum-free medium containing 2 g of plasmid DNA, 0.3 g of a ␤-galactosidase expression plasmid (pSV-gal, Promega, France), and 5 g of Lipofectin (Life Technologies, Inc., France) for 16 h. The medium was then replaced by fresh medium containing 10% fetal calf serum for 24 h. Cells were harvested in lysis buffer (Promega) and centrifuged, and the supernatants were assayed for luciferase activity using a microplate reader luminometer (Victor, Wallac, France). ␤-Galactosidase activity was determined by a colorimetric method (Promega). Each transfection assay included control transfections with no DNA, and with pGL3-basic.
Electrophoretic Mobility Shift Assay-HepG2 nuclear extracts were  Functional Analysis of the Chimpanzee apo(a) Promoter prepared from confluent 75-mm flasks by the method described by Dignam et al. (23). Aliquots of nuclear extracts were stored at Ϫ70°C. Protein concentration was determined with the bicinchoninic acid protein assay reagent (BCA, Pierce). For electrophoretic mobility shift assay (EMSA), 0.25 pmol of 32 P-end-labeled double-strand oligonucleotide was mixed with 6 g of nuclear extract in a final volume of 20 l containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 3 mM MgCl 2 , 0.5 mM EDTA, 1 mM dithiothreitol, 5% glycerol, 2 g of poly(dI-dC)⅐poly(dI-dC), 4 mM spermidine, and 1 g of bovine serum albumin. The appropriate competitor was added to the reaction mixture before the addition of the end-labeled probe. Samples were incubated for 15 min on ice, loaded onto a 6% polyacrylamide gel, and electrophoresed at 200 V for 3 h. The protein-DNA complex were visualized by autoradiography of the dried gels on Hyperfilm MP (Amersham Pharmacia Biotech; Life Science) at Ϫ70°C. The sequences of the oligonucleotides were as follows: 5Ј-TAATGTTTGAATTCTGCTGAGTCAG-3Ј (CH probe) and 5Ј-TAAT-GTTTGAACCCTGCTGAGCCAG-3Ј (HU probe) corresponding to the Ϫ14 to ϩ11 region of the chimpanzee and human apo(a) genes, respectively. The nonspecific competitor had the following sequence: 5Ј-GGATCCAGCGGGGGCGAGCGGGGGCGA-3Ј.

RESULTS
Sequence Analysis- Fig. 1 shows a comparison of the sequence of the 1.4-kb 5Ј-flanking region of the chimpanzee apo(a) gene with that of the corresponding region of the human gene. Sequence analysis revealed a high degree of homology between chimpanzee and human apo(a) genes (98%). Several differences are of note. The first one concerns the TTTTA repeat present at position Ϫ1400 bp in humans. The number of repeats varies from 5 to 12 in human populations (15,16,18). In the 5Ј-flanking region of apo(a) that we cloned, the number of repeats was four. To determine whether there was also a polymorphism in the chimpanzee apo(a) promoter at this position, we amplified a fragment spanning this repeat in 50 animals. In every animal, we found the same size for this fragment, indicating that the number of TTTTA repeats was constant in the chimpanzee (Fig. 2). Another interesting difference concerns position ϩ93 (relative to the start site of transcription). In humans, a C/T polymorphism has been described at this position that creates an additional ATG start codon (13). In chimpanzee, every clone that was sequenced had a C at this position. Moreover, the next position was also different from that in the human sequence. Consequently, an ATG could not be present in the chimpanzee sequence. It is also of note that the corresponding sequence in baboon apo(a) is identical to that of the chimpanzee in this region (24).
Analysis of the Chimpanzee apo(a) Promoter Activity-We next compared the promoter activity of the 1.4-kb fragment of 5Ј-flanking sequence in the chimpanzee apo(a) gene to that of the corresponding human sequence. As shown in Fig. 3, the chimpanzee apo(a) promoter construct (pCH) exhibited 5-fold greater activity than that of the human (pHU) promoter. In vitro promoter analyses of this 1.4-kb 5Ј-region of the human apo(a) gene had previously shown that the essential regulatory elements were located in the very proximal 5Ј-flanking sequence. Furthermore, deletion of sequences Ϫ1301 to Ϫ98 had little to no effect on promoter activity (25). Therefore, we compared the transcriptional activities of the Ϫ98 to ϩ141 apo(a) regions of the chimpanzee and human promoters to evaluate the role of this minimal promoter fragment in the marked difference in activity observed with the 1.4-kb apo(a) fragments. The 5Ј-deletion mutants of pCH and pHU were generated using the PvuII site located at position Ϫ98. Transient transfection assays revealed that the chimpanzee short construct 5Ј⌬pCH had approximately 5-fold higher transcriptional activity than the corresponding human plasmid 5Ј⌬pHU (Fig.  3). Therefore, similar differences in transcriptional activity were observed between both species using either the 1.4-kb apo(a) fragments or constructs containing the minimal apo(a) promoter regions Ϫ98 to ϩ141. These results provided a firm indication that base differences located specifically in the very proximal 5Ј-region of the gene were responsible for the high transcription level of the chimpanzee apo(a) sequence as compared with its human counterpart. As a first step in the identification of these nucleotides, we compared the chimpanzee apo(a) promoter sequence to the corresponding human and baboon sequences. Lp(a) concentrations in baboon are not sig- nificantly different from those observed in Caucasians (26). Therefore, assuming that the baboon and human apo(a) promoters possess similar transcriptional activities, we hypothesized that the identification of base changes specific to the chimpanzee apo(a) sequence could be relevant to the increased apo(a) promoter activity observed in this species. Nine positions were found to be altered in the chimpanzee apo(a) 5Ј sequence, whereas they were conserved in both humans and baboon (boxed in Fig. 1). Among these nine differences, only two adjacent base changes (CC3 TT), at positions Ϫ3 and Ϫ2 from the start site of transcription, were present in the Ϫ98 to ϩ141 region (in boldface characters in Fig. 1). Consequently, site-directed mutagenesis was used to evaluate the effect of this set of change (CC7TT) on the promoter activities of the chimpanzee and human apo(a) sequences. Mutagenesis of the chimpanzee apo(a) promoter to replace nucleotides TT at positions Ϫ3 and Ϫ2 with CC resulted in a significant reduction in transcriptional activity to the level of its human counterpart (Fig. 4, see plasmid 5Ј⌬CHmut.-3,-2). This result suggests that either one or both T bases are implicated in the increase of transcriptional activity observed with the chimpanzee sequence. However, the reverse change, i.e. the human promoter mutated to TT, did not result in an increase but rather in a small reduction in luciferase expression (Fig. 4, see plasmid 5Ј⌬HUmut.-3,-2). Therefore, the thymidine residues at positions Ϫ3 and/or Ϫ2 are essential for high level transcription of the chimpanzee apo(a) promoter, but it appears that one or several other nucleotides may also be involved. In addition to positions Ϫ3 and Ϫ2, the two species differ at five additional positions (Ϫ51, Ϫ24, ϩ8, ϩ65, and ϩ94, see Figs. 1 and 4) in the Ϫ98 to ϩ141 region. Each one of these bases was mutated independently in the short human construct 5Ј⌬pHU along with positions Ϫ3 and Ϫ2. Transient transfection assays revealed that substituting the nucleotides at positions Ϫ3, Ϫ2, and ϩ8 in 5Ј⌬pHU by those found at the corresponding positions in the chimpanzee sequence increased the transcriptional activity of the promoter about 5-fold, to a level similar to that observed for the wild type chimpanzee construct 5Ј⌬pCH (Fig.  4). This effect was observed only with these combined mutations, but in contrast, was not detected when any of the four other different positions was mutated along with positions Ϫ3 and Ϫ2 (Fig. 4).
Electrophoretic Mobility Shift Analysis of the Ϫ14 to ϩ11 Region of the apo(a) Promoter-Transient transfection experiments indicated that positions Ϫ3/Ϫ2 and ϩ8 contribute significantly to the high level of activity of the chimpanzee apo(a) promoter. We therefore investigated protein-DNA interactions in this region. An electrophoretic mobility shift assay (EMSA) was performed with nuclear extracts from HepG2 cells and synthetic oligonucleotides spanning the Ϫ14 to ϩ11 region of both human and chimpanzee apo(a) genes. Interaction with the CH probe corresponding to the chimpanzee sequence in the absence of competitor resulted in the formation of three complexes (Fig. 5, lane 1); the band designated C1 specifically competed with a molar excess of unlabeled CH probe (Fig. 5,  lane 3), but not with a nonspecific competitor (Fig. 5, lane 2) or with the human HU probe (Fig. 5, lane 4). The incubation of nuclear extracts with the HU probe resulted in the formation of three retarded complexes in the absence of competitor (Fig. 5,  lane 5). Bands H1 and H2 disappeared after competition with a molar excess of unlabeled HU probe (Fig. 5, lane 7), but not after competition with an unrelated oligonucleotide (Fig. 5,  lane 6) or with the unlabeled CH probe (Fig. 5, lane 8). These results suggest that distinct specific complexes were formed with the two probes corresponding either to the chimpanzee or the human apo(a) sequences. Interestingly, the same results for EMSA were obtained when position ϩ8 was substituted with a C or a T in the CH and HU probes, respectively, thereby suggesting that positions Ϫ3 and/or Ϫ2 exert major influence on the formation of the specific complexes observed for each sequence (data not shown).

DISCUSSION
Elevated plasma levels of Lp(a) are a significant independent risk factor for cardio-and cerebrovascular diseases in humans (27)(28)(29)(30)(31). The understanding of the molecular mechanisms that dictate the level of apo(a) gene expression appears to be essential, because the apo(a) gene locus has been shown to be the major genetic determinant of Lp(a) levels (7,32). In the present study, we cloned, sequenced, and functionally characterized the 5Ј-flanking sequence of the chimpanzee apo(a) gene, a species exhibiting severalfold higher mean Lp(a) levels than humans. Our findings reveal that the chimpanzee apo(a) sequence exhibited a 5-fold elevation in activity as compared with its human counterpart. Furthermore, base differences located in the very proximal 5Ј-region of the apo(a) genes at positions Ϫ3/Ϫ2 and ϩ8 accounted for these findings. Our results provide evidence that the level of apo(a) gene transcriptional activity may represent a key factor in determining plasma Lp(a) concentrations.
Sequence analysis of the 1.4-kb 5Ј-flanking region of the chimpanzee apo(a) gene revealed a high degree of homology with its human counterpart (98%). This value corresponds to the overall level of nucleotide variation detected between both species. Consequently, it can be considered as a rather low degree of variation, because non-coding regions are usually more subject to variations than coding regions. By comparison, we have reported 1.4% of variation between the coding sequences of the human and chimpanzee apo(a) genes (33).
Apo(a) size polymorphism is a major component of the variability of plasma Lp(a) concentrations. However, size polymorphism does not explain the overall variance of Lp(a) levels in any population analyzed to date nor the differences observed across ethnic groups (for review see Ref. 34). A limited number of apo(a) sequence variants that could affect human Lp(a) levels (other than the number of kringle IV) have been identified until now. Notably, a pentanucleotide (TTTTA) 5-12 repeat polymorphism (PNRP) present in the 5Ј-flanking region of the apo(a) gene and a CϾT polymorphism at position ϩ93 that introduces an additional upstream ATG initiation codon with its own in-frame stop codon (12,13). High repeat lengths (n ϭ 10, 11) of the PNPR and the T allele at position ϩ93 have been shown to be significantly associated with low levels of Lp(a) in human populations (15)(16)(17)(18). The mechanism of action of the PNRP has not been determined. However, in vitro reporter assays have revealed that the number of TTTTA repeats does not affect the promoter activity of the human gene (15,35). Moreover, the lack of association of the PNRP and Lp(a) levels in Africans does not support a direct role of the PNRP, but rather has led to the suggestion that this polymorphic region is in allelic association with a functional but unknown sequence FIG. 2. Representative example of the size determination of the apo(a) 5-region encompassing the TTTTA repeats in chimpanzee. PCR products generated by amplification of genomic DNA from eight unrelated animals by using REPD and REPR primers were loaded on a 10% polyacrylamide gel. After electrophoresis, the amplified fragments were revealed by ethidium bromide staining. M is the molecular weight DNA marker pBR322 digested by MspI.
variant of the human apo(a) gene (15). Therefore, the constant and low copy number (n ϭ 4) of the pentanucleotide (TTTTA) in the chimpanzee apo(a) promoter (Figs. 1 and 2) cannot be considered as a potential determinant of high Lp(a) levels observed in this species. On the other hand, the impossibility of having an additional unproductive ATG start codon at position ϩ93 in the chimpanzee apo(a) promoter ( Fig. 1) could partly contribute to elevated mean Lp(a) levels in chimpanzee as compared with humans. However, the difference in mean Lp(a) levels between the two species that results from the lowering effect of the ϩ93 C/T polymorphism in humans is probably limited in view of the low frequency of the T variant (0.09 -0.15) in human populations and its general impact on mean Lp(a) levels (17,18).
We found the same 5-fold difference in promoter activity using either constructs containing the 1.4-kb or the minimal apo(a) Ϫ98 to ϩ141 regions when comparing the chimpanzee and human sequences (Fig. 3). This finding strongly suggested that the difference in activity observed between apo(a) sequences mainly originated from base differences located in the proximal promoter regions. The human apo(a) constructs used in our experiments contained a C at position ϩ93. Therefore, the levels of luciferase expression obtained with the human constructs were not diminished by the presence of the additional ATG, which has been shown to reduce in vitro translational efficiency by 60% (13). In the minimal promoter region, two base changes in the chimpanzee sequence are found within footprint regions previously identified in the human apo(a) promoter (25). The nucleotide change  5. Electromobility shift assays of apo(a) ؊14 to ؉11 regions. Gel shift studies were performed using either the chimpanzee Ϫ14 to ϩ11 apo(a) sequence (probe CH) or the corresponding human sequence (probe HU). HepG2 nuclear extracts (6 g) were used in the absence of competitor (lanes 1 and 5) or in the presence of a 100-fold molar excess of cold nonspecific oligonucleotide (lanes 2 and 6), of unlabeled CH probe (lanes 3 and 8) or unlabeled HU probe (lanes 4 and  7). C1 and H1/H2 are the specific retarded complexes formed with the CH and HU probes, respectively. at position Ϫ51 affects a consensus site for the binding of CAAT/ enhancer-binding protein. However, Wade et al. (25) showed by transient transfection experiments that mutations at this site did not modify promoter activity. The other base change concerns position ϩ94 located at the very end of the footprint B region. Point mutations in this footprint were associated with a moderate increase in human apo(a) promoter activity, whereas deletion of the all footprint region in the baboon apo(a) sequence had no effect (24,25). Consequently, these two positions did not appear as probable candidates to explain the high transcription efficiency of the chimpanzee promoter. We therefore focused our attention on the only nucleotides (Ϫ3T, Ϫ2T), which were specific to the chimpanzee sequence and absent from the human and baboon apo(a) sequences. This strategy was straightforward, because reverting these two nucleotides TT at positions Ϫ3 and Ϫ2 in the chimpanzee promoter to the CC found in the human sequence, reduced its activity to a level typical of that of the human promoter (Fig. 4). However, even if these results demonstrated that positions Ϫ3 and/or Ϫ2 are essential to the high level of transcription observed with the chimpanzee promoter, our experiments showed that position ϩ8 was also required. We had to mutate the 3 positions (Ϫ3/Ϫ2/ϩ8) in the human apo(a) construct to increase its activity to a level similar to that obtained with the chimpanzee apo(a) sequence (see 5Ј⌬pHUmut.-3,-2,ϩ8, Fig. 4). Positions Ϫ3/Ϫ2/ϩ8 flank the major transcription start site of the human apo(a) mRNA (12). Because the TATA box is conserved in the chimpanzee sequence, it is likely that transcription is initiated in the same region for both apo(a) genes. It is conceivable that the Ϫ3/Ϫ2/ϩ8 base changes in the chimpanzee apo(a) sequence improve the strength of the promoter by increasing the ratio of productive to abortive initiation of transcription by the RNA polymerase. Indeed, variations in promoter sequence occurring in the vicinity of the transcription start site have been shown to influence strongly the abortive initiation process (the synthesis and release of abortive short (2-15 bases) transcripts) and therefore transcription efficiency (36). Alternatively, base changes at positions Ϫ3/Ϫ2/ϩ8 in the chimpanzee apo(a) sequence could favor the binding of a specific transcription activator. This hypothesis would be consistent with the results of the EMSA experiments showing that the DNA-protein complexes formed with the Ϫ14 to ϩ 11 apo(a) region were different when using either the chimpanzee or the human sequence (Fig. 5). This factor could cooperate with the transcription factor HNF-1␣, which binds immediately downstream of the mRNA start site and ensures optimal transcriptional activity of the human apo(a) promoter in vitro (25). The exact mechanism(s) by which positions Ϫ3/Ϫ2/ϩ8 potentiate the transcriptional activity of the chimpanzee promoter remains to be determined.
Sequence variations in the regulatory regions of the apo(a) gene are thought to predominantly determine the difference in plasma Lp(a) levels observed between individuals exhibiting similar apo(a) size isoforms. Notably, Suzuki et al. (14) have observed that certain haplotypes defined in the 1.4-kb 5Ј-region of the human apo(a) gene were associated with specific in vitro promoter activities that correlated with the in vivo levels of Lp(a). These results support a role of the 5Ј-flanking region of the apo(a) gene in determining, to a significant degree, variations in plasma Lp(a) levels. The results of our study are also in good agreement with this hypothesis, because elevated mean Lp(a) concentrations in the chimpanzee (2-to 4-fold higher than in humans) are associated with marked elevation of the transcriptional activity of the chimpanzee apo(a) promoter. However, the relatively weak in vitro activity of the human 1.4-kb apo(a) promoter has suggested that other genetic elements may play a role in regulating apo(a) expression. Recent studies have notably revealed the presence of potential enhanc-ers located 20 -28 kb 5Ј upstream of the apo(a) gene that significantly increased apo(a) basal promoter activity in vitro (37,38). The chimpanzee therefore appears to constitute a useful model in which to evaluate the importance of these enhancers, and studies are currently underway to determine whether such elements are also present upstream of the chimpanzee apo(a) gene.