From the Division of Basic Sciences, Section of
Biochemistry, Department of Medicine, University of Crete and the
Institute of Molecular Biology and Biotechnology, Herakleion 71110, Crete, Greece
We have identified the regulatory elements, some
of the factors and potential regulatory mechanisms which determine the
tissue specificity of the human apoC-II gene. The
545/+18 apoC-II
promoter directs high levels of expression of the reporter CAT gene in cells of hepatic origin (HepG2), low levels of expression in cells of
intestinal origin (CaCo-2) and basal expression in HeLa cells. Deletion
analysis identified negative regulatory elements within the
545/
388
region and positive regulatory elements within the
388/
55 region.
Linkage of different apoC-II promoter segments to the hepatic control
region-1 (HCR-1) enhanced the promoter activity 2.5-11-fold in HepG2
cells but did not affect its activity in CaCo-2 or COS-1 cells. DNase I
footprinting analysis using rat liver nuclear extracts identified five
protected regions within the
545/+18 apoC-II promoter as follows:
CIIA (
74/
44), CIIB (
102/
81), CIIC (
159/
116), CIID
(
288/
265), and CIIE (
497/
462). Elements CIIB and CIIC contain
hormone response elements. CIIB is recognized by hepatic nuclear
factor-4 (HNF-4) but not ARP-1 or EAR-2, whereas CIIC is recognized by
ARP-1 and EAR-2 but not by HNF-4. HNF-4 transactivated the apoC-II
promoter or the apoC-II promoter linked to the HCR-1 in COS-1 cells. A
double mutation in elements CIIB and CIIC that eliminated binding of
HNF-4 or ARP-1 and EAR-2, respectively, to these sites abolished the
enhancer activity of HCR-1. The combined data suggest that the apoC-II promoter/HCR-1 cluster can direct expression in cells of hepatic origin
and that optimal enhancer activity requires synergistic interactions
between factors bound to the distal HCR-1 and nuclear receptors bound
to the two proximal hormone response elements.
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INTRODUCTION |
Plasma apolipoprotein CII
(apoC-II)1 is a 79-amino acid
protein that plays an important role in the catabolism of
triglyceride-rich lipoproteins (1, 2). ApoC-II is a potent activator of
the lipoprotein lipase, the enzyme that hydrolyzes the triglycerides of
chylomicrons and very low density lipoproteins (3, 4). Patients with
inherited apoC-II deficiency are unable to clear triglyceride-rich
lipoprotein particles from their plasma, and develop type I
hyperlipidemia (2, 5, 6). The human apoC-II gene has been mapped on the
long arm of chromosome 19 in a gene cluster that contains the
apoE/CI/CI'/CIV/CII genes and spans 45 kb of chromosomal region (7-9).
The intergenic region between the apoC-II and apoC-IV genes is only
0.55 kb (1).
The major site of apoC-II mRNA and protein synthesis in mammalian
species is the liver and a minor site is the intestine (10-12). Studies using transgenic mice have provided evidence for the
existence of common regulatory regions within the 45 kb that control
the tissue-specific expression of the apoC-I and apoE genes (13-15). These regions were designated hepatic control region-1 and -2 (HCR-1 and HCR-2), respectively (13, 14).
The objective of the current study was to identify the promoter
elements which confer tissue specificity and assess the potential role
of HCR-1 in the regulation of the human apoC-II gene. We demonstrate
that the
545/+18 5'-intergenic sequence located between the apoC-IV
and apoC-II genes is a very strong hepatic promoter. This region is
sufficient to direct cell type-specific expression in cell cultures and
its strength in hepatic cells is enhanced by HCR-1. The proximal
apoC-II promoter contains two hormone response elements (HREs) which
have different binding specificities for orphan nuclear receptors.
Optimal activity of the promoter/enhancer cluster requires synergistic
interactions between hormone nuclear receptors which bind to these
elements and factors which bind to the HCR-1.
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EXPERIMENTAL PROCEDURES |
Materials--
All materials were obtained from sources
described previously (16).
Plasmid Constructions--
The 5'-flanking region between
nucleotides
545 and +18 of the human apoC-II gene was PCR amplified
from human genomic DNA and inserted into plasmids pUCSH-CAT (17) and
pBluescript KS(+) (Stratagene) at the
HindIII/SmaI and EcoRV sites,
respectively. The pBS apoC-II plasmid was used as a template for the
generation of the 5' deletions
388/+18,
205/+18,
104/+18, and
55/+18. The sequence of the 5' primers used in the PCR reactions is
shown in Table I. The 3'-primer was the
Universal primer that hybridizes outside the polylinker region of
plasmid pBluescript. PCR reactions were performed using the MJ Research
automated MiniCycler, according to specifications of the manufacturer.
Oligonucleotide primers were synthesized at the Microchemistry
Laboratory of the Institute of Moleculary Biology and Biotechnology,
Crete, Greece. The PCR products containing the apoC-II promoter
deletions were digested with HindIII and SmaI and
cloned into plasmid pUCSH-CAT, which is a modified version of pUC-CAT
(17). For the construction of the
545 CII CAT SV40 plasmid, the
545/+18 apoC-II fragment was excised from plasmid pBS apoC-II with
HindIII and PstI and cloned at the corresponding
sites of pCAT Enhancer vector (Promega) which contains the SV40
enhancer placed downstream of the CAT gene. Plasmids
256 apoA-I CAT,
872 apoC-III CAT, and
268 apoB CAT have been described previously
(17, 18). HCR-1 (17) was amplified by PCR from genomic DNA purified
from HeLa cells using the primers HCR-1 and HCR-1c. The sequence of
these primers is shown in Table I. Primer HCR-1 was designed in such a
way that it does not hybridize to HCR-2 sequence. The absence of HCR-2 in the PCR products was verified by DNA sequencing. The PCR product was
purified by agarose gel electrophoresis and electroelution, using the
Little Blue Tank apparatus from ISCO, digested with XbaI and
KpnI, and cloned into the XbaI/KpnI
sites of the apoC-II CAT plasmids at the 5' end of the apoC-II
promoter. The expression vectors pMT2-HNF4, pMT2-ARP1, and pMT2-EAR2
containing the full-length cDNAs of HNF4, ARP1, and EAR2 orphan
nuclear receptors in vector pMT2 have been described previously
(18).
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Table I
Oligonucleotides used as primers in the PCR-based deletion mutagenesis,
DNase I footprinting and sequencing
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The plasmids containing point mutations in the apoC-II promoter in
elements CIIB, CIIC, or both designated CII-388-CM CAT, CII-388-BM CAT,
and CII-388 C/BM CAT, respectively, were constructed by the two-step
PCR procedure. For instance, to generate the mutant CII-388-CM-CAT the
region upstream of nucleotide
130 was amplified by PCR using the
CII-388 5'-primer that contains a HindIII site and the
mutagenic CII-CM-AS 3'-primer (Table I) using the plasmid pBS apoC-II
as a template. The region downstream of nucleotide
166 was amplified
by PCR and the Univ-31 3'-primer that hybridizes downstream of pBS
polylinker (Table I) and a mutagenic CII-CM-S 5'-primer. An aliquot of
each of the two PCR products (1%) was mixed and used as a template for
the amplification of the full-length
388/+18 mutated apoC-II promoter
using the CII-388 and Univ-31 as 5'- and 3'-primers, respectively. A
similar procedure was utilized to generate the CII-388-BM-CAT and
CII-388-C/BM-CAT mutants using the CII-BM-AS 3' and CII-BM-S5' primers
(Table I). The amplified DNA was digested with HindIII and
SmaI and cloned into the corresponding sites of plasmid
pUCSH-CAT (18). The introduction of point mutations in the apoC-II
promoter was verified by DNA sequencing.
Cell Cultures, Transfections and CAT Assays--
HepG2, HeLa,
and COS-1 cells were cultured in Dulbecco's modified Eagle's medium
supplemented with fetal bovine serum (10%). CaCo-2 cells were grown in
Dulbecco's modified Eagle's medium + 20% fetal bovine serum.
Transient transfections were performed by the CaPO4
co-precipitation method as described previously (19). The CAT activity
of the cell extracts was determined as described previously (20). The
activity of
-galactosidase enzyme was measured to normalize for the
transfection efficiency (21).
Preparation of Nuclear Extracts and Whole Cell Extracts from
Transfected COS-1 Cells--
Rat liver nuclear extracts were prepared
from 10 rats (approximately 150 g of liver) following the protocol
of Gorski et al. (22). Whole cell extracts from COS-1 cells
transfected with vectors pMT2-HNF4, pMT2-ARP1, and pMT2-EAR2 were
prepared as described (18).
DNase I Footprinting and Electrophoretic Mobility Shift
Assays--
For DNase I footprinting assays, DNA fragments spanning
the
545/
388,
388/
205, and
205/+18 apoC-II promoter region
were obtained either by PCR amplification or restriction digestion, were labeled with [
-32P]ATP and purified by
nondenaturing polyacrylamide gel electrophoresis and electroelution.
The DNase I footprinting assay was performed as described previously,
using ~50 µg of rat liver nuclear extract and 70-350 ng of DNase I
(17, 23, 24). For electrophoretic mobility shift assays,
oligonucleotides corresponding to the two strands of the apoC-II
footprinted regions A-E were synthesized and annealed to generate the
double strands, labeled with Klenow and [
-32P]dCTP and
incubated with nuclear extracts (4 µg). Competitor oligonucleotides
were added to the reactions prior to the addition of the probe, at
25-200-fold excess. The sequence of the oligonucleotides used is shown
in Table II. Assays were performed as
described previously (17, 24, 25).
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RESULTS |
The ApoC-II Promoter Confers Cell Type-specific Expression--
To
identify the promoter elements that contribute to this tissue
specificity, the (
545/+18) apoC-II promoter region was linked to the
promoterless bacterial chloramphenicol acetyltransferase (CAT) gene and
the resulting plasmid (
545/+18)CII CAT (Fig.
1A) was transiently
transfected in HepG2, CaCo-2, and HeLa cells. To assess the relative
activity of the apoC-II promoter, these three cell lines were also
transfected in parallel with CAT containing vectors under the control
of the apoA-I and C-III gene promoters (17, 24) or the promoter and
enhancer of the SV40 virus (26).

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Fig. 1.
A-C, relative activity of the 545/+18
apoC-II promoter region. Panel A, schematic representation
of the apoC-II promoter region relative to the
apoE/apoC-I/apoC-I'/apoC-IV/apoC-II gene cluster on human chromosome
19. Double arrows show the intergenic distances in kb. Also
shown are the HCR-2 as well as the HCR-1 and the 545/+18 apoC-II
promoter region utilized in this study. Panel B, analysis of
the relative strength of the human apoC-II promoter. The indicated CAT
plasmids shown at the bottom of the panel (3 µg) were transiently
transfected together with plasmid CMV- gal (2 µg) in HepG2 cells
and CAT activity was determined as described under "Experimental
Procedures." Panel C, analysis of the relative strength of
the human apoC-II promoter in HepG2, CaCo-2, and HeLa cells. The
plasmid ( 545/+18)CII CAT was transiently transfected in parallel with
the pCAT control plasmid, under the control of the SV40 promoter and
enhancer in HepG2, CaCo-2, and HeLa cells. The relative activity of the
( 545/+18)CII CAT construct in the three cell lines is expressed as
the ratio of the apoC-II/SV40 activities. Note that the 545/+18
apoC-II promoter directs tissue-specific CAT expression. Plasmid
pUCSH-CAT (17), which contains only the promoterless CAT gene, was used
as a negative control. In both panels, vertical bars
represent the mean values (±S.E.) of at least two independent
experiments performed in duplicate.
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This analysis showed that the strength of the apoC-II promoter in HepG2
cells is comparable to that of the SV40 promoter/enhancer and it is 6.4 and 2 times higher than that of the apoA-I and the apoC-III promoter,
respectively (Fig. 1B) (17, 24). Fusion of the apoC-II
promoter to the SV40 enhancer increased slightly (1.2-fold) the
promoter activity in HepG2 and CaCo-2 cells (data not shown). The
activity of the apoC-II promoter in CaCo-2 cells is 28% of that
observed in HepG2 cells. In HeLa cells the apoC-II promoter activity is
very low. This can be deduced by comparison of the ratio of the
CII/SV40 promoter activity in the three different cell lines. This
ratio is 3.5 and 35 times higher in HepG2 cells as compared with CaCo-2
and HeLa cells, respectively (Fig. 1C). The findings
indicate that the 5'-flanking region (
545/+18) of the human apoC-II
gene is a strong promoter and the pattern of its expression in cell
cultures mimics the pattern of expression of the apoC-II gene in
vivo.
To further identify and characterize the regions which constitute the
apoC-II promoter activity, a series of progressive apoC-II promoter
deletions extending to nucleotides
388,
205,
104, and
55 were
generated and the truncated promoter segments were linked to the CAT
reporter gene. Transient transfection assays in HepG2 cells showed that
deletion of the
550 to
388 region increased 2-fold the apoC-II
promoter activity possibly due to the elimination of negative
regulatory elements (Fig. 2A).
Further deletion of the sequence extending to nucleotide
205
decreased the promoter activity to 75% as compared with the
545/+18
CII promoter (or 38% compared with the
388/+18 promoter). The
promoter activity was not affected further by deletion of the sequences upstream of nucleotide
104 but it was reduced to 10% compared with the
545/+18 promoter (or 5% compared with the
388/+18
promoter) by deletion of the sequence upstream of the nucleotide
55 (Fig. 2A).

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Fig. 2.
A and B, determination of the
minimal length of the apoC-II promoter required for transcription in
HepG2 and CaCo-2 cells. Panels A and B, promoter
segments extending to nucleotides 545, 388, 205, 104, and 55
were fused to the CAT reporter gene. Three µg of the resulting
plasmids along with 2 µg of plasmid CMV- gal were transfected in
HepG2 (panel A) and CaCo-2 (panel B) cells,
respectively, and the CAT activity was determined as described under
"Experimental Procedures." In each panel, the length of apoC-II
promoter segments is shown on the left, a representative CAT
assay is shown in the middle, and the mean values ± S.E. of at least two independent experiments performed in duplicate are shown
on the right as horizontal bars. The activity of
the ( 545/+18)CII CAT plasmid was arbitrarily set to 100% in both
panels.
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Similar results were obtained in transfection assays in cells of
intestinal origin (CaCo-2). Thus, deletion of the apoC-II promoter
region
545 to
388 increased the promoter strength 1.7-fold. Additional sequential deletions extending to nucleotides
205 and
104 reduced the apoC-II promoter activity to 70% compared with the
545/+18 CII promoter (or 41% compared with the
388/+18 promoter).
Finally, deletion of the sequence upstream of nucleotide
55 decreased
the promoter activity to 25% as compared with the
545/+18 promoter
(or 12% as compared with the
388/+18 promoter) (Fig.
2B).
The data of Fig. 2, A and B, indicate that a
short proximal promoter region between nucleotides
104 and +18
contains positive regulatory elements which are important for
transcription in cells of hepatic and intestinal origin and are the
binding sites for hepatic and intestinal nuclear factors. Two
additional sets of elements exist, one negative in the region
545 to
388 and one positive in the region
388 to
205. These elements
also contribute to the overall activity of the apoC-II promoter in
HepG2 and CaCo-2 cells.
The ApoC-II Promoter Contains Two Hormone Response Elements with
Different Specificity for Members of the Nuclear Receptor
Family--
The regulatory regions present in the apoC-II promoter
region
545/+18 were identified by DNase I footprinting using rat
liver nuclear extracts. This analysis identified the following five footprint regions designated: A (
74/
44), B (
102/
81), C
(
159/
116), D (
288/
265), and E (
497/
462) (Fig.
3, A-C). A summary of the footprints identified is shown in Fig. 3D. The DNase I
footprinting analysis is compatible with the functional CAT analyses
shown in Fig. 2A. Thus, the deletion of the apoC-II promoter
between nucleotides
104 and
55 that resulted in a dramatic drop in
promoter activity in HepG2 cells, is associated with the elimination of the footprint CIIB and part of footprint CIIA. Furthermore, the regions
545 to
388 and
388 to
205, the deletion of which resulted in
activation and repression of the apoC-II promoter, respectively, contain the footprints CIIE and CIID, respectively.

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Fig. 3.
A-D, definition of the binding
sites of hepatic nuclear activities on the apoC-II promoter by DNase I
footprinting. Panel A, DNase I footprinting analysis of the
apoC-II promoter segment 205/+18 labeled at position +18 with
[ -32P]ATP and T4 polynucleotide kinase. Reactions were
performed as described under "Experimental Procedures." In this as
well as in panels B and C, G + A is a Maxam and
Gilbert sequencing ladder of the same DNA fragment used in the
footprinting analysis. NE, footprinting reactions
performed in the absence of nuclear extracts using 40 and 80 ng of
DNase I. +NE, footprinting reactions performed in the
presence of 40 µg of rat liver nuclear extracts and 140 (1), 280 (2), and 420 (3) ng of DNase
I. Boxes show areas protected from DNase I digestion and
numbers refer to their positions relative to the
transcription initiation site (+1). Panel B, DNase I
footprinting analysis of the apoC-II promoter region 388/ 205
labeled at position 205 as described under "Experimental
Procedures." +NE represent footprinting reactions
performed with 140 and 280 ng of DNase I. Panel C, DNase I
footprinting analysis of the apoC-II promoter 545/ 388 region
labeled at position 545 as described under "Experimental Procedures." +NE represents footprinting reactions
performed as described in panel B. Panel D, summary of the
DNase I footprints on the apoC-II promoter region using rat liver
nuclear extracts.
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DNA binding and competition experiments using the CIIB (
102/
81)
region as probe and rat liver nuclear extracts showed that this probe
forms a broad DNA-protein complex designated CIIB1 (Fig.
4A). This complex could be competed out by excess of CIIB but not by excess of oligonucleotides CIIA, CIIC, CIID, and CIIE corresponding to the other regions identified by DNase I footprinting. Other competition experiments showed that the complex formed with oligonucleotide CIIB could be competed by an excess of oligonucleotide (BA1 Table II) shown previously to bind different members of the nuclear hormone receptor family (27) but not by other oligonucleotides which contain binding sites for C/EBP (17, 24) (Fig.
4A).

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Fig. 4.
A-C, gel electrophoretic mobility shift
and competition assays using the oligonucleotides CIIB ( 102/ 81),
CIIC ( 159/ 116) of the apoC-II promoter or BA1 ( 81/ 62) of the
apoB promoter as probes and rat liver nuclear extracts. Panel
A, a double stranded oligonucleotide corresponding to the apoC-II
footprint CIIB ( 102/ 81) was labeled at both ends with Klenow DNA
polymerase and [ -32P]dCTP and incubated with 4 µg of
rat liver nuclear extracts in the absence or presence of the competitor
oligonucleotides shown at the top at the indicated fold of
molar excess. The sequence of the competitor oligonucleotides is shown
in Table II. Oligonucleotides corresponding to footprints CIIA, CIIC,
CIID, and CIIE did not compete. The arrow shows the position
of a single broad complex designated CIIB1 that is formed by rat liver
nuclear extracts and the CIIB probe. Panel B,
oligonucleotide CIIC was labeled as described above, incubated with 4 µg of rat liver nuclear extracts in the absence or presence of the
indicated competitor oligonucleotides. Competition was performed at
100-fold molar excess of competitor relative to the probe unless
indicated otherwise. The arrow shows the position of a
single rat liver nuclear activity designated CIIC1, that interacts with
the CIIC probe. Panel C, the apoB promoter element BA1 ( 81
to 62) was labeled as described above, incubated with 4 µg of rat
liver nuclear extracts and tested in competition assays using 100-fold
molar excess of the indicated apoC-II and BA1 oligonucleotides.
Following electrophoresis, the gel was dried, exposed to x-ray film for
24 h, and the bands corresponding to the bound and free BA1 probe
were cut from the gel. The amount of radioactivity in each band was
estimated by scintillation counting. The results are plotted as percent
of the probe shifted in the presence of the competitor oligonucleotide,
relative to the percent of the probe shifted in the absence of any
competitor oligonucleotide. The percent of the probe shifted in the
absence of competitor oligonucleotide was arbitrarily set to
100%.
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Similar DNA binding and competition experiments showed that
oligonucleotide CIIC formed a single protein-DNA complex, designated CIIC1 (Fig. 4B). The formation of this complex could be
competed out very efficiently by 100-fold molar excess of unlabeled
CIIC competitor oligonucleotide but could not be competed out by cold oligonucleotides CIIA, CIIB, CIID, and CIIE suggesting that the CIIC1
binding activity recognizes a single site on the apoC-II promoter. The
binding of CIIC1 to oligonucleotide CIIC could not be competed out by
oligonucleotides BA1 which contains an HRE (27, 28) and BA2, AIC, and
CIIID (Table II) which contain binding sites for C/EBP (17, 24, 29,
30).
To assess the types of nuclear hormone receptors which can recognize
oligonucleotide CIIB, oligonucleotide BA1 was labeled and incubated
with rat liver nuclear extracts in the absence or presence of 100-fold
molar excess of unlabeled oligonucleotides BA1, CIIA, CIIB, CIIC, CIID,
and CIIE (Table II). This analysis showed that oligonucleotides CIIC
and CIIB competed 55 and 65% for the binding of nuclear factors which
recognize the regulatory element BA1 (Fig. 4C). The fact
that oligonucleotide CIIB does not compete for binding of the CIIC1
activity to the oligonucleotide CIIC and vice versa, whereas both
oligonucleotides compete partially for the binding of DNA-protein
complexes formed with oligonucleotide BA1 which contain binding sites
for nuclear receptors, suggested that oligonucleotides CIIB and CIIC
might bind different members of the nuclear hormone receptor
family.
Direct DNA binding and supershift assays were used to assess the
binding of orphan nuclear receptors HNF4, ARP1, and EAR2 to
oligonucleotides CIIB and CIIC. These receptors were shown earlier to
bind to the regulatory element BA1 of apoB (28). HNF4 is positive and
ARP1 and EAR2 are negative regulators of the apoB promoter activity in
HepG2 cells (27, 28). This analysis showed that oligonucleotides CIIB
and CIIC have a complementary pattern of binding to the three
receptors. Oligonucleotide CIIB is recognized by HNF4, but not ARP1 or
EAR2, whereas oligonucleotide CIIC is recognized by ARP1 and EAR2 but
not HNF4 (Fig. 5, A and B). Oligonucleotide CIIA did not recognize any of the three
receptors (data not shown). The findings indicate that the apoC-II
promoter contains two HREs that recognize different members of the
nuclear receptor gene superfamily. The DNA binding assays using CaCo-2 nuclear extracts showed that oligonucleotide CIIB forms a single protein-DNA complex with similar electrophoretic mobility as HNF-4 expressed in COS-1 cells (Fig. 5A, lane 6), whereas
oligonucleotide CIIC formed several protein-DNA complexes. The major
protein-DNA complex had similar mobility with the complex formed by
EAR2 expressed in COS-1 cells (Fig. 5A, last lane). The
similarity in electrophoretic mobility is consistent but not conclusive
evidence of the nature of nuclear activities which recognize the
regulatory elements CIIB and CIIC. The binding of HNF-4 present in rat
liver nuclear extracts to the regulatory element CIIB was further
verified by DNA binding supershift assays using anti-HNF-4 antibodies.
Addition of anti-HNF-4 resulted in the formation of a lower mobility
tertiary DNA-protein complex when either CIIB or BA1 oligonucleotides
were utilized as probes (Fig. 5B). The combined data
presented in Figs. 4 and 5 suggest that elements CIIB and CIIC contain
HREs that bind different members of the nuclear receptor superfamily
present in cells of hepatic and intestinal origin.

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Fig. 5.
A and B, DNA binding gel
electrophoretic mobility shift assays using the CIIB, CIIC
oligonucleotides as probes and extracts of COS-1 cells expressing
HNF-4, ARP-1, and EAR-2. Panel A, orphan nuclear receptors
HNF-4, ARP-1 and EAR-2 were transiently expressed in monkey-kidney
COS-1 cells and 36 h later whole cell extracts were prepared as
described under "Experimental Procedures." Panel A,
assays using the CIIB and CIIC oligonucleotides as probes and 4 µg of
rat liver nuclear extracts, 1 µl of CaCo-2 nuclear extracts or 1 µl
of the COS-1 extracts expressing nuclear receptors as indicated at the
top of the figure. Panel B, gel electrophoretic mobility shift assays using oligonucleotides CIIB ( 104/ 83) of the
apoC-II promoter and BA1 ( 82/ 61) of the apoB promoter as probes and
rat liver nuclear extracts (RLNE) or COS-1 extracts expressing HNF-4 in
the absence or presence of an anti-HNF-4 polyclonal antibody or control
anti-c-Jun and anti-NF B antibodies. The arrows show the positions of the complexes formed by the CIIB and BA1 probes
and HNF-4 in the presence or absence of the antibody.
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The HCR-1 Confers Cell Type-specific Enhancement of the ApoC-II
Promoter Which Requires Interactions between Hormone Nuclear Receptors
Bound to Elements CIIC and CIIB and Factors Bound to the
HCR-1--
Recent studies have established that the HCR-1 is necessary
for the hepatic expression of the apoE and apoC-I genes in transgenic mice (2, 7). HCR-1 also directs the hepatic expression of the apoA-IV
gene which is located at a different gene locus (31). To assess the
contribution of HCR-1 to the strength and the specificity of the
apoC-II promoter, a new plasmid was constructed which contains the CAT
gene under the control of the apoC-II promoter region fused to the
HCR-1 designated HCR-1/(
545/+18)CII-CAT. Cotransfection experiments
showed that HCR-1 enhanced 2.5-fold the strength of the
545/+18
apoC-II promoter in HepG2. This enhancement was hepatocyte-specific since it was not observed in CaCo-2 or COS-1 cells, where the activity
of the apoC-II promoter did not change significantly. In fact, HCR-1
caused a slight reduction of the apoC-II promoter activity in CaCo-2
cells (Fig. 6A). Thus, HCR-1
has the ability to enhance the activity of a liver-specific promoter,
such as apoC-II, in cells of hepatic origin.

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Fig. 6.
A and B, cell-specific
enhancement of apoC-II promoter strength by HCR-1. Panel A,
plasmid ( 545/+18)CII CAT alone or fused with HCR-1 (3 µg) was
transiently cotransfected into HepG2, CaCo-2, or COS-1 cells along with
3 µg of CMV- gal plasmid. The mean values (±S.E.) from at least
two independent transfections performed in duplicate are presented in
the form of bar graphs. The activity of the ( 545/+18)CII
CAT plasmid in HepG2 cells was arbitrarily set to 100%. The activity
of this plasmid is 3.5 times lower in CaCo-2 cells and approximately 35 times lower in COS-1 cells as compared with HepG2 cells (see Fig.
1C). The 545/+18 apoC-II promoter strength increased
2.5-fold in HepG2 when it was fused to the HCR-1 but was not altered in
COS-1 cells and slightly reduced in CaCo-2 cells. Panel B, 3 µg of CAT gene constructs under the control of the wild-type,
truncated, or mutated apoC-II promoter alone or fused to HCR-1 were
transiently transfected into HepG2 cells and the CAT activity was
determined 40 h later as described under "Experimental
Procedures." The activity of the ( 545/+18)CII CAT plasmid in HepG2
cells was arbitrarily set to 100%. HCR-1 increased 11-fold the
strength of the 205/+18 apoC-II promoter and 2.5-3-fold the strength
of the shorter or longer promoter segments. A double mutation,
( 388/+18)C/B MutCII CAT, abolished the HCR-1 mediated enhancer
activity. Bars represent mean values (±S.E.) from at least
two independent transfections performed in duplicate.
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To identify the regulatory elements of the apoC-II promoter responsible
for the HCR-1-mediated transcriptional enhancement, HCR-1 was fused to
truncated apoC-II promoter segments extending to nucleotides
388,
205, and
104. Transfection experiments using the truncated chimeric
promoters showed that HCR-1 enhanced the activity of the
205/+18
apoC-II promoters 11-fold whereas it enhanced 3- and 2.5-fold the
activity of the shorter (
104/+18) and longer (
388/+18) apoC-II
promoter regions (Fig. 6B). This observation suggested that
this proximal
205/+18 promoter region, which contains footprints
CIIA, CIIB, and CIIC, has the optimum capacity for enhancement by the
HCR-1.
To delineate further which regulatory regions of the proximal apoC-II
promoter were important for the observed cell type-specific transcriptional enhancement, we introduced point mutations at certain
positions within the HRE of footprints CIIC and CIIB which abolished
the binding of nuclear activities to this site (data not shown).
Chimeric CAT constructs were also generated which linked the HCR-1 to
the
388/+18 apoC-II promoter mutated in regions CIIB, CIIC, or both.
Transient transfection experiments showed that mutations within the
HREs of footprints CIIB, CIIC, and the double mutation in both regions
reduced the activity of the
388/+18 CII promoter to 36, 44, and 41%
respectively in HepG2 cells as compared with the wild-type promoter
activity (Fig. 6B), indicating that regions CIIB and CIIC
represent important regulatory elements of the apoC-II promoter.
Mutations in elements CIIB and CIIC reduced the ability of HCR-1 to
enhance the
388/+18 apoC-II promoter by approximately 42% as
compared with the wild-type promoter but did not affect the fold
enhancement of the activity of the mutated promoters. In contrast, the
activity of a chimeric
388/+18 apoC-II promoter containing the double
mutation in elements CIIB and CIIC could no longer be enhanced by HCR-1
in HepG2 cells (Fig. 6B). These data indicated that the cell
type-specific transcriptional enhancement conferred by HCR-1 depends on
synergistic interactions among factors bound to the distal HCR-1
elements and hormone receptors bound to the two proximal HREs.
HNF-4 Transactivates the ApoC-II Promoter and the ApoC-II
Promoter/HCR-1 Cluster in COS-1 Cells--
Hepatocyte nuclear factor-4
has been shown previously to transactivate several liver-specific
promoters such as apoC-III, albumin, and
1-antitrypsin
among others (32). Co-transfection of HepG2 cells with the
(
545/+18)CII CAT plasmid along with the expression vector pMT2-HNF-4
which carries the cDNA of human HNF-4 under the control of the
adenovirus major late promoter showed that HNF-4 could not
transactivate the apoC-II promoter (Fig. 7A). This could be due to the
presence of saturating amounts of endogenous HNF-4 in these cells, as
reported previously (32). To by-pass this limitation, co-transfection
experiments were performed in the monkey kidney COS-1 cells which lack
endogenous HNF-4 activity. As shown in Fig. 7B,
cotransfection of COS-1 cells with the (
545/+18)CII CAT plasmid along
with the HNF-4 expression vector resulted in a 9-fold transactivation
of the apoC-II promoter. Similar cotransfection experiments of COS-1
cells with shorter apoC-II promoter-CAT plasmids extending to
nucleotides
205,
104, and
55 showed that the
205/+18 promoter
which contains both elements CIIB and CIIC was transactivated by HNF-4
5.5-fold whereas the
104/+18 promoter which retains only element CIIB
was transactivated 9-fold by HNF-4. The findings suggest that factors
bound to the footprint CIID region contribute to the HNF-4 mediated
transactivation whereas factors bound to element CIIC hinder the HNF-4
mediated transactivation. Finally, the
55/+18 promoter which lacks
both elements CIIB and CIIC was transactivated only 1.8-fold by HNF-4.
The findings suggest that the interaction of HNF-4 with element CIIB is
essential for the efficient transactivation of the apoC-II promoter by
HNF4.

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Fig. 7.
A-C, transactivation of the apoC-II
promoter by HNF-4 in COS-1 cells. Panel A, HepG2 cells were
transiently cotransfected with 3 µg of the ( 545/+18)CII CAT plasmid
along with 2 µg of CMV- gal and 1 µg of expression vector
pMT2-HNF4 or the expression vector pMT2 alone. Forty hours following
transfection, cells were harvested and CAT activity was determined as
described under "Experimental Procedures." The mean values (±S.E.)
from at least two independent transfections performed in duplicate are
presented in the form of bar graphs. Panel B, COS-1 cells
were transiently co-transfected with 3 µg of the indicated apoC-II
promoter deletion plasmids along with 2 µg of CMV- gal and 1 µg
of expression vector pMT2-HNF4 or the expression vector pMT2 alone. The
mean values (±S.E.) from at least two independent transfections
performed in duplicate are presented in the form of bar
graphs. In both panels A and B the activity
of the HCR-1/( 545/+18)CII CAT plasmid was set arbitrarily to 100%.
Panel C, COS-1 cells were transiently cotransfected with
HCR-1/( 545/+18)CII CAT, (HCR-1(-388/+18)CII CAT, HCR-1 (-388/+18)Cmut CII CAT, HCR-1 (-388/+18)Bmut CII CAT, and HCR-1 (-388/+18)B/Cmut CII
CAT plasmids along with the pMT2 vector or the indicated concentrations (ng) of the pMT2-h HNF-4 vector and 2 µg of CMV- gal plasmid. Bars correspond to the mean values (±S.E.) of two
independent transfections performed in duplicate.
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As shown in Fig. 5, A and B, element CIIB is the
binding site of HNF-4. In addition it has been shown that HCR-1
contains an HRE which may bind different members of the hormone
receptor family including HNF-4 (14). To test whether the HREs present in the proximal promoter and/or the HCR-1 contribute to the HNF-4 mediated transcriptional enhancement, we performed co-transfection experiments using the reporter construct HCR-1/(
545/+18)CII CAT and
HNF-4 expression plasmid. This analysis showed that HNF-4 was unable to
transactivate this plasmid in HepG2 cells (data not shown). However,
HNF-4 transactivated the HCR-1/(
545/+18)CII promoter cluster 33-fold
in COS-1 cells in a dose-dependent fashion (Fig. 7C,
left side). This 33-fold transactivation is 3.6-fold higher than
the 9-fold transactivation achieved by the apoC-II promoter alone (Fig.
7B). The
388/+18 promoter linked to the HCR-1 was
transactivated by HNF-4 to the same extent (29-fold) as compared with
the
545/+18 promoter. Mutations in the regulatory elements CIIB and
CIIC reduced the HNF-4 mediated transactivation by approximately 40 and
60%, respectively. However, mutations in both elements reduced
the HNF-4-mediated transactivation by 83%. The findings suggested that
transactivation of the apoC-II promoter/HCR-1 enhancer cluster requires
synergistic interactions between HNF-4 bound to distal HCR-1
site(s) and HNF-4 or other nuclear receptors bound to the regulatory
elements CIIB and CIIC.
 |
DISCUSSION |
Background--
ApoC-II has been involved in the catabolism of
triglyceride-rich lipoproteins (5, 6). At the physiological plasma
concentrations, apoC-II activates lipoprotein lipase which hydrolyzes
the triglycerides of chylomicrons and very low density lipoproteins (3,
5, 6). In contrast, overexpression of the apoC-II gene in transgenic mice interferes with the catabolism of triglyceride-rich
lipoproteins and is associated with hypertriglyceridemia (33). This
indicates that correct regulation of apoC-II gene expression is
important for the catabolism of triglyceride-rich lipoproteins.
The 0.55-kb Long Intergenic Region between the ApoC-IV and ApoC-II
Genes Is a Strong Tissue-specific Promoter--
It was shown
previously that the 5'-region of the apoC-II gene (15) extending 6 kb
upstream of the initiation of transcription was able to promote
transcription in HepG2 cells. It was subsequently shown that this 6-kb
region contains a complete copy of the human apoC-IV gene (7). The
current study has established that the
545/+18 intergenic region
located 5' of the apoC-II gene and 3' of the apoC-IV gene had high
levels of promoter activity in cells of hepatic origin (HepG2) low
levels of activity in cells of intestinal origin (CaCo-2) and basal
activity in HeLa cells. The apoC-II promoter is the strongest
apolipoprotein promoter encountered to date and its activity is
comparable to the activity of the SV40 promoter/enhancer cluster. Thus,
this
545/+18 apoC-II promoter region displayed all the
characteristics of a tissue-specific promoter and was analyzed
further.
Positive and Negative Regulatory Elements Exist within the
545
and
205 ApoC-II Promoter Region--
A combination of deletion
mutagenesis and in vitro DNase I footprinting experiments
were employed to define the minimal region required for the apoC-II
promoter activity. The 5'-deletion mutagenesis established that optimal
promoter activity both in HepG2 and CaCo-2 cells could be achieved with
the
388 to +18 apoC-II promoter region. Extension of the promoter to
nucleotide
545 decreased the promoter activity to 50% of its optimal
value, indicating that factors recognizing this region exert a negative
effect on the promoter strength. This region contains a single
footprint, CIIE, between nucleotides
497 and
462, which contains
the sequence 5'-CAATGAGTAGAAG-3' between nucleotides
491 and
479.
This sequence is homologous to the consensus binding site for
transcription factor HNF-1 (5'-CAATNANNANNNG-3') (34, 35).
Deletion of the
388 to
205 promoter region decreased the promoter
strength to 38% of its optimal value that was achieved with the
388
to +18 promoter indicating the presence of positive regulatory elements
within the
388 to
205 region. This region contains a single
footprint, CIID, between nucleotides
288 and
265, and contains the
sequence 5'-TGACTC-3' that is homologous to the binding site for the
bZip transcription factor GCN4 in the UAS region of His3 and His4 genes
of Saccharomyces cerevisiae (36, 37). Preliminary
results2 showed that an
oligonucleotide corresponding to the CIID region forms five DNA-protein
complexes when incubated with rat liver nuclear extracts and three of
these complexes were competed out by oligonucleotides containing the
binding site for bZip family members such as C/EBP (38). The role of
HNF-1 C/EBP and related factors which bind to the footprints CIIE and
CIID, respectively, on apoC-II gene transcription is under
investigation.
The Proximal Regulatory Region
205 to +18 Contains Two HREs with
Different Specificities for Orphan Nuclear Receptors--
DNase I
footprinting analysis of the proximal promoter region identified three
footprints designated CIIC (
159 to
116), CIIB (
102 to
81), and
CIIA (
79 to
44). Deletion of footprint CIIC did not reduce further
the apoC-II promoter activity as compared with the
205 to +18
promoter in both HepG2 and CaCo-2 cells. This footprint contains two
direct repeats 5'-ACGTCC(CCCA)AGGTCA-3' between nucleotides
141 and
156 of the noncoding strand separated by four spacer nucleotides that
are included in parentheses. The first half-repeat is identical and the
second very homologous to the consensus half-repeat site
5'-AG(G/T)TCA-3' of the HREs (39-41). DNA-binding gel electrophoresis
assays using extracts of COS-1 cells transiently transfected with
plasmids expressing HNF-4, ARP-1 (COUP-TFII), or EAR-2 (COUP-TFI) (18,
42) showed that oligonucleotide CIIC binds ARP-1 and EAR-2 but does not
bind HNF-4, indicating that the region defined by footprint CIIC is an
important regulatory element.
A putative HRE with DR0 spacing has also been identified on the
regulatory element G (
669 to
648) of the human apoC-III enhancer
which binds selectively ARP-1 and EAR-3 but not HNF-4 (43, 46). It is
interesting that in the case of the apoC-III promoter, mutations or
deletions in element G reduced significantly the hepatic transcription,
and either had no effect or increased slightly the intestinal
transcription (43). In the case of the apoC-II promoter, deletion of
the HRE of element CIIC which has a DR4 spacing affected similarly the
hepatic and intestinal transcription. Studies with apolipoprotein as
well as other promoters have shown that ARP-1 and EAR-2 repress the
promoter activity (28, 44-47). Although repressor activity of a
transcription factor can be achieved by different mechanisms, it is
well documented that in all cases when ARP-1 and EAR-2 act as
repressors, the negative effect is being exerted by competition with
HNF-4 or other nuclear hormone receptors for the same binding site
(28). It is interesting that in the case of the apoC-II promoter
mutations in footprint CIIC, which prevent the binding of ARP-1, EAR-2
to this site reduced the
388/+18 promoter activity to 44% of its
value. The finding suggest that ARP-1 and EAR-2 or other nuclear
receptor which can occupy this site act as positive activators of the
apoC-II promoter. The identification of other nuclear hormone receptors
capable of interacting specifically with elements CIIB and CIIC, as
well as their ability to transactivate the apoC-II promoter in the presence or absence of their ligands is the focus of ongoing research.
Deletion of the promoter region
104 to
55 which contains the
footprint CIIB and portion of footprint CIIA reduced the promoter activity to 5 and 12% as compared with the
388/+18 promoter in HepG2
and CaCo-2 cells, respectively. This minimal promoter contains the TATA
box and 11 nucleotides of the regulatory element CIIA. It is possible
that the 11 nucleotides of footprint CIIA serve as the binding site for
nuclear factors which facilitate the recruitment of the basal
transcription machinery to the adjacent TATA box. This could explain
the residual activity of this promoter region which is higher in CaCo-2
cells.
The footprint CIIB contains a direct repeat 5'-AAGTCCTGGCCA-3' between
nucleotides
87 to
98 of the noncoding strand without spacer
oligonucleotides between the two half-repeats (DR0). Both half-repeats
have sequence homology with the consensus half-repeat site AG(G/T)TCA
of the hormone nuclear receptors (39-41). Using DNA binding and
supershift assays with HNF-4 as well as DNA binding assays with ARP-1
and EAR-2 it was shown that CIIB contains an HRE with binding
specificity for HNF-4 or related factors and is not recognized by
ARP-1 or EAR-2. The findings indicate that the region defined by
footprint CIIB is also an important regulatory element. Deletion or
point mutations in this element decreased the apoC-II promoter
strength, indicating that factors bound to this element act as positive
regulators of transcription.
Overall, the present study establishes that the 0.55-kb long intergenic
region between the apoC-IV and apoC-II gene is a very strong promoter
with strict cell type-specificity. This specificity is restricted to
cells of hepatic and intestinal origin and is consistent with the known
tissue-specific expression of the apoC-II gene (10-12). The HCR-1 acts
as a liver-specific transcriptional enhancer of the apoC-II promoter in
cell cultures. HNF-4 and possibly other nuclear receptors may play an
important role in the overall promoter activity and the transcriptional
enhancement.
Activation of the ApoC-II Promoter or the ApoC-II Promoter/HCR-1
Cluster in Nonhepatic Cells Requires the Presence of HNF-4--
The
potential contribution of the regulatory element CIIB in the apoC-II
gene transcription was assessed by transient co-transfection experiments using different apoC-II promoter constructs and plasmids expressing the human HNF-4. This analysis showed that co-transfection with HNF-4 did not alter the apoC-II promoter activity or the activity
of the apoC-II promoter/HCR-1 cluster in HepG2 cells, possibly due to
saturating amounts of HNF-4 in these cells. However, co-transfection of
HNF-4 in COS-1 cells transactivated different apoC-II promoter segments
5-9-fold. The different degree of transactivation of the
545/+18 as
compared with the
205/+18 promoter segments by HNF-4, as well as the
deletion analysis of the apoC-II promoter discussed previously,
indicates that factors bound to footprint regions CIID and CIIE
contribute independently to the transcriptional activation of the
apoC-II promoter in different cell types. The transactivation of the
apoC-II promoter was maintained when the element CIIC was deleted but
was diminished in the
55 to +18 apoC-II promoter construct, which
lacks the regulatory element CIIB.
HNF-4 also transactivated the apoC-II promoter/HCR-1 cluster in COS-1
cells 33-fold as compared with a maximum of 9-fold transactivation achieved with the apoC-II promoter alone. The mutagenesis of the HREs
of the apoC-II promoter indicated that both elements CIIB and CIIC are
essential for the HNF-4 mediated transactivation. When both elements
were mutated, transactivation was reduced by 83%. These data are
consistent with the hypothesis that transactivation of the apoC-II
promoter involves synergistic interactions of HNF-4 bound to a distal
HCR-1 site(s) and HNF-4 or other nuclear receptors bound to the
regulatory elements CIIB, CIIC, or both. One cannot exclude the
possibility, however, that the residual transactivation of the
388/+18 promoter mutated in both HREs involves direct protein-protein
interactions between HNF-4 non-bound to DNA and factors related to
ARP-1 or EAR-2 bound to the regulatory element CIIC (48). Similar
interactions may account for the activation of the
388/+18 apoC-II
promoter mutated in element CIIB, in COS-1 cells, by HNF-4 (Figs.
6B and 7C).
HCR-1 contains six footprints within a 580-nucleotide region. This
580-nucleotide region or a minimum of 319-nucleotide region containing
the first three footprints are sufficient to direct hepatic expression
of the apoE gene in transgenic mice (14). The present study showed that
this 319-base pair region enhanced 2.5-fold the strength of the
545/+18 apoC-II promoter in HepG2 cells and had no effect in CaCo-2
or COS-1 cells in the absence of HNF-4. The HCR-1-mediated
transcriptional enhancement of the
205/+18 apoC-II promoter was
reduced from 11- to 2.5-fold by deletion or point mutations of the
regulatory element CIIC and was abolished by mutations which eliminate
the binding of nuclear receptors to the HREs of both elements CIIC and
CIIB (48). Previous studies have shown that HNF-4 can synergize with a
wide variety of transcription factors including C/EBP (49), CREB (50), and HNF-1 (44). Similar to other systems, it is assumed that the
nuclear receptors which bind to proximal and distal HREs as well as
other factors which bind to the apoC-II promoter/HCR-1 enhancer
elements form a stereospecific DNA-protein complex (51). This complex
may interact directly or indirectly through TATA box-binding protein
associated factors or transcriptional mediators/intermediary factors
(52-55) with the factors of basal transcription complex, thus leading
to the transcriptional activation of the target gene (Fig.
8). Recent studies have established that
multiple interactions of transcription factors with different TATA box
binding-protein associated factors and the proteins of the TFIID
complex are responsible for the synergistic activation of the target
genes (54, 55). Mutations in the proximal promoter that prevent the
binding of HNF-4 and other nuclear receptors to their cognate sites are
expected to eliminate these interactions and abolish the synergistic
activation of the apoC-II promoter/HCR-1 enhancer cluster.

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Fig. 8.
Schematic representations showing putative
protein-protein interactions among the factors bound to the apoC-II
promoter/HCR-1 region and the proteins of the basal transcription
machinery. The figure is based on the findings of the present
study and previously published data. The model speculates that factors
that bind to the promoter and enhancer sites help to properly orient
activators (tentatively assumed to be nuclear hormone receptors), to
interact optimally with the proteins of the basal transcription complex through transcriptional mediators/intermediary factors,
(TIFs) thus leading to transcriptional synergism.
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We are grateful to Dr. John Taylor for advice
throughout these studies and for critically reviewing this manuscript.
We also thank Drs. Eileen Falvey, Margarita Hadzopoulou-Cladaras, Aris Moustakas, and Iannis Talianidis for helpful comments and Anne Plunkett
for typing the manuscript.