(Received for publication, November 10, 1995; and in revised form, January 16, 1996)
From the
Three proximal regulatory elements, AIB, AIC, and AID, of the
apoA-I gene are necessary and sufficient for its hepatic expression in vivo and in vitro. DNA binding and competition
assays showed that elements AIB and AID contain hormone response
elements composed of imperfect direct repeats that support the binding
of the hepatic nuclear factor-4, other nuclear orphan receptors, and
the ligand-dependent nuclear receptors retinoic X receptor (RXR),
RXR
/RAR
, and RXR
/T
R
. Substitution
mutations on repeats 1 and 2 in the hormone response sites of elements
AIB and AID, respectively, abolished the binding of all nuclear
receptors and reduced promoter activity to background levels,
indicating the importance of both hormone response elements for the
hepatic expression of the apoA-I gene. Cotransfection experiments in
HepG2 cells with normal and mutated promoter constructs and plasmids
expressing nuclear hormone receptors showed that RXR
homodimers
transactivated the wild type promoter 150% of control, in the presence
of 9-cis-retinoic acid (RA), whereas
RXR
/T
R
heterodimers repressed transcription to
60% of control, in the presence of T
. RXR
/RAR
and
hepatic nuclear factor-4 did not affect the transcription, driven by
the proximal apoA-I promoter. Potassium permanganate and dimethyl
sulfate interference experiments showed that RXR
homodimers,
RXR
/RAR
, and RXR
/T
R
heterodimers
participate in protein-DNA interactions with 12, 13, and 11 out of the
14 nucleotides, respectively, that span repeats 1 and 2 and the spacer
region separating them on the hormone response element of element AID.
The binding of RXR
homodimers and RXR
/T
R
heterodimers is associated with ligand-dependent activation by
9-cis-RA or repression by T
. Upon deletion or
mutation of repeat 1, homodimeric binding of RXR
is lost whereas
heterodimeric binding is retained. This heterodimeric binding to the
mutated element AID is mediated solely by interactions with repeat 2
and one adjacent nucleotide and is confined to a heptameric core
recognition motif. The interactions of the RXR
heterodimers with
repeat 2 are associated with low levels of ligand-independent
transcriptional activity. The findings suggest that the specific types
of homo- and heterodimers of nuclear hormone receptors occupying the
hormone response elements of apoA-I and the availability of the ligand
may play an important role in the transcriptional regulation of the
human apoA-I gene.
Epidemiological data and transgenic animal experiments have
shown that increased apoA-I and high density lipoprotein levels are
protective against atherosclerosis(1, 2) . Thus the
mechanisms which regulate the synthesis of apoA-I are important.
Previous studies have shown that the regulatory elements AIB
(-128/-77), AIC (-175/-148), and AID
(-220/-190) are sufficient for hepatic expression of the
human apoA-I gene in tissue culture (3, 4) and in
transgenic mice(5) . It has been shown that the regulatory
element AIC is recognized by heat stable factors related to
CCAAT/enhancer-binding protein (C/EBP) which act as positive regulators
and by heat labile activities, one of which acts as a negative
regulator(4) . In addition, the regulatory element AID contains
a hormone response element (HRE) ()that is recognized by
ARP-1, a transcriptional repressor of apoA-I(6) , hepatic
nuclear factor-4 (HNF-4)(7) , RXR
homodimers,
RXR
/RAR
, RXR
/ARP-1(8, 9) , and
RXR
/PPAR heterodimers(10) . All these factors are members
of the steroid/thyroid receptor superfamily, that includes receptors
for steroids, thyroid hormone, and retinoic acids as well as orphan
nuclear receptors with unidentified ligands. In the present study we
have demonstrated the existence of an additional HRE on the regulatory
element AIB between nucleotides -132/-119 and we have
investigated the role of both HREs and of different types of hormone
nuclear receptors on the transcriptional regulation of the human apoA-I
gene. We demonstrate that both HREs are recognized by homodimers of
HNF-4, ARP-1, EAR-2, EAR-3, and RXR
as well as heterodimers of
RXR
with RAR
or T
R
, and that both elements
are essential for the hepatic expression of the apoA-I gene. Binding of
the RXR
homodimers on the HRE of element AID requires direct
repeats 1 and 2 and leads to ligand-dependent transcriptional
activation whereas binding of the RXR
/RAR
and
RXR
/T
R
heterodimers on this HRE may occur on
direct repeats 1 and 2 or only on repeat 2. When bound on both repeats
1 and 2, the RXR
/T
R
heterodimers repress
transcription in the presence of T
, whereas the
RXR
/RAR
heterodimers and HNF-4 do not affect the
transcription. In addition, binding of the RXR
heterodimers to
only one repeat on the HRE of element AID is associated with low levels
of ligand-independent transcriptional activity. The findings
demonstrate that hormone nuclear receptors can modulate the
transcription of the human apoA-I gene and thus may affect plasma
apoA-I and high density lipoprotein levels.
T DNA ligase, polynucleotide kinase, Vent
polymerase, and restriction enzymes were purchased from New England
Biolabs. Transformation competent bacterial HB101 cells were purchased
from Life Technologies, Inc.. [
-
P]ATP (5000
Ci/mmol), [
-
P]dCTP,
[
H]acetyl coenzyme A (200 mCi/mmol),
[
-
P]dGTP (4000 Ci/mmol), and Econofluor
scintillation fluid were purchased from DuPont NEN. Chloramphenicol was
purchased from Sigma. Reagents for automated DNA synthesis were
purchased from Applied Biosystems, Inc. The sequencing kit was
purchased from U. S. Biochemical Corp. IB2 silica gels were purchased
from J. T. Baker Chemical Co. Bacto-tryptone and Bacto-yeast extract
were purchased from Difco. O-Nitrophenyl-
-D-galactopyranoside was purchased
from Sigma. Double-stranded poly(dI-dC) was purchased from Pharmacia
LKB Biotechnology Inc. Acrylamide, sodium dodecyl sulfate (SDS), urea,
Tris, and the anti-Flag antibody were purchased from International
Biotechnologies, Inc. The 5x Reporter Lysis Buffer was purchased from
Promega.
Figure 1:
Panel A, nucleotide sequence of the
regulatory element AID and part of the regulatory element AIB of the
human apoA-I gene. The arrows indicate the position of repeats
1 and 2 and the putative repeat 3 of the two HREs. The nucleotides of
the repeats are numbered 1 to 6 on the noncoding strand. Nucleotides in
the spacer region or nucleotides 5` of the first nucleotide of a repeat
are numbered -1 and -2. Panels B and C, DNA binding gel electrophoresis of orphan and ligand-dependent
nuclear receptors using the wild-type regulatory elements AIB (Panel B) and AID (Panel C) as probes. The probes
utilized are indicated in the bottom of the figure and their sequence
is shown in Table 2. The fast migrating bands which appear in the
RXR and T
R
lanes in Panel B represent
activities present in COS-1 extracts which bind weakly to the AIB
probe. The extracts utilized in the binding assays are indicated at the
top of the figure. NE indicates rat hepatic nuclear extracts.
HNF-4, RXR
etc. indicate extracts of COS-1 cells transfected with
vectors expressing HNF-4, RXR
, etc. Panel D, DNA binding
supershift assays using a monoclonal anti-RAR
antibody and an
anti-flag antibody against the flagged derivative of human
T
R
. Note that the monoclonal anti-RAR
antibody
supershifted the RXR
/RAR
heterodimer and the anti-flag
antibody supershifted the RXR
/T
R
heterodimer. The
electrophoresis experiment shown in D was performed at 150
volts, for 4 h, in order to better separate the supershifted bands. As
a result, the free probe has run out of the
gel.
To explore potential
differences in the binding requirements of different members of the
hormone nuclear receptor family we generated a series of nucleotide
substitution mutations in the elements AIB and AID, designated AIBM,
AIDM, AIDM1, AIDM2, AIDM3, AIDM4, and AIDM5 (Table 2). AIBM and
AIDM mutations span part of both repeats of element AIB or only the
second repeat of element AID, respectively. Mutation AIDM1 is localized
upstream of the HRE on element AID, whereas mutation AIDM2 contains
nucleotide substitutions in repeat 1. Finally, the AIDM3 and AIDM4
mutations contain substitutions in repeat 2, and the AIDM5 mutation
contains substitutions in the putative repeat 3. We also generated two
deletion mutations, one of repeat 1 and one of putative repeat 3, in
the HRE of element AID, which were designated AIREP1 and
AI
REP3, respectively. The oligonucleotides containing these
mutations, shown in Table 2, were tested in DNA binding and
competition experiments. These analyses showed that the AIDM1 mutation
had no effect on the binding of rat hepatic nuclear extracts (compare lane 1 with lane 7 of Fig. 2A). The
AIDM2 mutation increased substantially the binding of the slower
migrating activity present in the rat liver nuclei (compare lane 1 with lane 9 of Fig. 2A). The AIDM3 and
AIDM4 mutations did not bind substantially or compete for the binding
of hepatic nuclear activities to element AID (Fig. 2A, lanes
5, 6, 11, and 13). The DNA-protein complexes formed with
AIDM4 are not competed out by the wild type AID sequences (data not
shown). Thus, these complexes must originate from the binding to the
mutated probe of activities unrelated to nuclear hormone nuclear
receptors which do not normally bind to the wild type probe. The AIBM
mutation and the AIDM mutation which altered drastically the HREs of
element AIB and AID, respectively, abolished the binding of all nuclear
activities to element AID (Fig. 2D, lanes 8 and 16), whereas deletion or mutation of the putative repeat 3 and
deletion of repeat 1 (AI
REP3, AIDM5, and AI
REP1) did not
affect qualitatively the binding of hepatic nuclear activities to this
site (Fig. 2, E and F).
Figure 2:
A-D,
DNA binding gel electrophoresis assay of orphan and ligand-dependent
nuclear hormone receptors using various mutated sequences of the
regulatory element AID as probe. The probes utilized are indicated at
the bottom of each figure and their sequences are shown in Table 2. The extracts utilized are indicated on the top of the
figure and have been described in the legend to Fig. 1. Panel A, binding and competition assays of hepatic nuclear
extracts to the wild type (AID) and the mutated (AIDM1, AIDM2, AIDM3,
and AIDM4) probes. Panel B, binding of orphan nuclear
receptors to the wild type (AID) and the mutated (AIDM1, AIDM2, AIDM3,
and AIDM4) probes. Panel C, binding of ligand-dependent
nuclear receptors to the wild-type (AID) and the mutated (AIDM2, AIDM3,
and AIDM4) probes. Panel D, binding of rat liver nuclear
extracts, orphan and ligand-dependent nuclear receptors to probes
having mutations in both repeats of element AIB (AIBM) or in the second
repeat of element AID (AIDM). Panel E, binding of rat liver
nuclear extracts, orphan and ligand-dependent nuclear receptors to
probes having deletions of either the putative repeat 3 (A1REP3)
or repeat 1 (A1
REP1) of element AID. Panel F, binding of
rat liver nuclear extracts, orphan, and ligand-dependent nuclear
receptors to a probe having nucleotide substitution in the putative
repeat 3 (AIDM5) of element AID.
We have also tested
the effects of the deletion and oligonucleotide substitution mutations
within the two regulatory elements AID and AIB (Table 2) on the
binding of orphan and ligand-dependent nuclear receptors. This analysis
showed that the AIDM1 mutation did not affect the binding of the
homodimers of the orphan receptors HNF-4, ARP-1, EAR-2, and EAR-3 (Fig. 2B). The remainer of the mutations affected the
binding of the orphan receptors differentially. Binding of HNF-4 was
moderately affected by changes in repeat 1 (AIDM2 mutation) and it was
greatly affected by changes in the first half of repeat 2 (AIDM3
mutation). Finally, binding was diminished by changes in the second
part of repeat 2 (AIDM4 mutation). Binding of ARP-1, a known negative
regulator of apoA-I(6) , was not greatly affected by the first
three mutations (AIDM1, AIDM2, and AIDM3), whereas binding of the
orphan receptors EAR-2 and EAR-3 was mainly affected by the AIDM3
mutation (Fig. 2B). Deletion of repeat 1 of element AID
(AIREP1) reduced the binding of HNF-4 but did not affect
qualitatively the binding of the other orphan receptors ARP-1, EAR-2,
and EAR-3 (Fig. 2E), whereas deletion or mutations in
the putative repeat 3 of element AID (AI
REP3 and AIDM5) did not
affect the binding of any of the orphan receptors (Fig. 2, E and F). Finally the mutations AIBM and AIDM, which
altered drastically the HREs of elements AIB and AID, respectively,
abolished the binding of all the orphan nuclear receptors to this site (Fig. 2D). The findings suggest that the orphan nuclear
receptors have different binding specificities for the regulatory
element AID of apoA-I, although in most cases binding is affected
considerably by changes in repeat 2. The analysis with the
ligand-dependent nuclear receptors showed that the AIDM2 and
AI
REP1 mutations which affect the first repeat and the AIDM3 and
AIDM4 mutations which affect the second repeat in the HRE of element
AID abolished the binding of RXR
homodimers (Fig. 2, C and E). The AIDM4 mutations affected mainly the binding
of RXR
/RAR
heterodimers. The AIDM2, AIDM3, and AIDM4
mutations decreased the binding of RXR
/T
R
heterodimers. Deletion of repeat 1 of element AID (AI
REP1) did not
affect qualitatively the binding of RXR
/RAR
and
RXR
/T
R
heterodimers (Fig. 2E).
Deletion or mutations in the third repeat of the element AID (AIDM5 and
AID
REP3) did not affect qualitatively the binding of RXR
homo- and heterodimers (Fig. 2, E and F). In
addition, the AIDM2 mutation formed a DNA-protein complex of higher
mobility than the complex formed with the wild type oligonucleotide or
those carrying the AIDM3 and AIDM4 mutations (Fig. 2C, lane
6). Finally, the AIBM and AIDM mutations, which altered
drastically the HREs of the regulatory elements AIB and AID,
respectively, eliminated the binding of all combinations of
ligand-dependent nuclear receptors to the mutated probe (Fig. 2D).
In summary, the findings of Fig. 2, A-F, demonstrate that binding of RXR
homodimers requires intact repeats 1 and 2 in the HRE of element AID,
whereas binding of RXR
/RAR
and RXR
/T
R
heterodimers can still occur when short mutations are introduced in
either repeat 1 or repeat 2. Similarly, the binding of the orphan
nuclear receptors is differentially affected by the various mutations.
Binding of HNF-4 requires the intact second part of repeat 2, whereas
binding of EAR-2 and EAR-3 requires the intact first part of repeat 2
of element AID. Additionally, binding of ARP-1 is affected only by
extensive mutagenesis of repeat 2. Finally, the binding of crude rat
liver nuclear extracts parallels that of HNF-4.
Figure 3: Effect of deletion or nucleotide substitutions in the HRE of elements AID and AIB on the apoA-I promoter strength in HepG2 cells. The localization of the promoter mutations is shown in Table 2.
More
detailed analysis of the effect of nucleotide substitution or deletion
mutations in the regulatory element AID on the promoter strength showed
that the AIDM2 mutation which altered nucleotides within the first
repeat and the deletion (AIREP1) of the first repeat of element
AID reduced the promoter strength to approximately 50 and 35% of
control, respectively. The AIDM3 and AIDM4 mutations which altered
nucleotides within the second repeat reduced the promoter strength to
30 and 15% of control, respectively. A mutation 5` of repeat 1 (AIDM1)
and a mutation within the putative third repeat (AIDM5) reduced the
promoter strength to 75 and 90% of control, respectively, and deletion
of the putative repeat 3 (AI
REP3) increased slightly the promoter
strength to 115% of control (Fig. 3). The combined data of Fig. 2and Fig. 3indicate that mutations in the
regulatory element AID and AIB, which diminish the binding of hepatic
nuclear activities and all types of nuclear receptors to the two HREs
also decrease proportionally the strength of the apoA-I promoter.
Mutations in repeat 2 of the HRE of element AID affected more severely
the promoter strength as compared to those in repeat 1 (Fig. 3).
Figure 4:
A-I, KMnO and dimethyl sulfate (DMS) modification pattern of the DNA-protein complexes formed
with the RXR
homo- and heterodimers using the wild type element
AID as probe (Table 2). The RXR
homo- and heterodimers were
produced by expression of the corresponding cDNAs in COS-1 cells. The
KMnO
and dimethyl sulfate modification pattern of RXR
homodimers with the coding and noncoding strand of element AID is shown
in Panels A and B, respectively. Panel C is
a summary of the interference pattern deduced from the findings of Panels A and B. The KMnO
and dimethyl
sulfate modification pattern of RXR
/RAR
heterodimers with the
coding and noncoding strand of element AID is shown in Panels D and E, respectively. Panel F is a summary of the
interference pattern deduced from the findings of Panels D and E. The KMnO
and dimethyl sulfate modification
pattern of RXR
/T
R
heterodimers with the coding
and noncoding strand of element AID is shown in Panels G and H, respectively. Panel I is a summary of the
interference pattern deduced from the findings of Panels G and H. F indicates free probe; and B indicates probe
recovered from the DNA-protein complex after chemical treatment. Strong
interactions are illustrated with large rectangles for the
RXR
homodimers, ovals for the RXR
/RAR
heterodimers, and diamonds for the
RXR
/T
R
heterodimers. Weak interactions are
illustrated with small rectangles, ovals, or diamonds. The nucleotide sequence of the coding and noncoding
strand of element AID is indicated on each side of Panels A, B, D,
E, G, and H. Nucleotides which participate in DNA protein
interactions are indicated by an asterisk (*).
The KMnO and dimethyl sulfate
modification analysis was also utilized to determine the mode of
binding of RXR
/RAR
or RXR
/T
R
heterodimers on the wild type element AID. The analysis with the
RXR
/RAR
heterodimers showed that all the nucleotides of
repeats 1 and 2, which participate in DNA-protein interactions with the
RXR
homodimers also participate in DNA-protein interactions with
the RXR
/RAR
heterodimers. In addition, nucleotide 4 of the
noncoding strand of repeat 1 participates in weak DNA-protein
interactions with the RXR
/RAR
heterodimers (Fig. 4, D-F). Overall 13 of the 14 nucleotides that form repeats 1 and
2 and the putative spacer region between them participate in
interactions with this heterodimer. Six of the nucleotides are in
repeat 1, six in repeat 2, and one in the spacer region between repeats
1 and 2. Ten of the nucleotides participate in strong, and three
participate in weak DNA-protein interactions. The nucleotides which
participate in weak interactions are nucleotides 3 and 4 of the
noncoding strand and nucleotide -1 of the coding strand. Repeat 3
is not involved in the binding of RXR
/RAR
heterodimers (Fig. 4F). The analysis with the
RXR
/T
R
heterodimers showed that all but one of
the nucleotides of repeats 1 and 2 which participate in DNA-protein
interactions with the RXR
homodimers also participate in
interactions with the RXR
/T
R
heterodimers. The
oligonucleotide which does not participate in DNA-protein interactions
with the RXR
/T
R
heterodimers is nucleotide 3 of
the noncoding strand of repeat 1 (Fig. 4, G and H). Overall, four nucleotides of repeat 1 and seven
nucleotides of repeat 2 and the putative spacer region between them
participate in DNA-protein interactions with the
RXR
/T
R
heterodimers. Six of the nucleotides
participate in strong and the remaining five in weak DNA-protein
interactions. The nucleotides which participate in strong interactions
are nucleotides 5 of repeat 1 and 5 and 6 of repeat 2 of the coding
strand and nucleotides 1, 2, and 3 of repeat 2 of the noncoding strand.
The remaining nucleotides as well as the nucleotide -1 in the
putative spacer region between repeats 1 and 2 participate in weak
DNA-protein interactions. Residues of the putative repeat 3 are not
involved in the binding of the RXR/T
R
heterodimers (Fig. 4, G-I).
Figure 5:
A-H, KMnO and dimethyl sulfate
modification pattern of the DNA-protein complexes formed with the
RXR
/RAR
or RXR
/T
R
heterodimers using
the mutated element AIDM2 or AI
REP1 as probes (Table 2). The
RXR
/RAR
and RXR
/T
R
heterodimers were
produced by expression of the corresponding cDNAs in COS-1 cells. The
KMnO
and dimethyl sulfate modification pattern of
RXR
/RAR
heterodimers with the coding and noncoding strand of
element AIDM2 is shown in Panels A and B,
respectively. Panel C is a summary of the interference pattern
deduced from the findings of Panels A and B. The
KMnO
and dimethyl sulfate modification pattern of
RXR
/T
R
heterodimers with the coding and noncoding
strand is shown in Panels D and E, respectively. Panel F is a summary of the interference pattern deduced from
the findings of Panels D and E. The KMnO
and dimethyl sulfate modification pattern of RXR
/RAR
heterodimers with the coding and noncoding strand of the AI
REP1
probe is shown in Panels G and H. Panel I is
a summary of the interference pattern deduced from the findings of Panels G and H. F indicates the free probe, and B indicates the probe recovered from the DNA-protein complex after
chemical treatment. Strong interactions are illustrated with ovals for the RXR
/RAR
heterodimers, and diamonds for
the RXR
/T
R
heterodimers. Weak interactions are
illustrated with small ovals or diamonds. The
nucleotide sequence of the coding and noncoding strand of the mutated
element AID is indicated on each side of Panels A, B, D, E, G,
and H. Nucleotides which participate in DNA-protein
interactions are indicated by an asterisk (*).
The KMnO and dimethyl sulfate modification pattern of
the RXR
/T
R
heterodimers with the mutated AIDM2
probe is shown in Fig. 5, D and E,
respectively, and summarized in Fig. 5F. This analysis
showed that the binding of the RXR
/T
R
heterodimers to the mutated AIDM2 probe is confined to a heptameric
core recognition motif. The seven nucleotides of the coding and
noncoding strand which participate in DNA-protein interactions with the
RXR
/T
R
heterodimers are identical to those which
participate in DNA-protein interactions with the RXR
/RAR
heterodimers. The exception here is that only 2 residues, nucleotides 5
and 6 of the coding strand of repeat 2, participate in strong
DNA-protein interactions and the remaining in weak interactions. The
putative repeat 3 does not participate in any DNA-protein interactions
with the RXR
/T
R
heterodimers. The KMnO
and dimethyl sulfate modification pattern of the
RXR
/RAR
heterodimers with the mutated AID
REP1 probe is
shown in Fig. 5, G and H, and summarized in Fig. 5I. The pattern is similar to that obtained with
the AIDM2 probe which carries mutations in the first repeat. The only
exception is that nucleotide 5 on the coding strand of the putative
third repeat also participates in weak DNA-protein interactions.
The
combined data of Fig. 4, A-I, and 5, A-I,
indicate that a heptameric core recognition motif is the minimum
sequence required for the binding of RXR/RAR
and
RXR
/T
R
heterodimers to the regulatory element
AID. The binding of the RXR
homodimers requires the presence of
both direct repeats of element AID. Finally, the putative repeat 3 has
minimal participation in DNA-protein interactions with homo- or
heterodimers of RXR
. Fig. 6A shows a summary of
all the nucleotides which participate in DNA-protein interactions with
the various combinations of the ligand-dependent nuclear receptors. Fig. 6B summarizes the mode of binding of the
ligand-dependent nuclear receptors RXR
, RXR
/RAR
, and
RXR
/T
R
on the wild type and the mutated
regulatory element AID of the human apoA-I promoter. This summary is
based on the DNA binding data of Fig. 1and Fig. 2and
KMnO
and dimethyl sulfate interference analyses pattern of Fig. 4, A-I, and 5, A-I.
Figure 6:
Panel A, schematic representation of the
nucleotides in the HRE of regulatory element AID which participate in
DNA-protein interactions with the RXR homodimers and the
RXR
/RAR
or RXR
/T
R
heterodimers. Panel B, schematic representation of the mode of interaction
of the RXR
homodimers and RXR
/RAR
and
RXR
/T
R
heterodimers with the normal and the
mutated HREs of the regulatory element AID. The figure is deduced from
the data of Fig. 4and 5.
Figure 7:
A-D, effect of RXR homo- and
heterodimers and HNF-4 on the transactivation of the
(-264/+5) apoA-I promoter in HepG2 cells, in the presence or
absence of ligands. Panel A shows transactivation by RXR
homodimers in the presence of 10
M 9-cis-RA (closed squares) or the absence of any
ligand (closed diamonds). Panel B shows lack of
transactivation by RXR
/RAR
heterodimers in the presence of
10
M 9-cis-RA (closed
squares), 10
M all-trans-RA (closed circles), and a trend toward repression in the absence
of any ligand (closed diamonds). Panel C shows
repression by RXR
/T
R
heterodimers in the presence
of 10
M T
(closed
circles), transactivation at low T
R
concentrations in the presence of 10
M 9-cis-RA, lack of transactivation at higher
T
R
concentration (closed squares), and lack
of transactivation in the absence of any ligand (closed
diamonds). Panel D shows lack of transactivation by low
concentrations of HNF-4 and a trend toward repression by higher
concentrations of HNF-4.
Figure 8:
Effect on selected mutations in the HRE of
element AID (Table 2) on the apoA-I promoter strength and its
transactivation by HNF-4 and ligand-dependent nuclear receptors. Panel A, effect of the AIDM1 to AIDM4 mutations on the apoA-I
promoter strength (gray bars) and its transactivation by HNF-4 (black bars). The mutations tested are indicated at the bottom
of the figure and are described in Table 2. Panel B, effect of the AIDM2 and AIREP1 mutations on the
transactivation of the apoA-I promoter by RXR
homodimers. Panel C, effect of the AIDM2 and AI
REP1 mutations on the
transactivation of the apoA-I promoter by RXR
/RAR
heterodimers. Panel D, effect of the AIDM2 and AI
REP1
mutations on the transactivation of the apoA-I promoter by
RXR
/T
R
heterodimers.
Cotransfection experiments with RXR/RAR
heterodimers
showed that the promoter which lacks repeat 1 (AI
REP1) exhibited
30-35% activity and was not affected by RXR
/RAR
heterodimers in the presence or absence of 9-cis-RA or
all-trans-RA. The promoter which carries a mutation in the
first repeat of element AID (AIDM2) was transactivated 2-fold by
RXR
/RAR
heterodimers in the presence or absence of any of the
two ligands (Fig. 8C). Finally, cotransfection
experiments with RXR
/T
R
heterodimers showed that
the promoter which lacks the first repeat had the same 30-35%
activity and was not affected by the RXR
/T
R
heterodimers and their ligands. The promoter which carries a mutation
in the first repeat of element AID (AIDM2) was transactivated 1.3-fold
by RXR
/T
R
heterodimers in the absence of any of
the two ligands, was transactivated 2.1-fold in the presence of
9-cis-RA, and was not affected by T
(Fig. 8D). It should be noted that binding of the
RXR
/T
R
heterodimers to this mutated promoter
sequence generates a slow migrating DNA-protein complex (Fig. 2C).
The combined data of Fig. 4, Fig. 5, and Fig. 8indicate that binding of
RXR/RAR
and RXR
/T
R
heterodimers to
repeat 2 of element AID is associated with low levels of
ligand-independent transcriptional activity whereas binding of homo- or
heterodimers of RXR
to repeats 1 and 2 can lead to
ligand-dependent activation or repression of transcription.
Drastic mutations on the second
repeat in the HRE of element AID which eliminated the binding of orphan
and ligand-dependent nuclear receptors also diminished the apoA-I
promoter strength and its ability to be transactivated or repressed by
them. On the other hand, limited mutations in repeat 2 (AIDM3 and
AIDM4) which reduced the promoter strength to 30 and 15% of control,
respectively, resulted in the diminished binding of liver nuclear
extracts and of HNF-4 as well as other orphan receptors and
ligand-dependent nuclear receptors to the mutated sites. The findings
suggest that an intact repeat 2 in the HRE of element AID is required
for optimal hepatic expression of the apoA-I gene. Interestingly,
certain mutations which diminished the binding of HNF-4 did not affect
considerably the binding of ARP-1, and of EAR-2 and EAR-3 to the
mutated probes. ARP-1, EAR-2, and EAR-3 which usually act as
repressors, exhibit a wider tissue distribution than HNF-4 but are also
expressed, at lower concentrations, in liver and intestinal cells as
compared to HNF-4 (20) . The promoter mutations which allow
preferential binding of ARP-1, EAR-2, and EAR-3 but diminish binding of
liver nuclear extracts and HNF-4 decreased substantially the apoA-I
promoter strength, thus supporting further the role of HNF-4 as a
positive regulator of the hepatic expression of the human apoA-I gene.
The mutagenesis analysis also established that the binding RXR
homodimers have a strict requirement for intact repeats 1 and 2 in the
HRE of element AID. On the other hand, binding of the RXR
/RAR
heterodimers is affected mostly by alterations in repeat 2, and binding
of the RXR
/T
R
heterodimers is affected by
alterations in both repeats 1 and 2 of this HRE.
Interestingly,
mutations in repeat 1 (AIDM2) of element AID produced a slower
migrating DNA-RXR/T
R
complex, compared to that
formed with the wild type or other mutated AID probes. The mobility of
this complex is similar to that formed with hepatic nuclear extracts
using the same mutated probe. The origin of this complex remains
unclear. DNA binding assays with the wild type element AID and 1:1
ratio of COS-1 extracts enriched in RXR
or T
R
have provided a tentative explanation for the observed higher mobility
DNA-RXR
/T
R
complex. We have found that
supplementation of the COS-1 extracts with 9-cis-RA favors the
formation of both the higher mobility RXR
/T
R
complex and a RXR
homodimeric complex that displays intermediate
mobility between the high and the low DNA-RXR
/T
R
complex. In contrast, supplementation of the COS-1 extract mixed with
T
favors the formation of the faster migrating complex
(data not shown). Thus, it is possible that the fast-migrating complex
may represent a DNA-RXR
/T
R
dimer which is
accessible to T
(21) and the slow-migrating complex
may represent a higher order complex between
RXR
/T
R
and a third protein, and that the
formation of this complex is enhanced by the presence of
9-cis-RA(22, 23) .
An important feature of the HREs is the
number of nucleotides separating the two repeats (spacer region). It
has been proposed that spacing determines the type of homo- or
heterodimers of receptors that bind to an
HRE(15, 16, 24) . It has been suggested that
RXR homodimers require direct repeats with a spacing of one nucleotide
(DR1) for binding(25, 26, 27, 28) ,
RXR/RAR heterodimers can bind to DR1, DR2, or DR5s (28, 29, 30, 31) , whereas
RXR/TR heterodimers prefer DR4s for
binding(16, 32, 33) . Nevertheless,
exceptions to this rule have been noted(15, 18) .
Repeats 1 and 2 on element AID of apoA-I have been classified as a DR2,
whereas repeats 2 and 3 can be classified as a DR1 (15) .
Similarly, repeats 1 and 2 of element AIB can be classified as a DR1.
As shown in this study, contrary to the rule, the HREs of apoA-I are
recognized both by homodimers of RXR
as well as heterodimers of
RXR
with RAR
or T
R
. Furthermore, the
nucleotides which participate in the binding of these homo- and
heterodimers are nearly identical. It has been shown that binding of
homodimeric RXR
is dramatically influenced by the nature of the
nucleotide preceding both AGG/TTCA motifs(34) . According to
the data, RXR
homodimers preferentially interact with direct
repeats containing either an A or a G immediately upstream of the
AGG/TTCA motif, whereas repeats which contain either a T or a C at the
same position have greatly reduced binding(34) . As shown in Fig. 1A, both repeat 1 and repeat 2 in the HRE on the
noncoding strand of element AID are preceded by a G nucleotide.
Similarly repeats 1 and 2 of the HRE on the noncoding strand of element
AIB are preceded by G and A, respectively. In contrast, the putative
repeat 3 of the HRE on the noncoding strand of element AID which does
not participate appreciably in protein-DNA interactions is preceded by
a C nucleotide. Interestingly, the RXR
/RAR
and
RXR
/T
R
heterodimers also have the ability to
interact with repeat 2 and one or two neighboring nucleotides,
suggesting that their minimum requirement for binding to this HRE is a
heptameric core recognition motif rather than a hexameric motif, as
suggested previously(18) . In this latter case, one should not
exclude the possibility of extensive nonspecific but nevertheless
stabilizing interactions of the heterodimers with the phosphate
backbone of the mutated probe(35, 36) .
It has
been shown previously that the HRE in element AID is a RXR response
element(8, 9, 19) . This element, when placed
in front of a minimal promoter was transactivated 12-fold by RXR,
in the presence of 9-cis-RA in CV-1 cells (19) and
8-fold by all-trans-RA in HepG2 cells(9) . The minimal
thymidine kinase promoter carrying the HRE of element AID was
transactivated 10-fold by the RXR
/RAR
heterodimers, in the
presence of all-trans-RA, and 25-fold in the presence of
9-cis-RA in CV-1 cells(19) . HREs from other promoters
linked to the minimal thymidine kinase promoter could also confer
transactivation of the heterologous promoter by RXR
/RAR
heterodimers, whereas larger promoter constructs containing other HREs
could be either activated or repressed by RXR
/RAR
heterodimers (19) . In general, it has been proposed that the
RXR/RAR complexes can function both as repressors or activators of gene
expression, depending upon the nature of the direct repeats. Repression
was generally observed by DR1s, whereas activation can be observed by
certain DR1 as well as by DR2 and DR5
motifs(22, 23, 28, 29) . Finally the
minimal thymidine kinase promoter under the control of the HRE of
element AID was not transactivated by RXR/T
R heterodimers.
These heterodimers in the presence of T
(37) were
shown previously to bind mainly on DR4 motifs (38, 39, 40) and to activate transcription in
the presence of T
.
Our findings show that the extent of
transactivation observed using minimal promoters carrying the HRE of
element AID are much greater than the extent of transactivation
observed by the RXR homodimers and the RXR
/RAR
heterodimers, when this HRE exists in the context of the proximal human
apoA-I promoter (-264/+5). Consistent with these findings, a
recent study has shown that retinoids can increase 100 to 150% above
control the apoA-I promoter activity and by 25 to 30% the apoA-I mRNA
and protein in HepG2 cells(41) . Similar to other promoter
systems we suggest that in the context of the entire apoA-I promoter
the nuclear hormone receptors which occupy elements AIB and AID form
stereospecific DNA-protein complexes which interact directly or
indirectly via the TATA box binding protein associated factors with the
proteins of the basal transcription system(42, 43) .
Other proteins bound to the proximal apoA-I promoter may help to orient
properly the nuclear receptor molecules bound to the two sites and
optimize their interactions as well as their interactions with the
proteins of the basal transcriptional machinery. It is expected that
the complex formed with the heterologous promoters which contain the
HRE are different than the complexes formed with the intact apoA-I
promoter and this may explain the relatively large increase in
transactivation observed in the heterologous promoter constructs. We
must also point out that the minimal reporter constructs containing the
HREs utilized in the previous studies have very low levels of promoter
activity. Thus the 8- to 15-fold transactivation achieved by the
RXR
homodimers and RXR
/RAR
heterodimers using the
heterologous promoters is low compared to the activity of the intact
apoA-I promoter. Previous studies have established that the proximal
-264/+5 apoA-I promoter region is sufficient for hepatic
transcription both in vivo(5) and in
vitro(3, 4) , therefore the transactivation
levels observed in this study using the proximal apoA-I promoter by
homo- and heterodimers of RXR
in hepatic cells, reflect more
closely the physiological situation.
The present
as well as previous studies demonstrate that a large number of
transcription factors that belong to the steroid/thyroid receptor
superfamily bind to the HREs on the regulatory elements AIB and AID of
apoA-I promoter. It is believed that the major role of RXR is to
modulate a number of different hormonal signaling pathways through the
formation of heterodimers. In addition, RXR has the ability to form
homodimers that respond to 9-cis-RA. The dual function of RXR
would always be under the tight control of intracellular signals such
as the retinoids, thyroid hormone, or other unidentified ligands, that
will ensure the activation of one or more specific signaling pathways.
Regarding apoA-I gene regulation the present study indicates that
increase in the concentration of RXR and 9-cis-RA will
accelerate transcription. On the other hand, at a given RXR
concentration, an increase in the concentration of T
R
and T
or an increase in ARP-1, EAR-2, and EAR-3 will
repress transcription. Repression in transcription may occur either by
sequestering RXR or by displacing the binding of other positive
regulators(46) . Finally, an increase in the concentration of
RAR
and all-trans-RA or of HNF-4 will lead to
intermediate levels of transcription. Overall, the specific types of
homo- and heterodimers which can occupy the HREs of apoA-I and the
availability of ligands in the cell nucleus may determine the extent of
transcriptional activation of the human apoA-I promoter.