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J. Biol. Chem., Vol. 275, Issue 39, 30423-30431, September 29, 2000

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A Hormone Response Element in the Human Apolipoprotein CIII (ApoCIII) Enhancer Is Essential for Intestinal Expression of the ApoA-I and ApoCIII Genes and Contributes to the Hepatic Expression of the Two Linked Genes in Transgenic Mice^*

Horng-Yuan Kan, Spiros Georgopoulos, and Vassilis Zannis

From the Section of Molecular Genetics, Whitaker Cardiovascular Institute of the Department of Medicine and the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

Received for publication, June 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have generated transgenic mice carrying wild-type promoters of the human apolipoprotein A-I (apoA-I)-apoCIII gene cluster or promoters mutated in their hormone response elements. The wild-type cluster directed high levels of apoA-I gene expression in liver and intestine, moderate expression in kidney, and low to minimal expression in other tissues. It also directed high levels of chloramphenicol acetyltransferase (CAT) expression (used as a reporter for the apoCIII gene) in liver, low levels in intestine and kidney, and no expression in other tissues. Mutations in the apoCIII promoter and enhancer abolished the intestinal and renal expression of the apoA-I gene, reduced hepatic apoA-I expression by 80%, and abolished CAT expression in all tissues. A similar pattern of expression was obtained by mutations in the apoCIII enhancer alone. Mutations in the proximal apoA-I promoter reduced by 85% hepatic and intestinal apoA-I expression and did not affect CAT expression. The findings suggest that a hormone response element within the apoCIII enhancer is essential for intestinal and renal expression of apoA-I and apoCIII genes and also enhances hepatic expression. The hormone response elements of the proximal apoA-I promoter or the apoCIII enhancer can promote independently low levels of hepatic and intestinal expression of the apoA-I gene in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ApoA-I¹ is the major protein component of HDL and is responsible for its biological functions. Epidemiological and genetic data in humans have shown convincingly that high levels of HDL or apoA-I are associated with decreased risk of developing atherosclerosis, indicating the importance of apoA-I in cellular cholesterol homeostasis and the protection against atherosclerosis (1-3). Similarly, transgenic mice overexpressing apoA-I are protected against diet-induced atherosclerosis (4). ApoCIII is the major component of very low density lipoprotein and a minor component of HDL (5). ApoCIII has been shown to modulate the catabolism of triglyceride-rich lipoproteins in vitro (6-8). This concept was further supported by in vivo studies, which showed that transgenic mice overexpressing the human apoCIII gene develop severe hypertriglyceridemia (9, 10).

Previous studies have established that there is a linkage (11) and common regulatory mechanism of the apoA-I/apoCIII/apoA-IV gene cluster (15-19). Each of the three genes contains hormone response elements (HREs) in its proximal promoter that bind orphan and ligand-dependent nuclear receptors with different specificities (15, 19). The distal regulatory region of the apoCIII promoter acts as a common enhancer for the three genes of the cluster (16-18). The enhancer also contains two HREs and three SP1 binding sites (12-16). Mutagenesis analysis showed that alterations, which prevent the binding of nuclear receptors to HREs, abolished the activity of these promoters in cell cultures (17-21). Mutations in the HREs and some of the SP1 sites of the enhancer also significantly affected the enhancer activity in cell cultures (16-19). Other transcription factors bind to proximal promoters of the apoA-I/apoCIII genes and may affect their overall activity and tissue specificity (16-18, 21-22). In vitro observations regarding gene regulation are meaningful only if they can mimic in vivo events. As part of the current study, we have generated transgenic mice containing the WT and mutant regulatory regions of the apoA-I/apoCIII gene cluster. Mutations were created to destroy the HREs of the apoA-I promoter or the apoCIII promoter and enhancer region. The transgenic mouse lines were designed to address the following questions. Which regulatory elements control the tissue-specific expression of the human apoA-I and apoCIII gene in vivo? How do mutations in the proximal HREs affect the promoter strength in vivo? How do mutations in the HREs of the enhancer affect the tissue-specific expression of the two genes in vivo?

Our findings suggest that the HREs of the apoA-I or apoCIII promoter and enhancer that bind HNF-4 control the overall expression of the apoA-I and apoCIII genes. When the proximal HREs are mutated, the apoCIII enhancer alone may independently direct the expression of the apoA-I gene at lower levels in liver, intestine, and other tissues. Mutations in the proximal HREs reduce, but do not eliminate, the hepatic expression of the apoA-I and apoCIII genes. In contrast, mutations in the HRE of the apoCIII enhancer abolished the intestinal and renal expression and reduced the hepatic expression of the two closely linked genes in vivo.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

Reagents were purchased from the following sources: the Klenow fragment of the DNA polymerase I restriction enzymes, T₄ ligase, T₄ polynucleotide kinase, and Vent polymerase from New England Biolabs (Beverly, MA); [-³²P]ATP (5000 Ci/mmol), [¹⁴C]chloramphenicol, [-³²P]dCTP, and [-³²P]dGTP (3000 Ci/mmol) from NEN Life Science Products; Bacto®-Tryptone and bacto yeast extracts from Difco (Detroit, MI; acrylamide, SDS, urea, and Tris from International Biotechnologies, Inc. (Rochester, NY); bacterial XL-1 Blue cells from Stratagene (La Jolla, CA); custom-made oligonucleotides from Life Technologies, Inc.; plasmid pSPORT-1 from Life Technologies, Inc.; S1 nuclease from Roche Molecular Biochemicals; proteinase K, spermine, phenylmethylsulfonyl fluoride, and ammonium acetate from Sigma.

Methods

Plasmid Constructions-- The human -890/+24 apoCIII promoter was amplified using the pUC-19 CIII promoter plasmid as a template and the CIII 890K and CIII 24E oligonucleotides (Table I) as primers. The pUC-19 CIII plasmid used as template in the polymerase chain reaction contains a 3-kb EcoRI-EcoRI fragment containing the N-terminal region and 5' regulatory sequence that encompasses the apoCIII gene (23). The primers introduced KpnI and EcoRI sites at the 5' and 3' ends of the -890/+24 apoCIII promoter and enhancer region. Following digestion with KpnI and EcoRI, the amplified fragment was cloned into the corresponding sites of the pSPORT-1 plasmid (Life Technologies, Inc.). The EcoRI site of this plasmid was subsequently blunted by EcoRI digestion and filling of the 5' protruding end using the Klenow fragment of the DNA polymerase I to generate the modified pSPORT-1 CIII WT plasmid.

A 5.5-kb region containing the entire apoA-I gene and a 2.1-kb promoter segment was excised from the pUC19 A-I CIII plasmid. This plasmid contains an 11-kb fragment that encompasses the apoA-I gene and the C-terminal region of the apoCIII gene, as obtained by EcoRI digestion of the genomic apoA-I 6 clone (24). The 5.5-kb XbaI apoA-I gene fragment was then cloned into the XbaI site of the modified pSPORT-1 CIII WT plasmid. The resulting pSPORT-1A-I WT-890/+24 CIII WT plasmid was digested with NotI and ligated with the CAT cDNA flanked by NotI sites to generate the pSPORT-1 A-I WT CIII WT CAT plasmid (Fig. 1A). The CAT cDNA sequence containing NotI sites at the 5' and 3' ends was obtained by DNA amplification using the pUCSH CAT plasmid as a template (21) and the pCAT-1N and pSV40A2 oligonucleotides (Table I) as 5' and 3' primers respectively. CAT cDNA sequence contains a unique EcoRI site that allows identification of the transgenic mice.

The mutations in the HREs in element I₄ of the apoCIII enhancer or in both elements B and I₄ in the apoCIII promoter and enhancer region were generated by amplification and mutagenesis of the -890/+24 apoCIII promoter region using the oligonucleotides PN2 and PBN1 as 5' and 3' external primers and either the oligonucleotides CIII BP-1 and CIII BP-2 as 5' and 3' mutagenic primers for element B or the same external primers and the oligonucleotides CIII IP-1 and CIII IP-2 as 3' and 5' mutagenic primers for element I₄ (Table I), and the pSPORT-1 CIII WT (-890/+24) plasmid as a template.

In a typical amplification/mutagenesis reaction, the 5' external primer and the 3' mutagenic primer are used to amplify the upstream region of interest, and the 5' mutagenic primer and 3' external primer are used to amplify the downstream region of interest. Following amplification, an aliquot of 5 containing the two amplification products were mixed and amplified using the 5' and 3' external primers. The amplified mutant -890/+24 apoCIII promoter region mutated in element I₄ was excised via KpnI/EcoRI digestion and cloned into the corresponding sites of pSPORT-1 plasmid. This intermediate plasmid was used as a template for further mutagenesis of the promoter region using as external primers the PN2 and PBN1 oligonucleotides and as mutagenic primers the CIII BP1 and CIII BP2 oligonucleotides (Table I). The amplified -890/+24 apoCIII promoter region containing the mutations in elements B and I₄ was digested with KpnI and EcoRI and cloned into the corresponding sites of the pSPORT-1 plasmid to generate the pSPORT-1 CIII (B+ I₄) plasmid. The EcoRI site of this plasmid was eliminated as described for the WT construct, and the modified derivative was digested with SalI and BamHI.

Similarly, a 6.89-kb fragment encompassing the 2.1-kb apoA-I promoter region, the entire apoA-I gene, and the CAT cDNA was obtained by BamHI and SalI digestion of the pSPORT-1A-I WT CIII WT CAT plasmid (Fig. 1A). The mutated -890/+24 apoCIII promoter and enhancer sequence and the 6.89-kb apoA-I gene segment were introduced by triple ligation into the BamHI site of the pSPORT-1 plasmid to generate the pSPORT-1 A-I WT CIII (B+ I₄) CAT plasmid (Fig. 1B). Similarly, the mutated -890/+24 apoCIII enhancer sequence and the 6.89-kb apoA-I gene segment were introduced by triple ligation into the BamHI site of the pSPORT-1 plasmid to generate the pSPORT-1 A-I WT CIII (I₄) CAT plasmid (Fig. 1C).

The mutations in elements B and D of the -255/+5 apoA-I promoter were generated as described above (20). The -255/+5 apoA-I promoter region containing mutations in the HREs of elements B and D (15) was amplified using as primers oligonucleotides Rev5-16 and 3 CAT-26 (Table I). A pBluescript A-I plasmid containing the 5.5-kb XbaI apoA-I gene segment described above was digested with XbaI, EcoRI, and AlfII to obtain a 3.9-kb fragment containing the apoA-I gene and a 1.6-kb fragment representing the intergenic sequence between the apoA-I and apoCIII genes. The same plasmid was also digested with XbaI and EcoRI to obtain a 6.3-kb XbaI/EcoRI sequence containing the 5' upstream apoA-I promoter region along with the pBluescript plasmid. The XbaI/AlfII proximal apoA-I promoter region containing the two mutations in elements B+D and the XbaI/EcoRI and AlfII/EcoRI fragments containing the upstream apoA-I promoter and the coding apoA-I gene sequence were joined in a triple ligation to generate the pBluescript-1 A-I (B+D) plasmid. The insert of this plasmid was excised via XbaI digestion and was used to replace the 5.5-kb XbaI fragment containing the wild-type sequence from the pSPORT-1 A-I WT CIII WT CAT plasmid, thereby generating the mutant pSPORT-1 A-I (B+D) CIII WT CAT plasmid (Fig. 1D). In a separate construction, the insert of the pBluscript-1 A-I (B+D) plasmid was excised via XbaI digestion and was used to replace the 5.5-kb XbaI fragment containing the wild-type sequence from the pSPORT-1 A-I WT CIII(I₄) CAT plasmid to generate a construct with mutations in the HREs on element B+D of the apoA-I promoter and I₄ of the apoCIII enhancer (not shown). The correct sequence of these plasmids was confirmed by DNA sequencing. The inserts of the WT plasmids and the corresponding mutant pSPORT-1 plasmids containing mutations in the apoCIII promoter and enhancer or the proximal promoter were excised by BamHI digestion and used for microinjections into fertilized mouse embryos.

Generation and Characterization of the Transgenic Mice-- Heterozygous transgenic mice used in these studies were generated using standard transgenic mice methodologies (25, 26). To obtain fertilized oocytes, C57BL/6J female embryo donor mice at 6-8 weeks of age were injected with pregnant mare serum followed by injection of human chorionic gonadotropins 48 h later. The female embryo donors were mated with C57BL/6J males, and on the following day, the fertilized oocytes were recovered in M2 medium (24). DNA of the construct of interest was microinjected into the pronuclei of the embryo at a concentration of 2 µg/ml in injection buffer (10 mM Tris, pH 7.4, 0.1 mM EDTA). Embryos that survived microinjection were cultured for 30 min in M2 medium (26) and then implanted into the oviduct of pseudopregnant female mice (Swiss Webster) mated previously with vasectomized male mice (Swiss Webster). Thirty embryos were implanted in each female pseudopregnant mouse. Recipient mice progressed through gestation, and approximately 70% of them gave birth to transgenic mice. Typically, about 20-30% of the littermates were identified as transgenic founders (Fo) by Southern blotting of DNA isolated from tail biopsies of 4-week-old mice. Identification was confirmed by Southern blotting of mouse genomic DNA following EcoRI digestion and hybridization with the 978-bp apoA-I probe. This analysis detected an 8.2-kb band in the transgenic mice that corresponds to microinjected transgene. The apoA-I probe used for a hybridization represented 978 bp of the apoA-I gene restriction fragment, comprising 540 bp of exon 4 and 438 bp of the intergenic sequence between the apoA-I and apoCIII genes. F1 progeny of transgenic mice were obtained through breeding of the Fo founders. Three or four transgenic mouse lines were generated per construct to overcome positional effects and ensure that the pattern of expression is characteristic of a specific construct.

Lipid, Lipoprotein, and ApoA-I Profile of Transgenic Mice-- Levels of total serum cholesterol and serum TG were determined using commercially available enzymatic kits (Roche Molecular Biochemicals). ApoA-I levels were determined by enzyme-linked immunosorbent assay.

RNA Isolation, Northern Blotting and S1 Nuclear Mapping-- Total cellular RNA was isolated from liver and other tissues using the guanidine isothiocyanate method. The RNA was purified using the Qiagen RNA/DNA midi-kit. RNA was eluted with high salt buffer, precipitated by isopropanol, and dissolved in RNase-free water. Equal quantities of RNA (10 µg) were separated by electrophoresis in 1.0% agarose-formaldehyde gels. Following transfer to a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech), the RNA was cross-linked to the filter by UV irradiation (Stratalinker, Stratagene) at 0.12 J/cm² for 1 min. The apoA-I probe for hybridization is 438 bp in length. This probe contains 290 bp of exon 4 of human apoA-I and 148 bp of the intergenic sequence between the apoA-I and apoCIII genes and does not cross-hybridize with the mouse apoA-I mRNA. The mouse 28 S rRNA probe was obtained from Ambion (Austin, TX). This oligonucleotide probe contains 43 nucleotides of highly conserved sequences among higher eukaryotes. The 28 S rRNA probe was labeled by 5' end labeling using T4 polynucleotide kinase method. The apoA-I mRNA of each sample was normalized by dividing with the 28 S rRNA signal. Hybridization reactions contained 1-2 × 10⁶ cpm ³²P-labeled DNA per ml of buffer. Unhybridized probe was removed by washing at 68 °C with 2× SSC, 0.1% SDS, followed by 15-30-min washes with 1× SSC and then with 0.5× SSC, as needed. Quantitation of x-ray film was performed using a Molecular Dynamics PhosphorImager using the Image Quant program. The relative levels (%) of apoA-I mRNA represents the ratio of apoA-I to 28 S ribosomal RNA signal or the ratio of apoA-I to mouse -actin signal when the RNA levels of a single tissue from different mouse lines were compared.

For S1 nuclease mapping (27), RNA was prepared from liver and other tissues using the lithium chloride method (28). A 30-s sonication step was added after homogenization of the tissue (27). The actin probe (ATCC) represented a 240-bp AvaI/BamHI restriction fragment of the mouse -actin gene and contained 100 bp of exon I and 140 bp of the 5' end of the gene (29). The 978-bp apoA-I probe has been described above. A specific 540-bp apoA-I mRNA signal and a 100-bp mouse -actin signal was obtained by this analysis.

CAT Assays-- For the CAT assay, F1 mice were sacrificed at 2 months of age. Tissues were collected and used immediately or were frozen and stored at 80 °C. CAT assays were performed as described (30). Briefly, the tissue was homogenized in polytron homogenizer on ice using 5 µl of 15 mM Tris·Cl buffer, pH 8.0 (containing 1 mM dithiothreitol and 0.4 mM phenylmethylsulfonyl fluoride), per mg of tissue, 50 mM KCl, 15 mM NaCl, 2 mM EDTA, 0.15 mM spermine. The supernatant of the homogenate was heated at 65 °C for 8 min and centrifuged in a microcentrifuge for 3 min. The supernatant was used for CAT and protein determination assays. The specific activity was calculated as pmol of product/mg of protein.

The concentration of soluble protein was determined by the Bio-Rad protein assay. The percentage of chloramphenicol converted to acetylated forms was determined either by densitometric scanning of autoradiograms or by scraping individual spots from the TLC and counting in a scintillation counter. CAT activities were expressed as pmol of acetyl chloramphenicol generated per min per mg of protein, after subtracting the background for each tissue from control mice that did not express the CAT gene.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Generation and Characterization of Transgenic Mice Harboring WT and Mutant Promoter Regions of the ApoA-I/ApoCIII Gene Cluster-- We have generated and studied in detail four sets of mouse lines expressing the constructs of Fig. 1. The top construct designated AI WT C-III WT contains 2.1 kb of the 5' regulatory sequence along with 1.81 kb containing the entire coding sequence of apoA-I gene, a 1.6-kb segment containing the intragenic sequence, and the CAT gene in front of the -890/+24 regulatory sequence of the apoCIII gene. In this construct, the apoCIII gene was replaced by the CAT cDNA sequence (Fig. 1A). The second construct, designated A-I WT CIII (B+I₄) Mut, contains mutations in the two HREs of elements B and I₄ respectively, of the apoCIII promoter and enhancer (Fig. 1B). The third construct, designated AI WT CIII I₄ Mut, contains a mutation in element I₄ of the apoCIII enhancer (Fig. 1C). The fourth construct, designated A-I (B+D)Mut CIII WT, contains mutations in elements B and D of the proximal apoA-I promoter (Fig. 1D). Transgenes containing the WT and the three mutant apoA-I/CIII promoter constructs were excised from the corresponding pSPORT-1 constructs, separated by agarose gel electrophoresis, electroeluted, and purified on QIAEXII gel extraction system (Qiagen, Valencia, CA). The names of the transgenes are shown in parentheses in Fig. 1. These names are utilized throughout the text. The purified DNA fragments were dissolved in 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, at a concentration of 2 ng/µl, and were microinjected into fertilized eggs from C57BL/6J females mated with males of the same strain.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   pSPORT-1 plasmid derivatives containing WT (A) and mutated apoCIII (B and C) or apoA-I (D) promoter segments. The names of the resulting transgenic lines harboring these transgenes are indicated in parentheses. The WT and mutant sequences of the HREs of the apoC-III promoter and enhancer and apoA-I promoter are shown in B and D, respectively. Nucleotide substitutions in the mutated sequences are depicted in boldface.

Transgenic founders were identified by Southern blotting analysis using as probe the 978-bp apoA-I fragment described under "Experimental Procedures." The number of transgene copies incorporated into the genome of each transgenic founder was determined by Southern blotting and by comparison of the intensity of the bands formed when increasing amounts of the transgene were diluted in nontransgenic DNA (Fig. 2). The four transgenic lines expressing the apoA-I WT CIII WT promoter enhancer cluster contain 3, 5, 25, and 30 copies of the transgene, respectively. Finally, the transgenic lines carrying A-I (B+D) Mut CIII WT construct contain 1, 15, and 30 copies of the transgene, respectively. The transgenic lines carrying the A-I WT CIII (B+I₄) Mut construct mutated in the apoCIII promoter and enhancer contain 10, 20, and 30 copies of the transgene, respectively. The transgenic lines carrying AI WT CIII I₄ Mut contain 1, 10, and 10 copies of the transgene, respectively (Fig. 2).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Gene copy number of mouse lines expressing WT and mutated apoA-I/apoCIII promoter enhancer constructs. The names of transgenic mouse lines analyzed are described in Fig. 1. Results are summarized in Tables III and V.

Lipid and ApoA-I levels of Mice Expressing the ApoA-I Gene under the Control of the WT and Mutant ApoA-I/ApoCIII Promoter and Enhancer Regions-- The plasma lipid and apoA-I levels of mice expressing the human apoA-I gene were determined as explained under "Experimental Procedures" (Table II). The triglyceride levels were within the normal range. The total cholesterol HDL and apoA-I levels of the transgenic mice expressing the WT construct were, in general, increased relative to the lines expressing the mutant transgenes (Table II).

Expression of the CAT Gene under the Control of the WT and Mutated ApoCIII Promoter and Enhancer-- The activity of the apoCIII promoter and enhancer was measured by the activity of the reporter CAT gene under the control of WT apoCIII promoter and enhancer as described under "Experimental Procedures." Four different mouse lines carrying the WT construct displayed high levels of CAT activity in the liver, low levels of expression in the intestine and kidney, and no expression in the lung, spleen, heart, brain, stomach, and skeletal muscle (Fig. 3A; Table III). The levels of CAT activity in the intestine and kidney in mice expressing the WT construct were approximately 6 and 4%, respectively, of that observed in the liver. The findings indicate that the elements of the proximal apoA-I promoter are not required for the activity of the apoCIII promoter and enhancer. This pattern of expression mimics the expression of the apoCIII gene in fetal human tissues and rat and rabbit tissues (31, 32). The only difference is the ectopic expression of the apoCIII gene in the kidney (Fig. 3A; Tables III and IV). CAT expression in three mouse lines carrying the construct mutated in the two proximal HREs was similar to that of the lines carrying the WT constructs (compare Fig. 3D and Table III with Fig. 3A). The findings indicate that the elements of the proximal apoA-I promoter do not interfere with the activity of the apoCIII promoter and enhancer.

View larger version (59K): [in this window] [in a new window]   Fig. 3.   CAT assays of transgenic mouse tissues expressing constructs containing the WT and mutated apoA-I and apoCIII promoter segments shown in Fig. 1A. A, mice expressing CAT gene under the control of the WT apoA-I promoter/WT apoCIII promoter and enhancer. B, mice expressing the CAT genes under the control of the WT apoA-I promoter/mutant apoCIII promoter and enhancer. C, mice expressing the CAT gene under the control of WT apoA-I promoter/mutant apoCIII enhancer. D, mice expressing the CAT gene under the control of the mutant apoA-I promoter/WT apoCIII promoter and enhancer. The names of the transgenic lines are described in Fig. 1. CAT assays were performed in triplicate as described under "Experimental Procedures."

View this table: [in this window] [in a new window]   Table IV ApoC-III mRNA expression in fetal human tissues and adult mouse and rabbit tissues + and  are the same as described for Table V.

Consistent with the in vitro findings, mutations in the HREs of elements B and I₄ of the apoCIII promoter and enhancer abolished the expression of the CAT gene in all of the tissues tested (Fig. 3B). Mutations in element I₄ of the apoCIII enhancer alone abolished the intestinal expression and reduced dramatically the hepatic expression by approximately 98% as compared with mice carrying the WT apoA-I promoter/WT apoCIII enhancer construct. The expression in kidney was barely detectable (Fig. 3C; Table III). The findings indicate that the HRE present on element I₄ of the apoCIII enhancer (which, as shown previously, binds HNF-4 (18, 19)) is essential for the intestinal and renal expression of the apoCIII gene. In addition, the HRE of element B of the proximal apoCIII promoter can permit very low levels of hepatic expression of the apoCIII gene.

Expression of the ApoA-I Gene under the Control of the WT and Mutated ApoA-I Promoter/ApoCIII Enhancer Regions-- The expression of apoA-I gene was determined by Northern blotting and S1 nuclease mapping, as described under "Experimental Procedures." Analysis of four different mouse lines showed that in general, the WT apoA-I promoter/WT apoCIII enhancer directs high levels of expression in the liver and intestine, moderate levels of expression in the kidney, and low levels of expression in the lung (Fig. 4A). S1 nuclease mapping also detected very low levels of expression in the stomach, heart, spleen, and muscle (Fig. 4E; Table V). Very low levels of expression in the brain were observed in one out of four transgenic mouse lines. As shown in Tables V and VI, this pattern of expression mimics the expression of apoA-I gene in fetal human tissues and adult rat tissues (30-34).

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Northern (A-D) and S1 nuclease protection analysis (E) of apoA-I mRNA obtained from tissues of transgenic mice expressing constructs containing the WT and mutated apoA-I and apoCIII promoter and enhancer constructs shown in Fig. 1. A and E, mice expressing the apoA-I gene under the control of the WT apoA-I promoter/WT apoCIII promoter and enhancer. B, mice expressing the apoA-I gene under the control of the WT apoA-I promoter/mutant apoCIII promoter and enhancer. C, mice expressing the apoA-I gene under the control of WT apoA-I promoter mutant/apoCIII enhancer. D, mice expressing the apoA-I gene under the control of the mutant apoA-I promoter/wild-type apoCIII promoter and enhancer. The names of the transgenic lines are described in Fig. 1. Northern blotting and S1 nuclease protection analysis was performed as described under "Experimental Procedures." The human apoA-I and mouse -actin probes gave bands of 540 and 100 bp, respectively (23, 29). These panels show the relative apoA-I mRNA levels in different tissues of a specific mouse lines. Comparative levels of apoA-I mRNA in liver and intestine of different mouse lines are shown in Fig. 5.

View this table: [in this window] [in a new window]   Table VI ApoA-I mRNA levels in fetal human tissues and adult mouse and rabbit tissues + and  are the same as described for Table V.

Mutations in the HRE of elements B and I₄ of the apoCIII promoter and enhancer respectively suppressed the intestinal and renal expression of the apoA-I gene and reduced the levels of the hepatic expression by 80% as compared with mice carrying the WT apoA-I promoter WT apoCIII enhancer construct (Figs. 4B and 5). Only one of the three mouse lines carrying 30 copies of the transgenes showed very low levels of apoA-I expression in the intestine and kidney (Table V).

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Northern blotting analysis of 10 µg of total RNA obtained from liver (A) and intestine (B) of transgenic mice expressing constructs containing the WT and mutated apoA-I and apoCIII promoter and enhancer shown in Fig. 1. The nylon membrane was hybridized simultaneously with the apoA-I mouse 28 S RNA (A) or mouse -actin probe (B) as described under "Experimental Procedures." The names of the transgenic lines are described in Fig. 1. The ratio of apoA-I to 28 S signal (A) or apoA-I to -actin signal (B) of samples obtained from mice carrying the WT apoA-I WT apoCIII construct was arbitrarily set to 100%. The ratios of apoA-I/28 S or apoA-I/-actin mRNA for the other samples provide a measure of their abundance relative to those of the WT control.

The contribution of element I₄ of the apoCIII enhancer on the expression of the apoA-I gene was tested by analysis of three additional mouse lines mutated in this element. This analysis showed that the presence of a mutation in element I₄ that precludes the binding of HNF-4 and other nuclear receptors to this site (19) abolished the intestinal and renal expression of the apoA-I gene in intestine and kidney and reduced the hepatic expression by approximately 80% as compared with mouse lines carrying the WT apoA-I WT apoCIII enhancer construct (Fig. 4C). The pattern of expression of the apoA-I gene in the liver was quantitatively similar to that observed in the double mutant in elements B and I₄ (Fig. 5A). This indicates that the mutation in the HRE of element B of the proximal apoCIII promoter does not further affect the expression of the neighboring apoA-I gene. The expression pattern of the apoA-I gene carrying mutation in the element I₄ in other tissues with very low levels of expression, as determined by S1 nuclease mapping, remained qualitatively similar to those observed in tissues of mice expressing the WT apoA-I promoter WT apoCIII enhancer construct, with the exception of the stomach, in which the expression was lost (Tables V and VI).

Mutations in the two HREs on elements B and D of the proximal apoA-I promoter did not alter qualitatively the expression of the apoA-I gene in the major tissues (Fig. 4D; Table V). However, quantitation of the relative levels of expression showed that the steady state apoA-I mRNA levels in the liver and the intestine in the mice carrying the B and D mutation in the proximal apoA-I promoter were reduced by 85% as compared with the expression in mice carrying the WT apoA-I construct (Fig. 5). The pattern of expression of the apoA-I gene in the lung and heart in mice carrying the B+D mutation in the proximal apoA-I promoter was similar to that of mice carrying the WT construct apoA-I promoter WT apoCIII enhancer. No expression was detected in muscle and brain (Tables V and VI). A single mouse line carrying a triple mutation that altered the two HREs of elements B and D of the apoA-I promoter and the HRE on element I₄ of the apoCIII enhancer abolished the expression of the apoA-I gene in all tissues tested (data not shown). The findings indicate that the apoA-I promoter cannot function in the absence of the intact HREs in the proximal promoters and the apoCIII enhancer regions. Comparison of the in vivo and in vitro data of apoA-I and apoCIII gene regulation are shown in Fig. 6. Putative transcriptional mechanism that explain the finding of Figs. 3-5 are shown in Fig. 7 and are considered further below.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   A, schematic representation of the WT and mutated -890/+24 apoCIII promoter enhancer and the -255/-5 apoA-I promoter/-500/-800 apoCIII enhancer regions showing the binding specificity of the regulatory sites in vitro. Numbers in parentheses indicate the percent activity of the promoter enhancer cluster when it is mutated such that the indicated factors do not bind to the mutated site (15, 16, 18, 19). Factors are symbolized by ovals. *, present study; **, expression studies in transgenic mice carrying a 300 bp apoA-I promoter (44); ***, expression studies in transgenic mice carrying a 200 bp apoCIII promoter. (10) - indicates nucleotide sequences upstream of the transcription initiation site.

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Mechanisms by which the apoA-I promoter and enhancer cluster is activated in transgenic mice. A, transcriptional enhancement when all the HREs of both the apoA-I promoter and the apoCIII enhancer are intact (WT). B, gene expression when the HREs on element B and I4 of the apoCIII promoter and enhancer are mutated. C, gene expression when the HRE on element I4 of the apoCIII enhancer is mutated. D, gene expression when the HREs on elements B and D of the apoA-I promoter are mutated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Background-- Previous studies using deletion, nucleotide substitution, and footprinting analysis of the apoA-I promoter as well as CAT assays localized the promoter elements required for hepatic transcription of the apoA-I gene in cell cultures downstream of nucleotide -239. This region contains three regulatory elements, designated B, C, and D. Elements B and D bind to their DR1 sites (number indicates the number of nucleotides between the half AC(G/T)TCA repeats), orphan nuclear receptors HNF-4, ARP-1, EAR-2, and EAR-3 (12-15). Both elements also bind homodimers of RXR and heterodimers of RXR with RAR and T₃R. Mutations in either of the two HREs of either element B or D sites, which precluded the binding of nuclear receptors to these sites, abolished promoter activity in cell cultures (15). Regulatory element C of apoA-I is recognized by heat-stable factors related to CCAAT enhancer box-binding protein, which act as positive regulators, and another activity, designated AIC1 (20) (Fig. 6B).

Similarly, deletions, nucleotide substitutions, footprinting analysis, and CAT assays using the apoCIII promoter localized the promoter elements required for hepatic and intestinal transcription downstream of nucleotide -890 (21, 35). This region contains 10 regulatory elements, designated A-J. The regulatory elements B, I, and G contain HREs, which are recognized by various orphan and ligand-dependent nuclear receptor families (12, 19, 36). Elements C and D bind CCAAT enhancer box-binding protein. New activity, designated CIIIC1 (factor 1 bound to the regulatory region C of the apoCIII gene), also binds to element C. The distal regulatory region contains two HREs on elements G and I₄ as well as three binding sites for the ubiquitous transcription factor SP1 (16) on elements F, H, and I. Regarding the nuclear receptor specificity of elements B, G, and I₄, it was shown previously that element B, which contains a DR1 site, binds strongly HNF-4, ARP-1, EAR-2, and EAR-3 (heterodimers of RXR with RAR); it binds less efficiently homodimers of RAR and heterodimers of RXR with T₃R or PPAR (12, 18, 19, 37). Element G, which contains both DR0 and DR5 sites, binds strongly on DR0 sites ARP-1 and EAR-3. It also binds strongly on DR5 sites heterodimers of RXR with either RAR or T₃R, and it does not bind HNF-4. Finally, element I₄, which contains a DR1 site, binds strongly HNF-4, ARP-1, EAR-3, and RXR/RAR heterodimers ,and it binds less efficiently RXR/T₃R heterodimers (19) (Fig. 6). Cotransfection experiments have shown that for apoA-I and apoCIII genes, ARP-1, EAR-2, EAR-3, and heterodimers of RXR with T₃R in the presence of T3 act as negative regulators, whereas HNF-4 and RXR homo- or heterodimers in the presence of 9-cis RA act as positive regulators or do not alter the activity of the apoA-I and apoCIII promoters (12, 15, 19).

Initial in vitro (16-18, 22, 36) and subsequent in vivo experiments (21, 38-40) showed that the distal regulatory region -590/-800 of the apoCIII promoter is a common enhancer of the apoA-I/apoCIII/apoA-IV gene cluster. As discussed above, the enhancer contains two HRE binding sites and three SP1 binding sites (15-19, 22) (Fig. 6). The current study using transgenic mice represents the first attempt to elucidate which regulatory elements and corresponding factors are essential for the activity and tissue specificity of the apoA-I and apoCIII promoters and what is the contribution of the apoCIII enhancer to the tissue-specific expression of the two genes. The study has focused on the HREs present in the proximal apoA-I and apoCIII promoters and the apoCIII enhancer, which bind HNF-4 and other nuclear receptors (18, 19). Although the transgenic approach represents an approximation as compared with the knock-in methodologies (41), our findings, combined with other published information, provide clear answers on the mechanisms that control the transcription of the apoA-I and apoCIII genes in vivo.

The Proximal ApoA-I and ApoCIII Promoters, in Combination with the ApoCIII Enhancer, Confer Correct Tissue-Specific Expression of the ApoA-I and ApoCIII Genes in Vivo-- In humans and nonhuman primates, the genes of the human apoA-I/apoCIII/apoA-IV cluster are expressed at different levels in the liver and the intestine. ApoA-I is expressed abundantly in both the liver and intestine in most species. ApoCIII is expressed predominantly in the liver and to a lesser extent in the intestine, whereas apoA-IV is expressed predominantly in the intestine and to a lesser extent in the liver (30, 32, 33, 42). A previous study has shown that the apoCIII enhancer was sufficient to direct the intestinal expression of the apoA-I gene but did not restrict gene expression to the villus cells (38). In contrast, another preliminary report has shown that the full-length intergenic region between the apoCIII and apoA-IV genes allowed intestinal expression of the apoA-IV gene in transgenic mice, specifically the villus enterocytes, with a cephalocaudal gradient (39). Our subsequent work has suggested that the -700/+10 apoA-IV promoter sequence, in combination with the apoCIII enhancer, directed the expression of a reporter gene in enterocytes in a pattern similar to the expression pattern of the endogenous apoA-IV gene (25, 33, 42). In the current study we have established that regulatory elements representing the -890/+24 apoCIII promoter and the +5/-2100 apoA-I promoter directed expression of the apoA-I and apoCIII gene in a pattern similar to that observed in fetal human and adult rat and rabbit tissues (31, 32). The only difference was that in mice expressing the CAT gene and the control of the WT apoCIII promoter and enhancer, there was a low level of ectopic expression in the kidney. It is possible that the construct utilized lacks a silencer, which may be involved in the repression of the expression of the apoCIII gene in the kidney. Analysis of the relative expression of the reporter gene under the control of the apoCIII promoter and enhancer in proximal middle and distal segments of the intestine indicated that compared of the proximal segment, the expression was decreased slightly (to 77%) in the middle segment and significantly (to 25%) in the distal segment, indicating a gradient along the cephalocaudal axis of expression (data not shown).

The HREs of the Proximal ApoCIII and ApoA-I Promoter and the ApoCIII Enhancer Are Essential for Their in Vivo Activity for All Tissues-- Previous mutagenesis studies have established that mutations in the HREs of the regulatory element B of the proximal apoCIII promoter and element I₄ of the apoCIII enhancer diminished the activity of the apoCIII promoter and enhancer cluster in cell cultures (16, 19). Similar reduction in the activity in cell cultures was observed in constructs containing the apoA-I promoter/apoCIII enhancer, by mutations in either the HRE of element D of the proximal A-I promoter or element I₄ of the apoCIII enhancer (15, 18). In the current study, the simultaneous mutation in the HRE of element B of the apoCIII promoter and the HRE of element I₄ of the apoCIII enhancer abolished the activity of the apoCIII promoter in all the tissues tested. We have found that simultaneous mutations of elements B and D of the proximal apoA-I promoter and element I₄ of the apoCIII enhancer in one mouse line that we have generated recently abolished the expression of the apoA-I and apoCIII genes in all tissues tested (data not shown). Consistent with these findings, a recent report showed that the expression of the apoA-I and apoCIII genes is abolished in the embryonic liver of mice in which the HNF-4 gene was inactivated by homologous recombination (43). The findings indicate the importance of these two HREs of the proximal apoA-I promoter and the apoCIII enhancer for the overall apoCIII and apoA-I promoter activity in vivo.

The HRE of the ApoCIII Enhancer Controls the Expression of the ApoA-I and ApoCIII Genes in the Intestine and Kidney-- To dissect the contribution of the proximal and distal HRE on the apoCIII promoter, activity was clarified by generation of an additional mouse line carrying a mutation only in the element I₄ of the apoCIII enhancer. This element was shown previously to bind HNF-4 and other hormone nuclear receptors (19). This mutation abolished the expression of the CAT gene (which serves as a reporter of the apoCIII gene) in intestine and kidney and diminished the expression in liver by 98% as compared with mice carrying the WT apoA-I WT apoCIII promoter construct. The observation that the hepatic expression of the apoCIII gene persists when the HRE in the element I₄ of the enhancer is mutated, albeit at very low levels, is consistent with previous findings that showed that hepatic expression of apoCIII could be achieved by a construct containing 200 bp of the apoCIII promoter (10). Similarly, a previous transgenic study established that 300 bp of the proximal apoA-I promoter were sufficient to direct hepatic expression of the apoA-I gene (40, 44), suggesting that this region contains at least some of the necessary elements for the hepatic expression. Equally revealing in this study was the observation that either mutations in both HREs of the apoCIII promoter and enhancer or a single mutation in the HRE of the enhancer abolished selectively the intestinal and renal expression of the apoA-I gene and reduced the hepatic expression by 80% as compared with the expression in mice carrying the WT apoA-I promoter/WT apoCIII enhancer construct. Our findings clearly establish that the intestinal expression of the apoA-I and apoCIII gene requires the HRE, located within the regulatory element I₄ of the apoCIII enhancer (18), which can bind HNF-4 (19). Most likely, the inability of HNF-4 to bind to this element due to the mutations we introduced prevents intestinal expression of the apoA-I and apoCIII genes.

The HRE of the ApoCIII Enhancer Alone May Independently Contribute to the Expression of the ApoA-I Gene in the Liver, Intestine, and Kidney-- Mutagenesis studies have established that mutations in the HREs of the regulatory element D of the apoA-I promoter or element I₄ of the apoCIII enhancer diminish the activity of the promoter/enhancer cluster in HepG2 cells (18, 37). A single mutation in the HREs of elements D and/or B, or both, also diminishes the activity of the proximal (-255/+5) apoA-I promoter in HepG2 cells (15). Our in vivo studies showed that mutations in the two proximal HREs of the apoA-I promoter permitted expression of the apoA-I gene in the liver, intestine, kidney, and some of the minor tissues (Fig. 4D and Table V). The levels of the intestinal and hepatic expression of the apoA-I gene in the mouse lines carrying the mutations in the two HREs of the apoA-I promoter was approximately 15% of those observed in mice carrying the WT apoA-I promoter WT apoCIII enhancer.

The findings indicate that hepatic and intestinal expression is still possible, albeit at lower levels, when the proximal apoA-I promoter is inactivated by mutations that prevent the binding of hormone nuclear receptors to the two proximal HREs. The most probable interpretation of these findings is that in vivo either the proximal apoA-I promoter alone (Fig. 7D) or the apoCIII enhancer alone (Fig. 7B) can drive independently the hepatic and intestinal transcription of the apoA-I gene.

Potential Mechanisms of Transcriptional Enhancement in Vivo-- The present study shows that the apoA-I promoter and the enhancer individually contribute 20 and 15% to the overall transcriptional activity. When both the promoter and the enhancer are functional, the activity of the apoA-I promoter/apoCIII enhancer cluster is 100%. The combined effects of the promoter and enhancer are not additive (0.15 + 0.20 = 0.35 activity) but rather synergistic, leading to 100% activity. This represents a 2.9-fold enhancement of activity (100:.35 = 2.9). Our findings suggest that enhancement of the hepatic transcription of the apoA-I gene may be achieved by independent recruitment of the proteins of the basal transcription machinery by factors that bind either to the proximal apoA-I promoter or the apoCIII enhancer (Fig. 7, B and C). Transcriptional synergism is achieved through the concerted action of both the promoter and the enhancer (Fig. 7A). A similar mechanism may also apply for the closely linked apoCIII and apoA-IV genes (Fig. 7, B and D). The transcriptional mechanism described for the liver is somehow different in the intestine and the kidney. In these tissues, transcription cannot occur by the proximal promoters alone. For these tissues, promotion of transcription requires synergy between the proximal promoter and the apoCIII enhancer.

Numerous studies have established that increases in plasma apoA-I and HDL concentration are associated with protection from cardiovascular disease (1-4). In addition, alteration in apoCIII levels has been shown to affect the catabolism of triglyceride-rich lipoproteins (6-10). Thus, the in vivo transcriptional regulatory mechanisms that emerge from this and similar studies may provide rational approaches for correcting low plasma HDL levels and reducing the plasma levels of atherogenic triglyceride-rich lipoprotein particles in humans.

	ACKNOWLEDGEMENTS

We thank Markella Zanni for editorial corrections and comments, Dr. Catherine Reardon for performing lipid and HDL analysis, Anne Plunkett for preparing the manuscript, andDr. Jose Ordovas of Tufts University for determining the plasma apoA-I levels in mice.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HL33952 and by Kos Pharmaceuticals (Miami, FL).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.: 617-638-5085; Fax: 617-638-5141.

Published, JBC Papers in Press, July 12, 2000, DOI 10.1074/jbc.M005641200

	ABBREVIATIONS

The abbreviations used are: apo, apolipoprotein; ARP-1, apolipoprotein AI regulatory protein; CAT, chloramphenicol acetyltransferase; EAR-2, vErb-A related protein 2; HDL, high density lipoprotein; HNF, hepatocyte nuclear factor; HRE, hormone response element; RAR, all-trans retinoic acid receptor ; RXR, 9-cis retinoic acid receptor ; T₃R, thyroid hormone receptor ; WT, wild-type; DR, direct repeat; Mut, mutated; Fo, founder(s); bp, base pair(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES