A Short Proximal Promoter and the Distal Hepatic Control Region-1 (HCR-1) Contribute to the Liver Specificity of the Human Apolipoprotein C-II Gene
HEPATIC ENHANCEMENT BY HCR-1 REQUIRES TWO PROXIMAL HORMONE RESPONSE ELEMENTS WHICH HAVE DIFFERENT BINDING SPECIFICITIES FOR ORPHAN RECEPTORS HNF-4, ARP-1, and EAR-2*

Pelagia Vorgia, Vassilis I. ZannisDagger , and Dimitris Kardassis§

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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
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Discussion
References

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

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 beta -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 [gamma -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 [alpha -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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Table II
Oligonucleotides used in the DNA binding assays

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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-beta 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.

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-beta 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.

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 [gamma -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.

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 [alpha -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%.

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-NFkappa 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.

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-beta 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.

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 alpha 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-beta 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-beta 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-beta gal plasmid. Bars correspond to the mean values (±S.E.) of two independent transfections performed in duplicate.

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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported in part by grants from the Greek General Secretariat for Science and Technology and the Greek Ministry of Health.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.

Dagger Supported by National Institutes of Health Grant HL-33952.

§ To whom correspondence should be addressed: Division of Basic Science, Section of Biochemistry, Dept. of Medicine, University of Crete, P. O. Box 1393, Heraklion 71110, Crete, Greece.

1 The abbreviations used are: apoC-II, apolipoprotein C-II; HCR-1, hepatic control region-1; HCR-2, hepatic control region-2; HRE, hormone response element; apoC-I, apolipoprotein C-I; apoC-III, apolipoprotein C-III; HNF-4, hepatic nuclear factor 4; EAR-2, v-erbA-related-2; ARP-1, apoA-I regulatory protein 1; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; CMV, cytomegalovirus; C/EBP, CAT enhancer-binding protein; kb, kilobase pair(s).

2 D. Kardassis unpublished results.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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