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INTRODUCTION |
Although apolipoprotein A-II
(apoA-II)1 is the second most
abundant apolipoprotein in high density lipoprotein (1), its physiological functions are still unclear. Inactivation of the apoA-II gene in mice suggests a complex role for apoA-II in
atherosclerosis, with both antiatherogenic and proatherogenic
properties (2). Clinical observations and tissue culture studies
suggest that an increase in apoA-II production may be less protective
against atherosclerosis by raising the proportion of a high density
lipoprotein subclass containing both apoA-I and apoA-II (LpAI:AII),
thereby decreasing the proportion of the antiatherogenic subclass of
high density lipoprotein containing only apoA-I (LpAI) (3, 4). ApoA-II
is expressed in the liver and, to a much lesser extent, in the human
fetal intestine (5). Recent kinetic data in normolipidemic humans
indicate that human apoA-II production rate is the major factor
determining the distribution of apoA-I between LpAI and LpAI:AII (6,
7). Understanding the molecular mechanisms involved in hepatic
transcription of apoA-II may, therefore, lead to the development of new
tools to control the ratio between LpAI and LpAI:AII.
We have already reported that the human (
911/+29) apoA-II promoter is
sufficient for liver-restricted expression of a reporter gene in
transgenic mice (8). The
911/
614 base pair distal region exhibits
enhancer properties and potentiates the strength of the homologous
proximal promoter AB (
65/
33 base pairs) (9-12) as well as the
strength of heterologous promoters (13). The element AB is important
for the function of the apoA-II promoter and binds an activity
designated CIIIB1 (12). CIIIB1 was first described as a heat-stable
factor that binds to element B of the apoC-III promoter (14, 15). The
CIIIB1 binding site GTCACCTG contains the CANNTG consensus E-box motif
(16, 17). E-box motifs are recognized by basic helix/loop/helix
transcription factors, which contain a basic (b) DNA binding domain and
a helix/loop/helix (HLH) dimerization domain. In the case of
bHLH/ZIP-related proteins, the bHLH domain is contiguous with a second
dimerization domain, a leucine zipper (ZIP). The latter group includes
a wide variety of transcription factors, such as c-Myc, Max (18), TFE3
(19), TFEB (20), SREBP1 (21), and upstream stimulatory factor (USF) (16). USF appears to be the predominant bHLH/ZIP factor in liver nuclear extracts (22). Human USF was first designated as major late
transcription factor (16). Three USF isoforms have been described,
USF1, 2a, and 2b, with apparent molecular masses of 43, 44, and 38 kDa,
respectively (23-25). USF1 and USF2a and 2b are encoded by two
different genes, and USF2a and 2b are generated by differential
splicing. Although ubiquitously expressed, USF has been involved in
transcription of genes with tissue specificity (25, 26).
In the present study, we purified CIIIB1 from rat liver nuclear
extracts by DNA sequence-specific affinity chromatography. We found
that CIIIB1 is immunologically related to USF using anti-USF1 and
anti-USF2a. DNA binding and competition experiments showed that USF
binds to the regulatory elements AB, K, and L. We have established in
HepG2 cells the functional significance of USF in apoA-II promoter
activity. Finally, cotransfection experiments in COS-1 cells and DNA
binding assays demonstrated synergism between USF2a and HNF-4 in the
transactivation of the apoA-II promoter and a cooperative binding of
the two factors to their cognate sites of the apoA-II promoter.
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EXPERIMENTAL PROCEDURES |
Synthetic Oligonucleotides--
The following oligonucleotides
were used for plasmid construction in this work. KJ Kpn
Hindc contains the sequence of the domains K and J from
position
768 to
712 (9) with two restriction sites, KpnI
and HindIII. KJ Kpn Hindnc is the
complementary strand. L Sal Kpnc contains the domain
L from position
807 to
769 (9) with SalI and
KpnI restriction sites. L Sal Kpnnc represents
the complementary strand. For gel-shift assays, the used
double-stranded oligonucleotides of element AB (
68 to
32) and
mutated element AB have been previously described (12) (see Fig.
2A). Oligonucleotides J (
738 to
712), K (
762 to
741), K/J (
764 to
712) and L (
805 to
771) represent the J, K,
K/J, and L regions of the apoA-II promoter (9). Oligonucleotide CIIIB
represents the element B of the human apoC-III promoter from
92 to
67 (14). Oligonucleotides NFY, MLP, and HNF-4 contain the binding
site for NFY proteins (27), a consensus binding site for USF (25), and
a binding site for HNF-4, respectively (14).
Plasmid Construction--
The AB plasmid, containing the
chloramphenicol acetyl transferase (CAT) gene under the control of the
80/+29 minimal human apoA-II promoter, has been previously described
(9), as have the ABM1 and ABM6 plasmids with mutations M1 and M6 in
element AB (12). The E-AB and E-ABM1 plasmids containing the distal enhancer (
911/
614) and the proximal promoter (
80/+29) with either
nonmutated element AB or mutation M1 in element AB have also been
described (12). To join the K/J and AB apoA-II promoter regions, the AB
plasmid was digested by KpnI and HindIII and
ligated with the annealed KJ Kpn Hindc and nc
oligos, thus generating the K/J-AB plasmid (see Fig. 3A).
Similarly, the AB plasmid was digested with SalI and
KpnI and ligated to the annealed L Sal Kpnc and
nc oligos to generate the L-AB plasmid (see Fig.
3A). The K/J-AB plasmid was subsequently digested by
SalI and KpnI to ligate the annealed L Sal
Kpnc and nc oligos, thus generating the
L-K/J-AB plasmid (see Fig. 3A). After
HindIII/XhoI digestion of E-ABM1 plasmid, the
ABM1 element was gel-purified. The L-K/J-AB plasmid was then digested
by HindIII and XhoI, and the AB element was
removed and replaced by the gel-purified ABM1 element, thus generating
the L-K/J-ABM1 plasmid (see Fig. 3A). These plasmids were sequenced.
Purification of CIIIB1--
All buffers contained 1 mM dithiothreitol, 0.1 mM benzamidine, 2 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin, which
were added just before use. All purification steps were carried out at
4 °C. Nuclear extracts were prepared from rat livers, as described
previously (28). The extracts (1.6 g of protein) in nuclear dialysis
buffer (NDB) containing 25 mM Hepes, pH 7.6, 0.1 mM EDTA, 40 mM KCl, and 10% glycerol were
heated at 85 °C for 5 min, and protein aggregates were removed by
centrifugation for 5 min at 13,000 rpm. The supernatant was adjusted to
5 mM MgCl2 and to 125 µg/ml sonicated herring
sperm and was used for affinity chromatography purification. DNA
sequence-specific affinity chromatography was performed according to
Kadonaga and Tjian (29), using as ligand the double-stranded
oligonucleotide that represents the element B (
93 to
71) of the
human apoC-III promoter region (14). The column was eluted with a step
gradient of 0.5 M and 1 M KCl in NDB. CIIIB1
activity was recovered in 0.5 M KCl fractions. Three
successive affinity chromatographies were performed.
Partial Amino Acid Sequencing--
After the third affinity
chromatography, fractions containing CIIIB1 were further purified by
preparative SDS-PAGE (30). The gel was stained briefly in 0.25%
Coomassie Blue R-250. A digestion was performed in an excised gel slice
with a lysyl endopeptidase. The resulting peptides were separated on a
reverse phase C18 column. Amino acid sequence analysis of the selected
peaks was performed by automated Edman degradation using an Applied
Biosystems model 473A.
Western Blot Analysis--
Affinity chromatography purified
CIIIB1 from rat liver nuclear extracts and whole COS-1 cell extract
overexpressing USF2a were separated by SDS-PAGE in 10% gel, then
electrotransferred on a nitrocellulose membrane in a Bio-Rad apparatus,
according to the instructions of the manufacturer. After blocking for
2 h with 3% bovine serum albumin in TBS (20 mM
Tris-HCl, pH 7.5, 150 mM NaCl), rabbit anti-USF1 or -USF2a
antibodies, diluted 200- and 1,000-fold, respectively, in TBS with 1%
bovine serum albumin, were incubated overnight and washed three times
(TBS, TBS with 0.1% Tween, TBS). Antisera against human USF1 (43 kDa)
and USF2a (44 kDa) were kindly provided by Michel Raymondjean (U129
INSERM) (25, 31). Then anti-rabbit antibodies conjugated with
peroxidase diluted 1,000-fold in TBS with 1% bovine serum albumin were
incubated for 1 h and washed three times (TBS, TBS with 0.1%
Tween, TBS), and peroxydase-conjugated antibodies were detected using
4-chloro-1-naphtol enzyme as substrate.
Cell Transfection and CAT Assay--
The various CAT gene
reporter plasmids were cotransfected with the plasmid
RSV-
gal in HepG2 cells and COS-1 cells using the calcium
phosphate-DNA coprecipitation method (32).
-Galactosidase activity
was determined to normalize the variability in transfection efficiency
(33). CAT assays were performed in a liquid phase, as described
previously (34). The initial velocity of the enzymatic reaction was
estimated by measuring the amount of labeled acetylchloramphenicol, which diffused directly into the liquid phase in the scintillation counting vial.
Overexpression in COS-1 Cells--
cDNAs encoding USF1,
USF2a, and TDU2 (USF2a that lacks the NH2-terminal
activation domain) subcloned into the eucaryotic expression vector pCMV were kindly provided by Michel Raymondjean (U129 INSERM) (25, 31). These vectors and an HNF-4 eucaryotic expression vector (35)
were transfected into COS-1 cells using the calcium phosphate
coprecipitation method. Whole-cell extracts were prepared as described
previously (35) to be used in gel retardation assays.
Electrophoretic Mobility Shift Assays (EMSA)--
Annealing and
labeling of synthetic oligonucleotides were performed as described
previously (14). EMSA were performed according to Fried and Crothers
(36) as described by Lacorte et al. (12). Briefly, 3 µl of
rat liver nuclear extract (2 to 3 µg/µl of total proteins) and 6 µl of the same extract, heated for 5 min at 85 °C were used in
EMSA. Aliquots of 6 µl and 4 µl of the first and second affinity
elution fractions, respectively, were also used in EMSA. For supershift
assays, anti-USF1, -USF2a, or -HNF-4 antibodies were diluted 10-fold,
and 1 µl was added to the reaction mix. Anti-HNF-4 antibody was
kindly provided by F. M. Sladek (37).
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RESULTS |
CIIIB1 Is Immunologically Related to USF--
To further
characterize CIIIB1, which is essential for apoA-II promoter activity,
the protein was purified to near homogeneity from rat liver nuclear
extracts by heat treatment at 85 °C for 5 min followed by three
cycles of DNA sequence-specific affinity chromatography with the
element B of the human apoCIII promoter (Fig.
1). The presence of CIIIB1 in purified
fractions was assessed by EMSA analysis using the CIIIB probe (Fig.
1C, lanes 1-4). SDS-PAGE analysis of the
affinity-purified CIIIB1 revealed two predominant proteins, with
molecular masses of 45 and 97 kDa, respectively (Fig. 1A).
The 97-kDa protein was not a dimer of the 45-kDa protein, since
affinity samples were loaded on SDS-PAGE under reducing conditions.
Digestion with lysyl endopeptidase of the 97-kDa protein generated five
peptides, which were microsequenced. The sequences obtained matched
perfectly those of the human polypyrimidine tract binding
protein-associated splicing factor (PSF) (Table
I) (38). Peptide sequences also shared
significant homology with the non-Pou domain-containing,
octamer-binding protein (NonO) (39, 40).

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Fig. 1.
Affinity purification and immunological
relationship of rat CIIIB1 complex to USF. CIIIB1 is predominantly
a USF1/2a heterodimer. A, analysis of CIIIB1 eluted from
affinity chromatography by 12% SDS-PAGE. Proteins were stained with
Coomassie Brillant Blue. N.E., crude rat liver nuclear
extracts. N.E. 85°C, crude rat liver nuclear extract
heated at 85 °C for 5 min. 1st to 3rd AFF.,
fractions eluted in 0.5 M KCl from the first, second, and
third DNA-specific affinity chromatographies. M.W.,
molecular mass markers are as noted. Arrows indicate the two
main proteins, with apparent molecular masses of 97 and 45 kDa,
respectively. B, analysis of purified CIIIB1 by Western
blotting after two affinity chromatographies (2nd AFF.).
FT, flow-through of the second affinity column.
USF2a, whole-cell extract of COS-1 cells overexpressing
USF2a. Proteins were electrophoresed on a 10% SDS-polyacrylamide gel
and analyzed by Western blotting, as described under "Experimental
Procedures." Anti-USF1 and anti-USF2a were diluted 200- and
1,000-fold, respectively. The position of USF1 and USF2a polypeptides
are indicated as a closed circle and open
triangle, respectively. C and D, analysis of
CIIIB1 by EMSA using element B ( 92 to 67) of the human apoC-III
promoter (CIIIB probe) (B) or the element AB ( 68 to 33)
of the human apoA-II promoter (AIIAB probe) (C) as probes,
in the presence or absence of anti-USF1 or anti-USF2a, as indicated in
the figure. CIIIB1 activity was obtained from from rat liver nuclear
extract (N.E.), from rat liver nuclear extract heated 5 min
at 85 °C (N.E.85°C), from the first affinity column
fraction (1st AFF.), and from the second affinity column
fraction (2nd AFF.). USF1 and USF2a
contain whole-cell extract of COS-1 cells overexpressing USF1 or USF2a,
respectively. Oligo CIIIB and oligo AIIAB, corresponding, respectively,
to CIIIB and AIIAB probes, are competitors added in 100-fold molar
excess. The asterisk indicates USF1/2a heterodimer, the
open triangle indicates the USF2a/2a homodimer, and the
closed circle indicates the USF1/1 homodimer. EMSA were
carried out as described under "Experimental Procedures".
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Table I
Comparison of the amino sequence of the peptides obtained from the
97-kDa purified protein with PSF and NonO (38, 39)
Residues in NonO that differ from PSF are underlined.
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The 45-kDa polypeptide might correspond to the 41-kDa protein observed
by Ogami et al. (15). We investigated whether this protein
corresponds to USF because the CIIIB1 binding site, GTCACCTG, contains
an E-box motif, CANNTG. This motif binds heat-stable b/HLH/ZIP proteins
such as USF, which is expressed abundantly in the liver. An immunoblot
analysis was performed with specific antibodies raised against the
transactivation domain of USF1 (M domain) or USF2a (G domain) (25)
(Fig. 1B). Anti-USF1 or anti-USF2a (Fig. 1B,
left and right panels) cross-reacted with a
43/44-kDa polypeptide in the purified affinity fraction (Fig.
1B, 2nd AFF.). When both antibodies were used
(Fig. 1B, middle panel), two polypeptides were
observed in the affinity-purified fraction (Fig. 2B,
2nd AFF.). The upper polypeptide comigrates with the 44-kDa
isoform USF2a in the USF2a control (Fig. 2B, USF2a), and the
lower polypeptide corresponds to the 43-kDa isoform USF1. To further
confirm the identity of CIIIB1, EMSA and supershift assays were
performed with anti-USF1 and anti-USF2a (Fig. 1, C and
D). The CIIIB1 complex is competed out by a 100-fold excess
of unlabeled oligonucleotides CIIIB and AIIAB (Fig. 1C,
lanes 5 and 6, and Fig. 1D,
lanes 15 and 16). When both antibodies were added
to the reaction, the CIIIB1 complex was totally supershifted (Fig.
1C, lane 9, and Fig. 1D, lane
19). A residual complex remained after addition of each antibody.
The mobility of the remaining complex corresponds to the homodimer
USF1/1 (Fig. 1C, lanes 8 and 10, and
Fig. 1D, lanes 18 and 20) or to the
homodimer USF2a/2a (Fig. 1C, lanes 7 and
12, and Fig. 1D, lanes 17 and 22). These findings provide strong evidence that CIIIB1
is mostly the heterodimer USF1/2a.
USF Binds to Element AB and Transactivates the Proximal ApoA-II
Promoter--
USF1 and USF2a overexpressed in COS-1 cells formed
specific complexes with the AIIAB probe (Fig.
2B). Oligonucleotides
containing specific mutations in the element AB were used as probes
that either avoid or do not affect the binding of CIIIB1(12) (Fig. 2A). Fig. 2C shows that binding of USF2a to
element AB was not competed out by oligonucleotides ABM1 and ABM2
containing mutations in the E-box motif, which abolish the binding of
CIIIB1 (12). In contrast, mutations ABM3 to ABM6, which do not affect
CIIIB1 binding (12), competed out USF2a binding. Furthermore, as
expected, USF1 and USF2a bound to oligonucleotide ABM6 but not to
oligonucleotide ABM1 (Fig. 2D).

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Fig. 2.
USF binding to element AB; analysis of
specific DNA binding pattern. A shows the sequence of
the wild type and mutated oligonucleotides of the element AB that were
used as probes or competitors in B and C. The
CIIIB1 binding site is represented by an opened square. The
mutant sequences are underlined. B, binding of
USF1 and USF2a overexpressed in COS-1 cells to AIIAB probe.
C, competition assays of the binding of USF2a to AIIAB probe
using mutated oligonucleotides as competitors. ABM1 and ABM2 are
specific mutations in the E-box motif. Competitors were added in 10- and 100-fold molar excess. D, DNA binding assays of USF1 and
USF2a overexpressed in COS-1 cells to the wild type AIIAB probe or
mutated ABM1 and ABM6 probes. EMSA and supershift assays were performed
as described under "Experimental Procedures" and in Fig. 1.
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To evaluate the potential role of USF in the transactivation of the
human apoA-II promoter, cotransfection experiments were performed in
HepG2 cells using USF1 or USF2a expression vectors and a vector
expressing the CAT gene under the control of the wild-type proximal
apoA-II promoter (AB plasmid) shown in Fig. 3A, as well as AB plasmid
derivatives containing the mutated forms ABM1 and ABM6 (Fig.
3B). This analysis showed that USF2a transactivated 4.5-fold
and 13-fold the proximal AB promoter with 50 ng and 500 ng of plamid,
respectively (Fig. 3B). Similar results were obtained by
cotransfection with USF1 (data not shown). Cotransfection with a
truncated mutant USF2a protein, TDU2, that lacks the
NH2-terminal activation domain but contains normal DNA
binding and dimerization domains had no effect on the minimal apoA-II
promoter activity. Mutation within the E-box motif (ABM1 plasmid)
diminished the USF2a-mediated transactivation, whereas mutation outside
the E-box motif (ABM6 plasmid) resulted in transactivation by USF2a
similar to that obtained with the wild type construct (Fig.
3B). The combined data of Figs. 2 and 3 demonstrate that USF
binds to element AB and transactivates the proximal apoA-II
promoter.

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Fig. 3.
Transactivation of various constructs of the
apoA-II promoter by USF in HepG2 cells. A, schematic
representation of the ( 911/+29) apoA-II promoter (upper
panel) and of the various CAT reporter plasmids used for transient
transfection experiments (lower panel). B,
comparison of the transactivation effect of USF2a or its mutated form,
TDU2 (lacking the transactivation domain), on the wild type or mutated
apoA-II proximal promoter, which contains one E-box motif. Transient
transfections in HepG2 cells and CAT assays were performed as described
under "Experimental Procedures." For each experiment, CAT activity
was calculated by linear regression as the slope of the reaction
velocity and was expressed in cpm/min. CAT activities are expressed as
a percentage of the activity of the reference plasmid AB, arbitrarily
fixed at 1%. C, effect of the distal E-box motifs present
in elements K and L on the activity of the proximal AB apoA-II promoter
and its transactivation by USF1 and USF2a. CAT activities are expressed
as a percentage of the activity of the reference plasmid E-AB,
arbitrarily fixed at 100%. D, inhibition of the activity of
the L-K/J-AB and E-AB promoters by the mutated USF2a derivative, TDU2.
CAT activities are expressed as described in C. Experiments
were performed in triplicate and repeated three to five times. Results
represent means ±S.D. Statistical significance was determined by
Student's t test. a, p 0.001 relative to the AB reporter with control DNA. b,
p > 0.05 relative to the AB reporter with control DNA.
c, p > 0.05 relative to the ABM1 reporter
with control DNA. d, p 0.01 relative to
the ABM6 reporter with control DNA.
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Synergistic Interaction of USF Bound to Proximal and Distal
Elements Containing E-box Motifs--
Similarly to element AB,
elements K and L bind CIIIB1 (10). The addition of anti-USF1 or -USF2a
antibodies supershifted the CIIIB1 complex formed with the AIIK probe
and crude nuclear extracts obtained from HepG2 cells or
affinity-purified CIIIB1 fractions (Fig.
4A, N.E. and
2nd AFF.). The AIIL probe formed with HepG2 nuclear extract
multiple DNA-protein complexes related to NFY and C/EBP family, giving
a smeary pattern (Fig. 4B, lanes 7-9). After
competition with an oligonucleotide containing the binding site of NFY,
CIIIB1 became the predominant activity (Fig. 4B,
lane 10) and was specifically supershifted by anti-USF1 and -UFS2a (Fig. 4B, lanes 11 and 12).
Fig. 4B (lanes 13-16) shows that
affinity-purified CIIIB1 binds to the AIIL probe and is supershifted by
both antibodies. In addition, EMSA with USF1 and USF2a, overexpressed in COS-1 cells, gave direct evidence of the binding of USF1 or USF2a to
K and L elements (Fig. 4C).

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Fig. 4.
The apoA-II enhancer region 911/ 614
contains two USF binding sites located in the elements K and L. A, EMSA using AIIK double-strand oligonucleotide ( 762 to
741) as probe and nuclear extract obtained from HepG2
(N.E.) or purified second affinity CIIIB1 fraction
(2nd AFF.). Supershift assays were performed with the
addition of anti-USF1 or anti-USF2a as described in Fig. 1.
B, EMSA using the oligonucleotide AIIL ( 805 to 771) as
probe. Oligo NFY, an oligonucleotide that contains a binding
site of NFY, is used as competitor added in 100-fold molar
excess. C, direct binding of USF1 and USF2a overexpressed in
COS-1 cells on oligonucleotides AIIK or AIIL as probes. EMSA and
supershift assays were performed as described in Fig. 1.
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To evaluate the respective contribution of distal E-box motifs within
elements K and L in the apoA-II promoter activity, vectors placing the
distal elements L and/or K/J upstream of the proximal AB promoter were
generated (Fig. 3A). The addition of elements L or K/J to
the proximal AB promoter did not modify the transcriptional activity of
the proximal apoA-II promoter and the level of transactivation by USF2a
(Fig. 3C). However, the addition of both distal elements L
and K/J (Fig. 3A) increased 8-fold the activity of the
proximal promoter and resulted in an 8.5-fold transactivation by USF2a. Moreover, mutation within the proximal E-box motif diminished the
activity of the promoter (L-K/J-ABM1 promoter) as well as its
transactivation by USF2a. Given that CIIIB1 mostly consists of a
heterodimer USF1/2a (Fig. 1), we analyzed the role of the different
isoforms of USF in the transactivation of the apoA-II promoter.
Cotransfection of HepG2 cells with USF1 and USF2a expression vectors
led to a similar transactivation of L-K/J-AB promoter as cotransfection
with USF2a (Fig. 3C). These findings suggest that distal
elements L and K/J and proximal element AB are all required for the
transactivation of the apoA-II promoter by USF homo- or heterodimers.
The role of USF in the transactivation of the human apoA-II promoter
was further evaluated using a dominant negative form of USF2a, TDU2,
that lacks the transactivation domain. This analysis showed that the
activity of the L-K/J-AB promoter is decreased by 40 and 67% by 50 and
500 ng of TDU2 expression vector, respectively, as compared with 50 and
500 ng of the control expression vector, pCMV (Fig. 3D,
left panel). The right panel of the Fig.
3D shows a similar 20, 60, and 53% inhibition of the E-AB
promoter activity by 50 ng, 500 ng, and 1 µg of TDU2, respectively.
Synergism between HNF-4 and USF in Transactivation of the ApoA-II
Promoter--
We have previously shown that element L binds
C/EBP
and that element K/J contains an E-box motif
adjacent to an HNF-4 binding site (10, 35). To determine potential
synergism of the liver-enriched transcription factors
C/EBP
and HNF-4 with the ubiquitously expressed USF, we
performed cotransfection experiments of HNF-4, C/EBP
, and USF2a with
the E-AB reporter plasmid in non-hepatic COS-1 cells. This promoter was
transactivated 21-fold by USF2a (Fig. 5A). The transactivation was
specific because deletion of the activation domain of USF2a (in the
mutant TDU2) abolished the USF2a-mediated transactivation, and mutation
of the proximal E-box motif (in the E-ABM1 reporter) was not
specifically transactivated by USF2a (Fig. 5A). HNF-4 or
C/EBP
alone transactivated 2-fold the E-AB promoter
activity (Fig. 5B). This transactivation was additive when
COS-1 cells were transfected simultaneously with HNF-4 and C/EBP
,
yielding a 4-fold increase in promoter activity. Cotransfection of
COS-1 cells with C/EBP
and USF2a resulted in similar
levels of transactivation of the apoA-II promoter to those achieved
with USF2a alone. In contrast, when COS-1 cells were cotransfected with USF2a and HNF-4, we observed a synergistic 36-fold
transactivation of the apoA-II promoter activity (Fig. 5B).
Cotransfection of COS-1 cells with the TDU2 mutant of USF2a and HNF-4
resulted in similar transactivation as that of HNF-4 alone (Fig.
5B). These findings clearly establish a synergism between
USF and HNF-4 on the transactivation of the apoA-II promoter.

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Fig. 5.
Synergism of the liver-enriched factor HNF-4
with the ubiquitous USF in the transactivation of the apoA-II promoter
in COS-1 cells. A, transactivation of the E-AB reporter
by USF2a. B, effect of liver-enriched transcription factors
C/EBP and HNF-4 on the transactivation of the E-AB
promoter by USF2a. CAT activity is expressed as fold activation of the
activity of the reference plasmid E-AB, arbitrarily fixed at 1. CAT
assays were carried out as described in Fig. 3. Statistical
significance was determined by Student's t test.
a, p 0.01 relative to the USF2a
activator.
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The binding of USF2a and HNF-4 to elements K and J of the apoA-II
promoter was further studied by DNA binding assays. Fig. 6A shows that element K binds
USF2a but not HNF-4 (left panel, lane 1 versus lane 5), whereas element J binds HNF-4 but not USF2a (middle panel, lane 9 versus lane 8).
As expected, the K/J probe was able to bind both factors (Fig.
6A, right panel, lanes 15 and
17). The simultaneous addition of USF2a and HNF-4 in the
reaction (Fig. 6A, right panel, lane
19) generated a slower migrating band, which was competed out by
USF-specific oligonucleotides, MLP, AIIAB, and AIIK (Fig.
6A, right panel, lanes 22-24) and by
an HNF-4-specific oligonucleotide (Fig. 6A, right
panel, lane 25). This slower migrating band
corresponds to the simultaneous binding of USF2a and HNF-4. To
better characterize the binding of USF2a and HNF-4, we performed EMSA
with either increasing amounts of USF2a and a constant amount of HNF-4
or increasing amounts of HNF-4 and a constant amount of USF2a. Fig.
6B shows that when the USF2a/HNF-4 ratio increased
(lanes 4-6), the slower migrating band corresponding to the
simultaneous binding of HNF-4 and USF2a increased, whereas the band
corresponding to HNF-4 binding decreased. When the USF2a/HNF-4 ratio
decreased (lanes 7-9), the slower migrating band
corresponding to the simultaneous binding of HNF-4 and USF2a increased,
whereas the band corresponding to USF2a binding decreased. These
results demonstrate a cooperative binding of HNF-4 and USF2a on the
element K/J of the apoA-II promoter.

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Fig. 6.
Cooperative binding of USF2a and HNF-4 on the
element K/J of the apoA-II promoter. A, binding of
USF2a and HNF-4 overexpressed in COS-1 cells on the ( 762 to 741)
AIIK probe (left panel), ( 738 to 712) AIIJ probe
(middle panel) and the ( 764 to 712) AIIK/J probe
(right panel). Oligos AIIAB, AIIK, and AIIK/J correspond to
AIIAB, AIIK, and AIIK/J probes, respectively. Oligos MLP and HNF-4
correspond to oligonucleotide sequences containing the binding site of
USF and HNF-4, respectively. Competitors are added in 100-fold molar
excess. B, binding of USF2a and HNF-4 on the probe K/J. EMSA
and supershift assays were performed as described in the Fig. 1.
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 |
DISCUSSION |
The distal region (
911/
614) of the human apoA-II promoter
displays an enhancer-type activity (10, 11, 13), which is synergized by
the proximal regulatory element AB (12). An important regulatory role
is exerted by CIIIB1, a heat-stable transcription factor with an
Mr of 41 kDa that binds to the elements AB, K, and L (10, 15). Nucleotide substitutions, which prevent the binding of
CIIIB1 to element AB, abolished communication between the proximal
region and the enhancer (12). CIIIB1 was purified to homogeneity.
However, its relationship with existing factors was not established
(15). In the present study, purification of CIIIB1 led to the isolation
of two thermostable proteins with relative masses of 45 and 97 kDa,
respectively. The relationship between USF and CIIIB1 was investigated
because the CIIIB1 binding site, GTCACCTG, contains an E-box motif
CANNTG, a binding site recognized by b/HLH/ZIP proteins. USF family
members are thermostable (41) and are expressed predominantly in liver
(22). Three major USF isoforms, USF1, 2a, and 2b, have been described,
which differ in the NH2-terminal transactivation domain and
can form homo- and heterodimers. The USF1/2a heterodimer is predominant in liver and the hepatic cell lines HepG2 and ATF3 (25). Using specific
antibodies against USF1 and USF2a and AB, K, and L probes, we showed by
Western blotting and EMSA that the affinity-purified 45-kDa protein was
immunologically related to USF1 and USF2a and that the heterodimer
USF1/2a is the predominant form. The optimal binding sequence of USF to
E-box motifs has been defined as
RYCAC
1G+1TGRY (42). E-box motifs within
elements AB, K, and L mainly contain variations in the central portion
1/+1 of the consensus sequence. This may account for the difference
in their relative binding affinities for CIIIB1 reported by Cardot
et al. (10).
Sequencing of proteolytic peptides of the 97-kDa protein, co-purified
with CIIIB1, shared identity with PSF, another heat-stable protein
involved in early spliceosome formation (38). PSF shares significant
sequence identity with NonO, a protein that binds to DNA via a
helix/turn/helix domain and to RNA via ribonuclear protein-binding
motifs (RNP) (39). The sequence homology between these two proteins
lies within the adjacent RNP and the helix/turn/helix regions. It is
interesting to note that NonO is able to enhance the binding of E47, a
bHLH protein, to a noncanonical E-box motif (40). This E-box motif is
identical to that of the element AB of the apoA-II promoter. Thus, it
is possible that PSF could likewise increase binding of USF to element
AB via a mechanism similar to that by which NonO enhances E47 binding.
Similarly, the heat-stable protein PC5 has been described as a cofactor
that stimulates transcriptional activity of USF (43).
We demonstrated the functional relevance of USF in the control of
apoA-II promoter activity by the analysis of the transactivation properties of USF on various apoA-II promoter constructs. The minimal
promoter containing the proximal E-box motif (AB) and schematic
promoters containing various combinations of distal and proximal E-box
motifs were specifically transactivated by USF2a. A negative dominant
mutant of USF2a, TDU2, that lacks the transactivation domain but
contains the DNA binding domain and the dimerization domain, inhibits
apoA-II promoter activity. Mutation in the E-box motif within the
element AB, preventing communication between proximal and distal E-box
motifs (12), strongly reduces transactivation of the mutant L-K/J-AB
promoter by USF2a. It is possible that USF molecules bound to two or
more independent sites form multimeric complexes and, thus, promote DNA
looping (44). Such a mechanism might be involved in apoA-II promoter
activity. Our study shows that CIIIB1 is principally a heterodimer
USF1/2a. USF1/2a was shown to transactivate the proximal apoA-II
promoter (AB) and the L-K/J-AB promoter with the same efficiency as USF2a.
The ubiquitously expressed USF has been found to regulate a wide
variety of genes involved in different specialized functions, such as
tissue specificity, development, and metabolic regulation (26).
Association of USF with other transcriptional factors has been reported
(45). Specifically, C/EBP
promotes USF binding and
activation of the C/EBP
promoter (46). Liver-enriched
transcriptional factors, C/EBP
and HNF-4, bind to the
enhancer of the apoA-II promoter. We investigated potential synergistic
interactions between USF, HNF-4, and C/EBP
. Cotransfection
experiments have established synergistic activation of the apoA-II
promoter by a combination of USF and HNF-4 but neither by a combination
of C/EBP
and HNF-4 nor by a combination of USF and
C/EBP
. The K/J region of the apoA-II promoter contains E-box motifs
followed by an HNF-4 binding site. We demonstrated by DNA binding
experiments a cooperative binding of USF2a and HNF-4 on the
element K/J. This cooperative binding might account for the observed
synergism between USF and HNF-4 in the transactivation of apoA-II
promoter. This is the first time that such cooperative binding between
HNF-4 and USF has been reported. It would appear that the optimal
transcription of apoA-II gene is determined by the combined
action of ubiquitously expressed factor, USF, and a liver-enriched
transcription factor, HNF-4.
The glucose response element or carbohydrate response element of the
L-type pyruvate kinase (L-PK) promoter contains two E-box motifs
adjacent to an HNF-4 binding site located between nucleotides
168 and
144 (47, 48). These cis-elements are responsible for mediating the
transcriptional regulation of L-PK by carbohydrate metabolism and
glucagon/cAMP (49). USF was shown to bind the glucose response element
of the L-PK promoter. However, the role of USF in mediating glucose
response is still under discussion (31, 48, 50, 51). Recently, HNF-4
has been shown to be phosphorylated by PKA and is involved in cAMP
transcriptional effects on L-PK gene (52). Using
transgenic mice expressing the CAT reporter gene under the control of
the (
911/+29) human apoA-II promoter, we demonstrated that the
apoA-II gene is transcriptionally regulated at the weaning
period (8), during which the high fat diet (milk) shifts to a high
carbohydrate (chow) diet, and the insulin/glucagon ratio is modified.
Such metabolic regulation could involve USF and HNF-4. It has also been
demonstrated that USF binding to a proximal E-box motif is required for
insulin regulation of the fatty acid synthase (53). Interestingly, this E-box motif is surrounded by two overlapping sterol regulatory element-binding protein (SREBP) binding sites. USF and SREBP bind on
the fatty acid synthase promoter, independently of their respective binding sites, suggesting that distinct signaling transduction pathways
are probably involved in recruiting USF or SREBP. It has recently been
shown that SREBP-1 and -2 and USF bind to overlapping sites in elements
AB and K of the apoA-II promoter (54), suggesting that these elements
may also play a role in the regulation of apoA-II gene in
response to extracellular cholesterol levels.
In conclusion, ubiquitously expressed USF plays a crucial role in the
constitutive expression of the liver-restricted apoA-II gene. The binding of USF to proximal and distal E-box motifs of the
apoA-II promoter and the synergistic interaction with the liver-enriched factor HNF-4 result in a synergistic transactivation of
the apoA-II promoter. The cooperative binding of USF an HNF-4 may be
responsible for this synergy. A potential role of USF in the metabolic
regulation of the apoA-II gene is currently being investigated.