From the Division of Basic Sciences, Section of Biochemistry, Department Of Medicine, University Of Crete and Institute Of Molecular Biology and Biotechnology, Heraklion, Crete, Greece
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ABSTRACT |
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The regulatory elements CIIC
(159/
116) and CIIB (
102/
81) of the apolipoprotein CII (apoCII)
promoter have distinct specificities for orphan nuclear receptors
(Vorgia, P., Zannis, V. I., and Kardassis, D. (1998) J. Biol. Chem. 273, 4188-4199). In this communication we
investigated the contribution of ligand-dependent and
orphan nuclear receptors on the transcriptional regulation of the human apoCII gene. It was found that element CIIC in
addition to ARP-1 and EAR-2 binds RXR
/T3R
heterodimers strongly,
whereas element CIIB binds hepatic nuclear factor 4 (HNF-4)
exclusively. Binding is abolished by mutations that alter the HRE
binding motifs.
Transient cotransfection experiments showed that in the presence of T3,
RXR/T3R
heterodimers transactivated the
205/+18 apoCII promoter
1.6- and 11-fold in HepG2 and COS-1 respectively. No transactivation
was observed in the presence of 9-cis-retinoic acid.
Transactivation requires the regulatory element CIIC, suggesting that
this element contains a thyroid hormone response element. HNF-4 did not
affect the apoCII promoter activity in HepG2 cells. However, mutations
in the HNF-4 binding site on element CIIB and inhibition of HNF-4
synthesis in HepG2 cells by antisense HNF-4 constructs decreased the
apoCII promoter activity to 25-40% of the control, indicating that
HNF-4 is a positive regulator of the apoCII gene. ARP-1 repressed the
205/+18 but not the
104/+18 apoCII promoter activity in HepG2
cells, indicating that the repression depends on the regulatory element
CIIC. In contrast, combination of ARP-1 and HNF-4 transactivated
different apoCII promoter segments as well as a minimal adenovirus
major late promoter driven by the regulatory element CIIB. Mutagenesis
or deletion of elements CIIB or CIIC established that the observed
transactivation requires DNA binding of one of the two factors and may
result from HNF-4-ARP-1 interactions that elicit the transactivation
functions of HNF-4.
The combined data indicate that RXR/T3R
in the presence of T3 and
HNF-4 can upregulate the apoCII promoter activity by binding to the
regulatory elements CIIC and CIIB, respectively. In addition, ARP-1 can
either have inhibitory or stimulatory effects on the apoCII promoter
activity via different mechanisms.
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INTRODUCTION |
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Plasma apolipoprotein CII (apoCII)1 is a potent activator of the lipoprotein lipase, has known protein and gene sequence, and plays an important role in the catabolism of triglyceride-rich lipoproteins (1-8).
We have shown recently that the 0.55-kilobase intergenic region between
the apoCII and apoCIV genes is a strong cell
type-specific promoter, and its activity is enhanced by hepatic control
region 1 (9, 10). The apoCII promoter contains five footprints defined by hepatic nuclear extracts and designated CII-A (74/
44), CII-B (
102/
81), CII-C (
159/
116), CII-D (
288/
265), and CII-E
(
497/
462). An important role in apoCII gene regulation
and transcriptional enhancement is mediated by two hormone response
elements, which map within the footprinted regions CIIB (
102/
81)
and CIIC (
159/
116) and have different specificities for orphan
nuclear receptors. CIIC is recognized by ARP-1, EAR-2, but not HNF-4,
whereas CIIB is recognized exclusively by HNF-4 (10).
Orphan nuclear receptors as well as receptors for retinoids and
thyroids are members of a nuclear receptor superfamily that controls
diverse biological functions including growth, development, and
homeostasis (11-15). They recognize specific hexameric AG(G/T)TCA motifs with variations in sequence, spacing, and orientation, designated hormone response elements (HREs) (16-20). In the current study, we demonstrate that in the presence of T3, RXR/T3R
heterodimers bind to a thyroid hormone response element (TRE) present
in element CIIC and transactivate the human apoCII promoter. Binding of
ARP-1 to the same site repressed the promoter activity. Furthermore, antisense methodologies and promoter mutagenesis established that HNF-4
is a positive activator required for optimal activity of the apoCII
promoter in HepG2 cells. Finally, combination of ARP-1 and HNF-4
superactivate the apoCII promoter via novel mechanisms that may require
interaction of the two factors on the apoCII promoter.
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EXPERIMENTAL PROCEDURES |
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Materials-- The sources of materials utilized have been described (10).
Plasmid Constructions--
The construction of the apoCII
promoter plasmids 545/+18 CII CAT,
388/+18 CII CAT,
388/+18 CII
Bmut CAT,
388/+18 CII Cmut CAT,
388/+18 CII C/B mut CAT,
205/+18
CII CAT,
104/+18 CII CAT as well as the pUCSHCAT vector have been
described previously (10, 21). Plasmids pMT2, pMT2-hHNF-4, pMT2-hARP-1,
pMT2-hEAR-2, pMT2-hRXR
, pMT2-hRAR
, pMT2-hT3R
, and pMT2-hRAR
have been described previously (22, 23). The plasmid
205/+18 CII Cmut
CAT was constructed as follows. The
388/+18 CII Cmut CAT plasmid was digested by EcoRI, and the excised fragment corresponding to
the apoCII promoter
205/+18 was subcloned into the vector pBluescript at the EcoRI site, thus creating the intermediate plasmid
pBS
205/+18 CII Cmut. The apoCII promoter was subsequently excised from pBluescript by HindIII and SmaI digestion
and was cloned into the corresponding sites of vector pUCSHCAT
(21).
Transfections and CAT Assays--
Maintenance and conditions of
transfections of human hepatoma (HepG2) and monkey kidney COS-1 have
been described (10, 26). In the hormone induction experiments, the
hormone (107 M T3 or 10
6
M 9-cis-retinoic acid) was added to the cells
18 h after transfections, and cells were cultivated for 22 h
in Dulbecco's modified Eagle's medium supplemented with 5% charcoal
stripped and delipidated fetal bovine serum. For the generation of
permanent cell lines expressing antisense HNF-4, HepG2 cells were
seeded at a density of 5 × 105 cells/60-mm diameter
dishes, the day before transfection. The cells were transfected with 17 µg of pcDNA-neo anti-HNF-4 ribozyme, pCDNA-neo anti-HNF-4 or
the empty pCDNA-1-neo vector by the
Ca3(PO4)2 precipitation method.
Eighteen h after transfection, cells were trypsinized, diluted 1/20 and
1/40 and replated in 100-mm diameter dishes in a Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum and 500 µg/ml Geneticin (Life Technologies, Inc.). The concentration of
Geneticin in the cell culture medium was increased to 1000 µg/ml 2 weeks later to minimize the possibility of selection of false positive
clones. Resistant clones were selected using a cloning cylinder were
grown in Dulbecco's modified Eagle's medium + 10% fetal bovine serum
containing 1000 µg/ml Geneticin, characterized, and stored in liquid
nitrogen. Clones expressing the antisense HNF-4 were utilized in
transfection experiments in parallel with control HepG2 cultures.
Expression of Proteins in COS-1 Cells--
COS-1 cells were
transfected with 32 µg each of the following expression vectors:
pMT2-HNF-4, pMT2-ARP-1, pMT2-EAR-2, pMT2-hRXR, pMT2-hRAR
,
pMT2-hT3R
, and pMT2-hPPAR
(22, 23). Eighteen h later, cells were
fed with fresh medium and cultured for an additional 48 h. For the
preparation of whole cell extracts, cells were washed 2× with
phosphate-buffered saline and scraped into 1 ml of TEN buffer (40 mM Tris, pH 7.9, 1 mM EDTA, 150 mM
NaCl). Cells were pelleted by low speed centrifugation, resuspended in 200 µl of WCE lysis buffer (500 mM KCl, 50 mM
Hepes, pH 7.6, 1 mM EDTA, 1 mM dithiothreitol,
100 µM phenylmethylsulfonyl fluoride, 10% glycerol), and
lysed by three cycles of freeze-thawing. Undissolved material was
removed by high speed centrifugation at 4 °C, and the supernatant
was aliquoted and stored at
80 °C until use. The concentration of
the proteins in the whole cell extracts was approximately 0.5-1
µg/ml.
Gel Electrophoretic Mobility Shift Assays-- Assays using rat liver nuclear extracts or COS-1 whole cell extracts were performed as described (21, 27).
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RESULTS |
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Heterodimers of RXR/T3R
Bind to a TRE on Element CIIC and
Transactivate the Human apoCII Promoter in HepG2 and COS-1
Cells--
DNA binding experiments were performed using
oligonucleotide CIIC as a probe (Table I)
and extracts from COS-1 cells expressing RXR
, RAR
, T3R
, and
PPAR
. This analysis showed that element CIIC strongly binds
RXR
/T3R
, less efficiently binds T3R
, and weakly binds
RXR
/RAR
and RXR
/PPAR
heterodimers (Fig.
1A-D). Element CIIC contains
two direct repeats 5' ACGTCC(CCCA)AGGTCA 3' in the noncoding strand
between nucleotides
140 to
155 separated by four spacer nucleotides
(included in parentheses) (Fig. 1A). Mutations in both half
repeats of element CIIC abolished the binding of orphan and
ligand-dependent nuclear receptors to this site (Fig.
1A-D). Cotransfection experiments in HepG2 cells using the
205/+18 apoCII promoter mutated in element CIIC showed that this mutation reduced the apoCII promoter activity to approximately 40% of
the control value (Fig.
2A).
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HNF-4 Binds Exclusively to the Regulatory Element CIIB and Is
Required for Optimal Activity of the apoCII Promoter in Cells of
Hepatic Origin--
A previous study showed that the regulatory
element CIIB binds HNF-4 but not ARP-1 or EAR-2 (10). This element
contains a direct AAGTCCTGGCCA repeat between nucleotides 87 to
98
of the noncoding strand without spacer nucleotides between the two half
repeats (DR0). Mutations within the two half repeats (Fig. 1A) abolish the binding of HNF-4 to this site (Fig.
3A). DNA binding experiments
using the oligonucleotide CIIB as probe (Table I) and extracts from
COS-1 cells expressing different hormone nuclear receptors showed that
homodimers of RXR
and heterodimers of RXR
with RAR, T3R
, and
PPAR
do not bind to this element, indicating that the HRE of element
CIIB is an exclusive HNF-4 binding site (Fig. 3A).
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ARP-1 Can Repress the apoCII Promoter Activity by Binding to the
Regulatory Element CIIC. Combination of ARP-1 and HNF-4 Superactivates
the apoCII Promoter--
Cotransfection of HepG2 cells with the
205/+18 apoCII promoter CAT construct along with an ARP-1 expression
vector repressed the apoCII promoter activity by 50%. Repression could
be reversed by cotransfection with HNF-4 (Fig.
4A). This result indicated that ARP-1 plays a negative role in apoCII gene regulation.
ARP-1 was unable to repress the activity of the -104/+18 apoCII
promoter, which lacks element CIIC in HepG2 cells, indicating that
repression depends on the presence of the regulatory element CIIC (Fig.
4B). Unexpectedly, cotransfection of HepG2 cells with ARP-1
and HNF-4 transactivated the
205/+18 or the
104/+18 apoCII promoter
4.7- and 2.2-fold respectively, despite the fact that the activity of
these promoters in HepG2 cells is not affected by HNF-4 (10) (Fig. 4,
A and B). In addition, the combination of ARP-1
and HNF-4 transactivated the synthetic CIIB-AdML promoter, which
contains a single copy of element CIIB, whereas HNF-4 had no effect,
and ARP-1 increased 2-fold the activity of this promoter (Fig.
4C). Transactivation occurs despite the fact that ARP-1
cannot bind to either the
104/+18 apoCII promoter or the CIIB AdML
promoter. The findings suggest that the observed transactivation may be the result of direct protein-protein interactions, which increase the
transactivation potential of one or both of these factors. The
possibility that ARP-1 squelches negative regulators is less likely
since element CIIB is an exclusive HNF-4 binding site. In addition,
interactions of negative regulators such as ARP-1 with components of
the basal transcription complex are expected to exert negative rather
than positive effects on transcription (30). The positive effect of
ARP-1 on the apoCII promoter activity was further demonstrated by
co-transfection experiments in COS-1 cells that lack or contain very
low amounts of endogenous HNF-4 and ARP-1. This analysis showed that
HNF-4 transactivated the -104/+18 apoCII promoter 4-fold. The same
promoter, in the presence of increasing concentrations of ARP-1, was
transactivated up to 15-fold (Fig. 4D). Transactivation of
the
205/+18 apoCII promoter by the combination of ARP-1 and HNF-4
also occurred when the regulatory element CIIB, which is the binding
site of HNF-4, was mutated (Fig. 4E). The combined data of
Fig. 4, D and E indicate putative HNF-4-ARP-1
interactions on the apoCII promoter when either one of the two factors
is bound to the DNA. A schematic representation of the putative
mechanisms of activation or repression of the apoCII promoter
activity by HNF-4, ARP-1, and RXR
/T3R
heterodimers is shown in
Fig. 5.
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DISCUSSION |
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The Regulatory Element CIIC Is a TRE--
Thyroid hormone
receptors recognize specific hexameric half repeat motifs AG(G/T)TCA,
preferably separated by four spacer nucleotides (16-20). The
regulatory element CIIC, which is recognized by ARP-1 and EAR-2,
contains two direct repeats with DR4 spacing on the noncoding strand
between nucleotides 140 to
155. Previous studies have suggested
that this motif is the preferred binding site of RXR
/T3R
heterodimers (28). Indeed, the DNA binding data confirmed that
T3R
/RXR
heterodimers bind very strongly to this element. T3R
binds less efficiently, and homodimers of RXR
or heterodimers of
RXR
with RAR
and PPAR bind weakly, thus establishing that element
CIIC contains a TRE.
HNF-4 Is an Important Activator of the apoCII Promoter-- The DNA binding data of this and a previous study (10) established that the HRE present in element CIIB recognizes HNF-4 exclusively but does not recognize other orphan or ligand-dependent nuclear receptors that bind to the regulatory element CIIC (Fig. 3A). The importance of HNF-4 for the function of the apoCII promoter could not be assessed by cotransfection experiments, possibly due to the saturating amounts of HNF-4 in HepG2 cells (10). In the current study, utilization of antisense methodologies very convincingly established that HNF-4 is an important activator of the apoCII promoter. It is interesting that both the mutagenesis of the DNA recognition motif of HNF-4 as well as the antisense HNF-4 constructs reduced the apoCII promoter activity by approximately 60 to 75% of the control. The finding suggests that HNF-4 contributes to optimal promoter strength, but other factors, such as CCAAT enhancer binding protein C/EBP or related activities that bind to element CIID, may account for the remaining 25 to 40% of the promoter activity in the absence of HNF-4. The fact that HNF-4 is an activator of the apoCII promoter strength is also supported by cotransfection experiments in COS-1 cells, where the promoter is transactivated 9-fold in the presence of HNF-4 (10). The preservation of partial promoter activity in the absence of HNF-4 also differentiates the apoCII promoter from the promoters of the apoA-I, CIII, and AIV gene cluster, where intact HREs are essential for promoter activity (23, 34).
ARP-1 May either Repress or Transactivate the apoCII Promoter
Activity. Repression Requires Binding of ARP-1 to the TRE of Element
CIIC. Transactivation Requires the Presence of HNF-4 and Is Independent
of DNA Binding--
The regulatory element CIIC that contains the TRE
is required for optimal promoter activity, since mutagenesis of the TRE that abolishes binding of hormone nuclear receptors to this site reduces the apoCII promoter activity to approximately 40% of the control (Fig. 2A). On the other hand, this element is the
binding site of orphan nuclear receptors ARP-1 and EAR-2. Previous
studies have shown that ARP-1 and EAR-2 can usually but not always (36, 37) repress the promoter activity of other genes by competing for
binding to the same HRE (38-43). In the case of RXR and T3R
, transrepression may also be involved (30). The DNA binding data, in
combination with the transactivation data of this study, established that the transcriptional repression is caused by the binding of ARP-1
to the regulatory element CIIC, which in addition to orphan nuclear
receptors, strongly binds RXR
/T3R
heterodimers. In the absence of
this element, the activity of the apoCII promoter is not affected by
ARP-1.
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ACKNOWLEDGEMENTS |
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We thank Dr. Hinrich Gronemeyer for providing
us with 9-cis-retinoic acid and Dr. Fulvio Mavilio for
providing the phosphoglycerol kinase -galactosidase plasmid. We also
thank Dr. Helen Dell, Margarita Hadzopoulou-Cladaras, Horng-Yuan Kan,
and Aris Moustakas for helpful comments and Anne Plunkett for typing
the manuscript.
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FOOTNOTES |
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* This work was supported by Greek General Secretariat for Science and Technology Grants PENE 1663 and EPET 646 and Greek Ministry of Health Grant KESY E396.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed.Tel.: 3081-394564; Fax:
3081-394530.
§ Supported by National Institute of Health Grant HL-33952.
1
The abbreviations used are: apoCII,
apolipoprotein CII; apoCI', apoCI pseudogene; HRE, hormone response
element; TRE, thyroid hormone response element; hHNF-4, human hepatic
nuclear factor 4; hEAR-2, human v-erbA-related factor 2; hARP-1, human
apoA-I regulatory protein 1; CAT, chloramphenicol acetyltransferase; AdML, adenovirus major late promoter; hRXR, human retinoid X receptor
; hT3R
, human thyroid hormone receptor
; hRAR
,
human retinoic acid receptor
; hPPAR
, human peroxisome
proliferator-activated receptor
; T3, triiodothyronine; CREB, cAMP
response element binding protein.
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REFERENCES |
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