From the INSERM, Unité 551, Dyslipoprotéinémies, Athérosclérose:
Génétique, Métabolisme et Thérapeutique,
Hôpital de la Pitié, 83 Boulevard de l'Hôpital,
Paris 75651 Cedex 13, France, § CV Therapeutics, Palo Alto,
California 94304, and the ¶ International Center for Medical
Research, Franceville BP769, Gabon
Received for publication, March 12, 2001, and in revised form, April 10, 2001
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ABSTRACT |
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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 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 (<0.001 to >1 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 (7-9). 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 5'-flanking region of the gene may also contribute to variance in
plasma Lp(a) levels (12-14). 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 5'-region of
the apo(a) gene, are associated with Lp(a) levels
(15-18).
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 = 0.61 mg/ml
versus 0.18 mg/ml) or in African populations for which the
highest mean Lp(a) levels (~0.30-0.40 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 ± 121 kDa versus
789 ± 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 5'-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
5'-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.
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 5'-Region of the Chimpanzee apo(a)
Gene--
The 5'-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
5' 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-µl 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
MgCl2 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 5'- and 3'-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 Plasmid Constructs--
The 1.4-kb fragment of the chimpanzee
apo(a) 5'-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 5'-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 5'-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 +93, was equally cloned in the
pGL3-basic expressing vector (plasmid pHU). To generate the 5'-deletion
constructs 5' Site-directed Mutagenesis--
Substitution mutants for
positions 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 × 105 cells
were plated into six-well dishes, grown for 24 h, and then incubated with 1 ml of serum-free medium containing 2 µg of plasmid DNA, 0.3 µg of a Electrophoretic Mobility Shift Assay--
HepG2 nuclear extracts
were prepared from confluent 75-mm flasks by the method described by
Dignam et al. (23). Aliquots of nuclear extracts were stored
at Sequence Analysis--
Fig. 1 shows
a comparison of the sequence of the 1.4-kb 5'-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 Analysis of the Chimpanzee apo(a) Promoter Activity--
We next
compared the promoter activity of the 1.4-kb fragment of 5'-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 5'-region of the human apo(a) gene had previously shown that the essential regulatory elements were located in the very
proximal 5'-flanking sequence. Furthermore, deletion of sequences Electrophoretic Mobility Shift Analysis of the Elevated plasma levels of Lp(a) are a significant independent risk
factor for cardio- and cerebrovascular diseases in humans (27-31). 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 5'-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 5'-region of the apo(a) genes at positions Sequence analysis of the 1.4-kb 5'-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
5'-flanking region of the apo(a) gene and a C>T polymorphism at position +93 that introduces an additional upstream ATG
initiation codon with its own in-frame stop codon (12, 13). High repeat
lengths (n = 10, 11) of the PNPR and the T allele at position
+93 have been shown to be significantly associated with low levels of
Lp(a) in human populations (15-18). 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 variant of the human apo(a) gene (15).
Therefore, the constant and low copy number (n = 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 +93 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 +93 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) 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 5'-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
5'-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 enhancers located 20-28 kb 5' 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.
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Oligonucleotides and conditions for amplification
1410 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-µl
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.
pCH and 5'
pHU, in which the luciferase gene is
driven by the minimal apo(a) promoter sequence (
98 to
+141), plasmids pCH and pHU were digested by MluI (5'
cloning site, see above) and PvuII (
98) 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).
3 and
2 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 (CC
TT) and the chimpanzee (TT
CC)
promoters. Briefly, the mutagenic oligonucleotides (mutTT-2-3:
5'-TAATGTTTGAATTCTGCTGAGCCAG-3' or mutCC-2-3:
5'-TAATGTTTGAACCCTGCTGAGTCAG-3') 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
5'
pCHmut.-3,-2 and 5'
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 5'
pHU as described above to simultaneously substitute
positions
3,
2 (CC
TT) along with a third position (
51 G
C,
or
24 A
T, or +8 C
T, or +65 G
A, or +94 G
A) by the
nucleotides found in the chimpanzee sequence at the respective
positions. Mutations at positions
2,
3, and +8 were introduced
using the mutagenic oligonucleotide mut-2-3+8 (5'-TAATGTTTGAATTCTGCTGAGTCAG-3'). 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
51:
5'-ATCTATTGACATTCCACTCTC-3', position
24:
5'-TATAAGACTCTTTATTCAAGG-3', position +65:
5'-GGTTTGTGGATGCGTTTACTC-3', position +94:
5'-GTCAACAACATCCTGGGATTG-3'). After a first screening of the mutated constructs by restriction analysis, the sequences of the selected mutants (5'
pHUmut.-3,-2,-51,
5'
pHUmut.-3,-2,-24, 5'
pHUmut.-3,-2,+8, 5'
pHUmut.-3,-2,+65, and
5'
pHUmut.-3,-2,+94) were verified by sequencing.
-galactosidase expression plasmid (pSV-gal, Promega, France), and 5 µg 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.
70 °C. Protein concentration was determined with the
bicinchoninic acid protein assay reagent (BCA, Pierce). For
electrophoretic mobility shift assay (EMSA), 0.25 pmol of
32P-end-labeled double-strand oligonucleotide was mixed
with 6 µg of nuclear extract in a final volume of 20 µl containing
10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 3 mM MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol, 5% glycerol, 2 µg of
poly(dI-dC)·poly(dI-dC), 4 mM spermidine, and 1 µg 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
70 °C. The sequences
of the oligonucleotides were as follows:
5'-TAATGTTTGAATTCTGCTGAGTCAG-3' (CH probe) and 5'-TAATGTTTGAACCCTGCTGAGCCAG-3' (HU probe)
corresponding to the
14 to +11 region of the chimpanzee and human
apo(a) genes, respectively. The nonspecific competitor had
the following sequence: 5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3'.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1400 bp in humans. The number of repeats varies
from 5 to 12 in human populations (15, 16, 18). In the 5'-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 +93 (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).
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Fig. 1.
Comparison of the 5'-flanking regions of the
chimpanzee (CH) and human (HU)
apo(a) genes. Nucleotide numbering starts from
the transcription initiation site (+1). The TATA box and the binding
site for the transcription factor HNF-1 as described by Wade et
al. (25) for the human apo(a) gene are shown by
heavy lines above the sequence. The star
indicates the position of the +93 C/T polymorphism present in the human
gene. The boxes indicate base changes in the
apo(a) chimpanzee sequence at positions that are conserved
in humans and baboon.
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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.
1301 to
98 had little to no effect on promoter activity (25). Therefore, we compared the transcriptional activities of the
98 to
+141 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 5'-deletion mutants of pCH and pHU were generated using
the PvuII site located at position
98. Transient
transfection assays revealed that the chimpanzee short construct
5'
pCH had approximately 5-fold higher transcriptional activity than
the corresponding human plasmid 5'
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
98 to +141. These results provided a firm indication that base
differences located specifically in the very proximal 5'-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 significantly 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) 5' sequence, whereas they were conserved in both
humans and baboon (boxed in Fig. 1). Among these nine
differences, only two adjacent base changes (CC
TT), at positions
3
and
2 from the start site of transcription, were present in the
98 to +141 region (in boldface characters in Fig. 1).
Consequently, site-directed mutagenesis was used to evaluate the effect
of this set of change (CC
TT) 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
3 and
2 with CC resulted in a significant reduction in
transcriptional activity to the level of its human counterpart (Fig.
4, see plasmid
5'
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 5'
HUmut.-3,-2). Therefore, the thymidine residues
at positions
3 and/or
2 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
3 and
2, the two species differ at five additional
positions (
51,
24, +8, +65, and +94, see Figs. 1 and 4) in the
98
to +141 region. Each one of these bases was mutated independently in
the short human construct 5'
pHU along with positions
3 and
2.
Transient transfection assays revealed that substituting the nucleotides at positions
3,
2, and +8 in 5'
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
5'
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
3 and
2 (Fig.
4).
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Fig. 3.
Comparison of the transcriptional activity of
the 5'-flanking sequences of the human (HU) and
chimpanzee (CH) apo(a) genes in HepG2
cells. Plasmid constructs contain either 1.4-kb of
apo(a) 5'-flanking sequence (pCH and
pHU) or the minimal 98 to +141 apo(a) promoter
region (5'
pHU and 5'
pCH) cloned upstream of the firefly
luciferase reporter gene. Each vector was co-transfected with a
-galactosidase expression plasmid. Error bars represent
standard error of the mean of at least four independent experiments
performed in duplicate or triplicate. Luciferase activity is normalized
to
-galactosidase activity. Results obtained with the 1.4-kb
fragments or the minimal promoters are presented relative to the values
obtained with the human constructs (pHU and
5'
pHU, respectively) set arbitrarily at
100%.
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Fig. 4.
Mutational analysis of the chimpanzee and
human apo(a) minimal promoter regions. Expression
vectors 5' pHU and 5'
pCH contain the
98 to +141 regions of the
human and chimpanzee apo(a) genes, respectively. The base
differences between the human and chimpanzee apo(a)
sequences and their respective positions to the transcription start
site (+1) are indicated. Substitution mutants of 5'
pHU and 5'
pCH
were obtained by site-directed mutagenesis as described under
"Experimental Procedures," and the nucleotides mutated are
underlined. Normalized luciferase activities are relative to
5'
pHU set arbitrarily at 100%.
14 to +11 Region
of the apo(a) Promoter--
Transient transfection experiments
indicated that positions
3/
2 and +8 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
14 to +11 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 +8 was substituted with a C or a T
in the CH and HU probes, respectively, thereby suggesting that
positions
3 and/or
2 exert major influence on the formation of the
specific complexes observed for each sequence (data not shown).
View larger version (62K):
[in a new window]
Fig. 5.
Electromobility shift assays of
apo(a) 14 to +11 regions. Gel shift studies
were performed using either the chimpanzee
14 to +11
apo(a) sequence (probe CH) or the corresponding human
sequence (probe HU). HepG2 nuclear extracts (6 µg) 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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3/
2 and +8 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.
98 to +141 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 +93. 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 at
position
51 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 +94 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 (
3T,
2T), 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
3 and
2 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
3 and/or
2 are essential to the
high level of transcription observed with the chimpanzee promoter, our
experiments showed that position +8 was also required. We had to mutate
the 3 positions (
3/
2/+8) in the human apo(a) construct
to increase its activity to a level similar to that obtained with the
chimpanzee apo(a) sequence (see 5'
pHUmut.-3,-2,+8, Fig.
4). Positions
3/
2/+8 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
3/
2/+8 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
3/
2/+8 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
14 to + 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
3/
2/+8 potentiate the transcriptional activity of the chimpanzee
promoter remains to be determined.
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FOOTNOTES |
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* This work was supported by INSERM and by a Contrat de valorisation INSERM/BAYER-PHARMA (92094).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AY028467.
To whom correspondence should be addressed: Tel.:
33-1-42-17-78-78; Fax: 33-1-45-82-81-98; E-mail:
thillet@ext.jussieu.fr.
Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M102204200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Lp(a), lipoprotein (a); apo(a), apolipoprotein (a); PCR, polymerase chain reaction; kb, kilobase(s); bp, base pair(s); EMSA, electrophoretic mobility shift assay; PNRP, pentanucleotide repeat polymorphism.
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REFERENCES |
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