©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of an Enhancer Element in the Human Apolipoprotein C-III Gene That Regulates Human Apolipoprotein A-I Gene Expression in the Intestinal Epithelium (*)

(Received for publication, March 27, 1995; and in revised form, May 26, 1995)

Joseph G. Bisaha (1) Theodore C. Simon (2)(§) Jeffrey I. Gordon (2) Jan L. Breslow (1)(¶)

From the  (1)Laboratory of Biochemical Genetics and Metabolism, Rockefeller University, New York, New York 10021 and the (2)Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Studies using transgenic mice indicate that expression of the human apolipoprotein (apo) A-I gene in the liver and small intestine is controlled by spatially distinct cis-acting DNA elements; hepatic expression is controlled by a domain defined by nucleotides -256 to -1, while small intestinal expression requires elements positioned 9 kilobases 3` to the gene, between nucleotides -1300 and -200 of the convergently transcribed apoC-III gene. In this report we have mapped this enhancer to a 260-base pair (bp) region of the apoC-III promoter spanning nucleotides -780 to -520. The elements contained within this 260-bp apoC-III domain are sufficient to direct a pattern of expression in villus-associated enterocytes distributed along the duodenal-to-ileal axis that resembles that of mouse and human apoA-I. However, the elements produce inappropriate activation of apoA-I expression in proliferating and nonproliferating crypt epithelial cells, and in subpopulations of cholecystokinin- and serotonin-producing enteroendocrine cells. Cis-acting suppressors of these inappropriate patterns of expression are located outside of nucleotides -1300 to -200 of the human apoC-III gene. DNase I protection and gel mobility gel shift assays identified two 21-bp sequences, nucleotides -745 to -725 and -700 to -680 of human apoC-III, which bind nuclear proteins present in a human enterocyte-like cell line (Caco-2). These sequences are conserved in the orthologous mouse apoC-III gene. The 260-bp apoC-III element is the first intestinal enhancer that has been identified in an in vivo system and should provide insights about how cell lineage-specific, differentiation-dependent, and cephalocaudal patterns of gene expression are established and maintained in the perpetually renewing gut epithelium. In addition, novel intestinal transcription factors may bind to the enhancer and regulate its activity.


INTRODUCTION

Apolipoprotein (apo) (^1)A-I is the major protein constituent of human high density lipoproteins (HDL). Numerous epidemiological studies have determined that low levels of plasma HDL are correlated with increased risk of atherosclerosis and increased incidence of coronary heart disease (see (1) , and references therein). Plasma HDL levels are directly related to the plasma concentration of apoA-I(2) . Transgenic mice that express human apoA-I in their livers have increased circulating levels of HDL particles (comparable to human HDL and HDL)(3, 4) . C57Bl/6 transgenic mice that produce high levels of human apoA-I in their livers have a reduced incidence of fatty streak formation, compared to their normal littermates, when exposed to a high fat, atherogenic diet(5) . In addition, apoE-deficient mice that overexpress a human apoA-I transgene have decreased atherosclerosis compared to apoE-deficient mice when fed a chow diet(6) .

ApoA-I is produced in hepatocytes and in the principal epithelial cell lineage of the small intestine: the polarized absorptive enterocytes. Immunocytochemical studies of duodenal/proximal jejunal biopsies obtained from normal adult humans after a 12-h fast or 1 h after a corn oil meal indicated that apoA-I is not detectable in cells that populate the proliferative unit of the intestine: the crypt of Lieberkühn. Rather, apoA-I is first detectable after nonproliferating, differentiating enterocytes exit the crypt. Expression persists as enterocytes complete their rapid upward migration to the apical extrusion zone of an adjacent villus(7) .

The human apoA-I gene is located on chromosome 11, in a cluster that contains the apoC-III and A-IV genes (Fig. 1). Cis-acting DNA elements necessary for apoA-I expression in cultured human hepatoma cells (HepG2) and in the hepatocytes of transgenic mice appear to be located between nucleotides at -256 bp and the start of transcription(3, 8, 9, 10, 11, 12, 13) . However, studies in transgenic mice indicate that a genomic fragment containing up to 5.5 kb of 5` nontranscribed domain of apoA-I, its 4 exons and 3 introns, plus 4 kb of 3` flanking sequence is not expressed in the small intestine. The determinants of intestinal expression appear to reside in a 1.1-kb fragment that extends from nucleotides -200 to -1300 of the adjacent, convergently transcribed human apoC-III gene(14) . This fragment can function in either orientation to direct intestinal expression when it is placed 1.7 kb 3` to apoA-I's terminal exon. Intestinal expression, defined as accumulation of human apoA-I mRNA in total RNA prepared from the proximal third of the small bowel of adult mice, is sustained in the absence of apoC-III exonic and intronic sequences(14) . However, the cell lineage-specific and spatial patterns of apoA-I/C-III transgene expression were not defined in this study.


Figure 1: Transient transfection analysis of the intestinal control domain contained in the human apoC-III gene. Schematic depiction of the human apolipoprotein A-I/C-III/A-IV gene cluster and recombinant DNAs used for transfection analysis. The 1.1-kb PstI (P) fragment of the apoC-III promoter was digested with SacI (S), ApaI (A), BsaI (B), and StuI (St) and subcloned into pA-I-CAT. The element I deletion mutant was created by digesting the apoC-III -780 to -520 containing plasmid with EcoNI, which removes a 50-bp fragment containing element I, followed by religation. Recombinant DNAs were transfected into near confluent, proliferating Caco-2 cells and CAT assays were performed. Results are presented as the mean activity (± S.D.) relative to that obtained with pA-I-CAT alone (n = 3 independent transfections for each construct).



There is very limited information available about the cis- or trans-acting factors that determine axial, temporal, and cell lineage-specific patterns of transcription in the intestinal epithelium, in part because in vivo assays are required to define these parameters. There are three ``axes'' (two spatial and one temporal) that need to be considered when evaluating endogenous gene or transgene expression in the mouse gut epithelium. The intestine's four principal epithelial cell lineages, enterocytic, goblet, enteroendocrine, and Paneth cell, are all derived from multipotent stem cells located near the base of each crypt(15, 16, 17) . The descendants of these stem cells undergo several rounds of cell division in the midportion of the crypt, creating a transit cell population. Each lineage subsequently completes its differentiation program during an orderly migration. Paneth cells differentiate during a downward migration to the base of the crypt(18) . Enterocytes, goblet cells, and enteroendocrine cells all differentiate as they migrate in vertical coherent bands from each crypt up an adjacent villus to an apical extrusion zone located near the villus tip(19, 20) . The migration-associated differentiation and exfoliation programs of these three lineages are completed in 2-5 days(21, 22, 23, 24) . The differentiation program of a given cell lineage varies along the duodenal-to-ileal axis (25, 26) . Thus, gene expression must be evaluated as a function of cell type, the state of differentiation of that cell (i.e. its position along the crypt-to-villus unit) and its location along the cephalocaudal axis.

We have now used transgenic mice and cultured cell lines to map further the intestinal enhancer contained in the human apoA-I/C-III/A-IV gene cluster, to define its effects on gene expression along the crypt-villus and duodenal-colonic axes, and to characterize nuclear proteins that may modulate its activity.


EXPERIMENTAL PROCEDURES

Transient Transfection Assays Using Established Human Cell Lines

DNA fragments derived from nucleotides -1300 to -200 of the human apoC-III gene were isolated after digestion with the restriction enzymes shown in Fig. 1. The DNA fragments were then blunt-ended with T4 DNA polymerase or the Klenow fragment of DNA polymerase I, and SacI linkers were added. The resulting DNAs were ligated to SacI-digested pA-I-CAT. pA-I-CAT, a gift of Dr. Jonathan Smith (Rockefeller University), contains nucleotides -256 to +22 of the human apoA-I gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene(27) . Plasmids were then purified by cesium chloride density gradient centrifugation.

Cell lines were obtained from the American Type Tissue Collection and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (HepG2 and HeLa) or minimal essential medium with 20% fetal calf serum (Caco-2). Cells were plated on 60-mm plastic dishes and incubated for 24 h at 37 °C under an atmosphere containing 95% air, 5% CO(2). Duplicate plates were transfected with 10 µg of each test plasmid, 5 µg of pCMV-betagal (27) , or 5 µg of pLEN4S (an hepatic nuclear factor 4, HNF-4, expression plasmid)(28) . Cells were shocked with 15% glycerol 24 h after transfection and harvested 24 h later. beta-Galactosidase and CAT assays were performed as described(27) . Equivalent amounts of extracted total cell protein were used in each CAT assay (equivalence was determined by defining the levels of beta-gal expression in cleared cell lysates). Relative CAT activity was determined by comparing the percentage of acetylated to unacetylated chloramphenicol obtained with each apoA-I/C-III-containing recombinant DNA to the percentage obtained with the pA-I-CAT control that lacked apoC-III sequences.

Generation of Transgenic Mice

Plasmid pH-A-I-14 contains an insert consisting of nucleotides -300 to -1 of the human apoA-I gene, its exons and introns, and 1.7 kb of 3` flanking sequence. This insert DNA was used to generate a pedigree of transgenic mice that expressed human apoA-I in the liver only(14) .

BamHI linkers were added to a 260-bp ApaI/SacI fragment that contains nucleotides -780 to -520 of the human apoC-III gene. It was subcloned into pH-A-I-14 at its unique BamHI site. A plasmid (pH-A-I-16) was identified containing this fragment in an orientation opposite to the direction of apoA-I gene transcription, just as it is the apoA-I/apoC-III/apoA-IV cluster on chromosome 11 (Fig. 1). BamHI linkers were also added to a 90-bp ApaI/StuI fragment that encompasses nucleotides -780 to -690 of apoC-III. The linker-containing DNA was ligated to BamHI-digested pH-A-I-14 and a recombinant plasmid (pH-A-I-17) was identified where the apoC-III fragment was in an orientation opposite to that of apoA-I.

The inserts from pH-A-I-16 and pH-A-I-17 were released with EcoRI/SalI, gel-purified, and microinjected into fertilized oocytes obtained from (C57Bl/6J CBA/J)F1 females that had been mated to (C57Bl/6J CBA/J)F1 males. Founders containing each construct were identified by probing Southern blots of PstI-digested tail DNA with a radiolabeled 0.7-kb DNA fragment that extends from the SacI site in exon 4 of the human apoA-I gene to a PstI site 300 bp 3` to this terminal exon(14) . Pedigrees were established and maintained by crosses to nontransgenic littermates. Animals were caged in microisolators under a strict light cycle (lights on at 0600 and off at 1800) and fed a standard chow diet ad libitum (no. 5001; Ralston Purina, St. Louis, MO).

Characterization of Transgenic Mice

Transgene copy number - Copy number was estimated by quantitative Southern blot analysis (14) of PstI-digested tail DNA prepared from F1 transgenic mice belonging to each pedigree.

Ribonuclease Protection Assays

Total cellular RNA was isolated from liver, the proximal third of the small intestine, and brain using guanidinium thiocyanate-phenol-chloroform(29) . RNA integrity was confirmed by denaturing formaldehyde-agarose gel electrophoresis(30) . The pattern of transgene expression in these tissues was defined by incubating a P-labeled, 230-nucleotide human apoA-I cRNA (5 10^6 cpm) with 10 µg of tissue RNA for 3 h at 65 °C(31) . The samples were then digested with RNase T1 (EC 3.1.27.3; Sigma; final concentration = 2 µg/ml) for 40 min at 30 °C. The reaction products were analyzed either by gel electrophoresis through a 8% polyacrylamide gel containing 8 M urea followed by autoradiography, or by a quantitative filter counting assay(31) . Note that previous studies verified that the human apoA-I cRNA probe does not produce a protected 230-nucleotide fragment when incubated with non-transgenic mouse liver or intestinal RNA(14) .

Single and Multilabel Light Microscopic Immunohistochemical Studies of Normal and Transgenic Mouse Tissues

Two hours prior to sacrifice, transgenic mice and their normal littermates received an intraperitoneal injection of an aqueous mixture of 5`-bromo-2`-deoxyuridine (BrdUrd, dose = 120 mg/kg body weight) and 5`-fluoro-2`-deoxyuridine (Sigma, dose = 12 mg/kg) to label proliferating cells in S-phase(32) . Immediately after sacrifice, the entire small intestine was removed en bloc, flushed with Bouin's solution, and then placed in this fixative for 6 h at room temperature. The intestine was washed three times with 70% ethanol, opened with an incision placed down the length of its cephalocaudal axis, rolled up around a forceps, and the resulting Swiss roll embedded in paraffin. Five-micrometer sections were cut, then deparaffinized in Hemo-De (Fisher Scientific), rehydrated in isopropanol and water, and placed in PBS-blocking buffer (phosphate-buffered saline (PBS, pH 7.4), 1% (w/v) bovine serum albumin (Sigma), 0.2% powdered skim milk (0.2%), and 0.3% (v/v) Triton X-100) for 15 min at room temperature. Primary antisera were diluted 1:1000 in PBS-blocking buffer. These well characterized antisera included goat anti-human apoA-I (supplied by Peter Herbert, Miriam Hospital, Providence, RI; (33) ), rabbit anti-mouse apoA-I (gift of N. Azrolan, Rockefeller University), rabbit anti-serotonin (Incstar Corp., Stillwater, MN; cf. (25) ), rabbit anti-cholecystokinin (CCK; residues 1-38; Peninsula Laboratories, Belmont, CA; cf. 25), rabbit anti-human lysozyme (Dako, Santa Barbara, CA;), and goat anti-BrdUrd(32, 33) . Following an overnight incubation at 4 °C, slides were washed in PBS followed by PBS-blocking buffer. Antigen-antibody complexes were detected using CY3-labeled donkey anti-goat secondary sera (human apoA-I; final dilution in PBS-blocking buffer = 1:500) or a fluorescein isothiocyanate (FITC)-labeled donkey anti-rabbit sera (mouse apoA-I, serotonin, CCK; final dilution = 1:100). (All secondary antibodies were obtained from Jackson Immunoresearch.) Goat anti-BrdUrd was labeled with biotin using a kit from Boerhinger Mannheim before it was applied to tissue sections. These antigen-antibody complexes were detected using fluorescein-labeled streptavidin (Sigma; 5 µg/ml).

Control experiments revealed that (i) none of the labeled secondary antibodies bound to tissue sections in the absence of primary antibody or in the presence of preimmune sera, (ii) none of the normal littermate tissues gave a detectable signal when incubated with the anti-human apoA-I sera followed by a fluoroprobe-tagged secondary antibody, and (iii) overnight incubation of a 1:1000 dilution of anti-human apoA-I sera with purified human apoA-I (Sigma; 10 µg/ml antisera) completely blocked its ability to react with gut epithelial cell lineages in mice that expressed a human apoA-I/C-III transgene (data not shown).

Sections were also incubated with FITC-labeled soybean agglutinin (Glycine max; final concentration = 5 µg/ml; obtained from Sigma; carbohydrate specificity = GalNAcalpha3GalNAc; GalNAcalpha/beta3/4Gal; cell specificity = goblet and Paneth cells plus enterocytes)(26) . The protocol for staining with soybean agglutinin was similar to that described above for antibodies, except that powdered skim milk was omitted from PBS-blocking buffer and the sections were incubated with the lectin for 16 h at 4 °C. After three washes in PBS, followed by one wash with PBS, coverslips were mounted on the sections with PBS/glycerol (1:1, v/v) containing 5% 1,4-diazabicyclo-[2.2.2]octane (Sigma).

Confocal Microscopy

A Molecular Dynamics Multiprobe 2001 inverted confocal microscope system, equipped with a 60 oil immersion objective lens, was used to scan sections of the Swiss rolls. These sections were stained as described above and scanned using a 50-µm aperture.

DNase I Protection Assays

Nuclear extracts were prepared from cells, 1 day after they had reached confluence, according to a protocol described by Dignam et al.(34) . The 260-bp SacI/ApaI fragment of the apoC-III gene containing intestinal expression activity was subcloned into the SacI site of pUC18. This fragment was then excised by digestion with EcoRI and PstI (sites in the vector's polylinker) and labeled at one end with [alpha-P]dATP using the Klenow fragment of DNA polymerase I. Probe (1 10^5 cpm) was mixed with 25 µg of nuclear protein extracted from various cultured cell lines or with bovine serum albumin (negative control) in a 20-µl reaction volume that contained KCl (final concentration 60 mM), HEPES (20 mM, pH 7.9), Ficoll (4% w/v), MgCl(2) (1 mM), dithiothreitol (1 mM), and poly(dI-dC) (0.25 mM). Following a 20-min incubation at room temperature, DNase I (EC 3.1.21.1; Sigma; prepared in 25 mM CaCl(2)) was added to a final concentration of 0.4 unit/µl. After a 5-min incubation at room temperature, the reaction was stopped by adding 4 µl of a solution containing 125 mM Tris, pH 8.0, 125 mM EDTA, and 3% SDS. Forty micrograms of proteinase K and 5 µg of yeast tRNA were then added and the reactions incubated at 65 °C for an additional 30 min. The mixture was extracted twice with phenol/chloroform, and nucleic acids were precipitated with ethanol, resuspended in loading dye, and subjected to electrophoresis through 6% polyacrylamide gels containing 6 M urea.

Gel Mobility Shift Assays

Complementary oligonucleotide probes corresponding to regions of the apoC-III promoter protected from DNase I digestion by Caco-2 nuclear extract proteins were synthesized, annealed, and end-labeled using [alpha-P]dATP and Klenow fragment of DNA polymerase I. Element I corresponds to nucleotides -742 to -725 of the human apoC-III gene. Element II corresponds to nucleotides -700 to -673. Element I-II was a combination of these two oligonucleotides. The coding strand sequences of these oligonucleotides were as follows: Element I, 5`-aattcTGGGTCCAGAGGGCAAAActgca-3`; Element II, 5`-aattCAAAGGCCTCGGGCTCTGAGCGGCCTTG-3`; Element I-II, 5`-aattcTGGGTCCAGAGGGCAAAAGGCCTCGGGCTCTG-3` (Underlined nucleotides were changed to C to produce mutant competitor oligonucleotides as shown in Fig. 5. Lowercase letters represent nucleotides used for labeling and cloning purposes.) Binding reactions were performed as described above for the footprinting assays, except that 1 µg of nuclear protein was used and reaction volumes were brought to 10 µl with 100 µg/ml salmon sperm DNA. P-Labeled oligonucleotide probe (1 10^5 cpm) was included in each reaction. The mixtures were incubated at room temperature for 20 min, and the products were analyzed by polyacrylamide gel electrophoresis. For supershift assays, 1 µl of undiluted rabbit anti-HNF-4 (W. Zhong, Rockefeller University) or ARP-1 sera (F. Gaudet, Harvard Medical School) was added and the solution incubated for an additional 20 min prior to gel electrophoresis.


Figure 5: DNase I protection analysis of nucleotides -780 to -520 of the human apoC-III gene. PanelA, a 260-bp SacI/ApaI apoC-III fragment was end-labeled and incubated with bovine serum albumin (lane1) or nuclear protein extracts prepared from proliferating HepG2 (lane2) and Caco-2 (lane3) cells; panelB, comparison of the 5` nontranscribed domains of the orthologous human and mouse apoC-III genes. Boxed areas correspond to footprinted elements I and II. The positions of G C substitutions made in each hexamer of elements I or II are shown (see text and Fig. 6C).




Figure 6: Gel mobility shift analysis using elements I and II contained in the apoC-III gene promoter. PanelA, double-stranded oligonucleotides corresponding to elements I, II, and I-II were synthesized, radiolabeled, and incubated with no protein (1) or nuclear proteins prepared from near-confluent, proliferating HeLa (2), HepG2 (3), and Caco-2 (4) cells. b represents a complex formed with HepG2 and Caco-2 extracts, while complex a is Caco-2-specific. Other bands are not specific. Panel B, a 100-fold molar excess of unlabeled oligonucleotide was used as competitor in assays containing Caco-2 nuclear extracts. Competitors used were elements I, II, I-II, and a nonspecific oligonucleotide (ns). Panel C, a 100-fold molar excess of unlabeled mutant competitor oligonucleotide (see Fig. 5B and ``Experimental Procedures'') were mixed with Caco-2 nuclear extracts as described in panelB. Mutants are designated Im1 and 2 (mutations in the first and second putative hexamers of element I, respectively) and IIm1 and 2 (mutations in the first and second hexamers of element II, respectively). PanelD, nuclear extracts prepared from Caco-2 cells were incubated with double-stranded oligonucleotides corresponding to element I. HNF-4-specific (lane2) and ARP-1 specific (lane3) antibodies were then added and incubation continued. Complex b is supershifted by the HNF-4 antibody (ss).




RESULTS

Transient Transfection Assays

To define the element(s) that control apoA-I gene expression in the intestine, transient transfection assays were performed. Different fragments from the 1.1-kb apoC-III fragment, previously shown to be necessary for intestinal expression (14) , were placed upstream of nucleotides -256 to +22 of the human apoA-I gene linked to a CAT reporter (apoA-I-CAT), and the resulting recombinant plasmids were used for transient transfections of an established human enterocyte-like cell line (Caco-2), a nonintestinal epithelial cell line (HeLa), and a hepatocyte-like cell line (HepG2). Transfections were performed in proliferating cells when they had achieved 80-90% confluence. Earlier studies had established that the human apoA-I gene is expressed in both HepG2 and Caco-2 cells under these growth conditions(8) .

Addition of nucleotides -1300 to -520 of human apoC-III to apoA-ICAT produces a 4-fold increase in reporter expression in near confluent Caco-2 cells (Fig. 1). A 90-bp fragment spanning nucleotides -780 to -690 produce a comparable increase in CAT levels (6-fold). Nucleotides -760 to -710 appear necessary for this enhancement, since their deletion eliminates the effect (Fig. 1). The positive effects produced by nucleotides -780 to -690 are cell-specific; no increase in CAT activity was detectable in HepG2 or HeLa cells over that produced by apoA-ICAT alone (data not shown).

Studies in Transgenic Mice

To confirm that the elements controlling apoA-I gene expression in cultured cells also control intestinal expression in vivo, a number of pedigrees of transgenic mice were generated (Fig. 2A). Four lines were established with a construct that consisted of nucleotides -300 to -1 of the human apoA-I gene, its exons and introns, and 1.7 kb of its 3` flanking sequences, followed by a 260-bp fragment containing nucleotides -780 to -520 of the human apoC-III gene (in its ``normal'' opposite orientation to the apoA-I gene). One of these lines, A16, was used for further analysis. Similarly, two pedigrees were established with a related construct lacking nucleotides -689 to -520 of apoC-III (i.e. the only apoC-III gene sequences represented were contained in the 90-bp element spanning nucleotides -780 to -690). One line with this construct, A17, was used for further analysis. Two previously characterized pedigrees of transgenic mice were used as reference controls: (i) members of line 427 contain nucleotides -256 to -1 of human apoA-I, its exons and introns, plus 80 bp of 3` flanking sequence and express human apoA-I mRNA only in the liver(14) ; (ii) members of line A14 contain nucleotides -300 to -1 of human apoA-I, its exons and introns, and 1.7 kb of 3` flanking sequence, followed by nucleotides -200 to -1300 of the human apoC-III gene and, as noted above, express human apoA-I mRNA in their livers and small intestine(14) .


Figure 2: RNase protection analysis of transgene expression in adult mouse liver, intestine, and brain. A, schematic representation of the recombinant DNAs used to produce mouse lines 427 (transgene expression confined to the liver), A14 (transgene expression in liver and small intestine), A16, and A17. B, a human apoA-I specific riboprobe was hybridized to yeast tRNA (tRNA), total cellular RNA isolated from 1-day post-confluent HepG2 cells (HG2), and total cellular RNA isolated from the liver (L), proximal third of the small intestine (I), and brain (B) from four lines of adult transgenic mice. Human apoA-I mRNA yields a protected fragment of 230 bases (arrow).



Adult mice belonging to all four pedigrees produce human apoA-I mRNA in their livers (Fig. 2B and Table 1). The steady state levels of human apoA-I mRNA in liver RNA prepared from adult members of each pedigree are similar (data not shown). This is not the case in the proximal third of the small intestine, where the steady state levels of human apoA-I mRNA vary between comparably aged members of the four lines. Nonetheless, the range of mRNA concentrations in duodenum/proximal jejunum is similar to that observed in adult transgenic mice with nucleotides -200 to -1300 of apoC-III (compare lines A14 and A16 in Fig. 2B).



Deletion of nucleotides -689 to -520 from the 260-bp apoC-III sequence that supports small intestinal expression abolishes human apoA-I expression in the duodenum and proximal jejunum but has little effect on steady state human apoA-I mRNA levels in liver (Table 1). This reduction is not an insertion site effect since it was observed in adult members of two transgenic pedigrees (data not shown). No apoA-I expression was detectable in the brains of any transgenic mice from any of these lines (Fig. 2B).

Single and multilabel immunocytochemical surveys were conducted to compare the cell lineage-specific, differentiation-dependent, and regional patterns of expression of the various transgenes with the pattern of expression of the endogenous mouse apoA-I gene. Adult, nontransgenic littermates contain immunoreactive apoA-I in villus-associated enterocytes. No immunoreactive protein is detectable in proliferating and nonproliferating crypt epithelial cells (Fig. 3A). Multilabel studies with antibodies directed against mouse apoA-I and a Paneth cell-specific marker (lysozyme) or a lectin (soybean agglutinin) that reacts with glycoconjugates produced in the majority of adult (C57Bl/6J CBA/J)F1 small intestinal goblet cells indicated that neither of these lineages contains detectable levels of the apolipoprotein (data not shown).


Figure 3: Multilabel immunocytochemical analysis reveals that nucleotides -1300 to -200 and nucleotides -780 to -520 of the human apoC-III gene direct human apoA-I expression to proliferating and nonproliferating jejunal crypt epithelial cells as well as villus-associated enterocytes. The patterns of apoA-I expression along the crypt-to-villus axis of adult mice belonging to pedigrees A14 (containing nucleotides -1300 to -200 of the apoC-III gene) and A16 (containing nucleotides -780 to -520 of the apoC-III gene) are indistinguishable. Panels A-C, a section of the proximal jejunum of a 10 month-old mouse from pedigree A14 (apoC-III). The section was incubated with rabbit anti-mouse and goat anti-human apoA-I sera. Antigen-antibody complexes were visualized with FITC-conjugated donkey anti-rabbit and CY3-conjugated donkey anti-goat sera. Panel A, mouse apoA-I is confined to villus-associated enterocytes (solidarrowheads). Immunoreactive protein is seen associated with the Golgi-apparatus of these cells and in the lamina propria (openarrow), reflecting basolateral secretion of the apolipoprotein. Panel B, the same section as in panelA, showing the distribution of human apoA-I in villus-associated enterocytes, the lamina propria, as well as the crypt. PanelC, a double exposure of the same section as shown in A and B, demonstrating co-expression of mouse and human apoA-I in villus-associated enterocytes (yellow). However, only human apoA-I is apparent in crypt epithelial cells (red-orange, closedarrows). Panels D-F, a 10-month-old mouse from pedigree A16 (apoC-III) received an intraperitoneal injection of BrdUrd 2 h before sacrifice. A section of the proximal jejunum was incubated with goat anti-human apoA-I and biotinylated goat anti-BrdUrd sera. Antigen-antibody complexes were visualized with CY3-conjugated donkey anti-goat sera and FITC-conjugated streptavidin. Panel D, proliferating S-phase cells in jejunal crypts detected with the anti-BrdUrd sera (green nuclear staining, openarrows). Panel E, same section as in D but stained with anti-human apoA-I sera. Note that the distribution of human apoA-I along the crypt-to-villus axis is identical to that observed in comparably aged mice belonging to pedigree A14 (see panelB). PanelF, a double exposure directly demonstrating human apoA-I (orange) in proliferating (green, solidarrows) and nonproliferating crypt epithelial cells. Bar = 25 µm.



Multilabel immunocytochemical studies of enteroendocrine cells in the proximal small intestine of adult mice (20, 25) suggest that a committed enteroendocrine cell progenitor yields descendants that follow one of three principal differentiation pathways during their migration from the crypt to the villus tip. One pathway yields cells that initially express substance P alone, followed by substance P and serotonin, followed by secretin alone or serotonin alone. Another pathway produces cells that sequentially express gastrin plus CCK, followed by gastrin plus CCK plus glucagon-like peptide-1 (GLP-1), followed by gastrin plus CCK or secretin alone. A third pathway yields cells that only express gastric inhibitory peptide. Serotonin- and CCK-producing cells are the most abundant enteroendocrine cell subpopulations in the adult mouse duodenum(25) . Multilabel studies using anti-serotonin or anti-CCK sera revealed that enteroendocrine cells in the first two pathways do not contain detectable levels of mouse apoA-I (data not shown).

Immunocytochemical analyses indicated that the expression domain of the mouse apoA-I gene in nontransgenic adult (C57Bl/6J CBA/J)F1 mice extends from villus-associated enterocytes in the proximal boundary of the duodenum to the distal jejunum (duodenum, jejunum, and ileum are operationally defined in this study as the proximal, middle, and distal thirds of the small intestine).

Comparably aged adult transgenic littermates containing the 260-bp apoC-III element (nucleotides -780 to -520) also express human apoA-I in their villus-associated enterocytes. Moreover, the regional pattern of human apoA-I expression along the duodenal-to-ileal axis mimics that of mouse apoA-I. Unlike the intact endogenous mouse apoA-I gene, the transgene is expressed in proliferating and nonproliferating duodenal and jejunal crypt epithelial cells (Fig. 3, D-F). Multilabel light microscopic studies using lysozyme and the soybean agglutinin failed to detect human apoA-I in duodenal, jejunal, or proximal ileal Paneth cells or in lectin-positive goblet cells (data not shown). However, 20% of CCK-producing and 0.1% of serotonin-producing enteroendocrine cells co-express the transgene (Fig. 4, A-C). These cells do not produce detectable amounts of mouse apoA-I in transgenic mice or, as noted above, in their normal littermates.


Figure 4: Confocal microscopic studies disclose anomalous expression of human apoA-I in serotonin-producing enteroendocrine cells of adult transgenic mice. A section of the proximal intestine of a 10-month-old transgenic mouse containing apoA-I/apoC-III was incubated with goat anti-human apoA-I and rabbit anti-serotonin sera. Antigen-antibody complexes were visualized with CY3-conjugated donkey anti-goat and FITC-conjugated donkey anti-rabbit sera. Panel A, human apoA-I is evident in the Golgi apparatus of enterocytes (closedarrowheads) and in the lamina propria (closedarrow). Diffuse cytoplasmic staining is also evident in a single cell (openarrowheads). Panel B, view of the same section at the same focal plane as shown in panelA but stained with anti-serotonin sera. A single enteroendocrine cell with diffuse cytoplasmic staining is evident. Panel C, overlaid images demonstrate co-expression of human apoA-I and serotonin in the enteroendocrine cell (yellow-green). Bar = 1 µm.



Precocious expression of human apoA-I in crypt epithelial cells and inappropriate expression in CCK- and serotonin-producing enteroendocrine cells is also evident in transgenic mice that contain the construct with nucleotides -1300 to -200 of the human apoC-III gene. The distribution of human apoA-I along the crypt-to-villus axis and the fractional representation of apoA-I-positive CCK and serotonin cells in adult mice belonging to these pedigrees are indistinguishable from the patterns observed in mice that only contain nucleotides -780 to -520 (Fig. 3, B, C, E, and F, plus data not shown). Light microscopic surveys also indicated that adult mice with the 1.2-kb apoC-III fragment do not produce human apoA-I in members of the Paneth or goblet cell lineages (data not shown).

Together, these findings allow us to conclude that nucleotides -780 to -520 of the human apoC-III gene contain cis-acting sequences that are sufficient to direct a duodenal-to-jejunal pattern of expression of the upstream human apoA-I gene in villus-associated enterocytes of adult transgenic mice which mimics that of the intact endogenous mouse apoA-I gene. However, nucleotides -780 to -520 lack cis-acting sequences that suppress precocious expression of human apoA-I in proliferating and nonproliferating crypt epithelial cells and in subpopulations of CCK- and serotonin-producing enteroendocrine cells. Moreover, these suppressors of crypt and enteroendocrine cell expression are not represented in the region encompassed by nucleotides -1300 to -200.

Elements Contained within Nucleotides -780 to -520 of Human ApoC-III Bind Caco-2 Nuclear Proteins

Nuclear extracts, prepared from Caco-2 and HepG2 cells 1 day after they reached confluence, were incubated with the 260-bp apoC-III fragment. Footprinting studies using DNase I indicated that two 21-bp sites are protected by proteins present in Caco-2 extracts (Fig. 5A, lane3). One site extends from nucleotides -745 to -725 (element I), while the other site spans nucleotides -700 to -680 (element II). Element I is also protected by HepG2 nuclear proteins. Element II is not protected (Fig. 5A, lane2).

The orthologous human and mouse apoC-III genes show a high degree of sequence similarity within, but not outside of, these two protected sites (Fig. 5B). The sequences of elements I and II were used to search the transcription factor binding site data base (fastsa.tfd; (35) ). Element I (GGGTCCAGAGGGCAAAATAGGG) shows significant similarity to the consensus recognition site of the nuclear hormone receptor superfamily(36) . The consensus sequence is a direct repeat (DR) of the hexamer AGGTCA with a variable number of intervening nucleotides from 0 to 5. The sequence of element I most closely resembles a DR-2 motif with the half-sites GGGTCC and AGGGCA and two spacer nucleotides (Fig. 5B). Element II has no obvious sequence similarity to known transcription factor binding sites.

Double-stranded oligonucleotides corresponding to elements I, II, and I plus II (I-II) were then synthesized, radiolabeled, and incubated with nuclear extracts from HeLa, HepG2, and Caco-2 cells. When element I was used as the template DNA, one complex forms after incubation with HepG2 and Caco-2 extracts (labeled b in Fig. 6A). Another complex (a in Fig. 6A) is Caco-2- specific. One Caco-2-specific complex forms with element II (nucleotides -700 to -680) (Fig. 6A, a). A similar complex forms when Element I-II is used (Fig. 6A). Element II blocks assembly of the radiolabeled complex a formed between Caco-2 nuclear proteins and element I. Conversely, element I blocks formation of complex a that forms when element II binds Caco-2 nuclear proteins (Fig. 6B).

Mutational analysis revealed that two of the direct repeat half-sites are important for complex a formation. Oligos were synthesized where the second G in each hexamer of element I or II was changed to a C (see Fig. 5B). When nucleotides -740 or -689 were mutated, complex a was not formed (Fig. 6C).

Several members of the superfamily of nuclear hormone receptor transcription factors have been shown to be involved in regulating apolipoprotein gene expression(10, 37, 38) . Gel mobility shift assays were performed using antibodies to two of these factors, hepatic nuclear factor 4 (HNF-4) and apolipoprotein regulatory protein 1 (ARP-1), Caco-2 nuclear extracts, and radiolabeled element I to determine whether complex a or b was formed by interactions between this element and either of these proteins. Complex b, which forms with both Caco-2 or HepG2 nuclear extracts, is supershifted by anti-HNF-4 (Fig. 6D, lane2), indicating the presence of HNF-4 or an HNF-4-like protein. The Caco-2-specific complex a is not affected by addition of this antibody preparation. Moreover, none of the complexes were supershifted after addition of anti-ARP-1 (lane3). Comparable studies were also performed with element II. Neither anti-HNF-4 nor anti-ARP-1 produced a supershift of complex a formed when element II was incubated with HepG2 or Caco-2 extracts (data not shown).

We performed transient transfection assays to examine the effects of HNF-4 on apoA-I expression in near-confluent, proliferating Caco-2 cells. Introduction of an HNF-4 expression vector into cells containing pA-I-CAT is associated with a 3-fold increase in CAT activity (Fig. 7). This increase reflects an interaction with a previously described HNF-4 binding site located at nucleotides -214 to -192 of the human apoA-I gene(39) . When the HNF-4 expression plasmid was co-transfected into cells containing either (i) the 260-bp apoC-III fragment encompassing elements I and II linked to apoA-I-CAT or (ii) the 90-bp apoC-III fragment containing element I and only half of element II linked to apoA-I-CAT, CAT activity increased 20-26-fold compared to apoA-I-CAT (Fig. 7). The increase produced by the 260-bp apoC-III fragment was not significantly different from that produced by the 90-bp fragment. When element I was deleted from the 260-bp construct, HNF-4 responsiveness was not observed (Fig. 7). The effect was cell-specific, i.e. overexpression of HNF-4 had no effect on CAT activity in proliferating HepG2 or HeLa cells containing either of the two apoC-III/apoA-I-CAT DNAs (data not shown). In addition, when the 260- and 90-bp apoC-III fragments were linked to an adenovirus major late promoter-CAT reporter construct and transient transfection assays were performed as described above, CAT activity was greatly increased when cotransfection of HNF-4 expression plasmids occurred (data not shown).


Figure 7: Effect of HNF-4 on expression of human apoA-I in near confluent Caco-2 cells. HNF-4 expression plasmids were co-transfected (+) with plasmids containing human apoC-III nucleotides -780 to -520 (I+II), -780 to -690 (I), or an apoC-III deletion mutant (see Fig. 1), linked to apoA-I-CAT. Results are presented as mean activity (± S.D.) relative to that obtained with pA-I-CAT alone (n = 3 independent transfections for each construct).



Together, these data suggest that HNF-4 binding to element I (nucleotides -745 to -725) increases apoA-I transcription in Caco-2 cells and may be one interaction that contributes to the ability of nucleotides -780 to -520 of apoC-III to direct human apoA-I gene expression in the small intestine of transgenic mice. It is apparently not the only interaction required given the inability of nucleotides -780 to -690 to perform the same function in vivo. Element II may also be required for intestinal expression in vivo. Although it does not appear to bind HNF-4, element II's ability to compete with element I for binding to Caco-2-specific nuclear proteins suggests that the two sites may either be recognized by a common or similar transcription factor(s) and/or that factor binding to one site may influence binding at the other site.


DISCUSSION

The experimental results presented in this report indicate that intestinal expression of the apoA-I gene is dependent upon the presence of an enhancer element located 9.2 kb 3` to its promoter. The human apoC-III gene is expressed predominantly in hepatocytes and villus-associated enterocytes, whereas apoA-IV is expressed exclusively in villus-associated enterocytes(40, 41, 42) . Preliminary studies conducted in transgenic mice indicate that intestinal expression of human apoC-III and A-IV also requires the presence of sequences located in the 5` nontranscribed domain of the apoC-III gene(43) . Thus, the enhancer or enhancers positioned in this region may operate to effect intestinal expression of the entire 17-kb A-I/C-III/A-IV locus, i.e. they represent a locus control region. It will be particularly important to assess the ability of either nucleotides -1300 to -200 or -780 to -520 of human apoC-III to direct appropriate lineage-specific, differentiation-dependent, and cephalocaudal patterns of human apoC-III and apoA-IV gene expression in transgenic mouse gut. Specifically, do the 260- or 1100-bp sequences produce precocious activation of apoC-III or apoA-IV synthesis in proliferating crypt epithelial cells and/or support expression in cell lineages other than enterocytic?

Two features of the ``inappropriate'' pattern of apoA-I gene expression noted in the small intestine of apoA-I/C-III transgenic mice have been encountered during the course of functional mapping studies of transcriptional regulatory elements in other nonhomologous genes that are expressed in villus-associated enterocytes, lending additional support to several evolving concepts about regulation of gene expression in this epithelial lineage. First, distinct elements appear to control expression in enterocytes distributed along the crypt-to-villus and duodenal-to-ileal axes. This is true for the rat intestinal fatty acid binding protein gene (Fabpi; (32) ), the rat liver fatty acid-binding protein gene (Fabpl; (44) ), the mouse ileal lipid-binding protein gene (Ilbp; (45) ), and the human sucrase-isomaltase gene(46) . Correct cephalocaudal patterns of expression appear to be determined by cis-acting activators as well as spatial suppressors (Fabpi, Fabpl, Ilbp). Prohibition of precocious activation of expression in proliferating and nonproliferating crypt epithelial cells requires the presence of cis-acting suppressors (Fabpi, Fabpl, sucrase-isomaltase). Second, precocious activation of transgene expression in the crypt is often associated with inappropriate expression in other cell lineages. There is an apparent correlation between the cell stratum where transgene expression is first initiated and the extent of inappropriate expression in these lineages: the closer to the stem cell zone (postulated to be at stratum 5; (47) and (48) ), the greater the number of lineages, other than the enterocytic, that support expression (32, 44) . This may reflect differing degrees of commitment of the cells that support initiation of transgene expression; inappropriate expression in nonenterocytic lineages could arise due to the absence of cis-acting elements in the various transgenes that normally bind lineage-specific repressors produced by cells after they are allocated to enteroendocrine, goblet, and Paneth cell lineages. Transgenes offer a set of instruments for measuring where and defining how lineage allocation occurs in the crypt. By crossing mice containing the human apoA-I/C-III fusion gene, described in this study, with mice that contain nucleotides -596 to +21 of rat Fabpl linked to a human growth hormone reporter (a transgene which is expressed deeper in the crypt and in all four lineages; cf. (44) and (49) ), it may be possible to determine which crypt epithelial cell populations support expression of one or both transgenes using double label electron microscopic immunocytochemistry (50) . The results could be correlated with previously published transmission electron microscopic studies of the morphologic differentiation of the multipotent stem cells' descendants(18, 21, 22, 23, 24) . Moreover, these bi-transgenic mice could be used for isolation of gut stem cells, their early descendants, and/or members of specific lineages if the reporters encode gene products that allow for positive and negative selection (e.g. by fluorescence-activated cell sorting) ex vivo.

Finally, it is important to emphasize the difficulty in using established enterocyte-like cell lines such as Caco-2 to predict the activity of specific cis-acting transcriptional regulatory sequences in vivo, or the functional importance of trans-acting factors that bind to these sites. For example, the similarities in activities of the 90- and 260-bp apoC-III elements in Caco-2 cells contrasts with their markedly different capacities to support transgene expression in the mouse gut epithelium. Defining the state of proliferation and differentiation of Caco-2 cells (and whether they support expression of the intact endogenous copy of the gene being studied) is certainly important when designing and analyzing experiments conducted in this line. Nonetheless, a previous study that correlated the pattern of expression of various Fabpi reporter transgenes along the crypt-to-villus axis of mice with their relative activities in proliferating/preconfluent and ``differentiated''/postconfluent Caco-2 cells suggested that ``differentiated''/postconfluent cells have a transcriptional regulatory environment which resembles that of enterocytes positioned in the upper crypt rather than more differentiated descendants located on the villus(51) . It appears that transgenic mice will remain a requisite final audit of the significance (relevance) of transient transfection studies conducted in cell lines derived from the gut epithelium.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL08695 (to J. G. B.), HL33714 (to J. L. B.), and DK30292 (to J. I. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a postdoctoral fellowship from the American Heart Association, Missouri Affiliate.

To whom correspondence should be addressed: Laboratory of Biochemical Genetics and Metabolism, Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-7700; Fax: 212-327-7165.

(^1)
The abbreviations used are: apo, apolipoprotein; ARP-1, apolipoprotein regulatory protein-1; CAT, chloramphenicol acetyltransferase; CCK, cholesytokinin; DR, direct repeat; FITC, fluorescein isothiocyanate; HDL, high density lipoproteins; HNF-4, hepatic nuclear factor-4; PBS, phosphate-buffered saline; BrdUrd, 5`-bromo-2`-deoxyuridine; bp, base pair(s); kb, kilobase pair(s).


ACKNOWLEDGEMENTS

We thank Bill Coleman for technical assistance with the confocal microscopic studies.


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