Volume 271,
Number 14,
Issue of April 5, 1996 pp. 8402-8415
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Binding
Specificity and Modulation of the ApoA-I Promoter Activity by Homo- and
Heterodimers of Nuclear Receptors (*)
(Received for publication, November 10, 1995; and in revised form, January 16, 1996)
Iphigenia
Tzameli,
Vassilis
I.
Zannis (§)
From the Section of Molecular Genetics, Center for Advanced
Biomedical Research, Cardiovascular Institute and the Departments of
Medicine and Biochemistry, Boston University Medical Center, Boston,
Massachusetts 02118-2394
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- ACKNOWLEDGEMENTS
- REFERENCES
ABSTRACT 
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/TR 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/TR 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/TR
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.
INTRODUCTION
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 TR, 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/TR 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/TR 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.
MATERIALS AND METHODS
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.
Plasmid Constructions and CAT Assays
The apoA-I promoter
region was derived from an apoA-I genomic clone as described previously (11) . The proximal promoter region spanning nucleotides
-264/+5 was subcloned into the Asp718 and SmaI restriction sites of the pUCSH-CAT plasmid to produce the
CAT derivative designated
[-264/+5]apoA-I-CAT(4) . This reporter
construct was used to create mutated apoA-I promoter constructs. The
mutated -264/+5 CAT constructs containing deletions or
nucleotide substitutions in elements AIB and AID were generated by
amplification of the parent pUCSH[-264/+5] apoA-I
CAT construct(4) . For example, in order to generate the
mutation AIDM1, the region upstream of nucleotide -186 was
amplified by PCR using the PCR-264AI 5` primer which contains an Asp718 and EcoRV restriction sites and the mutagenic
AIDM1 primer (Table 1). The region downstream of nucleotide
-225 was amplified by using the AIDM1C mutagenic primer and the
PCR-19AI 3` primer which contains an SspI restriction site. An
aliquot containing 5% of the two amplified regions was used as a
template for further amplification by the 5` and 3` primers PCR-264AI
and PCR-19AI. The amplified DNA was digested with Asp718 and SspI and cloned into the Asp718 and SmaI
sites of pUCSH-CAT(4) . The remaining mutations were
constructed in a similar manner using the amplification primers shown
in Table 1. PCR reactions were performed using the Perkin Elmer
automated thermocycler according to the manufacturer's
specifications. The sequence of the final constructs was determined by
DNA sequencing. Oligonucleotides used were synthesized by the
solid-phase phosphite triester method using an automated AB-380B
oligonucleotide synthesizer.
Preparation of Nuclear Extracts and Whole Cell Extracts
from Transfected COS-1 Cells
Nuclear extracts were prepared from
livers of 10 rats (approximately 120 g of liver) as
described(12) . Extracts from COS-1 cells transfected with the
pMT2 vector carrying full-length cDNAs for HNF-4, ARP-1, EAR-2, EAR-3,
RXR, and TR were prepared as
described(6) . Similarly prepared were extracts for COS-1 cells
transfected with the pSG5 expression vector carrying a flag epitope
fused to the NH
-terminal of TR.
Gel Electrophoretic Mobility Shift Assay
This
analysis was performed using either crude hepatic nuclear extracts or
COS-1 whole cell extracts as described(13) . Competitors were
used at 50-100-fold excess. In supershift assays, various
dilutions of polyclonal or monoclonal antibodies were added to the
reaction mixture prior to the addition of the probe.
Dimethyl Sulfate and Potassium Permanganate Interference
Assays
For the methylation interference assay, single stranded
DNA (5 pmol) was end-labeled with T polynucleotide kinase
and annealed to its complementary unlabeled strand. Double stranded DNA
(10
cpm) was treated with dimethyl sulfate for 3 min at
room temperature in the presence of 2 µg of salmon sperm DNA. For
permanganate interference assay, single-stranded DNA (10 cpm) was treated with potassium permanganate (KMnO)
for 10 min at room temperature in the presence of 4 µg of salmon
sperm DNA followed by annealing to its complementary
strand(14) . The treated probes were incubated with COS-1 whole
cell extracts expressing the indicated nuclear receptors; the complexes
were analyzed by a preparative mobility shift assay. Following
electrophoresis the protein-DNA complexes and the free probe were
excised from the gel, electroeluted, and treated with 1 M piperidine for 30 min at 95 °C. The samples were then dried,
the dry pellets were counted and dissolved in 98% formamide dye. Equal
counts from all the samples were fractionated by electrophoresis and
the bands were visualized by autoradiography.
Transient Transfection Experiments and CAT
Assays
Monolayers of COS-1 or HepG2 cells were maintained as
stocks in Dulbecco's modified Eagle's medium supplemented
with either 10% fetal calf serum or charcoal stripped 5% fetal calf
serum, respectively. 0.5 
10
HepG2 cells were plated
on 30-mm dishes and the following day were transfected using the
calcium-phosphate DNA co-precipitation method. A total of 6 µg of
plasmid DNA was used, containing the wild type or mutant apoA-I-CAT
plasmids, phosphoglycerol kinase -galactosidase plasmid (generous
gift of Dr. F. Mavilio) as internal control, and various concentrations
of pMT2 expression plasmids carrying the cDNAs of different hormone
nuclear receptors. Post-transfection HepG2 cells were subjected to 30
and COS-1 cells to 60 s of glycerol shock. Cotransfection experiments
with the ligand-dependent nuclear receptors RXR, RAR, and
TR were performed either in the absence or presence of
10
M 9-cis-RA (generous gift of
Dr. H. Gronemyer), 10M all-trans-RA, or 10
M T. The cells were harvested 40 h post-transfection and
lysed in 200 µl of 1 reporter lysis buffer. An aliquot of
the cell extracts was heated at 65 °C for 10 min prior to the CAT
assays. The assays were performed in a 7-ml plastic scintillation vial,
in a total volume of 250 µl, containing 100 mM Tris-HCl,
pH 7.8, 1 mM chloramphenicol, 0.25 µCi of H-labeled acetyl coenzyme A, and 20-30 µl of
extracts. The reaction mixture was also supplemented with 100
µM cold acetyl coenzyme A. One blank sample and one sample
containing 5 milliunits of purified CAT enzyme were always included.
The reaction mixture was overlaid with 4 ml of water-immiscible
scintillation fluid and incubated at 42 °C for a maximum of 45 min
before it was counted in a liquid scintillation counter. The units of
active CAT enzyme in the cell extracts was determined by comparison of
the radioactivity counts obtained in the samples containing the cell
extracts and the counts obtained in samples containing 5 milliunits of
purified CAT enzyme. The background values of the blank sample were
always subtracted from the counts/min values of the different samples.
Each experiment was repeated two to three times in quadruplicate and
the mean values were calculated. The -galactosidase activity of
the cell lysates was determined spectrophotometrically by monitoring
the hydrolysis of the synthetic substrate o-nitrophenyl
galactoside at 410 nM and was utilized to normalize for
variability in the efficiency of transfection. Control samples
containing 5, 7.5, and 10 milliunits of purified -galactosidase
allowed the conversion of the OD units of the different samples into
-galactosidase units.
RESULTS
Binding of Orphan and Ligand-dependent Nuclear
Receptors to the Regulatory Elements AIB and AID of the Human ApoA-I:
Effect of Promoter Mutations on Binding
The regulatory element AID (-220/-190) contains
sequences that share high similarity with an AGG/TTCA motif (half-site)
found in HREs on the promoter regions of a variety of
genes(15, 16, 17) . Examination of the HRE on
element AID showed the presence of three putative direct repeats
between nucleotides -190 to -210, whereas the element AIB
contains two putative direct repeats between nucleotides -132 to
-119 (Fig. 1A). DNA binding assays have shown
that both regulatory elements AIB and AID can support the homodimeric
binding of HNF-4, ARP-1, EAR-2, EAR-3, and RXR as well as the
heterodimeric binding of RXR/RAR and RXR/TR.
Monomers of TR or homodimers of either
TR or RAR do not bind to either regulatory
element (Fig. 1, B-C). The findings establish that the
regulatory elements AIB and AID of apoA-I contain functional HREs which
can bind a variety of orphan and ligand-dependent nuclear receptors. We
have performed DNA binding supershift experiments to characterize the
putative RXR/RAR and RXR/TR
heterodimers which bind to the regulatory element AID. For this
analysis we have utilized a monoclonal anti-RAR antibody and an
anti-flag antibody against TR which contains an
amino-terminal flag sequence encoding for
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys. The expression vector carrying a
flagged version of TR was transiently expressed in
COS-1 cells. These COS-1 extracts, as well as extracts of COS-1 cells
expressing RXR or RAR were utilized in the supershift assays. Fig. 1D shows that the monoclonal anti-RAR
antibody supershifted the RXR/RAR heterodimers bound to the
wild type oligonucleotide AID. Similarly, the anti-flag antibody
supershifted the RXR/TR heterodimers, bound to
oligonucleotide AID (Table 1).
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 TR 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
TR. Note that the monoclonal anti-RAR antibody
supershifted the RXR/RAR heterodimer and the anti-flag
antibody supershifted the RXR/TR 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 AI
REP1 and
AIREP3, 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 (AIREP3, AIDM5, and AIREP1) 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 (A1REP1) 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 (AIREP3 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
AIREP1 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/TR
heterodimers. Deletion of repeat 1 of element AID (AIREP1) did not
affect qualitatively the binding of RXR/RAR and
RXR/TR heterodimers (Fig. 2E).
Deletion or mutations in the third repeat of the element AID (AIDM5 and
AIDREP3) 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/TR
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.
Contribution of the HREs to the Strength of the ApoA-I
Promoter in HepG2 Cells
To assess the importance of the two HREs for the apoA-I
promoter strength we introduced the mutations of Table 2in the
apoA-I promoter and generated mutant -264/+5 A-I CAT
constructs. Transient transfection assays in HepG2 cells showed that
drastic mutations in the regulatory element AIB (AIBM mutation) or the
regulatory element AID (AIDM mutation) or both elements (AIBDM
mutation) reduced the promoter activity to 3-7% of the control (Fig. 3). This finding, combined with the DNA binding data of Fig. 2D establishes the importance of both HREs for the
hepatic transcription of the apoA-I gene since promoter mutations which
preclude the binding of hormone nuclear receptors to any of the two
HREs essentially abolish the hepatic expression of apoA-I.
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 (AIREP3) 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).
Mode of Binding of RXR Homodimers,
RXR/RAR, and RXR/TR Heterodimers on the
Wild Type Regulatory Element AID as Determined by Dimethyl Sulfate and
KMnO Interference Assays
The observation that the RXR homo- and heterodimers have
different binding specificities on the regulatory element AID prompted
us to determine the nucleotides which are involved in these DNA-protein
interactions by permanganate (KMnO) and methylation
(dimethyl sulfate) interference analysis using the wild type element
AID as probe. This analysis showed that nucleotides at position 5 and 6
of repeat 1 and repeat 2, and nucleotides at position -1, located
in the spacer region between repeats 1 and 2 of the coding strand,
participate in strong DNA-protein interactions with RXR
homodimers. In addition, the nucleotide at position 6 of the putative
third repeat, of the coding strand, participates in weak DNA-protein
interactions with RXR. The KMnO and dimethyl sulfate
interference pattern of the noncoding strand showed that nucleotides at
position 1 and 2 of repeat 1 as well as nucleotides at position 1, 2,
3, and 4 of repeat 2 participate in strong DNA-protein interactions
with RXR, and the nucleotide at position 3 of repeat 1
participates in weak interactions with RXR (Fig. 4, A-C). Overall, 12 out of the 14 nucleotides that form repeats
1 and 2 and the putative spacer region on the HRE of element AID
participate in DNA-protein interactions with RXR homodimers. Five
of these nucleotides are on repeat 1, six on repeat 2, and one in the
spacer region between repeats 1 and 2. Eleven nucleotides participate
in strong and one in weak interactions. In addition, one nucleotide in
the putative third repeat participates in weak DNA-protein interactions
with the RXR homodimers (Fig. 4C). These findings
demonstrate the requirement of both repeat 1 and repeat 2 on the HRE of
element AID for the binding of RXR homodimers and the minimal
involvement of the putative third repeat in this binding. The findings
are also consistent with the DNA binding assays of homo- and
heterodimers of RXR to the mutated AID sequences (Fig. 2, C-F).
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/TR 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/TR 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/TR
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/TR 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/TR heterodimers. The
oligonucleotide which does not participate in DNA-protein interactions
with the RXR/TR 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/TR 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/TR heterodimers (Fig. 4, G-I).
Mode of Binding of the RXR/RAR and the
RXR/TR Heterodimers on the Regulatory Element AID
Carrying Mutation or Deletion of Repeat 1, as Determined by KMnO and Dimethyl Sulfate Interference Assays
As shown in Fig. 2, C and E,
mutation or deletion of the first repeat in the HRE of the regulatory
element AID (AIDM2 and AIDREP1 mutations) prevents the binding of
RXR homodimers but allows the binding of RXR/RAR and
RXR/TR heterodimers. To investigate further the
mode of binding of the heterodimers to the mutated sequence we
performed KMnO and dimethyl sulfate interference analysis
using the oligonucleotides AIDM2 and AIDREP1 as probes (Table 2). The KMnO and dimethyl sulfate modification
pattern of the RXR/RAR heterodimers with the mutated AIDM2
probe is shown in Fig. 5, A and B,
respectively, and summarized in Fig. 5C. This analysis
showed that the binding of the RXR/RAR heterodimers to the
mutated AIDM2 probe is confined to a heptameric core recognition motif.
The seven oligonucleotides which participate in DNA-protein
interactions are the six nucleotides of repeat 1 and the nucleotide
-1 localized in the putative spacer region between repeats 1 and
2. Nucleotides 5 and 6 of the coding strand and nucleotide 4 of the
noncoding strand participate in strong, and the remaining in weak,
DNA-protein interactions with the RXR/RAR heterodimers.
Figure 5:
A-H, KMnO and dimethyl sulfate
modification pattern of the DNA-protein complexes formed with the
RXR/RAR or RXR/TR heterodimers using
the mutated element AIDM2 or AIREP1 as probes (Table 2). The
RXR/RAR and RXR/TR 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/TR 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 AIREP1
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/TR 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/TR 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/TR
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/TR 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/TR heterodimers. The KMnO and dimethyl sulfate modification pattern of the
RXR/RAR heterodimers with the mutated AIDREP1 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/TR 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/TR 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/TR heterodimers. Panel B, schematic representation of the mode of interaction
of the RXR homodimers and RXR/RAR and
RXR/TR heterodimers with the normal and the
mutated HREs of the regulatory element AID. The figure is deduced from
the data of Fig. 4and 5.
Effect of Ligand-dependent Nuclear Receptors and HNF-4
on the ApoA-I Promoter Strength
Transactivation by RXR Homodimers in the Presence
of 9-cis-RA
We have performed cotransfection titration
experiments in HepG2 cells with the wild type or mutated
-264/+5 apoA-I CAT constructs and plasmids expressing
various combinations of ligand-dependent nuclear receptors in the
presence or absence of their corresponding ligands. The experiments
with RXR were performed in the presence or absence of its ligand
10M 9-cis-RA and increasing
amounts of an RXR expression plasmid, ranging from 50 to 750 ng.
This analysis showed that cotransfection with RXR transactivated
moderately (1.5-fold) the apoA-I promoter, in the presence of its
ligand 9-cis-RA. Optimal ligand-dependent transactivation was
observed in the range of 100-250 ng of plasmid. In the absence of
exogenously added ligand there was no significant transactivation in
this range of concentrations whereas at higher concentrations there was
a trend toward transcriptional repression (Fig. 7A).
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 10M 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
10M 9-cis-RA (closed
squares), 10M all-trans-RA (closed circles), and a trend toward repression in the absence
of any ligand (closed diamonds). Panel C shows
repression by RXR/TR heterodimers in the presence
of 10M T (closed
circles), transactivation at low TR
concentrations in the presence of 10M 9-cis-RA, lack of transactivation at higher
TR 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.
Lack of Transactivation by RXR/RAR
Heterodimers
The experiments involving RXR/RAR
heterodimers were performed with constant amounts (100 ng) of RXR
expression plasmid and increasing amounts of RAR, ranging from 50
to 500 ng. It is expected that the higher RAR concentrations will
favor the formation of heterodimers rather than homodimers of RXR.
The experiments were performed in the absence of any ligand or in the
presence of 10M 9-cis-RA or
10M all-trans-RA. This analysis
showed that the RXR/RAR heterodimers did not transactivate
significantly the apoA-I promoter in the presence of any of the
ligands. In the absence of both ligands and at higher concentrations of
RAR, there was a trend toward transcriptional repression (Fig. 7B). The findings suggest that the
RXR/RAR heterodimers abolished the 1.5-fold transactivation
achieved by RXR homodimers in the presence of 9-cis-RA.
Repression of Transactivation by
RXR/TR Heterodimers in the Presence of
T
The experiments involving
RXR/TR heterodimers were performed with constant
amounts of RXR (100 µg) and increasing amounts of
TR, ranging from 50 to 500 ng for the reasons
described above. The experiments were performed in the absence of any
ligand or in the presence of either 10M 9-cis-RA or 10M T. This analysis showed that the
RXR/TR heterodimers repressed transcription to
60% of control in the presence of T. In the presence of
9-cis-RA, cotransfection with 50 ng of TR and
100 ng of RXR expression plasmids caused a 1.5-fold increase in
transcription, similar to the increase observed with the RXR
homodimers (Fig. 7A). Most likely, this increase is the
result of the formation of RXR homodimers, promoted by the higher
concentration of RXR as compared to the TR
vector, in the presence of 9-cis-RA. When the two
receptor-expressing plasmids were used in equal concentration (100 ng),
this increase was no longer apparent.
Lack of Transactivation by HNF-4
Homodimers
Cotransfection experiments in HepG2 cells were also
performed with plasmids expressing HNF-4. This analysis showed that low
concentrations (25-100 ng) of HNF-4 did not increase the hepatic
expression of apoA-I beyond the levels of expression achieved in the
absence of exogenously added HNF-4. Higher concentrations of HNF-4
resulted in a gradual repression of transcription which reached 75% of
the control value at 750 ng of HNF-4 expression plasmid (Fig. 7D).
Effect of Selected Mutations within the Repeats of
the Regulatory Element AID on the ApoA-I Promoter Strength and Its
Transactivation by HNF-4 and the Ligand-dependent Nuclear Receptors
Cotransfection experiments with HNF-4 and the AIDM1 to AIDM4
mutated promoter constructs showed that the apoA-I promoter strength
remained similar in the presence and absence of HNF-4 (Fig. 8A). This indicates that diminished binding of
HNF-4 to the regulatory element AID impairs the promoter strength
despite the fact that other nuclear receptors may still bind to this
element. Similar cotransfection experiments were performed in HepG2
cells using the AIDM2, AIREP1, and AIREP3 mutated promoter
constructs and combinations of plasmids expressing RXR, RAR,
or TR. This analysis showed that the promoter which
lacks repeat 3 (AIREP3) behaves in all cases similarly to the wild
type promoter (data not shown). The promoter which lacks repeat 1
(AIREP1) had 30-35% activity and was not affected by
RXR in the presence or absence of 9-cis-RA. Interestingly
the promoter which carries a mutation in the first repeat of element
AID (AIDM2) was transactivated 2-fold by RXR in the presence or
absence of 9-cis-RA (Fig. 8B).
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 AIREP1 mutations on the
transactivation of the apoA-I promoter by RXR/RAR
heterodimers. Panel D, effect of the AIDM2 and AIREP1
mutations on the transactivation of the apoA-I promoter by
RXR/TR heterodimers.
Cotransfection experiments with RXR/RAR heterodimers
showed that the promoter which lacks repeat 1 (AIREP1) 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/TR heterodimers showed that
the promoter which lacks the first repeat had the same 30-35%
activity and was not affected by the RXR/TR
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/TR 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/TR 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/TR 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.
DISCUSSION
The Two Proximal HREs of the Human ApoA-I Promoter Are
Essential for Hepatic Expression
The present study has focused
on the functional significance of the regulatory elements AID and AIB
of apoA-I, and the potential contribution of hormone nuclear receptors
to the transcriptional activation of this promoter in hepatic cells.
Sequence comparisons showed that the regulatory elements AID and AIB
contain sequences with high similarity to an AGG/TTCA motif found on
the promoter sites of genes responsive to members of the
steroid/thyroid receptor
superfamily(15, 16, 18) . The HRE present on
element AID is composed of three putative direct repeats with the
sequence A/GGG/TTCA on the noncoding strand, whereas the HRE on element
AIB is composed of two putative direct repeats with the sequence
A/GGT/ATCA on the noncoding strand. In both cases there is a 1 to
2-nucleotide spacer region between the repeats. Drastic mutagenesis
which altered either part of both repeats in the HRE of element AIB or
repeat 2 and the adjacent spacer region in the HRE of element AID,
eliminated the binding of hepatic activities present in rat liver
nuclei and reduced the promoter strength to approximately 5-7% of
control. These findings suggest that both HREs are essential for
optimal hepatic expression of the apoA-I gene and that the factors
which occupy them act synergistically to increase transcription.
The HREs on the Regulatory Elements AID and AIB of ApoA-I
Are the Binding Sites of Orphan and Ligand-dependent Nuclear Receptors:
Mutations in the HRE of Element AID Affect Differently the Binding of
the Orphan and Ligand-dependent Nuclear Receptors
Previous
studies have identified some of the factors occupying the regulatory
element AID(3, 4, 6) . In the present study
we demonstrate that both regulatory elements AIB and AID of apoA-I are
the binding sites of the orphan nuclear receptors HNF-4, ARP-1, EAR-2,
and EAR-3, as well as of homodimers of RXR and heterodimers of
RXR with RAR or TR. Binding of the RXR
heterodimers was also verified by supershift assays. We did not observe
binding of RAR homodimers as suggested previously (8) as well
as binding of RAR/TR heterodimers shown
previously to recognize the direct repeat of myosin heavy chain (18) or monomers of homodimers of TR shown
previously to recognize inverted or palindromic
repeats(17, 19) .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/TR 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/TR 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 TR
have provided a tentative explanation for the observed higher mobility
DNA-RXR/TR complex. We have found that
supplementation of the COS-1 extracts with 9-cis-RA favors the
formation of both the higher mobility RXR/TR
complex and a RXR homodimeric complex that displays intermediate
mobility between the high and the low DNA-RXR/TR
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/TR dimer which is
accessible to T(21) and the slow-migrating complex
may represent a higher order complex between
RXR/TR and a third protein, and that the
formation of this complex is enhanced by the presence of
9-cis-RA(22, 23) .
Nucleotides in the HRE of Element AID Which Participate
in Protein-DNA Interactions with Homo- and Heterodimers of RXR:
Spacing Requirements for Binding to AID and Ability of
RXR/RAR and RXR/TR Heterodimers to Bind
to a Heptameric Core Motif
A series of KMnO and
dimethyl sulfate interference experiments using normal and mutated AID
sequences as probes demonstrated that the RXR homodimers and the
RXR/RAR and RXR/TR heterodimers share
almost identical contact points with the direct repeat 1 and 2 in the
HRE of element AID. These interactions involve four or five nucleotides
of repeat 1, six nucleotides of repeat 2, and one nucleotide in the
putative spacer region between repeat 1 and repeat 2. The putative
repeat 3 is not involved in binding of the RXR heterodimers and
shows only a weak contact point with the RXR homodimer. Overall,
the RXR homodimers and the RXR/RAR heterodimers appear
to bind more strongly to the intact HRE of element AID than the
RXR/TR. The RXR homodimers have 11 strong
and 2 weak DNA-protein contact points, the RXR/RAR
heterodimers have 10 strong and 3 weak DNA-protein contract points and
the RXR/T
