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INTRODUCTION |
Apolipoprotein(a) is the characteristic protein component of the
lipoprotein particle Lp(a). Lp(a) consists of a low density lipoprotein
covalently linked to apo(a) through its protein moiety, apolipoprotein
B-100 (1, 2). Most prospective studies and a recent
meta-analysis have identified Lp(a) excess as a major risk
factor of premature atherosclerotic vascular disease (3-6), although
there are clearly some exceptions (7). Apo(a) is a highly polymorphic
glycoprotein with a close sequence homology to plasminogen that
contains from 12 to 50 copies of a domain homologous to plasminogen
kringle 4 (8, 9). These sequence similarities constitute a basis to
explain the correlation between high Lp(a) concentration and
atherosclerosis. Through its apo(a) moiety, Lp(a) competes with
plasminogen for binding to fibrin. This prevents plasminogen conversion
to plasmin, which in turn hinders fibrinolysis and activation of latent
transforming growth factor-
, contributing to an atherogenic
phenotype (10-16).
In contrast to other lipoproteins, Lp(a) plasma concentration varies
widely (<0.1 to >100 mg dl
1) in individual humans, and
at least 90% of this variation is attributable to the apo(a) genetic
locus (17, 18), indicating that the regulation of apo(a) expression is
significant in controlling pathological levels of Lp(a). Generally,
Lp(a) plasma concentration is inversely proportional to the number of
apo(a) kringle repeats (19). However, other features of the apo(a)
locus contribute to determine Lp(a) plasma levels since Lp(a) plasma
concentration can vary several hundredfold in individuals with the same
number of kringles (20, 21). Sex steroid hormones and particularly estrogens are known to lower Lp(a) levels. Population studies have
shown that Lp(a) levels increase 8-13% in postmenopausal women
relative to premenopausal controls (22, 23). Longitudinal studies have
reported that hormone replacement therapy lowers Lp(a) concentration as
much as 50% (24-27). Data from transgenic mice containing the human
apo(a) gene locus on a yeast artificial chromosome corroborate these
results (28). Pharmacological doses of 17
-estradiol lowered plasma
apo(a) protein concentration and hepatic apo(a) mRNA concentration
by ~80% in these mice (29).
In this study, we describe the identification of the site responsible
for estrogen regulation of apo(a) gene expression. Previous work from
this laboratory has shown that a DNA region located from
98 to +130
relative to the apo(a) transcription start site is sufficient to drive
apo(a) transcription in a reporter vector (30). This region binds to
the liver-enriched transcription factor hepatocyte nuclear factor-1
and partially accounts for the liver-specific synthesis of apo(a). The
homologous apo(a) and plasminogen genes are organized in a tandem
head-to-head configuration, with 35 kb1 of genomic DNA separating
their 5'-ends (31, 32). Sequences in this "intergenic" region
contribute to the regulation of apo(a) expression. Its deletion in the
above-mentioned genomic yeast artificial chromosome severely curtails
apo(a) expression in the transgenic
mouse.2 Several DNase
I-hypersensitive sites are located in this region ("DHI-DHIV")
(33), two of which correspond to apo(a) gene regulatory sequences. An
enhancer contained within a LINE retrotransposon lies 18 kb 5' of the
apo(a) promoter and corresponds to hypersensitive site DHIII (34, 35),
whereas an enhancer requiring the Sp1 and PPAR transcription factors
corresponds to the DHII element, located 26 kb upstream of the apo(a)
promoter (34). Here we report that the DHII element is also responsible
for estrogen inhibition of apo(a) expression. Estrogen receptor-
does not appear to bind directly to the DNA sequence element, but to
interfere with transcription factor(s) necessary for the DHII enhancer activity.
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MATERIALS AND METHODS |
Plasmid Construction--
The pGL3a reporter plasmid (35)
contains the Photinus pyralis (firefly) luciferase cDNA
driven by the apo(a)
98/+130 minimal promoter (30). The
apo(a)/plasminogen intergenic region was isolated from a bacterial
artificial chromosome clone containing ~150 kb of the
apo(a)/plasminogen locus, and restriction fragments were subcloned into
the pGL3a plasmid (35). The DHII fragment was generated by polymerase
chain reaction synthesis from the appropriate subclone with primers
DHII/L (5'-AAGGAGCCCTGAGCCTGAA-3') and DHII/R
(5'-TGCCATAAATATACAAGTCCCT-3') containing either an EcoRI or
a SacI restriction site. The polymerase chain reaction product was subcloned into the EcoRI site of pGL3a or into
the SacI site of either the pGL3-Promoter vector (Promega),
which contains the SV40 early promoter region, or the hsvTK vector. Constructs ee-I + ee-II and ee-II were generated by polymerase chain
reaction synthesis with primers derived from the sequence shown in Fig.
2A containing the EcoRI restriction site,
followed by ligation into the EcoRI site of pGL3a.
Constructions of the hsvTK vector, containing the
105/+10 herpes
simplex virus thymidine kinase promoter fragment, and of the expression
vectors for wild-type and mutant ER-
have been described and were
kindly provided by Sotirios Karathanasis (36). The vitERE plasmid
contains a copy of the vitellogenin gene ERE cloned in front of the
thymidine kinase promoter in the hsvTK vector. Site-specific
mutagenesis was carried out according to the mismatch primer protocol
(37) with oligonucleotides DHII/L and DHII/R as the external primers. The internal primers contained the mutations described in Fig. 3. The
polymerase chain reaction products were digested with EcoRI, subcloned into pGL3a, and confirmed by sequencing prior to the transfection assay.
Generation of HepG2-ER Cells--
To generate the HepG2-ER
cells, human HepG2 hepatoma cells (ATCC HB8065) were transfected with
the pSV2neo/CMV-ER-
expression vector as described (38), except that
the ER-
cDNA corresponds to the HEGO sequence (39). Cells were
cultured in Eagle's minimal essential medium (Life Technologies, Inc.)
supplemented with 1 mM HEPES, 2 mM glutamine,
0.1 mM minimal essential medium non-essential amino acids,
1.0 mM sodium pyruvate, 50 µg/ml gentamycin, 10% fetal
bovine serum, and 10 nM ICI 164,384 during selection in 1000 µg/ml G418 (Life Technologies, Inc.). Stable ER-
-expressing clones were identified by Western blotting (38) using anti-ER-
antibodies (kindly provided by G. Greene).
Cell Culture and Transfection Assays--
HepG2-ER cells were
maintained Eagle's minimal essential medium supplemented with 1 mM HEPES, 2 mM glutamine, 0.1 mM
minimal essential medium non-essential amino acids, 1 mM
sodium pyruvate, 1000 µg/ml G418, 50 µg/ml gentamycin, and 10%
charcoal/dextran-treated fetal bovine serum (Hyclone Laboratories).
Approximately 2 × 105 cells/well were seeded in a
12-well cell culture plate 24 h prior to transfection by a calcium
phosphate precipitation method according to the manufacturer's
protocol (Promega) as we described previously (30). Transfections were
carried out in triplicates. Briefly, 1.5 µg of pGL3a-based plasmid
and 0.15 µg of either pSV-
-galactosidase (Promega) or pRL-TK
(Promega) control plasmid were added to each well, covered with 2 ml of
maintenance medium. Following a 6-h transfection, fresh medium was
added containing either 100 nM 17
-estradiol (Sigma) in
ethanol or ethanol alone to a final concentration of 0.01%. After a
36-h incubation with one medium change, the cells were harvested and
lysed, and expression activity was determined using either the Dual
Light kit (Tropix Inc.) or the Dual Luciferase Reporter kit (Promega)
according to the protocols provided. To account for transfection
efficiency, results are reported as the ratio of the sample to control
plasmid activity.
Gel-shift Analysis--
Nuclear extracts from HepG2 or HepG2-ER
cells were prepared as described (30). The following oligonucleotide
probes were used: ee-II,
5'-CATGTTGACACAGGTCAAATCCTtgaacTCTGTTGCCCAAATA-3'; and ee-IImut,
where the nucleotides indicated in lowercase in ee-II were changed to
CCTAG. A complementary oligonucleotide was synthesized, and the probe
was end-labeled with [
-32P]ATP. The procedure for gel
mobility shift assay was as described (40), except that the binding
buffer contained 60 mM KCl and 10% (w/v) Ficoll. Human
recombinant estrogen receptor-
was from PanVera (Madison, WI).
Polyclonal antibodies against human ER-
and ARP-1
(apoA-I regulatory protein) were
from Santa Cruz Biotechnology (Santa Cruz, CA). The sequence of the
ARP-1 oligonucleotide used for gel-shift competition was
5'-CTAGCGATATCATGACCTTTGTCCTAGGCCTC-3' (41).
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RESULTS |
Identification of an Apo(a) Gene Estrogen-responsive
Element--
The 35-kb region between the apo(a) and plasminogen genes
was isolated in a bacterial artificial chromosome clone, and fragments were subcloned in front of the apo(a) minimal promoter and luciferase cDNA as reported (35). The plasmids were tested in HepG2 cells that
had been modified to produce estrogen receptor-
(HepG2-ER cells).
Following transfection, the cells were treated with 100 nM
17
-estradiol (E2) for 36 h, and luciferase activity
was determined. The expression level of the minimal apo(a) promoter
reporter (construct pGLa3) (Fig. 1) was
not affected by E2 treatment. No constructs showed
significant up-regulation by E2. Only the construct
spanning the DHII enhancer showed significant reduction of luciferase
activity in the presence of E2 (construct 20). This genomic
region was tested in both orientations (constructs 20+ and 20
) (Fig.
1) because its enhancer activity had been found to be
orientation-dependent (34). The enhancer activity and
E2 response were larger with the insert cloned in the same
orientation relative to the apo(a) minimal promoter as it is in the
chromosomal locus (defined as the positive orientation). It is
noteworthy that expression from the reporter plasmids containing the
two other regions previously shown to have enhancer properties,
constructs 14 and 24, was not significantly affected by E2
(34, 35, 42).

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Fig. 1.
Apolipoprotein(a)/plasminogen intergenic
region. The chromosomal region separating the apo(a) and
plasminogen (plg) genes is shown at the top. Their
respective direction of transcription is indicated by the
arrows at the extremities of the map. The numbering
indicates the fragments subcloned into the luciferase reporter vector.
Fragments 1 and 24 contain the first EcoRI site upstream of
the apo(a) and plasminogen gene transcription start sites, respectively
(cf. Ref. 33). DNase I-hypersensitive sites are indicated by
vertical arrows. Below is shown the transcriptional activity
of the reporter constructs in HepG2-ER cells. HepG2-ER were transfected
with 1.5 µg of reporter construct/well (of a 12-well plate) together
with 0.15 µg of pSV- -galactosidase. Following transfection, the
cells were grown in the absence (stippled bars) or presence
(black bars) of 100 nM 17 -estradiol for
36 h. Transcriptional activities, normalized to
pSV- -galactosidase activity, are expressed relative to the activity
of the pGL3a construct, the plasmid containing the luciferase reporter
driven by the 98/+130 apo(a) minimal promoter. The data represent the
mean ± S.D. of three transfections.
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Identification of the Minimal Estrogen-responsive Unit--
To
define the minimal estrogen-responsive unit (ERU) located within
fragment 20, we characterized the KpnI-SduI
subfragment shown in Fig. 2. When
transfected into HepG2-ER cells, this fragment was capable of
increasing by 10-fold the luciferase expression driven by the apo(a)
minimal promoter in a orientation-independent fashion. After
E2 treatment, luciferase expression was reduced by ~70%.
Fig. 2A highlights the DNA sequences required for the enhancer activity (ee-I and ee-II) (34). Reporter vectors containing both enhancer elements I and II exhibited significant enhancer activity, which was sharply reduced by E2. Enhancer element
II alone had a small effect compared with enhancer elements I + II, but
was still E2-responsive. However, when enhancer element II was cloned as a tandem duplicate in the reporter vector, enhancer activity and E2 response were the most dramatic. All the
responses were essentially independent of the orientation of the cloned construct.

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Fig. 2.
Identification of the minimal
estrogen-responsive unit. A shows a blow-up of region
20 of Fig. 1. The KpnI-SduI fragment is the DHII
region containing the essential elements of the enhancer, shown in the
boxes underneath. B shows the transcriptional
activity of the reporter constructs in HepG2-ER cells. The cloning
orientation is shown in the construct diagram on the left. Details of
the transfection and data analysis are as described in the legend to
Fig. 1. Stippled and black bars indicate that the
cells were grown in the absence and presence of 100 nM
17 -estradiol, respectively. plg, plasminogen;
luc, luciferase.
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The relationship between the ERU and the apo(a) minimal promoter was
studied by subcloning the DHII enhancer in both orientations in front
of two heterologous promoters, the SV40 early promoter and the hsvTK
promoter (data not shown). Upon transfection of HepG2-ER cells, the
DHII enhancer increased 3-fold the transcription driven by the SV40
promoter, but had no effect on the hsvTK promoter. The SV40 promoter
construct showed a small reduction in luciferase activity following
E2 treatment. A similar reduction was observed for the
DHII/SV40 construct, suggesting that there is no additional contribution by the DHII insert to E2 response mediated by
the SV40 promoter. These results with heterologous promoters suggest that the DHII enhancer/ERU requires a synergistic interaction with
factors bound to the apo(a) minimal promoter.
The estrogen responsiveness induced by the DHII element is dependent on
both ER-
and E2. In fact, when normal HepG2 cells (lacking ER-
) were transfected with the DHII/apo(a) minimal promoter reporter vector, there was no change in luciferase activity in response
to E2 treatment. Cotransfection with an expression vector for ER-
made normal HepG2 cells estrogen-responsive. This
responsiveness was partially competed by the estrogen receptor
antagonist ICI 182,780 at a concentration of 1 µM (data
not shown).
Mutation Analysis of Enhancer Element II--
Inspection of the
sequence of ee-II reveals a very close match to the consensus ERE
(shown in boldface in Figs. 2A and 3), the
DNA-binding site of ER-
(43). An ERE is characterized by a 6-base
pair palindromic repeat separated by three nucleotides. The palindromic
repeat in ee-II is separated by six nucleotides. We carried out
extensive mutagenesis of this site (shown in Fig. 3) and tested the constructs by
transfecting HepG2-ER cells. Mutations in the right arm of the ERE
reduced the enhancer activity, but retained estrogen responsiveness, as
did inserting six nucleotides between the left and right arms of the
potential binding site. Mutations in the left arm eliminated both the
enhancer activity and estrogen response. The observation that only
mutations in the left arm of the potential ERE are effective in
impairing estrogen response suggests that ER-
might not be bound to
the full palindromic site in a canonical manner. However, binding of
some trans-acting factor to the left arm sequence is
suggested. Furthermore, the observation that estrogen response
impairment is accompanied by enhancer activity impairment suggests that
ligand-bound ER-
interferes with the function of factors necessary
to ee-II activity.

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Fig. 3.
Mutation analysis of the estrogen-responsive
unit. The sequence at the top highlights the potential ERE in
ee-II (cf. Fig. 2). The nucleotide changes in the mutated
constructs are shown underneath. Details of the transfection and data
analysis are as described in the legend to Fig. 1. Stippled
and black bars indicate that the cells were grown in the
absence and presence of 100 nM 17 -estradiol,
respectively.
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ER-
Domain Requirement for E2
Responsiveness--
To gain more insight on the mechanism of
E2-dependent repression of the DHII enhancer,
we carried out an analysis of which ER-
domains are required to that
effect. To this end, normal HepG2 cells were cotransfected with the
DHII/apo(a) minimal promoter reporter vector or a control vector
carrying the luciferase gene driven by the vitellogenin ERE/hsvTK
promoter (Fig. 4, shaded and
white bars, respectively) plus either wild-type or mutant ER-
. As the first group of bars reiterates, estradiol treatment increases activity from the vitellogenin ERE, but reduces activity from
the apo(a) gene element. ER-
is composed of one DNA-binding domain
(DBD), responsible for binding to the ERE (44), and two transactivation
domains, N-terminal AF1 and C-terminal AF2 (39). Changing the DBD
binding selectivity from an ERE to a glucocorticoid receptor element by
a point mutation (mutant AF1-X-AF2) completely destroys
E2 responsiveness from the ERE/hsvTK plasmid as expected, but does not eliminate the inhibitory effect of E2 on the
DHII enhancer. Deletion of the AF1 domain also nearly fully impairs response from the ERE/hsvTK plasmid, but not the E2
inhibitory response from the DHII enhancer (mutant
X-DBD-AF2). Inactivation of the AF2 domain reduces ERE/hsvTK
plasmid activation, but retains the E2 effects the DHII
enhancer (mutant AF1-DBD-X). Finally, inactivation of both
transactivation domains reduces ERE/hsvTK plasmid activation and
completely abolishes DHII enhancer repression (mutant
X-DBD-X). The requirement for intact
transactivation domains supports the hypothesis that ER-
interacts
with other transcription factors involved in the DHII enhancer
activity, thus interfering with their function.

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Fig. 4.
Analysis of the ER-
domain requirement for estrogen responsiveness. Normal HepG2
cells were cotransfected with 1.5 µg of either the DHII/pGL3a
construct (shaded bars) or the vitERE/hsvTK construct
(white bars) together with 0.15 µg of pRL-TK (expressing
Renilla reniformis luciferase) and 0.15 µg of expression
vector encoding ER- with the indicated truncations and point
mutations (indicated by X). Following transfection, the
cells were grown in the absence (unhatched bars) or presence
(hatched bars) of 100 nM 17 -estradiol for
36 h. Growth medium was changed once. Transcriptional activities,
normalized to pRL-TK activity, are expressed relative to the activity
of the vitERE/hsvTK construct, the activity of which (cut off in the
figure) was set to 100%. The data represent the mean ± S.D. of
three transfections.
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That a functional ER-
DNA-binding domain is not necessary for
E2 responsiveness is a strong indication that ER-
exerts
its activity without a direct interaction with DNA. To confirm this hypothesis, we subjected an oligonucleotide probe encompassing the
sequence of ee-II (cf. Fig. 2A) to
electrophoretic mobility shift assay. The formation of a complex
between the DNA probe and nuclear protein extracts made from HepG2
cells (Fig. 5, lane 2) was
competed by the addition of purified human recombinant ER-
(lane 5). Similar results were obtained by incubating the probe with nuclear as well as whole cell protein extracts made from
HepG2-ER cells in the presence or absence of 100 nM
E2 (data not shown). The addition of polyclonal antibodies
against ER-
to the incubation mixture failed to supershift any band
in the assay (lane 7). These findings support the conclusion
that ER-
is not bound to ee-II DNA. Similar results were obtained by
using as a probe an oligonucleotide containing the GTTCA
CTAGG
mutation, which abolishes the enhancer and estrogen response activity
of the ERU (cf. Fig. 3). Inspection of the sequence around
this site reveals a potential binding site for the nuclear orphan
receptor ARP-1, a member of the steroid receptor superfamily (41).
Competition gel-shift experiments with a consensus ARP-1-binding
sequence (Fig. 5, lane 4) and gel-supershift experiments
with polyclonal antibodies against ARP-1 (lane 8) imply that
ARP-1 does not bind the probe sequence either.

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Fig. 5.
Gel-shift analysis of the ERU. An
oligonucleotide spanning the ee-II sequence (see Fig. 2A)
was used as a probe for a gel-shift assay (lanes 1-8). The
same probe was used in lanes 9-16 except for a GTTCA ctagg mutation, which inactivates enhancer/estrogen response activity.
Lanes 1 and 9 contain probe only, whereas all
other lanes contain 5 µg of HepG2 nuclear extract. Lanes 3 and 11 and lanes 4 and 12 contain a
100-fold excess of unlabeled probe and ARP-1 oligonucleotide
competitor, respectively. Lanes 5 and 13 contain
0.3 µg of purified human recombinant ER- . Lanes 6 and
14 contain 0.2 µg of goat immunoglobulin G. Lanes
7 and 15 and lanes 8 and 16 contain 0.2 µg of goat anti-ER- and anti-ARP-1 immunoglobulin G,
respectively.
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DISCUSSION |
Sex steroid hormones are the most potent known regulators of
apo(a) gene expression and concomitant plasma concentration of Lp(a).
Previous studies have described chromosomal elements involved in apo(a)
expression, a 300-base pair promoter encompassing the apo(a)
transcription start site and putative apo(a) enhancers located 18 and
26 kb upstream (30, 34, 35). Control elements located at large
distances from the promoter are involved in the regulation of numerous
other genes, including other apolipoproteins (45-48). Here we report
that the DNA region containing the enhancer element at 26 kb from the
apo(a) promoter also promotes 17
-estradiol responsiveness in a
reporter vector in transfected hepatic cells. Its position coincides
with a DNase I-hypersensitive site (33). Such sites are normally
associated with an open chromatin structure, an indication that the DNA
region is in a favorable conformation to interact with transcription
factors (49). The activity of the 700-base pair fragment containing the
DHII enhancer (construct 20 in Fig. 1) was
orientation-dependent, the activity being stronger when the
fragment was cloned in the same orientation relative to the apo(a)
promoter as it is in the chromosomal locus. A larger 2-kb fragment was
reported to lack enhancer activity when cloned in the opposite
orientation to the apo(a) promoter (34). For this reason, we failed to
detect this enhancer in our previous screening for apo(a) regulatory
elements. It is not clear what gives rise to this phenomenon. It is
possible that additional negative regulatory or insulator elements are
present downstream of the DHII enhancer, to ensure proper separation
and differential regulation of the apo(a) and plasminogen genes.
The orientation dependence is essentially lost when the
enhancer/estrogen-responsive region is deleted down to its core
elements. The DHII enhancer was shown to require a 186-base pair region encompassing two footprints (ee-I and ee-II in Fig. 2). The Sp1 and
PPAR transcription factors are required for ee-I activity, and the PPAR
for ee-II activity (43). (Such PPAR sites could be involved in the
reported effect of retinoids on apo(a) expression (50).) We found that
the ee-II region alone was sufficient to confer E2
responsiveness to the apo(a) minimal promoter reporter vector. Although
this region contains a potential ERE sequence, our data indicate that
direct binding of ER-
to this DNA site does not seem to be involved.
Mutations in the right arm of the ERE do not impair E2
responsiveness. ER-
mutants lacking a functional ERE DNA-binding
domain are still efficient, and gel shifts and supershifts fail to
yield any evidence of ER-
binding. On the other hand, the
requirement for functional transactivation domains suggests that ER-
interacts with a transcription factor necessary for the enhancer
activity, interfering in this way with its function. The gel-shift data
indicate that the presence of ER-
prevents the interaction between
other nuclear proteins and the ERU. This interaction is crucial to its
function. In fact, an oligonucleotide bearing a mutation that destroys
enhancer and estrogen response activity is impaired in its ability to
form the same complexes that are inhibited by ER-
.
Interaction of ER-
with other transcription factors has been
reported for a number of gene regulatory units. In some cases, no
direct binding of the receptor to a canonical ERE sequence is involved.
For example, in erythroid precursor cells, ER-
binds to the
transcription factor GATA-1 through its AF2 domain without direct or
indirect DNA binding, preventing the activation of GATA-1-requiring genes (51, 52). Another well characterized mechanism by which nuclear
receptors have been shown to modulate gene expression is competition
for common non-DNA-binding cofactors with other transcription factors
(36, 53). This possibility is unlikely due to the observation that
binding of a transcription factor to a DNA target seems to be involved
in the DHII enhancer/ERU activity. In fact, mutations at a specific
site of the ERU destroy all DHII activity. This observation also argues
against the possibility that ER-
exerts its effect indirectly by
inhibiting the expression of factors required for DHII activity.
The identity of the factor interacting with ER-
is not determined.
The sequence spanning the nucleotides whose mutation was crucial
contains a potential binding site for the nuclear orphan receptor
ARP-1, but gel-shift competition and gel-supershift experiments did not
yield evidence to support its role in ERU function.
The physiological relevance of the DHII enhancer/ERU remains to be
established in vivo. Experiments are under way to delete this region from a yeast artificial chromosome transgenic mouse carrying the apo(a) genomic locus. It is interesting to note that, although apo(a) expression is responsive to estrogen, plasminogen expression is not. The homologous apo(a) and plasminogen genes are
separated by only ~35 kb of genomic DNA. Their minimal promoter regions have significant sequence and functional similarities, including the requirement for hepatocyte nuclear factor-1
. It will
be of interest to discover how the two genes are differentially regulated. It can be suggested that some form of insulator element (54,
55) separates the last apo(a) control region (probably the DHII region)
from the plasminogen promoter.